BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY Basic Genetics for ART Practitioners BASIC CYTOGENETICS AND CYTOGENETICS OF INFERTILITY INTRODUCE MYSELF TALK ABOUT TYPES OF CHROMOSOME ABNORMALITIES SEEN DURING PREGNANCY THE PHENOTYPES ASSOCIATED WITH THESE ABNORMALITIES THE WAYS IN WHICH WE DETECT THEM Richard Hall BSc SRCS Cytogenetics Department, Guy's & St Thomas' NHS Foundation Trust

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

G-banded karyotype Groups Size and position of centromere

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 MICROARRAYS 8p del 4p dup 10p del MLPA CGH FISH

Preparation of metaphases CULTURE SYNCHRONISE HARVEST 72 hours to 14 days ANALYSE CHROMOSOMES STAIN SLIDES PREPARE SLIDES

Traditional microscopy METAPHASE Low power x100 High power x1000

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.

Chromosome abnormalities Aneuploidy too many chromosomes too few chromosomes Rearrangements translocations balanced unbalanced inversions

Chromosome abnormalities Chromosome abnormalities seen in adults referred for: infertility mostly sex chromosome aneuploidy rearrangements involving sex chromosomes recurrent miscarriage balanced chromosome rearrangements e.g. translocations and inversions 2.5% 6% However, up to 50% of first trimester loss is due to foetal chromosome abnormality – mostly de novo

Spontaneous abortion products 15% of first trimester pregnancies are lost 46,N +16 45,X Triploidy Other autosomal trisomy 50% abnormal TRISOMY FOR ALL C’SOMES SEEN (EXCEPT 1) – ACCOUNTS FOR 60% OF ALL ABNORMALITIES MOST COMMON T16 AND 45,X (APPROX 20%) POLYPLOIDY (MOSTLY TRIPLOIDY) 15% REMAINING ABNORMALITIES (5%) ARE MOSTLY STRUCTURAL REARRANGEMENTS 50% normal

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.

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.

Meiosis I non-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.

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 lag The 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 lost 47,XXY/46,XY

Turner syndrome High mortality in first trimester foetuses Oedema of extremities Coarctation of the aorta Webbed neck NECK WEBBING AND OEDEMA Classical karyotype = 45,X (45%)

Turner syndrome Phenotype very variable, often mild and dependant on karyotype Short stature Increased carrying angle Infertility 7% mosaic, eg 45,X/46,XX 45% structural abnormality, eg 46,X,i(X)(q10)

Structural abnormalities of the X-chromosome Monosomy for short arm is associated with features of Turner syndrome or primary ovarian failure The location of the breakpoint in the X may influence gonadal function Partial monosomy for, or balanced rearrangements with, breakpoint in long arm more likely to be associated with premature ovarian failure

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 Example karyotypes = 47,XXY 47,XXY/46,XY 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,XXY 47,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 translocations Exchange of material between chromosomes Two types Robertsonian reciprocal FIRST TALK ABOUT ROBERTSONIAN TRANSLOCATIONS INVOLVES THE ACROCENTRIC CHROMOSOMES

Normal male karyotype: 46,XY Acrocentric chromosomes IN BALANCED CARRIERS THE CHROMOSOME NUMBER IS USUALLY 45 LOSS OF P-ARM MATERIAL CAN BE NON-HOMOLOGOUS OR HOMOLOGOUS

Robertsonian translocations der(14;21)(q10;q10) MOST COMMON ROB IS DER(13;14) 75% OF ALL ROBS ARROWS INDICATE BREAKPOINTS ON THE NORMAL HOMOLOGUES

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.

Robertsonians and miscarriage Behaviour at meiosis Female carriers of der(14;21) have 10% risk of Down’s syndrome child Alternate segregation Adjacent segregation Robertsonians and miscarriage

Robertsonian translocations Summary Result from fusion of two acrocentric chromosomes (13, 14, 15, 21, 22) Prevalence of 1 in 1000 Balanced carriers have reproductive risks present as: recurrent miscarriage Patau syndrome Down’s syndrome male infertility 14;21 ACCOUNTS FOR APPROX 10% OF ROBS HOMOLOGOUS ROBS WILL ALWAYS RESULT IN IMBALANCE UPD RISK

Reciprocal translocations Exchange of material between two non-homologous chromosomes Prevalence of 1 in 500 Balanced carriers are generally phenotypically normal Reproductive consequences because of behaviour at meiosis

The homologous chromosomes cannot pair properly Behaviour at meiosis The homologous chromosomes cannot pair properly Instead they must form a quadrivalent

Alternate segregation Normal Balanced (like carrier parent)

Adjacent-1 segregation Unbalanced trisomy and monosomy Unbalanced monosomy and trisomy

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

Reciprocal translocations Summary 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. This happens by chance, is more likely to happen as maternal age increases and does not require analysis of parental blood

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