Chapter 5: Principles of Clinical Cytogenetics
Clinical indications for chromosome analysis
• Problems of early growth and development
• Stillbirth or neonatal death
• Fertility problems
• Family history
• Neoplasia
• Pregnancy in female of advanced age (>35y)
Methods
• Peripheral blood is mixed with heparin to prevent clotting
• WBCs are collected, placed in tissue culture medium and stimulated to divide
• After few days cells are arrested in metaphase by chemicals, then collected and treated
with a hypotonic solution to release the chromosomes
• Chromosomes are then fixed, spread on slides and stained: ready for analysis
NB: cell cultures from peripheral blood have disadvantage of being short-lived (3-4 days),
but can be transformed to lymphoblastoid cell lines. Alternative is skin fibroblast cell lines.
Bone marrow is invasive, but requires little or no culture.
Chromosome identification
Karyotyping
• G-banding (Giemsa) most widely used in metaphase 400 bands per haploid karyotype
(minimum resolution of aberration detected is 5 Mb)
o G dark bands are AT rich, gene poor, late replicating and chromatin is more
condensed
o G pale bands are GC rich, gene rich and early replicating
• Alternative banding techniques:
o Q banding (limited use because fluorescent microscope needed)/ R banding
(inverse of G banding)/ C (centromere) banding/ high-resolution banding (in
prometaphase 550 bands or prophase 850 bands)/ NOR staining (analysis of
acrocentric short arms)
C-banding and NOR staining substituted by FISH
• 3 chromosome types are distinguished, depending on position of centromere:
o (Sub)metacentric: arms of approx. equal length
o Acrocentric: centromere near one end and small masses of chromatin known as
satellites, containing ribosomal RNA + repetitive sequences (human chr
13,14,15,21,22)
13
, Satellite regions can be involved in polymorphism as CNV
o Telocentric: centromere at one end and only a single arm (does not occur in
normal human karyotype
FISH
• See earlier
• Confluence of genomic and cytogenetic approach molecular cytogenetics
• Deletion is easy to spot; but tandem duplication not visible in metaphase visible in
interphase
• Fiber FISH: used mainly in research to characterize very small fragments by stretching
the DNA
Microarrays (aCGH)
• provides much more sensitive, high-resolution assessment of the genome, but without
info on translocation or rearrangements (e.g. inversion)
• Deletion = 1 copy versus 2 = 1/2 log2(1/2)=-1
• Duplication = 3 copies versus 2 = 3/2 log2(3/2)=0.58
• detects lots of benign CNVs/ polymorphisms, particularly common in subtelomeric and
centromeric regions
these collectively underscore uniqueness of an individual genome
• SNP array: applied for indels and detecting LOH-regions and UPD
QMPSF
= Quantitative Multiplex PCR of Short Fluorescent Fragments
MLPA
• = Multiplex Ligation-dependent Probe Amplification
• Applications: telomeres screening, multiple genomic disorders screening, aneuploidy
screening and high resolution screening for specific disease
Chromosome abnormalities
Abnormalities in chromosome number
• Aneuploidy = abnormal number of chromosomes (5% of pregnancies)
o Commonly caused by meiotic nondisjunction in meiosis I (see fig 5-7)
o ~15% of all pregnancies result in spontaneous abortion 50% of these have an
aneuploidy
o Monosomies are generally not viable (except 45,X). Trisomies are viable for chr
13,18,21,X,Y
Selection in utero abortion in >90% of aneuploidies
14
, • reciprocal translocation = exchange of segments between non-homologous
chromosomes usually no phenotypic effect, but may be associated with higher risk of
abnormal offspring [chromosome # can be normal or changed)
• polyploidy = increase in the multiple of a haploid set (3n,4n)
o arises through error at or post fertilization
• Triploidy (3n): can be liveborn, but don’t survive long
o If extra set is paternal: abnormal placenta and partial hydatidiform moles
o If extra set is maternal: early spontaneous abortion
o Very rarely live birth, but then death within a day
• Tetraploid (4n): occasionally observed
Abnormalities of chromosome structure
• Present in ~1 in 375 newborns (less common than aneuploidy pregnancies)
• Stable (functional centromere+2 telomeres) versus unstable
• Balanced versus unbalanced
Balanced aberrations:
• Inversion
Usually normal phenotype, but risk of offspring with unbalanced
karyotype (resulting from loop in meiosis)
E.g. inv(9)(p11q12): present in ~1% of population and considered
normal variant
o Paracentric (not incl. centromere) both breaks on one arm (freq 0.009-
0.049%)
Very low risk of life-born unbalanced offspring
o Pericentric (incl centromere) a break in each arm (freq 0.12-0.7%)
Small inversions produce recombinants with large deletions/duplications
high risk of miscarriage
Large inversions produce recombinants with small
deletions/duplications higher risk of viable abnormal offspring
• Balanced translocation
Carriers can produce high frequency of unbalanced gametes offspring
with unbalanced karyotype (risk 1-20%; most imbalances will not
implant or will be spontaneously lost during gestation)
Chromosome break can disrupt a gene phenotype
Cave microdeletion/-duplication phenotype!
o Reciprocal translocation
15
Clinical indications for chromosome analysis
• Problems of early growth and development
• Stillbirth or neonatal death
• Fertility problems
• Family history
• Neoplasia
• Pregnancy in female of advanced age (>35y)
Methods
• Peripheral blood is mixed with heparin to prevent clotting
• WBCs are collected, placed in tissue culture medium and stimulated to divide
• After few days cells are arrested in metaphase by chemicals, then collected and treated
with a hypotonic solution to release the chromosomes
• Chromosomes are then fixed, spread on slides and stained: ready for analysis
NB: cell cultures from peripheral blood have disadvantage of being short-lived (3-4 days),
but can be transformed to lymphoblastoid cell lines. Alternative is skin fibroblast cell lines.
Bone marrow is invasive, but requires little or no culture.
Chromosome identification
Karyotyping
• G-banding (Giemsa) most widely used in metaphase 400 bands per haploid karyotype
(minimum resolution of aberration detected is 5 Mb)
o G dark bands are AT rich, gene poor, late replicating and chromatin is more
condensed
o G pale bands are GC rich, gene rich and early replicating
• Alternative banding techniques:
o Q banding (limited use because fluorescent microscope needed)/ R banding
(inverse of G banding)/ C (centromere) banding/ high-resolution banding (in
prometaphase 550 bands or prophase 850 bands)/ NOR staining (analysis of
acrocentric short arms)
C-banding and NOR staining substituted by FISH
• 3 chromosome types are distinguished, depending on position of centromere:
o (Sub)metacentric: arms of approx. equal length
o Acrocentric: centromere near one end and small masses of chromatin known as
satellites, containing ribosomal RNA + repetitive sequences (human chr
13,14,15,21,22)
13
, Satellite regions can be involved in polymorphism as CNV
o Telocentric: centromere at one end and only a single arm (does not occur in
normal human karyotype
FISH
• See earlier
• Confluence of genomic and cytogenetic approach molecular cytogenetics
• Deletion is easy to spot; but tandem duplication not visible in metaphase visible in
interphase
• Fiber FISH: used mainly in research to characterize very small fragments by stretching
the DNA
Microarrays (aCGH)
• provides much more sensitive, high-resolution assessment of the genome, but without
info on translocation or rearrangements (e.g. inversion)
• Deletion = 1 copy versus 2 = 1/2 log2(1/2)=-1
• Duplication = 3 copies versus 2 = 3/2 log2(3/2)=0.58
• detects lots of benign CNVs/ polymorphisms, particularly common in subtelomeric and
centromeric regions
these collectively underscore uniqueness of an individual genome
• SNP array: applied for indels and detecting LOH-regions and UPD
QMPSF
= Quantitative Multiplex PCR of Short Fluorescent Fragments
MLPA
• = Multiplex Ligation-dependent Probe Amplification
• Applications: telomeres screening, multiple genomic disorders screening, aneuploidy
screening and high resolution screening for specific disease
Chromosome abnormalities
Abnormalities in chromosome number
• Aneuploidy = abnormal number of chromosomes (5% of pregnancies)
o Commonly caused by meiotic nondisjunction in meiosis I (see fig 5-7)
o ~15% of all pregnancies result in spontaneous abortion 50% of these have an
aneuploidy
o Monosomies are generally not viable (except 45,X). Trisomies are viable for chr
13,18,21,X,Y
Selection in utero abortion in >90% of aneuploidies
14
, • reciprocal translocation = exchange of segments between non-homologous
chromosomes usually no phenotypic effect, but may be associated with higher risk of
abnormal offspring [chromosome # can be normal or changed)
• polyploidy = increase in the multiple of a haploid set (3n,4n)
o arises through error at or post fertilization
• Triploidy (3n): can be liveborn, but don’t survive long
o If extra set is paternal: abnormal placenta and partial hydatidiform moles
o If extra set is maternal: early spontaneous abortion
o Very rarely live birth, but then death within a day
• Tetraploid (4n): occasionally observed
Abnormalities of chromosome structure
• Present in ~1 in 375 newborns (less common than aneuploidy pregnancies)
• Stable (functional centromere+2 telomeres) versus unstable
• Balanced versus unbalanced
Balanced aberrations:
• Inversion
Usually normal phenotype, but risk of offspring with unbalanced
karyotype (resulting from loop in meiosis)
E.g. inv(9)(p11q12): present in ~1% of population and considered
normal variant
o Paracentric (not incl. centromere) both breaks on one arm (freq 0.009-
0.049%)
Very low risk of life-born unbalanced offspring
o Pericentric (incl centromere) a break in each arm (freq 0.12-0.7%)
Small inversions produce recombinants with large deletions/duplications
high risk of miscarriage
Large inversions produce recombinants with small
deletions/duplications higher risk of viable abnormal offspring
• Balanced translocation
Carriers can produce high frequency of unbalanced gametes offspring
with unbalanced karyotype (risk 1-20%; most imbalances will not
implant or will be spontaneously lost during gestation)
Chromosome break can disrupt a gene phenotype
Cave microdeletion/-duplication phenotype!
o Reciprocal translocation
15
