d-scholarship.pitt.edud-scholarship.pitt.edu/18464/1/Anatalia_Labilloy_Master_… · Web...

LYSOSOMAL STORAGE DISORDERS: FROM BASIC SCIENCE TO PUBLIC HEALTH

by

Anatália Labilloy

MD, Universidade Federal do Piauí, Brazil, 2010

Submitted to the Graduate Faculty of

Department of Human Genetics

Graduate School of Public Health in partial fulfillment

of the requirements for the degree of

Master of Public Health

University of Pittsburgh

2013

University of Pittsburgh

Graduate School of Public Health

This essay is submitted

by

Anatália Labilloy

on

April 8th 2013

and approved by

Essay Advisor: ________________________David Finegold, MDProfessorDepartment of Human GeneticsGraduate School of Public HealthUniversity of Pittsburgh

Essay Reader: ________________________Karen Cuenco, PhDAssistant ProfessorDepartment of Oral BiologyCenter for Craniofacial Human GeneticsSchool of Dental MedicineUniversity of Pittsburgh

2

Copyright © by Anatália Labilloy

2013

3

David Finegold, MD

LYSOSOMAL STORAGE DISORDERS: FROM BASIC SCIENCE TO PUBLIC HEALTH

Anatália Labilloy, MPH

University of Pittsburgh, 2013

ABSTRACT

According to the first Principle of the Ethical Practice of Public Health by the

Public Health Leadership Society, “Humans have a right to the resources necessary for

health.” When dealing with rare inheritable conditions, oftentimes disease mechanisms

remain largely unknown. The uniqueness of rare conditions render healthcare

professionals unaware of their existence or of ways to deliver best medical care

adapted to individual patients’ needs. Lysosomal Storage Disorders constitute a group

of more than 50 rare heritable conditions that share some pathological and biochemical

features and result in significant morbidity and early death. Most of the

pathophysiological aspects of individual disorders of this group remain uncharacterized.

Advances in clinical development of therapies in the past two decades have improved

clinical outcomes for patients with some of these diseases, but progress still needs to

be made for better management of some of the complications of these disorders. The

cost associated with some of these therapeutic options still represents a limiting factor

to access, especially in developing countries. This study’s primary aim was to foster

basic science research in the field of lysosomal storage disorders by developing and

4

Karen T.Cuenco, 01/12/13,

Insert who came up with the set of principle’s here. APHA? I am not sure who it is, but you need the citation here.

characterizing a cell model for Fabry disease that can be used to understand disease

pathogenesis for drug development. An additional aim was to develop a strategic plan

for a public health genetics program with focus on Lysosomal Storage Disorders in an

underprivileged state in a developing country. This study’s public health significance lies

in the continuation of efforts to understand disease mechanisms, which potentially allow

development of new therapeutic approaches through basic and clinical science

research. In addition, efforts to increase awareness of these disorders among

healthcare professionals, and to integrate research findings into clinical practice,

fostering the delivery of evidence-based medical care in the field are of public health

relevance and necessary to improve health of patients with Lysosomal Storage

Disorders.

5

TABLE OF CONTENTS

ACKNOWLEDGEMENTS…………………………………………….………………………XII

1. DEVELOPMENT OF A CELL MODEL OF FABRY DISEASE IN RENAL TUBULAR

EPITHELIAL CELLS……………………………………………………………….……………1

1.1 INTRODUCTION AND BACKGROUND………….………..………………………….1

1.1.1 FABRY DISEASE DIAGNOSIS AND TREATMENT…………………………..9

1.1.2 MODELS OF FABRY DISEASE…………………….………………………....12

1.1.3 PUBLIC HEALTH RELEVANCE……………….………………………………14

1.2 METHODS……………………………………….……………………………………..16

1.3 RESULTS……………………………………………………………………………….18

1.4 DISCUSSION……………………………….…………………………………..………21

2. A PUBLIC HEALTH GENETICS PLAN FOR PIAUÍ STATE WITH FOCUS ON

LYSOSOMAL STORAGE DISORDERS…………………………………………………….23

2.1INTRODUCTION AND BACKGROUND……………………………………………...23

2.1.1 HEALTHCARE IN BRAZIL……………..….…………………………………...23

2.1.2 PIAUÍ STATE DEMOGRAPHICS…….………………………………………..24

2.1.3 PIAUÍ STATE HEALTH DEPARTMENT……………..……………………….26

2.1.4 GENETICS IN BRAZIL……………….………………..……………………….28

2.1.4.1 NEWBORN SCREENING IN BRAZIL………………………………29

2.1.4.2 CURRENT GENETICS SERVICES IN PIAUÍ STATE………….…32

2.1.4.3 LYSOSOMAL STORAGE DISORDERS IN BRAZIL AND PIAUÍ

STATE….........................................................................................................................33

2.2 NEEDS ASSESSMENT AND DEVELOPMENT OF A PUBLIC HEALTH

GENETICS STATE PLAN IN LYSOSOMAL STORAGE DISORDERS………...……….34

2.2.1 MISSION………………....…………………………………………………..…...34

6

2.2.2 ASSUMPTIONS……………………………………………….…………………35

2.2.3 NEEDS ASSESSMENT……………………………………………………………..35

2.2.4 GOALS, OBJECTIVES AND ACTIVITIES…..…………………………………….37

2.2.4.1 GOAL 1…………………………………………………………………….......37

2.2.4.2 GOAL 2…………………………………………………………………………39

2.2.4.3 GOAL 3…………………………………………………………………………40

2.2.5 PROGRAM DEVELOPMENT STRATEGY………………………………………..40

2.2.6 EVALUATION PLAN…………………………………………………..…………….41

2.2.7 EXPECTED OUTCOMES AND IMPACT………………………………………….44

2.2.8 CHALLENGES……………………………………………………………………….45

3. FINAL CONSIDERATIONS AND FUTURE DIRECTIONS……..……………………..45

APPENDIX A: LYSOSOMAL STORAGE DISORDERS……………………………….….48

APPENDIX B: GOVERNMENTAL AND NON-GOVERNMENTAL ORGANIZATIONS

PROVIDING CARE FOR INDIVIDUALS WITH DISABILITIES/SPECIAL NEEDS IN

PIAUÍ STATE, BRAZIL………………………………………………………………………..50

BIBLIOGRAPHY…………………………………………………………………………….....52

7

LIST OF ABBREVIATIONS

α-Gal A Alpha-galactosidase A

CNS Central Nervous System

eGFR estimated Glomerular Filtration Rate

ERT Enzyme replacement therapy

Gb3 Globlotriaosylceramide

GSL Glycosphingolipid

HIV human immunodeficiency virus

IFN Interferon

LSD Lysosomal Storage Disorder

M6P Mannose-6-phosphate

MDCK Madin-Darby canine kidney

MHC major histocompatibility complex

MRI Magnetic Resonance Imaging

mRNA messenger ribonucleic acid

siRNA small interfering ribonucleic acid

SRT Substrate Reduction Therapy

SUS Brazilian Unified Health System

TIA Transient Ischemic attack

8

NPHPSP National Public Health Performance Standards Program

9

LIST OF TABLES

Table 1: Lysosomal Storage Disorders with available treatment ………………………….6

Table 2: The Essential Public Health Services………..……………………………………14

10

LIST OF FIGURES

Figure 1: Fabry disease progresses with age, resulting in multisystem involvement and

early death.................................................................................………..………………….2

Figure 2: Disorders of GSL and ganglioside metabolism and their relationship...........…5

Figure 3: Efficient silencing of α-Gal A expression in MDCK cells by siRNA transfection

......................................................................................................................................18

Figure 4: α-Gal A silenced MDCK cells have increased levels of Gb3...........................19

Figure 5: Electron micrographs of MDCK cells treated with control and α-Gal A siRNA.

....................................................................................................................................... 21

Figure 6: Accumulation of "zebra bodies in α-Gal A siRNA treated MDCK cells.............21

Figure 7: Strategy for the Program Development phase of a Public Health Genetics Plan

for Piauí State with Focus on LSDs................................................................................40

11

ACKNOWLEDGEMENTS

Another cycle has ended for others to begin. The accomplishments that I have

achieved during my academic studies leading to a Master in Public Health in Public

Health Genetics at the University of Pittsburgh were unparalleled. The difficulties and

barriers existed, but with determination, hard work and full support from the professors

of the Department of Human Genetics and from my wonderful classmates the path was

smooth and the professional and personal achievements were very fruitful.

I would like to express my sincere gratitude to my thesis advisor Dr David

Finegold for guiding and supporting me over the progress of my graduate studies, and

for being an example of passionate scientist and caring doctor. I would also like to thank

Karen Cuenco for accepting to be part of my thesis committee and her helpful advices,

Dr Robert Ferrell for inspiring conversations and my academic advisor Dr Candance

Kammerer for the patience and invaluable guidance.

I am especially thankful to Dr Ora Weisz for introducing me to key concepts in

basic science research in cell biology, a field that was new to me. She has encouraged

me and served as a role model of a successful scientist, leader and woman. Jennifer

Bruns was also essential to this work for her friendship and technical support with the

experiments in developing the renal cell model. I couldn’t have worked in a better

environment than the one I found at Dr Weisz’s lab.

12

Foremost I would like to thank my family for always being there for me, my

husband Guillaume for his love, patience and understanding and my parents for having

always invested in my education and for showing full support even being so far away.

You are the foundation for my academic success.

Lastly, I would also like to acknowledge the financial support from Shire HGT as

a grant to the Graduate School of Public Health for a scholarship in the field of

Lysosomal Storage Disorders.

13

1. DEVELOPMENT OF A CELL MODEL OF FABRY DISEASE IN RENAL TUBULAR

EPITHELIAL CELLS

1.1 Introduction and Background

Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by

abnormal function of the lysosomal hydrolase α-galactosidase A (α-Gal A), encoded by

the gene GLA. [1] Deficiency of α-Gal A leads to progressive accumulation of its

substrates, neutral glycosphingolipids (GSLs) with terminal α-D-galactosyl residues,

mainly globotriaosylceramide (Gb3), in a variety of cell types. [2] Fabry disease occurs

in both males and females, and classic clinical presentation of the disease includes

renal, cardiovascular, cerebrovascular, neurological, gastroenterological,

dermatological, ocular, and auditory findings, among others, resulting in significant

morbidity and early death. [3, 4] Figure 1 shows a diagram with some of the clinical

findings of Fabry disease and its disease progression with age.

The first signs and symptoms of Fabry disease often appear in the first decade of

life, mostly presenting as neuropathic pain, gastrointestinal symptoms, and impaired

quality of life. Some pediatric patients may also present with serious complications of

Fabry disease and multi-system organ involvement. Data from Fabry registry have show

a mean age of onset of symptoms of 6 years for boys and 9 years for girls. [5-7]

14

Anatalia Ribeiro, 03/17/13,

I was missing the in between double space, fixed!


Double check formatting for citations. Is this supposed to superscripted for UPitt format?

Figure 1: Fabry disease progresses with age, resulting in multisystem involvement and early death. Progression in any organ system can proceed independently from the others. MRI: magnetic resonance imaging; TIA: transient ischemic attack. Adapted from [8]

Some patients may never display the classic phenotype, presenting a milder form

of the disease. In fact, “cardiac variants” and “renal variants” of Fabry disease have

been described, where patients present disease limited to only one or few organs. [9]

The kidney is one of the most affected organs in individuals with Fabry disease.

While advanced stages of renal chronic disease usually present in middle adulthood,

the first histological signs of Fabry nephropathy have been seen as early as in utero. [5]

Additionally, children with Fabry disease can present early signs of kidney involvement

such as hyperfiltration, microalbuminuria, and overt proteinuria [10, 11]. Within the

Progression with age

Considerable morbidity and mortality

TIA and strokes

MRI white matter lesions, vertebrobasilar dolichoectasia, pulvinar sign

Systolic and diastolic

dysfunction, myocardial infarction

hypertrophic cardiomyopathy,

short PR interval,

arrhythmias

End-Stage renal Disease

microalbuminuria, proteinuria hyperfiltration, isosthenuria

neuropathic pain, exercise intolerance, gastrointestinal symptoms, cornea verticillata

and angiokeratomas

glycosphingolipid accumulation

alpha-galactosidase A deficiency

15

kidney, podocytes seem to be the most involved cell type, showing a greater degree of

vacuolization. However, Gb3 inclusions are also observed in proximal and distal tubules

as well as mesangial, endothelial, and vascular smooth muscle cells. As Fabry

nephropathy progresses, mesangial expansion, interstitial fibrosis, tubular atrophy, and

glomerulosclerosis are observed. [12].

Fabry disease is the second most common disease of its group of LSDs, behind

only Gaucher disease, an autosomal recessive form of LSD characterized in its most

common form mainly by spleen, liver, lungs, bone and bone marrow involvement. [13]

Currently, more than 50 disorders have been clinically categorized as LSDs, resulting

from deficient activity of specific lysosomal enzymes or abnormalities in transmembrane

proteins as well as in proteins involved in lysosomal biogenesis. [14] Appendix A

presents a comprehensive list of LSDs. Although individually rare, the estimated

combined prevalence of this group of conditions ranges from 1:4,000 to 1:8,000 births.

[15] In spite of being considered panethnic, Individual estimates of incidence vary from

study population and some disorders of this group are thought to be underdiagnosed;

some disorders of this group such as Gaucher disease, mucolipidosis type IV, Tay-

Sachs disease and Niemann-Pick disease have increased incidence in Ashkenazi

Jews, attributed to historical founder effects and genetic drift.[16]

In spite of their genetic heterogeneity, LSDs share the phenotype of

accumulation of one or several substrates within lysosomes and in non-lysosomal

compartments, as well as some clinical and biochemical features [17, 18]. Figure 2

shows the biochemical link among several LSDs. As lysosomal sphingolipid degradation

occurs in a step-wise manner, the product of degradation of one enzyme serves as the

16

substrate for the subsequent reaction. In addition, two or more pathways can be

interconnected, presenting the same final product. As an example, lactosylceramide

acts as a product of both alpha-galactosidase A degradation of Gb3 and of sialidase

degradation of GM3. Moreover, the same sphingolipid activator protein (SAP), which

are proteins required for hydrolysis of sphingolipids, can participate in different steps of

GSL metabolism. [19]

Specific treatment is available for several LSDs, and early diagnosis and

management translates into better outcomes for patients. Treatment options either

clinically available or under research development for disorders of this group include

enzyme replacement therapy (ERT), substrate reduction therapy (SRT), hematopoietic

stem cell therapy (HSCT), chemical chaperones, cell therapy and gene therapy. [27]

Table 1 shows a list of treatable LSDs with their respective estimated incidence and

treatment options available or under development.

17


Add a sentence commenting on the comparative diseases and their incidence. Which is most common? Any trends?

Figure 2: Disorders of GSL and ganglioside metabolism and their relationship. Abbreviations: DHCer (dihydroceramide), S1P (sphingosine 1-phosphate), SM (sphingomyelin) . [19]

Several studies have estimated the prevalence of Fabry disease to range

between 1:40,000 and 1:117,000 live births. [20] With recent advances in high-

throughput multiplex enzyme assays using tandem mass spectrometry, allowing

feasibility for larger scale screening tests, some countries have started pilot newborn

screening programs for some LSDs, including Fabry disease. [21, 22] Surprisingly,

18

newborn screening data suggests a much higher incidence of Fabry disease than

initially thought, ranging from 1:3,859 newborns in Austria [23] and 1:3,100 in Italy [24]

to as high as 1:557 births in Taiwan [25]. This suggests that the disease might be

greatly under-diagnosed worldwide. In the United States, pilot newborn screening

studies for Fabry disease and other LSDs are currently being performed. If expanded to

the whole country, more than 1,000 babies are expected to be diagnosed with the

disease each year in the United States, based upon estimates from European countries

[23-24]. However, newborn screening for LSDs remains controversial, since specific

treatment is unavailable for some conditions of the group. In addition, the onset of

symptoms for some LSDs may not occur until adulthood, in those circumstances

neonatal diagnosis may not interfere with the clinical course of the disease. [26]

Table 1: Lysosomal Storage Disorders with available treatment. Disease Treatment available

Aspartylglucosaminuria HSCT available

Fabry disease ERT available, Chaperones in CD

Fucosidosis HSCT available

Gaucher disease ERT, SRT and HSCT available

Krabbe disease HSCT available

α-Mannosidosis HSCT available, ERT in CD

Metachromatic leukodystrophy

IT-ERT in CD, gene therapy in CD

MPS I ERT and HSCT available

MPS II ERT available; IT-ERT in CD

MPS III (all types) IT- ( in CD or MPS IIIA; SRT in CD; GT in CD

19


Add a list indicating what the abbreviations mean.

Table 1 continued

MPS IV A ERT in CD

MPS VI ERT available, HSCT available

Mucolipidosis II/III HSCT in CD

Neuronal ceroid lipofuscinosis

Cell therapy in CD, gene therapy in CD

Niemann-Pick B ERT in CD

Niemann-Pick C SRT available, HSCT in CD for NPC2

Pompe disease ERT available

Sandhoff disease Chaperones in CD for late-onset forms

Tay-Sachs disease Chaperones in CD for late-onset forms

Adapted from ref. [27]

The lysosomal hydrolase α-Gal A enzyme is encoded by the gene GLA, located

in Xq22. The GLA gene is comprised of seven exons and 12,436 base pairs. [1] In the

lysosome lumen, α-Gal A is found as a homodimer, each monomer presenting a N-

terminal, which contains the active site, and a C-terminal. [28] According to the Human

Gene Mutation Database, over 600 mutations have been described for Fabry disease.

(http://www.hgmd.org). The majority of mutations in GLA gene leading to Fabry disease

are missense and translate into a disruption of the hydrophobic core of α-Gal A, which

makes Fabry disease primarily a disease of protein-folding. [29]

Gb3, also known as CD77 and Pk, is the primary glycosphingolipid degraded by

α-Gal A. Biosynthesis of Gb3 from lactosylceramide is performed by the type II

membrane protein alpha-1,4-galactosyltransferase (A4GALT) in the Golgi apparatus.

[30] After its synthesis, Gb3 is then incorporated into the plasma membrane and

intracellular membranes, with a preference for sphingolipid and cholesterol enriched

20


You can put this in your list of references and put the number for it here.

https://sremote.pitt.edu/,DanaInfo=www.hgmd.org+

microdomains (aka, lipid rafts). This glycosphingolipid can also shed from the plasma

membrane, being found in the plasma associated with lipoproteins, preferentially LDL.

[31] Gb3’s deacylated form, globotriaosyl-sphingosine, aka lyso-Gb3, is also elevated in

Fabry disease. Lyso-Gb3 is degraded by α-Gal A at a rate 50 times lower than Gb3, and

presents inhibitory properties over both native and recombinant α-Gal A. [32] Thus,

lyso-Gb3 might have additional direct contribution to the pathogenesis of the disease

and in organ-response to therapy.

Gb3 has been implicated in a variety of cellular processes. As an example, Gb3

is the receptor for the subunit B of Shiga and Shiga-like toxins (Stx) produced by

bacteria as Escherichia coli and Citrobacter freundii. [33, 34] Gb3 is also one of the

several glycolipids that act as co-receptor for gp120 in HIV entry, being required in

CD4/CXCR4-dependent fusion. [35, 36] In fact, this interaction is being further explored

in the development of new therapies for HIV, as the Gb3 analog adamantylGb3 inhibits

HIV infection in vitro. [37]

As a component of sphingolipid- cholesterol enriched microdomains, Gb3 is

thought to play a role in signal transduction, with major involvement in inflammation and

immune responses. Gb3 is involved in CD19-mediated adhesion and is required for

both interferon-alpha induced growth inhibition and for the plasma membrane

localization of Hsp70 [38, 39]. In addition, MHC class II and type 1 IFN receptor

molecules present binding domains for Gb3, corroborating with its role in immune

response [40, 41]. Moreover, Gb3, along with other globo series glycosphingolipids, can

be a mediator of the cSrc kinase, beta-catenin, and caspase signaling pathways [42].

21

Increase in levels of Gb3 is not a specific finding of Fabry disease. Although to a

lesser extent than in Fabry disease, Gb3 levels are elevated in Niemann-Pick C,

another LSD, which is consistent with the fact that these conditions share some of their

pathophysiology. [43] Greater Gb3 levels can also be seen in more common disorders.

Cells in pre-cancerous and cancerous states, such as colorectal adenoma, Burkitt’s

lymphoma, breast cancer, and testicular carcinoma may also present increased levels

of Gb3. [44] Interestingly, this increase in Gb3 levels is considered a sign of bad

prognosis in these conditions, being associated with multidrug resistance and greater

invasiveness. [45] However, both the underlying mechanism for such an increase in

Gb3 levels and the cellular consequences culminating in greater tumor aggressiveness

remain unclear. Some cytokines such as TNF-alpha and Interleukin-1 are shown to up-

regulate Gb3 levels [46, 47] nevertheless the pathophysiological impact of such

increases also needs to be better understood.

1.1.1 Fabry disease Diagnosis and Treatment

Due to the presence of several signs and symptoms that mimic common

disorders, the absence of pathognomonic clinical features, and marked phenotypical

heterogeneity, clinical diagnosis of Fabry disease is usually delayed and involve visits to

several medical specialists and misdiagnoses. [48] After onset of clinical manifestation,

overall diagnostic delays are about 15 years for both genders [49], with possiblly even

longer delays in developing countries. Some of the misdiagnoses include rheumatoid

arthritis, rheumatic fever, systemic lupus erythematous, Raynaud’s disease, celiac

disease, and multiple sclerosis, among others. [9] After clinical suspicion is raised,

demonstration of deficient α-Gal A in leukocytes in males is sufficient to establish a

22

diagnosis of Fabry disease in males, and may be followed by mutational analysis of

GLA gene. An initial screening through plasma enzyme activity in dried blood spots can

be performed in males, but should be confirmed by leukocyte enzyme

activity/genotyping. For females, disease status should be determined by mutation

analysis of GLA since α-Gal A enzyme activity may be within the normal range in

females even in the presence of overt disease. [50]

In the recent years, there have been increased screening efforts for diagnosis of

Fabry disease in high-risk adult populations. These efforts included screening for Fabry

disease among patients with end-stage renal disease [51], unexplained hypertrophic

cardiomyopathy [52, 53] and young patients presenting with stroke but no apparent

predisposing factor [54]. Most of the studies focused in male patients due to the low

sensitivity of the enzyme activity analysis in females, who usually require genotyping, a

more expensive and laborious method, for definite diagnosis. These studies have

shown prevalence of Fabry disease in high-risk populations ranging from 0 to 12

percent.

Although the presence of residual α-Gal A activity is associated with slower

progression of Fabry nephropathy, α-Gal A in plasma or leukocyte enzyme activity

levels do not always correlate with disease phenotype. Similarly, conservative amino

acid changes in GLA gene may be associated with slower progression of the disease,

but genotype-phenotype correlations have not been fully established for Fabry disease.

Phenotype heterogeneity may play a role even within a single family of individuals

sharing the same genotype. [55]

23

Currently, the only commercially available treatment option for patients with

Fabry disease consists of recombinant enzyme replacement therapy (ERT)

administered intravenously. There are two approved forms of recombinant enzyme:

agalsidase alfa and agalsidase beta. Agalsidase alfa is engineered in a human

fibroblast cell line and presents amino acid sequence identical to that of native human

α-Gal A, being glycosylated with both sialic acid and mannose-6-phosphate (M6P)

residues. Its administration occurs intravenously at a dose of 0.2 mg/Kg every other

week. [56] Agalsidase beta is produced in Chinese hamster ovary cells, also presenting

the same amino acid sequence as the native human α-Gal A. The approved dose for its

intravenous administration is 1 mg/Kg every other week. [57] The pattern of

glycosylation as well as M6P receptor binding and M6P receptor mediated endocytosis

slightly differs between the two recombinant enzymes. Agalsidase beta presents a

higher number of fully sialylated oligossacharides and a higher level of phosphorylation

in comparison to agalsidase alfa. However, these discrepancies do not seem to

translate in functionally relevant differences. [58, 59]

Long-term ERT significantly improves overall clinical status of individuals with the

disease, mainly by reducing neuropathic pain, decreasing cardiomyopathy, and

improving quality of life. [60-62] However, both ERT regimens are only able to slightly

retard the progression of chronic kidney disease, and a steady decline of eGFR is still

observed despite long-term treatment. [63, 64] Moreover, as the intravenously

administered enzyme does not cross the blood brain barrier, disease progression in the

CNS resulting in white matter lesions and strokes remains unchanged in spite of ERT.

[65] A better comprehension of the pathophysiology of Fabry disease could provide the

24

means to develop better clinical approaches and novel pharmacological targets in the

management of Fabry disease patients.

Other treatment options are currently under investigation. Results of a phase II

clinical trial for the pharmacological chaperone Migalastat HCl were recently published,

showing that its oral administration in patients with responsive mutations increased

blood α-Gal A activity by 50 percent. [66] The iminosugar binds and stabilizes the

nascent enzyme in the ER, restoring its efficient trafficking to lysosomes in the cases

where proper trafficking of the misfolded enzyme is the main contributing factor for

enzyme deficiency. [67] A phase III clinical trial for migalastat is currently ongoing.

A plant cell-expressed recombinant α-Gal A, PRX-102, is also under a phase I/II

clinical trial study for treatment of Fabry disease. The recombinant enzyme has

undergone chemical modifications to improve enzyme activity and stability and to

decrease its immunogenicity. [68]

1.1.2 Models of Fabry Disease

Although naturally occurring animal models have been reported for other LSDs,

no animals have been identified with Fabry disease. A knockout mouse model for Fabry

disease with lack of α-Gal A activity was developed and described by Oshima et al. [69]

However, use of this knockout mice model in translational research has been

controversial; while the mice present some histological changes compatible with Fabry

disease, both male and female mice appear to be clinically normal,[69] or present mild

phenotype as they age [70, 71], not reflecting what occurs in humans with classic

phenotype.

25


Repeat that it is the GLA gene (or specific mutation) being knocked out here.

Establishment of cell models have also been an useful as a tool for testing

hypotheses of disease pathogenesis, for identifying biomarkers of disease progression

and clinical prognosis, and for early-stages drug development and testing for several

human diseases.

One of the technologies being used to study protein function in the context of a

living cell and to generate knockdown of proteins of interest for human disease is RNA

interference (RNAi). Small interfering RNA (siRNA) molecules serve as a template to

the multi-protein complex RISC (RNA induced silencing complex) for recognition of

complementary mRNA of interest, which results in activation of RNAse, culminating in

cleavage and degradation of the mRNA and consequent loss of protein translation. [72]

siRNA mediated transient silencing of α-Gal A could mimic the loss of protein activity

observed in Fabry disease in different cell types. This technology would be of particular

significance for kidney cells, as the pathophysiology of Fabry nephropathy is poorly

understood and the kidneys of Fabry patients have little response to therapy, making

kidney cells a good target for the studies on disease pathogenesis and drug discovery.

Madin-Darby canine kidney (MDCK) cells, isolated from dog kidney cortex and

easily cultivated as polarized monolayers in vitro, have been studied and characterized

extensively as a model for the study of renal cell function. [73].

In this study, we have designed, developed and characterized a renal epithelial

tubular cell model for the study of the effects of deficiency of α-Gal A.

1.1.3 Public Health Relevance

26


I think you mean that so little is understood that we need to study it. Is it really a good drug target? Can you argue that it would be easier to implement, to study, etc? The next sentence suggest that the animal model is there. Is that the main plus to looking at the kidney?


This is a big concept that you are proposing. You may need some additional explanation of this here, or perhaps later…maybe it is explained later in the text?

The National Public Health Performance Standards Program (NPHPSP) through

the Core Public Health Functions Steering Committee has established in 1994 the “Ten

Essential Public Health Services”, which constitute the essential framework for the core

public health activities. Error: Reference source not found shows a list of the Essential

Public Health Services. The 10th essential public health service, “research for new

insights and innovative solutions to health problems” can be interpreted as the

responsibility of public health systems to investigate human diseases and their

mechanisms, search for elucidation of their pathophysiology as well as promote means

of developing new therapies.

Table 2: The Essential Public Health Services

1. Monitor health status to identify and solve community health problems

2. Diagnose and investigate health problems and health hazards in the community

3. Inform, educate, and empower people about health issues

4. Mobilize community partnerships and action to identify and solve health problems

5. Develop policies and plans that support individuals and community health efforts

6. Enforce laws and regulations that protect health and ensure safety

7. Link people to needed personal health services and assure the provision of health care when otherwise available

8. Assure competent public and personal health care workforce

Progress in basic science research involving the development of cell models for

human diseases are an essential first step for understanding the cellular dysfunctions

underlying disease mechanisms, for identifying prognostic factors and biomarkers of

disease progression, and for evaluating therapeutic approaches to cure diseases.

Table 2 continued

27

1. Evaluate effectiveness, accessibility, and quality of personal and population-based health services

2. Research for new insights and innovative solutions to health problems


Mention this group in your abstract

These models are especially crucial in cases where mouse models do not replicate

human disease phenotypes, such as in Fabry disease.

Moreover, a better understanding of the pathophysiology of this relatively rare

condition can potentially help unravel pathogenic mechanisms underlying some

common diseases. In fact, Fabry nephropathy resembles diabetic nephropathy in

humans. [74] Moreover, a link between glycosphingolipid metabolism and development

and progression of diabetic nephropathy and metabolic syndrome has been suggested.

[75] Furthermore, as previously mentioned, Gb3 is also elevated in certain cancers. This

increase is associated with metastasis and tumor aggressiveness and a decreased

response to chemotherapy drugs. In addition, some LSDs within the group of

sphingolipidosis may also play a role in complex disorders. For instance, mutations in

GBA, the gene encoding beta-glucocerebrosidase and causing Gaucher’s disease,

have been found to be the most common risk factor ever described for Parkinson’s

disease. [76] As this disease also presents elevated levels of sphingolipids, elucidation

of disease mechanisms for Fabry disease may lead to new avenues of investigation of

the link between Parkinson disease and the LSD Gaucher disease.

1.2 Methods

Cell culture. Madin-Darby canine kidney (MDCK) cells were incubated on 10 cm plastic

dishes in MEM with 10% fetal bovine serum (FBS) at 37oC with 5% CO2. Nonpolarized

cells were cultured on 12 mm permeable supports with 0.4 m pores for four to six days

prior to the experiments.

28


Somewhere right before 1.2 Methods, you need a clear statement of purpose of experiments. The previous sections describe the disease and that you need a model. But, what exactly is the purpose of the experiments?

siRNA knockdown. We designed a small interfering RNA (siRNA) for transient

knockdown of the canine α-Gal A targeting the following sequence 5’-

GATAGATCTGCTGAAATT-3’. siRNA against firefly luciferase targeting the sequence

5’-GAATATTGTTGCACGATTT-3’ was used as a control. The custom siRNA duplex

and control siRNA constructs were purchased from Dharmacon. MDCK cells were

transfected with either α-Gal A or control siRNA using Lipofectamine™ 2000

transfection reagent (Invitrogen, Inc) and Opti-MEM® I Reduced Serum Media

(Invitrogen, Inc). Briefly, 2.6 μg of either α-Gal A or control siRNA were incubated with

10 μL of lipofectamine and 250 μL of Opti-MEM for 30 minutes at room temperature.

125 μL of the transfection mix and 5 x 105 subconfluent MDCK cells suspended in 333

μL of MEM were added to upper chamber of a 12-well transwell filter and briefly

homogenized. Growth media was replaced within 6 to 8 hours of transfection and every

other day subsequently. Experiments were performed three to six days later.

RT-PCR. Isolation of total RNA was performed using kit RNAquous (Ambion) according

to manufacture’s recommendations. Purified total RNA underwent reverse transcription

by incubation with Moloney murine leukemia virus reverse transcriptase (Ambion) at

42°C for 1 hour. PCR for canine alpha-galactosidase was carried out using Phusion ®

High-Fidelity PCR kit (New England BioLabs, Inc.), having as sense primer 5’-

TGTGCAACGTTGACTGCCAAGAAG-3’ and the anti-sense primer 5 ’-

TCCTGCAGGTTTACCATAGCCACA-3’. As a control, RT-PCR for canine beta-actin

was also carried out using the sense primer 5’-CTGCTGGAAGGTGGACAG-3’ and the

anti-sense primer 5’-ACCTTCAACTCCATCATGAAG-3’. Denaturing temperature was

29


I don’t know much about this. Was the media replaced to reach a certain volume/concentration? Is this replacement done once?

95oC, annealing temperature was 58oC, and extension temperature of 68oC, with 25

cycles of amplification.

Indirect Immunofluorescence and confocal microscopy. Immunofluores-cence was

performed according to the protocol described by Mattila et al. [77] Anti-CD77

monoclonal antibody (clone 38-13, Abcam), which recognizes Gb3, was used at a

dilution of 1:10. Goat anti-rat secondary antibodies (Alexa 488 and Cy5) were used at a

dilution of 1:500. Capture of images was performed on an Olympus Fluoview FV1000

laser-scanning confocal microscope in the Renal-Electrolyte Division, University of

Pittsburgh School of Medicine.

Electron microscopy. Membranes containing monolayers of α-Gal A knockdown and

control MDCK cells were fixed in 2.5% of glutaraldehyde, 2% of paraformaldehyde in

PBS and post-fixed in 1% Osmium tetroxide. Samples were then dehydrated in graded

series of ethanol solutions and embedded in EPON according to standard procedures.

Ultra-thin sections were stained with uranyl acetate and lead citrate and examined using

a JEOL electron microscope, in collaboration with Dr Willi Halfter, University of

Pittsburgh.

1.3 Results

Silencing of α-Gal A in MDCK cells

In order to confirm knockdown of the protein of interest at the mRNA level, RT-

PCR for α-Gal A was performed. Canine beta-actin was used as a PCR control.

30

Significant reduction of α-Gal A mRNA was observed in the α-Gal A silenced MDCK

cells compared to control, without significant differences in beta-actin levels (Figure 3).

Figure 3: Efficient silencing of α-Gal A expression in MDCK cells by siRNA transfection. MDCK cells were transfected with siRNA targeting canine α-Gal A or firefly luciferase (control) and processed for RT-PCR after three days to determine the extent of GLA mRNA reduction.

Accumulation of Gb3 in MDCK cells silenced for α-Gal A

In order to evaluate the effect of α-Gal A silencing in MDCK cells on metabolism of neutral D-α-galactosyl GSL, levels of Gb3 were investigated using specific monoclonal antibody against CD77, its membrane-bound form, followed by indirect immunofluorescence. Intensity and pattern of Gb3 staining were compared between cells transfected with either α-Gal A or non-silencing siRNA using fluorescence microscopy. A drastic increase in Gb3 staining intensity, in a punctate pattern, was observed in α-Gal A silenced cells after three days of transfection compared to non-silenced MDCK cells (

).

31


Move to methods section.


Move to methods section


This goes to the methods section. If you want to keep these comments here, summarize all of the sentences on methods (that are currently in the results section) into a short paragraph that will go into the methods section. Think of it as a methods overview of what you do with all of the components described in the methods section.


The heading for this section and these sentences should go into your methods section.

Figure 4 α-Gal A silenced MDCK cells have increased levels of Gb3.. MDCK cells were treated with control (left panel) or α-Gal A (right panel) siRNA for six days, then processed for indirect immunofluorescence with anti-CD77 (Gb3) antibody (red). Immunofluorescence microscopy images were acquired and processed using identical settings.

Ultrastructural changes induced by α-Gal A silencing

Light and electron microscopy of biopsied tissues of patients with Fabry disease

usually show cell-type specific ultra-structural changes that are characteristic of LSDs.

In cell types with greater degree of lipid deposition, electron microscopy shows several

degrees of intracellular vacuolization and the presence of membranous concentric or

parallel shapes of packing of lipid aggregates in the lysosomes, also called “zebra

bodies”, are especially found. [78, 79] To further investigate ultrastructural changes

induced by α-Gal A transient silencing, electron microscopy was performed after six

days of transient α-Gal A knockdown. We observed a greater number of round electron-

dense structures in α-Gal A silenced MDCK cells, corresponding to lysosomes (Figure

32


Move to methods section. You can rephrase this and keep it in the results section, but the details should be in Methods.

5). Furthermore, We have visualized in the α-Gal A silenced cells some lamellar myelin-

like structures, corresponding to “zebra bodies” ().

Figure 5: Electron micrographs of MDCK cells treated with control and α-Gal A siRNA. MDCK cells were transfected with control (left panel) or α-Gal A siRNA (right panel) and plated on permeable support for 3 days before processing for transmission electron microscopy. Increased osmiophilic bodies (arrows) are seen in the GLA-siRNA treated MDCK cells. 6,000x magnification.

Figure 6: Accumulation of "zebra bodies in α-Gal A siRNA treated MDCK cells. Electron micrograph of a lysosome containing transversely-stacked, osmiophilic myelin-like membranes, also called a “zebra body”, in MDCK cells transiently silenced with α-Gal A siRNA. 60,000x magnification.

33

1.4 Discussion

In this study, we have developed an in vitro model of Fabry disease in renal

tubular epithelial cells using RNAi technology. The α-Gal A silenced cells present a

dramatic increase in membrane-bound Gb3 levels, as well as ultra-structural changes

compatible with Fabry disease, such as increased number of electron-dense structures

corresponding to lysosomes and the characteristic presence of “zebra bodies”. Due to

its effectiveness in showing disease phenotype in a short period of time, as well as its

reproducibility, this model might be of great utility for testing hypotheses on disease

pathogenesis and in high-throughput initial screening in drug development not only for

Fabry disease, but also for common disorders in which overexpression or decreased

metabolism of the GSL may also be found, such as certain types of cancer.

Post-transcriptional gene silencing using RNAi technologies has proven to be

effective and easily reproducible. In the past few years, it has been widely used for

developing disease models such as well as for targeted therapy for a variety of

disorders. [80] However, some limitations are foreseen. Although presenting less

potential biological risks than the use of lentiviral vectors for stable expression, silencing

for a particular gene using siRNA molecules remains effective only for up to few days

after the administration of their transfection in cell culture systems due to successive

cell divisions and the action of cellular nucleases. [80] Therefore, for maintenance of

disease status and study of effects of chronic exposure to substrates, successive

transfections might be necessary.

34

The extensive use of MDCK cells in research has made them a well-

characterized cell line in the study of intracellular events and renal cell function.

However, a foreseen limitation in the use of MDCKs in translational research, as it is

with other species, is that although a considerable homology between the human and

canine genome exists, there might be some traits that are species-specific and cannot

be extrapolated to humans. [81]

Although presenting limitations, cell models continue to represent essential tools for

understanding the fundamental bases of disease, for identifying prognostic factors and

biomarkers of disease progression, and for the initial steps in the development and

evaluation of therapeutic approaches to treat human disease. These models are

especially crucial in cases where knockout mice do not present the same phenotype as

humans, presenting challenges for translational research.

2. A PUBLIC HEALTH GENETICS PLAN FOR PIAUI STATE WITH FOCUS ON

LYSOSOMAL STORAGE DISORDERS

2.1 Introduction and Background

2.1.1 Healthcare in Brazil

Brazil is the fifth most populous country in the world, and the sixth biggest world

economy by nominal GDP, according to the United Nations. According to the World

Bank, Brazil’s gross national income per capita is 11,420 USD, (compared to 48,820

USD in the United States) which categorizes Brazil as an upper middle-income country

by the World Bank. In spite of the recent economic growth, the country’s development

35

indicators still classify it as a developing economy according to the International

Monetary Fund. [82, 83]

Provision of public health care in Brazil is considered a right to all citizens and a

responsibility and obligation of the State, being provided in public institutions free of

charge. The national health policy had its principles and directives set by the

Constitution of 1988, which established the Brazilian Unified Health System (in

Portuguese, SUS). Although private hospitals and private health insurances are

available in the country, according to the National Health Agency, 74.9 percent of the

Brazilian population rely only on SUS to receive medical care. [84, 85]

Some of the difficulties faced by the population in regard to public healthcare

services in Brazil include: lack of access to services in remote areas due to scarce

number of healthcare professionals, delays in obtaining medical appointments with

specialists, lower number of beds than the demand in tertiary care hospitals, especially

at Intensive Care Units and significant overcrowding of Emergency Care Units. [86]

In 1998, the Brazilian National Health Council approved a National Drug Policy

Act that aims to promote access to and rational use of medicines that are considered

essential. The National Drug Policy Act sets which drugs classify as essential, as well

as the standards for policies and regulation in drug discovery, clinical trials and drug

approval in the country. The act assigns individual states the responsibility of promoting

access to medications that are considered exceptional, which are medicines that are the

standard of care for a condition but constitute an economic burden to individual patients.

[87]

36

2.1.2 Piauí State Demographics

Piauí State is composed of 224 counties and covers an area of 251,577.738

square kilometers (equivalent to 97,134.7 square miles) with a density of 32.1 persons

per square mile. According to the Brazilian Institute of Statistics and Geography (IBGE)

demographic census, the 2010 population for Piauí State was 3,118,360. The economy

is primarily based on the teritary sector (services), industry (chemical and textile),

agriculture (soybean, cotton, rice, sugar cane, and cassava) and free-range livestock

farming. Teresina is the capital of the state and the most populous city. Other major

cities include Parnaíba, Picos, Piripiri and Floriano.[88]

Piauí state has the lowest gross state domestic product in Brazil. Data from 2009

show that the per capita gross state domestic product for Piaui was 6,051.10 Brazilian

reais (BRL), which currently corresponds to 2,964.63 USD1. Data from 2003 by IBGE

have shown that 53.11 percent of the population in Piaui state lives below poverty level

taking into account the micro-level estimation of poverty and inequality by Elbers,

Lanjouw and Lanjouw [89]. In 2010, median household income for Piauí state was 900

BRL per month, corresponding to a yearly income of 5,296.2 USD per household1. In

rural areas of the state, the median monthly household income is 518 BRL (3,045.24

USD/year1). In 79.41 percent of the households in Piauí state, per capita income was

less than minimal wage for Brazil, which was 510 BRL per month (2,987.52 USD/year)

for the country in 2010. [88]

The majority of the population (69 percent) is multi-ethnic, mainly due to

miscegenation between individuals with European lineage and Amerindians (originating

1 Convertion based on exchange rate data obtained on 01/02/2013, 1 BRL = 0.49 USD. Provided by Citibank N/A.

37

“caboclos”). Caucasians represent 24 percent of the population. Black individuals

represent only seven percent of the population, mainly due to the economic activity

during the colonization period, which did not favor African slavery.[88]

According to IBGE in 2009 there were 2,093 healthcare units in Piaui state, 75.5

percent of which had public healthcare coverage through SUS. There are 3,125

registered physicians in the State, representing a physician density rate of ten

physicians for each 10,000 people. 83.1 percent of the households rely on the state for

healthcare, and only 12.6 percent of state population has some sort of private health

insurance. [88]

2.1.3 Piauí State Health Department

According to the Brazilian National Health Council, the following actions constitute

duties of Brazilian State Health Departments, according to the Basic Operational Norms

of the Brazilian Unified Health System (published in the Official Gazette on 11/06/1996)

[90]:

- Planning and implementation of a State Health Plan containing strategies,

priorities and goals of actions and services, including the integration of plans of

the several county health departments;

- Structuring and operation of the state component of the National Audit Office;

- Structuring and operation of epidemiological data processing systems for delivery

of services and for critical inputs;

- Structuring and operation of epidemiological surveillance systems, including also

health surveillance and monitoring food and nutrition;

- Structuring and operation of human resources and of science and technology;

38

- Elaboration of the state component of nationwide public health programs for

health threats that have a risk of spreading beyond its territorial boundary;

- Elaboration of the state component of the network of public health laboratories;

- Structuring and operation of the state component of pharmaceutical care;

- State responsibility regarding the provision of outpatient and hospital services of

high cost, treatment away from home and the availability of exceptional

medicines and supplies;

- Setting and operating policies regarding the use of blood and blood components,

- Hiring and support of qualified permanent technical staff, able to fully accomplish

the duties of the state health department;

- Implementation of mechanisms to integrate the policies and actions of relevance

to the health of the population; examples of which are those related to sanitation,

water, housing and environmental health.

The Piauí State Health Department stated mission is to coordinate and implement

the operations of the National Health System in the State in order to ensure increased

and timely access to healthcare services and appropriate and humanized care in the

context of health promotion, prevention and recovery, as well as to ensure quality

management of local health departments, aiming to improve health indicators and

quality of life of the state population.[91]

The Piauí State Health Department stated values include [91]:

- Assurance of universality, thoroughness, equity and accessibility to healthcare

services and actions, with continuity, responsibility, humanization and

commitment;

39

- Exercise of participatory management, fostering transversality of health policies,

programs, actions and services and strengthening the social Participation.

2.1.4 Genetics in Brazil

Genetic diseases and birth defects are currently the second most common cause

of infant mortality in Brazil. As health indicators consistent with developing countries

have significantly improved in the last few years, the country has observed a drastic

decrease in infant morbidity and mortality related to poor sanitary conditions,

malnutrition and infectious diseases. Therefore, genetic diseases arose as a significant

public health issue in the country. [86]

Genetics as a medical specialty is still considerably young in the country. The

first Medical Genetics residency was established in 1977 at Hospital das Clínicas de

Ribeirão Preto, University of São Paulo. In 1983, the Brazilian Federal Council of

Medicine recognized Medical Genetics as a medical specialty. Currently, there are 11

Medical Genetics residency programs in Brazil, offering a total of 21 new positions per

year. There are no genetic counseling programs in the country, and medical geneticists

perform this role. However, there are only 121 medical geneticists in Brazil, 85 percent

of which are concentrated in the South and South East regions. [86] Dr Raskin,

president of the Brazilian Society of Medical Genetics, estimates that the waiting time

for a first time appointment in medical genetics in the country can be as long as one

year. [92]

In October of 2004 the Brazilian Ministry of Health established a workgroup in

Clinical Genetics, which was responsible to plan and implement a National Public

40

Health Policy in Clinical Genetics and to incorporate genetics within the Brazilian public

health system, which became effective in 2009. [93, 94]

2.1.4.1 Newborn Screening in Brazil

On June 6th 2001, the Brazilian Ministry of Health implemented the National

Newborn Screening Program, through the act GM/MS n. 822. The specific aims of the

program included [95]:

- Detection of metabolic conditions in a pre-symptomatic stage;

- Coverage of 100 percent of live newborns;

- Active search of screened newborns in the case of a positive initial result;

- Diagnostic confirmation, as well as follow-up and appropriate early intervention

measures of all identified newborns, in order to minimize their morbidity and

mortality.

In order to select which conditions should be screened for, the Brazilian Ministry

of Health considered the need to select conditions that could be screened and managed

taking into account the health system diversity throughout the country. Conditions that

could be diagnosed early in their course, through screening tests with high sensitivity

and confirmatory tests with high specificity should be priority. Additionally conditions

should have an economically feasible and cost-effective available treatment that

positively interferes with the disease natural history. [96]

The Ministry of Health also established that the program should be carried out

and articulated in a partnership with the State Health Departments. Consequently, at

least one Newborn Screening Reference Center was implemented in each of the 27

41

states in Brazil. These centers are responsible for coordinating the local networks of

newborn screening present in each state and are composed of a multi-disciplinary team

for medical care, composed of physicians, dietitians, psychologists, and social workers.

As different states presented different healthcare system organizations, the Ministry of

Health felt the need to implement the national program in phases. Consequently, the

following phases of operation were established [96]:

● Phase I: screening, confirmatory tests, follow-up and management of PKU and

congenital hypothyroidism;

● Phase II: screening, confirmatory tests, follow-up and management of PKU,

congenital hypothyroidism, sickle cell disease and other hemoglobinopathies.

● Phase III: screening, confirmatory tests, follow-up and management of PKU,

congenital hypothyroidism, sickle cell disease and other hemoglobinopathies and

cystic fibrosis.

Private laboratories also provide newborn screening, however, these are not

integrated into a public health program, thus lacking the coordinated structure for follow-

up and management of newborns with positive screening tests. There are three different

groups of screening tests: basic, plus and master. The basic screening comprises the

conditions covered by the phase III of the National Newborn Screening Program, the

plus performs screening for ten conditions while the master performs screening of 46

conditions. Health insurance reimbursement policies are not uniform. The cost of the

screening for phase I is approximately 7.50 US dollars per sample, while for phases II

and III cost 12 USD and 20 USD per sample, respectively. For confirmatory exams, the

42

cost can vary from 15 to 40 USD per sample. [96] The cost to the parents for the master

screening in case of non-coverage by private health insurance is about 120 USD.

In 2005, through the act 6197/05, the screening for biotinidase deficiency was

also introduced in the public health system. [97] However, the screening for this

condition, although established by law, has not yet been introduced in most of the states

in Brazil.

The Newborn screening program in Brazil has still suboptimal achievements.

Only three out of 27 states have achieved phase III program, which allows screening for

only four conditions. Newborn screening coverage varies from 52.41 to 100 percent,

with an average of 78.92 percent for the year of 2007. In the same year, for 3,035,096

live-births and approximately 2,400,000 screened newborns, 108 cases of PKU were

detected, 1,231 cases of congenital hypothyroidism, 1,140 cases of

hemoglobinopathies and 58 cases of cystic fibrosis, totalizing 2,537 cases. Data from

2007 also showed that 16,408 children and adults have been followed so far after a

diagnosis was received through the newborn screening program, but those values can

be underestimate since they represent only results from the governmental funded

healthcare and no integrative effort exist with private newborn screening. [98]

2.1.4.2 Current Genetics Services in Piauí State

According to the Brazilian Society of Medical Genetics, currently there no

medical geneticists or clinical genetics laboratories are presented in Piaui State. [99]

The genetic-related resources available in Piauí State are the following:

43

- Hospital Infantil Lucidio Portela (Lucidio Portela Children’s Hospital), located in

Teresina, serves as the Newborn Screening Reference Center for the State and

is overseen by the State Office for Healthcare for Individuals with Disabilities

(CAPD).

- Central Laboratory of Piauí State (LACEN) located in Teresina, is the only

laboratory responsible for running the laboratory tests for the State Newborn

screening program, and is overseen by the State Office for Healthcare for

Individuals with Disabilities (CAPD).

A comprehensive list of not-for-profit organizations in the state that provide care

and services to people with mental and physical disabilities, including those with

hereditary causes, can be seen in Appendix B.

2.1.4.3 Lysosomal Storage Disorders in Brazil and Piauí State

The exact prevalence of LSDs in Brazil and Piauí State are not known due to the

lack of an integrated epidemiological surveillance service for genetic diseases in the

country. Gaucher disease, the most prevalent LSD, affects more than 600 individuals in

Brazil according to the Brazilian Gaucher Disease Patient Association. [100]

Several individual states, through resolution by the Brazilian Ministry of Health,

have included imiglucerase, one of the treatment options of ERT for Gaucher disease,

in the list of treatments covered by the Brazilian Unified Health System, which ensures

treatment for this condition without any additional cost to the patients and their families.

Some individual states, such as Sao Paulo State, have set up guidelines for indications

of therapy and follow-up evaluations and tests for patients with the disease. In case of

no appropriate follow-up insignificant clinical response to the medication, the patient

44

undergoes additional evaluation prior to continue with therapy, in an attempt to optimize

the treatment since the cost is considerable for Brazilian’s public health system. [101,

102]

At present, treatments for other LSDs have not been included in the list of

covered “exceptional drugs”. Besides Gaucher disease, specific treatment through

either ERT or substrate reduction therapy (SRT) is available for Fabry disease,

Mucopolysaccharidosis type I, Mucopolysaccharidosis type II, Mucopolysaccharidosis

type IV, Pompe Disease and Niemann-Pick disease type C in developed countries.

[103] Some of the diagnosed patients have been seeking access to these treatments

through legal measures.

According to the Fabry Registry 2007 Annual Report, age of diagnosis for Fabry

patients in Brazil averages 53 years old in males and 70 years old in females, which

represents an extremely high frequency of late diagnosis of the disease in the country.

[104]

Screenings for Fabry disease were performed in high-risk populations in Brazil in

some individual states, with special focus on individuals with end-stage renal disease of

undetermined cause. [105, 106] In Piauí State, screening among 350 male

hemodialysis patients resulted in diagnosis of Fabry disease in two individuals. The age

of the patients was 57 and 74 years, and the first signs and symptoms of Fabry disease

started decades prior to the diagnosis. One of the individuals had a classic form of

Fabry disease and died due to a massive myocardial infarction several days after

receiving the diagnosis of Fabry disease. A diagnosis of Fabry disease was not

suspected in any of the individuals [107] Further family screening in one of the

45

individuals resulted in diagnosis of Fabry disease in additional 61 individuals, the

majority of them living in a remote village in the South of Piaui State with scarce access

to medical care [unpublished data from the author].

2.2 Needs Assessment and Development of a Public Health Genetics State Plan in

Lysosomal Storage Disorders

2.2.1 Mission

Our mission is to improve health and quality of life of individuals with LSDs living

in Piaui State, an underprivileged state in Brazil, by developing the needed

infrastructure for proper clinical management and prompt diagnosis of patients with

LSD, delivering state-of-the-art medical care, and by educating physicians and the

public in genetics and LSDs.

2.2.2 Assumptions

- A significant delay of diagnosis and marked underdiagnosis of LSDs occur in

Piaui State, resulting in preventable morbidity and mortality;

- This delay in diagnosis occurs in part due to:

o Lack of awareness of LSDs among healthcare professionals and of

proper training of primary care physicians and specialists in recognizing

disease phenotypes that could be compatible with LSDs.

o Lack of genetics laboratory and clinic for diagnosis and management of

patients with LSDs

o

2.2.3 Needs Assessment

46

Some of the following facts were observed by analyzing the current healthcare

situation in Piauí State and in Brazil, with focus on the provision of genetics services,

especially the ones with direct or indirect impact in delivery of medical care for patients

with LSDs:

- Lack of coordinated national genetics infrastructure

- Lack of genetics infrastructure in the State,

- Although some genetics-related activities take place in Piauí state, there is no

centralized state genetics department or program.

- Genetics expertise among healthcare providers is insufficient to meet patients’

needs.

- Lack of effective public policies in genetics that take place uniformly across the

country, including appropriate coverage by public or private health insurances for

diagnostic and follow-up tests, medical appointments and genetic counseling, as

well as for treatment of genetic disorders, when indicated.

A thorough needs assessment will be performed through focus group interviews

involving healthcare professionals, social workers, patients with LSD and their family

members. Additionally, surveys disseminated to physicians of different specialties will

be conducted prior to the implementation of the plan and after its establishment, and

these resources will be used to guide program planning and implementation.

2.2.4 Goals, Objectives and Activities:

47

2.2.4.1 Goal 1 - Build the public health infrastructure needed to diagnose and provide

appropriate clinical care to individuals with LSDs in Piauí State, Brazil

- Objective 1.1: Engage key stakeholders

o Some of the foreseen stakeholders for the development of a LSD program

in Piauí State include: patients with LSDs and their families, healthcare

professionals, hospitals, clinical laboratories, schools of Medicine, Piauí

State Medical Council, Piauí State Health Department, Brazilian Ministry

of Health, health insurance companies and SUS (Unified Healthcare

System).

- Objective 1.2: Build State Capacity and Self-Efficacy

o Workforce: recruit and train staff and volunteers

o Seek for short and long-term funding for program staff and activities

o Ensure quality and sustainability of LSD genetics services

Consult with LSD experts to set standards of care

Consult with policy makers

o Establish and coordinate partnerships

Partner with existing public clinical laboratory in establishing the

required equipment and supplies for running diagnostic and follow-

up tests for the different LSDs.

Partner with reference university hospital for creating and

establishing a LSD medical genetics clinic and a program office

48

o Reimbursement and coverage – Seek for implementation of the legislation

that regulates the coverage of genetics services by both public and private

healthcare systems.

o Promote universal access for those in need

Establish regular outreach clinic in key cities of the state

Facilitate scheduling of medical appointments through the use of a

1-800 hotline and online appointments

- Objective 1.3: Surveillance

o Obtain data from the laboratories that perform clinical diagnosis of

LSDs in Brazil to estimate data on the current prevalence of the

disease in Piauí State.

o Annually assess incidence data for LSDs in Piauí State by number

of individuals diagnosed with LSD in Piauí State both through and

outside the program, as well as monitor health outcomes for

individuals being followed through the program.

o Maintain accessible epidemiological data in the program website

2.2.4.2 Goal 2: Increase awareness and foster knowledge in the field of LSD among

healthcare professionals and priority populations in Piaui State, Brazil

- Objective 2.1: Educate physicians

49


Given the earlier objectives will impact surveillance, how long will it take to get accurate prevalence and incidence estimates?


Any issue of access to phones & internet in this region?

o Develop and maintain a program website, where we will maintain up-to-

date information on recommendations and guidelines in diagnosis, follow-

up and treatment of patients with LSD.

o Deliver talks in medical conferences and meetings

- Objective 2.2: Educate medical and allied health sciences students

o Deliver talks as part of the current academic curriculum of the schools

o Discuss with course directors the need of the introduction of a Medical

Genetics course or of improvement of the syllabus in the curriculum of

Schools of Medicine.

- Objective 2.3: Educate laboratory personnel in technical performance of LSD

diagnostic and follow-up tests

o Provide workshops to laboratory personnel in biochemical and molecular

approaches for the diagnosis of LSDs

- Objective 2.4: Educate patients and family members

o Provide culturally appropriate learning material in the disease

o Provide genetic counseling

o

2.2.4.3 Goal 3: Reduce mortality and morbidity from LSDs in Piauí State

- Objective 3.1: improve access to genetics services

o Establish the first genetics clinics with focus on LSD in Piauí State

- Objective 3.2: promote early and appropriate diagnosis of LSDs

50

o Establish a Biochemical and Molecular Genetics Laboratory with focus on

LSDs

- Objective 3.3: promote use of up-to-date evidence-based medical practice in the

management of LSDs.

2.2.5 Program Development Strategy

Figure 7 represents a diagram with a summary of the planned strategy for the

development phase of the LSD program in Piauí State.

Figure 7. Strategy for the Program Development phase of a Public Health Genetics Plan for Piauí

State with Focus on LSDs. Adapted from [108]

2.2.6 Evaluation plan

We will perform both process and outcome evaluations throughout the

establishment of the program, in the following issues: relevance/needs assessment of

LSDs in the State, design and delivery of the program and success of the program and

Formation of working group

Determination of Program

Strategy

Stakeholders and Needs

Assessment

Program logic and evaluation

plan

Medical Talks on LSD

Establishment of Genetics laboratory

Establishment of Genetics

clinic

Seek funding

51

its activities. Some of the evaluation questions to be addressed, as well as the

indicators and data sources that will be utilized in the evaluation plan are described

bellow.

Evaluation issues:

1. Relevance/Needs assessment

a. What is the magnitude of LSDs in Piaui State?

i. We will investigate along with the only two clinical laboratories that

perform diagnostic tests in LSD in Brazil the actual number of

individuals diagnosed with LSDs in the past ten years.

b. Is the current prevalence along with what is expected based in worldwide

prevalence data?

i. Based on reported data of incidence for the different LSDs, we will

compare obtained prevalence with expected prevalence for LSDs

based on reports from other populations.

c. What is the existing laboratory structure for running LSDs diagnosis and

follow-up tests in Piaui State?

i. In consultancy with experienced Biochemical and Molecular

Genetics Laboratory directors, we will determine the current

structure and what are the needs in terms of equipment, supplies

and specialized training for running diagnostic and follow-up tests in

LACEN, the reference laboratory for newborn screening in Piauí

State.

52

d. What is the current level of knowledge in LSDs among physicians and

students in the health sciences?

i. Prior to the delivery of medical conferences for healthcare

professionals and students in the health sciences, we will

administer pre-tests with general questions in genetics, diagnosis

and treatment of lysosomal storage disorders in order to obtain a

baseline on the general knowledge among these professionals in

regard to proper diagnosis and management of LSDs.

ii. We will also administer pre-tests along with the surveys to

physicians from different specialties during the needs assessment

process.

2. Design and delivery

a. Has the program changed awareness of relevance of LSDs?

i. Immediately after the delivery of medical conferences for

healthcare professionals and students in the health sciences, we

will administer post-tests to evaluate the acquired knowledge in the

field of LSD through our talks. If agreed, we will also contact the

professionals by email one year after the participation in our talks

and re-administer the post-test to these professionals in order to

determine a longer-term assimilated knowledge.

b. Does the program follow updated recommendations and guidelines for

diagnosis, follow-up and treatment of LSDs?

53


Is there a general timeline between pre-test, education, and final post-test?


What will be in the pre-test?

i. We will have external consultancy from LSD experts, which will

evaluate the recommendations and guidelines found in our website,

and will also observe and evaluate the activities of the clinic during

one week a year to ensure delivery of updated medical care.

c. Is the program integrated to healthcare systems and adequate to reality

and community needs?

i. We will evaluate the incorporation of the genetic clinic into the

health system of the state based on the number of referrals from

different medical specialties and also evaluate the number and

quality of the outreach clinic services.

d. Which improvements could be made to the program to better serve the

community and healthcare professionals?

i. We will perform periodic surveys among program staff, patients and

family members and healthcare professionals and gather

information on what could be improved in the delivery of care in

LSD.

3. Success

a. Were the expected outcomes achieved?

i. We will annually assess and publically report:

1. Attendance rate to education sessions performed throughout

the program

2. Pre- and post-test results applied before and after each

education session

54

3. Number of screening and diagnosis tests run in the

biochemical and molecular genetics laboratory

4. Number of referral visits to the Medical Genetics LSD clinic

5. Number of diagnosed individuals with LSDs

6. Health outcomes for individuals being followed through the

program, with elaboration of periodic reports

2.2.7 Expected Outcomes and Impact

Short-Term Outcomes:

- Infra-structure for a Medical Genetics clinic and a Biochemical and Molecular

Genetics Laboratory with focus on LSD

- Increased awareness in the relevance of the field of LSDs among the medical

community

- Increased knowledge in current recommendations on the diagnosis and

management of patients with LSDs among the medical community

Medium-Term Outcomes:

- Increased screening and diagnosis of patients with LSDs

- Early diagnosis and intervention of patients with LSDs

- Proper management of the comorbidities frequently found in patients with LSDs

- Proper genetic counseling of patients with LSD and their family members at risk

- Patient and family education on the disease

Long-Term Outcomes:

55

- Decreased morbidity and mortality and increased quality of life of patients with

manageable LSDs in Piauí State

2.2.8 Challenges

Some of the foreseen challenges in the development and execution of a Public

Health Genetics Plan with Focus on LSDs include:

- Scarcity of public funding for program development and implementation in

genetics in Brazil

o We will also seek funding through public-private partnerships and apply for

international grants, always optimizing the use of already existing

resources in the state in order to reduce costs.

- Scarcity of well-trained professionals in the field of LSD and genetics in general

to compose the program staff

o Healthcare professionals recruited for the program will be trained in a

reference center in LSD prior to the start of the activities

- Some of the remote areas of the State have difficult access (i.e. crumbling roads

and highways)

The outreach clinics will be planned ahead, allowing extra time for

possible intercurrences.

3.0 Final Considerations and Future Directions

In this study, we developed and fully characterized a cell model for Fabry

disease, a Lysosomal Storage Disorder, in renal tubular epithelial cells. Further studies

that could be performed in these cells include proteomics and cell signaling arrays to

56

investigate possible qualitative and quantitative changes in protein expression and

signal transduction in renal tubular cells silenced for α-Gal A. We could also study

intracellular events and function of specific organelles in the lieu of Fabry disease. As

the methods become fully optimized and expertise becomes available, other cell models

of interest could also be developed, especially from the main organs of involvement of

the disease, such as cardiomyocytes for the study of cardiomyopathy and glial cells for

the study of the CNS involvement. The effect of novel forms or SRTs or ERTs or

manipulation of new pathways could also be studied in a high-throughput manner.

Additionally, here we design the framework for a Public Health Genetics Plan for

Piauí State, the lowest income state in Brazil, a developing country. We have discussed

relevant background information, and important policy issues. We purpose a

comprehensive program aiming to increase awareness and knowledge in the field,

promptly diagnose and promote health and quality of life for individuals with LSD in

Piauí State, including components of needs assessment and a robust evaluation plan. A

Public Health Genetics Plan for Piauí State could serve as a pilot for implementation of

similar programs in developing countries.

57

Appendix A – Lysosomal Storage disorders by subgroup

Protein DiseaseDEFECTS IN GLYCAN DEGRADATIONDefects in glycoprotein degradationα-Sialidase SialidosisGalactosialidosis Cathepsin A

α-Mannosidase α-Mannosidosisβ-Mannosidase β-Mannosidosis

Glycosylasparaginase Aspartylglucosaminuriaα-Fucosidase Fucosidosis

α-N-Acetylglucosaminidase SchindlerDefects in glycolipid degradationA. GM1 Gangliosideβ-Galactosidase GM1 gangliosidosis / MPS IVB

β-Hexosaminidase α-subunit GM2-gangliosidosis (Tay-Sachs)β-Hexosaminidase β-subunit GM2-gangliosidosis (Sandhoff)

GM2 activator protein GM2 gangliosidosisGlucocerebrosidase Gaucher disease

Saposin C Gaucher diseaseB. Defects in the degradation of sulfatideArylsulfatase A Metachromatic leukodystrophy

Saposin B Metachromatic leukodystrophy

58

Formyl-Glycin generating enzyme Multiple sulfatase deficiency

β-Galactosylceramidase (Krabbe) Globoid cell leukodystrophyC. Defects in degradation of globotriaosylceramideα-Galactosidase A Fabry

Defects in degradation of Glycosaminoglycan (Mucopolysaccharidoses)A. Degradation of heparan sulphateIduronate sulfatase MPS II (Hunter)α-Iduronidase MPS 1 (Hurler, Scheie)

Heparan N-sulfatase MPS IIIa (Sanfilippo A)Acetyl-CoA transferase MPS IIIc (Sanfilippo C)

N-acetyl glucosaminidase MPS IIIb (Sanfilippo B)β-glucuronidase MPS VII (Sly)

N-acetyl glucosamine 6-sulfatase MPS IIId (Sanfilippo D)Degradation of other mucopolysaccharidesN-Acetylgalactosamine 4-sulfatase MPS VI

Galactose 6-sulfatase MPS IVA (Morquio A)Hyaluronidase MPS IX

Defects in degradation of glycogenα-Glucosidase Pompe

DEFECTS IN LIPID DEGRADATIONDefects in degradation of sphingomyelinAcid sphingomyelinase Niemann Pick type A and BAcid ceramidase Farber lipogranulomatosis

Defects in degradation of triglycerides and cholesteryls esterAcid lipase Wolman and cholesteryl ester storage

disease

DEFECTS IN PROTEIN DEGRADATIONCathepsin K Pycnodystostosis

Tripeptidyl peptidase Ceroide lipofuscinosis 2Palmitoyl-protein thioesterase Ceroide lipofuscinosis 1

DEFECTS IN LYSOSOMAL TRANSPORTERSCystinosin (cystin transport) Cystinosis

59

Sialin (sialic acid transport) Salla disease

DEFECTS IN LYSOSOMAL TRAFFICKING PROTEINSUDP-N-acetylglucosaminePhosphotransferase γ-subunit Mucolipidosis III (I-cell)

Mucolipin-1(cation channel) Mucolipidosis IVLAMP-2 Danon

NPC1 Niemann Pick type CCLN3 Ceroid lipofuscinosis

CLN 6 Ceroid lipofuscinosis 6CLN 8 Ceroid lipofuscinosis 8

LYST Chediak-HigashiMYOV Griscelli Type 1

RAB27A Griscelli Type 2Melanophilin Griscelli Type 3

AP3 β-subunit Hermansky Pudliak 2Adapted from ref [109]

Appendix B – Governmental and non-governmental organizations providing care for

individuals with disabilities/special needs in Piauí state, Brazil.*

Special need Resource CityAutism Associação de amigos dos autistas do Piauí TeresinaBlindness Associação dos cegos do Piauí Teresina

CAP - Centro de apoio pegagógico às pessoas portadoras de deficiência visual

Teresina

ASCEB - Núcleo Associação dos Cegos de Água Branca

Água Branca

Physical disability

Associação dos deficientes físicos de Teresina Teresina

60

Sociedade de Apoio ao Deficiente Físico TeresinaAssociação dos Cadeirantes do Município de Teresina

Teresina

SADEFINP - Sociedade de Apoio ao Deficiente Físico do Norte do Piauí

Barras

Intellectual disability

CIES - Centro integrado de educação especial Teresina

Fundação Viver com digndade - Lar Renascer - Pastoral da Esperança

Teresina

APAE - Centro de Recuperação e Profissionalização Integrado Cristina Leite

Teresina

CHAC - Centro de Habilitação Ana Cordeiro TeresinaInstituto Panda - Núcleo de Apoio à Pessoa com Deficiência/Paralisia Cerebral

Teresina

Associação Pestalozzi "Casa Odylo Costa Filho" TeresinaEscola de Educação Especial Prof Cordão Campo

MaiorDeafness APADA - Associação de pais e amigos dos

deficientes auditivosTeresina

ASTE - Associação dos surdos de Teresina TeresinaCAS - Centro de Apoio Pedagógico ao Surdo TeresinaAPAE - Escola de Educação Especial Prof Consuelo Pinheiro

Teresina

Others AMH - Associação de mielomeningocele e/ou hidrocefalia de Teresina

Teresina

AOSEPI - Associação dos ostomizados do Estado do Piauí

Teresina

FCD - Fraternidade Cristã de Doentes e Deficientes TeresinaPastoral da Amizade TeresinaCEPI - Centro de Profissionalização Integrado TeresinaUEESPI - Unidade de Educação Especial TeresinaPNES - Programa TEC NEP - Educação, tecnologia e profissionalização para pessoas com necessidades especiais

Teresina

SINE - Serviço Nacional de Empregos TeresinaCONEDE-PI Conselho Estadual de Defesa dos Direitos da Pessoa com Deficiência

Teresina

CONEDE-TE Conselho Municipal de Defesa dos Direitos da Pessoa Com Deficiência

Teresina

FUNDECAP - Fundação dos Deficientes de Capitão de Campos

Capitão de Campos

CIEE- Centro de Integração Empresa Escola TeresinaNúcleo de Reabilitação Profissional TeresinaSEINT - Ministério do Trabalho e Emprego - Seção de Inspeção do Trabalho - Grupo Pro Igualdade

Teresina

Instituto Professor Magalhães TeresinaIEDE - Instituto Educacional de Desenvolvimento Teresina

61

IntegradoEscola Viva TeresinaEscola Castelinho TeresinaColégio Base Dez Teresina

*Adapted from: http://www.seid.pi.gov.br/entidades.php

Bibliography

1. Bishop, D.F., et al., Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme. Proc Natl Acad Sci U S A, 1986. 83(13): p. 4859-63.

2. Desnick, R.J., Y.A. Ioannou, and C.M. Eng, eds. alpha-galactosidase A deficiency: Fabry disease. 8th ed. The Metabolic and Molecular Bases of Inherited Disease, ed. C.R. Scriver, et al.2001, McGraw-Hill: New York. 3733-3774.

3. Eng, C.M., et al., Fabry disease: baseline medical characteristics of a cohort of 1765 males and females in the Fabry Registry. Journal of inherited metabolic disease, 2007.

62

http://www.seid.pi.gov.br/entidades.php

30(2): p. 184-92.

4. Waldek, S., et al., Life expectancy and cause of death in males and females with Fabry disease: findings from the Fabry Registry. Genetics in medicine : official journal of the American College of Medical Genetics, 2009. 11(11): p. 790-6.

5. Hopkin, R.J., et al., Characterization of Fabry disease in 352 pediatric patients in the Fabry Registry. Pediatr Res, 2008. 64(5): p. 550-5.

6. Ramaswami, U., et al., Fabry disease in children and response to enzyme replacement therapy: results from the Fabry Outcome Survey. Clin Genet, 2011.

7. Tondel, C., et al., Prominence of glomerular and vascular changes in renal biopsies in children and adolescents with Fabry disease and microalbuminuria. Clin Ther, 2008. 30 Suppl B: p. S42.

8. Zarate, Y.A. and R.J. Hopkin, Fabry's disease. Lancet, 2008. 372(9647): p. 1427-35.

9. Germain, D.P., Fabry disease. Orphanet J Rare Dis, 2010. 5: p. 30.

10. Hulkova, H., et al., [Postmortem diagnosis of Fabry disease in a female heterozygote leading to the detection of undiagnosed manifest disease in the family]. Cas Lek Cesk, 1999. 138(21): p. 660-4.

11. Ramaswami, U., et al., Assessment of renal pathology and dysfunction in children with Fabry disease. Clin J Am Soc Nephrol, 2010. 5(2): p. 365-70.

12. Fogo, A.B., et al., Scoring system for renal pathology in Fabry disease: report of the International Study Group of Fabry Nephropathy (ISGFN). Nephrol Dial Transplant, 2010. 25(7): p. 2168-77.

13. Cox, T.M. and J.P. Schofield, Gaucher's disease: clinical features and natural history. Bailliere's clinical haematology, 1997. 10(4): p. 657-89.

14. Fuller, M., P.J. Meikle, and J.J. Hopwood, Epidemiology of lysosomal storage diseases: an overview. 2006.

15. Poupetova, H., et al., The birth prevalence of lysosomal storage disorders in the Czech Republic: comparison with data in different populations. J Inherit Metab Dis, 2010. 33(4): p. 387-96.

16. Risch, N., et al., Geographic distribution of disease mutations in the Ashkenazi Jewish population supports genetic drift over selection. American journal of human genetics, 2003. 72(4): p. 812-22.

63

17. Cox, T.M. and M.B. Cachon-Gonzalez, The cellular pathology of lysosomal diseases. J Pathol, 2012. 226(2): p. 241-54.

18. Ballabio, A. and V. Gieselmann, Lysosomal disorders: from storage to cellular damage. Biochim Biophys Acta, 2009. 1793(4): p. 684-96.

19. Xu, Y.H., et al., Multi-system disorders of glycosphingolipid and ganglioside metabolism. Journal of lipid research, 2010. 51(7): p. 1643-75.

20. Meikle, P.J., et al., Prevalence of lysosomal storage disorders. JAMA, 1999. 281(3): p. 249-54.

21. Dajnoki, A., et al., Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry. Clin Chim Acta, 2010. 411(19-20): p. 1428-31.

22. Spacil, Z., et al., Comparative triplex tandem mass spectrometry assays of lysosomal enzyme activities in dried blood spots using fast liquid chromatography: application to newborn screening of Pompe, Fabry, and Hurler diseases. Anal Chem, 2011. 83(12): p. 4822-8.

23. Mechtler, T.P., et al., Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria. Lancet, 2012. 379(9813): p. 335-41.

24. Spada, M., et al., High incidence of later-onset fabry disease revealed by newborn screening. Am J Hum Genet, 2006. 79(1): p. 31-40.

25. Chien, Y.H., et al., Fabry Disease: Incidence of the Common Later-Onset alpha -Galactosidase A IVS4+919G>A Mutation in Taiwanese Newborns -- Superiority of DNA-Based to Enzyme-Based Newborn Screening for Common Mutations. Mol Med, 2012.

26. Ross, L.F., Newborn screening for lysosomal storage diseases: an ethical and policy analysis. Journal of inherited metabolic disease, 2012. 35(4): p. 627-34.

27. Giugliani, R., Newborn screening for lysosomal diseases: current status and potential interface with population medical genetics in Latin America. Journal of inherited metabolic disease, 2012. 35(5): p. 871-7.

28. Garman, S.C. and D.N. Garboczi, The molecular defect leading to Fabry disease: structure of human alpha-galactosidase. J Mol Biol, 2004. 337(2): p. 319-35.

29. Garman, S.C., Structure-function relationships in alpha-galactosidase A. Acta Paediatr Suppl, 2007. 96(455): p. 6-16.

30. Okuda, T., et al., Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to

64

verotoxins. J Biol Chem, 2006. 281(15): p. 10230-5.

31. Dawson, G., A.W. Kruski, and A.M. Scanu, Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias. J Lipid Res, 1976. 17(2): p. 125-31.

32. Aerts, J.M., et al., Elevated globotriaosylsphingosine is a hallmark of Fabry disease. Proc Natl Acad Sci U S A, 2008. 105(8): p. 2812-7.

33. Warnier, M., et al., Trafficking of Shiga toxin/Shiga-like toxin-1 in human glomerular microvascular endothelial cells and human mesangial cells. Kidney Int, 2006. 70(12): p. 2085-91.

34. Nyholm, P.G., J.L. Brunton, and C.A. Lingwood, Modelling of the interaction of verotoxin-1 (VT1) with its glycolipid receptor, globotriaosylceramide (Gb3). Int J Biol Macromol, 1995. 17(3-4): p. 199-204.

35. Fantini, J., et al., Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion. Glycoconj J, 2000. 17(3 -4): p. 199-204.

36. Puri, A., et al., Role of glycosphingolipids in HIV-1 entry: requirement of globotriosylceramide (Gb3) in CD4/CXCR4-dependent fusion. Biosci Rep, 1999. 19(4): p. 317-25.

37. Lund, N., et al., A novel soluble mimic of the glycolipid, globotriaosyl ceramide inhibits HIV infection. AIDS, 2006. 20(3): p. 333-43.

38. Gehrmann, M., et al., Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS One, 2008. 3(4): p. e1925.

39. Maloney, M.D., B. Binnington-Boyd, and C.A. Lingwood, Globotriaosyl ceramide modulates interferon-alpha-induced growth inhibition and CD19 expression in Burkitt's lymphoma cells. Glycoconj J, 1999. 16(12): p. 821-8.

40. George, T., et al., MHC class II proteins contain a potential binding site for the verotoxin receptor glycolipid CD77. Cell Mol Biol (Noisy-le-grand), 2001. 47(7): p. 1179-85.

41. Ghislain, J., C.A. Lingwood, and E.N. Fish, Evidence for glycosphingolipid modification of the type 1 IFN receptor. J Immunol, 1994. 153(8): p. 3655-63.

42. Liu, Y.Y., et al., Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and beta-catenin signaling. Mol Cancer, 2010. 9: p. 145.

65

43. Harzer, K. and B. Kustermann-Kuhn, Quantified increases of cholesterol, total lipid and globotriaosylceramide in filipin-positive Niemann-Pick type C fibroblasts. Clin Chim Acta, 2001. 305(1-2): p. 65-73.

44. Kovbasnjuk, O., et al., The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer. Proc Natl Acad Sci U S A, 2005. 102(52): p. 19087-92.

45. De Rosa, M.F., et al., Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog. J Biol Chem, 2008. 283(8): p. 4501-11.

46. Van de Kar, N.C., L.A. Monnens, and V.W. Van Hinsbergh, Tumor necrosis factor and interleukin 1 induce expression of the glycolipid verotoxin receptor in human endothelial cells. Implications for the pathogenesis of the haemolytic uraemic syndrome. Behring Inst Mitt, 1993(92): p. 202-9.

47. Eisenhauer, P.B., et al., Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin. Infect Immun, 2001. 69(3): p. 1889-94.

48. Lidove, O., et al., Fabry disease 'The New Great Imposter': results of the French Observatoire in Internal Medicine Departments (FIMeD). Clin Genet, 2011.

49. Wilcox, W.R., et al., Females with Fabry disease frequently have major organ involvement: lessons from the Fabry Registry. Mol Genet Metab, 2008. 93(2): p. 112-28.

50. Martins, A.M., et al., Guidelines to diagnosis and monitoring of Fabry disease and review of treatment experiences. The Journal of pediatrics, 2009. 155(4 Suppl): p. S19-31.

51. Wallin, E.F., M.R. Clatworthy, and N.R. Pritchard, Fabry disease: results of the first UK hemodialysis screening study. Clin Nephrol, 2011. 75(6): p. 506-10.

52. Elliott, P., et al., Prevalence of Anderson-Fabry disease in patients with hypertrophic cardiomyopathy: the European Anderson-Fabry Disease survey. Heart, 2011. 97(23): p. 1957-60.

53. Hagege, A.A., et al., Screening patients with hypertrophic cardiomyopathy for Fabry disease using a filter-paper test: the FOCUS study. Heart, 2011. 97(2): p. 131-6.

54. Baptista, M.V., et al., Mutations of the GLA gene in young patients with stroke: the PORTYSTROKE study--screening genetic conditions in Portuguese young stroke patients. Stroke, 2010. 41(3): p. 431-6.

66

55. Branton, M.H., et al., Natural history of Fabry renal disease: influence of alpha-galactosidase A activity and genetic mutations on clinical course. Medicine, 2002. 81(2): p. 122-38.

56. Beck, M., Agalsidase alfa--a preparation for enzyme replacement therapy in Anderson-Fabry disease. Expert Opin Investig Drugs, 2002. 11(6): p. 851-8.

57. Wilcox, W.R., et al., Long-term safety and efficacy of enzyme replacement therapy for Fabry disease. Am J Hum Genet, 2004. 75(1): p. 65-74.

58. Lee, K., et al., A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease. Glycobiology, 2003. 13(4): p. 305-13.

59. Clarke, J.T., et al., The pharmacology of multiple regimens of agalsidase alfa enzyme replacement therapy for Fabry disease. Genet Med, 2007. 9(8): p. 504-9.

60. Caballero, L., et al., Enzyme replacement therapy in Fabry disease: influence on cardiac manifestations. Curr Med Chem, 2010. 17(16): p. 1679-89.

61. Ramaswami, U., Update on role of agalsidase alfa in management of Fabry disease. Drug Des Devel Ther, 2011. 5: p. 155-73.

62. Watt, T., et al., Agalsidase beta treatment is associated with improved quality of life in patients with Fabry disease: findings from the Fabry Registry. Genet Med, 2010. 12(11): p. 703-12.

63. Feriozzi, S., et al., The Effectiveness of Long-Term Agalsidase Alfa Therapy in the Treatment of Fabry Nephropathy. Clin J Am Soc Nephrol, 2012. 7(1): p. 60-69.

64. Warnock, D.G., et al., Renal outcomes of agalsidase beta treatment for Fabry disease: role of proteinuria and timing of treatment initiation. Nephrol Dial Transplant, 2011.

65. Buechner, S., et al., Central nervous system involvement in Anderson-Fabry disease: a clinical and MRI retrospective study. J Neurol Neurosurg Psychiatry, 2008. 79(11): p. 1249-54.

66. Germain, D.P., et al., Safety and pharmacodynamic effects of a pharmacological chaperone on alpha-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies. Orphanet journal of rare diseases, 2012. 7: p. 91.

67. Benjamin, E.R., et al., The pharmacological chaperone 1-deoxygalactonojirimycin increases alpha-galactosidase A levels in Fabry patient cell lines. J Inherit Metab Dis, 2009. 32(3): p. 424-40.

67

68. Protalix-Biotherapeutics. PRX-102. Available from: http://www.protalix.com/product-development/prx-102.asp. Accessed on 01/03/2013.

69. Ohshima, T., et al., alpha-Galactosidase A deficient mice: a model of Fabry disease. Proc Natl Acad Sci U S A, 1997. 94(6): p. 2540-4.

70. Eitzman, D.T., et al., Fabry disease in mice is associated with age-dependent susceptibility to vascular thrombosis. Journal of the American Society of Nephrology : JASN, 2003. 14(2): p. 298-302.

71. Ohshima, T., et al., Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(11): p. 6423-7.

72. Bartz, S. and A.L. Jackson, How will RNAi facilitate drug development? Science's STKE : signal transduction knowledge environment, 2005. 2005(295): p. pe39.

73. Simmons, N.L., Cultured monolayers of MDCK cells: a novel model system for the study of epithelial development and function. Gen Pharmacol, 1982. 13(4): p. 287-91.

74. Ortiz, A., et al., Recommendations and guidelines for the diagnosis and treatment of Fabry nephropathy in adults. Nature clinical practice. Nephrology, 2008. 4(6): p. 327-36.

75. Mather, A.R. and L.J. Siskind, Glycosphingolipids and kidney disease. Adv Exp Med Biol, 2011. 721: p. 121-38.

76. Lesage, S., et al., Large-scale screening of the Gaucher's disease-related glucocerebrosidase gene in Europeans with Parkinson's disease. Hum Mol Genet, 2011. 20(1): p. 202-10.

77. Mattila, P.E., et al., Multiple Biosynthetic Trafficking Routes for Apically Secreted Proteins in MDCK Cells. Traffic, 2012. 13(3): p. 433-42.

78. Fischer, E.G., M.J. Moore, and D.J. Lager, Fabry disease: a morphologic study of 11 cases. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 2006. 19(10): p. 1295-301.

79. Thurberg, B.L., et al., Cardiac microvascular pathology in Fabry disease: evaluation of endomyocardial biopsies before and after enzyme replacement therapy. Circulation, 2009. 119(19): p. 2561-7.

80. Sindhu, A., P. Arora, and A. Chaudhury, Illuminating the gateway of gene silencing: perspective of RNA interference technology in clinical therapeutics. Molecular biotechnology, 2012. 51(3): p. 289-302.

68

http://www.protalix.com/product-development/prx-102.asp

http://www.protalix.com/product-development/prx-102.asp

81. Ostrander, E.A. and R.K. Wayne, The canine genome. Genome research, 2005. 15(12): p. 1706-16.

82. World-dataBank. World Development indicators. Available from: http://databank.worldbank.org/ddp/home.do. Accessed on 01/05/2013.

83. Nielsen, L. International Monetary Fund. Classifications of Countries Based on Their Level of Development: How it is Doen and How it Could be Done. . Available from: : http://www.imf.org/external/pubs/ft/wp/2011/wp1131.pdf. Accessed on 01/05/2013.

84. ANS. Agência Nacional de Saúde Complementar - Dados Gerais. 2012; Available from: http://www.ans.gov.br/index.php/materiais-para-pesquisas/perfil-do-setor/dados-gerais. Accessed on 01/06/2013.

85. Saúde, M.d. Lei n 8.080 - Lei Orgânica da Saúde. 1990; Available from: http://portal.saude.gov.br/portal/arquivos/pdf/lei8080.pdf. Accessed on 01/07/2013.

86. Melo, D.G. and J. Sequeiros, The challenges of incorporating genetic testing in the unified national health system in Brazil. Genetic testing and molecular biomarkers, 2012. 16(7): p. 651-5.

87. Saúde, M.d. Portaria MS n. 3.916 - Política Nacional de Medicamentos. 1998; Available from: http://pfdc.pgr.mpf.gov.br/atuacao-e-conteudos-de-apoio/legislacao/saude/portarias/portaria_MS_3.916.1998. Accessed on 01/07/2013.

88. Estatística, I.B.d.G.e. Censo Demográfico. Estados: Piauí. 2010; Available from: http://www.ibge.gov.br/estadosat/perfil.php?sigla=pi. Accessed on 01/07/2013.

89. Elbers, C.L., J.O.; Lanjouw, P., Micro-level Estimation of Poverty and Inequality. Econometrica, 2002. 71(1): p. 355-364.

90. Saúde, C.N.D. Norma Operacional Básica do Sistema Único de Saúde - SUS. 1996; Available from: http://conselho.saude.gov.br/legislacao/nobsus96.htm - 6. Accessed on 01/03/2013.

91. Piauí, S.d.E.d.S.d. Portal da Saúde: Missão e Valores. 2012; Available from: http://www.saude.pi.gov.br/paginas/37-missao-e-valores. Accessed on 01/04/2013.

92. Raskin, S. Raras e Subdiagnosticadas: Um Dia para as Raras (para a Associação Brasileira de Pacientes Portadores de Doença de Fabry e Seus Familiares). 2010; Available from: http://www.fabry.org.br/noticias-imprensa-integra.php?cd=13. Accessed on 01/04/2013.

69

http://www.fabry.org.br/noticias-imprensa-integra.php?cd=13

http://www.saude.pi.gov.br/paginas/37-missao-e-valores

http://conselho.saude.gov.br/legislacao/nobsus96.htm#6

http://www.ibge.gov.br/estadosat/perfil.php?sigla=pi

http://pfdc.pgr.mpf.gov.br/atuacao-e-conteudos-de-apoio/legislacao/saude/portarias/portaria_MS_3.916.1998

http://pfdc.pgr.mpf.gov.br/atuacao-e-conteudos-de-apoio/legislacao/saude/portarias/portaria_MS_3.916.1998

http://portal.saude.gov.br/portal/arquivos/pdf/lei8080.pdf

http://www.ans.gov.br/index.php/materiais-para-pesquisas/perfil-do-setor/dados-gerais

http://www.ans.gov.br/index.php/materiais-para-pesquisas/perfil-do-setor/dados-gerais

http://www.imf.org/external/pubs/ft/wp/2011/wp1131.pdf

http://databank.worldbank.org/ddp/home.do

93. Saúde, M.d. Portaria n. 2.380/GM - Grupo de Trabalho de Genética Clínica . 2004; Available from: http://dtr2001.saude.gov.br/sas/PORTARIAS/Port2004/GM/GM-2380.htm. Accesssed on 01/05/2013.

94. Saúde, M.d. Portaria n. 81- Política Nacional de Atenção Integral em Genética Clínica . 2009; Available from: http://portal.saude.gov.br/portal/saude/Gestor/visualizar_texto.cfm?idtxt=30616. Accessed on 01/03/2013.

95. Carvalho, T.M. Histórico do Programa Nacional de Triagem Neonatal - Apresentação. Sociedade Brasileira de Triagem Neonatal. Available from: http://www.sbtn.org.br/pg_historico_apresentacao.htm. Accessed on 01/05/2013.

96. Ambulatorial, S.d.A.à.S.C.-G.d.M.C., Manual de Normas Técnicas e Rotinas Operacionais do Programa de Triagem Neonatal2003, Brasília, DF: Ministério da Saúde.

97. Jurídica, C. Teste do Pezinho pode trazer mais um diagnóstico . 2006; Available from: http://www.centraljuridica.com/click.php?mid=3377. Accessed on 01/05/2013.

98. Saúde, M.d. Indicadores do Programa Nacional de Triagem Neonatal. 2008; Available from: http://portal.saude.gov.br/portal/arquivos/pdf/INDICADORES_TRIAGEM_NEONATAL.pdf. Accessed on 01/05/2013.

99. Sociedade Brasileira de Genética Médica. Available from: http://www.sbgm.org.br/. Accessed on 01/05/2013.

100. Martins, A.M., et al., Recommendations on diagnosis, treatment, and monitoring for Gaucher disease. The Journal of pediatrics, 2009. 155(4 Suppl): p. S10-8.

101. Secretaria de Saúde do Estado de Mato Grosso do Sul - Portaria n. 1.318/GM . 2002; Available from: http://pfdc.pgr.mpf.gov.br/atuacao-e-conteudos-de-apoio/legislacao/saude/portarias/portaria_MS_1318.2002. Accessed on 01/06/2013.

102. Saúde, M.d. Secretaria de Assistência a Saúde do Estado de São Paulo. Protocolos Clínicos e Diretrizes Terapêuticas. Medicamentos Excepcionais. Doença de Gaucher. . 2002; Available from: http://dtr2001.saude.gov.br/sas/dsra/protocolos/do_d11_00.htm. Accessed on 01/06/2013.

103. Wyatt, K., et al., The effectiveness and cost-effectiveness of enzyme and substrate replacement therapies: a longitudinal cohort study of people with lysosomal storage disorders. Health technology assessment, 2012. 16(39): p. 1-543.

70

http://dtr2001.saude.gov.br/sas/dsra/protocolos/do_d11_00.htm

http://pfdc.pgr.mpf.gov.br/atuacao-e-conteudos-de-apoio/legislacao/saude/portarias/portaria_MS_1318.2002

http://pfdc.pgr.mpf.gov.br/atuacao-e-conteudos-de-apoio/legislacao/saude/portarias/portaria_MS_1318.2002

http://www.sbgm.org.br/

http://portal.saude.gov.br/portal/arquivos/pdf/INDICADORES_TRIAGEM_NEONATAL.pdf

http://portal.saude.gov.br/portal/arquivos/pdf/INDICADORES_TRIAGEM_NEONATAL.pdf

http://www.centraljuridica.com/click.php?mid=3377

http://www.sbtn.org.br/pg_historico_apresentacao.htm

http://portal.saude.gov.br/portal/saude/Gestor/visualizar_texto.cfm?idtxt=30616

http://dtr2001.saude.gov.br/sas/PORTARIAS/Port2004/GM/GM-2380.htm

http://dtr2001.saude.gov.br/sas/PORTARIAS/Port2004/GM/GM-2380.htm

104. Genzyme. Fabry RADAR 2007: The Fabry Registry Aggregate Data Annual Report. 2007; Available from: http://www.fabry.org/fsig.nsf/PDFs/PDFsR/$File/2007_RADAR.pdf. Accessed on 01/03/2013.

105. Marinho, L.A.L.R., J.F.M.; Oliveira, T.C.; Sabóia, T.M. , Prevalência da doença de fabry em pacientes portadores de doençarenal crônica submetidos à hemodiálise em Natal – RN / Prevalence of fabry disease in patients with chronic kidney disease onhemodialysis in Natal – RN, Brazil. J Bras Nefrol, 2007. 29(4): p. 235-238.

106. Porsch, D.B., Doença de Fabry em Pacientes Submetidos à Hemodiálise no Rio Grande do Sul: Estudo de Prevalência e Relato Clínico, in Medicine2008, Universidade Federal do Piauí: Porto Alegre.

107. Vale, N.F.D.S., A.B.R.; Veras, A.B.; Monteiro, F.M.R.; Sousa, J.L.M.; Bezerra, V.L.; Martins, A.M.; D'Almeida, V.; Castro, J.A.F.; Monte Neto, J.T.; Melo, A.P.R.; Monte, S.J.H., Diagnosis of Fabry disease in Subjects on Hemodialysis in Piauí State: Role of Screening and Case Studies. . J Bras Nefrol, 2008. 30(4): p. 259-63.

108. Gaff, C.L., et al., A model for the development of genetics education programs for health professionals. Genetics in medicine : official journal of the American College of Medical Genetics, 2007. 9(7): p. 451-7.

109. Greiner-Tollersrud, O.K.B., T., Lysosomal Storage Disorders, in Madame Curie Bioscience Database [Internet]2000, Landes Bioscience: Austin (TX).

71

http://www.fabry.org/fsig.nsf/PDFs/PDFsR/$File/2007_RADAR.pdf

Date post:	27-Sep-2018
Category:	Documents
Upload:	trandang
View:	212 times
Download:	0 times

d-scholarship.pitt.edud-scholarship.pitt.edu/18464/1/Anatalia_Labilloy_Master_… · Web...

Documents