Cytogenetics Flashcards
History of Cytogenetics
- “Dark Ages”: before 1952; chromosomes visualized by number in humans unknown
- “Hypotonic Period”: 1952-1959; # chromosomes identified in 1956, DS/TS/KS discovered in 1959, karyotyping popular
- “Banding Era”: 1974-1989; Giemsa staining used and chromosomes identified based on banding pattern
- “Molecular Era”: 1989-present; FISH, CGH, array CGH used
Number of Genes in a Chromosome
500-4,000
Number of Genes in a Metaphase Band
50-100
Number of Genes in a High-Res Prophase Band
50
General Phenotype of Chromosomal Abnormality
developmental delays, dysmorphology, birth defects, other medical problems
Chromosomal Dosage
more imbalance present worse problems are; extra dosages much better tolerated than missing ones
What percentage of all spontaneous 1st trimester abortions have a chromosomal condition?
50%
What percentage of all live births have a chromosomal condition?
1%
What is the most common aneuploidy resulting in live birth?
T21
What is the most common aneuploidy in 1st trimester SAbs?
T16
What percentage of pregnancies in women >35yrs have a chromosomal condition?
2%
Indications for Testing Chromosomes
- problems in early growth/development
- stillbirth and neonatal death
- fertility problems
- family history suspicious of chromosomal abnormality
- AMA/abnormal prenatal screen
- neoplasms
Anatomy of a Chromosome
- comprised of two sister chromatids
- centromere
- telomeres
- p arm (short)
- q arm (long)
Chromosomal Shapes
- defined by location of centromere
- metacentric
- submetacentric
- acrocentric
Group A Chromosomes
1, 2, 3
Group B Chromosomes
4, 5
Group C Chromosomes
6, 7, 8, 9, 10, 11, 12, X
Group D Chromosomes
13, 14, 15
Group E Chromosomes
16, 17, 18
Group F Chromosomes
19, 20
Group G Chromosomes
21, 22, Y
Metacentric Chromosomes
Groups A and F (1, 2, 3, 19, 20)
Submetacentric Chromosomes
Groups B, C, E (4, 5, 6, 7, 8, 9, 10, 11, 12, X, 16, 17, 18)
Acrocentric Chromosomes
Groups D and G (13, 14, 15, 21, 22, Y)
Giemsa Bands
400-500
High-Res Prophase Bands
800-1200
Dark Giemsa Bands
more condensed, less transcriptionally active, late replicating, AT rich, heterochromatin
Light Giemsa Bands
more transcriptionally active, CG rich, euchromatin
C-banding
- selectively stains heterochromatin around centromere
- inherited polymorphisms of 1, 9, 16, and Yq make it look like there’s more material
- identifies dicentric chromosomes
Silver Staining
detects NORs present at end of acrocentric chromosomes, which contain rRNA genes
Karyotyping Methodology
- Blood sample drawn
(WBCs, skin, amniocytes, chorionic villi, umbilical cord blood can also be used) - RBCs separated out and WBCs stimulated to divide with phytohemagluttinin (PHA)
- WBCs cultured and incubated for 3 days
- Colchicine added to destroy spindle fibers and arrest cell in metaphase
- WBCs separated off and hypotonic solution added
- Cells fixed and dropped onto slide
- Slide is stained with banding solution and photographed (from photographed chromosome spread, karyotype generated by cutting out chromosomes and arranging them by classification)
- Look at ~20 cells and takes a week to finish (if mosaicism is suspected, more cells need to be looked at (50-100))
Band Nomenclature
- arms divided into p and q
- q10 and p10 = centromeres
- each arm separated into regions (1-n) starting from centromere to telomere
- each region separated into bands starting with landmark band closest to centromere (1-n)
- band further divided into sub-bands (represented by a decimal)
- telomere referred to as qter/pter
- Example: 6p23 -> short arm of chr 6, region 2, band 3
ISHC Karyotype Nomenclature
- cen = centromere
- del = deletion
- der = derivative chromosome
- dup = duplication
- inv = inversion
- mar = marker chromosome
- mat = maternal origin
- pat = paternal origin
- t = translocation
- ter = end of chromosome arm
- = gain of material
- = loss of material
- / = mosaic
When to Order Karyotype
- patients with obvious chromosomal syndromes
- family history of chromosomal rearrangements
- history of multiple miscarriages
- prenatal diagnosis of aneuploidy
FISH Methodology
- DNA probe prepared that is fluorescently labeled and complementary to target region
- chromosome denatured and exposed to probe, which attaches to complementary sequence
- slide examined under fluorescent lighting (presence of signal indicates probe attachment; lack of signal indicates missing target sequence)
- results available within 48 hours
- if abnormal, needs to be followed up with karyotype or microarray
When is FISH Used
- rapid diagnosis of aneuploidy (T13/18/21, X, Y)
- rapid diagnosis of triploidy
- specific microdeletions syndromes via locus-specific probes
- specific duplications
- identification of translocations/marker chromosomes
Limitations of FISH
- need to know chromosomal region of interest
- probe may hybridize even if there is a small change in region of interest; appears as though nothing is wrong
- does not distinguish trisomy due to aneuploidy vs chromosome rearrangement
- not efficient for genome-wide analysis
Limitations of Karyotype
- resolution limited to large structural differences >5Mb; cannot detect small deletions/duplications/insertions/point mutations
- may not be able to identify marker chromosomes or other small structural rearrangements
- may be unable to tell if rearrangement is truly balanced or not
- unable to detect UPD, ROH
Array CGH Methodology
- patient and control DNA labeled with fluorescent dyes and applied to microarray
- patient and control DNA compete to attach to probes
- microarray scanner measures fluorescent signals
- computer software analyzes data and generates plot
- if patient DNA > control DNA, duplication
- if control DNA > patient DNA, deletion
- if no del/dup, patient and control DNA hybridize equally
Array CGH Limitations
- cannot detect balanced rearrangements
- VUSs possible
- probe spacing (where in genome) vs probe resolution (how small to detect); if large probe used, may not be able to detect small deletions
- may not detect low-level mosaicism (<20%)
- cannot detect triploidy or ROH/UPD
SNP Array Methodology
- looks at variation in nucleotide bases of patient DNA vs database/reference DNA
- detects SNPs in heterozygous (AB) and homzygous states (AA, BB)
- detects number of alleles present
- able to detect ROH, UPD, copy neutral variations, triploidy
SNP Array Limitations
- unable to detect balanced rearrangements
- potential VUSs
- may not detect low-level mosaicism (<20%)
Chromosomal Duplications
- section of chromosome has made extra copy of itself
- direct = tandem duplication
- inverted = mirror duplication
Chromosomal Deletions
- loss of genetic material
- interstitial = in the middle of chromosome
- terminal = at end of chromosome
Chromosomal Insertions
- piece of one chromosome inserted into completely different chromosome
- results from at least three breaks in two chromosomes
Chromosomal Inversions
- chromosome breaks in two places and piece reinserts itself in opposite orientation
Pericentric inversions
- involve centromere
- may produce chromosomally unbalanced gametes resulting from recombination
- homologues form inversion loop during meiosis
- crossover leads to 4 possible gametes: normal homologue, inverted homologue, homologue with p arm dup and q arm del, homologue with p arm del and q arm dup
- only heterozygotes with small distal (non-inverted) segments will have viable abnormal offspring
Genetic Counseling Points for Pericentric Inversions
- risk of having abnormal child after having a recombinant child is 5-15%
- risk of having abnormal child w/o fhx of recombinant child is 1%
- actual risks depend on specific inversion
- prenatal diagnosis offered to any heterozygotes whose family had a recombinant child; any heterozygote with specific inversions; any heterozygotes with inversions involving chr13/18/21; molecular analysis to exclude deletion in PWS/AS region of 15q11-13
Paracentric Inversions
- do not involve centromere
- theoretically cannot produce unbalanced offpsring
- recombinant gametes formed are acentric or dicentric, both of which are nonviable
Genetic Counseling Points for Paracentric Inversions
- hard to detect on classic karyotype; need at least high-res prophase banding
- risk of abnormal offpsring is 0.1-0.5%
- prenatal diagnosis may not be offered except when parent carries inversion shown in literature to result in abnormal offspring
Isochromosomes
- chromosome breaks at centromere and kinetochore micotubules separate short and long arms as opposed to separating chromatids
- complete duplication of p arm or q arm
- ISOp or ISOq
Reciprocal Translocations
- two different chromosomes exchange segments with each other, typically between non-homologous chromosomes
- generates atypical derivative chromosomes
- translocated segments and centric segments
- chromosome number indicated by which centromere it contains
- balanced translocations have no loss or gain of genetic material
Single-Segment Exchange
- one of translocated segments is very small and comprises only telomeric region of chromosome
- more likely to result in live-born child with problems
Double-Segment Exchange
- both translocated segments are large
Alternate Segregation
- 2:2
- A&B; A’&B’
- only way to get phenotypically normal offspring
- assume that this is frequent
Adjacent-1 Segregation
- 2:2
- A&B’; A’&B
- segregation involves different centromeres
- more common
- most likely when translocated segments are small
Adjacent-2 Segregation
- 2:2
- A&A’, B&B’
- segregation involves same centromeres
- rarer and less likely to result in liveborn child
- most likely when centric segments are small
Tertiary Segregation
- 3:1
- most likely when quadrivalent is lop-sided
- 2 of 3 chromosomes are normal
Interchange Segregation
- 3:1
- most likely when quadrivalent is lop-sided
- 2 of 3 chromosomes are derivative
4:0 Segregation
- all four chromosomes go to one cell
- most likely when translocated and centric segments are both large
- not viable
Genetic Counseling Points for Reciprocal Translocations
- a little bit of monosomy but more of trisomy means there’s less imbalance and less material rearranged but more likely to have baby with problems
- the larger the segments involved, may continue to have miscarriages but less likely to bring a baby with problems to term
- liveborn aneuploid offspring demonstrates viability for that abnormal combination to recur (recurrence risks high)
- ascertainment by miscarriage or infertility is more difficult to predict
Four Factors Determining Magnitude of Risk (Reciprocal Translocations)
- mode of ascertainment (presence of liveborn with problems or miscarriages)
- predicted type of segregation leading to potential viable gametes
- Sex of transmitting parent (risk always high if from mother than from father; selection process against abnormal sperm; whatever egg is ovulated, whether normal or not, is the one that’s available)
- the assessed imbalance of the potentially viable gamete (risks range from 0% to 30% and depend on actual cytogenetic imbalance)
Frequency of Autosomal Translocation Carriers
1/500
Robertsonian Translocations
- translocations involving chr 13, 14, 15, 21, 22, Y
- typically results in loss of NORs
- balanced carriers have 45 chromosomes
Frequency of Robertsonian Translocation Carriers
1/1000
Three Mechanisms for Robertsonian Translocations
- centric fusion: part of centromere from one chromosome and part from other join together
- union following breakage in one short arm and one long arm
- union following breakage in both short arms (dicentric chromosomes; more common)
Alternate Segregation (RT)
- 2:1
- produces normal and balanced gametes
- favored in male and female carriers
- females have 20% risk to have abnormal offspring
Adjacent Segregation (RT)
- 2:1
- produces two types of disomic and nullisomic gametes
Segregation of Homologous Robertsonian Translocations
- no gametes are balanced
- 1:0 segregation lead to disomic or nullisomic gamete
- 100% chance for abnormal offspring, 0% chance for normal offspring
- only T13 or T21 viable
Genetic Counseling Points for Robertsonian Translocations
- only balanced conceptuses with T13 or T21 survive
- fetal T14/15/22 miscarry
- translocations involving chr 14/15 have concerns for UPD
- if child has der(21) due to rob (21q;21q) and a normal sib, translocation is de novo
Empiric Risks for Robertsonian Translocations
Chance for Unbalanced Offspring:
13;13 - 100% mother, 100% father
13;14 - 1% mother, 1% father
13;15 - 1% mother, 1% father
13;21 - 15% mother, 1% father
13;22 - 1% mother, 1% father
14;21 - 15% (amnio)/10% (liveborn) mother, 1% father
15;21 - 10% mother, 1% father
21;21 - 100% mother, 100% father
21;22 - 10% (liveborn) mother, 1% father
14;15, 14;22, 15;22 lethal in utero
3:0 Segregation (RT)
rare
Y-Chromosome Rearrangements
- Inversions: not usually clinically significant
- Translocations: 70% of the time, the other chromosome involved is acrocentric (chr 15, 22) w/o gain/loss of euchromatin and normal fertility; reciprocal exchange between Yq and autosome typically de novo, leads to balanced male, infertility
X-Chromosome Rearrangements
- relative tolerance for X chromosome abnormalities compared to autosomes d/t X-inactivation
- Inversions: breakpoints in X critical region may influence female phenotype; inheritance of recombinant X leads to variant form of Turner syndrome (del(Xq)/dup(Xp) characterized by normal/tall stature and ovarian dysgenesis; del(Xp)/dup(Xq) associated with short stature and intact ovarian function); recombinant X in males is lethal in utero; paracentric inversions typically don’t have phenotypic consequence unless breakpoints in critical region
- Translocations: when there are X;autosome translocations, normal X is preferentially inactivated; in unbalanced offspring, der(x) preferentially inactivated; adjacent-1 segregation results in functional X disomy or X deletion as well as autosomal deletion or duplication; 3:1 segregation results in tertiary trisomy -> risk of X duplication, X duplication/autosome duplication or tertiary monosomy -> risk of X deletion/autosome deletion; adjacent-2 segregation results in X deletion/autosome duplication or X duplication/autosome deletion; 50% females carriers and all male carriers infertile; fertile females at risk of having offspring with Turner/Klinefelter variants
Common Inversions (no phenotypic effect)
- inv(2)(p11.2q13)
- inv(Y)(p11q13)
- inversions of 1, 9, 16, and Y heterochromatin
