Lecture 6 - Chromosomal Aberrations- Dr. Faisal Al-Allaf -md-y3-2009

transcript

١٤٤٤/٠٩/٢٨ Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 1

Dr. Faisal Al-AllafAssistant Professor of Genetics and Molecular Medicine

Umm Al-Qura University Faculty of Medicine, Makkah, Saudi Arabia

fallaf@uqu.edu.saTel/Fax: 5270000 Ext: 4198

Chromosomal Aberrations

Chromosomal Aberrations Numerical aberrations

Aneuploidy, Polyploidy, Monosomy, Trisomy Trisomy 21

Structural aberrations Deletions, ring chromosome, duplications, inversions, Isochromosomes, fragile site, translocations

Mosaicism and chimarism Lyonization Effect of maternal age Indications for chromosome analyses

Cytogenetics Preparation of karyotyping Banding (G, Q, R, and C) FISH

Chromosomal disorders Chromosomal disorders involve:

Numerical abnormalities (Abnormal numbers of chromosomes (Aneuploidy)

Structural aberrations Different chromosome constitution

in two or more cell line (Mixoploidy).

Chromosome abnormalities are a principal cause of pregnancy loss, an estimated 10-15% of conception having a chromosome abnormality of which 95% are lost before term

Close to 95% of the abnormalities are aneuploidies, the remainder being chromosome structural abnormalities

Numerical aberrations Euploidy means that the chromosome number

per body cell is an integral multiple of the haploid number, N=23, aneuploidy that it is other than an integral multiple.

Diploidy describes the normal situation, 2N = 46 chromosomes.

1. Extra haploid sets (Polyploidy) refers to multiples of haploid number (e.g. triploidy, 3N =69).

2. Extra single chromosome (Trisomy) (2N+1) is presence of three copies of one chromosome, (2N-1) is called monosomy.

3. Missing single chromosome (monosomis) are nearly always incompatible with survival (lethal) except for XO.

Mosaicism and Chimerism (Mixoploidy)

Mosaicism: presence within an individual of 2 or more genetically different cells issued from a single zygote (following a single fertilization event)

A mosaic is an individual with multiple cell lines (which exhibit different genotypes) that arise from a single zygote

Chimerism: presence within an individual of 2 or more genetically different cells issued from different zygotes (result from multiple fertilizations )

Both yield incidence patterns that depart from general rules

Mosaicism Mosaicism arises when a genetic

mutation occurs in post-zygotic mitotic division. All progeny of the mutant cell and, therefore, the cell types derived from it will exhibit the new mutation

X chromosome inactivation creates a mosaic for expression of two X chromosomes in normal women, which in heterozygote can allow pathological expression of X-linked recessive alleles

Clinical presentations are generally more pronounced the earlier a mutation occurs

For example, a woman heterozygous for recessive colour blindness is colour blind if normal X is inactivated.

Mechanisms leading to numerical chromosomal abnormalities

Polyploidy arises as a result of:

1. Fertilization by two sperms

2. A diploid sperm due to failure in meiosis

3. A diploid ovum due to a failure in meiosis

Monosomies and Trisomies may result from

1. Non-disjunction or

2. Anaphase lag

Mechanisms leading to numerical chromosomal abnormalities

Non disjunction: this is caused by duplication of a chromosome from one parent with loss of the corresponding homologue from the other parent. For example, uniparental disomy of maternal chromosome 15 can result in the same phenotype as Prader—Willi.

Anaphase lag: occurs when there is a delay in the movement of one chromosome from the metaphase plate during ansphase. This may result in the loss of a chromosome if it fails to reach the pole of the cell before the nuclear membrane reforms. Eg: a few cases of cystic fibrosis with severe growth retardation have two homologous chromosomes 7 of maternal origin

Autosome disorders and conception

Chromosomal abnormalities are present in at least 10% of spermatozoa and 25% of oocytes

Approximately 50% of spontaneous first trimester miscarriages have a chromosome abnormality, including probably 95% Trisomy 13 (T13) and T18 fetuses. The most common is T16, not seen in live births

The only autosomal trisomies compatible with survival to birth are

Trisomy 13, causing Patau syndrome (PS) Trisomy 18, causing Edward syndrome (ES) Trisomy 21, causing Down syndrome (DS)

The majority (95% of DS and ES, 80% of PS) have complete trisomy and a severe clinical phenotype

Recurrence risk of chromosomal disorders depends on maternal age

The birth frequency (recurrence risk) of this class increases with maternal age, especially after 35 years of age. Nevertheless 75% of DS babies are born to women under 35, since most babies are born to younger mothers

Around 4% of DS and ES and close to 20% of PS have major translocations involving the relevant chromosomes. Around 1-4% of patients with each syndrome show mosaic expression

Translocation DS almost always involves another acrocentric chromosome, i.e. 13, 14, 15, or 22; t14,21 is most common

Translocations that lead to partial trisomy are associated with milder manifestation and longer survival

Non-disjunctionNullisomic, monosomic and disomic gametes

Down Syndrome Problems in infancy and childhood

Hearing deficit is a problem in 60-80% and epilepsy in 5-10%. About 30% have either an overactive or under active thyroid gland

There is risk of obesity, sleep apnoea and skeletal problems including dislocation of cervical vertebrae

Acute lymphocytic leukaemia accounts for 5% of deaths in childhood. Upper respiratory tract infections are common

Congenital heart disease affects around 40% of DS babies

There is usually significant intellectual delay, with specific deficits in speech and auditory short-term memory

Down Syndrome Problems in adolescence and adulthood

More than 80% of DS patients survive beyond 10 years

Average adult height ~150 cm Average intelligent quotient (IQ) of young adults 40-

Males are nearly always sterile and about 40% of females fail to ovulate

Life expectancy is reduced and there are Alzheimer–like features in half those over 40 years of age

47, XY, +21

Sex chromosome disordersTurner syndrome: 45, X karyotype

It is usually diagnosed at birth. The frequency is 1 in 2000-3000 live born female but there is high incidence of intrauterine mortality 98-99%

Lack of ovarian development leading to deficient secretion of sex steroid

Increased incidence of diabetes mellitus, inflammatory bowel disease, and autoimmune disease

Body cells contain NO Barr bodies

Normal intelligence, webbed neck, coarctation of aorta, high arched palate, shield-like chest with widely spaced nipples, renal abnormalities, short metatarsals, gonadal dysgenesis, may also have edema of ands and feet and micrognathia

Sex chromosome disordersTurner syndrome: 45, X karyotype

50% of turners patients are due to monosomy; 45, X

30-40% mosaicism: mostly 45, X/ 46,XX; a few 45X / 45, XY

10-20% isochromosomes, ring chromosomes, deletions … etc.

Treatment includes Short stature: GH therapy Hypothyroidism: Thyroxine therapy Hand–eye coordination: physical

therapy Sexual development: Estrogen

therapy at 12-13 years Aorta: surgical correction

Sex chromosome disordersKlinefelter syndrome: 47, XXY

47 XXY karyotype can also be 48XXXY, 49XXXXY and 50, XXXXXY

The frequency is 1 in 500- 1 in 1000 live-born males

Abnormal presence of Barr bodies

Life expectancy is Normal, but 50% die before birth

Phenotype is basically male, tall with elongated lower legs and forearms, but with a feminine body shape and low muscle mass

Increased risk of osteoporosis if untreated

Increased risk of breast cancer and extragonadal germ cell tumors

Sex chromosome disordersKlinefelter syndrome: 47, XXY

Prepuberty features: small testes, disproportonality long legs, personality and behavioral disorder

Postpuberty features: small testes, gynaecomastia, and other signs of androgen deficiency (diminished body hair, small phallus, poor muscular development taller than average height)

Management of the disease: Testesterone therapy by long-term

implants at beginning of puberty

Fertility can be achieved using testicular aspiration and intra-cytoplasmic sperm in injection (ICSI)

Sex chromosome disordersTriple-X syndrome: 47, XXX

Triple X syndrome: this is a female with an extra X chromosome, Karyotype 47, XXX (and 48, XXXX, 49, XXXXX). The additional X is of maternal origin in 95% of cases

Abnormal number of Barr bodies

Frequency of 1/1000-1/1500 female births

Generally tall with slender body shape, 25% are infertile and some have mild mental retardation, otherwise there are no distinguishing features

Sex chromosome disorders47, XYY

This male has an extra Y chromosome, the incidence being in 1000 males.

2-3% of males institutionalized because of learning problems or antisocial criminal behavior

XYY males are often tall, but has normal body proportions. Large teeth, and normal fertility. Although often asymptomatic, there may be subtle motor incoordination and behavior problems, with aggression in childhood.

It is debatable whether an increase in criminal behavior is seen among XYY adults, though the majority do not fall foul of the law.

Structural aberrations Structural aberrations include:

Translocations Deletions Ring chromosomes Duplications Inversions Isochromosomes Fragile sites

Most structural aberrations result from Chromosomal breakage occurs close together and

enzymatic repair mechanisms link the wrong ends

Unequal exchange between homologous repeated sequences on the same or different chromosomes

Mechanisms leading to structural chromosomal disorders

Around two-thirds of the structural abnormalities arise in oocytes, one-thirds in sperm, the latter increasing with paternal age

Chromosomal damage is increased by Environmental conditions (e.g. mutagenic chemicals, radiation) Genetic chromosome instability disorders (e.g. ataxia

telangiectasia and Fanconi’s syndrome)

Deletions (code: del)

Deletion of part of a chromosome can be Interstitial Terminal

Interstitial deletions can arise from two breaks, followed by faulty repair, from unequal crossing–over in a previous meiosis, or as a consequence of a translocation in a parent

The error is described using the code ‘del’ followed by a description of the missing region in a separate set of brackets. For example, DiGeorg syndrome, caused by a deletion at 22q 11.22, is formulated: 46,XX, del(22)(q11.22)

Deletions (code: del)

A terminal deletion of the long arm of chromosome 1 from band 21 would be formulated: 46,XX,del(1)(q21;qter), CAN ALSO BE pter

Deletions are generally characterized by mental handicap, multiple congenital malformations, and sometimes associated with growth failure

Several syndromes are ascribed to microscopically invisible microdeletions

When several genes are deleted together, the term contiguous gene syndrome is applied to the corresponding phenotype

Ring chromosomes (code: r) If two breaks occur in the same

chromosome the broken ends can fuse as a ring

Acentric are lost, but if the ring contains a centromere it can survive subsequent cell division

Clinically a ring represents two deletions. They can double by sister chromatid exchange, leading to effective trisomy, or be lost , resulting in monosomy

46,XY,r(21)

Duplications (code: dup) Duplication is the presence of two

adjacent copies of chromosomal segment and can be either ‘direct’ (or ‘tandem’ ), or ‘inverted’.

Duplications may originate by unequal crossing – over in a previous meiosis, or as a consequence of translocation, inversion, or presence of an isochromosome in a parent

Duplications are more common, but generally less harmful than deletions. As example is cat eye syndrome involving iris coloboma formulated: 46, XX, dup(22) (q13; q11)

Inversions (code: inv) Inversions arise from two chromosomal

breaks with end– to –end switching of the intervening segment.

If inversion includes the centromere it is pericentric (A), if not, the inversion is paracentric (B).

Inversion can lead to chromosomally unbalanced gametes following crossing–over.

46,XX, inv(9), (p12q12)

Isochromosomes (code: i) An isochromosome has one

chromosome arm deleted and the other duplicated

In live births the commonest involves the long arm of the X, resulting in Turner syndrome due to short arm monosomy

Most isochromosomes cause spontaneous abortion

46 X, i(Xq)

Fragile site (code: fra) A fragile site is an apparent gap in

a chromosome

Some are common (or ‘universal’), others are rare and sensitive to folate levels in the medium in which the cells under examination are cultured.

These are inherited in a Mendelian fashion, a well known example being the defect associated with fragile X syndrome

46,XY,fraX

Translocations (code: ‘rob’ or ‘t’ or ‘ins’)

A translocation involves transposition of chromosome material usually between chromosomes (non homologous).

Three types are recognized: 1. Centric fusion or ‘Robertsonian’ 2. Reciprocal 3. Insertional

Centric fusion or ‘Robertsonian translocations’ (code: ‘rob’) arises from breaks at or near the centromeres of two chromosomes, followed by their fusion

46,XY,t(2;4)(p23;q25), male with receprocal translocation involving the short arm of chromosome 2 at region 2 band 3 and the long arm of chromosome 4 at region 2 band 5

Centric fusion or Robertsonian translocations (rob)

The long arms of chromosomes 13, 14, 15, 21 and 22 only are involved, especially 13 with 14 (rob13;14), and 14 with 21 (rob 14;21)

These are all acrocentric chromosomes with very small short arms

In the example to the right… Mom’s Ch 21 is attached to Ch 14 She shows NO symptoms However, her children can be severely affected

1/4 Trisomy 21 (Down’s Syndrome) Accounts for 9% of Down syndrome

children born to mothers who are less than 30 years of age

1/4 Normal 1/4 Carrier like herself 1/4 Monosomy 21 (Always lethal)

Reciprocal translocations (t) Reciprocal translocation involves inter-

chromosomal exchange

Either arm of any chromosome can be involved and carriers are usually healthy

The medical significance is therefore usually for future generations, as carriers can produce chromosomally unbalanced fetuses

Reciprocal translocation can also activate genes in cancer

Insertional translocations (ins) Insertional translocation

involves insertion of a deleted segment interstitially at another location.

It is extremely rare and balanced carriers are usually healthy, but may produce chromosomally unbalanced offspring with either a duplication or a deletion.

XX maleness syndrome XX maleness syndrome: sequence of Y

(SRY) is transferred to the X chromosome

The incidence is 1 in 20,000 males. These males may have a similar appearance to males with Klinefelter’s syndrome

They may looks normal, have a normal IQ, and get married. They are sterile

XX males usually present at the infertility clinic or when a prenatally predicted female appears to be male

Meiosis in a 14/21 translocation carrier produces familial down syndrome

For translocation cases: 1-3% for male carriers; 10-15% for female

Maternal serum screening in the second trimester detects 75-80% of DS pregnancies

Sceening is based on ‘triple‘ or ‘’quadruple’ testing, followed by chromosomal analysis or FISH

Indications for chromosome analysis1. Suspected chromosome

abnormality

2. Multiple congenital anomalies and/or developmental retardation

3. Disorders of sexual function

4. Undiagnosed mental retardation

5. Certain malignancies

6. Infertility or multiple miscarriage

7. Stillbirth and neonatal death

Cytogenetics The study of number and structure of

chromosomes is called cytogenetics

Traditionally it is performed on compacted chromosomes at a magnification of about 1000X providing resolution to around 3 million base pairs, or one narrow chromosome band.

By incorporation of molecular techniques, the resolution can be reduced to 2 kb (2000 bp)

Karyotyping and banding Fluorescent in situ hybridization (FISH) Comparative genomic hybridization (CGH)

Preparation of karyotype A visual karyotype is prepared

by arresting dividing cells at metaphase with a spindle inhibitor such as colchicine, spreading the cells on a glass slide and staining with Giemsa stain.

Traditionally a photographic positive is then made and the chromosomes cut out and assembled on a card , in pairs, in order of size, sometimes in conventional groups A – E.

In modern practice, this step is replaced by digital imaging.

Classification of human chromosome on the basis of centromere positions

Chromosomes are classified according to the position of the centromere: Chromosomes 1, 3, 16, 19 and 20, with

the centromere in the middle, are known as metacentric

Chromosomes 13, 14, 15, 21, 22 and Y, with the centromere near one end, are acroentric

The rest are submetacentric. The short arm is symbolized ‘p’ (for petite) and the long arm ‘q’

Individual chromosomes differ not only in the position of the centromere but also in their overall length.

Chromosomes are subdivided into groups labeled A-G on the basis of overall morphology. A = 1, 2, 3 B = 4, 5 C = 6-12 + X D = 13, 14, 15 E = 16, 17, 18 F = 19, 20 G = 21, 22 + Y

Classification of human chromosome on the basis of their morphology

Classification of human chromosome on the basis of their staining

Prophase and metaphase chromosomes exhibit alternating light and dark bands under appropriate staining conditions.

The bands reflect the differential folding of clusters of looped domains and also define regions of the genome that have different properties and functions.

The development of chromosome banding (staining) enables very precise recognition of individual chromosomes and the detection of chromosome abnormalities.

Chromosome banding (staining) also revealed that chromatin (the combination of DNA and histone proteins that comprise chromosomes) exists in two main forms:

Euchromatin stains lightly and consists of genes which are actively expressed.

Heterochromatin stains darkly and consists of chromatin that is either devoid of genes or has inactive genes (unexpressed repetitive DNA). Heterochromatin segments of the genome replicate very late in the S phase of the cell cycle.

G banding G banding is the most common procedure,

uses Giemsa stain after enzymatic treatment of metaphase chromosome

R bands are areas that Stain light with G banding Replicate DNA early in the S phase GC rich Have the highest gene density

G bands are areas that Stain dark with G banding Replicate DNA late in the S phase AT rich Have relatively fewer genes than R

Q banding Q banding uses quinacrine to stain chromosomes, which are

viewed with ultraviolet fluorescent microscopy.

Q banding gives a banding pattern similar to that obtained with Giemsa

R banding R banding or reverse banding, uses

Giemsa dyes under elevated temperatures to produce the reverse of the pattern seen in G banding or Q banding

Fluorescent dyes with a high-binding affinity to GC-rich DNA can also produce R banding

C banding C banding requires heating in

an alkali solution and staining with Giemsa. Or pretreated with acid followed by alkali then Giemsa.

C bands are area of constitutive heterochromatin located adjacent to the centromeres of all chromosomes and in the long arm of the Y chromosome (Yq)

Fluorescent in situ hybridization (FISH)

FISH enables the specific localization of genes and the direct visualization of abnormalities at the molecular level.

Hybridization with chromosome specific probes it allows rapid diagnosis or exclusion of trisomy in amniotic fluid cells.

Diagnosis of Microdeletions: probes are used in diagnosis of DiGeorge /VCFS AT

22q11 Wolf – Hirschhorn at

4p16.3 Prader – willi and

Angelman Williams syndrome Smith – Magenis

syndrome etc.

Fluorescent in situ hybridization (FISH)

Diagnosis of translocations: FISH probes directed at the bcr and abl sequences can be used to reveal the Philadelphia chromosome. As two fluorescent signals on the derivative Chromosome 22; in normal cells the signals are on separate chromosomes.

Diagnosis of sex chromosome rearrangements: In some phenotypic males lacking a Y chromosome , an SRY probe reveals the site to which the male – determining SRY locus has been translocated from its normal site at Yp11, often to the X.

Conventional abbreviations used in cytogenetics

A-G Chromosome groups M Monosomy1-22 Autosome number P Short armDel Deletion q long armDer Derivative chromosome r Ring

chromosomeDup Duplication t TranslocationI Isochromosome T TrisomyIns Insertion ter TerminalInv Inversion

International Standard of Chromosome nomenclature (ISCN)

Positions of genes along chromosome arms are defined by Region number (from the centromere

outwards) Band number Sub – band number Sub – sub – band number

Numerical disorders are described as follows Number of chromosomes, sex chromosomes,

+ or chromosome number, for example, boy with trisomy 21 is 47,XY +21

Structural disorders are described as follows Number of chromosomes, sex chromosomes,

mutation (chromosomes involved), (break points, margins or region) (p short arm; q, long arm).

References and Private Reading1. Emery’s Elements of Medical Genetics, 13th edition 2007, by Peter TURNPENNY and

Sian ELLARD. Churchill Livingstone ELSEVIER. ISBN: 978-0-7020-2917-2

2. Medical Genetics at a Glance, 2nd edition 2008, by Dorian PRITCHARD and Bruce KORF. Blackwell Publishing. ISBN: 978-1-4051-4846-7

3. Elsevier's Integrated Genetics, 2007, by Linda Adkison and Michael Brown. MOSBY ELSEVIER. ISBN: 978-0-323-04329-8

4. Genetics for Dummies, 2005, by Tara Robinson, Wiley Publishing, Inc. ISBN: 978-0-7645-9554-7

5. New Clinical Genetics, 2007, by Andrew Read and Dian Donnai. Scion. ISBN: 978-1-904842-31-6

6. Cell Biology and Genetics, Crash Course, 2nd edition 2006, by Manson, Jones, Morris, Michael STEEL and Dan HORTON-SZAR. MOSBY ELSEVIER. ISBN: 0-7234-3248-1

7. Human Molecular Genetics, 3rd edition, 2003, by STRACHAN T. and A. READ. Garland science/Taylor and Francis group. ISBN: 978-0-8153-4182-6

8. Human Genetics: Concepts and Applications, 7th edition 2007, by Ricki LEWIS. McGraw Hill international. ISBN: 978-0-07-110779-2

9. Genomes, 3rd edition 2006, by T.A. BROWN. Garland science, ISBN: 978-0-8153-4138-3

10. Colour Atlas of Genetics, 2007, by Eberhard Passarge. Thieme. ISBN: 978-1-58890-3365

Acknowledgments For the providers of all the educational materials

(video clips, pictures, diagrams and charts) including publishers, pharmaceutical companies or unknown internet users who made their material available for use, in this and other presentations, I offer heartfelt thanks and deep appreciation.

I feel particularly grateful to faculty, staff, and our brilliant students who provided a unique intellectual and wonderful environment for work.

Lecture 6 - Chromosomal Aberrations- Dr. Faisal Al-Allaf -md-y3-2009

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