BASIC CYTOGENETICS

AND CYTOGENETICS OF INFERTILITY

Richard Hall BSc SRCS

Cytogenetics Department, Guy's & St Thomas' NHS Foundation Trust

Basic Genetics for ART Practitioners

Chromosomes?• The most important objects in

the living world, for the genes they carry determine the existence and form of organisms.

G-banded karyotype

Cytogenetics?

• The study of the genetic constitution of cells through the visualisation and analysis of chromosomes.

– G-banding(and other traditional techniques)

– Fluorescence in situ hybridization (FISH)

– Molecular techniques(QF-PCR, MLPA)

Chromosome analysis techniques

QF-PCR

8p del

4p dup

10p del

MLPA

MICROARRAYS

FISH

CGH

Preparation of metaphasesCULTURE SYNCHRONISE HARVEST

PREPARE SLIDES

STAIN SLIDESANALYSE

CHROMOSOMES

72 hours

to14 days

Traditional microscopy

METAPHASE

Low power x100 High power x1000

Traditional microscopyHigh power (1000x) view. Next stage of analysis involves locating each chromosome pair and comparing them band for band.

Random distribution of chromosomes can hinder the accuracy and efficiency of the band comparison.

Typically 1000 bands per cell.

Chromosome abnormalitiesChromosome abnormalities

• AneuploidyAneuploidy– too many chromosomestoo many chromosomes– too few chromosomestoo few chromosomes

• RearrangementsRearrangements– translocationstranslocations

• balancedbalanced

• unbalancedunbalanced

– inversionsinversions

Chromosome abnormalities Chromosome abnormalities Chromosome abnormalities seen in Chromosome abnormalities seen in adults referred for:adults referred for:

•infertilityinfertility

mostly sex chromosome aneuploidymostly sex chromosome aneuploidy

rearrangements involving sex chromosomesrearrangements involving sex chromosomes

•recurrent miscarriagerecurrent miscarriage

balanced chromosome balanced chromosome rearrangementsrearrangements

e.g. translocations and inversionse.g. translocations and inversions

6%

2.5%

However, up to 50% of first trimester loss is due to foetal chromosome abnormality – mostly de novo

Spontaneous abortion productsSpontaneous abortion products

46,N46,N

+16+16

45,X45,X

TriploidyTriploidy

OtherOther

Other Other autosomalautosomaltrisomytrisomy

15% of first trimester pregnancies are lost

50% normal

50% abnormal

Aneuploidy

• Mostly from meiotic non-disjunction.

• Meiosis is the specialised cell division that generates haploid gametes.

• Errors in meiotic segregation occur frequently in human females, especially in MI.

clinically recognized pregnancies

05

101520253035

-20 20-24 25-29 30-34 35-39 40+

maternal age

% t

riso

mic

Chromosome abnormalities and maternal age

Meiosis Inon-disjunction

Meiosis I

Meiosis II

Disomic Nullisomic

Mosaicism

• The presence of two or more cell lines that are genetically identical, except for the chromosomal difference between them, in a single zygote.

• Frequently seen in patients with sex chromosome aneuploidy.

• Abnormal cell line may be in the minority.

47,XXY

47,XXY 46,XY

47,XXY 47,XXY 46,XY 46,XY

Anaphase lag – loss of one X

47,XXY/46,XY

Mosaicism

Turner syndromeTurner syndrome

High mortality in first trimester High mortality in first trimester foetusesfoetuses

• Oedema of extremitiesOedema of extremities

• Coarctation of the aortaCoarctation of the aorta

• Webbed neckWebbed neck

Classical karyotype = 45,X (45%)

Turner syndromeTurner syndromePhenotype very variable, Phenotype very variable, often mild and dependant often mild and dependant on karyotypeon karyotype

• Short statureShort stature

• Increased carrying angleIncreased carrying angle

• InfertilityInfertility

7% mosaic, eg 45,X/46,XX45% structural abnormality, eg 46,X,i(X)(q10)

Structural abnormalities of theX-chromosome

Monosomy for short arm is associated with features of Turner syndrome or primary ovarian failure

Partial monosomy for, or balanced rearrangements with, breakpoint in long arm more likely to be associated with premature ovarian failure

The location of the breakpoint in the X may

influence gonadal function

Structure of the X chromosome

• Xp11.2-p22.1– Ovarian failure (gonadal

dysgenesis)

• Xq13– X inactivation centre (XIST)

• Xq13-q26– ‘Critical region’ for ovarian

function– Breakpoints within this

region are associated with gonadal insufficiency

– Except breakpoints in Xq22

Klinefelter syndrome

• Incidence = 1/1000• Usually taller than average• Disproportionately long limbs• 30–50% gynaecomastia• Infertility/azoospermia• IQ may be reduced relative to

siblings

Example karyotypes = 47,XXY47,XXY/46,XY

Klinefelter syndrome

• Phenotype very variable – some patients are not diagnosed until they try for a family.

• Mosaics 47,XXY/46,XY may have milder phenotype and may be fertile.

• Therefore always carry out mosaicism check as infertility is the main clinical problem.

Chromosome translocationsChromosome translocations

• Exchange of material between chromosomesExchange of material between chromosomes

• Two typesTwo types– RobertsonianRobertsonian– reciprocalreciprocal

Acrocentric chromosomes

Normal male karyotype: 46,XYNormal male karyotype: 46,XY

Robertsonian translocationsRobertsonian translocationsder(14;21)(q10;q10)der(14;21)(q10;q10)

Robertsonians and infertility

• Some male carriers are infertile as they have spermatogenic arrest.

• Thought to be due to failure of pairing of the translocation in meiosis, which allows it to interfere with the X-Y bivalent.

• The more often this occurs the greater the effect on the sperm count.

• Prevalence of 1 in 1000.Prevalence of 1 in 1000.

• 10x excess in infertile men.10x excess in infertile men.

Alternate segregationAlternate segregation

Adjacent segregationAdjacent segregation

Robertsonians and miscarriage

Behaviour at meiosis

Female carriers of der(14;21) have 10%

risk of Down’s syndrome child

Robertsonian translocationsRobertsonian translocations

SummarySummary

• Result from fusion of two acrocentric Result from fusion of two acrocentric chromosomes (13, 14, 15, 21, 22)chromosomes (13, 14, 15, 21, 22)

• Prevalence of 1 in 1000 Prevalence of 1 in 1000

• Balanced carriers have reproductive risks Balanced carriers have reproductive risks present as:present as:– recurrent miscarriagerecurrent miscarriage– Patau syndromePatau syndrome– Down’s syndromeDown’s syndrome– male infertilitymale infertility

Reciprocal translocationsReciprocal translocations

• Exchange of material between two non-Exchange of material between two non-homologous chromosomeshomologous chromosomes

• Prevalence of 1 in 500Prevalence of 1 in 500

• Balanced carriers are generally phenotypically Balanced carriers are generally phenotypically normalnormal

• Reproductive consequences because of Reproductive consequences because of behaviour at meiosisbehaviour at meiosis

• The homologous chromosomes cannot pair properly

• Instead they must form a quadrivalent

Behaviour at meiosis

Balanced (like carrier parent)

Normal

Alternate segregation

Unbalanced

monosomy and trisomy

Unbalanced

trisomy and monosomy

Adjacent-1 segregation

Reciprocal translocations:Reciprocal translocations:reproductive risksreproductive risks

• For most translocations, ~50% of conceptions For most translocations, ~50% of conceptions will have either normal chromosomes or the will have either normal chromosomes or the balanced translocation.balanced translocation.

• Unbalanced products result in: Unbalanced products result in: – miscarriage (large segments)miscarriage (large segments)– dysmorphic delayed child (small segments).dysmorphic delayed child (small segments).

Reciprocal translocationsReciprocal translocationsSummarySummary

• Chromosome rearrangements are rare, but chromosome analysis is indicated if a couple have had three or more miscarriages of unknown aetiology.

• Essential that both partners are investigated as either the male or the female could carry a balanced rearrangement.

• Aneuploidy is the most common chromosomal cause of early miscarriage and requires no follow-up.

Suggested reading

• Gardner, RJM & Sutherland GR (2004). Chromosome abnormalities and genetic counseling. 3rd edn. Oxford University Press, New York.