Basic Genetics for ART Practitioners
BASIC CYTOGENETICS
AND CYTOGENETICS OF INFERTILITY
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.
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
CGH
MICROARRAYS
4p
dup
8p del
QF-PCR
MLPA
FISH
10p
del
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
2.5%
mostly sex chromosome aneuploidy
rearrangements involving sex chromosomes
• recurrent miscarriage
6%
balanced chromosome rearrangements
e.g. translocations and inversions
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
Other
autosomal
trisomy
+16
45,X
50% abnormal
Triploidy
Other
50% normal
46,N
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
% trisomic
clinically recognized pregnancies
35
30
25
20
15
10
5
0
-20
20-24
25-29
30-34
maternal age
35-39
40+
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.
Anaphase lag
– loss of one X
Mosaicism
47,XXY
46,XY
47,XXY
47,XXY
47,XXY
46,XY
47,XXY/46,XY
46,XY
Turner syndrome
High mortality in first trimester
foetuses
• Oedema of extremities
• Coarctation of the aorta
• Webbed neck
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
•
•
•
•
•
•
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
Normal male karyotype: 46,XY
Acrocentric chromosomes
Robertsonian translocations
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.
• 10x excess in infertile men.
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
Reciprocal translocations
• Exchange of material between two nonhomologous chromosomes
• Prevalence of 1 in 500
• Balanced carriers are generally phenotypically
normal
• Reproductive consequences because of
behaviour at meiosis
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
Unbalanced
trisomy and monosomy
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).
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.
Suggested reading
• Gardner, RJM &
Sutherland GR (2004).
Chromosome
abnormalities and genetic
counseling. 3rd edn.
Oxford University Press,
New York.