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BASIC CYTOGENETICS
AND CYTOGENETICS OF INFERTILITY
Richard Hall BSc SRCS
Cytogenetics Department, Guy's & St Thomas' NHS Foundation Trust
Basic Genetics for ART Practitioners
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Chromosomes?• The most important objects in
the living world, for the genes they carry determine the existence and form of organisms.
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G-banded karyotype
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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)
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Chromosome analysis techniques
QF-PCR
8p del
4p dup
10p del
MLPA
MICROARRAYS
FISH
CGH
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Preparation of metaphasesCULTURE SYNCHRONISE HARVEST
PREPARE SLIDES
STAIN SLIDESANALYSE
CHROMOSOMES
72 hours
to14 days
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Traditional microscopy
METAPHASE
Low power x100 High power x1000
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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.
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Chromosome abnormalitiesChromosome abnormalities
• AneuploidyAneuploidy– too many chromosomestoo many chromosomes– too few chromosomestoo few chromosomes
• RearrangementsRearrangements– translocationstranslocations
• balancedbalanced
• unbalancedunbalanced
– inversionsinversions
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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
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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
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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.
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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
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Meiosis Inon-disjunction
Meiosis I
Meiosis II
Disomic Nullisomic
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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.
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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
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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%)
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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)
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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
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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
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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
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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.
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Chromosome translocationsChromosome translocations
• Exchange of material between chromosomesExchange of material between chromosomes
• Two typesTwo types– RobertsonianRobertsonian– reciprocalreciprocal
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Acrocentric chromosomes
Normal male karyotype: 46,XYNormal male karyotype: 46,XY
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Robertsonian translocationsRobertsonian translocationsder(14;21)(q10;q10)der(14;21)(q10;q10)
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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.
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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
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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
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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
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• The homologous chromosomes cannot pair properly
• Instead they must form a quadrivalent
Behaviour at meiosis
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Balanced (like carrier parent)
Normal
Alternate segregation
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Unbalanced
monosomy and trisomy
Unbalanced
trisomy and monosomy
Adjacent-1 segregation
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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).
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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.
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Suggested reading
• Gardner, RJM & Sutherland GR (2004). Chromosome abnormalities and genetic counseling. 3rd edn. Oxford University Press, New York.