Presentation on theme: "BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY"— Presentation transcript:
1 BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY Basic Genetics for ART PractitionersBASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITYINTRODUCE MYSELFTALK ABOUT TYPES OF CHROMOSOME ABNORMALITIES SEEN DURING PREGNANCYTHE PHENOTYPES ASSOCIATED WITH THESE ABNORMALITIESTHE WAYS IN WHICH WE DETECT THEMRichard Hall BSc SRCSCytogenetics Department, Guy's & St Thomas' NHS Foundation Trust
2 Chromosomes?The most important objects in the living world, for the genes they carry determine the existence and form of organisms.You will spend the whole of your career examining the most important objects in the living world
3 G-banded karyotypeGroupsSize and position of centromere
4 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)
6 Preparation of metaphases CULTURESYNCHRONISEHARVEST72 hoursto14 daysANALYSE CHROMOSOMESSTAIN SLIDESPREPARE SLIDES
7 Traditional microscopy METAPHASELow power x100High power x1000
8 Traditional microscopy High 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.
9 Chromosome abnormalities Aneuploidytoo many chromosomestoo few chromosomesRearrangementstranslocationsbalancedunbalancedinversions
10 Chromosome abnormalities Chromosome abnormalities seen in adults referred for:infertilitymostly sex chromosome aneuploidy rearrangements involving sex chromosomesrecurrent miscarriagebalanced chromosome rearrangementse.g. translocations and inversions2.5%6%However, up to 50% of first trimester loss is due to foetal chromosome abnormality – mostly de novo
11 Spontaneous abortion products 15% of first trimester pregnancies are lost46,N+1645,XTriploidyOtherautosomaltrisomy50% abnormalTRISOMY FOR ALL C’SOMES SEEN (EXCEPT 1) – ACCOUNTS FOR 60% OF ALL ABNORMALITIESMOST COMMON T16 AND 45,X (APPROX 20%)POLYPLOIDY (MOSTLY TRIPLOIDY) 15%REMAINING ABNORMALITIES (5%) ARE MOSTLY STRUCTURAL REARRANGEMENTS50% normal
12 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.
13 Chromosome abnormalities and maternal age This is backed up by all major studies. This is a typical one showing a massive increase in the proportion of trisomic pregnancies among older women.
15 MosaicismThe 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.
16 Mosaicism Anaphase lag – loss of one X 47,XXY 46,XY 47,XXY 47,XXY When a chromosome fails to connect to the spindle apparatus or “lags behind” when chromosomes are segregating to the poles this is termed anaphase lagThe chromosome in question fails to be included in the reforming nuclear membrane and instead the single chromosome in cytoplasm forms a micronucleus which is then lost47,XXY/46,XY
17 Turner syndrome High mortality in first trimester foetuses Oedema of extremitiesCoarctation of the aortaWebbed neckNECK WEBBING AND OEDEMAClassical karyotype = 45,X (45%)
18 Turner syndromePhenotype very variable, often mild and dependant on karyotypeShort statureIncreased carrying angleInfertility7% mosaic, eg 45,X/46,XX45% structural abnormality, eg 46,X,i(X)(q10)
19 Structural abnormalities of the X-chromosome Monosomy for short arm is associated with features of Turner syndrome or primary ovarian failureThe location of the breakpoint in the X may influence gonadal functionPartial monosomy for, or balanced rearrangements with, breakpoint in long arm more likely to be associated with premature ovarian failure
20 Structure of the X chromosome Xp11.2-p22.1Ovarian failure (gonadal dysgenesis)Xq13X inactivation centre (XIST)Xq13-q26‘Critical region’ for ovarian functionBreakpoints within this region are associated with gonadal insufficiencyExcept breakpoints in Xq22
21 Klinefelter syndrome Example karyotypes = 47,XXY 47,XXY/46,XY Incidence = 1/1000Usually taller than averageDisproportionately long limbs30–50% gynaecomastiaInfertility/azoospermiaIQ may be reduced relative to siblingsExample karyotypes = 47,XXY47,XXY/46,XY
22 Klinefelter syndromePhenotype 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.
23 Chromosome translocations Exchange of material between chromosomesTwo typesRobertsonianreciprocalFIRST TALK ABOUT ROBERTSONIAN TRANSLOCATIONSINVOLVES THE ACROCENTRIC CHROMOSOMES
24 Normal male karyotype: 46,XY Acrocentric chromosomesIN BALANCED CARRIERS THE CHROMOSOME NUMBER IS USUALLY 45LOSS OF P-ARM MATERIALCAN BE NON-HOMOLOGOUS OR HOMOLOGOUS
25 Robertsonian translocations der(14;21)(q10;q10) MOST COMMON ROB IS DER(13;14)75% OF ALL ROBSARROWS INDICATE BREAKPOINTS ON THE NORMAL HOMOLOGUES
26 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 1000.10x excess in infertile men.
27 Robertsonians and miscarriage Behaviour at meiosisFemale carriers of der(14;21) have 10% risk of Down’s syndrome childAlternate segregationAdjacent segregationRobertsonians and miscarriage
29 Robertsonian translocations SummaryResult from fusion of two acrocentric chromosomes (13, 14, 15, 21, 22)Prevalence of 1 in 1000Balanced carriers have reproductive risks present as:recurrent miscarriagePatau syndromeDown’s syndromemale infertility14;21 ACCOUNTS FOR APPROX 10% OF ROBSHOMOLOGOUS ROBS WILL ALWAYS RESULT IN IMBALANCEUPD RISK
30 Reciprocal translocations Exchange of material between two non-homologous chromosomesPrevalence of 1 in 500Balanced carriers are generally phenotypically normalReproductive consequences because of behaviour at meiosis
31 The homologous chromosomes cannot pair properly Behaviour at meiosisThe homologous chromosomes cannot pair properlyInstead they must form a quadrivalent
33 Adjacent-1 segregation Unbalancedtrisomy and monosomyUnbalancedmonosomy and trisomy
34 Reciprocal translocations: reproductive risks For most translocations, ~50% of conceptions will have either normal chromosomes or the balanced translocation.Unbalanced products result in:miscarriage (large segments)dysmorphic delayed child (small segments).In fact the t(2;17) was first detected in an unbalanced form in a fetus that had died in utero. The fetus had inherited the derivative 17 via adjacent 1 segregation and had lissencephally associated with deletion of the LIS1 gene on the short arm of c’some 17
35 Reciprocal translocations SummaryChromosome 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.This happens by chance, is more likely to happen as maternal age increases and does not require analysis of parental blood
36 Suggested readingGardner, RJM & Sutherland GR (2004). Chromosome abnormalities and genetic counseling. 3rd edn. Oxford University Press, New York.