Epigenetics and Clinical Cytogenetics

Dr. Fardaei

10.2.92

22

Epigenetics Genomic Imprinting

Epigenetics refers to the study of heritable changes in gene expression that occur without a change in DNA sequence.

In some genetic disorders, the expression of the disease phenotype depends on whether the mutant allele has been inherited from the father or from the mother.

One important type of imprinting takes place during gametogenesis, before fertilization, and marks certain genes as having come from mother or father. Two mechanisms for initiating and sustaining epigenetic modifications :

DNA methylation, Histone modification (acetylation and methylation)acetylation and methylation)

33

Pedigree Presentation: Pedigree Presentation: ImprintingImprinting

44

PWS phenotype: Obesity, small hands and feet, short stature, hypogonadism, mental retardation

In 70% of cases, there is a deletion in the long arm of chr. 15inherited from the patient’s father.

In minority of cases (29%), they have two intact chr. 15s, bothinherited from the mother.

In 1% of cases defect in imprinting centre.

Prader-Willi Syndrome (PWS)

55

AS phenotype: Unusual facial appearance, short stature , severe mental retardation, spasticity, seizures

In 70% of cases, there is a deletion on the long arm of chr. 15 inherited from the patient’s mother.

In minority of cases (3-5%), they have two intact chr. 15s, bothinherited from the father.

In 5-10% of cases also mutation in a single gene (E6-AP ubiquitin-protein ligase), located on chromosome 15 that just expressed from maternal allele in the CNS can cause AS.

In 1% of cases defect in imprinting centre.

Angelman Syndrome (AS)

66

The study of chromosomes, their structure and their inheritance in medical genetics.

- 1% of live births,

- 2 % of pregnancies in women older than 35 years,

- 50 % of all spontaneous first-trimester abortions.

Clinical Cytogenetics

Clinical Indications for Chromosome Analysis

1- Problems of early growth and development Failure to thrive, Developmental delay, Dysmorphic facies, Multiple malformations, Short stature, Ambiguous genitalia, and Mental retardation

2- Stillbirth and neonatal death

3- Fertility problems

4- Family history A known or suspected chromosome abnormality in a first-degree

relative.

5- Neoplasia

6- Pregnancy in a woman of advanced age >30-35

Cells that can be used for chromosome analysis:

1- Cells from blood: short-lived (3 to 4 days)

White blood cells can also be transformed in culture to form lymphoblastoid cell lines that are potentially immortal.

2- Fibroblast cells: Long-term cultures, fibroblasts from skin for biochemical and chromosome analysis.

3- Fetal cells derived from amniotic fluid (amniocytes) or obtained by

chorionic villus biopsy can also be cultured successfully for cytogenetic, biochemical, or molecular analysis.

Chromosome Analysis

To perform a chromosome analysis, cells must be capable of growth and rapid division in culture .

T lymphocytes from heparinated blood stimulate to divide,

Chromosome Analysis

To perform a chromosome analysis, cells must be capable of growth and rapid division in culture .

T lymphocytes from heparinated blood stimulate to divide ,

After a few days the cells arrested in metaphase by chemicals that inhibit the mitotic spindle ,

Treated with a hypotonic solution to release the chromosomes, spread on slide and stained

Chromosome Analysis

Chromosome Identification

Commonly used staining method:

-Giemsa (G) banding: The most common method used in clinical laboratories. (400-500 bands in haploid, each band 6-8 MBP)

Classtaification of human chromosomes:

1- Metacentric, with a more or less central centromere

2- Submetacentric,

3- Acrocentric (chromosomes 13,14,15,21, and 22),

The stalks of these five chromosome pairs contain hundreds of copies of genes encoding ribosomal RNA.

4- Telocentric, with the centromere at one end. Not in human

Fragile Sites - Fragile sites are nonstaining observed occasionally on several chromosomes.

- Detection needs special growth conditions or chemicals that alter or inhibit DNA synthesis.

- Many fragile sites are heritable variants.

Fragile X Syndrome:

Diagnostic procedure:

1- Detection of CGG repeat in the FMR1 gene

2- Fragile site on the X

Fluorescence In Situ Hybridization (FISH)

Metaphase Interphase

A whole chromosome 'paint" probe specific for the X chromosome

A singlecopy DNA probe specific for the factor VIII gene on the X chromosome

Repetitive alpha satellite DNA probe specific for the centromere of chromosome 17

Detection of telomeres at the ends of each chromosome:

FISH using a repeated TTAGGG probe.

The two sister chromatids are evident

Use of different fluorochromes to detect multiple probes simultaneously.

Two-color and three-color applications are routinely used to diagnose specific deletions, duplications, or rearrangements.

Spectral Karyotyping (SKY). It is possible to detect and distinguish 24 different colors simultaneously.

Multiple probes simultaneously

CHROMOSOME ABNORMALITIES Numerical

Structural

Heteroploid: any chromosome number other than 46

Euploid: An exact multiple of the haploid chromosome number (n)

Aneuploid: any other chromosome number

TRIPLOIDY AND TETRAPLOIDY

Diploid (2n): normal

Triploid (3n): Triploid infants can be liveborn, they do not so long.

Tetraploid (4n): Occasionally reported.

Reason for Triploidy:

1- Most frequently results from fertilization by two sperm (dispermy).

2- Failure of one of the meiotic divisions, resulting in a diploid egg or sperm.

Triploidy:

The phenotype depends on the source of the extra chromosome set;

- An extra set of paternal chromosomes typically have an abnormal placenta and are classified as partial hydatidiform moles,

- An additional set of maternal chromosomes are spontaneously aborted earlier in pregnancy.

Reason for Tetraploidy:

Tetraploidy are always 92,XXXX or 92,XXYY; the absence of XXXY

or XYYY sex chromosome suggest that:

- Tetraploidy results from failure of completion of early cleavage division of the zygote.

ANEUPLOIDY

The most common human chromosome disorder (at least 3 to 4 percent of all pregnancies).

Most aneuploid patients have trisomy and less often, monosomy.

Trisomy for a whole chromosome is rarely compatible with life. By far the most common type of trisomy liveborn infants is trisomy 21 (karyotype 47,XX XY, + 21).

Monosomy for an entire chromosome is almost always lethal; an important exception is monosomy for the X chromosome, as seen in Turner syndrome.

The most common chromosomal mechanism: meiotic nondisjunction (During one of the two meiotic division usually during meiosis 1).

The five chromosomes that account for the vast majority of aneuploidy in liveborn individuals

Autosomes: 13, 18 and 21

Sex Ch. : X and Y

AUTOSOMAL DISORDERS

Three trisomy for an entire autosome: 21, 18, and 13.

Down Syndrome (Trisomy 21)

- The most common genetic cause of moderate mental retardation. - About 1 child in 800 is born with Down syndrome.

- Strong association between the incidence of Down syn. and advancing

maternal age.

DOWN SYNDROME (Trisomy 21)

-In 95%, Down syndrome resulting from meiotic nondisjunction

90% during maternal meiosis (predominantly in meiosis I)10% during paternal meiosis (usually in meiosis II)

- 4% of Down syndrome: Robertsonian translocation

- 21q21q Translocation:

Mosaic Down Syndrome

A small percentage of Down syndrome patients are mosaic:

Normal cells + cells with extra ch. 21

Milder and variable phenotype

Trisomy 18

About 60 percent of the patients are female.

Increased maternal age is the main factor

Incidence: 1 in 7500 in liveborns

Always mental retardation

Failure to thrive,

Severe malformation of the heart,

Incidence: 1 in 20,000 to 25,000 in liveborns.

Nondisjunction in maternal meiosis I.

Trisomy 13

Cytogenetic Abnormalities of the Sex Chromosomes

KLINEFELTER SYNDROME (47,XXY)

47,XYY SYNDROME

TRISOMY X (47,XXX)

TURNER SYNDROME (45,X)

46,XY

Trisomy 18 47,XX,+18 Trisomy 21

chromosome 13 green, chromosome 21 red

Application of multicolor FISH to interphase (do not need to culture cells)

A large number of prenatal cytogenetics laboratories are now performing prenatal interphase analysis using FISH (do not need to culture cells)

XY

21

X

21

13

21

Mosaicism

Mosaicism may be either numerical (the most common type) or structural.

A common cause: nondisjunction in an early postzygotic mitotic division.

For example, a zygote with an additional chromosome 21 might lose the extra chromosome in a mitotic division and continue to develop as a 46/47, + 21 mosaic.

Mosaics are less severely affected than nonmosaic individuals.

Chimerism (From Emery’s )

A human with more than one cell line derived from more than one zygotes.

In humans two kinds of chimera: Dispermic and Blood chimeras

Dispermic: two genetically different sperm fertilize two ova and the resulting two zygotes fuse to form one embryo. If two zygotes are of different sex: Hermaphroditism with an XX/XY karyotype.

Blood chimeras: Result from an exchange of cells, via the placenta, between non-identical twins in utero.