ANALYSIS AND DIAGNOSIS OF TARGETED MEDICAL AND …

ANALYSIS AND DIAGNOSIS OF TARGETED MEDICAL

AND GENETIC DISORDERS IN HUMAN POPULATION

OF KHYBER PAKHTUNKHWA, PAKISTAN

Submitted By

MUHAMMAD ISMAIL KHAN

Ph.D Scholar

Research Supervisor

Dr. MUHAMMAD ZAHID

Associate Professor

Co-Supervisor

Dr. MUSHARRAF JELANI

Associate Professor

DEPARTMENT OF ZOOLOGY

ISLAMIA COLLEGE, PESHAWAR PAKISTAN

SESSION: 2014-2017

` ii

ANALYSIS AND DIAGNOSIS OF TARGETED MEDICAL

AND GENETIC DISORDERS IN HUMAN POPULATION

OF KHYBER PAKHTUNKHWA, PAKISTAN

Submitted By


Reg #: 2010/ICP/MS-0183

Thesis submitted to the department of Zoology Islamia College Peshawar, for the

partial fulfillment of the requirement for the Degree of DOCTOR OF

PHILOSOPHY (Ph.D) in Zoology

DEPARTMENT OF ZOOLOGY

ISLAMIA COLLEGE, PESHAWAR PAKISTAN

SESSION: 2014-2017

` iii

APPROVAL CERTIFICATE

It is to certify that this thesis entitled “Analysis and Diagnosis of Targeted Medical

and Genetic Disorders in Human Population of Khyber Pakhtunkhwa,

Pakistan”submitted by Mr. Muhammad Ismail khan is hereby approved as partial

fulfillment for the award of degree of Doctor of Philosophy in Zoology

Supervisor: _______________________________

Dr. Muhammad Zahid Associate Professor

Department of Zoology

Islamia College Peshawar

Co-Supervisor: _______________________________

Dr. Musharraf Jelani Associate Professor

Centre of OMICs

Islamia College Peshawar

External Examiner 1: _______________________________

Dr. Sana Ullah Khan Chairman


University of Peshawar

External Examiner 2: _______________________________

Prof. Dr. Abdul Hamid Jan Ex-Chairman


University of Peshawar

Dated: 27/12/2019

` iv

DEDICATION

I dedicate these efforts to the World greatest man MUHAMMAD

(P.B.U.H) and His COMPANIONS (R.T.A) who is

the beacon house in the dark ages across

the universe.

` v

TABLE OF CONTENT

APPROVAL CERTIFICATE.................................................................................. III

DEDICATION........................................................................................................... IV

TABLE OF CONTENT .............................................................................................. V

LIST OF TABLES ................................................................................................. VIII

LIST OF FIGURES .................................................................................................. IX

LIST OF ACRONYMS USED ................................................................................... X

ACKNOWLEDGEMENTS .................................................................................... XII

ABSTRACT ............................................................................................................ XIII

CHAPTER - 1 ............................................................................................................... 1

INTRODUCTION........................................................................................................ 1

1.1 GENETIC DISORDERS .......................................................................................... 1

1.1.1 Types of Genetic Disorder ......................................................................... 2

1.2 RARE GENETIC DISEASES ................................................................................... 5

1.2.1 Prevalence of Rare Diseases and their Significance in Health Care ........ 6

1.2.2 Causes of Birth Defects of Rare Diseases.................................................. 7

1.2.3 Dysmorphology and Syndromology – history and general perspectives ... 7

1.3 DIFFERENT TYPES OF GENETIC DISEASES IN HUMANS ........................................ 8

1.3.1 Bone Deformities or Skeletal Dysplasias ................................................... 8

1.3.2 Classification of Bone Deformities ............................................................ 9

1.3.3 Group I: Bone Deformities Involving Defects in Extracellular Structural

Proteins ................................................................................................................ 10

1.3.4 Group II: Bone Deformities due to Defects in metabolic pathways ........ 11

1.3.5 Group III: Bone Diseases due to Abnormal Folding and Degradation of

Macromolecules ................................................................................................... 11

1.3.6 Group IV: Disorders of Imperfection in Hormones and Signal

Transduction Mechanisms ................................................................................... 19

1.3.7 Group V: Bone Disorders Involving Nuclear Proteins and Transcription

Factors ................................................................................................................. 24

1.4 HUMAN HEREDITARY DISORDERS OF INTELLECTUAL DISABILITY .................... 27

1.4.1 Etiology of intellectual disability ............................................................. 27

1.4.2 Classification of Id Based on Intelligence Quotient (Iq) ......................... 28

1.4.3 Prevalence................................................................................................ 28

1.4.4 X-Linked ID (XLID) ................................................................................. 29

1.4.5 Categories of X-Linked ID ....................................................................... 30

1.4.5.1 Syndromic XLID................................................................................... 30

1.4.5.2 Non-syndromic XLID ........................................................................... 31

1.4.6 Selected XLID Genes ............................................................................... 31

1.5 HUMAN HEREDITARY SKIN DISORDERS ............................................................ 34

1.5.1 Human Skin .............................................................................................. 35

1.5.2 Epidermis ................................................................................................. 35

1.5.3 Dermis ...................................................................................................... 35

1.5.4 Hypodermis .............................................................................................. 36

` vi

1.5.5 Other Ectodermal Appendages Involved In Human Hereditary Skin

Disorders.............................................................................................................. 36

1.5.6 Hair .......................................................................................................... 36

1.5.7 Sweat Glands ........................................................................................... 37

1.6 HUMAN SKIN DISORDER ................................................................................... 37

1.6.1 Alopecias .................................................................................................. 37

1.6.2 Isolated Alopecias .................................................................................... 38

1.6.3 Congenital Atrichia (Atrichia With Papular Lesions) ................................ 38

1.6.4 Monilethrix ............................................................................................... 38

1.6.5 Localized Autosomal Recessive Hypotrichosis ........................................ 39

1.6.6 Hypotrichosis Simplex ............................................................................. 40

1.6.7 Autosomal Dominant Woolly Hair/Hypotrichosis ................................... 41

1.7 ASSOCIATED ALOPECIAS .................................................................................. 41

1.7.1 Alopecia with Mental Retardation Syndrome .......................................... 41

1.7.2 Hypotrichosis with Juvenile Macular Dystrophy .................................... 42

1.7.3 Digenic Hereditary Hair Loss ................................................................. 42

1.7.4 Androgenetic Alopecia ............................................................................. 43

1.7.5 Alopecia Areata (Aa) ............................................................................... 43

1.7.6 Ectodermal Dysplasias ............................................................................ 44

1.7.7 Ectodermal Dysplasia of Hair, Nail and Teeth ....................................... 44

1.7.8 Ectodermal Dysplasia of Hair and Nail .................................................. 45

1.7.9 Hypohidrotic Ectodermal Dysplasia........................................................ 45

1.7.10 Odonto-Onycho-Dermal Dysplasia ..................................................... 46

1.7.11 Oligodontia .......................................................................................... 46

1.7.12 X-Linked Recessive Isolated Oligodontia ............................................ 47

1.7.13 Isolated Congenital Nail Dysplasiah ................................................... 47

1.7.14 Isolated Congenital Nail Clubbing ...................................................... 47

1.7.15 Cutis Laxa Syndrome ........................................................................... 48

1.7.16 Ectodermal Dysplasia-Cutaneous Syndactyly Syndrome. ................... 48

1.7.17 Trichorhinophalangeal Syndromes ...................................................... 48

1.7.18 Palmoplantar Keratodermas (Ppks) Syndrome ................................... 49

1.8 STRATEGIES OF GENETIC TESTING FOR VARIOUS GENETIC DISORDERS ........... 50

1.8.1 Hunting for the Causative Genes ............................................................. 50

1.8.2 Genome-Wide Mapping Phase ................................................................... 51

1.8.3 Genome-wide homozygosity mapping...................................................... 52

1.9 CURRENT ADVANCES IN THE AREA (WORLD WIDE) ......................................... 57

1.9.1 Fine-mapping and re-sequencing by next-generation sequencing (NGS)

57

CHAPTER - 2 ............................................................................................................. 61

MATERIALS AND METHODS .............................................................................. 61

2.1 STUDY SUBJECTS AND ETHICAL APPROVAL ..................................................... 61

2.2 CLINICAL SUMMARY AND INCLUSION CRITERIA FOR FAMILIES ........................ 61

2.3 EXCLUSION CRITERIA APPLIED ON FAMILIES. .................................................. 62

2.4 PEDIGREE CONSTRUCTION ................................................................................ 62

2.5 COLLECTION OF SAMPLES ................................................................................. 62

2.6 WHOLE EXOME SEQUENCING ........................................................................... 63

2.7 DATA QUALITY CHECK OR SEQUENCING .......................................................... 63

2.8 INITIAL DATA ANALYSIS PIPELINE ................................................................... 63

` vii

2.9 SECONDARY DATA ANALYSIS STRATEGY ......................................................... 64

2.10 VALIDATION BY SANGER SEQUENCING ......................................................... 64

2.11 COMPUTATIONAL PREDICTION FOR THE FILTERED VARIANTS ...................... 65

2.12 MENDELIAN INHERITANCE CHECK OR TRANSMISSION GENETICS ................ 66

2.13 POPULATION SCREENING OR ETHNICAL MATCH CONTROL ........................... 66

2.14 DATA REPORTING ......................................................................................... 66

CHAPTER - 3 ............................................................................................................. 67

RESULTS ................................................................................................................... 67

3.1 FAMILY A CLINICAL FINDINGS ......................................................................... 67

CHAPTER - 4 ............................................................................................................. 83

DISCUSSION ............................................................................................................. 83

CONCLUSION .......................................................................................................... 86

REFERENCES ........................................................................................................... 87

` viii

LIST OF TABLES

TABLE 2.1: THE LIST OF CANDIDATE VARIATIONS ....................................................... 65

` ix

LIST OF FIGURES

FIGURE 1.1: THE CONTINUOUS SPECTRUM OF DISEASE INTERCONNECTION ..................... 2

FIGURE 1.2: VARIANTS WITH WIDE RANGE OF DIFFERENT EFFECT SIZE (OR, ODDS

RATIO) AND MINOR ALLELE FREQUENCIES (MAF). ..................................... 4

FIGURE 2.1: PEDIGREE OF THE PAKISTANI FAMILY AFFECTED BY STRIATE

PALMOPLANTAR KERATODERMA (PPKS). ................................................. 71

FIGURE 2.2: THE CLINICAL PRESENTATION OF PATIENTS OF FAMILY (A) SHOWING

KERATODERMA OVER THE PALMS.............................................................. 74

` x

LIST OF ACRONYMS USED

GD Genetic Disorders

CM Cousin Marriage/ Consanguineous Marriage

CC Cross Cousin

AD Autosomal Dominant

AR Autosomal Recessive

DR Distantly Related

XLD X-linked Dominant

YLD Y-linked Dominant

ML Mitochondrial linked

DTC Double Third Cousin

MD Mendelian Disease

OM Online Mendelian

RGD Rare Genetic Diseases

FC First Cousin

BD Birth Defect

IGHD Isolated Growth Hormone Deficiency

HL Hearing Loss

HI Hearing Impairment

KP Khyber Pakhtunkhwa (Province-latest name)

KPK Khyber Pakhtunkhwa (Province-early name)

DS Down Syndrome

SEDC Spondyloepiphyseal Dysplasia Congenital

MPS Mucopolysaccharidosis

HS Hunter Syndrome

MBD Mothers‘ Brother Daughter

ISS Idiopathic Short Stature

IQ Intelligence Quotient

XLID X-Linked ID

HHSD Human Hereditary Skin Disorders

AA Associated Alopecias

` xi

COL Collagen

AA Alopecia Areata

OODD Odonto-Onycho-Dermal Dysplasia

EDCSS Ectodermal Dysplasia-Cutaneous Syndactyly Syndrome

TRPS Trichorhinophalangeal Syndromes

` xii

ACKNOWLEDGEMENTS

All commendations and praises for Almighty ALLAH, Who equipped the mankind

with wisdom and made him able to disclose the secrets of universe. All the reverence

and esteems for The Holy Prophet Hazrat Muhammad (SAW), who is the torchbearer

for humanity and the source of eternal Spring of Knowledge.

I am profound recognition of the support extended by family to complete the

strenuous journey of my doctoral studies.

I extend my cordial thanks to the professional and affectionate support of my

intellectual supervisor Dr. Muhammad Zahid, Associate professor, Department of

Zoology, Islamia College Peshawar, whose valid counselling, personal interest and

absolutely incredible supervision in spite of his numerous engagements and

responsibilities made me to complete this manuscript. I am happy to offer special

gratitude to my co-supervisor Dr. Musharraf Jelani, Associate professor at the Centre

of OMICs, Islamia College Peshawar for his valued guidance and encouragement. I

am grateful to, Dr. Changsoo Kang, from Department of Biology, Sungshin Women’s

University, Seoul, Republic of Korea for his unflinched support in sequencing and

investigations of this novel mutation.

Special regards to my esteemed colleagues at the Department of Zoology, Islamia

College Peshawar, for their encouragement in this whole journey.

And last but not least, my warmest thanks to my little soul-mate Brother Mr. Ibrahim

Khalil (Goldmedalist in Economics) who supported me a lot and took physical and

mental encumbrance in sampling and clinical diagnosis of families.


` xiii

ABSTRACT

Genes are essential elements of our genetic material “the DNA”. Normal function of

each gene is important for the daily works of our body. Any alteration in the structure

or function of genes is termed as “genetics” which may lead to structural or functional

abnormalities of our body and organs. Alterations that mainly affect DNA can be

transferred from parents to offspring’s and have to the potential to segregate in the

subsequent generations. Thus, a DNA change may be persistent and has the potential

to affect many generations continuously allowed to segregate.

Genetic diseases that affect less than 1/2,000 individuals in Western countries are

referred to “rare genetic diseases”. Pakistan in general, and Khyber Pakhtunkhwa in

specific, constitutes ofmany subgroups i.e. socio-economic, ethnic or culturally

isolated populations. In majority of cases, these people prefer to marry their closer

relatives for example first degree cousins, ethnically matched subgroups or with

whom residing for centuries in a same locality. These inbreeding brings DNA

similarity among the upcoming generations and have very little chance to be

genetically different from each other. This way once a person has got DNA alteration

it may prevail generations-after-generations and in case of disease variants may lead

to abnormalities that could not mend in the new-borns.

These genetic alterations are screened by DNA sequencing and compared with the

wild type DNA sequences to identify the causative variants. The field enormously

emended in the very recent past by using Sanger sequencing and next generation

sequencing tools and a basic tool for mutation detection in the disease carrying

patients. Sanger sequencing is implied to targeted sequencing for small range

screening analysing a few candidate genes, while whole exome sequencing and whole

genome sequencing is used for large scale, economic and time saving experiments.

The present dissertation describes clinical and molecular analysis of a consanguineous

family from a remote village of Khyber Pakhtunkhwa, Pakistan. There were two

` xiv

phenotypes a. palmoplantar keratoderma and b. intellectual disability presented by the

family in a fashion that either the affected individuals carried both or only one.

Palmoplantar keratoderma (PPK) phenotype was presented in three generations with

autosomal dominant mode of inheritance. The patients were characterized the

thickness of skin over the palms andsoles.They also had hyperkeratotic lesions

restricted only to the pressure regions of palms or extend longitudinally along the

fingers. Previously fourteen genes had been assigned to PPK phenotype. Whole-

exome sequencing followed by Sanger sequencing andin-silico bioinformatics

analyses we found a novel heterozygous variant in COL20A1 gene (NM_020882.2, c.

392C > G; p.Ser131Cys) in all the affected individuals of the three generations. The

alteration lied in the loop region close to fibronectin type III-1 domain of the collagen

20 α1 protein. This mutation was not found in the 219 unaffected healthy controls of

Pakhtun ethnicity collected from various districts of Khyber Pakhtunkhwa. The

variant was assigned “pathogenic” by in-silico prediction tools. To the best of our

knowledge, this gene had not been associated with any human genetic disorders

previously. Thus, we identified COL20A1 gene for the first time as a potential

candidate to segregate with the disease phenotype of PPK in Pakistani family.

The intellectual disability of segregated in autosomal recessive mode; however, no

causative variant was assigned in the whole exome sequencing data. The causative

variant might lie in deep intronic region for which we recommend whole genome

sequencing.

Next generation sequencing is becoming a powerful tool in molecular diagnostics and

helping families with genetic disorders on-time and accurate results. Most the

developed countries have already started WES analysis for newborn screening and

genetic counselling programs. WES has the potential to be used as one of the basic

genetic testing tool in the near future.

Chapter – 1 Introduction

Analysis And Diagnosis Of Targeted Medical And Genetic Disorders In Human Population Of Khyber

Pakhtunkhwa, Pakistan 1

CHAPTER - 1

INTRODUCTION

1.1 Genetic Disorders

There are about 25,000 protein-coding genes in the human genome and any variation

or mutation in these genes can potentially lead to a genetic disorder. The main

concept underlying a genetic disorder is that of inheritance; the disease may affect

other people related to the proband and it will co-segregate in families. One of the

first genetic diseases to be recognized was reported in the 2nd century. A family was

found, in which an infant and its three maternal cousins had died from extensive

bleeding, after medical procedures; this disorder is now known as hemophilia and

caused by mutations in genes regulating the blood coagulation (Ingram, 1976).

Some genetic disorders are strongly determined by a gene, these are called monogenic

or Mendelian disorders, while many other disorders result from multiple genes

interacting with environmental factors called multifactorial or complex disorders.

Thus, each disease can be placed at a different point along a continuous spectrum,

depending on whether the genetic or environmental effect is the strongest determining

factor. The majority of infectious diseases are triggered by environmental factors so

they lie at one extreme of the spectrum (Figure 1). Mendelian diseases, such as cystic

fibrosis, lie at the other extreme and are mainly determined by mutations in the

genome. Complex disorders lie in the middle of the spectrum as they are strongly

influenced by both types of factors (Figure 1).




Figure 1.1: The continuous spectrum of disease interconnection

Monogenic disorders are rare, and caused by mutations of a large effect size in one

gene (Figure 2). These mutations result in very low minor allele frequencies (MAF) of

the mutant alleles (MAF<1%). The mutations often occur in highly conserved regions

that are prone to negative evolutionary selection ((Ng et al., 2010b; Thomas and

Kejariwal, 2004)). Around 60% of the genetic mutations leading to monogenic

disorders have a missense or nonsense character, around 10% are splice sites, 7% are

insertions/ deletions (indels) and less than 1% occur in regulatory regions (Botstein

and Risch, 2003).

Gregor Mendel (1866) was one of the first to outline the inheritance of monogenic

traits in pea plants, and he described recessive and dominant models. In 1902, Garrod

showed that alkaptonuria (black urine disease) was inherited recessively, so that the

“inborn errors of metabolism” were present first time in the siblings but not present

before in the parents or family (Garrod, 1902).

1.1.1 Types of Genetic Disorder

The inheritance of monogenic disorders has been well established and follows six

main patterns:

1. Autosomal Dominant

Where the presence of one mutated allele on an autosome is enough to cause the

disease.




2. Autosomal Recessive

The presence of two mutated alleles in the same gene on two homologous autosomes

leads to disease.

3. X-Linked Recessive

X linked recessive is characterized by the presence of two mutated alleles on the X

chromosome (in females) or one (in males) are needed to cause any disease,

4. X-Linked Dominant

X-linked dominant diseases are characterized by the presence of one mutated allele on

the X chromosome is enough to cause syndrome or disease,

5. Y-linked

Y-linked where the presence of one mutated allele on the Y chromosome is needed to

cause any disease,

6. Mitochondrial-Linked

Mitochondrial-linked where maternal mitochondrial DNA carries the mutation

causing the disease. This is not typical Mendelian inheritance, as none of the parental

DNA is inherited, however, these disorders are also monogenic.

Complex diseases are fairly common in the general population. For example, the

prevalence of celiac disease, a complex, immune-related intolerance to gluten, is

around 1% in Caucasian populations (Mearin et al., 2005).

The first hypothesis on the character of causative variants for complex diseases was

based on their high prevalence in the population, and was called the “common

disease-common variant” (CD-CV) hypothesis. It states that complex diseases are

caused by a combination of multiple common polymorphisms, with MAF > 5%, of

small effect size, with each polymorphism explaining a small part of the heritability

(Figure 2) (Gibson, 2009; Schork et al., 2009). In total, from 2005 to 2010, some

5,854 common genetic single nucleotide polymorphisms (SNPs) were found to be




associated with 540 complex disorders (Hindorff et al., 2009). Celiac disease is

currently associated with 57 non-HLA SNPs representing 39 loci, and although no

causative gene was found, the majority of these SNPs map close to immune-related

genes, explaining some 15% of the heritability(Trynka et al., 2011).

Figure 1.2: Variants with wide range of different effect size (OR, odds ratio)

and minor allele frequencies (MAF).

The work of Thomas and Kejariwal showed that the majority of coding variants

associated through genome-wide association studies (GWAS) studies with complex

diseases map to less conserved regions than the severe mutations found for Mendelian

diseases, which may explain their more moderated effect size (Schork et al., 2009).

A second hypothesis, “common disease-rare variant” (CD-RV), states that complex

genetic diseases could be due to variants of low MAF (< 5%), and moderate effect

size (Schork et al., 2009) (Figure 2), with an incomplete penetrancein some families




(van Heyningen and Yeyati, 2004). The CD-RV hypothesis could explain part of the

“hidden heritability” of complex disorders.

Moderate and rare variants have so far remained largely undiscovered, as the DNA

chips used for genome-wide association studies (GWAS) do not contain variants with

a MAF < 5% (Manolio et al., 2009). These variants could be present within the

GWAS loci and collectively drive the association to the disease (“synthetic

association”)(Goldstein, 2011). For example, several rare variants in the NOD2 gene

are strongly associated with inflammatory bowel disease (IBD) and although the

variants show a familial clustering they do not follow a clear inheritance pattern (van

Heyningen and Yeyati, 2004).

The work of Cohen et al. in 2004 and 2005 showed that extreme phenotypes of

metabolic traits are also strongly correlated with rare variants in metabolic candidate

genes (Cohen et al., 2004; Cohen et al., 2006). The CD-RV hypothesis is very likely

to hold true for the majority of familial, Mendelian-like subtypes of complex

disorders. For example, a mutation in the VPS35 gene has been identified in a large

Austrian family segregating for Familial Parkinson’s disease (Zimprich et al., 2011),

and recent case-control studies on hyperglycemia, IBD and gout have shown an

excess of rare variants in loci mapped by GWAS (Rivas et al., 2011).

1.2 Rare Genetic Diseases

Diseases that affect less than 1/2000 individuals are referred to as rare; those with a

prevalence lower than 1/50 000 are referred to as ultra-rare. Increasing attention is

devoted to this group of patients for several reasons:

i. The recognition of a rare disease and confirmatory molecular/biochemical tests

may take years due to lack of knowledge of physicians, limited or no access to

certain diagnostic tests, and confusing patient routes.




ii. Numerous rare diseases are rapidly fatal or devastating, and a considerable ratio of

affected individuals die before even receiving a proper diagnosis.

iii. For 95% of rare diseases, no approved cure or definitive treatment exists.

Typically disabling, the patients’ quality of life is affected by the lack of

autonomy due to the prolonged, progressive, degenerative, and repeatedly life-

threatening aspects of their condition. To date, approximately 7000 rare diseases

are known.

1.2.1 Prevalence of Rare Diseases and their Significance in Health Care

80% of rare diseases are genetic of origin, and 80% of genetic disorders are rare. The

remaining 20% are caused by infections, environmental damage, or are

immunological, degenerative and proliferative by nature. Increasing evidence

supports the major role of genetic predisposition in this group of diseases, too. Rare

diseases are characterized by a broad diversity of symptoms that vary not only from

disease to disease but also from patient to patient affected by the same disease.

Because these diseases are so diverse and complex, there are inherent gaps that exist

in patient care and physician resources, leading to misdiagnosis and delay in

treatment.

The significance of rare diseases is especially high in the pediatric population as 50%

of rare diseases touch children, presenting often as birth defects or multiple congenital

anomalies. 20-30% of all neonatal deaths and 30-50% of post-neonatal deaths are due

to genetic disorders, and up to 71% of inpatient hospital admissions are for children

with a genetic defect - representing an 81% share of the total health care charges.

The majority of genetic disorders display mental retardation as a primary feature, thus

further increasing the burden of these conditions. The prevalence of mental

retardation in the population is 1-3% and that of multiple congenital anomalies is 2-

3%.




Due to steady improvements in general health care, many rare disease patients now

survive into adulthood and require medical help for chronic, age-related and

associating symptoms in addition to the primary genetic defect.

1.2.2 Causes of Birth Defects of Rare Diseases

The causes of birth defects are many and complex. According to Turnpenny and

Ellard, chromosomal anomalies visible by G-banding account for an approximate 6%

of congenital disorders (including Down syndrome representing half of the cases), an

additional 10-14% is caused by submicroscopic copy number changes. According to a

more recent report, chromosome abnormalities can be detected in one out of every 10

investigated patients with developmental delay. Mendelian disorders represent

another 7.5%, and environmental causes can be identified in 5- 10% of cases. In 20-

30%, the underlying genetic cause is multifactorial, and we have no exact data on the

prevalence of UPD (uniparental disomy) and imprinting defects in the genetically ill

population. In an estimated 38-40% of cases the genetic cause remains unknown.

1.2.3 Dysmorphology and Syndromology – history and general perspectives

Due to their low prevalence, the diagnosis of genetic rare diseases is often extremely

difficult, costly and time-consuming. In the past 10-15 years, the advent of genome-

wide studies, the use of array-based molecular cytogenetics on a routine diagnostic

basis and the increasingly widespread application of „panel-testing” with next

generation sequencing as well as of whole exome sequencing (WES) and whole

genome sequencing (WGS) have fundamentally changed today’s genetics, largely

contributing to the identification of new genes in syndromes previously of unknown

origin, of the recognition of new syndromes and of the better understanding of

genotype-phenotype correlations. The tremendous amount of information obtained by

these tests, however, require a whole new approach and refined interpretation of

genetic results. Choosing the right method with the highest possible diagnostic yield




and the lowest possible cost in a given case demands precise phenotyping, accurate

evaluation of morphological and functional symptoms, identification and

characterization of dysmorphic signs on a level more advanced than ever before.

“Nextgeneration sequencing requires next generation phenotyping” says the title of a

recent paper of one of today’s most acknowledged syndromologists, Prof. Raoul

Hennekam. For this, a profound knowledge in several fields of medicine

(embryology, anatomy, endocrinology, neurology, psychiatry) is needed.

The science of syndromology is therefore a more advanced application of

dysmorphology, where the right combination and correct identification of signal signs

result in the identification of the right syndrome, which then, of course, has to be

proved with appropriate genetic tests. According to the definition of Seemanova,

“Syndromology is a diagnostic method based on the analysis of phenotypic features,

by which seemingly separate symptoms that mark a common etiology can be

identified, the differential diagnostic spectrum can be narrowed and the true diagnosis

is delineated”.

1.3 Different Types of Genetic Diseases in Humans

1.3.1 Bone Deformities or Skeletal Dysplasias

Congenital bone disorders generally termed as skeletal dysplasias (SDs), are

hereditary abnormalities of bone and cartilage that affect their integrity, growth and

morphology (Offiah and Hall, 2003; Warman et al., 2011). Skeletal dysplasias are

heterogeneous group of deformities both genetically and clinically, which have been

divided into three subgroups; chondrodysplasias, dysostoses and osteodysplasias

(Rimoin et al., 2007).

The body systems which regulate the maintenance and growth of the skeleton may be

disrupted by several processes which leads to various diseases of bone and disorders.




Genetic abnormalities can affect the size and shape of the bones which cause bone

deformities or abnormal growth of bone and produce weak, thin bones, or bones that

are too dense. Growth and modelling of bones are under control of variety of genes

that express at different stages of life. Genetic disorders of bones originate from

anomalies in different process of skeletal development, homeostasis and growth and

remain a diagnostic impediment due to their variety. Inherited bone disorders may

follow autosomal dominant, recessive or X linked pattern of inheritance (Warman et

al., 2011). Significant improvements are seen in distinguishing of the molecular

abnormalities accountable for skeletal dysplasias (Deborah et al., 2009).

1.3.2 Classification of Bone Deformities

There is a generalized anomaly in growth of linear skeleton in osteochondrodysplasias

and there are associated anomalies in organ system in few disorders instead of

skeleton. Hereditary disorders involving the skeleton generate from anomalies in

different process of development of skeleton, homeostasis, growth and remain a

diagnostic obstacle due to their variety. Classification of 456 different conditions of

hereditary bone deformities were documented and placed in 40 groups that were

further positioned under ten large families according to their radiographic, molecular

and biochemical assessment. 316 of them were seen to be related to mutations found

in 1 or more than 226 different gene (Warman et al., 2011). In 2015 classification of

hereditary skeletal deformities: the groups of skeletal disorders were enlarged to 42

with 364 causative genes. On the other hand, the number of disorders has declined

from 456 to 436 (Bonafe et al., 2015). Despite the in progress evolvement in the

classification of hereditary bone deformities or skeletal dysplasias, still every

classification system has certain drawbacks because of the diversity of skeletal

disorders both in its clinical manifestations and pathogenesis. In fact, single gene

mutations may cause different types of disorders and a single disease may possibly

involve several genes which makes the classification more complicated and indistinct.




Rossi et al. (2001) suggested a simple and convenient classification system for

skeletal disorders based on their clinical features, molecular abnormalities, and

radiographic criteria. They classified the hereditary skeletal disorders into seven

groups according to the common discrepancies in, (i) Extracellular structural proteins,

(ii) Signal transduction mechanisms and hormones, (iii) Oncogenes and tumor

suppressor genes (iv) Metabolic pathways, (v) Transcription factors and Nuclear

proteins, (vi) Folding, processing and breakdown of macromolecules and (vii) RNA

and DNA processing and metabolism.

1.3.3 Group I: Bone Deformities Involving Defects in Extracellular Structural

Proteins

Extracellular structural proteins play a vital role skeletal development and

maintenance and mutations in the genes coding for proteins such as COL1A1,

COL1A2, COMP and MATN3 result in a variety bone deformities including

Osteogenesis imperfect, multiple epiphyseal dysplasia and pseudoachondroplasia

(Briggs and Chapman, 2002).

I. Multiple Epiphyseal Dysplasia

Multiple epiphyseal dysplasia (MED) is comparatively common type of skeletal

deformity with the involvement of epiphyses of long bones. It is characterized by mild

short stature, rotational or angular abnormality of the extremities, short feet and

hands, and/or precocious osteoarthritis. Radiologic features demonstrate delayed

ossification, irregular long bone epiphyses and hypoplasia and variable grades of

flattening of vertebral forms. It shows broad spectrum of severity (Unger et al., 2001;

Spranger et al., 2012). Genetically, MED comes in category of disorders that are

heterogenous with shared clinical and radiological phenotypes. Multiple epiphyseal

dysplasia is inherited in autosomal dominant pattern where genes encoding matrilin-3

(MATN3), cartilage oligomeric matrix protein (COMP), and alpha 1–3 chains of type

IX collagen are mutated (Unger et al., 2008). While multiple epiphyseal dysplasia,




also follows an autosomal recessive mode of transmission where gene encoding

diastrophic dysplasia sulfatetransporter (DTDST or SLC26A2) are mutated (Rossi

and Superti-Furga, 2001; Briggs and Chapman, 2002).

1.3.4 Group II: Bone Deformities due to Defects in metabolic pathways

Mutations in the genes encoding metabolic enzymes, ion channels and transporters

lead to a number of bone disorders. Cases in point are mutations in matrix

metalloproteinase 2 encoding gene causes Torg type osteolysis and mutations in CIC7

(Chloride channel 7) causes serious osteopetrosis (Kornak and Mundlos, 2003).

1.3.5 Group III: Bone Diseases due to Abnormal Folding and Degradation of

Macromolecules

This group includes principally the lysosomal storage disorders such as

mucopolysaccharidoses which are considered as the first skeletal dysplasias that were

described at biochemical level. Lysosomal disorders are group of heterogeneous

diseases with prenatal metabolic defects that manifest with deformities including the

joints or bones (Aldenhoven et al., 2009). A brief account of mucopolysaccharidoses

is conveyed in the following section.

I. Mucopolysaccharidosis or MPS

Mucopolysaccharidosis (MPS) is the group of disorders apprehensive with lysosomal

storage machinery. It results due to the deficiency of enzymes that degrade the

glycosaminoglycans (GAGs). In the animal cells the membrane-bound organelles are

found known as Lysosomes which play a vital role in the breakdown of

carbohydrates, lipids, proteins, nucleic acids, and cellular debris. Enzymes of

lysosomes are prepared in the endoplasmic reticulum; mannose-6-phosphate is tagged

to them in the Golgi apparatus and transported to the lysosomes. Protection of these

enzymes from degradation by an acidic environment upheld via an energy-dependent

pump is carried out by some proteins. Defects in the enzymes of lysosomes, cofactors,




or transport proteins result in MPS (Ashworth et al., 2006). Glycosaminoglycans

generally, stabilize fibre and provide support to cellular parts of the tissues while

helping maintenance of water in the body (Esko et al., 2009). The anomalous

accumulation of mucopolysaccharides in the lysosome cause inflammation of

lysosome that covers more area in cytoplasm, as a result the additional organelles of

the cell are obscured and distorting nuclear integrity which leads to disorder called

mucopolysaccharidosis (Coutinho et al., 2011). Mucopolysaccharides, the old name

of (GAGs) are the degradation products of proteoglycan with the exception of

hyaluronic acid are found in extracellular matrix. Proteoglycans give rise to GAGs by

the action of enzyme proteases and their breakdown occurs in lysosome. Four

pathways are responsible for glycosaminoglycans degradation in lysosome.

Depending upon the molecule which is to be degraded, (dermatan sulfate, keratan

sulfate, chondroitin sulfate and heparan sulfate) the pathway needs 10 enzymes: one

nonhydrolytic transferase, four glycosidases and five sulfatases (Neufeld and

Muenzer, 1995; Giugliani et al., 2011). The most representative clinical features of

MPS are short stature, pigeon chest, short neck, knock-knee deformity, lumbar

kyphosis, macrocephaly, dysostosis multiplex, broad mouth, compromised joint

mobility, organomegaly and craniofacial abnormalities (Coutinho et al., 2011).

Affected individuals may also have cardiovascular disease, hearing impairment,

skeletal disease, and significant intellectual and neurological problems. Formerly the

diseases were recognized due to their clinical manifestations, nevertheless, because of

biochemical advances, now these diseases have been classified by deficiencies of

specific enzyme. There are total 7 groups of MPS: type I, II, III, IV, VI, VII and IX.

Except the Hunter syndrome which is inherited in an X-linked recessive pattern all

others follow an autosomal recessive pattern of inheritance. There is an extensive

grade of variation in phenotype, as some are lethal in initial months of life while

others on life span have no significant impact (Ashworth et al., 2006).




II. Mucopolysacharidosis Type I or MPS Type I

MPS type I has 3 subtypes including Scheie, Hurler–Scheie and Hurler syndromes.

They are described by lysosomal hydrolase α-l-iduronidase deficiency that is required

to degrade the dermatan and heparin sulfate. As a consequence the accumulation of

these metabolites occurs in various tissues. Due to the lack of α-l-iduronidase the

phenotypic spectrum includes the most severe hurler syndrome, the mildest form

Scheie syndrome and Hurler-Scheie syndrome the intermediate form of MPS type I

(Ashworth et al., 2006). The estimated occurrence of severe MPS type I in 100,000

babies is one. While the attenuated MPS type I incidence is around 1 in 500,000

births. Scott and colleagues in 1992 cloned and purified the IDUA gene presenting

that it is of 19 kb and contain 14 exons. First two exons contain an intron of

approximately 566 bp, following an intron of 13 kb, while in 4.5 kb 12 exons lie. The

gene was located on chromosome 4p16.3 by southern blotting and unequivocal in situ

hybridization of mouse-human cell hybrids (Scott et al., 1992). There are three

subtypes of MPS type 1 which are explained in the following section.

III. Scheie Syndrome

The symptoms of Scheie syndrome includes, stiff joints, shortness of breath and poor

vision. They usually have mild cardiac and respiratory diseases and characteristic

facial abnormalities. They have normal life span and intelligence. Other

manifestations are umbilical and inguinal hernia, hepatosplenomegaly, cord

compression, dental caries and aortic valve stenosis. Optic nerve atrophy is not

common (Ashworth et al., 2006).

IV. Hurler–Scheie Syndrome: MPS I

Hurler–Scheie syndrome is attenuated form of MPS I which is characterized by mild

facial changes, premature death of the patients occur because of cardiorespiratory

disease. The intelligence is not affected as with scheie syndrome, the patients of

Hurler–Scheie syndrome may have retinopathy, diffuse corneal thickening and both




open-angle glaucoma and chronic angle-closure (Ashworth et al., 2006). Other

Clinical features include enlarged skull, mild mental retardation, corneal clouding,

thickened lips and stunted growth. (Bradbury et al., 1987).

V. Hurler Syndrome: MPS I

Hurler syndrome is the severe type of MPS I and development of clinical

manifestations occurs usually after first year of life. Hurler syndrome is characterized

by dysostosis multiplex, hepatosplenomegaly, multiple hernias, coarse facies, as well

as intellectual impairment. Ophthalmic findings include glaucoma, retinal pigmentary

degeneration, optic nerve head swelling and the most common is corneal clouding

(Haung et al., 1996).

VI. Hunter Syndrome: MPS Type II

Hunter syndrome’s incidence is estimated as 1 out of 100,000 to 150,000 male births

(Baehner et al., 2005; Fenzl et al., 2015). It follows the X-linked recessive inheritance

therefore it is unique among other types of MPS. The deposition of heparin and

dermatan sulfate is due to the insufficiency of enzyme iduronate-2-sulfatase

(Ashworth et al., 2006) encoded by IDS gene at locus Xq28 (Ganesh et al., 2013). It is

characterized by recurrent otitis media viral, upper respiratory infections and

abdominal hernias. These manifestations in one year old age group are common and

cannot alarm clinicians till the later sequelae arise, such as white skin lesions, learning

disabilities, abnormality of facial features, multiple joint stiffness and declining

cardiac function. Craniosynostosis and Seizures in the patient of MPS type II are also

common. (Ashworth et al., 2006; Ziyadeh et al., 2013). Affected males have 84%

neurologic involvement.

VII. Sanfilippo Syndromes: MPS Type III

Sanfilippo syndrome has higher prevalence than all other types of MPS. The

estimated incidence is 1 in 70,000 for all subtypes of MPS type III (Fenzl et al.,




2015). On the basis of gene mutated, there are four sub types of Sanfilippo syndromes

including Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo

syndrome type C and Sanfilippo syndrome type D. Characterization of each subtype

is based on deficiency of particular enzymes that are Type A, produced by deficiency

of heparin sulfamidase enzyme, type B occurs by deficiency of α-N-

acetylglucosaminidase enzyme, deficiency of acetyl-CoA: α-glucosaminide, and N-

acetylgalactosamine-6-sulfatase cause type C and D respectively. In Type A the

affected gene SGSH is located on chromosome 17q25.3 in type B the affected gene

NAGLU is located on chromosome 17q21 while in type C and D the affected gene

HGSNAT resides on chromosome 8p11.1 and GNS gene on chromosome 12p14

respectively (Ganesh et al., 2015). The most severe and common type of MPS type III

disease is Sanfilippo type A while heparan sulfate is accumulated in all subtypes.

MPS III subtypes are clinically indistinguishable. These affected individuals may

represent severe behavioral and learning disturbance, dental caries, joint contractures,

sleep problems and middle ear diseases (Ashworth et al., 2006). Patients mostly die

before their teenage years, however, survival of some patients subsequent to their

teenage in thirties is observed. It follows the autosomal inheritance pattern.

VIII. MPS type IV: Morquio Syndromes

Mucopolysaccharidosis IV is also called Morquio’s syndrome caused by improper

metabolism of keratansulphate. Deficiency of two different enzymes cause two

different subtypes of Morquio’s syndrome. Morquio’s A occurs as a result of N-

acetylglucosamine-6-sulfatase deficiency (MIM 253000) whereas β-galactosidase

deficiency causes Morquio’s B (MIM 253010). These both subtypes share some

common features namely short trunk dwarfism, spondyloepiphyseal dysplasia and

fine corneal deposits. Mucopolysaccharidosis type IV represents pertinence with

skeleton and the intelligence of the patients is mostly normal (Neufeld and Muenzer,

1995). Individuals with severe phenotypes do not pass thirty years of age (Montaño et




al., 2008). The frequency of Morquio syndrome is estimated as 1 in 200,000 births

(Fenzl et al., 2015).

IX. Mucopolysaccharidosis Type IV A or Morquio A Syndrome

Mucopolysaccharidosis IVA (Morquio A syndrome, MPS IVA (MIM #253000) is an

autosomal recessive lysosomal storage disease that is characterized by

disproportionate short stature, laxity of joints, anterior beaking of lumbar spine,

knock-knee, dysostosis multiplex with universal platyspondyly, rib cage flaring,

dental abnormalities and elevation of blood and urine KS (Hollister et al., 1975;

Neufeld and Muenzer, 2001; Tomatsu et al., 2003; 2005). The occurrence of MPS IV

A varies from 1 in 640,000 births in Western Australia and 1 in 76,000 births in

Northern Ireland (Hendriksz et al., 2013). Morquio A syndrome develops by

deficiency of an enzyme N-acetylgalactosamine-6- sulfatase (GALNS: N-acetyl-

galactosamine-6-sulfate sulfatase also called as galactosamine (N-acetyl)-6-sulfate

sulfatase because of mutation in the gene GALNS (OMIM 612222). Lacking GALNS

activity leads to deposition of the (GAGs) chondroitin-6-sulfate (C6S) and (GAGs)

keratin sulfate (KS) in several tissues, which causes skeletal and connective tissue

vagaries along with cardiac pathology and pulmonary constraints (Montano et al.,

2007; Harmatzet al., 2013; Yasuda et al., 2013). Molecular examination can confirm

the MPS IVA diagnosis and assist genetic counseling by spotting the contributory

mutations in the GALNS gene (Wood et al., 2013). Human GALNS gene (OMIM:

612222) is located on chromosome 16q24.3 (Tomatsu et al., 1992; Baker et al., 1993).

The length of GALNS gene is about 50 kb and contains 14 exons ranging from 67 to

791 bp and it contains introns of 380 bp to 14 kb. GALNS produce mRNA of 2,339

base pair which encodes a protein of 522 amino acids (Tomatsu et al., 1992). So far,

more than 275 mutations have been reported in the GALNS gene (Morrone et al.,

2014) and most common mutations are missense mutations (Tomatsu et al., 2005;

Hendriksz et al., 2013). Patients with Morquio A syndrome represent different




severity but individuals having severe phenotype die earlier the second or third decade

of life (Montano et al., 2008). For Morquio’s A or B animal model has not been

reported yet. Due to exon 2 disruption of GALNS a murine model was made for

Morquio’s type A. Excessive levels of glycosaminoglycans coupled with no enzyme

action were observed. Glycosaminoglycans were found accumulated in different body

tissues including heart, spleen, bone marrow, brain, liver and kidney (Tomatsu et al.,

2003).

X. Mucopolysaccharidosis Type IVB or Morquio B Syndrome

Mucopolysaccharidosis type IVB is an autosomal recessive disease categorized by

corneal clouding and skeletal dysplasia. Central nervous system is not involved and

has normal intelligence. (Santamaria et al., 2007). MPS type IVB has same clinical

features like type A but difference lies in aberrant gene. MPSIV B is caused by the

impairment in β-galactosidase enzyme that catalyzes the complex breakdown of

keratansulphate. Coded by GLB1, the hydrolases of β-linked galactose residues that is

found in oligosaccharides, glycoproteins, keratansulphate and GM1 ganglioside is

carried out by the enzyme β-galactosidase (Neufeld and Muenzer, 1995).

Chromosome 3p21.33 contains GLB1 gene. Its length is about 62.5kb and consists of

16 exons (Santamaria et al., 2007). GLB1 encodes a 667 residue protein having

molecular weight of 75 KD and 23 residue signal sequence residue. Moreover for

asparagines-linked glycosylation it possess 7 possible sites. The GLB1 gene

interestingly makes 2 mRNAs because of alternative splicing. The larger 2.5Kb

transcript produce 667 amino acid protein and found in lysosome. Smaller 2.0

produces beta galactosidase associated protein containing 546 amino acids without

catalytic action and possess several subcellular localization. Due to pre-mRNA’s

alternative splicing the exon 3, 4 and 6 are missing in smaller 2.0 Kb form (Privitera

et al., 1998). To date for MPS IVB there are no known animal models (Coutinho et

al., 2011).




XI. MPS Type VI or Maroteaus-lamy Syndrome

Maroteaus-lamy syndrome is a rare type of MPS with an incidence rate of 1 in

250,000 to 600,000 births. Few patients with this condition around the world have

been diagnosed. Transmitted in autosomal recessive form and is caused by mutation

in ARSB gene found on chromosome 5q11 (Ashworth et al., 2006; Kumar et al.,

2010). In MPS type VI the N-acetylgalactosamine-4-sulfatase enzyme, its deficiency

results in accumulation of dermatan sulfate. MPS VI is characterized by restriction of

joint movement, hepatosplenomegaly, coarse facies and cardiac diseases (Ashworth et

al., 2006).

XII. MPS Type VII or Sly Syndrome

Sly syndrome is an infrequent autosomal recessive disorder and is categorized by

macrocephaly, hepatosplenomegaly, frontal prominence, cardiomyopathy, spinal

kyphosis and learning difficulties (Ashworth et al., 2006). The incidence of sly

syndrome is 1 in 250,000 births. MPS type VII occurs because of β-glucuronidase

deficiency, leading to incapability of breaking down dermatan and heparin sulfate,

rare form of MPS VII causes hydrops fetalis and the patient may survive a few

months (Website 1). For the first time in 1973 sly and colleagues reported sly

syndrome; affected individuals granulocytes possess granular inclusions,

hepatomegaly and splenomegaly with the characteristics associated with

mucopolysaccharidosis. From normal control values the level of enzyme activity was

2% less. Both affected and carrier represented a middle level of enzyme activity (Sly

et al., 1973). The gene producing GUSB spans 21 Kb possessing 12 exons and makes

2 types of cDNAs by alternative splicing process. GUSB gene is located on 7q11.21-

q11.22 (Speleman et al., 1996).

XIII. Mucopolysaccharidosis Type IX or Natowicz Syndrome

Natowicz syndrome is an autosomal recessive lysosomal storage disorder. The mild

clinical phenotypes of MPS type IX include short stature, normal intelligence with




abnormal craniofacial features, due to the deficiency of hyaluronidase enzyme,

hyaluronic acid is accumulated with in lysosome which results in Natowicz syndrome.

The mutated HYAL1gene is located at locus 3p21, that encodes two lysosomal

hyaluronidases which act on different substrates (Triggs et al., 1999).

1.3.6 Group IV: Disorders of Imperfection in Hormones and Signal

Transduction Mechanisms

Defects in the genes encoding for hormones and signal transduction protein result in

several skeletal deformities which include various types of short stature and poles

apart syndromes. This group includes varied types of bone deformities ranging from

modest short stature to lethal thanatophoric dysplasia (Rossi et al., 2001).

I. Short Stature/ Dwarfism

Short stature (SS) is defined as a height of more than 2.0 standard deviations (SD)

below the average height recognized for a population with respect to their age and

sex. Short stature is a condition of stunted growth. It is hard to establish the incidence

of individuals with short stature at one particular moment, but it may be estimated to

be about 3–5%. The incidence of growth hormone deficiency (GHD) is about one in

4,000. Short stature is classified into 2 main groups i.e. known etiology or unknown

etiology. The known etiology is further divided into two subgroups: proportionate and

disproportionate, both of them may be congenital or acquired. While in children

unknown etiology includes idiopathic short stature (ISS), familial short stature (FSS).

There are several genes which cause hereditary Short stature SS, some of them are

(GH1, GHRH, GHRHR, GHR, EVC2, NPR2, NPPC, FGFR3, IGF1, PAPSS2) (De

Muinck Keizer-Schrama et al., 1998).

II. Proportionate Dwarfism

Proportionate dwarfism is a condition where all the body parts are proportionally

small. Proportionate dwarfism results because of non-skeletal deformities like growth

hormone deficiency as well as endocrine abnormalities etc.




III. Idiopathic Short Stature

A condition of short stature where height is below −2SD Scores for age, gender and

the analogous population, with no sign of a systemic disease, chromosomal,

psychological or nutritional disorder, or obvious hormonal abnormalities comes in the

definition of Idiopathic Short Stature (ISS). Short children with unknown aetiology

are categorized as ISS in which birth weight/length is >‐2SD and as Small for

Gestational Age (SGA) in which weight/length is <‐2SD (Clayton et al., 2013).

Idiopathic Short Stature genetic nature is until mostly unknown despite stature

existing in humans as one of the most transmissible traits. (Blair and Savage, 2002;

Walenkamp and Wit, 2006; Walenkamp and Wit 2007; Wit et al., 2012). The recent

studies have shown that epigenetic variation leads to the multifactorial complication

of height variability among children, by representing that a greater CG methylation

inside the IGF1 P2 promoter is associated with ISS (Yang et al., 1995; Rotwein, 2012;

Oberbauer, 2013).

IV. Isolated Growth Hormone Deficiency Disorder

Growth hormone (GH) is necessary for linear growth in childhood as well as in

adolescence. The production of IGF-I is regulated by GH, both IGF-I and GH

stimulate skeletal growth. The secretion of GH is stimulated by metabolic changes

and it has significant metabolic effects throughout life. (Jorgensen et al., 1995;

Jorgensen et al., 2007). Variation in growth hormone levels cause different disorders

among which the most prominent is isolated growth hormone deficiency (IGHD), and

it is classified in to 3 types on the basis of clinical features, including pattern of

inheritance and secretion of growth hormone.

V. Isolated Growth Hormone Deficiency type 1/ IGHD Type I

Isolated growth hormone deficiency type I (IGHD type 1) follows an autosomal

recessive inheritance pattern, where GH1 gene is mutated. It is further divided into

subtypes 1A and 1B.




VI. Isolated Growth Hormone Deficiency Type 1A

Isolated growth hormone deficiency type I A (IGHD type 1A) is identified by severe

short stature decreased birth length, extended jaundice associated with severe

subsequent childbirth growth retardation and appearance of characteristic facies after

six month of age (Phillips and Cogan et al., 1994; Kamijo et al., 1999). Associated

gene defects includes GH1 mutation, Deletions, microdeletions, nonsense mutations.

Individuals with (IGHD type 1A) have no detectable serum GH, and possess a strong

early response to therapy with GH.

VII. Isolated Growth Hormone Deficiency Type 1B

Isolated growth hormone deficiency 1B (IGHD type 1B) have milder phenotypes than

IGHD type 1A. Affected individuals have height ≥3 SD below the average. Serum

GH levels subsequent to provocative stimuli are less but detectable. Patients respond

well to growth hormone therapy and don not produce neutralizing antibodies. Isolated

growth hormone deficiency type 1B follows an autosomal recessive pattern of

inheritance, mutation in GH1 and GHRHR cause this disease.

VIII. Isolated Growth Hormone Deficiency Type 2

Isolated growth hormone deficiency type 2 (IGHD type 2) follows a dominant pattern

of inheritance. It is the most common type of isolated growth hormone deficiency and

is caused by mutation in GH1 gene. It is characterized by dwarfism with at least one

affected parent, the levels of GH are also low.

IX. Isolated growth hormone deficiency Type 3

IGHD type 3 follows X-linked recessive inheritance pattern with extremely variable

phenotype. It has been reported that many loci are responsible for etiology of IGHD

type 3 (Conley et al., 1991; Yokoyama et al.,1992).

X. Primordial Dwarfism

Primordial dwarfism is defined as growth disorders that appear in children having

intense intrauterine growth retardation (IUGR) and retain their deficiency for whole




life without any chromosomal or hormonal abnormality (Shaheen et al., 2014). This

definition excludes patients affected with IUGR showing good growth rate after birth

or those in which only abnormal growth parameter is stature (Alkuraya, 2015). A

number of genes have been reported which are mutated in different subtypes of

primordial dwarfism including PCNT, PLK4, CENPJ, CEP63, XRCC4, RBBP8,

DNA2, CEP152 (Dosari et al., 2010; Kalay et al., 2011; Qvist et al., 2011; Sir et al.,

2011; Shaheen et al., 2014 ) ORC1, ORC6, NSMCE2, CDT1, ORC4, BRCA2,

CRIPT, CDC6, RNU4ATAC, LIG4 (Murray et al., 2014) POC1A, LARP7 (Shaheen

et al., 2012; Alazami et al., 2012) OBSL1, CCDC8 and CUL7 (Al-Dosari et al.,

2012). All these genes show autosomal recessive inheritance. Clinically, the most

valuable trait for diagnosis of primordial dwarfism is the extent of involvement of

head circumference. However, subtypes are described based on additional features.

These include delay in development of child bearing microcephaly, the classical facial

expressions, optic-nerve hypoplasia, aberrant fundus pigmentation, skin pigmentation

abnormality, irregular digitization of feet, hands and Ichthyosis (Alkuraya, 2015).

XI. Disproportionate Dwarfism

Disproportionate dwarfism is a condition in which body parts are disproportionately

short. The most common type of disproportionate dwarfism is called Achondroplasia.

Disproportionate could be called either short trunk dwarfism or short limb dwarfism.

Following are the types of disproportionate dwarfism.

XII. Spondyloepiphyseal Dysplasia Congenital (SEDC)

Spondyloepiphyseal dysplasia congenital (SEDC, OMIM 183900) is member of the

type II collagenopathies (Li et al., 2014), with characteristic features: disproportionate

short-trunk dwarfism, vertebral and skeletal abnormalities, including thoracic

hyperkyphosis and scoliosis and various diseases of joints. Extraskeletal

manifestations include ocular anomalies, cleft palate and hearing loss.

Spondyloepiphyseal dysplasia congenital is a rare disease with prevalence of




3.4/1,000,000 (Spranger and Wiedemann, 1996; Mark et al., 2011; Xu et al., 2014)

and follows autosomal dominant mode of transmission. The causative gene of

spondyloepiphyseal dysplasia congenital is COL2A1 that is located on chromosome

12q13.11 (Givon et al., 1999).

XIII. Acromesomelic Dysplasias

Acromesomelic dysplasias are a group of skeletal deformities that predominantly

affects the distal segments (hands and feet) and middle segments (forearms and

forelegs) of the appendicular skeleton and leads to disproportionate shortening of

segments, there are bowed forearms, short tubular bones, short toes, very short and

broad fingers and large halluces. At birth there are no distinguishing signs, changes

include subsequent to birth shortening and bowing of the radius, dislocation or radial

head subluxation, very broad and short metatarsals, metacarpals, and phalanges

(Bartels et al., 2004; Tsuji and Kunieda, 2015).

Types of Acromesomelic Dysplasia

I. Acromesomelic Dysplasia, Maroteaux Type

Acromesomelic dysplasia Maroteaux Type (AMDM, MIM 602875) follows an

autosomal recessive inheritance pattern and mutation in NPR2 gene causes this

disorder. Kant et al. (1998) described that the severe type of disproportionate

dwarfism is Acromesomelic dysplasia Maroteaux Type (AMDM); particularly the

limb extremities are affected in this type of dysplasia.

II. Grebe Dysplasia or Acromesomelic Dysplasia, Grebe Type

Acromesomelic chondrodysplasias of the Grebe type (MIM 200700) is the severe

type and is characterized by small lower and upper limbs, bowed forearms, digits/toes

are stump like and very short. Patients are extremely short stature, with an adult

having height of about 100 cm. very short lower extremities, with limited flexion and

extension of the knee, overriding and very short toes. X-Ray of the upper limbs depict




a short bowed radius and ulna and a short humerus. Lower limbs radiographs depict a

missing fibula, a mildly short femur, Grebe type dysplasia follows autosomal

recessive mode of inheritance, and is caused by homozygous mutation in GDF5 gene

(Costa et al., 1998; Faiyaz-ul-Haque et al., 2002; Demirhan et al., 2005).

III. Hunter–Thompson Dysplasia

Hunter–Thompson Dysplasia (MIM 201250) is also known as acromesomelic

dwarfism and is characterized by dysplasia of the tibia, aplasia of the fibula, severe

type of brachydactyly with short metacarpals, aplastic or hypoplastic phalanges, and

fused carpal/ tarsal bones. In addition to this female patients may have genitourinary

anomalies along with hypoplasia of the uterus, dysfunction of ovary with consecutive

hyper gonadotropic hypogonadism may also occurs. Hunter–Thompson Dysplasia is

inherited in an autosomal recessive pattern. This type of dysplasia is also caused by

homozygous mutation in GDF5 gene (Costa et al., 1998; Faiyaz-ul-Haque et al.,

2002).

1.3.7 Group V: Bone Disorders Involving Nuclear Proteins and Transcription

Factors

A number of nuclear proteins and transcription factors (TFs) are implicated in the

development of skeleton of human which regulate the expression of the genes

involved in in bone, cartilage, or tooth growth. Mutations in the genes encoding for

these transcription factors like GLI3, SOX9, NEMO, SHOX, ZNF, and HOX D gene

clustere give rise to various types of bone deformities such as growth retardation,

short stature, polydactyly, syndactyly and split hand foot malformation (Wagner et al.,

1998;; Zhao et al., 2002; Um-e-Kalsoom et al., 2012).

I. Polydactyly

Polydactyly is the most common hereditary limb malformations featuring superfluous

fingers or toes, ranging from an integral completely developed superfluous digit(s) to




a small soft tissue with no bony structure. These malformations mostly have

rudimentary skin tags. (De and Simpson, 1998; Schwabe and Mundlos, 2004;

Christensen et al., 2011; Malik, 2014). The involvement of lower limbs are not more

common than that of upper limbs. Right hand and left foot are more common than the

left hand and right foot (Malik et al., 2014). Disturbance in the normal process of the

anterior–posterior axis of the developing limb causes polydactyly, with various

etiology and varied inter and intra-familial clinical features (Talamillo et al., 2005;

Biesecker, 2011; Materna-Kiryluk, 2013; Malik, 2014). The disorder may be in the

form of an isolated disorder i.e. Non syndromic polydactyly, or is combined with

other hand/foot abnormalities, or it can also be the part of a pleiotropic developmental

deformity syndrome: syndromic polydactyly (Biesecker, 2011). Currently, in human

four disease causing genes and ten loci have been reported. The genes include

ZNF141 (OMIM 194648), GLI3 (OMIM 194648), MIPOL1, (OMIM 606850) and

PITX1 (OMIM 602149). Recently, on the basis of location of the extra digit (s) in the

foot or hand non-syndromic polydactyly has been classified into following types.

Such as paraxial polydactyly (PPD), postaxial polydactyly (PAP) and axial (central)

polydactyly (Talamillo et al., 2005; Haber et al., 2007). Preaxial Polydactyly can be

defined as a superfluous digit which affects the first digits, postaxial polydactyly

includes the fifth digits, and the rare type, axial (central) polydactyly is involved in

the doubling of three central digits: the second, third or fourth digits (Talamillo et al.,

2005; Haber et al., 2007).

II. Syndactyly

Syndactyly is a digital deformity where neighbouring fingers and/or toes are webbed

because during the development of limb they fail to separate. Syndactyly is the most

common type of inherited limb deformities with the incidence of 3–10 in 10 000

births (Hay, 1971; Castilla et al., 1980). Syndactyly is clinically heterogeneous

developmental malformations. There are many possible combinations where the




adjacent fingers and/or toes are joined by a web. It may be bilateral or unilateral and

asymmetrical or symmetrical. Moreover, inter and intra-familial variability of

phenotype is more common. The condition is very changeable that the same person

may show asymmetrical phenotypes in the right and left, and upper and lower limbs.

The segregation of syndactyly may occurs as an isolated clinical phenotype. There are

many types of syndactyly most of them are non-syndromic, majority of these entities

follow Mendelian dominant fashion of segregation. However, an X-linked recessive

and two autosomal recessive types have also been designated. Commonly autosomal

dominant phenotypes are somewhat less acute and establish extensively different

expressivity and incomplete penetrance (Temtamy and McKusick, 1978).

III. Split Hand/Foot Malformation (SHFM)

Split-hand/foot malformation (SHFM) is also called as Ectrodactyly, and is

characterized by abnormalities of the central rays that affect the hands/feet which

leads to a variable phenotype including missing digits; aplasia/ hypoplasia of the

phalanges, metatarsals and metacarpals, as well as remaining digits syndactyly.

SHFM is inherited in autosomal dominant pattern with variable expressivity. People

with recessive inheritance are very rare. Many loci are associated with isolated

SHFM. Mutated p63 may leads to isolated SHFM (MIM 605289), however, p63 gene

is also culprit in a different conditions with Split-hand/foot malformation and

ectodermal dysplasia. 10q24-q25 duplication including the FBXW4 (dactylin) gene

parts, along with the entire BTRC, LBX1 and POLL genes are comparatively

common cause of Split-hand deformity (MIM 246560) (Rinne et al., 2007; Ferone et

al., 2012).

IV. Brachydactyly

Brachydactyly is a term used for disproportionately short toes and fingers.

Brachydactyly can be either isolated or as a part of syndromic malformation

(Temtamy and McKusick, 1978).




1.4 Human Hereditary Disorders of Intellectual Disability

Intellectual disability (ID) can be defined as restraint of an individual’s ability to

adapt, interconnect, and comprehend to learn (American Association on Intellectual

and Developmental Disabilities (AAIDD, 2014). Intelligence quotient (IQ) score can

be used as a scale to determine intellectual capacity. When IQ drops below ≥ 2

standard deviation under the age -appropriate mean then the subject will be

considered as intellectually disable (Tirosh and Jaffe, 2011). Intellectual disability

leads to mild, moderate, severe or profound quantifiable decrease in the individual’s

functional aptitude. Around 1-4 % among us are affected with ID, usually in the range

of mild to moderate. There exist higher occurrence rate which is (30 -40 %) more in

male population, may possibly because of X linked genes. (Leonard & Wen, 2002;

Nguyen & Disteche, 2006). Individuals bearing ID face various issues while living

independently, maintenance or upholding employment or caring for themselves. This

results in several financial and logistical dares for ID individuals, their relatives and

communities.

1.4.1 Etiology of intellectual disability

Both environmental and genetic factors lead to intellectual disability, and both

contribute equally to its origin (Menkes et al., 2006). 65-75% of ID cases are because

of these factors and 37-55% accounts for mild ID cases. Besides, other factors are

behavioral and social, such as nutritional deficiencies, childhood infection, poverty,

liquor and drug used by the mother, severe deficiency can also be causative factor

(McLaren and Bryson, 1987). Beside these, ID can also be caused by acquired

injuries. In the industrialised world, ID is observed in 1 in 100 births, which is

perhaps because of fetal alcohol syndrome, Incidence rate of 800 to 1000 live births

with Trisomy 21 (MIM 190685) is also a major cause (Brown, 2004). ID

pathogenicity involve multiple gene factors, including single gene disorder, epigenetic




modifications, mitochondrial factor, multiple metabolic factors, chromosomal

aberration and repeat expansion. Identifiable anomalies may be observed in

syndromic ID that starts from single origin like Fragile X syndrome, Down syndrome

and untreated Phenylketonuria (PKU). In case of non-syndromic form, ID may be the

only observable sign.

1.4.2 Classification of Id Based on Intelligence Quotient (Iq)

To measure intellectual disability, IQ is the basic scale. 5 different stages of ID has

been assigned by the American association of Intellectual disability, on the basis of

defined parameters. The individuals have IQ ≤ 20 exhibits most severe form of ID i.e.

Level 1. They are completely dependent on their parents and siblings, and cannot take

care of themselves.. This level of ID is termed as profound, and syndromic patterns

are shown by a greater fraction among these individuals. Those having IQ ranges

between 20 to 30 lie in next stage of ID i.e. Level 2, such individuals are also totally

dependent for daily chores on their parents. This stage is also termed as severe ID.

Individuals with an IQ 36 to 51 come under third level of ID, also known as

Moderate. The sense of self care and other normal concepts i.e. home recognition etc

is present in the affected individuals. Individuals with an IQ range of 52 to 67 lie in

the very next class of ID, as in this class, the affected individuals show an

independence for daily matters and the ID is known as mild. Then the last class comes

at the border-line scenario of ID, in which there exist a very good sense of

independent living with IQ score of 68 to 84. Such cases are very difficult to study,

analyze and interpret as their routine life examination exhibit a significant difference

from all above mentioned.

1.4.3 Prevalence

According to latest data gathered, it can be concluded that 1.5 to 2% of ID patients are

affected with moderate ID, while 0.3-0.5% affected show severe ID in the developed




countries (Leonard and Wen, 2002). On the other side, an ID of very high burden rate

has been observed in Low and Middle income contries suffering from low living

standards of Mass population (Maulik et al., 2011). Consanguineous marriages is the

second most important factor of very high burden of ID in low and middle income

countries (Bashi, 1977).

Mild and severe ID has been observed by 6.5 % and 1.9 % of ID patients in Pakistan

respectively (Durkin et al., 1998; Mirza et al., 2009). Such high rate of ID is because

of a number of reasons and factors, among which the crucial ones are low living

standards, low nutritional food, neo-natal infections, hard labour and basic education

(Bashir et al., 2002).

1.4.4 X-Linked ID (XLID)

Proteins that are usualy highly expressed in the brain, in the hippocampus, i.e. a

region that plays a key role in learning and memory are usualy encoded by XLID

genes (Boda et al., 2002). There are several mutations observed in these genes, the

most frequent among them are loss of function, affecting neuronal functioning by

damaging both inhibitory and excitatory transmission. The change of dendritic spine

shape, size, and density are well-known hallmarks of XLID (Purpura, 1974).

About 5% to 10% of all the ID in males and 1% to 3% of the population accounts for

XLID. It consists of reduced adaptive skills arising from mutations in X-chromosome

genes and a wide variety of disorders that are characterized by cognitive impairment.

On these basis, it can be concluded that XLID is more frequent in males rather than

females (Ropers HH and Hamel BC, 2005).

Both in syndromic (in which intellectual disability is only one of a large set of

symptoms, and non-syndromic forms) as well as in non-syndromic forms, X-linked

ID is observed. High burden Non-Syndromic cases which are (in which intellectual

disability is the only manifestation of the pathology) X-linked ID, has been detected




as compared to syndromic cases which have been reported to be about 160 in number

(Stevenson et al., 2012; Lubs et al., 2012). After a careful estimation, it was showed

that minimum 120 genes are included in the XLID among which more than 100 genes

have been known till date (Stevenson et al., 2012).

1.4.5 Categories of X-Linked ID

On the basis of phenotypic appearance & mode of inheritance XLID can be sub-

divided.

1.4.5.1 Syndromic XLID

Complex arrangement of phenotypic abnormalities are observed in syndromic XLID.

These involve behavioural abnormalities, characteristic cranio-facial appearance,

development differences or metabolic anomalies. These phenotypic variations of

affected individuals are mostly unremitting with an identifiable pattern (Lisik and

Aleksander, 2008).

The most prevalent form of ID is Fragile X Syndrome among all syndromic forms.

From literature, it was found that the incidence frequency is 1:6,000 in the females

and 1: 4,000 in males (Hagerman, 2002). Extension of CGG repeat sequences in 5’

UTR of FMR1 gene, is known to be the main cause of Fragile X- syndrome

(Hagerman, 2002).

Mild to severe phenotypes are because of most of the tandem repeat expansions.

Various effects are observed by the exact position of expansions of repetitive

sequences in the genome i.e. from transcription initiation reduction to protein toxicity

(Usdin and Grabczyk, 2000).

In humans, more than 20 unstable micro-satellite repeats has been identified so far,

which result in neurological anomalies (Judith et al., 2009). Loss as well as gain of

function is observed by the expansion of repeat sequences. If the gene is not




expressed at all or expression levels are very low to show any significant function, it

will lead to loss of functionality. Gain of function can be defined as the induction of

new cellular functions due to repeat expansions at mRNA or at protein level. If the

expansion of repeats occur within translated part of genome, it will cause abrupt

changes in the structure as well as function of that particular protein (Orr and Zoghbi,

2007).

mRNA will be affected if the repeat expansion occurs in translating genome region

(Judith et al., 2009). Epilepsy, autism, coffin Lowry syndrome and cerebellar ataxia

etc are syndromic forms of X linked ID.

1.4.5.2 Non-syndromic XLID

XLID is because of the several mutations which have been reported so far. The

proteins translated from these genes are involved in neuronal synapse, plasticity and

neuro-transmission (Ishizaki et al., 2000). Learning and memory functions are altered

by these genes as they follow a common pattern of early expression during

development as well as significant expression into the hippo-campus of adults

(Toniolo and D’Adamo, 2000). Thus a significant insight into the molecular as well

cellular pathways of these genes can be obtained by detailed study including

developmental expressional patterns as well as functional analysis of these gene. This

will also provide a better understanding of ID (Luscher et al., 2000).

1.4.6 Selected XLID Genes

I. ARX

Mild to severe XLID is observed by Mutations in ARX gene (Stromme, Mangelsdorf,

Scheffer, & Gecz, 2002), which are mostly syndromic, even though in some cases, it

may be inaccessible. ARX-related neurological features include seizures (infantile

spasms), dysarthria (motor speech disorder), dystonia (involuntary muscle

contractions) and autism (Shoubridge et al., 2010).




Physical variations from the norm, if introduced, are simply constrained to the mind

(hydranencephaly, lissencephaly and agenesis of the corpus callosum) and genitalia

(Kato et al., 2004). Perpetual looseness of the bowels is generally distinguished in

seriously influenced people (Kato et al., 2004). Disorders which are related with ARX

changes incorporate pleased disorder XLID, strange genitalia and agenesis of the

corpus callosum,) likewise named as X-connected lissencephaly unusual genitalia

disorder or XLAG (Kitamura et al., 2002; Proud, Levine and Carpenter, 1992) and

West disorder (X-connected juvenile fits) (Kato et al., 2003). Upto 7.5% of families,

demonstrating an evident XLID convey transformations in ARX quality. Among all

the distinguished change classes, the most well-known repeating transformation is 24-

base match duplication which causes an expansion in second polyalanine tract of

quality from 12 to 20 alanine buildups. This transformation (duplication) causes

gentle syndromic or non-syndromic ID, yet uncovers critical phenotypic variety inside

What's more, between families (Turner et al., 2002). By and large the relationship

between's ARX genotype-phenotype is relatively reliable (Olivetti and Noebels,

2012).

II. ATRX

ATRX is a huge gene, which included 350 kilo bases of genomic succession. In 90%

cases, zinc finger (exon 7-9) and helicase (exon 17-20) areas are changed (Gibbons et

al., 1995). ATRX change causes X-connected, alpha-thalassemia mental hindrance

(ATR-X) disorder (Gibbons et al., 1995), which incorporate serious ID, skeletal,

facial and urogenital anomalies, alongside mellow alpha-thalassemia (Gibbons et al.,

1995; McPherson et al., 1995). ATRX phenotypes incorporates gentle non-syndromic

ID at all influenced people (Guerrini et al., 2000; Yntema et al., 2002). Clinical

indications are once in a while seen in female bearers while, over 95% show changes

in X-inactivation (XI) designs (Stevenson, 2010).. The ATRX protein have part in

different organic procedures, for example, chromatin rebuilding, DNA replication and




gene articulation and include in certain sort of diseases (Clynes and Gibbons, 2013;

Clynes et al., 2013). Nonappearance of ATRX work cause strange chromatin direction

which brings about the unsettling influence of different genes and their pathways

(Clynes and Gibbons, 2013; Clynes et al., 2013). This has sound specific

inconvenience for cells which have Active ATRX changed X chromosomes and

furthermore give sensible clarification to skewed X-inactivation in unaffected female

bearers (Migeon, 2007).

III. CASK

CASK-related ID (not at all like numerous types of XLID) has been watched

regularly in females than in guys (Moog et al., 2011; Moog et al., 2013). The

phenotype of CASK, incorporates mellow to extreme ID joined by mind (counting

optic) abnormalities, huge microcephaly (inside the main year of life or beginning 21

prenatally) and particular facial highlights (Moog et al., 2011; Moog et al., 2013).

Mind imaging indicates pontocerebellar hypoplasia in which fourth ventricle is

expanded. There is diminished whirling in the cerebral cortex of a few cases however

not in all patients (Hackett et al., 2010). In the influenced guys, phenotype is variable

ID with or without innate nystagmus, miniaturized scale or macrocephaly and

cerebellar hypoplasia (Hackett et al., 2010; Piluso et al., 2009; Tarpey et al., 2009).

CASK transformations might be engaged with subset of FG disorder, hypotonia,

including formative postponement, macrocephaly, agenesis of the corpus callosum,

stomach related unsettling influences and trademark identity (Piluso et al., 2009).

In this case, mis-sense and splicing mutations are basic which might be hypomorphic

or asymptomatic/penetrant in female carriers (Hackett et al., 2010; Najm et al., 2008).

Nystagmus causing changes are situated at the C-terminal of protein (Hackett et al.,

2010). The CASK protein, a universal serine protein kinase is profoundly

communicated in fetal cerebrum and predictable with the ID phenotypes and mind

contortion (Hackett et al., 2010).




IV. KDM5C

KDM5C likewise named as JAR1D1C and SMCX), is situated at Xp11.22, encodes

lysine (K)- particular demethylase 5C which is a ubiquitously communicated protein,

and assume part in chromatin rebuilding by expelling the methyl group from histone

H3 at lysine 4, (Iwase et al., 2007; Tahiliani et al., 2007). In 2005, KDM5C was

perceived as a major factor of XLID and has been appeared to be associated with the

dendritic improvement and survival of neurons (Iwase et al., 2007; Jensen et al.,

2005). It likewise has been exhibited that loss of DNA methylation at numerous loci

is related with KDM5C mutations (Grafodatskaya et al., 2013), and its consumption

through RNA impedance causes articulation of various neuronal target genes

(Grafodatskaya et al., 2013; Tahiliani et al., 2007). Shockingly, KDM5C

downregulation is considered as an inert remedial approach in Huntington's ailment,

so the overexpression of neuronal genes can be turned around which is expected to

changed huntingtin (Vashishtha et al., 2013).

Like other XLID genes, KDM5C mutations can likewise bring about syndromic or

nonsyndromic types of ID. Transformation recurrence of KDM5C in XLID families

shifts from 0.6% to 2.8% (Goncalves et al., 2014; Jensen et al., 2005). The highlights

related with syndromic ID are; discourse delay, dysmorphic facial highlights, short

stature, genital inconsistencies, ataxia, adjusted muscle tone, spastic paraplegia and

forceful conduct (Goncalves et al., 2014; Jensen et al., 2005; Ounap et al., 2012). The

two guys and females might be influenced as KDM5C is one of numerous X

chromosome genes that escape from inactivation and have high articulation in females

when contrasted with guys (Johnston et al., 2008; Ounap et al., 2012).

1.5 Human Hereditary Skin Disorders

Skin is present on the outer surface of our body and it is the most protective and

largest integumentary organ of our body. In an average person, skin covers 1.7 m2 of




area and accounts of 16 % of total body weight (Wickett, 2004). Skin protect our

body form different external and internal encounters such as harmful UV radiation

from the sun and different temperature changes of our environment. It receives daily

assaults such as scratches and wounds. These damages are continuously replaced by

the continuous self-renewal property of skin. Skin continuous repair its damage

tissues by replacing old cells with the new ones (Fuchs, 2007). All these functions of

skin, that maintain the proper homeostasis of skin is maintained by controlled gene

expression (Yi and Fuchs, 2010).

Several genetic disorders of skin are well known due to the advance knowledge of

human genome and advances in molecular screening strategies. At molecular level,

over more than 300 single gene skin disorders are known till date.

1.5.1 Human Skin

Human skin is mainly consist of 3 layers. Epidermis is the outer layer of the skin,

followed by dermis which is consists of connective tissues and third layer is

hypodermis which is consist of fatty sub cutis tissues.

1.5.2 Epidermis

The outer most covering of the skin is known as epidermis. In epidermis the

differentiation and proliferation continues throughout the life. Epidermis is consist of

stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and

stratum basale (McGrath et al., 2004). The main function of epidermis is to provide

protection to organs, maintain homeostasis insulate the body, act as a water resistance

and regulate the temperature (Odland, 1991; Wickett and Visscher, 2006).

1.5.3 Dermis

Dermis is present below the epidermis, which is formed by the mesenchymal cells.

These mesenchymal cells forms the blood and connective tissues in the skin which




also includes fibroblast and mast cells of the dermis and fats cells present in the

hypodermis (Toma et al., 2001; McGrath et al., 2004).

1.5.4 Hypodermis

The hypodermis is the third layer which is also known as subcutis. It is present just

below the dermis and usually consist of fatty tissues. It is usually not considered as a

part of skin. This layer is consist of loose connective tissues and elastin and contains

adipocytes (fats), fibroblast and macrophages. Fat is helpful in the insulation of the

body (Odland, 1991; McGrath et al., 2004).

1.5.5 Other Ectodermal Appendages Involved In Human Hereditary Skin

Disorders

During development, any abnormality in the gene expression and epigenetic

mechanism due to mutation can results in the loss of function, and change in the

development and structure of the organism. Some of these genetic abnormalities can

be inherited to the next generation. In humans, these abnormalities are known as

human hereditary skin disorders. Along with the skin, some other ectodermal

appendages are usually involved in these skin disorders such as teeth, nails, hairs and

sweat glands.

1.5.6 Hair

Hairs are the fibres that consist of keratin and are usually covered with cuticles. Hairs

are formed by the intermediate filaments that are then surrounded by the matric

proteins. The diameter of hairs ranges from the 40-150 μm (Kajiura et al., 2006).

Hairs are produced by the hair follicles (HF) and the shape and appearance of hairs

depends upon the hair shaft’s structure (Schlake, 2007).




1.5.7 Sweat Glands

Sweat glands occur only in mammals, are exocrine in nature underlie the whole skin

(Biedermann et al., 2010) and responsible for performing thermoregulatory role in

body homeostasis (Nazzaro, 1989). Sweat glands are of two types, apocrine (at

puberty secreting into hair follicles in the armpits, groin and areoles) and eccrine

(distributed widely) (Folk and Semken, 1991; Biedermann et al., 2010). Sweat glands

are made up of myoepithelial tissues, which help in secretion of accumulation by

contractile squeezing under control of autonomous nervous system (Wilke et al.,

2007; Schlereth et al., 2009). A human possesses 2-4 millions of sweat glands

spreading throughout the whole skin. Following chest and scalp, soles and palms are

holding the highest concentration of eccrine sweat glands (620/cm2) weighing about

30-40 µg (Biedermann et al., 2010). Absence or anomalies in sweat glands disturb the

homeostasis and irregular temperature of the body in adults, while seizure and neural

disorders in children (Schlereth et al., 2009).

1.6 Human Skin Disorder

1.6.1 Alopecias

Any change in the sequence of genes responsible for the growth of hair results in to

complex disorder called as alopecia (Hardy 1992; Rosenquist and Martin, 1996). As a

consequence of such genetic alteration loss in the formation of hair may take place in

the life span of a person or it may occur congenitally. There are multitude number of

reasons for genetic hair loss that can be through alteration in genes or through any

foreign factor. Alopecia can be either syndromic or non-syndromic.




1.6.2 Isolated Alopecias

1.6.3 Congenital Atrichia (Atrichia With Papular Lesions)

The main cause for congenital Atrichia can be via atrichia at birth or flaking of

normal scalp hair several months after birth that may further result into sparse

eyelashes and eyebrows, and axillary or body hair (Ahmad et al., 1998a). The disorder

is referred as atrichia with papular lesions, when the age of affected person reaches to

1 to 26 years during the development of this disorder (Ahmad et al., 1998a;

Zlotogorski et al., 2002). The teeth, glands and nails of affected individual are normal

in this disorder.

The HR gene is responsible for APL which is located on chromosome 8p21.3. the

product of HR gene is mainly expressed in brain & skin and it is a transcriptional co-

regulator with a sole zinc-finger domain (Cachon-Gonzalez et al., 1994; Thompson,

1996; Ahmad et al., 1998b). Up till now 46 genetic changes have been found in

numerous families affected with APL (Azeem et al., 2011).

1.6.4 Monilethrix

It is an innate hair disorder which is autosomal dominant in its nature. Affected

people show usual thickness of hair nodes except unconnected frequently by

dystrophic limitations. The internodes have an elevated tendency to shatter, resulting

to hair loss, i.e., small stubble hair linked with “follicular keratosis and perifollicular

erythema”. The causal gene for the said disorder is present on chromosome 12q13

(Winter et al., 1997b). In helix termination or initiation motifs in type II hair keratins

hHb1, hHb3 and hHb6, usually, point mutations are drawn in, that are articulated in

hair cortex (Schweizer et al., 2007). koilonychias, lamellar splitting and brittleness

that are mainly nail deformities have also been seen in such disorders (Healy et al.,

1995).




1.6.5 Localized Autosomal Recessive Hypotrichosis

It is also a type of genetic hair loss in which scalp, chest and extremities and mainly

sparing the facial, axillary and pubic hair are severely affected. LAH has three main

types. Due to genetic changes in DSG 4 gene LAH 1 occurs. Furthermore, some other

symptoms have also been reported that are also associated with monilethrix hairs

Wajid et al., 2007). Desmoglein 4 is from desmosomal cadherins that shape the

central part of desmosomes (Delva et al., 2009). Desmoglein 4 is from desmosomal

cadherins that shape the central part of desmosomes (Delva et al., 2009). Total seven

desmosomal cadherins have been reported that include four desmogleins (DSG1-4)

and three desmocollins (DSC1-3), possessing five tandemly frequent extracellular

(EC) groups, a sole transmembrane area and a cytoplasmic group (Kljuic et al., 2003).

Hair follicle shaft cortex contains maximum numbers of DSG4 protein in the case of

people affected with LAH with abnormal phenotype (El-Amraoui and Petit, 2010).

Mutation in LIPH gene results in LAH2 (MIM 607365), whose causal gene is present

on chromosome 3q27.3. The gene “LIPH encodes a membrane-linked phosphatidic

acid-selective phospholipase A1 mPA-PLA1a, that forms 2-acyl lysophosphatidic

acid a lipid arbitrator with a diversity of biological characteristics” (Balazs et al.,

2001; Moolenaar et al., 2004; Naz et al., 2009). The reported genes for LIPh are

fifiteen. These genes are expressed in various organs of human body, such as hair

follicle, heart, spleen, ovary, prostate and kidney (Kazantseva et al., 2006).

Dsc3 (Chen et al., 2008) and Dsg3 (Chen et al., 2008) deletion is present in mice

(Koch et al., 1998) expand faulty hair anchoring in the telogen stage of development

sequence. A pemphigus vulgaris like physical features on skin are being shown by

such mice (Schmidt and Koch, 2007; Chen et al., 2008). Newly, on chromosome

10q11.23-22.3 and 7p21.3-p22.3 two new autosomal recessive hypotrichosis

positions are found (Naz et al., 2010; Basit et al., 2010). Alike characteristics have




been revealed by the affected family individuals on above two chromosomal areas

that are well-matched to other autosomal recessive hair trouncing diseases. Till now

no such important variants have been found apart from already identified two genes in

such families (Naz et al., 2010).

1.6.6 Hypotrichosis Simplex

Pathogenic mutations in monilethrix (MIM 158000) Autosomal dominant hereditary

hair loss diseases occurred as in as case of type II hair keratins hHb1, hHb3 and hHb6,

that have been found to be expressed in hair cortex (Zhang et al., 2009),

corneodesmosin (CDSN; MIM 602593) (Levy-Nissenbaum et al., 2003), in the

inhibitory upstream open reading frame (ORF) namely “APC-downregulated-by-1

APCDD1; MIM 607479” (Shimomura et al., 2010b), “(U2HR of the hairless HR)”

(Wen et al., 2009; Mansur et al., 2010), and Keratin-74 “(KRT74; MIM 608248)”

(Shimomura et al., 2010c) genes.

With variable degree of body and scalp hair both male and females are equally

harmed by a group of hereditary non-syndromic isolated alopecia known as

Hypotrichosis simplex “(HS; MIM 146520, MIM 605389). The possibility of

presence of HS may be localized (hair loss limited to the scalp only) or widespread

(Pasternack et al., 2008; Nahum et al., 2009).

The HS disease may appear either from birth or after the birth during growth and

development phase and it can be in autosomal dominant or recessive form (Sprecher,

2005; Horev et al., 2009; Nahum et al., 2009).

The autosomal recessive form of hypotrichosis simplex (MIM 241900) is

characterized, due to mutations in genes DSG4, LPAR6/P2RY5 and LIPH genes such

as DSG4, LPAR6/P2RY5 and LIPH (Sprecher, 2005) while mutations in the gene

CDSN (corneodesmosin) results in dominant HS (MIM 146520) that is comprised of

529 amino acids, articulated completely in epithelia (Davalos et al., 2005).




1.6.7 Autosomal Dominant Woolly Hair/Hypotrichosis

In humans, loss of hair function due to gene mutation can be transferred in autosomal

fashion, and up till now the mapped loci for such condition are four in number which

are located on various chromosomes and the similar genes for them have also been

identified. Keratins have been divided in to two types i.e. acidic type I and basic to

neutral type II and this classification is based on their biochemical character. The

group of genes for type I keratin possessing 28 keratins and are present on

chromosome 17q21.2, on the other end group of genes for type II keratin possessing

26 keratins and are present on chromosome 12q13.13 (Arin, 2009).

The position of autosomal dominant woolly hair/hypotrichosis is on chromosome

12q12-q14.1 (Shimomura et al., 2010c) & caused by pathogenic genetic changes in

gene KRT74 (Wasif et al., 2011).

Tightly twisted curled hair or sparse scalp hair are the main features of ADWH and it

is a very rare disease. Till now it has been found in 03 Pakistani families that are

connected to 12q12-q14.1 having type II keratin gene group (Shimomura et al.,

2010c; Wasif et al., 2011).

KRT74 is “encoding inner root sheath (IRS) specific epithelial keratin. Thus, mutant

proteins are resulting into disruption of keratin intermediate filament formation, most

likely in a dominant-negative” way (Shimomura et al., 2010c).

1.7 Associated Alopecias

1.7.1 Alopecia with Mental Retardation Syndrome

It is an uncommon autosomal recessive condition in which incomplete or whole hair

loss and minor to more complicated cerebral hindrance take place (John et al., 2006;

Wali et al., 2006b, 2007b).




APMR is a rare autosomal recessive “disorder, clinically characterized by partial or

complete hair loss and mild to severe mental retardation” (John et al., 2006; Wali et

al., 2006b, 2007b). It involves total alopecia of all areas of regular hair growth (scalp,

eyelashes, eyebrows, pubic and axillary hair) from birth. To date three APMR loci

have been mapped on chromosome 3q26.33-q27.3 (John et al., 2006), 3q26.2-q26.31

(Wali et al., 2006b) and 18q11.2-q12.2 (Wali et al., 2007b). However, genes causing

APMR have not been reported yet.

1.7.2 Hypotrichosis with Juvenile Macular Dystrophy

A rare autosomal recessive disease that is featured by small sparse scalp hair at birth

and advanced macular degeneration that subsequently leads to premature blindness

(Souied et al., 1995). The CDH3 gene is present on human chromosome 16q22.1, due

to genetic changes in it HJMD is caused. This gene is also accountable for calcium-

dpendent cell-cell a.dhesion (Sprecher et al., 2001).

1.7.3 Digenic Hereditary Hair Loss

In recent times, autosomal recessive form of hypotrichosis has been found and

mapped on chromosomes namely 12q21.2-q22 and 16q21-q23 in the two Pakistan

families (Basit et al., 2011). The CDH3 gene is present on 16q21-q23. Two various

types of physical changes (hypotrichosis with juvenile muscular dystrophy &

ectodermal dysplasia, ectrodactyly and macular dystrophy syndrome take place as a

result of genetic alteration of this gene (Shimomura et al., 2008b). Hair loss take place

in such affected families. Regular macular pigment epithelium and retina has been

shown by the affected families and these facts become evident after doing the fundus

examination, electroretinography and electrophysiological tests of affected individuals

retina (Basit et al., 2011). It was further revealed that a single base pair homozygous

insertion (c.1024_1025insG) in exon-9 and deletion of four base pair

(c.1859_1862delCTCT) in exon-13 have been identified in the gene CDH3 located on




chromosome 16q21-q23 were the two main genetic alterations that were present in the

two affected families. The change in the HJMD phenotype has been possible through

a modifier gene which is located on chromosome 12q21.2-q22. (Basit et al., 2011).

1.7.4 Androgenetic Alopecia

The alteration of terminal hairs into unspecified hairs and subsequently to vellus hairs

results in a genetic disease known as Androgenetic Alopecia. The hair pattern of such

affected individual is not regular (Richards et al., 2008). This disorder is associated

with both genders i.e. male pattern baldness and female pattern baldness. One of the

most important risk factor in this doisorder is the change in the gene receptor on X

chromosome (Hillmer et al., 2005). Chromosomes 3q21-q29, 11q14-q25, 18p11-q23

and 19p13-q1 are the susceptibility loci for AGA that has come forward through

genome wide studies (Hillmer et al., 2008).

1.7.5 Alopecia Areata (Aa)

It is a type of skin disease where hair follicles are attacked by body’s autoimmune

system suddenly (Martinez-Mir et al., 2007).

Alopecia areata is a condition where hairs are dropped in the form of patches on the

head and other parts of the body and may lead to complete loss of hairs from the head

called (alopecia totalis) and might progress to full hair loss from all party of the body

and head a condition known as (alopecia universalis) (Martinez-Mir et al., 2007).

In human beings Alopecia areata is another most common reason of hair loss, it is

about 2% (Gilhar and Kalish, 2006). All categories of AA histologically are identified

through the existence of diffused lymphocytic infiltrate all around the hair follicle.

Although Alopecia areata is autoimmune tissue-specific disorder but stills its

molecular mechanism is unidentified. Under normal conditions, hair follicle is




considered as an immune-privileged organ where major histocompatibility complex is

less expressed (Paus et al., 2005).

Though, AA depicts a breakdown of immune privilege, causing obliteration of hair

follicle through lymphocytes. Family oriented linkage analysis for alopecia areata has

resulted different loci on several chromosomes such as chromosome six, eight, sixteen

and eighteen (Martinez-Mir et al., 2007). One of such resemblance to the MHC locus

on p arm of chromosome 6 that supports the understanding that AA is an autoimmune

disease.

1.7.6 Ectodermal Dysplasias

Ectodermal dysplasias (EDs) is a genetically heterogeneous group of deformity

encompassing disorders in 2 or mote tissues of ectodermal origin including teeth,

nails, hair and sweat glands.

Ectodermal dysplasia can be non-syndromic identified by only ectodermal signs, or it

may be syndromic including ectodermal features along with other deformities.

TheEDs are categorized into two main groups. The pure ED, that is, the anhidrotic

ectodermal dysplasia (EDA) with no or less sweating on the other hand the hidrotic

ectodermal dysplasia (EDH) showing normal sweating (Clouston, 1939; Shigli et al.,

2005).

Currently, two hundred molecular and pathological conditions of ectodermal

dysplasias have been documented (Itin and Fistarol, 2004).

1.7.7 Ectodermal Dysplasia of Hair, Nail and Teeth

The rarest form of Ectodermal dysplasia of nail, hair, and teeth are inherited in

autosomal recessive pattern. Characterized by very fine or thin hairs on the head,

eyebrows and eyelashes maybe present or absent. Teeth are deformed and irregular

dystrophic toenails and fingers (Tariq et al., 2008a). On chromosome 18q22.1-q22.3 a




novel locus has been characterized with deformities of nail, hairs, and teeth along

with no other related deformity in Pakistani consanguineous families. 3 candidate

genes (ZNF407, CDH19, CDH9) from the mapped region were subjected to

sequencing in the family but it remains unsuccessful to find the functional sequence

variant in patients (Tariq et al., 2008a).

1.7.8 Ectodermal Dysplasia of Hair and Nail

ED of hair and nail type (MIM 602032) is a rarest form of congenital disorder

characterized by trichodysplasi (partial or total alopecia, hypotrichosis) along with toe

nail and finger dystrophy devoid of other related deformity. Several clinical kinds of

pure hair and nail dysplasia have been recognized on different chromosomes

including 17p12-q21.2 and 10q24.32-q25.1, 12q13.13 in consanguineous Pakistani

families. (Rafiq et al., 2005; Naeem et al., 2006a; Naeem et al., 2006b). Sequence

variants causing pathogenesis have been identified in the KRTHB5 genes (MIM

602767) present on chromosome 12q13 in consanguineous Pakistani families along

with ED of hair and nail type (MIM 602032), (Naeem et al., 2006a, Shimomura et al.,

2010d).

1.7.9 Hypohidrotic Ectodermal Dysplasia

In human beings a genetic disorder called Hypohidrotic ectodermal dysplasia (HED)

is a genetic disease with clinical features: thin hairs, thick lips, flattened nose,

hupohidrosis and teeth anomaly or hypodontia (Bayes et al., 1998; Chassaing et al.,

2006). The most common type of HED is X-linked HED (MIM 305100) results due

the mutation in EDA gene (MIM 300451). Which is located on chromosome Xq12–

q13.1 that encodes a family of tumornecrosis factor (TNF) called ectodysplasin.

(Tariq et al., 2007a; Zhang et al., 2009a; Clauss et al., 2010; Naqvi et al., 2011).

In X-linked HED (XLHED) most of the mutations are missense, but 1/5th is deletions

or insertions (Tariq et al., 2007a; Clauss et al., 2010). Mutations in EDA gene may




cause isolated hypodontia but it is very rare (Tao et al., 2006; Fan et al., 2008; Naqvi

et al., 2011). Some patients of HED show mixed inheritance autosomal recessive and

autosomal dominant patterns. Mutations in 2 genes that are functionally related;

(EDARADD) on chromosome 1q42.2-q43 and EDA-A1 receptor (EDAR) placed on

2q11-q13 chromosome are the reason of abovementioned inheritance patterns (Lind et

al., 2006; Tariq et al., 2007a; Azeem et al., 2009; Naqvi et al., 2011).

1.7.10 Odonto-Onycho-Dermal Dysplasia

Odonto-onycho-dermal dysplasia (OODD) is inherited in autosomal recessive fashion

and is the rarest from of ectodermal dysplasia showingsevere palmoplantar

hyperkeratosis, oligodontia, nail dystrophy and hyperhidrosis (Adaimy et al., 2007;

Bohring et al., 2009).

Mutations in WNT10A gene (MIM 606228) is the cause of OODD placed on

chromosome 2q35-36.2. WNT10A is the member of Wnt family of growth factors;

associates of a huge family of secreted proteins with rich in cysteine amino acids. It

is also involved in cell to cell regulation during embryogenesis. Small secreted

proteins containing 350-400 amino acids residues are encoded by Wnt genes (Adaimy

et al., 2007).

1.7.11 Oligodontia

Alteration in morphology and number of teeth is the most common congenital

deformities in human beings. The wordautosomal dominant oligodontia is restricted

to the missing of 6 or more teeth (Stockton et al., 2000; Lammi et al., 2003). The

Oligodontia is associated with several syndromes, like numerous forms of ectodermal

dysplasia. Haence in ED teeth are frequently small in size with cone-shape and

characteristically misplace their particular structure (Bailleul-Forestier et al., 2008).

Specific type of hypo/oligodontia results from mutations in MSX1 homeobox gene.

The effected teeth are 3rd molar and 2nd premolar (Vastardis et al., 1996). The location




of MSX1 is on the p16.1 arm of chromosome 4. And comprises of 2 exons (Kim et

al., 2006). A transcription factor (TF) gene PAX9 is placed on q12-q13 arm of

chromosome 14. Mutation in this gene causes the loss of the most perpetual molars

with or devoid of hypodontia in the primary teeth (Stockton et al., 2000; Bailleul-

Forestier et al., 2008).

1.7.12 X-Linked Recessive Isolated Oligodontia

Oligodontia (MIM 313500) is a type of inherited disease with the absence of teeth

follows the X-linked inheritance pattern that does not depict other HED feature (Han

et al., 2008). The corresponding locus has been mapped on Xq12-q13.1 (Tao et al.,

2006). Mutations in EDA genes causes X-linked recessive isolated oligodontia

(Tarpey et al., 2007; Han et al., 2008; Rasool et al., 2008; Azeem et al., 2009).

1.7.13 Isolated Congenital Nail Dysplasiah

Isolated congenital nail dysplasia (ICND; MIM 605779) follows the rare autosomal

dominant fashion of inheritance with the clinical features of thin and reduced

formation of the nail plates, most of the toenails and fingernails are affected (Hamm

et al., 2000). Krebsova et al. (2000) the culprit gene ICND has been mapped on p13

arm of chromosome 17.

1.7.14 Isolated Congenital Nail Clubbing

Hereditary nail clubbing is a rare genodermatosis with the clinical features: bulging of

nail plate, enlargement of terminal segments of toes and fingers, where connective

tissues are proliferated between nail matric and phalanges (Myers and Farquhar,

2001). This is caused because of abnormal activity of nail matrix at the time of

morphogenesis of nail. Nail clubbing can be related with systemic disorder or isolated

deformity (Gudbjartsson et al., 2005). The HPGD gene that is the cause of the

aforesaid disoreder is placed on q32.3-q34.2 arm of chromosome 4. Any mutation in

this gene causes nail clubbing (Uppal et al., 2008; Tariq et al., 2009b).




1.7.15 Cutis Laxa Syndrome

Cutis laxa syndrome is inherited in autosomal and X-linked pattern (Agha et al., 1978;

Zhang et al., 1999). It is a varied group of inborn deformities of elastic fibers which

contain microfibrillar and elastin constituents (Andiran et al., 2002). This syndrome

results from pathogenic sequence variants in about 8 genes comprising LTBP4, ELN,

FBLN4, RIN2, PYCR1, ATP6V0A2, ATP7A and FBLN5(Jelani et al., 2010).

Currently, in consanguineous Pakistani families genome scan using highly

polymorphic microsatellite markers, identified anovel locus located on chromosome

9q13-q21.32 involved in autosomal cutis laxa syndrome (Jelani et al., 2010).

Analysis of DNA sequence of different genes failed to recognise functional sequence

variant in individuals that are affected members of the family (Jelani et al., 2010).

1.7.16 Ectodermal Dysplasia-Cutaneous Syndactyly Syndrome.

The clinical features of the Ectodermal dysplasia-cutaneous syndactyly syndrome

(EDCS) are hypoplastic nails, palmoplantar keratoderma, hypotrichosis, tooth enamel

hypoplasia, hyperhidrosis and bilateral partial cutaneous syndactyly (Tariq et al.,

2009c). EDCS locus is located on chromosome 7p21.1-p14.3. Currently, there is not

any culprit gene identified on this locus (Brancati et al., 2010; Jelani et al., 2011). It

has been reported that the mutation in PVRL4 results in EDCS (Brancati et al., 2010;

Jelani et al., 2011).

1.7.17 Trichorhinophalangeal Syndromes

The trichorhinophalangeal syndromes (TRPSs) are uncommon genetic anomalies

inherited in dominant fashion (Tariq et al., 2008b). the clinical features of(TRPSs)

include facial developmental defects, including protruding ears, spare scalp hair, spare

eyebrows, thin upper vermilion border and pear-shaped nose (Rossi et al., 2007).

Skeletal deformities of the TRPS are short stature, hip malformations and cone-

shaped epiphyses at the phalanges (Momeni et al., 2000). 3 different kinds of TRPS




III have been documented: TRPS I (MIM 190350), TRPS II (MIM 150230) and TRPS

III (MIM 190351). Momeni et al. (2000). The TRPS I is located on chromosome

8q24.1. Various deletions, nonsense, and missense mutations have been reported in

the TRPS I gene (Gentile et al., 2003; Kaiser et al., 2004; Rossi et al., 2007). TRPS II,

is characterized by microcephaly, mental retardation and multiple cartilaginous

exostosis in addition to the signs of TRPS I. The cause of TRPS II is de novo deletion

of 2 genes that are contiguous including EXT1 and TRPS I placed on q24.1 arm of the

chromosome 8 (Buhler et al., 1987).

The TRPS III is characterized by the symptoms of TRPS I but related with short

stature, brachydactyly, reduced phalanges and metacarpals. Functionality of zinc

finger domain of TRPS I protein is affected by mutation in exon number 6 of the

TRPS 1 gene that results in causing TRPS III disorder (Kobayashi et al., 2002;

Piccione et al., 2009; Gai et al., 2011).

1.7.18 Palmoplantar Keratodermas (Ppks) Syndrome

Palmoplantar keratodermas (PPKs) are diverse group of genetic disorders causing

hyperkeratosis of soles and palms. The inheritance pattern is mixed; it may be

autosomal dominant or autosomal recessive, it can also be mitochondrial or probably

X-linked (Swensson et al., 1998; Bowden, 2010). Palmoplantar keratodermas (PPKs)

are diverse group of genetic disorders causing hyperkeratosis of soles and palms.

(Hennies et al., 1995).

On the other hand, striate palmoplantar keratoderma II is located on p24 arm of

chromosome number 6(Armstrong et al., 1999). Result due to mutation in DSP gene

(MIM 125647) (Armstrong et al., 1999). However, striate palmoplantar keratoderma

III is placed on chromosome 12q13.13 (Whittock et al., 2002) results due to mutation

in KRT1 gene (Whittock et al., 2002). On chromosome 3q27.2-q29 a novel locus has

been reported for autosomal recessive PPK in family with consanguineous marriages.




It is characterized by various bilateral participation of soles and palms with minor

involvement of nails, painful walking, and problems in grasping and severe fissuring

with bleeding (Khan et al., 2010).

1.8 Strategies of Genetic Testing for Various Genetic Disorders

1.8.1 Hunting for the Causative Genes

To understand and treat genetic disorders, it is essential to identify the causal gene or

variant. In general, there are two ways of looking for the causal gene: using a

hypothesis-driven (a) or a hypothesis-free approach (b). In a genome-wide approach,

we do not apply any hypothesis for searching for certain candidate genes or loci

(Hardy and Singleton, 2009). In a hypothesis-driven approach, on the other hand, the

candidate gene is investigated based on known, underlying disease mechanisms,

independent of the gene mapping strategy used (Kell and Oliver, 2004).

a. Hypothesis-driven approaches

In hypothesis-driven approaches, knowledge of underlying mechanisms of the disease

suggests protein products for possible involvement in the disease pathogenesis

(“reverse genetics”).(Hardy and Singleton, 2009)

For example, Sato et al. (2007) showed that Rab-8-deficient mice expressed a reduced

amount of the RAB8 protein, identical to what was observed in a patient with

microvillus inclusion disease (MVID). Although Rab8 turned out not to be the

causative gene for MVID, this experiment still suggested further candidate genes for

screening and led to the discovery of MYO5b as the causal gene that interacts with

RAB8 in the same pathway (Müller et al., 2008; AM Szperl et al., 2011). Using

reverse genetics without mapping the candidate locus has severe limitations,

especially in identifying genes for complex disorders, since these can have a broad

phenotypic spectrum and thus an unclear disease mechanism.




A candidate approach is therefore most effective when applied to a previously

mapped locus (so-called “positional candidate mapping”).

b. Hypothesis-free approach

The hypothesis-free approach can be divided into two phases: first a genome-wide

scan, in which appropriate statistical measures are applied to determine any

significant association between the gene’s position in the genome and the disease (by

either linkage analysis, homozygosity mapping, or a genome-wide association study),

or by sequencing the entire genome for variants matching the disease criteria (whole

genome re-sequencing). The second phase is replication, where the region, locus or

variant is investigated in an independent study designed to confirm the preliminary

findings.

1.8.2 Genome-Wide Mapping Phase

Genome-wide linkage scans These are mainly used to map candidate loci in families

segregating for disease, by finding significant linkage between a marker (for example,

a SNP or microsatellite) and the disease in the pedigree (family-based), while taking

sequencevariants (SVs) into account. There are two types of linkage analysis

first, model-based linkage (parametrical) in which a Mendelian model of inheritance

is assumed for analyzing the co-segregation. This model can be recessive or

dominant. In principle, co-segregation decreases if two loci are far away from each

other, so that recombination takes place more frequently (Haimila, 2009). The

significance of parametrical linkage is reported as a logarithm of the odds (LOD)

score; this is a function of recombination for each genotyped locus (or loci) at the

disease locus.

The larger the LOD score, the stronger the evidence for linkage with the disease, but a

negative LOD score means there is no co-segregation present. This approach was




successfully used in mapping Mendelian candidate loci with a strong genotype-

phenotype correlation.

The second type of linkage analysis is non-parametrical linkage in which no model

assumptions are made. This type of linkage is used in complex diseases, since there is

often an incomplete penetrance.

The analysis is performed in only the affected individuals from the family (“affected

only”); the linkage is based on the sharing of alleles, identical-by-descent, at the

disease locus. Both parametrical and model-free approaches have been used in

mapping the candidate regions in large families that segregate for a complex disease

(A Szperl et al., 2011).

Before the GWAS era (i.e. pre-2005), the most widely used method for mapping

complex disorders was a linkage-based analysis in sib-pairs, where both first-degree

relatives are affected (Dean, 2003). There is 0.25 probability of no identity-by-descent

sharing, 0.5 probability for one allele to be shared, and 0.25 for two alleles to be

shared as identityby- descent. A linkage approach would be adopted if both sibs share

more identity-by-descent than would be expected by chance. Such an approach was

used to map a candidate locus on chromosome 1 linked to celiac disease (Van Belzen

et al., 2003).

1.8.3 Genome-wide homozygosity mapping

This approach is used to study rare, autosomal recessive diseases by finding large,

homozygous regions spanning the causative mutation in consanguineous families

and in inbred populations (Lander and Botstein, 1987). In recessive diseases, the

children of related parents (for example, parents who are first cousins), often carry the

causative mutation within large, homozygous-by-descent loci (autozygosity). In

complete pedigrees, we can apply the same statistical techniques as for parametrical

linkage and the LOD score will indicate whether the linkage is significant. An




alternative approach is to genotype affected siblings from the family and to look for

the largest homozygosity region, as it has been shown that causative mutations often

map to one of the larger homozygous regions (Den Hollander et al., 2007).

Genome-wide association studies (GWAS)These studies are used to investigate

complex traits by finding associations between SNPs and the disease, mostly by using

a case-control design. In GWAS, the significance of the differences in allele

frequencies of genotyped markers is established in controls (unaffected individuals)

versus cases (affected individuals) (Cordell and Clayton, 2005). Markers used for

association studies are selected from common SNPs in HapMap, which is an

international catalogue of genetic variants in humans (Cordell and Clayton, 2005).

Hence, GWAS can be used to test the CV-CD hypothesis for any complex disease.

Due to the small effect size of the individual risk variants (OR < 1.5), a high number

of associated loci explain only a small part of the heritability (see Figure 2). For

example, around 71 loci have been associated with Crohn’s disease (a subtype of

inflammatory bowel disease), but explain only 20% of the heritable risk (Franke et al.,

2010). A standard GWAS is an indirect mapping approach where an associated,

common SNP (MAF > 5%) is tagging (tagging SNP) the unknown, causal variant.

This is possible since variants are in linkage disequilibrium (LD), which means they

are inter-dependent, create haplotypes, and are inherited together.

Thus, a genotyped SNP can predict and ‘tag’ other variants that have not been

genotyped (Goldstein and Cavalleri, 2005). As a GWAS can identify loci rather than

single genes, further fine-mapping of the associated loci is required to find the true

causal gene or variant (Wang et al., 2010). There are two hypotheses on the character

of causative, tagged variants: one is that the common

SNP is tagging the other common causative variant of small effect (CD-CV

hypothesis), when a causal mutation arose together with the common tagging SNP;




the other is where the common SNP tags rarer variants that arose after the tagging

SNP became common (synthetic association).

Replication phase

Replication of the findings in an independent study (either with a family-based or

case-control design) is an essential step and will provide the most convincing

evidence of a variant/gene causing the disease. Lack of replication may suggest a

false-positive finding, but may also be due to genetic heterogeneity or a too small

sample size.

Allelic heterogeneity

This occurs when different mutations in the same gene lead to the same disorder. This

type of heterogeneity is often observed and it is therefore necessary to replicate the

results.

An entire gene/locus needs to be screened rather than just the single candidate variant.

There are several examples of allelic heterogeneity underlying Mendelian and

complex diseases, such as in ICF, a recessive immunoglobulin deficiency syndrome,

which is caused by at least eleven different mutations in the same gene (Wijmenga et

al., 2000). MVID, an autosomal recessive disorder is caused by a wide spectrum of

mutations in the MYO5b gene, including missense, nonsense, deletion and splice-sites

mutations. Three rare mutations in the NOD2 gene have been associated with Crohn’s

disease, possibly explaining association of the NOD2 locus to this complex disorder .

Mutations located in different regions of the gene (functional domains, binding sites,

etc.) can have a different effect on the severity of the disease, and are observed as

genotype-phenotype correlations. For example, some genes influencing lipid levels

carry common variants with a modest effect size leading to complex traits, as well as

rare variants with a large effect size and causing Mendelian dyslipidemias (Kathiresan

et al., 2009).




Locus heterogeneity

This occurs when mutations in different genes cause the same disease. Lack of

replication of previously mapped candidate regions can be due to high locus

heterogeneity. For example, two different linkage regions were mapped for celiac

disease in two large, independent, families of Dutch origin. In the case of complex

diseases, it was proposed to investigate extreme phenotypes of the disease that would

be due to a more restricted group of loci and therefore easier to map and replicate. For

Mendelian disorders, the locus heterogeneity is not as common as for complex

diseases but it does occur on a regular basis. Griscelli syndrome, an autosomal

recessive disease, is caused by a mutation in one of three interacting genes, RAB27A-

MLPH-MYO5A a tripartite complex, leading to one of the three subtypes of this

syndrome (Van Gele et al., 2009).

Clinical heterogeneity

This is when a mutation in the same gene leads to different diseases. For example,

mutations in the RET gene can lead to two totally different diseases depending on

their position in the gene: Hirschsprung disease that affects the colon, and familial

medullary thyroid carcinoma(Hofstra et al., 1994). Classic examples of clinical

heterogeneity are two types of muscular dystrophies caused by different deletions in

the DMD gene. One is an out-of-frame deletion leading to Duchenne muscular

dystrophy (mostly fatal before adulthood), whereas the second is an in-frame deletion

leading to Becker muscular dystrophy, which is not life-threatening (Gillard et al.,

1989).

Population heterogeneity

Each population differs in terms of allelic frequency, biological adaptation risk, and

the prevalence of a disease. For severe Mendelian disorders, the mutated gene is more




likely to be the same and mutations will occur with a very low frequency in each

population as they undergo negative selection.

Therefore, monogenic disorders are fairly replicable between different populations.

The lack of replication of common SNPs associated to complex disorders in

independent, case-control studies can be the result of genetic heterogeneity within a

population due to geneticdrift. As SNPs chosen from HapMap are fairly transferable

between populations and create haplotypes due to LD, the transferability of a single

SNP out of a group of correlated SNPs, rather than direct replication of the associated

variant, might be a better way of replicating GWAS findings. This approach was

successfully applied by Shiner et al. (2009), who replicated the results of association

to a trait (adult height) in Europeans in an African population using two strategies:

direct replication, in which only 8% of the loci were replicated, and locus-wide

replication (transferability), in which 54% of the loci were replicated (Shriner et al.,

2009).

Fine-mapping

Once the candidate region is mapped and successfully replicated, the next step is to

fine-map it. Fine-mapping should narrow down the region or find the causative

variant. The success of this step depends on the type of disease (complex,

monogenic), the size of the region and its LD structure. The most common method is

direct re-sequencing of the region, by conventional Sanger sequencing or by next-

generation, high-throughput sequencing, which has been successfully applied to fine-

mapping of loci for monogenic diseases.

Fine-mapping of GWAS loci for complex diseases is much more complicated as all

variants from the associated loci are in rather high LD. Thus, cross-ethnic mapping is

one of the alternatives to break up large haplotypes (Seldin et al., 2011).




Cross-ethnic fine-mapping

The concept behind this approach is to use unrelated populations to fine-map regions

of high LD.Populations have different LD structures: older populations (Africans,

Indians) are expected to have smaller LD blocks, whereas younger populations such

as Europeans will have longer LD blocks.

In order to fine-map the region with this method the first step is to determine the risk

haplotypes in both populations and to check whether they share a common ancestor

and could possibly tag the same risk variant. This is crucial as some loci are

associated with disease by more than one independent signal, suggesting more than

one risk haplotype. The next step is to compare the LD block between the two

populations at the locus of the risk haplotype and to narrow down the region based on

the smallest LD block. This approach was successfully used to fine-map the IL2/IL21

region associated to celiac disease using Dutch and Indians populations. Such smaller

haplotypes are more amenable for resequencing studies and the availability of

haplotypes from different populations can assist in interpreting the results of such

studies.

1.9 Current Advances in the Area (World Wide)

1.9.1 Fine-mapping and re-sequencing by next-generation sequencing (NGS)

Next-generation sequencing (NGS) is used for highthroughput sequencing of the

entire exome or genome of individuals for family-based or case-control studies. With

the falling cost of NGS, it is rapidly becoming an alternative tool for the standard

mapping of genetic diseases with problematic phenotypes (Mendelian or complex) in

which GWAS simply does not tag the variant and a linkage method is not efficient

because of de novo changes or locus heterogeneity. Since 2009, NGS has acquired

high-throughput methods and become more affordable; a number of Mendelian

disorders have been solved (Ku et al., 2011; Ng et al., 2010a) and causative variants




have been found for some complex disorders. The difficulty of analyzing NGS data

depends on the disease itself: looking for homozygous mutations causing recessive

diseases is relatively easy compared to dominant disorders, as rare heterozygous

sequence variants are rather common in the genome (Consortium, 2010). Still, the

analysis for all study designs and disease types can be divided into two steps: the first

phase is a well-designed filtering method of good quality, well-covered SVs, and the

second phase is checking for co-segregation or the statistical relevance of the finding.

The first phase should lead to the discovery of one or more candidate variants.

Filtering steps will vary depending on the disease model (e.g. dominant or recessive)

and the study design (e.g. family based or case-control design) and might include: (a)

functionality of the SV, e.g. exon or coding regions, (b) the novelty or MAF of the

SV, based on the publicly available data sets like 1000 Genomes project, HapMap ,

or a private data set such as Genome of the Netherlands, (c) conservation of the region

containing the SV, (d) zygosity of the SV, (e) function of the gene containing the SV

and its likely involvement in the disease, etc. In a case-control design, the same

filtering needs to be applied for a representative number of cases and controls. If the

number of variants after filtering is still high, other information can be added in order

to filter out non-causative SVs. For example, Norton et al. (2011) applied expression

data from the heart, assuming that a causative gene for dilated cardiomyopathy must

be highly expressed in this organ (Norton et al., 2011). The second phase is to

investigate the correlation between the sequence variants and disease by co-

segregation of the selected SVs in families or testing for enrichment differences

between cases and controls by applying appropriate statistical tests that pool together

filtered SVs at a selected locus and testing them collectively (burden test). To prove

the significance of a finding, we need to demonstrate replication by screening a large

control panel and performing functional follow-up studies. NGS has been successfully

applied to investigate a wide spectrum of diseases, such as autosomal dominant




diseases, sporadic cases caused by de novo mutations, mitochondrial disease, X-

linked diseases, autosomal recessive diseases, complex diseases with Mendelian-like

segregation, and complex diseases in a case-control design.

Nowadays NGS rather than Sanger-type sequencing is used for the efficient and fast

screening of previously mapped regions. Candidate loci from linkage analysis are of

the standard size of around 10-30 cM (= 10-30 Mb) and contain some 100-300

candidate genes (Glazier et al., 2002). Loci associated in GWAS are smaller and

contain fewer genes, but each of the complex diseases is associated to many such

loci(Park et al., 2010). The high number of genes and lack of a high-throughput

screening method previously restricted the fine-mapping of candidate loci. But since

NGS technology has become feasible and affordable, researchers have been able to

screen for mutations in candidate regions by enriching for the entire exome and

further analyzing regions of interest, or by achieving a very high coverage from

enriching for selected regions of interest (Otto et al., 2010).

In conclusion, NGS is proving to be a very powerful approach, which can be used as a

screening method for hypothesis-driven as well as hypothesis-free approaches to

investigate both Mendelian and complex diseases. With targeted re-sequencing (e.g.

by exome enrichment), it is proving possible to solve most of the Mendelian disorders

in a fast and efficient way. In the future, whole genome sequencing could be the best

method for investigating complex diseases, as not only coding variants but regulatory

and miRNAs can be re-sequenced. Both methods are being continually improved:

exome enrichment capture is becoming more complete, containing more of the

expressed regions, although whole genome technology yields a better coverage at the

moment.

The study carried out in this project was to determine the genes that were responsible

for syndromic and some rare autosomal recessive disorders of genetic diseases and




their existent in the families in the population of province of KPK, Pakistan. This

project thus investigated many Syndromes involved in skin, eyes, bones and ID.

Autozygosity mapping strategy that involved positional candidate gene identification

approach was employed. Through determination of the genes responsible for

syndromic and non-syndromic genetic diseases, it would help in understanding the

processes involved in their formation alongside providing an accurate diagnosis,

timely genetic counselling, prenatal diagnosis and treatment, and in the future tailored

therapy in the families involved.

Chapter – 2 Materials and Methods



CHAPTER - 2

MATERIALS AND METHODS

2.1 Study Subjects and Ethical Approval

The samples were collected from the affected consanguineous a family residing in

Bannu city of Khyber Pakhtunkhwa province, Pakistan. Disease history and related

details were written after interviewing the family elders. Prior to start this study,

informed consent was signed either by the patients themselves or by their legal

guardians in case the participants were below the age of 18 years. The study was

further approved by a committee of and Medical Research and Ethics, Khyber

Pakhtunkhwa, Pakistan and institutional review board for medical genetics research

and ethics, King Abdul Aziz University, Jeddah, Saudi Arabia for genetic testing and

sharing the data for academic purposes.

2.2 Clinical Summary and Inclusion Criteria for Families

Detailed clinical parameters of the participants was taken including the radiology,

dysmorphology and all related clinical summary and reports were taken from the

Khalifa Gul Nawaz Hospital Bannu laboratory and presented to the research team. All

the potential of each clinical case or family were decided by the whole research team

involved in this study. Inclusion of any family from this study were done on the basis

of these parameters.

a. The family had more than two affected individuals presenting the disease

phenotype.

b. Patients had positive consanguinity in family.

c. Patients having clinical synopsis with a slightly different (severe/less severe)

clinical presentation were also marked as affected.




2.3 Exclusion Criteria Applied On Families.

The exclusion of any family was done based on following parameters

a. Sporadic cases were avoided to include in this study.

b. Similarly, the clinical diagnosis or negative disease history in the family were

excluded.

c. Patients without any effected members in the family or without availability of any

unaffected siblings or without both parents were also excluded.

d. Relatives of the affected individuals having some environmental or inconsistent

signs were marked as unaffected.

2.4 Pedigree Construction

Pedigree or the family tree drawing (Figure2.1) was prepared after the detailed

interviews with the family elders. The males were indicated by boxes and females by

circles. Affected members were indicated by filled symbols. Marriage lines were

drawn horizontal and sibling lines by verticals. Consanguinity was represented by the

double marriage lines.The generations were indicated by Romans and individuals’

numbers were indicated by Arabic numerals.

2.5 Collection of Samples

A10 ml of peripheral blood samples from five affected (IV-4, -6, -9, V-9, -10) and

eight unaffected (IV-8, -10, V-5, -6, -8 -11 and -13) family memberswerecollected in

the EDTA tubes. Genomic DNA was extracted using the standard protocols of kit

provided by the manufacturer, QIAamp Genomic DNA Extraction Kits (Qiagen,

USA). Standardization of the extracted genomic DNA was confirmed using the

nanodrop 2000 (Thermo Scientific, USA).




2.6 Whole Exome Sequencing

Whole exome sequencing (WES) was performed by a commercial company

Macrogen Inc. South Korea. The procedure followed was that 2 µg of total genomic

DNA from each sample was taken and subjected to human WES with paired-end

sequencing at 100x coverage. The libraries of 5.1 Mb were prepared using Sure Select

V4 kit kits (Agilent Technologies, Santa Clara, CA), and purified products of 100-bp

paired-end reads sequenced on the HiSeq 2000 platform (Illumina, San Diego, CA).

These methods had been tested as standard for previous WES analysis of molecular

diagnostics of rare genetic disorders (Ahmed et al., 2015; Alrayes et al., 2015; Jelani,

Ahmed, et al., 2015; Jelani, Jeon, et al., 2015; Lee et al., 2015; Serafi et al., 2015).

2.7 Data Quality Check or Sequencing

Raw data of WES was analysed and subjected to the identification of the causative

variants using the following parameters.

i. On averages, the bases with Phred score lower than 20 was excluded from the

study.

ii. All the variants with the mean depth of 105 were obtained in 100x coverage,

which was used for the identification of SNP quality.

iii. Mutations or variants that passes the quality control criteria having the minimum

Q call >20, minimum depth >10 was only considered for further studies.

2.8 Initial Data Analysis Pipeline

After assurance of data quality, we used Lasergene Genomic Suite V.12 software

package (DNASTAR, Madison, WI, USA) in the laboratory of our collaborator

Dr.Changoo Kang Sungshine Women University, Seoul, South Korea.It included the

following steps.




i. Alignment of FASTQ files to hg19 (NCBI build GRCh37) was done using

SeqMan NGen 12

ii. Annotation of variant alleles was done on the basis of dbSNP 142 using the

ArrayStar v.12.

iii. The mapped variations were linked using the 1000 Genomes

(http://www.1000genomes.org/) databases and dbSNP

(http://www.ncbi.nlm.nih.gov/snp/).

iv. Those variants that includes the single nucleotide mutation in all the effected

members of the family and not common in unaffected member of the family was

short listed using ArrayStar v.12 as mentioned in the literature (Jelani, Jeon, et al.,

2015; Lee et al., 2015).

2.9 Secondary Data Analysis Strategy

Those common mutations or variants that proved as non-pathogenic neutral

polymorphic in initial stage of data analysis was used as a reference for the exclusion

of the subsequent samples and analyses through Sanger validation.

2.10 Validation by Sanger Sequencing

The selected variants (Table 2.1)were subjected to the validation throughbidirectional

Sanger sequencing. This stage included the following steps.

i. Using the Ensemble Genome Browser (http://www.ensembl.org/), the

Genomic sequencing of the flanking regions was performed.

ii. For the selection of primers for the amplification by polymerase chain

reaction, Primer3Plus software. (http://www.bioinformatics.nl/cgi

bin/primer3plus/primer3plus.cgi) was used.

iii. Samples were prepared and run on ABI3500 Genetic Analyzer (Life

http://www.ncbi.nlm.nih.gov/snp/




Technologies, USA) according to standard procedures described by the

manufacture.

iv. BioEdit analysis software (http://www.mbio.ncsu.edu/bioedit/bioedit.html0)

was used for the analysis of the data obtained from ABI3500 sequencing.

Table 2.1: The list of candidate variations

Gene name Chromosome Amino acid change

RAPGEF2 Chr 04 p. E285G

ZNF705G Chr 08 p. A292V

PDGFRL Chr 08 p. G94D

KIF20B Chr 10 p. K1372E

KIF20B Chr 10 p. L397I

PPP6R2 Chr 22 p. G931R

SEMA3A Chr 07 p. N693S

DCTD Chr 04 p. K85N

EXTL3 Chr 04 p. S407G

COL20A1 Chr 20 p. S131

DLG5 Chr 10 N/A

ASPM Chr 01 p. H542Y

NRP2 Chr 02 p. N354K

FAM69C Chr 18 p. P305L

USP54 Chr 10 N/A

SH3RF2 Chr 05 p. K343N

C17orf70 Chr 17 p. P158L

LZTS1 Chr 08 p. V497I

GSN Chr 09 N/A

DCHS2 Chr 04 p. K1348Q

2.11 Computational Prediction for the Filtered Variants

Diseases causing or potential pathogenic effect of all the selected variants were

predicted by using the software; MutationTaster2 (Schwarz et al., 2014), Polyphen-2

(Adzhubei et al., 2013) and SIFT (Bendl et al., 2014).




2.12 Mendelian Inheritance Check Or Transmission Genetics

To check that inheritance pattern was followed correctly, all the available family

members were also subjected to Sanger sequencing. For autosomal recessive models

the homozygous and compound heterozygous variants and autosomal dominant cases

heterozygous variants was patterned for co-segregation with disease phenotypes.

Those variants that was not following the Mendelian inheritance was excluded.

2.13 Population Screening or Ethnical Match Control

Samples of ethnical matched healthy controls of 219adult Pakhtun individuals from

various districts of Khyber Pakhtunkhwa was used as control to calculate the minor

allele frequency of the identified variants (Table 2.1). The purpose of this step was to

exclude population specific neutral polymorphisms.

2.14 Data Reporting

The data or novel variants obtained from this study was only possible because of the

families who contributed to this work and the efforts of the physicians who managed

the patients. Data was published in the international Thomson Reuters Peer reviewed

journal without disclosing the privacy of the subjects(khan et al., 2018).

Chapter – 3 Results



CHAPTER - 3

RESULTS

3.1 Family-A Clinical Findings

First family was a three-generation Pakistani consanguineous family that was affected

with rare genetic syndrome known as PPKs. The pedigree and clinical features of the

family are shown in figure 2.1 and 2.2 respectively. The black-filled symbols denote

affected individuals whose symptoms are present over their palms and soles. In this

family, PPKS is inherited as dominant mode. Blood samples from the individuals

marked with asterisk were available for wholeexome sequencing and Sanger

sequencing. Each normal individual was homozygous for the normal C allele (serine)

at the 392th nucleotide position on COL20A1; on the contrary, all the affected

individuals were heterozygous for the G allele (cysteine) which replaces serine with

cystein at the amino position of 131th of the COL20A1 protein.




Genes & Genomics Online ISSN 2092-9293

https://doi.org/10.1007/s13258-018-0695-z Print ISSN 1976-9571

RESEARCH ARTICLE

Whole-exome sequencing analysis reveals co-segregation of a COL20A1 missense

mutation in a Pakistani family with striate palmoplantar keratoderma

Muhammad Ismail Khan1 · Soyeon Choi2 · Muhammad Zahid1 · Habib Ahmad1 ·

Roshan Ali3 · Musharraf Jelani4 ·Changsoo Kang2

Received: 12 October 2017 / Accepted: 14 April 2018

© The Genetics Society of Korea and Springer Science+Business Media B.V., part of

Springer Nature 2018

Abstract

Palmoplantar keratoderma (PPK) is a rare group of excessive skin disorder

characterized by thickness over the palms and soles. The striate palmoplantar

keratoderma (PPKS) is a form in which hyperkeratotic lesions are restricted to the

pressure regions extending longitudinally in the length of each finger to the palm.

Dominantly inherited mutations in genes including desmoglein 1, desmoplakin and

keratin 1 have been suggested as genetic causes of PPKS. In this study, we

investigated a three-generation Pakistani family segregating PPKS phenotype in

autosomal dominant fashion to identify genetic cause in this family. We have

performed whole-exome and Sanger sequencing followed by in silico bioinformatics

analysis to pinpoint candidate mutation associated with PPK. Revealed a novel

heterozygous mutation (NM_020882.2, COL20A1 c. 392C > G; p.Ser131Cys) in the

loop region close to fibronectin type III-1 domain of the c ollagen 20 α1. This variant

was not found in our in-house 219 ethnically matched Pakistani unaffected controls

and showed minor allele frequency of 3.4 × 10−5 in Exome Aggregation Consortium

database containing exome data of 59,464 worldwide individuals. It was assigned as

“pathogenic” by in silico prediction tools. Previously, association of mutation in the

COL14A1, one of the paralogous gene of COL20A1, with PPK was reported in the

https://doi.org/10.1007/s13258-018-0695-z




study with a Chinese family. Our study proposes COL20A1 gene as another potential

candidate gene for PPKS which expand the spectrum of collagen proteins in the

pathogenicity of PPK.

Keywords: Palmoplantar keratoderma · Exome sequencing · COL20A1 ·

Mutations · PPKS · Pakistani family

Muhammad Ismail Khan and Soyeon Choi have contributedequally to this work.

Musharraf Jelani

[email protected]

Changsoo Kang

[email protected]

1. Department of Zoology, Islamia College University,Peshawar, Pakistan

2. Department of Biology, Sungshin Women’s University, Seoul, Republic of Korea

3. Department of Molecular Biology and Genetics, Institute of Basic Medical

Sciences, Khyber Medical University, Peshawar, Pakistan

4. Department of Genetic Medicine, Faculty of Medicine and Princess Al‑Jawhara

Albrahim Center of Excellence in Research of Hereditary Disorders, King

Abdulaziz University, Jeddah 21589, Saudi Arabia

Introduction

Palmoplantar keratoderma (PPK) belongs to a group of common skin disorders

characterized by excessive thickening of the epidermis over the palms and soles of the

human body (Hennies et al. 1995). This disorder can be classified into hereditary and

acquired forms (Lucker et al. 1994), and multiple family-based studied revealed that

genetic factors are one of the causes increasing susceptibility to hereditary PPK. The

feature of hyperkeratosis may be isolated (the sole dominant clinical feature) or it may

be associated with other ectodermal abnormalities or extra cutaneous manifestations.

The striate (PPKS) is a form in which hyperkeratotic lesions are restricted to the

pressure regions extending longitudinally in the length of each finger to the palm. It

was revealed that mutations in three genes including desmoglein (DSG1),




desmoplakin (DSP) and keratin-1 (KRT1) increased susceptibility to the PPKS

(Armstrong et al. 1999; Rickman et al. 1999; Whittock et al. 2002).

In punctate type of PPK (PPPK), numerous hyperkeratotic papules are distributed

irregularly on the palms and soles, and mutations in the alpha and gamma

adaptinbinding protein (AAGAB) gene and collagen 14 α1 (COL14A1) have been

reported to be genetic causes of punctate type 1 and type 2 PPK, respectively (Giehl et

al. 2012; Guo et al. 2012). All of genetic mutations mentioned above co-segregated

with the disorders in dominant manner.

The prevalence of affected individuals may be underestimated in cases when mildly

affected individuals do not seek specialized medical care or they might be diagnosed

incorrectly (Schiller et al. 2014). Isolated PPK patients (n = 36) have been observed

without associated anomalies of skin or appendages including ichthyosis, ectodermal

dysplasia and epidermolysis bullosa (Has and Technau- Hafsi 2016).

Recently, genetic studies using whole-exome sequencing (WES) analysis is a

promising approach to identify genetic causes of hereditary PPK. In this study, we

performed whole-exome sequencing of a consanguineous Pakistani family presenting

clinical features of PPKS in order to pinpoint a causative mutation for it.

Materials and Methods

Study Subjects

A consanguineous family affected with PPKs (Fig. 1) was ascertained from a remote

village of Khyber Pakhtunkhwa province, Pakistan. The disease was assumed to

segregate in autosomal dominant form in this family. Informed consent form was

taken from the family elders or the legal guardians of the affected individuals under

the ages of 18. The study was approved by the institutional review board for medical

genetics research and ethics, King Abdulaziz University, Jeddah, Saudi Arabia under

project.




Whole-exome and Sanger Sequencing

Peripheral blood (10 ml) was drawn from five affected and eight unaffected family

members after informed consents were obtained, and genomic DNAs were isolated

using QIAamp DNA Blood Max kit (Qiagen, Hilden, Germany). Concentration of

gDNAswere measured by PicoGreen™ assay according to the manufacturer’s

instructions (Promega, Madison, WI, USA). Whole-exomes of five individuals were

captured using SureSelect V5 kit (Agilent Technologies, Santa Clara, CA, USA), and

were sequenced as 100-bp

Figure 2.1: Pedigree of the Pakistani family affected by striate palmoplantar

keratoderma (PPKS).

Fig. 1 Pedigree of the Pakistani family affected by striate palmoplantar keratoderma

(PPKS). The black-filled symbols denote affected individuals whose symptoms are

present over their palms and soles. In this family, PPKS is inherited as dominant

mode. Blood samples from the individuals marked with asterisk were available for

wholeexome sequencing and Sanger sequencing. Each normal individual was




homozygous for the normal C allele (serine) at the 392th nucleotide position on

COL20A1; on the contrary, all the affected individuals were heterozygous for the G

allele (cysteine) which replaces serine with cystein at the amino position of 131th of

the COL20A1 proteinpaired-end reads on an Illumina HiSeq2000 machine (Illumina,

San Diego, CA, USA). The genotype of candidate variants was confirmed in all

family members by Sanger sequencing.

Bioinformatic Analysis

To screen candidate variants which are likely to cause hyperkeratosis in the family,

we utilized several bioinformatic tools and followed a series of criteria to filter out

other variants less related to the disease. Using SeqMan NGen (Lasergene Genomic

Suite v.12, DNASTAR, Madison, WI, USA), we aligned the sequenced reads in

FASTQ file format to hg 19 (GRCh37, NCBI). Arraystar v. 12 (Rockvile, MD, USA)

annotated normal and variant alleles based on dbSNP 142 (UCSC).

Annotated normal and variant alleles were sorted out to screen the candidate

mutations triggering hyperkeratosis, following a set of criteria. (1) We hypothesized

that striate palmoplantar keratoderma inherits as an autosomal dominant mode of

inheritance in this family; that is, affected individuals (IV-4, -6, -9, V-9 and -10) are

heterozygous for the minor allele; unaffected parents (IV-8, -10, V-5, -6, -8 -11 and -

13) are homozygous for the major allele. (2) Variants showing a minor allele

frequency>0.003 in the ExAc browser (http://exac.broadinstitute.org) were excluded.

(3) Heterozygous variants in our in-house exome sequence data obtained from 45

normal unrelated Pakistani individuals were also excluded. (4) Synonymous and deep

intronic variants other than those present at splice junctions were excluded. The

remaining candidate variants were further analyzed in silico to predicted their

pathogenic effect using PolyPhen-2, PROVEAN, and MutationTaster softwares

(Adzhubei et al. 2010; Choi et al. 2012; Schwarz et al. 2014).




Three-Dimensional Protein Modeling

No X-ray crystal structure was available for the N-terminal of the protein where the

mutation of interest had occurred. As no suitable template was available for the N-

terminal region of the protein; therefore, we generated ab-initio three dimensional

protein model using online Robetta Server (Bystroff et al. 2000) and Quark (Xu and

Zhang 2011). The model was predicted only for the segment 23–165 (leaving the

signal peptide i.e. 1–22) due to the limitation of the servers. The predicted models

were assessed using the program Verify3D (Bowie et al. 1991), PROCHECK,

WHAT_CHECK (Hooft et al. 1996), Errat (Colovos and Yeates 1993), and Prove

(Pontius et al. 1996). The Ramachandran plot (Ramachandran et al. 1963) was

analyzed to find residues in forbidden regions. The selected model was refined using

ModRefiner (Xu and Zhang 2011). the effect of Serine to Cystein amino acid at

position 131 using PDB_Hydro (Azuara et al. 2006) was assessed in the predicted

wild type and mutant models. The structural difference was assessed using Biovia

Discovery studio visualizer.

Results

Clinical Characteristics of Subjects

Affected individuals in the Pakistani family were clinically diagnosed as striate type

of PPK. Both males and females were affected. All the affected individuals had mild

epidermal thickening over the pressure areas of palms and fingers (Fig. 2a, b);

however, soles were not affected. One (V-10) of the affected individuals had

intellectual disability, global developmental delay and language impairment and his

brain magnetic resonance imaging showed delayed myelination;




Figure 2.2: The Clinical presentation of patients of family (A) showing

keratoderma over the palms.

Fig. 2 Clinical presentation of patients showing keratoderma over the palms. Note the

specifically the yellowish appearance at the pressure areas and figures in patients IV-6

(a) and V-9 (b). c illustrates Sanger sequencing results of the normal individual (IV-

10) and the PPKS patient (V-10) who is a son of the former. Including IV-10, the

genotypes at the mutant loci (COL20A1 c.392 C>G) of all normal individuals are

homozygous for the C allele; those of the affected are heterozygous C and G alleles.

(Color figure online) however, these neurological features were not found in other

PPK patients of the family.

Whole-exome Sequencing and Bioinformatic Analysis

Among 13 genomic DNA samples available, whole exomes of five samples (the

affecteds: IV-6 and V-9, the unaffecteds: IV-8, V-5 and -8) were sequenced. On

average, the total number of bases identified in the reads was 9.2 Gb. It was 96.73%

of bases that acquired phred score over 20. The mean coverage of the target regions

was 100.

Including all the synonymous as well as non-synonymous variations, the five

individuals have 46,138 exonic variants that met the first quality control criteria,

which required that minimum Q call should be above 20 and minimum depth

coverage be above ten. Of these, 4137 variants were heterozygous, and 336 variants

remained after excluding the 3801variants which were found in 45 unrelated in-house




Pakistani controls. Further exclusion of variants that show minor allele frequencies

higher than 0.003 and that do not change amino acid sequence, 20 missense variants

remained (Table 1). To narrow down to causative mutation, we further performed

genotyping of all the 20 variants in the eight other family members (III-3, IV-4, -9, -

10, V-6, -10, -11, -13) by Sanger sequencing, and found that 19 variants except

COL20A1 p.Ser131Cys were carried by at least one normal individuals in the family.

Therefore, we could pinpoint that the COL20A1 variant replacing serine with cysteine

at 131th amino acid position of the COL20A1 gene (NM_020882.2, c.392C > G) at

chromosome 20 as the candidate mutation. Concurrently, this variant was not found in

the Sanger sequencing results healthy 219 normal Pakistani individuals. This gene is

known to encode collagen type XX alpha 1 chain, however, there has been no human

disease associated with disruptions in the normal function of this protein.

In silico analysis to simulate pathogenicity of the COL20A1 p.Ser131Cys mutation

suggested that this mutation was predicted to be “disease causing” by PolyPhen-2;

PROVEAN, however mutation taster predicted it to be polymorphism.

Analysis of the three-dimensional structural modeling of both the normal and mutated

COL20A1 proteins showed that two forms have almost identical topology except

some minor changes. Both the wild-type and mutation COL20A1 consist of seven

beta strands at N-terminal region, two small alpha helices and loop regions. The

mutated amino acid position of 131 resides in the loop region. The mutation

influenced the surface atom distributions as shown in the surface view of the models

(Fig. 3).




Fig. 3 a Schematic diagram of COL20A1 protein and position of the p.Ser131Cys

mutation. The domain information was obtained from ExPASy database

(http://www.unipr ot.org/unipr ot/Q9P21 8). The 3D models of COL20A1 (residues

23–165). Surface was added with atom color (Carbon:gray, Oxygen:red,

Nitrogen:blue, Slufer: yellow). normal protein (with Ser131) (b and d) and mutated

protein (with Cys131) shown in (c and e). The residue number 131 is shown in CPK

representation. f Superimposed mutated model (blue) over normal model (red). g




Blue, Serine (with side chain Oxygen in purple), Red: Cysteine (with side chain

Sulphur in yellow). (Color figure online)

Discussion

Clinically PPK can be classified into several subgroups, and there are 30 genes

associated with the term “palmoplantar keratoderma” in NCBI (https

://www.ncbi.nlm.nih.gov/gene). Recently, various clinical subtypes of PPK have been

assigned to their causative genes based on their functions as structural proteins

(keratins), cornified envelop (loricrin and transglutaminase) cell-to-cell adhesion

(plakophilin, desmoplakin, desmoglein 1), cell-to-cell communication (connexins)

and transmembrane signal transduction cathepsin C (Sakiyama and Kubo 2016;

Stypczynska et al. 2016). Here we suggest, COL20A1 p.Ser131Cys as a novel

candidate mutation for PPKS phenotype identified through WES analysis. Previously

its closest paralog, collagen 14α1 (COL14A1, MIM 120324) gene has been reported

to be the only collagen gene associated with PPK in a Chinese family (Guo et al.

2012).

The COL20A1 spans 41,665 base pairs on chromosome 20q13.33 and encodes for

135.84 kD protein consisting of 1284 amino acids. According to the UniProtKB

database COL20A1 has a signal peptide (1–22 aa) and a collagen alpha chain (23-

1284 aa) with six domains of fibronectin type III (1–6), two domains of collagen like

1 and 2 and a laminin-G like domain (http://www.unipr ot.org/unipr ot/Q9P21 8). Our

mutation p.Ser131Cys resides in the loop region after the fibronectin type III-1

domain (28–119 aa) (Fig. 3).

All our patients showed similar phenotype, although incomplete or age-dependent

penetrance of dominant mutations has previously been reported in punctate

palmoplantar keratoderma families (Guo et al. 2012; Martinez-Mir et al. 2003);

however, we did not observe this phenomenon in our patients. Although there were




additional neurological findings in one patient; however, they were not observed from

other affected individuals in our family and thus the neurological phenotypes were not

considered to be the results from COL20A1 p.Ser131Cys mutation.

The COL14A1 gene, a closest paralog of COL20A1, has been known as the only

collagen gene causing PPK phenotype in autosomal dominant fashion (Guo et al.

2012). The mutant COL14A1 was suggested to alter the normal keratinocyte

proliferation which could lead to the common features like hyperkeratosis in PPK

patients (Guo et al. 2012). The COL14A1 is a member of fibril-associated collagen

with an interrupted triple helix (FACIT) superfamily, interacting with the fibril

surface and regulates fibrillogenesis (Ansorge et al. 2009). FACITs are a subgroup

within the collagen family containing types IX, XII, and XIV collagens (Shaw and

Olsen 1991), based on sequence homology collagen XX has also been included in this

subgroup (Koch et al. 2001). The three-dimensional predicted protein structures of the

mutant COL20A1 showed different atom distribution on the surface. The amino acid

serine at 131st position resides at the surface of the protein and Ser-to-Cys alteration

might interfere the interactions between the monomers of collagen fibers. Oxygen in

serine residue can form hydrogen bond with neighboring chain of collagen which can

stabilize the structure; cysteine instead forms disulfide bridges. We suspect that the

mutated cysteine residue may have effect on interaction with another collagen

monomer as cysteines produce knots in collagens (Barth et al. 2003; Boulegue et al.

2008). Furthermore, the hydrophobic nature of cysteine (Nagano et al. 1999) as

compared to hydrophilic serine can affect protein structure while interacting with the

solvent water molecules.

Our genetic and in silico analyses suggest that COL20A1 p.Ser131Cys is the genetic

mutation underlying striate PPKin a consanguineous Pakistani family.




Acknowledgements:We acknowledge the volunteer participation of the family

members in this study. This study was supported to CK by intramural grant from

Sungshin Women’s University (2016-1-11-049/1).

Compliance with Ethical Standards

Conflict of Interest:Muhammad Ismail Khan, Soyeon Choi, Muhammad Zahid,

Habib Ahmad, Roshan Ali, Musharraf Jelani and Changsoo Kang declare that there is

no conflict of interest on the contents of the manuscript.

Ethical Approval:Written informed consent was obtained from all study participants

from this family. This study was approved by the Institutional Review Board

Committees at King Abdulaziz University (Jeddah, Saudi Arabia) and Sungshin

Women’s University (Seoul, South Korea).

References

Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P,

Kondrashov AS, Sunyaev SR (2010) A method and server for predicting

damaging missense mutations. Nat Methods 7:248–249

Ansorge HL, Meng X, Zhang G, Veit G, Sun M, Klement JF, Beason DP, Soslowsky

LJ, Koch M, Birk DE (2009) Type XIV collagen regulates fibrillogenesis:

premature collagen fibril growth and tissue dysfunction in null mice. J Biol

Chem 284:8427–8438

Armstrong DK, McKenna KE, Purkis PE, Green KJ, Eady RA, Leigh IM, Hughes AE

(1999) Haploinsufficiency of desmoplakin causes a striate subtype of

palmoplantar keratoderma. Hum Mol Genet 8:143–148

Azuara C, Lindahl E, Koehl P, Orland H, Delarue M (2006) PDB_ Hydro:

incorporating dipolar solvents with variable density in the Poisson-Boltzmann

treatment of macromolecule electrostatics. Nucleic Acids Res 34:W38–W42




Barth D, Kyrieleis O, Frank S, Renner C, Moroder L (2003) The role of cystine knots

in collagen folding and stability, part II. Conformational properties of (Pro-

Hyp-Gly)n model trimers with N- and C-terminal collagen type III cystine

knots. Chemistry 9:3703–3714

Boulegue C, Musiol HJ, Gotz MG, Renner C, Moroder L (2008) Natural and artificial

cystine knots for assembly of homoand heterotrimeric collagen models.

Antioxid Redox Signal 10:113–125

Bowie JU, Luthy R, Eisenberg D (1991) A method to identify protein sequences that

fold into a known three-dimensional structure. Science 253:164–170

Bystroff C, Thorsson V, Baker D (2000) HMMSTR: a hidden Markov model for local

sequence-structure correlations in proteins. J Mol Biol 301:173–190

Choi Y, Sims GE, Murphy S, Miller JR, Chan AP (2012) Predicting the functional

effect of amino acid substitutions and indels. PLoS ONE 7:e46688

Colovos C, Yeates TO (1993) Verification of protein structures: patterns of

nonbonded atomic interactions. Protein Sci 2:1511–1519

Giehl KA, Eckstein GN, Pasternack SM, Praetzel-Wunder S, Ruzicka T, Lichtner P,

Seidl K, Rogers M, Graf E, Langbein L et al (2012) Nonsense mutations in

AAGAB cause punctate palmoplantar keratoderma type Buschke–Fischer–

Brauer. Am J Hum Genet 91:754–759

Guo BR, Zhang X, Chen G, Zhang JG, Sun LD, Du WD, Zhang Q, Cui Y, Zhu J,

Tang XF et al (2012) Exome sequencing identifies a COL14A1 mutation in a

large Chinese pedigree with punctate palmoplantar keratoderma. J Med Genet

49:563–568

Has C, Technau-Hafsi K (2016) Palmoplantar keratodermas: clinical and genetic

aspects. J Dtsch Dermatol Ges 14:123–139, quiz 140

Hennies HC, Kuster W, Mischke D, Reis A (1995) Localization of a locus for the

striated form of palmoplantar keratoderma to chromosome 18q near the

desmosomal cadherin gene cluster. Hum Mol Genet 4:1015–1020




Hooft RW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature

381:272

Koch M, Foley JE, Hahn R, Zhou P, Burgeson RE, Gerecke DR, Gordon MK (2001)

alpha 1(Xx) collagen, a new member of the collagen subfamily, fibril-

associated collagens with interrupted triple helices. J Biol Chem 276:23120–

23126

Lucker GP, Van de Kerkhof PC, Steijlen PM (1994) The hereditary palmoplantar

keratoses: an updated review and classification. Br J Dermatol 131:1–14

Martinez-Mir A, Zlotogorski A, Londono D, Gordon D, Grunn A, Uribe E, Horev L,

Ruiz IM, Davalos NO, Alayan O et al (2003) Identification of a locus for type

I punctate palmoplantar keratoderma on chromosome 15q22-q24. J Med Genet

40:872–878

Nagano N, Ota M, Nishikawa K (1999) Strong hydrophobic nature of cysteine

residues in proteins. FEBS Lett 458:69–71

Pontius J, Richelle J, Wodak SJ (1996) Deviations from standard atomic volumes as a

quality measure for protein crystal structures. J Mol Biol 264:121–136

Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963) Stereochemistry of

polypeptide chain configurations. J Mol Biol 7:95–99

Rickman L, Simrak D, Stevens HP, Hunt DM, King IA, Bryant SP, Eady RA, Leigh

IM, Arnemann J, Magee AI et al (1999) N-terminal deletion in a desmosomal

cadherin causes the autosomal dominant skin disease striate palmoplantar

keratoderma. Hum Mol Genet 8:971–976

Sakiyama T, Kubo A (2016) Hereditary palmoplantar keratoderma “clinical and

genetic differential diagnosis”. J Dermatol 43:264–274

Schiller S, Seebode C, Hennies HC, Giehl K, Emmert S (2014) Palmoplantar

keratoderma (PPK): acquired and genetic causes of a notso rare disease. J

Dtsch Dermatol Ges 12:781–788

Schwarz JM, Cooper DN, Schuelke M, Seelow D (2014) Mutation- Taster2: mutation

prediction for the deep-sequencing age. Nat Methods 11:361–362




Shaw LM, Olsen BR (1991) FACIT collagens: diverse molecular bridges in

extracellular matrices. Trends Biochem Sci 16:191–194

Stypczynska E, Placek W, Zegarska B, Czajkowski R (2016) Keratinization disorders

and genetic aspects in palmar and plantar keratodermas. Acta Dermatovenerol

Croat 24:116–123

Whittock NV, Smith FJ, Wan H, Mallipeddi R, Griffiths WA, Dopping- Hepenstal P,

Ashton GH, Eady RA, McLean WH, McGrath JA (2002) Frameshift mutation

in the V2 domain of human keratin 1 results in striate palmoplantar

keratoderma. J Invest Dermatol 118:838–844

Xu D, Zhang Y (2011) Improving the physical realism and structural accuracy of

protein models by a two-step atomic-level energy minimization. Biophys J

101:2525–2534

Chapter – 4 ` Discussion



CHAPTER - 4

DISCUSSION

There are 30 genes which are linked to the term “palmoplantar keratoderma” in

national center for biotechnology information (NCBI library) and clinically PPK can

be categorized into numerous subcategories. In recent times different clinical subtypes

of PPK have been allocated to their contributing genes. It is based on functional and

structural proteins (keratins), cornified envelop (loricrin and transglutaminase), cell-

to-cell adhesion (plakophilin, desmoplakin, desmoglein 1), cell-to-cell communication

(connexins) and transmembrane signal transduction cathepsin C (Sakiyama and Kubo

2016; Stypczynska et al. 2016). At this study we propose, COL20A1 p.Ser131Cys as a

novel candidate mutation for PPKS phenotype identified WES analysis. Earlier its

closest paralog, which was reported to be the single collagen gene which is linked

with PPK in a Chinese family is collagen 14α1 (COL14A1, MIM 120324) gene (Guo

et al. 2012).

The COL20A1 is located on chromosome 20q13.33, spans 41,665 base pairs and

encodes for a protein which is 135.84kd in size and consist of 1284 amino acids. As

stated by UniProtKB database COL20A1 consist of a signal peptide which is 1–22

amino acid in length and a collagen alpha chain which is 23-1284 amino acids long

having six domains of fibronectin type III (1–6), two domains of collagen like 1 and 2

and and one domain like laminin-G (http://www.unipr ot.org/unipr ot/ Q9P21 8). Our

mutation is p.Ser131Cys is present in the loop region after the fibronectin type III-1

domain (28–119 aa) (Fig. 3). All patients showed same phenotypes though age-

dependent or incomplete penetrance of dominant mutation has been reported

previously in palmoplantar keratoderma families (Guo et al. 2012; Martinez-Mir et

al., 2003); yet we did not notice this phenomena in case of our patients. Though there

were added neurological findings in one patient but as they were not observed from




any other individual which are affected in our family therefore it neurological

phenotypes were not because of COL20A1 p.Ser131Cys mutation. The COL14A1

gene is a closest paralog of COL20A1 and it has been recognized as the only collagen

gene responsible for PPK phenotype in autosomal dominant manner (Guo et al. 2012).

The mutant COL14A1 was recommended to change the normal keratinocyte

propagation and it could lead to the common features such as hyperkeratosis in PPK

patients (Guo et al. 2012). The COL14A1 has an interrupted triple helix (FACIT)

superfamily and is a member of fibril-associated collagen. It interacts with the fibril

surface and helps to regulate fibrillogenesis (Ansorge et al. 2009). FACITs are a

subgroup within the collagen family. They contain types IX, XII, and XIV collagens

(Shaw and Olsen 1991), collagen XX has also been added in this subgroup based on

its sequence homology (Koch et al. 2001). The predicted three-dimensional structures

of the mutant COL20A1 protein showed different distribution of atom on the surface.

The amino acid serine positioned at 131st is found at the protein surface and Ser-to-

Cys alteration may affect the interactions between the monomers of collagen fibers.

Oxygen present in serine residue may develop hydrogen bond with adjoining chain of

collagen which can stabilize the structure; cysteine as an alternative forms disulfide

bridges. It is suspected that the presence of mutated cysteine residue might affect the

interactions with the other monomers of collagen because knots are produce by

cysteines in collagens (Barth et al. 2003; Boulegue et al. 2008). Moreover, as

compared to hydrophilic nature of serine, the hydrophobic nature of cysteine (Nagano

et al. 1999) may affect the structure of protein while interacting with the solvent water

molecules. Our genetic and analyses of in silico propose that COL20A1 p.Ser131Cys

is the genetic mutation and underlying striate PPK in a consanguineous Pakistani

family. Palmoplantar keratoderma (PPK) associated to a group of common disorders

of skin. It is characterized by extreme epidermis thickening in the palms and soles

areas of the human body (Hennies et al. 1995). This disorder can be categorized into




two forms: hereditary and acquired (Lucker et al. 1994). Various family-based

studied exposed that genetic aspects are among one of the reasons of the increasing

vulnerability to hereditary PPK. The feature of hyperkeratosis could be isolated (the

only dominant clinical feature) or it may also be linked with other ectodermal

anomalies or extra cutaneous signs. The striate (PPKS) is a type where hyperkeratotic

lesions are limited to the pressure regions spreading longitudinally in the length of

individual finger to the palm. It was discovered that the occurrence of mutations in

three genes which include desmoglein (DSG1), desmoplakin (DSP) and keratin-1

(KRT1) increased vulnerability to the PPKS (Armstrong et al. 1999; Rickman et al.

1999; Whittock et al. 2002). In punctate type of PPK (PPPK), several hyperkeratotic

papules are dispersed unevenly on the palms and soles, and mutations in the alpha

and gamma adaptinbinding protein (AAGAB) gene and collagen 14 α1 (COL14A1)

have been described to be the genetic reasons of punctate type 1 and type 2 PPK,

respectively (Giehl et al. 2012; Guo et al. 2012).

All of genetic mutations stated before are co-segregated with the disorders in

dominant fashion. The occurrence of affected individuals can be underrated in such

cases when slightly affected individuals do not pursue specific medical care or maybe

they are diagnosed wrongly (Schiller et al. 2014). Isolated PPK patients (n = 36) have

been observed without related abnormalities of skin or appendages which include

ichthyosis, ectodermal dysplasia and epidermolysis bullosa (Has and Technau- Hafsi

2016). Lately, genetic studies by using whole-exome sequencing (WES) analysis is a

hopeful method to recognize the genetic bases of hereditary PPK.

` Conclusion And Recommendations



CONCLUSION AND RECOMMENDATIONS

We experience WES analysis as a successful molecular diagnostic for rare disorders

in Pakistani population; however, there are also limitations of the test as we could not

find the causative variant of the intellectual disability in the same family segregating

in autosomal recessive fashion. We suggest whole genome sequencing for further

analysis of this family to locate the intellectual disability gene. Moreover, the

esteemed research team is working to identify the intellectual disability (ID) locus in

the same family, and another manuscript is duly submitted for publication in a

reputable international journal carrying forward the research and opening new vistas

in the biological & human genetics field.

Comprehending the whole research journey we are of the opinion to run awareness

campaigns regarding rare genetic disorders. We further suggest that families,

community and young college going students should be educated through seminars,

conferences and short training programs to highlight the hazards of rare genetic

disorders. In born screening could also be a better option to calculate the possible

effect of rare variants; however, they could only be introduced once the communities

are fully aware of the consequences of the rare genetic disorders which are becoming

very common in Pakistani population. We have proposed the establishment of a

screening centre and facilities for counselling of the would-be couples to prevent the

genetic ailment in future generations, based on a recommendation from a related but

different research project of the same research team.

References



REFERENCES

AAIDD (2010). Intellectual disability: definition, classification and system of

supports/The AAIDD Ad Hoc committee on terminology and classification (11th ed.).

Washington, DC: American Association on Intellectual and Developmental

Disabilities.

Abbasi-Moheb L, Mertel S, Gonsior M, Nouri-Vahid L, Kahrizi K, Cirak S,

Wieczorek D, Motazacker MM, Esmaeeli-Nieh S, Cremer K, Weissmann R,

Tzschach A, Garshasbi M, Abedini SS, Najmabadi H, Ropers HH, Sigrist SJ, Kuss

AW (2012). Mutations in NSUN2 cause autosomal-recessive intellectual disability.

Am J Hum Genet 90: 847-855.

Adaimy L, Chouery E, Megarbane H, Mroueh S, Delague V, Nicolas E, Belgeith H,

de Mazancourt P, Megarbane A (2007). Mutation in the WNT10A is associated with

an autosomal recessive ectodermal dysplasia: the odonto-onycho-dermal dysplasia.

Am J Hum Genet 81: 821-828

Agha A, Sakati NO, Higginbottom MC, Jones KL Jr, Bay C, Nyhan WL (1978). Two

forms of cutis laxa presenting in the newborn period. Acta Paediat Scand 67: 775-780

Ahmad W, Irvine AD, Lam H, Buckley C, Bingham EA, Panteleyev AA, Ahmad M,

McGrath JA, Christiano AM (1998a). A missense mutation in the zinc-finger domain

of the human hairless gene underlies congenital atrichia in a family of Irish travellers.

Am J Hum Genet 63: 984-991

Akarsu A, Ozbas F, Kostakoglu N (1997). Mapping of the second locus of postaxial

polydactyly type A (PAP-A2) to chromosome 13q21-q32. Am J Hum Genet 6:A265

Alazami AM, Al‐Owain M, Alzahrani F, Shuaib T, Al‐Shamrani H, Al‐Falki YH,

Al‐Qahtani SM, Alsheddi T, Colak D, Alkuraya FS (2012). Loss of function mutation

References



in LARP7, chaperone of 7SK ncRNA, causes a syndrome of facial dysmorphism,

intellectual disability, and primordial dwarfism. Hum Mutat 33(10): 1429-1434

Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA,

Masoodi TA (2013). Mutation in ADAT3, encoding adenosine deaminase acting on

transfer RNA, causes intellectual disability and strabismus. J Med. Genet 50: 425-

430.

Aldenhoven M, Sakkers R, Boelens J, De Koning T, Wulffraat N (2009).

Musculoskeletal manifestations of lysosomal storage disorders. Ann of the Rheu Dis

68(11): 1659-1665

Al-Dosari MS, Al-Shammari M, Shaheen R, Faqeih E, AlGhofely MA, Boukai A,

Alkuraya FS (2012). 3M syndrome: an easily recognizable yet underdiagnosed cause

of proportionate short stature. J Pediatrics 161(1): 139-145

Al-Dosari MS, Shaheen R, Colak D, Alkuraya FS (2010). Novel CENPJ mutation

causes Seckel syndrome. J Med Genet 47(6): 411-414

Ali G, Chishti MS, Raza SI, John P, Ahmad W (2007). A mutation in the lipase H

(LIPH) gene underlies autosomal recessive hypotrichosis. Hum Genet 121: 319-325

Alkuraya FS (2015). Primordial dwarfism: an update. Cur Opin Endocrinol Diabetes

Obes 22(1): 55-64

Alonso L, Fuchs E (2006). The hair cycle. J Cell Sci 119: 391-393

American Psychiatric Association (2000). Diagnostic and statistical manual of mental

disorders. American Psychiatric Association, Washington, fourth 2000, text revision.

Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY (1999). Rett

syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding

protein 2. Nat Genet 23: 185-188.

References



Andiran N, Sarikayalar F, Saraçlar M, Caglar M (2002). Autosomal recessive form of

congenital cutis laxa: more than the clinical appearance. Pediatr Dermatol 19

Arin MJ (2009). The molecular basis of human keratin disorders. Hum Genet 125:

355-373

Armstrong DK, McKenna KE, Purkis PE, Green KJ, Eady RAJ, Leigh IM, Hughes

AE (1999). Haploinsufficiency of desmoplakin causes a striate subtype of

palmoplantar keratoderma. Hum Mol Genet 8: 143-148

Ashworth JL, Biswas S, Wraith E, Lloyd IC (2006). Mucopolysaccharidoses and the

eye. Surv Ophthalmol 51(1): 1-17

Aslam M, Chahrour MH, Razzaq A, Haque S, Yan K, Leal SM, Ahmad W (2004). A

novel locus for autosomal recessive form of hypotrichosis maps to chromosome

3q26.33-q27.3. J Med Genet 41: 849-852

Ayesh SK, Suheir M, Nassar WA, Al-Sharef BY, Abu- Libdeh H (2005). Genetic

screening of familial Mediterranean fever mutations in the Palestinian population.

Saudi Med J 26: 732-737.

Ayub M, Basit S, Jelani M, Ur Rehman F, Iqbal M, Yasinzai M, Ahmad W (2009). A

homozygous nonsense mutation in the human desmocollin-3 (DSC3) gene underlies

hereditary hypotrichosis and recurrent skin vesicles. Am J Hum Genet 85: 515-520

Azeem Z, Jelani M, Naz G, Tariq M, Wasif N, Naqvi SK, Ayub M, Yasinzai M,

Amin-ud-din M, Wali A, Ali G, Chishti MS, Ahmad W (2008). Novel mutations in G

protein-coupled receptor gene (P2RY5) in families with autosomal recessive

hypotrichosis (LAH3). Hum Genet 123: 515-519

Azeem Z, Wasif N, Basit S, Razak S, Waheed RA, Islam A, Ayub M, Kafaitullah,

Kamran-Ul-Hassan Naqvi S, Ali G, Ahmad W (2011). Congenital atrichia with

References



papular lesions resulting from novel mutations in human hairless gene in four

consanguineous families. J Dermatol 38: 1-6

Ba W, van der Raadt J, Nadif Kasri N (2013). Rho GTPase signaling at the synapse:

implications for intellectual disability. Exp Cell Res 319: 2368-74.

Baehner F, Schmiedeskamp C, Krummenauer F, Miebach E, Bajbouj M, Whybra C,

Kohlschütter A, Kampmann C, Beck M (2005). Cumulative incidence rates of the

mucopolysaccharidoses in Germany. J Inherit Metab Dis 28(6): 1011-1017

Bailleul-Forestier I, Molla M, Verloes A, Berdal A (2008). The genetic basis of

inherited anomalies of the teeth. Part 1: clinical and molecular aspects of non-

syndromic dental disorders. Eur J Med Genet 51: 273-291

Bailleul-Forestier I, Molla M, Verloes A, Berdal A (2008). The genetic basis of

inherited anomalies of the teeth. Part 1: clinical and molecular aspects of non-

syndromic dental disorders. Eur J Med Genet 51: 273-291

Baker E, Guo X-H, Orsborn A, Sutherland G, Callen DF, Hopwood J, Morris C

(1993). The morquio A syndrome (mucopolysaccharidosis IVA) gene maps to

16q24.3. Am J Hum Gene 52(1): 96

Balazs L, Okolicany J, Ferrebee M, Tolley B, Tigyi G (2001). Topical application of

the phospholipid growth factor lysophosphatidic acid promotes wound healing in

vivo. Am J Physiol Regul Integr Comp Physiol 280: 466-472

Bartels CF, Bükülmez H, Padayatti P, Rhee DK, van Ravenswaaij-Arts C, Pauli RM,

Mundlos S, Chitayat D, Shih L-Y, Al-Gazali LI (2004). Mutations in the

transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause

acromesomelic dysplasia, type Maroteaux. Am J Hum Gene 75(1): 27-34

Basel-Vanagaite (2007). Genetics of autosomal recessive non-syndromic mental

retardation: recent advances. Clin Genet 72: 167-174.

References



Basel-Vanagaite L, Attia R, Yahav M, Ferland RJ, Anteki L, Walsh CA, Drasinover

V (2006). The CC2D1A, a member of a new gene family with C2 domains, is

involved in autosomal recessive nonsyndromic mental retardation. J Med Genet 43:

203-210.

Basel-Vanagaite L, Taub E, Halpern GJ, DrasinoverV, Magal N, Davidov B,

Zlotogora J, Shohat M (2007) Genetic screening for autosomal recessive

nonsyndromic mental retardation in an isolated population in Israel. Eur J Hum Genet

15: 250-253.

Basit S, Ali G, Wasif N, Ansar M, Ahmad W (2010b). Genetic mapping of a novel

hypotrichosis locus to chromosome 7p21.3-p22.3 in a Pakistani family and screening

of the candidate genes. Hum Genet 128: 213-220

Basit S, Wali A, Aziz A, Muhammad N, Jelani M, Ahmad W (2011). Digenic

inheritance of an autosomal recessive hypotrichosis in two consanguineous pedigrees.

Clin Genet 79: 273-281

Baujat G, Le Merrer M (2007). Ellis-van Creveld syndrome. Orphanet J Rare Dis 2:

27

Bayes M, Hartung AJ, Ezer S, Pispa J, Thesleff I, Srivastava AK, Kere J (1998). The

anhidrotic ectodermal dysplasia gene (EDA) undergoes alternative splicing and

encodes ectodysplasin-A with deletion mutations in collagenous repeats. Hum Mol

Genet 7: 1661-1669

Bazzi H, Christiano AM (2007). Broken hearts, woolly hair, and tattered skin: when

desmosomal adhesion goes awry. Curr Opin Cell Biol 19: 515-520

Bener A, Hussain R, Teebi AS (2007). Consanguineous marriages and their effects on

common adult disease: studies from an endogamous population. Med Princ Pract 166:

262-267.

References



Bergmann C, Wobser M, Morbach H, Falkenbach A, Wittenhagen D, Lassay L, Ott

H, Zerres K, Girschick HJ, Hamm H (2011). Primary hypertrophic osteoarthropathy

with digital clubbing and palmoplantar hyperhidrosis caused by 15-PGHD/HPGD

loss-of-function mutations. Exp Dermatol 20: 531-533

Berman, E.R. (1991) Biochemistry Of The Eye. Plenum Press

Betz RC, Lee YA, Bygum A, Brandrup F, Bernal AI, Toribio J, Alvarez JI, Kukuk

GM, Ibsen HH, Rasmussen HB, Wienker TF, Reis A, Propping P, Kruse R, Cichon S,

Nothen MM (2000). A gene for hypotrichosis simplex of the scalp maps to

chromosome 6p21.3. Am J Hum Genet 66: 1979-1983

Biedermann T, Pontiggia L, Böttcher-Haberzeth S, Tharakan S, Braziulis E, Schiestl

C, Meuli M, Reichmann E (2010). Human eccrine sweat gland cells can reconstitute a

stratified epidermis. J Invest Dermatol 130: 1996-2009







Bienvenu T, Poirier K, Friocourt G, Bahi N, Beaumont D, Fauchereau F, Couvert P

(2002). ARX, a novel Prd-class-homeobox gene highly expressed in the

telencephalon, is mutated in X-linked mental retardation. Hum Mol Gen 11: 981-991.

Biesecker LG (2011). Polydactyly: how many disorders and how many genes? 2010

update. Dev Dyn 240(5): 931-942

Biggerstaf RH, Mazaheri M (1968). Oral manifestations of the Ellis-van Creveld

syndrome. J Am Dent Assoc 77: 1090-1095

References



Bittles AH (2001). Consanguinity and its relevance to clinical genetics. Clin Genet

60: 89-98.

Bittles AH, Black ML (2010a). Evolution in health and medicine Sackler colloquium:

consanguinity, human evolution, and complex diseases. Proc Natl Acad Sci USA 107:

1779-1786.

Blaydon DC, Ishii Y, O'Toole EA, Unsworth HC, Teh MT, Ruschendorf F, Sinclair

C, Hopsu-Havu VK, Tidman N, Moss C, Watson R, de Berker D, Wajid M,

Christiano AM, Kelsell DP (2006). The gene encoding R-spondin 4 (RSPO4), a

secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat

Genet 38: 1245-1247

Blaydon DC, Ishii Y, O'Toole EA, Unsworth HC, Teh MT, Ruschendorf F, Sinclair

C, Hopsu-Havu VK, Tidman N, Moss C, Watson R, de Berker D, Wajid M,

Christiano AM, Kelsell DP (2006). The gene encoding R-spondin 4 (RSPO4), a

secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat

Genet 38: 1245-1247

Bohring A, Stamm T, Spaich C, Haase C, Spree K, Hehr U, Hoffmann M, Ledig S,

Sel S, Wieacker P, Röpke A (2009). WNT10A mutations are a frequent cause of a

broad spectrum of ectodermal dysplasias with sex-biased manifestation pattern in

heterozygotes. Am J Hum Genet 85: 97-105

Bonafe L, Cormier‐Daire V, Hall C, Lachman R, Mortier G, Mundlos S, Nishimura

G, Sangiorgi L, Savarirayan R, Sillence D (2015). Nosology and classification of

genetic skeletal disorders: 2015 revision. Am J Med Gene 167(12): 2869-2892

Botstein, D., & Risch, N. (2003). Discovering genotypes underlying human

phenotypes: past successes for mendelian disease, future approaches for complex

disease. Nature genetics, 33, 228.

References



Bourgeron T (2016). Bourgeron, T. (2016). The genetics and neurobiology of

ESSENCE: The third Birgit Olsson lecture. Nord J Psychiat 70: 1-9.

Bowden PE (2010). Mutations in a keratin 6 isomer (K6c) cause a type of focal

palmoplantar keratoderma. J Invest Dermatol 130(2): 336-338

Bradbury J, Martin L, Strachan I (1989). Acquired Brown's syndrome associated with

Hurler-Scheie's syndrome. Br J Ophthalmol 73(4): 305-308

Brancati F, Fortugno P, Bottillo I, Lopez M, Josselin E, Boudghene-Stambouli O,

Agolini E, Bernardini L, Bellacchio E, Iannicelli M, Rossi A, Dib-Lachachi A,

Stuppia L, Palka G, Mundlos S, Stricker S, Kornak U, Zambruno G, Dallapiccola B

(2010). Mutations in PVRL4, encoding cell adhesion molecule nectin-4, cause

ectodermal dysplasia-syndactyly syndrome. Am J Hum Genet 87: 265-273

Brandon NJ, Sawa A (2011). Linking neurodevelopmental and synaptic theories of

mental illness through DISC1. Nat Rev Neurosci 12: 707-722.

Briggs MD, Chapman KL (2002). Pseudoachondroplasia and multiple epiphyseal

dysplasia: mutation review, molecular interactions, and genotype to phenotype

correlations. Hum Mutat 19(5): 465

Bruchle NO, Frank J, Frank V, Senderek J, Akar A, Koc E, Rigopoulos D, van

Steensel M, Zerres K, Bergmann C (2008). RSPO4 is the major gene in autosomal-

recessive anonychia and mutations cluster in the furin-like cysteine-rich domains of

the Wnt signaling ligand R-spondin 4. J Invest Derm 128: 791-796

Brzezicha B, Schmidt M, Makalowska I, Jarmolowski A, Pienkowska J,

Szweykowska-Kulinska Z (2006). Identification of human tRNA: m5C

methyltransferase catalyzing intron-dependent m5C formation in the first position of

the anticodon of the pre-tRNA Leu (CAA). Nucleic Acids Res 34: 6034-6043.

References



Buhler EM, Buhler UK, Beutler C, Fessler R (1987). A final word on the tricho-rhino-

phalangeal syndromes. Clin Genet 31: 273-275

Bundey, S. and Alam, H. (1993) A five-year prospective study of the health of

children in different ethnic groups, with particular reference to the effect

ofinbreeding. Eur J Hum Genet. 1 (3): 206-219

Bundy A, Silver B, Plummer D (1985). An analytical comparison of some rule-

learning programs. Artif Intell 27:137-181.

Cachon-Gonzalez MB, Fenner S, Coffin JM, Moran C, Best S, Stoye JP (1994).

Structure and expression of the hairless gene of mice. Proc Nat Acad Sci 91: 7717-

7721

Cai LQ, Wang PG, Gao M, Lu WS, Xu SX, Fang QY, Zhou WM, Lin D, Du WH,

Zhang SM, Yang S, Zhang XJ (2009). A novel U2HR non-synonymous mutation in a

Chinese patient with Marie Unna Hereditary Hypotrichosis. J Dermatol Sci 55: 125-

127

Castilla EE, Paz JE, Orioli‐Parreiras IM, Opitz JM, Hermann J (1980). Syndactyly:

frequency of specific types. Am J Med Gene 5(4): 357-364

Chassaing N, Bourthoumieu S, Cossee M, Calvas P, Vincent MC (2006). Mutations

in EDAR account for one-quarter of non-ED1-related hypohidrotic

ectodermaldysplasia. Hum Mutat 27: 255-259

Chelly J, Khelfaoui M, Francis F, Cherif B, Bienvenu T (2006). Genetics and

pathophysiology of mental retardation. Eur J Human Genet 14: 701-713.

Chen J, Den Z, Koch PJ (2008). Loss of desmocollin 3 in mice leads to epidermal

blistering. J Cell Sci 121: 2844-2849

Chiang, C., Litingtung, Y., Lee, E.(1996) Cyclopia and defective axial patterning in

mice lacking Sonic hedgehog gene function. Nature. 383 (6599): 407-413

References



Chishti MS, Kausar N, Rafiq MA, Amin M, Ahmad W (2008). A novel missense

mutation in RSPO4 gene underlies autosomal recessive congenital anonychia in a

consanguineous Pakistani family. Br J Dermatol 158: 621-623

Chong A (2010). Common congenital hand conditions. Singapore Med J 51(12): 965-

971

Chow, R.L., Altmann, C.R., Lang, R.A. and Hemmati-Brivanlou, A. (1999) Pax6

induces ectopic eyes in a vertebrate. Development. 126: 4213-4222

Christensen JC, Leff FB, Lepow GM, Schwartz RI, Colon PA, Arminio ST, Nixon P,

Segel D, Leff S (2011). Congenital polydactyly and polymetatarsalia: classification,

genetics, and surgical correction. J Foot and Ankle Sur 50(3): 336-339

Clarke B (2008). Normal bone anatomy and physiology. Clin J Am Soc Nephrol 3(3):

S131-S139

Clauss F, Chassaing N, Smahi A, Vincent MC, Calvas P, Molla M, Lesot H, Alembik

Y, Hadj-Rabia S, Bodemer C, Manière MC, Schmittbuhl M (2010). X-linked and

autosomal recessive Hypohidrotic Ectodermal Dysplasia: genotypic-dental phenotypic

findings. Clin Genet 78: 257-266

Clayton P, Bonnemaire M, Dutailly P, Maisonobe P, Naudin L, Pham E, Zhang Z,

Grupe A, Thiagalingam A, Denèfle P (2013). Characterizing short stature by insulin-

like growth factor axis status and genetic associations: results from the prospective,

cross-sectional, epidemiogenetic EPIGROW study. J Clin Endocrinol Metab 98(6):

E1122-E1130

Clouston HR (1939). The major forms of hereditary ectodermal dysplasia. Can Med

Assoc J 40: 1-7

References



Coelho D, Kim JC, Miousse IR, Fung S, Du Moulin M, Buers I, Nürnberg P (2012).

Mutations in ABCD4 cause a new inborn error of vitamin B12 metabolism. Nat

Genet 44: 1152-1155.

Cohen, J. C., Kiss, R. S., Pertsemlidis, A., Marcel, Y. L., McPherson, R., & Hobbs, H.

H. (2004). Multiple rare alleles contribute to low plasma levels of HDL cholesterol.

Science, 305(5685), 869-872.

Cohen, J. C., Pertsemlidis, A., Fahmi, S., Esmail, S., Vega, G. L., Grundy, S. M., &

Hobbs, H. H. (2006). Multiple rare variants in NPC1L1 associated with reduced sterol

absorption and plasma low-density lipoprotein levels. Proceedings of the National

Academy of Sciences, 103(6), 1810-1815.

Conley ME, Burks AW, Herrod HG, Puck JM (1991). Molecular analysis of X-linked

agammaglobulinemia with growth hormone deficiency. J Pediat 119(3): 392-397

Consortium, G. P. (2010). A map of human genome variation from population-scale

sequencing. Nature, 467(7319), 1061.

Cooper S-A, Smiley E, Morrison J, Williamson A, Allan L (2007). Mental ill-health

in adults with intellectual disabilities: prevalence and associated factors. Br J

Psychiatry 190: 27-35.

Cordell, H. J., & Clayton, D. G. (2005). Genetic association studies. The Lancet,

366(9491), 1121-1131.

Cormack DH (2001). Essential histology. Lippincott Williams and Wilkins. 2nd edn,

Baltimore, Maryland, USA Pp: 292

Costa T, Ramsby G, Cassia F, Peters KR, Soares J, Correa J, Quelce‐Salgado A,

Tsipouras P (1998). Grebe syndrome: clinical and radiographic findings in affected

individuals and heterozygous carriers. Am J Med Gene 75(5): 523-529

References



Costa-Mattioli M, Monteggia LM (2013). mTOR complexes in neurodevelopmental

and neuropsychiatric disorders. Nat Neurosci 16:1537-1543.

Cotsarelis G (2006). Epithelial stem cells: a folliculocentric view. J Invest Dermatol

126:1459-1468

Coutinho MF, Lacerda L, Alves S (2011). Glycosaminoglycan storage disorders: a

review. Biochem Res Int 2012

Darr A, Model B (1988). The Frequency of consanguineous marriage among British

Pakistanis. J Med Genet 25: 186-190.

Davalos NO, García-Vargas A, Pforr J, Davalos IP, Picos-Cardenas VJ, García-Cruz

D, Kruse R, Figuera LE, Nothen MM, Betz RC (2005). A non-sense mutation in the

corneodesmosin gene in a Mexican family with hypotrichosis simplex of the scalp. Br

J Dermatol 153: 1216-1219

De la Torre J, Simpson RL (1998). Complete digital duplication: a case report and

review of ulnar polydactyly. Ann Plast Surg 40(1): 76-79

De Muinck Keizer-Schrama S (1998). Diagnosis Of Short Stature In Childhood. Ned

Tijdschr Geneeskd 142(46): 2519-2525

Dean, M. (2003). Approaches to identify genes for complex human diseases: lessons

from Mendelian disorders. Human mutation, 22(4), 261-274.

Delva E, Tucker DK, Kowalczyk AP (2009). The desmosome. Cold Spring Harb

Perspect Biol 19: a002543

Demirhan O, Türkmen S, Schwabe G, Soyupak S, Akgül E, Taştemir D, Karahan D,

Mundlos S, Lehmann K (2005). A homozygous BMPR1B mutation causes a new

subtype of acromesomelic chondrodysplasia with genital anomalies. J Med Genet

42(4): 314-317

References



Den Hollander, A. I., Koenekoop, R. K., Mohamed, M. D., Arts, H. H., Boldt, K.,

Towns, K. V., . . . McKibbin, M. (2007). Mutations in LCA5, encoding the ciliary

protein lebercilin, cause Leber congenital amaurosis. Nature genetics, 39(7), 889.

Deshaies RJ, Joazeiro CA (2009). RING domain E3 ubiquitin ligases. Annu Rev

Biochem, 78: 399- 434.

Duncan, M.K., Haynes, J.I. 2nd, Cvekl, A. and Piatigorsky, J. (1998) Dual roles for

Pax-6: a transcriptional repressor of lens fiber cell-specific beta-crystallin genes. Mol

Cell Biol. 18 (9): 5579-5586

Duzenli S, Redler S, Müller M, Polat M, Dogruer D, Pasternack SM, Betz RC (2009).

Identification of a U2HR gene mutation in Turkish families with Marie Unna

hereditary hypotrichosis. Clin Exp Dermatol 34: e953-956

El-Amraoui A, Petit C (2010). Cadherins as targets for genetic diseases. Cold Spring

Harb Perspect Biol 2: a003095

Endele S, Rosenberger G, Geider K, Popp B, Tamer C, Stefanova I, Milh M, Kortüm

F, Fritsch A, Pientka FK, Hellenbroich Y, Kalscheuer VM, Kohlhase J, Moog

U, Rappold G, Rauch A, Ropers HH, von Spiczak S, Tonnies H, Villeneuve

N, Villard L, Zabel B, Zenker M, Laube B, Reis A, Wieczorek D, Van Maldergem L,

Kutsche K (2010). Mutations in GRIN2A and GRIN2B encoding regulatory subunits

of NMDA receptors cause variable neurodevelopmental phenotypes. Nat Genet 42:

1021-1026.

Esko JD, Kimata K, Lindahl U (2009). Proteoglycans and sulfated

glycosaminoglycans. J Med Genet 6(1): 344-349

Everman DB, Bartels CF, Yang Y, Yanamandra N, Goodman FR, Mendoza‐Londono

JR, Savarirayan R, White SM, Graham JM, Gale RP (2002). The mutational spectrum

of brachydactyly type C. Am J Med Genet 112(3): 291-296

http://www.ncbi.nlm.nih.gov/pubmed?term=Popp%20B%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Tamer%20C%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Stefanova%20I%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Milh%20M%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Kort%C3%BCm%20F%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Kort%C3%BCm%20F%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Fritsch%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Pientka%20FK%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Hellenbroich%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Kalscheuer%20VM%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Kohlhase%20J%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Moog%20U%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Moog%20U%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Rappold%20G%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Rauch%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Ropers%20HH%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=von%20Spiczak%20S%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=T%C3%B6nnies%20H%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Villeneuve%20N%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Villeneuve%20N%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Villard%20L%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Zabel%20B%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Zenker%20M%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Laube%20B%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Reis%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Wieczorek%20D%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Van%20Maldergem%20L%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

http://www.ncbi.nlm.nih.gov/pubmed?term=Kutsche%20K%5BAuthor%5D&cauthor=true&cauthor_uid=20890276

References



Ezer S, Bayes M, Elomaa O, Schlessinger D, Kere J (1999). Ectodysplasin is a

collagenous trimeric type II membrane protein with a tumor necrosis factor-like

domain and co-localizes with cytoskeletal structures at lateral and apical surfaces of

cells. Hum Mol Genet 8: 2079-2086

Fahiminiya S, Almuriekhi M, Nawaz Z, Staffa A, Lepage P, Ali R, Hashim L,

Schwartzentruber J, Abu Khadija K, Zaineddin S, Gamal H, Majewski J, Ben-Omran

T (2013). Whole exome sequencing unravels disease-causing genes in

consanguineous families in Qatar. Clin Genet 86: 134-141.

Faiyaz‐Ul‐Haque M, Ahmad W, Zaidi S, Haque S, Teebi A, Ahmad M, Cohn D, Tsui

LC (2002). Mutation in the cartilage‐derived morphogenetic protein‐1 (CDMP1) gene

in a kindred affected with fibular hypoplasia and complex brachydactyly (DuPan

syndrome). Clin Genet 61(6): 454-458

Fan H, Ye X, Shi L, Yin W, Hua B, Song G, Shi B, Bian Z (2008). Mutations in the

EDA gene are responsible for X-linked hypohidrotic ectodermal dysplasia and

hypodontia in Chinese kindreds. Eur J Oral Sci 116: 412-417

Fantes, J., Ragge, N.K., Lynch, S.A.(2003) Mutations in SOX2 cause anophthalmia.

Nat Genet. 33 (4): 461-463

Fedulov V, Rex CS, Simmons DA, Palmer L, Gall CM, Lynch G (2007). Evidence

that long-term potentiation occurs within individual hippocampal synapses during

learning. J Neurosci 27: 8031-8039.

Ferone G, Thomason HA, Antonini D, De Rosa L, Hu B, Gemei M, Zhou H,

Ambrosio R, Rice DP, Acampora D (2012). Mutant p63 causes defective expansion

of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

EMBO Mol Med 4(3): 192-205

References



Fishburn J, Turner G, Daniel A, Brookwell R, Opitz JM (1983). The diagnosis and

frequency of X‐linked conditions in a cohort of moderately retarded males with

affected brothers. Am J Med Genet 14: 713-724.

Folk GE, Semken HA (1991). The evolution of sweat glands. Int J Biometeorol 35:

180-186

Franke, A., McGovern, D. P., Barrett, J. C., Wang, K., Radford-Smith, G. L., Ahmad,

T., . . . Roberts, R. (2010). Genome-wide meta-analysis increases to 71 the number of

confirmed Crohn's disease susceptibility loci. Nature genetics, 42(12), 1118.

Freude K, Hoffmann K, Jensen LR, Delatycki MB, des Portes V, Moser B, Chelly J

(2004). Mutations in the FTSJ1 gene coding for a novel S-adenosylmethionine–

binding protein cause nonsyndromic X-linked mental retardation. Am J Hum

Genet 75: 305-309.

Frints SGM, Froyen G, Marynen P, Fryns JP (2002). X‐linked mental retardation:

vanishing boundaries between non‐specific (MRX) and syndromic (MRXS)

forms. Clin Genet 62: 423-432.

Froyen G, Corbett M, Vandewalle J, Jarvela I, Lawrence O, Meldrum C, Chelly J

(2008). Submicroscopic duplications of the hydroxysteroid dehydrogenase

HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental

retardation. Am J Hum Genet 82: 432-443.

Fuchs E (2007). Scratching the surface of skin development. Nature 445: 834-842

Fuchs E, Merrill BJ, Jamora C, DasGupta R (2001). At the roots of a never-ending

cycle. Dev Cell 1: 13-25

Fujioka H, Ariga T, Horiuchi K, Otsu M, Igawa H, Kawashima K, Yamamoto Y,

Sugihara T, Sakiyama Y (2005). Molecular analysis of non‐syndromic preaxial

References



polydactyly: preaxial polydactyly type‐IV and preaxial polydactyly type‐I. Clin Genet

67(5): 429-433

Gai Z, Gui T, Muragaki Y (2011). The function of TRPS1 in the development and

differentiation of bone, kidney, and hair follicles. Histol Histopathol 26: 915-921

Galjaard R-JH, Smits AP, Tuerlings JH, Bais AG, Avella AMB, Breedveld G, de

Graaff E, Oostra BA, Heutink P (2003). A new locus for postaxial polydactyly type

A/B on chromosome 7q21–q34. Eur J Hum Genet 11(5): 409-415

Ganesh A, Bruwer Z, Al-Thihli K (2013). An update on ocular involvement in

mucopolysaccharidoses. Curr Opin Ophthalmol 24(5): 379-388

Gao B, Guo J, She C, Shu A, Yang M, Tan Z, Yang X, Guo S, Feng G, He L (2001).

Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1. Nat

Genet 28(4): 386-388

Gao B, Hu J, Stricker S, Cheung M, Ma G, Law KF, Witte F, Briscoe J, Mundlos S,

He L (2009). A mutation in Ihh that causes digit abnormalities alters its signalling

capacity and range. Nature 458(7242): 1196-1200

Garrod, A. E. (1902). About alkaptonuria. Medico-chirurgical transactions, 85, 69.

Garshasbi M, Hadavi V, Habibi H, Kahrizi K, Kariminejad R, Behjati F, Tzschach A,

Najmabadi H, Ropers HH, Kuss AW (2008). A defect in the TUSC3 gene is

associated with autosomal recessive mental retardation. Am J Hum Genet 82: 1158-

1164.

Garshasbi M, Kahrizi K, Hosseini M, Nouri Vahid L, Falah M, Hemmati S, Hu H,

Tzschach A, Ropers HH, Najmabadi H, Kuss AW (2011). A novel nonsense mutation

in TUSC3 is responsible for non syndromic autosomal recessive mental retardation in

a consanguineous Iranian family. Am J Med Genet 155A: 1976-1980.

References



Gentile M, Fiorente P, Buonadonna AL, Macina F, Cariola F (2003). A novel

mutation in exon 7 in a family with mild tricho-rhino-phalangeal syndrome type I.

Clin Genet 63: 166-167

Gibson, G. (2009). Decanalization and the origin of complex disease. Nature Reviews

Genetics, 10(2), 134.

Gilhar A, Kalish RS (2006). Alopecia Areata: A tissue specific autoimmune disease

of the hair follicle. Autoimmun Rev 5: 64-69

Gillard, E., Chamberlain, J., Murphy, E., Duff, C., Smith, B., Burghes, A., . . .

Bodrug, S. (1989). Molecular and phenotypic analysis of patients with deletions

within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene.

American journal of human genetics, 45(4), 507.

Gillberg C (2010). The ESSENCE in child psychiatry: early symptomatic syndromes

eliciting neurodevelopmental clinical examinations. Res Dev Disabil 31: 1543-1551.

Giugliani R, Gutierrez Carvalho C, Herber S, Lapagesse de Camargo Pinto L (2011).

Recent advances in treatment approaches of mucopolysaccharidosis VI. Curr Pharm

Biotech 12(6): 956-972

Givon U, Kumar SJ, Scott Jr CI (1999). Involvement of the humerus in two

generations with spondyloepiphyseal dysplasia. Clin Orthop 366: 174-177

Glaser, T., Jepeal, L., Edwards, J.G. et al. (1994) PAX6 gene dosage effect in a

family with congenital cataracts, aniridia, anophthalmia and central nervous system

defects. Nature Genet. 7: 463-471

Glazier, A. M., Nadeau, J. H., & Aitman, T. J. (2002). Finding genes that underlie

complex traits. Science, 298(5602), 2345-2349.

Goldstein, D. B. (2011). The importance of synthetic associations will only be

resolved empirically. PLoS biology, 9(1), e1001008.

References



Goldstein, D. B., & Cavalleri, G. L. (2005). Genomics: understanding human

diversity. Nature, 437(7063), 1241.

Graw, J. (2003) The genetic and molecular basis of congenital eye defects. Nat Rev

Genet. 4 (11): 876-888

Gudbjartsson DF, Thorvaldsson T, Kong A, Gunnarsson G, Ingolfsdottir A (2005).

Allegro version 2. Nat Genet 37: 1015-1016

Guo B, Lee SK, Paksima N (2013). Polydactyly. Bull Hosp Joint Dis 71(1)

Haack TB, Hogarth P, Kruer MC, Gregory A, Wieland T, Schwarzmayr T, Cuno SM

(2012). Exome sequencing reveals de novo WDR45 mutations causing a

phenotypically distinct, X-linked dominant form of NBIA. Am J Hum Genet 91:

1144-1149.

Haber LL, Adams HB, Thompson GH, Duncan LS, Didomenico LA, McCluskey WP

(2007). Unique case of polydactyly and a new classification system. J Pediat

Orthopaedr 27(3): 326-328

Haimila, K. (2009). Genetics of T cell co-stimulatory receptors-CD28, CTLA4, ICOS

and PDCD1 in immunity and transplantation.

Halder, G., Caliaerts, P. and Gehring, W.J. (1995) Induction of ectopic eyes by

targeted expression of the eyeless gene in Drosophila. Science. 267: 1788-1762

Han D, Gong Y, Wu H, Zhang X, Yan M, Wang X, Qu H, Feng H, Song S (2008).

Novel EDA mutation resulting in X-linked non-syndromic hypodontia and the

patternof EDA-associated isolated tooth agenesis. Eur J Med Genet 51: 536-546

Hanson, I. and Van Heyningen, V. (1995) Pax6: more than meets the eye. Trends

Genet. 11 (7): 268-272

Hardy MH (1992). The secret life of the hair follicle. Trends Genet 8: 55-60

References



Hardy MH (1992). The secret life of the hair follicle. Trends Genet 8: 55-60

Hardy, J., & Singleton, A. (2009). Genomewide association studies and human

disease. New England Journal of Medicine, 360(17), 1759-1768.

Harmatz P, Mengel KE, Giugliani R, Valayannopoulos V, Lin S-P, Parini R, Guffon

N, Burton BK, Hendriksz CJ, Mitchell J (2013). The Morquio A Clinical Assessment

Program: baseline results illustrating progressive, multisystemic clinical impairments

in Morquio A subjects. Mol Genet Metab 109(1): 54-61

Harris JC (2006). Intellectual disability: understanding its development, causes,

classification, evaluation, and treatment. New York : Oxford University Press 42-98.

Hart DB (1983). Menkes' syndrome: an updated review. J Am Acad Dermatol 9: 145-

152

Harvey K, Duguid IC, Alldred MJ, Beatty SE, Ward H, Keep NH, Smart TG (2004).

The GDP-GTP exchange factor collybistin: an essential determinant of neuronal

gephyrin clustering. J Neurosci 24: 5816-5826.

Hashimoto K (1970). The ultra-structure of the skin of human embryos. IX.

Formation of the hair cone and intra-epidermal hair canal. Arch Clin Exp Dermatol

238: 333-345

Hattersley, K., Laurie, K.J., Liebelt, J.E. et al. (2010) A novel syndrome of paediatric

cataract, dysmorphism, ectodermal features, and developmental delay in Australian

Aboriginal family maps to 1p35.3-p36.32. BMC Med Genet. 11:165

HAY S (1971). Incidence of selected congenital malformations in Iowa. Am J

Epidemiol 94(6): 572-584

Healy E, Holmes SC, Belgaid CE, Stephenson AM, McLean WHI, Rees JL, Munro

CS (1995). A gene for monilethrix is closely linked to the type II keratin gene cluster

at 12q13. Hum Mol Genet 4: 2399-2402

References



Hejtmancik, J.F. (2008) Congenital cataracts and their molecular genetics. Semin

Cell Dev Biol. 19 (2): 134-149

Hennies HC, Kuster W, Mischke D, Reis A (1995). Localization of a locus for the

striated form of palmoplantar keratoderma to chromosome 18q near the desmosomal

cadherin gene cluster. Hum Mol Genet 4: 1015-1020

Herculano-Houzel S (2009). The human brain in numbers: a linearly scaled-up

primate brain. Front Hum Neurosci 3: 31.3-31.11.

Higgins JJ, Pucilowska J, Lombardi RQ, Rooney JP (2004). A mutation in a novel

ATP-dependent Lon protease gene in a kindred with mild mental

retardation. Neurology 63: 1927-1931.

Hillmer AM, Flaquer A, Hanneken S, Eigelshoven S, Kortum AK, Brockschmidt FF,

Golla A, Metzen C, Thiele H, Kolberg S, Reinartz R, Betz RC, Ruzicka T, Hennies

HC, Kruse R, Nothen MM (2008). Genome-wide scan and fine-mapping linkage

study of androgenetic alopecia reveals a locus on chromosome 3q26. Am J Hum

Genet 82: 737-743

Hillmer AM, Hanneken S, Ritzmann S, Becker T, Freudenberg J, Brockschmidt FF,

Flaquer A, Freudenberg-Hua Y, Jamra RA, Metzen C, Heyn U, Schweiger N, Betz

RC, Blaumeiser B, Hampe J, Schreiber S, Schulze TG, Hennies HC, Schumacher J,

Propping P, Ruzicka T, Cichon S, Wienker TF, Kruse R, Nothen MM (2005). Genetic

variation in the human androgen receptor gene is the major determinant of common

early-onset androgenetic alopecia. Am J Hum Genet 77: 140-148

Hills CB, Kochilas L, Schimmenti LA, Moller JH (2011). Ellis-van Creveld syndrome

and congenital heart defects: presentation of an additional 32 cases. Pediatr Cardiol

32: 977-82

References



Hindorff, L. A., Sethupathy, P., Junkins, H. A., Ramos, E. M., Mehta, J. P., Collins, F.

S., & Manolio, T. A. (2009). Potential etiologic and functional implications of

genome-wide association loci for human diseases and traits. Proceedings of the

National Academy of Sciences, 106(23), 9362-9367.

Hofstra, R. M., Landsvater, R. M., Ceccherini, I., Stulp, R. P., Stelwagen, T., Luo, Y.,

. . . Romeo, G. (1994). A mutation in the RET proto-oncogene associated with

multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma.

Nature, 367(6461), 375.

Holbrook KA, Odland GF (1978). Structure of the human fetal hair canal and initial

hair eruption. J Invest Dermatol 71: 385-390

Hollister D, Cohen A, Rimoin D, Silberberg R (1975). The Morquio syndrome

(mucopolysaccharidosis IV): Morphologic and biochemical studies. Johns Hopkins

Med 137(4): 176-183

Homan CC, Kumar R, Nguyen LS, Haan E, Raymond FL, Abidi F, Jolly LA (2014).

Mutations in USP9X are associated with X-linked intellectual disability and disrupt

neuronal cell migration and growth. Am J Hum Genet 94: 470-478.

Horev L, Tosti A, Rosen I, Hershko K, Vincenzi C, Nanova K, Mali A, Potikha T,

Zlotogorski A (2009). Mutations in lipase H cause autosomal recessive hypotrichosis

simplex with woolly hair. J Am Acad Dermatol 61(5): 813-818

Horev L, Tosti A, Rosen I, Hershko K, Vincenzi C, Nanova K, Mali A, Potikha T,

Zlotogorski A (2009). Mutations in lipase H cause autosomal recessive hypotrichosis

simplex with woolly hair. J Am Acad Dermatol 61(5): 813-818

Hsü C (1965). Hereditary syndactylia in a Chinese family. Chin Med J 84(7): 482

Hu H, Eggers K, Chen W, Garshasbi M, Motazacker MM, Wrogemann K, Kahrizi K,

Tzschach A, Hosseini M, Bahman I, Hucho T, Muhlenhoff M, Gerardy-Schahn R,

References



Najmabadi H, Ropers HH, Kuss AW (2011). ST3GAL3 mutations impair the

development of higher cognitive functions. Am J Hum Genet 89: 407-414.

Hu JC, Simmer JP (2007). Developmental biology and genetics of dental

malformations. Orthod Craniofac Res 2: 45-52

Huang Y, Bron A, Meek K, Vellodi A, McDonald B (1996). Ultrastructural study of

the cornea in a bone marrow-transplanted Hurler syndrome patient. Exp Eye Res

62(4): 377-388

Hurst JA, Firth HV, Smithson S (2005). Skeletal dysplasias. Semin Fetal Neonatal

Med 10: 233-241

Hussain R, Bittles AH (1988). The prevalence and demographic characteristics of

consanguineous marriages in Pakistan. J Biosoc Sci 30: 261-275.

Hussain S, Benavente SB, Nascimento E, Dragoni I, Kurowski A, Gillich A,

Humphreys P, Frye M (2009). The nucleolar RNA methyltransferase Misu (NSUN2)

is required for mitotic spindle stability. J Cell Biol 186: 27-40.

Igoillo-Esteve M, Genin A, Lambert N, Desir J, Pirson I, Abdulkarim B,

Vanderhaeghen P (2013). tRNA methyltransferase homolog gene TRMT10A

mutation in young onset diabetes and primary microcephaly in humans. PLoS

Genet 9: e1003888.

Ingram, G. (1976). The history of haemophilia. Journal of clinical pathology, 29(6),

469.

Insel PA, Head BP, Patel HH, Roth DM, Bundey RA, Swaney JS (2005).

Compartmentation of G-protein-coupled receptors and their signalling components in

lipid rafts and caveolae. Biochem Soc Trans 33: 1131-1134.

References



Iqbal Z, Neveling K, Razzaq A, Shahzad M, Zahoor MY, Qasim M, De Brouwer AP

(2012). Targeted next generation sequencing reveals a novel intragenic deletion of the

TPO gene in a family with intellectual disability. Arch Med Res 43: 312-316.

Irvine AD, Christiano AM (2001). Hair on a gene string: recent advances in

understanding the molecular genetics of hair loss. Clin Exp Dermatol 26: 59-71

Ishii Y, Wajid M, Bazzi H, Fantauzzo KA, Barber AG, Blaydon DC, Nam JS, Yoon

JK, Kelsell DP, Christiano AM (2008). Mutations in R-spondin 4 (RSPO4) underlie

inherited anonychia. J Invest Dermatol 128: 867-870

Itin PH, Fistarol SK (2004). Ectodermal dysplasias. Am J Med Genet C 131: 45-51

Jelani M, Chishti MS, Ahmad W (2011). Mutation in PVRL4 gene encoding nectin-4

underlies ectodermal-dysplasia-syndactyly syndrome (EDSS1). J Hum Genet 56: 352-

357

Jelani M, Tariq M, Jan IA, Ullah H, Naeem M, Ahmad W (2010). Congenital cutis

laxa syndrome maps to a novel locus on chromosome 9q13-q21.32. J Dermatol Sci

61:134-6

John P, Ali G, Chishti MS, Naqvi SM, Leal SM, Ahmad W (2006). Localization of a

novel locus for alopecia with mental retardation syndrome to chromosome 3q26.33-

q27.3. Hum Genet 118: 665-667

Jørgensen JO, Møller L, Krag M, Billestrup N, Christiansen JS (2007). Effects of

growth hormone on glucose and fat metabolism in human subjects. Endocrinol Metab

Clin North Am 36(1): 75-87

Jørgensen JOL, Møller N, Wolthers T, Møller J, Grofte T, Vahl N, Fisker S, Ørskov

H, Christiansen JS (1995). Fuel metabolism in growth hormone-deficient adults.

Metabolism 44: 103-107

References



Kaiser FJ, Brega P, Raff ML, Byers PH, Gallati S, Kay TT, de Almeida S,

Horsthemke B, Ludecke HJ (2004). Novel missense mutations in the TRPS1

transcription factor define the nuclear localization signal. Eur J Hum Genet 12: 121-

126

Kajiura Y, Shunichi W, Takashi I, Koichi N, Atsuo I, Katsuaki I, Naoto Y, Yuya S,

Yoshiyuki A (2006). Structural analysis of human hair single fibres by scanning

microbeam SAXS. J Struc Bio 155: 438-444

Kalay E, Yigit G, Aslan Y, Brown KE, Pohl E, Bicknell LS, Kayserili H, Li Y,

Tuysuz B, Nurnberg G, Kiess W, Koegl M, Baessmann I, Buruk K, Toraman B,

Kayipmaz S, Kul S, Ikbal M, Turner DJ, Taylor MS, Aerts J, Scott C, Milstein K,

Dollfus H, Wieczorek D, Brunner HG, Hurles M, Jackson AP, Rauch A, Nurnberg P,

Karaguzel A, Wollnik B (2011). CEP152 is a genome maintenance protein disrupted

in Seckel syndrome. Nat Genet 43(1): 23-26

Kalscheuer VM, Freude K, Musante L, Jensen LR, Yntema HG, Gecz J, Gurok U

(2003). Mutations in the polyglutamine binding protein 1 gene cause X-linked mental

retardation. Nat genet 35: 313-315.

Kalsoom UE, Basit S, Kamran-ul-Hassan Naqvi S, Ansar M, Ahmad W. Genetic

mapping of an autosomal recessive postaxial polydactyly type A to chromosome

13q13.3-q21.2 and screening of the candidate genes. Hum Genet 2012: 131: 415–422.

Kalsoom UE, Wasif N, Tariq M, Khan S, Hecht J, Krawitz P, Mundlos S, Ahmad W

(2013). Whole exome sequencing identified a novel zinc-finger gene ZNF141

associated with autosomal recessive postaxial polydactyly type A. J Med Genet 50(1):

47-53

Kamijo T, Hayashi Y, Seo H, Ogawa M (1999). Hereditary isolated growth hormone

deficiency caused by GH1 gene mutations in Japanese patients. Growth Horm IGF 9:

31-36

References



Kant SG, Polinkovsky A, Mundlos S, Zabel B, Thomeer RT, Zonderland HM, Shih

LY, van Haeringen A, Warman ML (1998). Acromesomelic dysplasia Maroteaux

type maps to human chromosome 9. Am J Hum Genet 63(1): 155-162

Kanzler MH, Rasmussen JE (1986). Atrichia with papular lesions. Arch Dermatol

122:565-567

Karaveg K, Siriwardena A, Tempel W, Liu ZJ, Glushka J, Wang, B. C, Moremen KW

(2005). Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases

involved in N-glycan processing and endoplasmic reticulum quality control. J Biol

Chem 280: 16197-16207.

Karsenty G, Kronenberg HM, Settembre C (2009). Genetic control of bone formation.

Ann Rev Cell Dev 25: 629-648

Kathiresan, S., Willer, C. J., Peloso, G. M., Demissie, S., Musunuru, K., Schadt, E. E.,

. . . Tanaka, T. (2009). Common variants at 30 loci contribute to polygenic

dyslipidemia. Nature genetics, 41(1), 56.

Kaufman L, Ayub M, Vincent JB (2010). The genetic basis of non syndromic

intellectual disability: a review. Neurodevelop Disord 2: 182-209.

Kazantseva A, Goltsov A, Zinchenko R, Grigorenko AP, Abrukova AV, Moliaka YK,

Kirillov AG, Guo Z, Lyle S, Ginter EK, Rogaev EI (2006). Human hair growth

deficiency is linked to a genetic defect in the phospholipase gene LIPH. Science 314:

982-985

Kell HJ, Lubinski D, Benbow CP Steiger JH (2013). Creativity and technical

innovation spatial ability’s unique role. Psychol Sci 24: 1831-1836.

Kell, D. B., & Oliver, S. G. (2004). Here is the evidence, now what is the hypothesis?

The complementary roles of inductive and hypothesis‐driven science in the

post‐genomic era. Bioessays, 26(1), 99-105.

References



Kelleher RJ, Bear MF (2008). The autistic neuron: troubled translation ?. Cell 135:

401-406.

Kere J, Srivastava AK, Montonen O, Zonana J, Thomas N, Ferguson B, Munoz F,

Morgan D, Clarke A, Baybayan P, Chen EY, Ezer S, Saarialho-Kere U, de la

Chapelle A, Schlessinger D (1996). X-linked anhidrotic (hypohidrotic) ectodermal

dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 13: 409-

416

Kesby JP, Eyles DW, Burne TH, McGrath JJ (2011). The effects of vitamin D on

brain development and adult brain function. Mol Cell Endocrinol 347: 121-127.

Khan MA, Rafiq MA, Noor A, Ali N, Ali G, Vincent JB, Ansar M (2011). A novel

deletion mutation in the TUSC3 gene in a consanguineous Pakistani family with

autosomal recessive non syndromic intellectual disability. BMC Med Genet 12: 56.

Khan MA, Rafiq MA, Noor A, Hussain S, Flores JV, Rupp V, Vincent AK, Malli R,

Ali G, Khan FS, Ishak GE, Doherty D, Weksberg R, Ayub M, Windpassinger C,

Ibrahim S, Frye M, Ansar M, Vincent JB (2012). Mutation in NSUN2, which Encodes

an RNA Methyltransferase, Causes Autosomal-Recessive Intellectual Disability. Am

J Hum Genet 90: 856-863.

Khan S, Muzaffar S, Tariq M, Khan A, Basit S, Ahmad W (2010). Mapping of a

novel locus for an autosomal recessive form of palmoplantar keratoderma on

chromosome 3q27.2-q29. Br J Dermatol 163: 711-718

Kim JK, Kim E, Baek IC, Kim BK, Cho AR, Kim TY, Song CW, Seong JK, Yoon

JB, Stenn KS, Parimoo S, Yoon SK (2010). Overexpression of Hr links excessive

induction of Wnt signaling to Marie Unna hereditary hypotrichosis. Hum Mol Genet

19: 445-53

References



Kim KA, Zhao J, Andarmani S, Kakitani M, Oshima T, Binnerts ME, Abo A,

Tomizuka K, Funk WD (2006a). R-spondin proteins: a novel link to beta-catenin

activation. Cell Cycle 5: 23-26

Kjaer KW, Hansen L, Eiberg H, Utkus A, Skovgaard LT, Leicht P, Opitz JM,

Tommerup N (2005). A 72‐year‐old Danish puzzle resolved—comparative analysis of

phenotypes in families with different‐sized HOXD13 polyalanine expansions. Am J

Med Genet 138(4): 328-339

Kjaer KW, Hansen L, Schwabe GC, Marques-de-Faria AP, Eiberg H, Mundlos S,

Tommerup N, Rosenberg T (2005). Distinct CDH3 mutations cause ectodermal

dysplasia, ectrodactyly, macular dystrophy (EEM syndrome). J Med Genet 42: 292-

298

Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O'Shaughnessy R, Mahoney MG,

Levy M, Montagutelli X, Ahmad W, Aita VM, Gordon D, Uitto J, Whiting D, Ott J,

Fischer S, Gilliam TC, Jahoda CA, Morris RJ, Panteleyev AA, Nguyen VT,

Christiano AM (2003). Desmoglein 4 in hair follicle differentiation and epidermal

adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris.

Cell 113: 249-260

Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O'Shaughnessy R, Mahoney MG,

Levy M, Montagutelli X, Ahmad W, Aita VM, Gordon D, Uitto J, Whiting D, Ott J,

Fischer S, Gilliam TC, Jahoda CA, Morris RJ, Panteleyev AA, Nguyen VT,

Christiano AM (2003). Desmoglein 4 in hair follicle differentiation and epidermal

adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris.

Cell 113: 249-260

Klopocki E, Hennig BP, Dathe K, Koll R, de Ravel T, Baten E, Blom E, Gillerot Y,

Weigel JF, Krüger G (2010). Deletion and point mutations of PTHLH cause

brachydactyly type E. Am J Hum Genet 86(3): 434-439

References



Klopocki E, Wasif N, Tariq M, Khan S, Hecht J, Krawitz P, Mundlos S, Ahmad W

(2013). Whole exome sequencing identified a novel zinc-finger gene ZNF141

associated with autosomal recessive postaxial polydactyly type A. J Med Genet 50(1):

47-53

Kobayashi H, Hino M, Shimodahira M, Iwakura T, Ishihara T, Ikekubo K, Ogawa Y,

Nakao K, Kurahachi H (2002). Missense mutation of TRPS1 in a family of tricho-

rhino-phalangeal syndrome type III. Am J Med Genet 107: 26-29

Koch PJ, Mahoney MG, Cotsarelis G, Rothenberger K, Lavker RM, Stanley JR

(1998). Desmoglein 3 anchors telogen hair in the follicle. J Cell Sci 111: 2529-2537

Kornak U, Mundlos S (2003). Genetic disorders of the skeleton: a developmental

approach. Am J Hum Genet 73(3): 447-474

Kornak, U., & Mundlos, S. (2003). Genetic disorders of the skeleton: a developmental

approach. [Review]. Am J Hum Genet, 73(3), 447-474.

Krahn GL, Hammond L, Turner A (2006). A cascade of disparities: health and health

care access for people with intellectual disabilities. Ment Retard Dev Disabil Res

Rev 12: 70-82.

Krebsova A, Hamm H, Karl S, Reis A, Hennies HC (2000). Assignment of the gene

for a new hereditary nail disorder, isolated congenital nail dysplasia, to chromosome

17p13. J Invest Dermatol 115: 664-667

Ku, C.-S., Naidoo, N., & Pawitan, Y. (2011). Revisiting Mendelian disorders through

exome sequencing. Human genetics, 129(4), 351-370.

Kumar R, Neilsen PM, Crawford J, McKirdy R, Lee J, Powell JA, Saif Z, Martin JM,

Lombaerts M, Cornelisse CJ, Cleton-Jansen AM, Callen DF (2005). FBXO31 is the

chromosome 16q24.3 senescence gene, a candidate breast tumor suppressor, and a

component of an SCF complex. Cancer Res 65: 11304-11313.

References



Kumar V, Abbas AK, Fausto N, Aster JC. (2014). Robbins and cotran pathologic

basis of disease, Professional Edition: Expert Consult-Online: Elsevier Health

Sciences.

Kurban M, Ghosn S, Abbas O, Shimomura Y, Christiano AM (2010a). A missense

mutation in the P2RY5 gene leading to autosomal recessive woolly hair in a Syrian

patient. J Dermatol Sci. 57: 132-134

Kurban M, Michailidis E, Wajid M, Shimomura Y, Christiano AM (2010b). A

common founder mutation in the EDA-A1 gene in X-linked hypodontia. Dermatology

221: 243-247

Lammi L, Halonen K, Pirinen S, Thesleff I, Arte S, Nieminen P (2003). A missense

mutation in PAX9 in a family with distinct phenotype of oligodontia. Eur J Hum

Genet 11: 866-871

Lander, E. S., & Botstein, D. (1987). Homozygosity mapping: a way to map human

recessive traits with the DNA of inbred children. Science, 236(4808), 1567-1570.

Lander, E.S. and Botstein, D. (1987) Homozygosity mapping: a way to map human

recessive traits with the DNA of inbred children. Science. 236 (4808):1567-1570

Lander, E.S. and Botstein, D. (1987) Homozygosity mapping: a way to map human

recessive traits with the DNA of inbred children. Science. 236 (4808):1567-1570

Langbein L, Schweizer J (2005). The keratins of the human hair follicle. Int Rev

Cytol 243: 1-78


Cytol 243: 1-78


Cytol 243: 1-78

References



Larti F, Kahrizi K, Musante L, Hu H, Papari E, Fattahi Z, Wienker TF (2015). A

defect in the CLIP1 gene (CLIP-170) can cause autosomal recessive intellectual

disability. Eur J Human Genet 23: 331-336.

Law R, Dixon-Salazar T, Jerber J, Cai N, Abbasi AA, Zaki MS, Nguyen M (2014).

Biallelic truncating mutations in FMN2, encoding the actin-regulatory protein Formin

2, cause nonsyndromic autosomal-recessive intellectual disability. Am J Hum Genet

95: 721-728.

Lehmann K, Seemann P, Boergermann J, Morin G, Reif S, Knaus P, Mundlos S

(2006). A novel R486Q mutation in BMPR1B resulting in either a brachydactyly type

C/symphalangism-like phenotype or brachydactyly type A2. Eur J Hum Genet 14(12):

1248-1254

Lehmann K, Seemann P, Silan F, Goecke T, Irgang S, Kjaer K, Kjaergaard S,

Mahoney M, Morlot S, Reissner C (2007). A new subtype of brachydactyly type B

caused by point mutations in the bone morphogenetic protein antagonist NOGGIN.

Am J Hum Genet 81(2): 388-396

Lehmann K, Seemann P, Stricker S, Sammar M, Meyer B, Süring K, Majewski F,

Tinschert S, Grzeschik K-H, Müller D (2003). Mutations in bone morphogenetic

protein receptor 1B cause brachydactyly type A2. Proc Natl Acad Sci 100(21): 12277-

12282

Leonard H, Wen X (2002). The epidemiology of mental retardation: challenges and

opportunities in the new millennium. Ment Retard Dev Disabil Res Rev 8: 117-134.

Lettice LA, Horikoshi T, Heaney SJ, van Baren MJ, van der Linde HC, Breedveld GJ,

Joosse M, Akarsu N, Oostra BA, Endo N (2002). Disruption of a long-range cis-

acting regulator for Shh causes preaxial polydactyly. Proc Natl Acad Sci 99(11):

7548-7553

References



Levy-Nissenbaum E, Betz RC, Frydman M, Simon M, Lahat H, Bakhan T, Goldman

B, Bygum A, Pierick M, Hillmer AM, Jonca N, Toribio J, Kruse R, Dewald G,

Cichon S, Kubisch C, Guerrin M, Serre G, Nothen MM, Pras E (2003). Hypotrichosis

simplex of the scalp is associated with nonsense mutations in CDSN encoding

corneodesmosin. Nat Genet 34: 151-153

Li S, Zhou H, Qin H, Guo H, Bai Y (2014). A novel mutation in the COL2A1 gene in

a Chinese family with Spondyloepiphyseal dysplasia congenita. Joint Bone Spine

81(1): 86-89

Lind LK, Stecksen-Blicks C, Lejon K, Schmitt-Egenolf M (2006). EDAR mutation in

autosomal dominant hypohidrotic ectodermal dysplasia in two Swedish families.

BMC Med Genet 7:80

Loddo S, Parisi V, Doccini V, Filippi T, Bernardini L, Brovedani P, Ricci F, Novelli

A, Battaglia A (2013). Homozygous deletion in TUSC3 causing syndromic

intellectual disability: A new patient. Am J Med Genet 161A: 2084-2087.

Malik S (2014). Polydactyly: phenotypes, genetics and classification. Clin Genet

85(3): 203-212

Malik S, Schott J, Ali SW, Oeffner F, Amin-ud-Din M, Ahmad W, Grzeschik K-H,

Koch MC (2005). Evidence for clinical and genetic heterogeneity of syndactyly type

I: the phenotype of second and third toe syndactyly maps to chromosome 3p21. 31.

Eur J Hum Genet 13(12): 1268-1274

Malik S, Ullah S, Afzal M, Lal K, Haque S (2014). Clinical and descriptive genetic

study of polydactyly: a Pakistani experience of 313 cases. Clin Genet 85(5): 482-486

Manolio, T. A., Collins, F. S., Cox, N. J., Goldstein, D. B., Hindorff, L. A., Hunter, D.

J., . . . Chakravarti, A. (2009). Finding the missing heritability of complex diseases.

Nature, 461(7265), 747.

References



Mark PR, Torres‐Martinez W, Lachman RS, Weaver DD (2011). Association of a p.

Pro786Leu variant in COL2A1 with mild spondyloepiphyseal dysplasia congenita in a

three‐generation family. Am J Med Genet 155(1): 174-179

Martinez-Mir A, Zlotogorski A, Gordon D, Petukhova L, Mo J, Gilliam TC, Londono

D, Haynes C, Ott J, Hordinsky M, Nanova K, Norris D, Price V, Duvic M, Christiano

AM (2007). Genome-wide scan for linkage reveals evidence of several susceptibility

loci for alopecia areata. Am J Hum Genet 80: 316-328

Materna-Kiryluk A, Jamsheer A, Wisniewska K, Wieckowska B, Limon J,

Borszewska-Kornacka M, Sawulicka-Oleszczuk H, Szwalkiewicz-Warowicka E,

Latos-Bielenska A (2013). Epidemiology of isolated preaxial polydactyly type I: Data

from the Polish Registry of Congenital Malformations (PRCM). BMC Pediatr 13(1):

26

Matise TC, Chen F, Chen W, Francisco M, Hansen M, He C, Ziegle JS (2007). A

second-generation combined linkage–physical map of the human genome. Genome

Res 17(12): 1783-1786.

Matsuda A, Suzuki Y, Honda G, Muramatsu S, Matsuzaki O, Nagano Y, Doi T,

Shimotohno K, Harada T, Nishida E, Hayashi H, Sugano S (2003) Large-scale

identification and characterization of human genes that activate NF-kappaB and

MAPK signaling pathways. Oncogene 22: 3307-3318.

Maulik PK, Mascarenhas MN, Mathers CD, Dua T, Saxena S (2011). Prevalence of

intellectual disability: a meta-analysis of population-based studies. Res Dev

Disabil 32: 419-436.

McGrath JA, Eady RA, Pope FM (2004). Rook's Textbook of Dermatology (7th Ed)

Blackwell Publishing Pp: 3.1-3.6

References



McGrath JA, Hoeger PH, Christiano AM, McMillan JR, Mellerio JE, Ashton GHS,

Dopping-Hepenstal PJC, Lake BD, Leigh IM, Harper JI, Eady RAJ (1999). Skin

fragility and hypohidrotic ectodermal dysplasia resulting from ablation of plakophilin

1. Br J Dermatol 140: 297-307

McGrath JA, McMillan JR, Shemanko CS, Runswick SK, Leigh IM, Lane EB,

Garrod DR, Eady RAJ (1997). Mutations in the plakophilin 1 gene result in

ectodermal dysplasia/skin fragility syndrome. Nat Genet 17: 240-244

Mearin, M. L., Ivarsson, A., & Dickey, W. (2005). Coeliac disease: is it time for mass

screening? Best practice & research Clinical gastroenterology, 19(3), 441-452.

Menkes JH, Alter M, Steigleder GK, Weakley DR, Sung JH (1962). A sex-linked

recessive disorder with retardation of growth, peculiar hair, and focal cerebral and

cerebellar degeneration. Pediatrics 29: 764-779

Merlob P, Grunebaum M, Reisner S (1985). Familial opposable triphalangeal thumbs

associated with duplication of the big toes. J Med Genet 22(1): 78-80

Miano, M.G., Jacobson, S.G., Carothers, A.(2000) Pitfalls in homozygosity mapping.

Am J Hum Genet. 67 (5): 1348-1351

Millar SE (2002). Molecular mechanisms regulating hair follicle development. J

Invest Dermatol 118: 216-225

Millar SE (2002). Molecular mechanisms regulating hair follicle development. J


Mir A, Sritharan K, Mittal K, Vasli N, Araujo C, Jamil T, Rafiq MA, Anwar Z,

Mikhailov A, Rauf S, Mahmood H, Shakoor A, Ali S, So J, Naeem F, Ayub M,

Vincent JB (2014). Truncation of the E3 ubiquitin ligase component FBXO31 causes

non-syndromic autosomal recessive intellectual disability in a Pakistani family. Hum

Genet 133: 975-984.

References



Mir A, Kaufman L, Noor A, Motazacker MM, Jamil T, Azam M, Naeem F (2009).

Identification of mutations in TRAPPC9, which encodes the NIK-and IKK-β-binding

protein, in nonsyndromic autosomal-recessive mental retardation. Am J Hum

Genet 85: 909-915.

Modell B, Darr A (2002) Science and society: genetic counselling and customary

consanguineous marriage. Nat Rev Genet 3: 225-229.

Modell B. Kuliev AM (1992). Social and genetic implications of customary

consanguineous marriages among British Pakistanis. Occasional papers, second

series, No.4, London, Galton Institute.

Mohan J (1969). Postaxial polydactyly in three Indian families. J Med Genet 6(2):

196

Mohorko E, Glockshuber R, Aebi M (2011). Oligosaccharyltransferase: The central

enzyme of N-linked protein glycosylation. J Inherit Metab Dis 34: 869-878.

Molinari F, Rio M, Meskenaite V, Encha-Razavi F, Augé J, Bacq D, Sonderegger P

(2002). Truncating neurotrypsin mutation in autosomal recessive nonsyndromic

mental retardation. Science 298: 1779-1781.

Mollica F, Volti SL, Sorge G (1978). Autosomal recessive postaxial polydactyly type

A in a Sicilian family. J Med Genet 15(3): 212-216

Momeni P, Glockner G, Schmidt O, von Holtum D, Albrecht B, Gillessen-Kaesbach

G, Hennekam R, Meinecke P, Zabel B, Rosenthal A, Horsthemke B, Ludecke HJ

(2000). Mutations in a new gene, encoding a zinc-finger protein, cause tricho-rhino-

phalangeal syndrome type I. Nat Genet 24: 71-74

Montano A, Tomatsu S, Gottesman G, Smith M, Orii T (2007). International Morquio

A Registry: clinical manifestation and natural course of Morquio A disease. J Inherit

Metab Dis 30(2): 165-174

References



Montaño AM, Tomatsu S, Brusius A, Smith M, Orii T (2008). Growth charts for

patients affected with Morquio A disease. Am J Med Genet 146(10): 1286-1295

Moolenaar WH, van Meeteren LA, Giepmans BN (2004). The ins and outs of

lysophosphatidic acid signaling. Bioessays 26: 870-881

Morgan, N.V., Westaway, S.K., Morton, J.E. et al. (2006) PLA2G6, encoding a

phospholipase A2, is mutated in neurodegenerative disorders with high brain iron. Nat

Genet. 38 (7): 752-754

Morrone A, Caciotti A, Atwood R, Davidson K, Du C, Francis‐Lyon P, Harmatz P,

Mealiffe M, Mooney S, Oron TR (2014). Morquio A Syndrome‐Associated

Mutations: A Review of Alterations in the GALNS Gene and a New Locus‐Specific

Database. Hum Mutat 35(11): 1271-1279

Mortier GR (2001). The diagnosis of skeletal dysplasias: a multidisciplinary

approach. Eur J Radiol 40(3): 161-167

Mortier, G. R. (2001). The diagnosis of skeletal dysplasias: a multidisciplinary

approach. [Review]. Eur J Radiol, 40(3), 161-167.

Motazacker MM, Rost BR, Hucho T, Garshasbi M, Kahrizi K, Ullmann R, Abedini

SS, Nieh SE, Amini SH, Goswami C, Tzschach A, Jensen LR, Schmitz D, Ropers

HH, Najmabadi H, Kuss AW (2007). A defect in the ionotropic glutamate receptor 6

gene (GRIK2) is associated with autosomal recessive mental retardation. Am J Hum

Genet 81: 792-798.

Motazacker MM, Rost BR, Hucho T, Garshasbi M, Kahrizi K, Ullmann R, Tzschach

A (2007). A defect in the ionotropic glutamate receptor 6 gene (GRIK2) is associated

with autosomal recessive mental retardation. Am J Hum Genet 81: 792-798.

Mueller, R.F. and Bishop, D.T. (1993) Autozygosity mapping, complex

consanguinity, and autosomal recessive disorders. J Med Genet. 30 (9): 798-799

References



Müller, T., Hess, M. W., Schiefermeier, N., Pfaller, K., Ebner, H. L., Heinz-Erian, P.,

. . . Köhler, H. (2008). MYO5B mutations cause microvillus inclusion disease and

disrupt epithelial cell polarity. Nature genetics, 40(10), 1163.

Murray JE, Bicknell LS, Yigit G, Duker AL, Kogelenberg M, Haghayegh S,

Wieczorek D, Kayserili H, Albert MH, Wise CA (2014). Extreme growth failure is a

common presentation of ligase IV deficiency. Hum Mutat 35(1): 76-85

Myers KA, Farquhar DRE (2001). Does this patient have clubbing? JAMA 286: 341-

347

Naeem M, Jelani M, Lee K, Ali G, Chishti MS, Wali A, Gul A, John P, Hassan MJ,

Leal SM, Ahmad W (2006b). Ectodermal dysplasia of hair and nail type: mapping of

a novel locus to chromosome 17p12-q21.2. Br J Dermatol 155: 1184-1190

Naeem M, Muhammad D, Ahmad W (2005). Novel mutations in EDAR gene in two

Pakistani consanguineous families with autosomal recessive hypohidrotic ectodermal

dysplasia. Br J Dermatol 153: 46-50

Naeem M, Wajid M, Lee K, Leal SM, Ahmad W (2006a). A mutation in the hair

matrix and cuticle keratin KRTHB5 gene causes ectodermal dysplasia of hair and nail

type. J Med Genet 43: 274-279

Nahum S, Pasternack SM, Pforr J, Indelman M, Wollnik B, Bergman R, Nöthen MM,

König A, Khamaysi Z, Betz RC, Sprecher E (2009). A large duplication in LIPH

underlies autosomal recessive hypotrichosis simplex in four Middle Eastern families.

Arch Dermatol Res 301: 391-393

Nahum S, Pasternack SM, Pforr J, Indelman M, Wollnik B, Bergman R, Nöthen MM,

König A, Khamaysi Z, Betz RC, Sprecher E (2009). A large duplication in LIPH

underlies autosomal recessive hypotrichosis simplex in four Middle Eastern families.

Arch Dermatol Res 301: 391-393

References



Nakao K, Morita R, Saji Y, Ishida K, Tomita Y, Ogawa M, Saitoh M, Tomooka Y,

Tsuji T (2007). The development of a bioengineered organ germ method. Nat Meth 4:

227-230

Nakashima Y, Tomatsu S, Hori T, Fukuda S, Sukegawa K, Kondo N, Suzuki Y,

Shimozawa N, Orii T (1994). Mucopolysaccharidosis IV A: molecular cloning of the

human N-acetylgalactosamine-6-sulfatase gene (GALNS) and analysis of the 5′-

flanking region. Genomics 20(1): 99-104

Naqvi Syed Kamran-Ul-Hassan, Wasif N, Javed H, Ahmad W (2011). Two novel

mutations in the gene EDAR causing autosomal recessive hypohidrotic ectodermal

dysplasia. Orthd Cranf Res 14: 156-159

Naruse I, Ueta E, Sumino Y, Ogawa M, Ishikiriyama S (2010). Birth defects caused

by mutations in human GLI3 and mouse Gli3 genes. Cong Anom 50(1): 1-7

Naz G, Ali G, Kamran-Ul-Hassan Naqvi S, Azeem Z, Ahmad W (2010). Mapping of

a novel autosomal recessive hypotrichosis locus on chromosome 10q11.23-22.3. Hum

Genet 127: 395-401

Naz G, Ali G, Kamran-Ul-Hassan Naqvi S, Azeem Z, Ahmad W (2010). Mapping of

a novel autosomal recessive hypotrichosis locus on chromosome 10q11.23-22.3. Hum

Genet 127: 395-401

Nazzaro V (1989). Normal development of human fetal skin. G Ital Dermatol

Venereol 124: 421-427

Nelson J (1997). Incidence of the mucopolysaccharidoses in Northern Ireland. Hum

Genet 101(3): 355-358

Nelson J, Crowhurst J, Carey B, Greed L (2003). Incidence of the

mucopolysaccharidoses in Western Australia. Am J Med Genet 123(3): 310-313

References



Neufeld E, Muenzer J. The Mucopolysaccharidoses, Scriver CR, Beaudet AL, Sly

WS, Valle D, The Metabolic Basis of Inherited Disease (1995) McGraw-Hill, New

York, 2465-2494

Ng BG, Buckingham KJ, Raymond K, Kircher M, Turner EH, He M, Chong JX

(2013). Mosaicism of the UDP-galactose transporter SLC35A2 causes a congenital

disorder of glycosylation. Am J Hum Genet 92: 632-636.

Ng, S. B., Buckingham, K. J., Lee, C., Bigham, A. W., Tabor, H. K., Dent, K. M., . . .

Nickerson, D. A. (2010a). Exome sequencing identifies the cause of a mendelian

disorder. Nature genetics, 42(1), 30.

Ng, S. B., Nickerson, D. A., Bamshad, M. J., & Shendure, J. (2010b). Massively

parallel sequencing and rare disease. Human molecular genetics, 19(R2), R119-R124.

Norton, N., Li, D., Rieder, M. J., Siegfried, J. D., Rampersaud, E., Züchner, S., . . .

McGee, S. (2011). Genome-wide studies of copy number variation and exome

sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy. The

American Journal of Human Genetics, 88(3), 273-282.

O’Leary T, Wyllie DJ (2011). Neuronal homeostasis: time for a change ? J Physiol

589: 4811-4826.

O’Roak BJ, Vives L, Fu W, Egertson JD, Stanaway IB, Phelps IG (2012). Multiplex

targeted sequencing identifies recurrently mutated genes in autism spectrum disorders.

Science 338: 1619-22.

Oberbauer A (2013). The regulation of IGF-1 gene transcription and splicing during

development and aging. Front Endocrinol 4

Odland GF (1991). Structure of the skin. In: Goldsmith LA, editor.

Physiology,biochemistry and molecular biology of the skin. New York: Oxford

University Press Pp: 3-62

References



Oeseburg B, Dijkstra GJ, Groothoff JW, Reijneveld SA, Jansen DEC (2011).

Prevalence of chronic health conditions in children with intellectual disability: a

systematic literature review. J Intellect Dev Disabil 49: 59-85.

Offiah AC, Hall CM (2003). Radiological diagnosis of the constitutional disorders of

bone. As easy as A, B, C? Pediatr Radiol 33(3): 153-161

Oldridge M, Ana M, Maringa M, Propping P, Mansour S, Pollitt C, DeChiara TM,

Kimble RB, Valenzuela DM, Yancopoulos GD (2000). Dominant mutations in ROR2,

encoding an orphan receptor tyrosine kinase, cause brachydactyly type B. Nat Genet

24(3): 275-278

Orioli IM, Castilla EE (1999). Thumb/hallux duplication and preaxial polydactyly

type I. Am J Med Genet 82(3): 219-224

Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y (2001). Morphogenesis

and renewal of hair follicles from adult multipotent stem cells. Cell 104: 233-245

Otto, E. A., Hurd, T. W., Airik, R., Chaki, M., Zhou, W., Stoetzel, C., . . . Murga-

Zamalloa, C. A. (2010). Candidate exome capture identifies mutation of SDCCAG8

as the cause of a retinal-renal ciliopathy. Nature genetics, 42(10), 840.

Ouni M, Gunes Y, Belot M-P, Castell A-L, Fradin D, Bougnères P (2015). The IGF1

P2 promoter is an epigenetic QTL for circulating IGF1 and human growth. Clin

Epigenet 7(1): 22

Pakkenberg B, Pelvig D, Marner L, Bundgaard MJ, Gundersen HJG, Nyengaard JR

(2003). Aging and the human neo-cortex. Exp Gerontol 38: 95-99.

Pan S, Cheng X, Sifers RN (2013). Golgi situated endoplasmic reticulum alpha 1,2

mannosidase contributes to the retrieval of ERAD substrates through a direct

interaction with gamma-COP. Mol Biol Cell 24:1111‐1121.

References



Park, J.-H., Wacholder, S., Gail, M. H., Peters, U., Jacobs, K. B., Chanock, S. J., &

Chatterjee, N. (2010). Estimation of effect size distribution from genome-wide

association studies and implications for future discoveries. Nature genetics, 42(7),

570.

Pasternack SM, von Kugelgen I, Aboud KA, Lee YA, Rüschendorf F, Voss K,

Hillmer AM, Molderings GJ, Franz T, Ramirez A, Nürnberg P, Nöthen MM, Betz RC

(2008). G protein-coupled receptor P2Y5 and its ligand LPA are involved in

maintenance of human hair growth. Nat Genet 40: 329-334

Pasternack SM, von Kugelgen I, Aboud KA, Lee YA, Rüschendorf F, Voss K,

Hillmer AM, Molderings GJ, Franz T, Ramirez A, Nürnberg P, Nöthen MM, Betz RC

(2008). G protein-coupled receptor P2Y5 and its ligand LPA are involved in

maintenance of human hair growth. Nat Genet 40: 329-334

Pasternak JJ (2005). An introduction to human molecular genetics. John Wiley and

Sons Inc. New Jersey.

Pastor PN, Reuben CA (2009). Emotional/behavioral difficulties and mental health

service contacts of students in special education for non–mental health problems. J

Sch Health 79: 82-89.

Paus R, Cotsarelis G (1999). The biology of hair follicles. N Engl J Med 341: 491-497

Paus R, Foitzik K (2004). In search of the "hair cycle clock": a guided tour.

Differentiation 72: 489-511

Paus R, Nickoloff BJ, Ito T (2005). A 'hairy' privilege. Trends Immunol 26: 32-40

Piccione M, Niceta M, Antona V, Di Fiore A, Cariola F, Gentile M, Corsello G

(2009). Identification of two new mutations in TRPS1 gene leading to the tricho-

rhino-phalangeal syndrome type I and III. Am J Med Genet A 149: 1837-1841

References



Plöger F, Seemann P, Schmidt-von Kegler M, Lehmann K, Seidel J, Kjaer KW, Pohl

J, Mundlos S (2008). Brachydactyly type A2 associated with a defect in proGDF5

processing. Hum Mol Genet 17(9): 1222-1233

Pollitt RJ, Jenner FA, Davies M (1968). Sibs with mental and physical retardation and

trichorrhexis nodosa with abnormal amino acid composition of the hair. Arch Dis

Child 43: 211-216

Price VH, Odom RB, Ward WH, Jones FT (1980). Trichothiodystrophy: sulfur-

deficient brittle hair as a marker for a neuroectodermal symptom complex. Arch

Dermatol 116: 1375-1384

Privitera S, Prody CA, Callahan JW, Hinek A (1998). The 67-kDa enzymatically

inactive alternatively spliced variant of β-galactosidase is identical to the

elastin/laminin-binding protein. J Biol Chem 273(11): 6319-6326

Qvist P, Huertas P, Jimeno S, Nyegaard M, Hassan MJ, Jackson SP, Borglum AD

(2011). CtIP Mutations Cause Seckel and Jawad Syndromes. [Research Support, Non-

U S Gov't]. PLoS Genet 7(10): 6

Radhakrishna U, Bornholdt D, Scott HS, Patel UC, Rossier C, Engel H, Bottani A,

Chandal D, Blouin J-L, Solanki JV (1999). The phenotypic spectrum of GLI3

morphopathies includes autosomal dominant preaxial polydactyly type-IV and

postaxial polydactyly type-A/B; No phenotype prediction from the position of GLI3

mutations. Am J Hum Genet 65(3): 645-655

Rafiq MA, Faiyaz-Ul-Haque M, Ud Din MA, Malik S, Sohail M, Anwar M, Haque S,

Paterson AD, Tsui LC, Ahmad W (2005). A novel locus of ectodermal dysplasia

maps to chromosome 10q24.32-q25.1. J Invest Dermatol 124: 338-342

References



Rafiq MA, Kuss AW, Puettmann L, Noor A, Ramiah A, Ali G, Khan, MA (2011).

Mutations in the alpha 1,2-mannosidase gene, MAN1B1 , cause autosomal-recessive

intellectual disability. Am J Hum Genet 89: 176-182.

Rafique MA, Ansar M, Jamal SM, Malik S, Sohail M, Faiyaz-Ul-Haque M, Haque S,

Leal SM, Ahmad W (2003). A locus for hereditary hypotrichosis localized to human

chromosome 18q21.1. Eur J Hum Genet 11: 623-628

Rafiullah R, Aslamkhan M, Paramasivam N, Thiel C, Mustafa G, Wiemann S, Berkel

S (2016). Homozygous missense mutation in the LMAN2L gene segregates with

intellectual disability in a large consanguineous Pakistani family. Am J Med

Genet 53: 138-144.

Ramot Y, Horev L, Smolovich I, Molho-Pessach V, Zlotogorski A (2010). Marie

Unna hereditary hypotrichosis caused by a novel mutation in the human hairless

transcript. Exp Dermatol 19: 320-322

Ramser J, Abidi FE, Burckle CA, Lenski C, Toriello H, Wen G, Schwartz CE (2005).

A unique exonic splice enhancer mutation in a family with X-linked mental

retardation and epilepsy points to a novel role of the renin receptor. Hum Mol Gen 14:

1019-1027.

Rasool M, Schuster J, Aslam M, Tariq M, Ahmad I, Ali A, Entesarian M, Dahl N,

Baig SM (2008). A novel missense mutation in the EDA gene associated with X-

linked recessive isolated hypodontia. J Hum Genet 53: 894-898

Rauch A, Wieczorek D, Graf E, Wieland T, Endele S, Schwarzmayr T, Dufke A

(2012). Range of genetic mutations associated with severe non-syndromic sporadic

intellectual disability: an exome sequencing study. Lancet 380: 1674-1682.

Raynaud M, Moizard MP, Dessay B, Briault S, Toutain A, Gendrot C, Ronce N,

Moraine C (2000). Systematic analysis of X-inactivation in 19 XLMR families:

References



extremely skewed profiles in carriers in three families. Europ J Hum Genet 8: 253-

258.

Ress A, Graham T (1998). Finger polydactyly. Hand Clin 14(1): 49-64

Richards JB, Yuan X, Geller F, Waterworth D, Bataille V, Glass D, Song K, Waeber

G, Vollenweider P, Aben KK, Kiemeney LA, Walters B, Soranzo N, Thorsteinsdottir

U, Kong A, Rafnar T, Deloukas P, Sulem P, Stefansson H, Stefansson K, Spector TD,

Mooser V (2008). Male-pattern baldness susceptibility locus at 20p11. Nat Genet 40:

1282-1284

Richardson R, Donnai D, Meire F, Dixon M (2004). Expression of Gja1 correlates

with the phenotype observed in oculodentodigital syndrome/type III syndactyly. J

Med Genet 41(1): 60-67

Riley EP, Infante MA, Warren KR (2011). Fetal alcohol spectrum disorders: an

overview. Neuropsychol Rev 21: 73-80.

Rimoin DL, Cohn D, Krakow D, Wilcox W, Lachman RS, Alanay Y (2007). The

skeletal dysplasias. Ann N Y Acad Sci 1117(1): 302-309

Rindermann H Thompson J, (2011). Cognitive capitalism the effect of cognitive

ability on wealth, as mediated through scientific achievement and economic

freedom. Psychol Sci 22: 754-763.

Rinne T, Brunner HG, van Bokhoven H (2007). p63-associated disorders. Cell Cycle

6(3): 262-268

Risheg H, Graham JM, Clark RD, Rogers RC, Opitz JM, Moeschler JB, Stevenson

RE (2007). A recurrent mutation in MED12 leading to R961W causes Opitz-

Kaveggia syndrome. Nature genet 39: 451-453.

References



Rivas, M. A., Beaudoin, M., Gardet, A., Stevens, C., Sharma, Y., Zhang, C. K., . . .

Burtt, N. (2011). Deep resequencing of GWAS loci identifies independent rare

variants associated with inflammatory bowel disease. Nature genetics, 43(11), 1066.

Roberts JL, Whiting DA, Henry D, Basler G, Woolf L (1999). MarieUnna congenital

hypotrichosis: Clinical description, histopathology, scanning electron microscopy of a

previously unreported large pedigree. J Invest Dermatol Symp Proc 4: 261-267

Roessler, E. and Muenke, M. (2003) How a Hedgehog might see holoprosencephaly.

Hum Mol Genet. 12 Spec No 1: R15-25

Rope AF, Wang K, Evjenth R, Xing J, Johnston JJ, Swensen JJ, Carey JC (2011).

Using VAAST to identify an X-linked disorder resulting in lethality in male infants

due to N-terminal acetyltransferase deficiency. Am J Hum Genet 89: 28-43.

Ropers HH (2008). Genetics of intellectual disability. Curr Opin Genet Dev 18:241-

50.

Ropers HH (2010). Genetics of early onset cognitive impairment. Annu Rev

Genomics Hum Genet 11: 161-187.

Ropers HH, Hoeltzenbein M, Kalscheuer V, Yntema H, Hamel B, Fryns JP, Moraine

C (2003). Nonsyndromic X-linked mental retardation: where are the missing

mutations? TIG 19: 316-320.

Rosenquist TA, Martin GR (1996). Fibroblast growth factor signalling in the hair

growth cycle: expression of the fibroblast growth factor receptor and ligand genes in

the murine hair follicle. Dev Dyn 205: 379-386

Rossi A, Devirgiliis V, Panasiti V, Borroni RG, Carlesimo M, Gentile M, Cariola F,

Calvieri S (2007). Missense mutation in exon 7 of TRPS1 gene in an Italian family

with a mild form of trichorhinophalangeal syndrome type I. Br J Dermatol 157: 1021-

1024

References



Rossi A, Superti-Furga A (2001). Mutations in the diastrophic dysplasia sulfate

transporter (DTDST) gene (SLC26A2): 22 novel mutations, mutation review,

associated skeletal phenotypes, and diagnostic relevance. Hum Mutat 17(3): 159

Rotanova TV, Botos I, Melnikov EE, Rasulova F, Gustchina A, Maurizi MR,

Wlodawer A (2006). Slicing a protease: structural features of the ATP-dependent Lon

proteases gleaned from investigations of isolated domains. Protein Sci 15: 1815-1828.

Rotwein P (2012). Mapping the growth hormone–Stat5b–IGF-I transcriptional circuit.

Trends Endocrinol Metabol 23(4): 186-193

Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot, A, Li N, Berriz GF,

Gibbon FD, Dreze M, Ayivi-Guedehoussou N (2005). Towards a proteome-scale map

of the human protein-protein interaction network. Nature 437: 1173-1178.

Ruiz-Perez VL, Tompson SWJ, Blair HJ, Espinoza-Valdez C, Lapunzina P, Silva EO,

Hamel B, Gibbs JL, Young ID, Wright MJ, Goodship JA (2003). Mutations in two

non-homologous genes in a head-to-head configuration cause Ellis-van Creveld

syndrome. Am J Hum Genet 72: 728-732

Ruiz-Perez VL, Tompson SWJ, Blair HJ, Espinoza-Valdez C, Lapunzina P, Silva

EO, Hamel B, Gibbs JL, Young ID, Wright MJ, Goodship JA (2003). Mutations in

two non-homologous genes in a head-to-head configuration cause Ellis-van Creveld

syndrome. Am J Hum Genet 72: 728-73

Russell LJ, Weaver DD, Bull MJ, Weinbaum M, Opitz JM (1984). In utero brain

destruction resulting in collapse of the fetal skull, microcephaly, scalp rugae, and

neurologic impairment: the fetal brain disruption sequence. Am J Med genet 17: 509-

521.

References



Santamaria R, Chabás A, Callahan JW, Grinberg D, Vilageliu L (2007). Expression

and characterization of 14 GLB1 mutant alleles found in GM1-gangliosidosis and

Morquio B patients. J Lipid Res 48(10): 2275-2282

Sarfarazi M, Akarsu AN, Sayli BS (1995). Localization of the syndactyly type II

(synpolydactyly) locus to 2q31 region and identification of tight linkage to HOXD8

intragenic marker. Hum Mol Genet 4(8): 1453-1458

Sartaj R, Sharpe P (2006). Biological tooth replacement. J Anat 209: 503-509

Sato D, Liang D, Wu L, Pan Q, Xia K, Dai H, Wang H, Nishimura G, Yoshiura K-I,

Xia J (2007). A syndactyly type IV locus maps to 7q36. J Hum Genet 52(6): 561-564

Savarirayan R, Rimoin DL (2002). The skeletal dysplasias. Best Practice & Research

Clin Endocrinol Metabol 16(3): 547-560

Schaffer JV, Bazzi H, Vitebsky A, Witkiewicz A, Kovich OI, Kamino H, Shapiro LS,

Amin SP, Orlow SJ, Christiano AM (2006). Mutations in the desmoglein 4 gene

underlie localized autosomal recessive hypotrichosis with monilethrix hairs and

congenital scalp erosions. J Invest Derm 126: 1286-1291

Schalock RL, Luckasson RA, Shogren KA, Shogren KA (2007). The renaming of

mental retardation: understanding the change to the term intellectual disability. J

Intellect Dev Disabil 45: 116-124.

Schlake T (2007). Determination of hair structure and shape. Semin Cell Dev Biol 18:

267-273

Schlereth T, Dieterich M, Birklein F (2009). Hyperhidrosis--causes and treatment of

enhanced sweating. Dtsch Arztebl Int 106: 32-37

Schlereth T, Dieterich M, Birklein F (2009). Hyperhidrosis--causes and treatment of

enhanced sweating. Dtsch Arztebl Int 106: 32-37

References



Schmidt A, Koch PJ (2007). Desmosomes: just cell adhesion or is there more? Cell

Adh Migr 1: 28-32

Schork, N. J., Murray, S. S., Frazer, K. A., & Topol, E. J. (2009). Common vs. rare

allele hypotheses for complex diseases. Current opinion in genetics & development,

19(3), 212-219.

Schramm T, Gloning K, Minderer S, Daumer‐Haas C, Hörtnagel K, Nerlich A,

Tutschek B (2009). Prenatal sonographic diagnosis of skeletal dysplasias. Ultra Obstet

Gynecol 34(2): 160-170

Schramm, T., Gloning, K. P., Minderer, S., Daumer-Haas, C., Hortnagel, K., Nerlich,

A., & Tutschek, B. (2009). Prenatal sonographic diagnosis of skeletal dysplasias.

Ultrasound Obstet Gynecol, 34(2), 160-170.

Schwabe GC, Mundlos S (2004). Genetics of congenital hand anomalies.

Handchirurgie Mikrochirurgie Plastische Chirurgie 36(3): 85-97

Schwabe GC, Tinschert S, Buschow C, Meinecke P, Wolff G, Gillessen-Kaesbach G,

Oldridge M, Wilkie AO, Kömec R, Mundlos S (2000). Distinct mutations in the

receptor tyrosine kinase gene ROR2 cause brachydactyly type B. Am J HumGenet

67(4): 822-831

Schwabe GC, Türkmen S, Leschik G, Palanduz S, Stöver B, Goecke TO, Mundlos S

(2004). Brachydactyly type C caused by a homozygous missense mutation in the

prodomain of CDMP1. Am J Med Genet 124(4): 356-363

Schweizer J (2006). More than one gene involved in monilethrix: intracellular but

also extracellular players. J Invest Dermatol 126: 1216-1219

Schweizer J, Bowden PE, Coulombe PA, Langbein L, Lane EB, Magin TM, Maltais

L, Omary MB, Parry DA, Rogers MA, Wright MW (2006). New consensus

nomenclature for mammalian keratins. J Cell Biol 174: 169-174

References



Scott HS, Guo X-H, Hopwood JJ, Morris CP (1992). Structure and sequence of the

human α-L-iduronidase gene. Genomics 13(4): 1311-1313

Scriver CR. (2001). The metabolic & molecular bases of inherited disease (Vol. 4):

New York; Montreal: McGraw-Hill.

Seemann P, Schwappacher R, Kjaer KW, Krakow D, Lehmann K, Dawson K,

Stricker S, Pohl J, Plöger F, Staub E (2005). Activating and deactivating mutations in

the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2.

J Clin Invest 115(9): 2373

Seldin, M. F., Pasaniuc, B., & Price, A. L. (2011). New approaches to disease

mapping in admixed populations. Nature Reviews Genetics, 12(8), 523.

Shaheen R, Faqeih E, Ansari S, Abdel-Salam G, Al-Hassnan ZN, Al-Shidi T, Alomar

R, Sogaty S, Alkuraya FS (2014). Genomic analysis of primordial dwarfism reveals

novel disease genes. Genome Res 24(2): 291-299

Shaheen R, Faqeih E, Ansari S, Abdel-Salam G, Al-Hassnan ZN, Al-Shidi T, Alomar

R, Sogaty S, Alkuraya FS (2014). Genomic analysis of primordial dwarfism reveals

novel disease genes. [Research Support, Non-U S Gov't]. Genome Res 24(2): 291-299

Shaheen R, Faqeih E, Shamseldin HE, Noche RR, Sunker A, Alshammari MJ, Al-

Sheddi T, Adly N, Al-Dosari MS, Megason SG (2012). POC1A truncation mutation

causes a ciliopathy in humans characterized by primordial dwarfism. Am J Med

Genet 91(2): 330-336

Shen W, Han D, Zhang J, Zhao H, Feng H (2011). Two novel heterozygous mutations

of EVC2 cause a mild phenotype of Ellis-van Creveld syndrome in a Chinese

family.Am J Med Genet 155A: 2131-2136

Shen Y, Nilsson SK (2012). Bone, microenvironment and hematopoiesis. Curr Opin

Hematol 19(4): 250-255

References



Shevell MI, Ashwal S, Donley D, Flint J, Gingold M, Hirtz D, Sheth RD (2003).

Practice parameter: Evaluation of the child with global developmental delay Report of

the Quality Standards Subcommittee of the American Academy of Neurology and

The Practice Committee of the Child Neurology Society. Neurology, 60: 367-380.

Shigli A, Reddy RP, Hugar SM, Deshpande D (2005). Hypohidrotic ectodermal

dysplasia: a unique approach to esthetic and prosthetic management: a case report. J

Indian Soc Pedod Prev Dent 23: 31-34

Shimomura Y, Agalliu D, Vonica A, Luria V, Wajid M, Baumer A, Belli S,

Petukhova L, Schinzel A, Brivanlou AH, Barres BA, Christiano AM (2010b).

APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex. Nature

464: 1043-1047

Shimomura Y, Christiano AM (2010a). Biology and genetics of hair. Annu Rev

Genomics Hum Genet 11: 109-132

Shimomura Y, Wajid M, Ishii Y, Shapiro L, Petukhova L, Gordon D, Christiano AM

(2008a). Disruption of P2RY5, an orphan G protein-coupled receptor, underlies

autosomal recessive woolly hair. Nat Genet 40: 335-339

Shimomura Y, Wajid M, Kurban M, Sato N, Christiano AM (2010d). Mutations in the

keratin 85 (KRT85/hHb5) gene underlie pure hair and nail ectodermal dysplasia. J


Shimomura Y, Wajid M, Petukhova L, Kurban M, Christiano AM (2010c).

Autosomal-dominant woolly hair resulting from disruption of keratin 74 (KRT74), a

potential determinant of human hair texture. Am J Hum Genet 86: 632-638

Shimomura Y, Wajid M, Shapiro L, Christiano AM (2008b). P-cadherin is a p63

target gene with a crucial role in the developing human limb bud and hair follicle.

Development 135: 743-753

References



Shoichet SA, Hoffmann K, Menzel C, Trautmann U, Moser B, Hoeltzenbein M, Fryns

JP (2003). Mutations in the ZNF41 gene are associated with cognitive deficits:

identification of a new candidate for X-linked mental retardation. Am J Hum

Genet 73: 1341-1354.

Shriner, D., Adeyemo, A., Gerry, N. P., Herbert, A., Chen, G., Doumatey, A., . . .

Rotimi, C. N. (2009). Transferability and fine-mapping of genome-wide associated

loci for adult height across human populations. PLoS One, 4(12), e8398.

Sir JH, Barr AR, Nicholas AK, Carvalho OP, Khurshid M, Sossick A, Reichelt S,

D'Santos C, Woods CG, Gergely F (2011). A primary microcephaly protein complex

forms a ring around parental centrioles. Nat Genet 43(11): 1147-1153

Slutsky I, Abumaria N, Wu LJ, Huang C, Zhang L, Li B, Zhao X, Govindarajan A,

Zhao MG, Zhuo M, Tonegawa S, Liu G (2010). Enhancement of learning and

memory by elevating brain magnesium. Neuron 65: 165-177.

Sly WS, Quinton BA, McAlister WH, Rimoin DL (1973). Beta glucuronidase

deficiency: report of clinical, radiologic, and biochemical features of a new

mucopolysaccharidosis. J Pediatr 82(2): 249-257

Smirle J, Au CE, Jain M, Dejgaard K, Nilsson T, Bergeron J (2013). Cell biology of

the endoplasmic reticulum and the Golgi apparatus through proteomics. Cold Spring

Harb Perspect Biol 5: a015073.

Smith, C.A.B. (1953) The detection of linkage in human genetics. JR Stat Soc B. 15:

153-184

Souied E, Amalric P, Chauvet ML, Chevallier C, Hoang PL, Munnich A, Kaplan J

(1995). Unusual association of juvenile macular dystrophy with congenital

hypotrichosis: occurrence in two siblings suggesting autosomal recessive inheritance.

Ophthal Genet 16: 11-15

References



South AP (2004). Plakophilin 1: an important stabilizer of desmosomes. Clin Exp

Dermatol 29: 161-167

Speleman F, Vervoort R, Van Roy N, Liebaers I, Sly W, Lissens W (1996).

Localization by fluorescence in situ hybridization of the human functional β-

glucuronidase gene (GUSB) to 7q11. 21→ q11. 22 and two pseudogenes to 5p13 and

5q13. Cytogenet Genome Res 72(1): 53-55

Spence MA, Sparkes RS, Curtis RK, Tideman S, Sparkes MC, Crist M (1979).

Linkage analysis of one large pedigree segregating autosomal dominant monilethrix.

Cytogenet Cell Genet 25: 208

Spiegel R, Pines O, Ta-Shma A, Burak E, Shaag A, Halvardson J, Shalev S (2012).

Infantile cerebellar-retinal degeneration associated with a mutation in mitochondrial

aconitase, ACO2. Am J Hum Genet 90: 518-523.

Spranger J (1977). Catabolic disorders of complex carbohydrates. Postgrad Med J

53(622): 441-449

Spranger J, Wiedemann H-R (1966). Dysplasia spondyloepiphysaria congenita. The

Lancet 288(7464): 642

Sprecher E (2005). Genetic hair and nail disorders. Clin Dermatol 23: 47-55

Sprecher E (2005). Genetic hair and nail disorders. Clin Dermatol 23: 47-55

Sprecher E, Bergman R, Richard G, Lurie R, Shalev S, Petronius D, Shalata A,

Anbinder Y, Leibu R, Perlman I, Cohen N, Szargel R (2001). Hypotrichosis with

juvenile macular dystrophy is caused by a mutation in CDH3, encoding P-cadherin.

Nat Genet 29: 134-136

Sprengel MM, Rost BR, Hucho T, Garshasbi M, Kahrizi K, Ullmann R, Abedini SS,

Nieh SE, Amini SH, Goswami C, Tzschach A, Jensen LR, Schmitz D, Ropers HH,

References



Najmabadi H, Kuss AW (2007). A defect in the ionotropic glutamate receptor 6 gene

(GRIK2) is associated with autosomal recessive mental retardation. Am J Hum Genet

81: 792-798.

Stevenson RE, Massey PS, Schroer RJ, McDermott S, Richter B (1996). Preventable

fraction of mental retardation: analysis based on individuals with severe mental

retardation. Mental retardation 34: 182.

Stockton DW, Das P, Goldenberg M, D'Souza RN, Patel PI (2000). Mutation of PAX9

is associated with oligodontia. Nat Genet 24: 18-19

Stromme P, Diseth TH (2000). Prevalence of psychiatric diagnoses in children with

mental retardation: data from a population-based study. Dev Med Child Neuro 42:

266-270.

Swanson AB, Brown KS (1962). Hereditary triphalangeal thumb. J Heredity 53(6):

259-265

Swensson O, Langbein L, McMillan JR, Stevens HP, Leigh IM, McLean WH, Lane

EB, Eady RA (1998). Specialized keratin expression pattern in human ridged skin as

an adaptation to high physical stress. Br J Dermatol 139(5): 767-775

Szperl, A. M., Golachowska, M. R., Bruinenberg, M., Prekeris, R., Thunnissen, A.-M.

W., Karrenbeld, A., . . . Ksiazyk, J. (2011). Functional characterization of mutations

in the myosin Vb gene associated with microvillus inclusion disease. Journal of

pediatric gastroenterology and nutrition, 52(3), 307.

Szperl, A., Ricaño‐Ponce, I., Li, J., Deelen, P., Kanterakis, A., Plagnol, V., . . .

Mulder, C. (2011). Exome sequencing in a family segregating for celiac disease.

Clinical genetics, 80(2), 138-147.

Talamillo A, Bastida M, Fernandez‐Teran M, Ros M (2005). The developing limb and

the control of the number of digits. Clin Genet 67(2): 143-153

References



Tanaka A, Lai-Cheong JE, Café ME, Gontijo B, Salomao PR, Pereira L, McGrath JA

(2009). Novel truncating mutations in PKP1 and DSP cause similar skin phenotypes

in two Brazilian families. Br J Dermatol 160: 692-697

Tao R, Jin B, Guo SZ, Qing W, Feng GY, Brooks DG, Liu L, Xu J, Li T, Yan Y, He

L (2006). A novel missense mutation of the EDA gene in a Mongolian family with

congenital hypodontia. J Hum Genet 51: 498-502

Tao R, Jin B, Guo SZ, Qing W, Feng GY, Brooks DG, Liu L, Xu J, Li T, Yan Y, He

L (2006). A novel missense mutation of the EDA gene in a Mongolian family with

congenital hypodontia. J Hum Genet 51: 498-502

Tariq M, Ahmad S, Ahmad W (2008b). A novel missense mutation in the TRPS1

gene underlies trichorhinophalangeal syndrome type III. Br J Dermatol 159: 476-478

Tariq M, Ayub M, Jelani M, Basit S, Naz G, Wasif N, Raza SI, Naveed AK, ullah

Khan S, Azeem Z, Yasinzai M, Wali A, Ali G, Chishti MS, Ahmad W (2009a).

Mutations in the P2RY5 gene underlie autosomal recessive hypotrichosis in 13

Pakistani families. Br J Dermatol 160: 1006-1010

Tariq M, Azeem Z, Ali G, Chishti MS, Ahmad W (2009b). Mutation in the HPGD

gene encoding NAD+-dependent 15-hydroxyprostaglandin dehydrogenase underlies

isolated congenital nail clubbing (ICNC). J Med Genet 46: 14-20

Tariq M, Chishti MS, Ali G, Ahmad W (2008a). A novel locus for ectodermal

dysplasia of hairs, nails and teeth type maps to chromosome 18q22.1-22.3. Ann Hum

Genet 72: 19-25

Tariq M, Khan MN, Ahmad W (2009c). Ectodermal dysplasia-cutaneous syndactyly

syndrome maps to chromosome 7p21.1-p14.3. Hum Genet 125: 421-429

Tarpey P, Pemberton TJ, Stockton DW, Das P, Ninis V, Edkins S, Futreal PA,

Wooster R, Kamath S, Nayak R, Stratton MR, Patel PI (2007). A novel gln358glu

References



mutation in ectodysplasin A associated with X-linked dominant incisor hypodontia.

Am J Med Genet 143: 390-394

Tarpey PS, Smith R, Pleasance E, Whibley A, Edkins S, Hardy C, Stephens P (2009).

A systematic, large-scale resequencing screen of X-chromosome coding exons in

mental retardation. Nature Genet 41: 535-543.

Tarpey PS, Stevens C, Teague J, Edkins S, O’Meara S, Avis T, Dicks E (2006).

Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex,

AP1S2, cause X-linked mental retardation. Am J Hum Genet 79: 1119-1124.

Taylor GA, Jordan CE, Dorst SK, Dorst JP (1984). Polycarpaly and other

abnormalities of the wrist in chondroectodermal dysplasia: the Ellis-van Creveld

syndrome. Radiology 151: 393-396

Teebi AS (2010). Genetic disorders among Arab populations. Springer Science and

Business Media.

Temtamy SA, McKusick VA (1977). The genetics of hand malformations. Birth

Defects Orig Artic Ser 14(3): i-xviii, 1-619

Thesleff I (2003). Developmental biology and building a tooth. Quintessence Int 34:

613-620

Thomas GM, Huganir RL (2004). MAPK cascade signalling and synaptic

plasticity. Nat Rev Neurosci 5: 173-183.

Thomas, P. D., & Kejariwal, A. (2004). Coding single-nucleotide polymorphisms

associated with complex vs. Mendelian disease: evolutionary evidence for differences

in molecular effects. Proceedings of the National Academy of Sciences, 101(43),

15398-15403.

Thompson CC (1996). Thyroid hormone-responsive genes in developing cerebellum

include a novel synaptotagmin and a hairless homolog. J Neurosci 16: 7832-7840

References



Timal S, Hoischen A, Lehle L, Adamowicz M, Huijben K, Sykut-Cegielska J,

Gilissen C (2012). Gene identification in the congenital disorders of glycosylation

type I by whole-exome sequencing. Hum Mol Gen 21: 4151-4161.

Toma JG, Akhavan M, Fernandes KJ, Barnabé-Heider F, Sadikot A, Kaplan DR,

Miller FD (2001). Isolation of multipotent adult stem cells from the dermis of

mammalian skin. Nat Cell Biol 3: 778-784

Tomatsu S, Fukuda S, Masue M, Sukegawa K, Masuno M, Orii T (1992).

Mucopolysaccharidosis type IVA: characterization and chromosomal localization of

N-acetylgalactosamine-6-sulfate sulfatase gene and genetic heterogeneity. Am J Hum

Genet 51(Suppl 4): A178

Tomatsu S, Gutierrez M, Nishioka T, Yamada M, Yamada M, Tosaka Y, Grubb JH,

Montaño AM, Vieira MB, Trandafirescu GG (2005). Development of MPS IVA

mouse (Galnstm (hC79S· mC76S) slu) tolerant to human N-acetylgalactosamine-6-

sulfate sulfatase. Hum Mol Genet 14(22): 3321-3335

Tomatsu S, Orii KO, Vogler C, Nakayama J, Levy B, Grubb JH, Gutierrez MA, Shim

S, Yamaguchi S, Nishioka T (2003). Mouse model of N-acetylgalactosamine-6-

sulfate sulfatase deficiency (Galns−/−) produced by targeted disruption of the gene

defective in Morquio A disease. Hum Mol Genet 12(24): 3349-3358

Toro R, Konyukh M, Delorme R, Leblond C, Chaste P, Fauchereau F (2010). Key

role for gene dosage and synaptic homeostasis in autism spectrum disorders. Trends

Genet 26: 363-72.

Triggs-Raine B, Salo TJ, Zhang H, Wicklow BA, Natowicz MR (1999). Mutations in

HYAL1, a member of a tandemly distributed multigene family encoding disparate

hyaluronidase activities, cause a newly described lysosomal disorder,

mucopolysaccharidosis IX. Proc Natl Acad Sci 96(11): 6296-6300

References



Troost T, Jaeckel S, Ohlenhard N, Klein T (2012). The tumour suppressor Lethal 2

giant discs is required for the function of the ESCRT-III component Shrub/CHMP4. J

Cell Sci 125: 763-776.

Trynka, G., Hunt, K. A., Bockett, N. A., Romanos, J., Mistry, V., Szperl, A., . . .

Castillejo, G. (2011). Dense genotyping identifies and localizes multiple common and

rare variant association signals in celiac disease. Nature genetics, 43(12), 1193.

Tsuji T, Kunieda T (2005). A loss-of-function mutation in natriuretic peptide receptor

2 (Npr2) gene is responsible for disproportionate dwarfism in cn/cn mouse. J Biol

Chem 280(14): 14288-14292

Tucker A, Sharpe P (2004). The cutting-edge of mammalian development; how the

embryo makes teeth. Nat Rev Genet 5: 499-508

Umm-E-Kalsoom, Wasif N, Tariq M, Ahmad W (2010). A novel missense mutation

in the EVC gene underlies Ellis-van Creveld syndrome in a Pakistani family. Pediatr

Int 52: 240-246

Unger S, Bonafé L, Superti-Furga A (2008). Multiple epiphyseal dysplasia: clinical

and radiographic features, differential diagnosis and molecular basis. Best Pract Res

Clin Rheumat 22(1): 19-32

Unger SL, Briggs MD, Holden P, Zabel B, Ala-Kokko L, Paassilta P, Lohiniva J,

Rimoin DL, Lachman R, Cohn DH (2001). Multiple epiphyseal dysplasia:

radiographic abnormalities correlated with genotype. Pediatr Radiol 31(1): 10-18

Uppal S, Diggle CP, Carr IM, Fishwick CWG, Ahmed M, Ibrahim GH, Helliwell PS,

Latos-Bielenska A, Phillips SEV, Markham AF, Bennett CP, Bonthron DT (2008).

Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic

osteoarthropathy. Nat Genet 40: 789-793

References



Van Belzen, M. J., Meijer, J. W., Sandkuijl, L. A., Bardoel, A. F., Mulder, C. J.,

Pearson, P. L., Wijmenga, C. (2003). A major non-HLA locus in celiac disease maps

to chromosome 191. Gastroenterology, 125(4), 1032-1041.

Van Gele, M., Dynoodt, P., & Lambert, J. (2009). Griscelli syndrome: a model system

to study vesicular trafficking. Pigment cell & melanoma research, 22(3), 268-282.

Van Heyningen, V. and Williamson, K.A. (2002) PAX6 in sensory development.

Hum Mol Genet. 11 (10):1161-1167

van Heyningen, V., & Yeyati, P. L. (2004). Mechanisms of non-Mendelian

inheritance in genetic disease. Human molecular genetics, 13(suppl_2), R225-R233.

van Steensel MA, Steijlen PM, Bladergroen RS, Vermeer M, van Geel M (2005). A

missense mutation in the type II hair keratin hHb3 is associated with monilethrix. J

Med Genet 42: e19

Vastardis H, Karimbux N, Guthua SW, Seidman JG, Seidman CE (1996). A human

MSX1 homeodomain missense mutation causes evidence tooth agenesis. Nat Genet

13: 417-421

Ventruto V, Theo G, Celona A, Fioretti G, Pagano L, Stabile M, Cavaliere M (1980).

A and B postaxial polydactyly in two members of the same family. Clin Genet 18(5):

342-347

Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Eussen BE (1991).

Identification of a gene (FMR-1) containing a CGG repeat coincident with a

breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65:

905-914.

Vogt A, Hadam S, Heiderhoff M, Audring H, Lademann J, Sterry W, Blume-Peytavi

U (2007). Morphometry of human terminal and vellus hair follicles. Exp Dermatol

16:946-950

References



Voronina, V.A., Kozhemyakina, E.A., O'Kernick, C.M. (2004) Mutations in the

human RAX homeobox gene in a patient with anophthalmia and sclerocornea. Hum

Mol Genet. 13: 315-322

Wagner JK, Eblé A, Hindmarsh PC, Mullis PE (1998). Prevalence of human GH-1

gene alterations in patients with isolated growth hormone deficiency. Pediatr Res

43(1): 105-110

Wajid M, Bazzi H, Rockey J, Lubetkin J, Zlotogorski A, Christiano AM (2007).

Localized autosomal recessive hypotrichosis due to a frameshift mutation in the

desmoglein 4 gene exhibits extensive phenotypic variability within a Pakistani family.

J Invest Dermatol 127: 1779-1782

Walenkamp M, Wit J (2007). Genetic disorders in the GH–IGF-I axis in mouse and

man. Eur J Endocrinol 157(1): S15-S26

Walenkamp MJ, Wit JM (2006). Genetic disorders in the growth hormone–insulin-

like growth factor-I axis. Horm Res Paediatr 66(5): 221-230

Wali A, Chishti MS, Ayub M, Yasinzai M, Kafaitullah, Ali G, John P, Ahmad W

(2007a). Localization of a novel autosomal recessive hypotrichosis locus (LAH3) to

chromosome 13q14.11-q21.32. Clin Genet 72: 23-29







References



Wali A, John P, Gul A, Lee K, Chishti MS, Ali G, Hassan MJ, Leal SM, Ahmad W

(2006b). A novel locus for alopecia with mental retardation syndrome (APMR2) maps

to chromosome 3q26.2-q26.31. Clin Genet 70: 233-239

Wallis, D.E., Roessler, E., Hehr, U.(1999) Mutations in the homeodomain of the

human SIX3 gene cause holoprosencephaly. Nat Genet. 22 (2): 196-198

Wang, K., Dickson, S. P., Stolle, C. A., Krantz, I. D., Goldstein, D. B., &

Hakonarson, H. (2010). Interpretation of association signals and identification of

causal variants from genome-wide association studies. The American Journal of

Human Genetics, 86(5), 730-742.

Warman ML, Cormier‐Daire V, Hall C, Krakow D, Lachman R, LeMerrer M, Mortier

G, Mundlos S, Nishimura G, Rimoin DL (2011). Nosology and classification of

genetic skeletal disorders: 2010 revision. Am J Med Genet155(5): 943-968

Wasif N, Ahmad W (2012). A Novel Nonsense Mutation in RSPO4 Gene Underlies

Autosomal Recessive Congenital Anonychia in a Pakistani Family. Pediatr Dermatol

[In press]

Wasif N, Tariq M, Ali G, Hassan MJ, Ahmad W (2010). A novel splice site mutation

in the EDAR gene underlies autosomal recessive hypohidrotic ectodermal in a

Pakistani family. Pediatr Dermatol 27: 106-108

Wen Y, Liu Y, Xu Y, Zhao Y, Hua R, Wang K, Sun M, Li Y, Yang S, Zhang XJ,

Kruse R, Cichon S, Betz RC, Nöthen MM, van Steensel MA, van Geel M, Steijlen

PM, Hohl D, Huber M, Dunnill GS, Kennedy C, Messenger A, Munro CS, Terrinoni

A,Hovnanian A, Bodemer C, de Prost Y, Paller AS, Irvine AD, Sinclair R, Green J,

Shang D, Liu Q, Luo Y, Jiang L, Chen HD, Lo WH, McLean WH, He CD, Zhang X

(2009). Loss-of-function mutations of an inhibitory upstream ORF in the human

hairless transcript cause Marie Unna hereditary hypotrichosis. Nat Genet 41: 228-233

References



White, D.R., Ganesh, A., Nishimura, D. (2007) Autozygosity mapping of Bardet-

Biedl syndrome to 12q21.2 and confirmation of FLJ23560 as BBS10. Eur J Hum

Genet. 15 (2): 173-178

Whiting DA, Dy LC (2006). Office diagnosis of hair shaft defects. Semin Cutan Med

Surg 25: 24-34

Whittock NV, Smith FJ, Wan H, Mallipeddi R, Griffiths WA, Dopping-Hepenstal P,

Ashton GH, Eady RA, McLean WHI, McGrath JA (2002). Frameshift mutation in the

V2 domain of human keratin 1 results in striate palmoplantar keratoderma. J Invest

Dermatol 118: 838-844

Wickett RR (2004). Basic of skin structure. J Cosmet Sci 55: 132-133

Wickett RR, Visscher MO (2006). Structure and function of the epidermal barrier.

Am J Infect Control 34: 98-110

Wijmenga, C., Hansen, R. S., Gimelli, G., Björck, E. J., Davies, E. G., Valentine, D., .

. . Van Den Heuvel, L. P. (2000). Genetic variation in ICF syndrome: evidence for

genetic heterogeneity. Human mutation, 16(6), 509-517.

Wilke K, Martin A, Terstegen L, Biel SS (2007). A short history of sweat gland

biology. Int J Cosmet Sci 29: 169-179

Williams SR, Aldred MA, Der Kaloustian VM, Halal F, Gowans G, McLeod DR,

Zondag S, Toriello HV, Magenis RE, Elsea SH (2010). Haploinsufficiency of

HDAC4 causes brachydactyly mental retardation syndrome, with brachydactyly type

E, developmental delays, and behavioral problems. Am J Med Genet 87(2): 219-228

Wilson C, Cotsarelis G, Wei ZG, Fryer E, Margolis-Fryer J, Ostead M, Tokarek R,

Sun TT, Lavker RM (1994). Cells within the bulge region of mouse hair follicle

transiently proliferate during early anagen: heterogeneity and functional differences of

various hair cycles. Differentiation 55: 127-136

References



Wimplinger I, Morleo M, Rosenberger G, Iaconis D, Orth U, Meinecke P, Kutsche K

(2006). Mutations of the Mitochondrial Holocytochrome c–Type Synthase in X-

Linked Dominant Microphthalmia with Linear Skin Defects Syndrome. Am J Hum

Genet 79: 878-889.

Winter H, Rogers MA, Gebhardt M, Wollina U, Boxall L, Chitayat D, Babul-Hirji R,

Stevens HP, Zlotogorski A, Schweizer J (1997b). A new mutation in the type II hair

cortex keratin hHb1 involved in the inherited hair disorder monilethrix. Hum Genet

101: 165-169

Winter H, Rogers MA, Langbein L, Stevens HP, Leigh IM, Labrèze C, Roul S, Taieb

A, Krieg T, Schweizer J (1997a). Mutations in the hair cortex keratin hHb6 cause the

inherited hair disease monilethrix. Nat Genet 16: 372-374

Wit J, Van Duyvenvoorde H, Scheltinga S, De Bruin S, Hafkenscheid L, Kant S,

Ruivenkamp C, Gijsbers A, Van Doorn J, Feigerlova E (2012). Genetic analysis of

short children with apparent growth hormone insensitivity. Horm Res Paediatr 77(5):

320-333

Wong, F.L., Cantor, R.M. and Rotter, J.I. (1986) Sample-size considerations and

strategies for linkage analysis in autosomal recessive disorders. Am J Hum Genet.

39:25-37

Wood TC, Harvey K, Beck M, Burin MG, Chien Y-H, Church HJ, D’Almeida V, van

Diggelen OP, Fietz M, Giugliani R (2013). Diagnosing mucopolysaccharidosis IVA. J

Inherit Metab Dis 36(2): 293-307

Wood VE (1970). Duplication of the index finger.J Bone Jt Surg 52(3): 569-573

Wright, S. (1922) Coefficients of inbreeding and relationship. Am Nat. 56: 330

References



Wrzosek M, Łukaszkiewicz J, Wrzosek M, Jakubczyk A, Matsumoto H, Piątkiewicz

P, Nowicka G (2013). Vitamin D and the central nervous system. Pharmacol Rep 65:

271-278.

Xin B, Puffenberger EG, Turben S, Tan H, Zhou A, Wang H (2010). Homozygous

frameshift mutation in TMCO1 causes a syndrome with craniofacial dysmorphism,

skeletal anomalies, and mental retardation. Proc Natl Acad Sci USA 107: 258-263.

Xin HS, Schaefer DM, Liu QP, Axe DE, Meng QX (2010). Effects of polyurethane

coated urea supplement on in vitro ruminal fermentation, ammonia release dynamics

and lactating performance of Holstein dairy cows fed a steam-flaked corn-based

diet. Asian-Aust J Anim Sci 23: 491-500.

Xu L, Qiu X, Zhu Z, Yi L, Qiu Y (2014). A novel mutation in COL2A1 leading to

spondyloepiphyseal dysplasia congenita in a three-generation family. Eur Spine J

23(2): 271-277

Yang CC, Cotsarelis G (2010). Review of hair follicle dermal cells. J Dermatol Sci

57: 2-11

Yang H, Adamo ML, Koval AP, McGuinness MC, Ben-Hur H, Yang Y, LeRoith D,

Roberts Jr CT (1995). Alternative leader sequences in insulin-like growth factor I

mRNAs modulate translational efficiency and encode multiple signal peptides. Mol

Endocrinol 9(10): 1380-1395

Yasuda E, Fushimi K, Suzuki Y, Shimizu K, Takami T, Zustin J, Patel P, Ruhnke K,

Shimada T, Boyce B (2013). Pathogenesis of Morquio A syndrome: an autopsied case

reveals systemic storage disorder. Mol Genet Metab 109(3): 301-311

Yi JJ, Ehlers MD (2007). Emerging roles for ubiquitin and protein degradation in

neuronal function. Pharmacol Rev 59: 14-39.

Yi R, Fuchs E (2010). Micro-RNA-mediated control in the skin. Cell Death Differ 17:

References



Yokoyama Y, Narahara K, Tsuji K, Moriwake T, Kanzaki S, Murakami M, Namba H,

Ninomiya S, Higuchi J, Seino Y (1992). Growth hormone deficiency and empty sella

syndrome in a boy with dup (X)(q13. 3→ q21. 2). Am J Med Genet 42(5): 660-664

Yu TW, Chahrour MH , Coulter ME, Jiralerspong S, Okamura-Ikeda K, Ataman B

(2013). Using whole-exome sequencing to identify inherited causes of autism. Neuron

77: 259-73.

Zaias N (1963). Embryology of the human nail. Arch Dermatol 87: 37-53

Zelzer E, Olsen BR (2003). The genetic basis for skeletal diseases. Nature 423(6937):

343-348

Zhang MC, He L, Giro M, Yong SL, Tiller GE, Davidson JM (1999). Cutis laxa

arising from frameshift mutations in exon 30 of the elastin gene (ELN). J Biol Chem

274: 981-986

Zhang SD, Meng J, Zhao JJ, Tian W (2009). Mutation E402K of the hHb6 in a

Chinese Han family with monilethrix. Eur J Dermatol 19: 508-509

Zhang, J., Chiodini, R., Badr, A. and Zhang, G. (2011) The impact of next-generation

sequencing on genomics. J Genet Genomics. 38 (3): 95-109

Zhao H, Tian Y, Breedveld G, Huang S, Zou Y, Jue Y, Chai J, Li H, Li M, Oostra BA

(2002). Postaxial polydactyly type A/B (PAP-A/B) is linked to chromosome 19p13.

1-13.2 in a Chinese kindred. Eur J Hum Genet 10(3): 162-166

Zhou H, Clapham DE (2009). Mammalian MagT1 and TUSC3 are required for

cellular magnesium uptake and vertebrate embryonic development. Proc Natl Acad

Sci USA 106: 15750-15755.

Zhu J, Qin Y, Zhao M, Aelst LV, Malinow R (2002). Ras and Rap control AMPA

receptor trafficking during synaptic plasticity. Cell 110: 443-455.

References



Zhu, C.C., Dyer, M.A., Uchikawa, M.(2002) Six3-mediated auto repression and eye

development requires its interaction with members of the Groucho-related family of

co-repressors. Development. 129 (12): 2835-2849

Zimprich, A., Benet-Pagès, A., Struhal, W., Graf, E., Eck, S. H., Offman, M. N.,

Lichtner, P. (2011). A mutation in VPS35, encoding a subunit of the retromer

complex, causes late-onset Parkinson disease. The American Journal of Human

Genetics, 89(1), 168-175.

Ziyadeh J, Le Merrer M, Robert M, Arnaud E, Valayannopoulos V, Di Rocco F

(2013). Mucopolysaccharidosis type I and craniosynostosis. Acta Neurochir 155(10):

1973-1976

Zlotogorski A, Marek D, Horev L, Abu A, Ben-Amitai D, Gerad L, Ingber A,

Frydman M, Reznik-Wolf H, Vardy DA, Pras E (2006). An autosomal recessive form

of monilethrix is caused by mutations in DSG4: clinical overlap with localized

autosomal recessive hypotrichosis. J Invest Dermatol 126: 1292-1296

Zlotogorski A, Panteleyev AA, Aita VM, Christiano AM (2002). Clinical and

molecular diagnostic criteria of congenital atrichia with papular lesions. J Invest

Dermatol 118: 887-890

Appendices



CONSENT FORM

تحقیقی کام میں شمولیت کا اجازت نامہ

پاکستان کے صوبہ خیبر پختونخواہ کے انسانی عنوان: ابادی میں مخصوص طبی وجنیاتی بیماریوں کی تشخیص

کرنااور ان پر سائنسی تجزیہ

رابطہ نمبر: ،محمد اسماعیل خان : تحقیق کنندہ کا نام

اسلامیہ کالج پشاور 03348802406

اہد ، ڈیپارٹمنٹ آف زوالوجی زڈاکٹر محمد کا نام:نگران

اسلامیہ کالج پشاور

مشرف جیلانی ، اومکس سینٹر ڈاکٹر شریک نگران کا نام :

اسلامیہ کالج پشاور

۔۔۔۔۔۔۔۔نمبر۔۔۔۔۔۔۔ ۔۔قومیت۔۔۔۔۔۔۔۔۔رابطہ۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔ریض کا نامم

۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔۔پتہ۔۔۔۔۔۔

تعارف:

ہم آپ کو ایک تحقیقی پروگرام میں شامل ہونے کی دعوت دے رہے

ہیں۔ پروگرام میں شمولیت سے پہلے ضروری ہے کہ آپ اس سے

سمجھیں۔ اس فارم میں ہمارے ورھیں امتعلق ضروری معلومات پڑ

تحقیقی کام کا مقصد فوائد خطرات درج ہیں۔ اس پروگرام میں شمولیت

سے انکار م گر اوبلکل رضا کارانہ ہے۔ آپ کسی بھی وقت اس پر

سکتے ہیں۔ اور اس کے لئے کوئی جرمانہ یا سزا نہیں ہوگا۔

تحقیقی پروگرام کا مقصد:

وذہنی بیماریاں جیسے کہ کیرا ٹو ڈرما طبیمختلف قسم کے جنیاتی ،

لٹر سینڈ روم ، ٹرنر سینڈ روم ، لی سینڈ روم ، ڈاون سینڈ روم ، کلینف

اور ذہنی معذوری پر تحقیق شامل ہے۔ ، کوفن لوری سینڈ روم

تحقیقی کام میں شامل کرنیکی وجہ:

کے وںبیماریاور اپ کے خاندان میں مختلف جنیاتی چونکہ آپ

Appendices



اس لئے آپ کو اس تحقیقی مطالعہ میں شامل علامات پائے جاتے ہیں

کیا جاتا ہے۔

تحقیقی کام کے فوائد:

وں بیمارییں مختلف جنیاتی کہ ہم ہوں گے یہ فوائد کے اس تحقیقی کام

کے علاج میں استعمال کی تشخیص اور ان پر تجز یہ کے بعد اس

ملتی ہے جس کے ذریعے ان میں مدد کی دریافت ہونے والی ادویات

۔ اس بنائی جا سکتی ہےکی زندگی کو بہتر بیماریوں میں مبتلا لوگوں

راست فائدہ ہوسکتا ہ تحقیقی کام میں حصہ لینے سے آپ کو بھی برا

ہے اور تحقیق سے آئندہ نسلوں کو بھی فائدہ ہوگا اور یہ نقصان دہ بھی

نہیں ہے۔

رح خفیہ رکھی جائے گی:آپ سے متعلق معلومات کس ط

آپ کی فراہم کردہ معلومات محقق کی ذاتی فائل میں محفوظ رکھا

جائیگا۔ اس تحقیقی مطالعہ کیلئے جانے والی تمام نمونوں اور معلومات

کو ایک خاص کوڈ نمبر دیا جائے گا۔ نتائج کی معلومات صرف محقق

کے اور ڈاکٹر مشرف جیلانی اہدز، ڈاکٹر محمد محمد اسماعیل خان

۔ تمام معلومات کو تالا بند جگہ پر یعلاوہ کسی اور کو نہیں دی جائے گ

رکھا جائیگا۔ جن تک صرف محقق کی رسائی ہوگی اس تحقیقی کام سے

حاصل ہونے والی معلومات کو شائع بھی کیا جاسکتا ہے۔ لیکن اس سے

م کی معلومات آپ کی شناخت نہیں ہوگی۔ آپ سے متعلق کسی بھی قس

۔یجائے گدی آپ کی اجازت کے بغیر کسی کو بھی نہیں

تحقیقی اخراجات:

برداشت کرے گا۔ اس کے لئے آپ سے تحقیق کے تمام اخراجات محقق

اس تحقیق کام میں شمولیت پر آپ کو کوئی بھی خرچہ نہیں لیا جائے گا۔

امل ہونے کوئی معاوضہ نہیں دیا جائے گا۔ اگر آپ اس تحقیق کام میں ش

کے لئے رضا مند ہے تو اس کے لئے مندرجہ ذیل معلومات درکار ہیں۔

ذاتی معلومات مثلا نام، جنس، پتہ، قومیت اور خاندانی معلومات .1

ضروری ہیں جو کہ ٹیسٹ مختلف تحقیقی کام کے لئے خون کے .2

کرینگے۔ ہم خود اس کا خرچہ ادا

ہمیں اس تحقیقی مطالعے کے لئے آپ کے خون سے یہ ٹسٹ کرنے

نکال کر معائنہ کیا جائے گا۔ جس DNAہونگے۔ خون کے نمونے سے

Appendices



وں کے میں جین کے مختلف اقسام کی وجہ سے پشتون قوم کے بیماری

۔یجائے گ بارے میں معلومات حاصل کی

اس کے نمائندہ میں نے مندرجہ بالا تمام معلومات پڑھ لی ہیں۔ محقق یا

نے مجھے اس تحقیقی مطالعے کے بارے میں سمجھا دیا ہے۔ اور تمام

سوالات کے جوابات بھی دیئے ہیں۔ میں یہا ں اپنی اجازت دیتا ہوں کہ

میں اس تحقیقی پروگرام میں سبجکٹ کے طور پر شامل ہوں گا۔

دستخط / تاریخ امید وار / سبجکٹ

انگھوٹا

دستخط تاریخ کنندہ کا نامتحقیق

Appendices



CONSENT FORM

1. Study Title: Analysis and Diagnosis of Targeted Medical and Genetic

Disorders in Human Population of Khyber Pakhtunkhwa, Pakistan

2. Introduction

Our body is made of unique building blocks, called “cells”. Group of cells make an

organ for example heart, brain, kidney etc. Every cell has a control room type content

called DNA, which lies in the form of 23 paired structures or molecules called

chromosomes. We receive 23 chromosomes from father and 23 from mother, and thus

every human has 46 or 23 pairs of chromosome in each body cell.

DNA is the materials that has the information of our all developmental programs

including skin color, height, eyes color, hair etc. Thus if our DNA has normal

structure and function then our normal developmental processes are followed in cells.

In contrast, any abnormality in the DNA will lead to abnormalities in developmental

processes and thus some babies are born with intellectual disability, some with heart

malformation, some with may having walking, hearing, speaking or vision

abnormalities etc.

The total content of our DNA in our cells is called “genome”. About one to two per

cent of this genomic DNA is the part that codes for various proteins or enzymes and is

termed as “exome” which carries necessary units called “genes” for our normal

functions of cells in an organ. For example, heart cells have pumping, kidneys have

filtering, stomach has digestion, bone have support and muscles have contraction

properties. All the properties are retained by cells due to normal structure and function

of assigned genes, if altered will ultimately lead to altered functions.

In the current time, various populations throughout the world are trying to understand

structural and functional organization of their genomes and exomes and are trying to

predict the risk of their populations specific diseases.

3. Methodology

Appendices



In this study, we also propose to analyze at least 14 subjects from different ethnic

groups of Khyber Pakhtunkhwa. The steps will be involved in this project

a. A few milliliters of samples (blood or saliva) collection from each participant.

b. Genomic DNA extraction from the blood and saliva.

c. DNA sequencing

d. Data analysis, comparing DNA sequencing results with other populations, by

sharing with national and international collaborators.

e. Identifying or calculating potential risks of a genetic disease in a sample under

analysis.

f. Concluding and communicating these results with the study participants.

g. Making these results public without disclosing the privacy of the study

participants.

4. Privacy of your DNA samples

Every research laboratory has recommendations and protocols to assign a unique

entry number to a sample, so that it could be distinguished for its identity during

storage or analysis and cannot be recognized without permissions.

5. Benefits or advantages

All genomics studies have the advantages of having informations about our genetic

makeup. We could know whether a specific genetic disease can have a chance in our

family or not? Similarly, if some disease risk is their then the risk could be reduced

through marriage planning. If some diseases are treatable or manageable at early

stages, then physicians could make necessary measures and the subject could be

guided for proper medications. Our sequencing data could benefit to be used as a

reference for diagnostics purposes and may help others or their future generations.

6. Disadvantages

By knowing some specific disease genes or risks in our DNA we may feel upset. This

may become a stigma for our family or social life for example a social pressure for

keeping you and your family aside from marriages of your choices. In some specific

situations if the young girls are boys are once known for a potential carrier mutation

that could lead to homozygous pathogenic alteration in coming generation. That is

Appendices



why some young ladies or men may be refused for some proposed marriages. The

sequencing data may remain lifelong for comparison and diagnostic purposes of other

patients.

7. Signatures of the study participant(s)

I Mr/Mrs/Ms_______________________s/o, d/o, w/o

_____________________willingly participate in this study. My age is ____years,

belong to ___________________ tribe/ethnic group, and live in ____________

district of Khyber Pakhtunkhwa, Pakistan.

I have read all the details of this project and am clearly aware of the advantages and

disadvantages of this research study.

I am convinced to provide my blood/saliva sample for genomic DNA extraction and

sequencing.

I agree that the data generated in this project can be shared for academic

collaboration, whenever needed without disclosing my personal details or related

informations.

I further declare that my participation to this study is totally on my own will (I am not

forced, not influenced and not pressurized by any mean).

SIGNATURE________________ DATE: ________________

Appendices



ANTI-PLAGERISM REPORT

Date post:	14-Feb-2022
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

ANALYSIS AND DIAGNOSIS OF TARGETED MEDICAL AND …

Documents