Cytogenetics Review Questions

Lecture 1

List the three broad categories of clinical indications for chromosomal analysis.
Prenatal, Constitutional, Cancer/Acquired

Which family members should have chromosomal analysis?
1. Both parents of a child with structural chromosome rearrangement, deletion, duplication, and 2. all family members at risk of having a chromosome rearrangement.

List 5 prenatal indications for cytogenetics analysis.
1. Advanced maternal age (Greater than 35 years old) 2. Previous pregnancy with chromosomal disorder 3. One parent is a known carrier (or other relative*) 4. Couples at risk of x-linked disorders for which a molecular test is not available 5. Fetal defects on ultrasound, 6. Prenatal screen high risk pregnancies 7. couples with 2+ spontaneous abortions 8. infertility.

What are the indications for chromosomal analysis of products of conception?
1)Abortuses (missed abortions) of unknown reason, 2)Malformed stillbirths, 3)Stillbirth of undetermined etiology

Compare amniocentesis and chorionic villus sampling with regards to gestational age, complication rate, turn around time, and false results

What are the clinical indications for tissue sampling instead of blood for cytogenetic analysis?
1)Suspicion of chromosomal mosaicism, 2) blood is not available (e.g. POC), 3) surgical or post-mortem tissue.

List 8 standard techniques for cytogenetics analysis.
1) Geimsa / G-Banding, 2) Quinacrin / Q-banding 3) Reverse / R-banding, 4)Centromere / C-banding, 5)NOR staining (nucleolus organizer regions), 6)DAPI staining, 7) Chromosomal breakage, 8) Sister chromatid Exchange (SCE)

List 5 Molecular cytogenetics techniques.
1)FISH (flourescence in situ hybridization), 2) Multi-colour FISH, 3) SKY (spectral karyotyping), 4) CGH (comparative genomic hybridization), 5) CGH array

What is g-banding?
Chromosomes are treated with trypsine and then stained with Geimsa (or wrights) which darkly stains the AT rich regions (heterochromatin), and lightly stains the GC rich regions of the chromosome.

Outline the general procedure for cytogenetics study.

1) cell culture at 37C 5%CO2 in medium (dividing and stimulation), 2) Chromosome elongation Thymidine BrdU, 3) Metaphase arrest with Colcemide, 4) Cell swelling with hypotonic KCl,* Hardening with acetic acid* 5) Fixation with Cournay's (Methanol: Acetic acid, 3:1), 6) Slide making (chromosome spread with ideal temperature and humidity), 7) Slide aging (air dry slide warmer), 8)Staining (G, Q, C, R-banding), 8) Molecular cytogenetic technique (FISH, multi-FISH, CGH, SKY, array CGH).

Broadly what at the three main morphological groups of chromosomes?
Metacentric, acrocentric, submetacentric.

What are the 4 minimum items included in a standard banding nomenclature?
1. Chromosome number, 2) short or long arm, 3) region on that arm, 4) band number within that region

What are the clinical indications for an individual to have chromosomal analysis?
1)suspected classic chromosome syndrome, 2) Mental retardation of undetermined etiology, 3) dysmophic features, 4) multiple congenital abnormalities, 5) abnormalities of sexual development, 6) ambiguous genitalia, 7)pubertal failure, 8)abnormalities of growth, 9) certain types of malignancies.

What is q-banding?
Chromosomes are prepared with quinacrine which produces flourescent bands in the AT rich regions, particularly useful in identifying polymorphisms on the acrocentric chromosomes ( ) and the Y chromosome.

What is R-banding?
Darkly stains the GC rich regions of the chromosome (Euchromatin), aka Reverse-banding, and is used to detect subtle deletions or rearrangements that may not be detected by Q or G banding.

What is C-banding?
C-Banding stains the constitutive heterochromatin that is localized to the pericentromeric regions of all chromosomes and on the distal long arm of Y. Used to identify pericentric inversions and polymorphisms in centromeric regions of 1,9,16, and Yq, as well as confirming translocations of Y

What is NOR?
NOR is a silver staining procedure which stains the nucleolus organizer regions of satellited chromosomes (used to study the size of stalks and satellites in the acrocentric chromosomes)

List the metacentric chromosomes.

List the submetacentric chromosomes.

List the acrocentric chromosomes.

What is Bloom syndrome?
Bloom syndrome is a rare AR genetic disorder with a defect in the BLM gene with a phenotype of short stature, tendency to sunburn, increased risk of malignancy, reduced or absent fertility, and prone to sister chromatid exchange [[1]]

What is SCE (Sister chromatid exchange?
SCE (sister chromatid exchange) is the interchange of homologous segments between two chromatids of one chromosome, grow the cells under special conditions to produce a differential staining of sister chromatids.

What is DAPI staining?
DAPI staining produces bright fluorescence of the heterochromatin regions of 1,9,16, and Y, as well as the centromere of 15, and is used to id marker chromosomes or translocations of Y.

Explain how chromosomal breakage studies are used to diagnose Fanconi's anemia.
Cultured cells are treated with Diepoxybutane, or mitomycin C to induce breakage, those cells with chromosomes prone to breakage are especially susceptible and this can be seen as gaps, breaks, deletions, triradial, quadriradial, dicentric, and complex figure in the metaphase.

Lecture 2

Describe the 4 steps of mitosis.
Prophase, metaphase, anaphase, telophase

List the 8 steps of meiosis.
Meiosis 1(Prophase 1, Metaphase 1, Anaphase 1, Telophase 1), Meiosis 2( Prophase 2, Metaphase 2, Anaphase 2, Telophase 2).

What is the main difference between constitutional and acquired chromosome anomalies.
Constitutional affects the whole patient, acquired usually limited to 1 organ.

What at the three main categories of patient features associated with unbalanced constitutional chromosomal anomalies?
1) dysmophy 2) Visceral malformations, 3) developmental/psychomotor delay.

What is meant by a homogeneous chromosomal anomaly?
Homogeneous chromosomal anomalies mean that all the cells STUDIED carry the anomaly, may be constitutional or acquired.

What is meant by a mosaic chromosomal anomaly?
Mosaic chromosomal anomalies mean that only some of the cells STUDIED carry the anomaly, may be constitutional or acquired.

What are chromosomal polymorphisms?
Chromosomal polymorphisms are variants of chromosomes that are widespread in a particular population which to date are not known to have any effect on the phenotype, they vary in size, position, and staining properties but must occur in heterochromatin regions usually near the centromere.

List 3 known chromosomal polymorphisms, according to ISCN 2013.
Chromosomal polymorphisms

Classify numerical abnormalities of chromosomes.
1) polyploidy (multiple complete sets of chromosomes, e.g. 3N), 2) Aneuploidy (monosomy (e.g. Turner's syndrome), trisomy (e.g. trisomy 18, 13, or 21), tetrasomy))

What are the four main types of abnormalities in chromosome structure?
1) Deletion, 2) Duplication, 3) Rearrangement (inversion or insertion), 4) Translocation

What is the key difference between a balanced and an unbalanced chromosomal rearrangement?
Balanced translocations imply that there is no missing or excess genetic material, while unbalanced translocations have either missing or excess genetic material from that of a normal genotype.

List three types of balanced chromosomal rearrangements.
Translocation, inversion, insertion.

List three unbalanced numerical chromosomal rearrangements.
trisomy, monosomy, multiploidy

List 5 structural unbalanced chromosomal rearrangements.
deletion duplication derivative chromsome recombination chromosome marker chromosome ring chromosome Dm & HSR

What is the karyotype for a female infant with cri-du-chat?
46,XX,del(5)(p15.1)

Lecture 3

What is FISH?
FISH is a molecular cytogenetic technique in which flourescently labelled DNA probes are hybridized to metaphase spreads or interphase nuclei.

When is interphase FISH more helpful than metaphase?
Interphase FISH is particularly useful in samples where there is poor culture growth such as bone marrow or cancer tissue.

What is the approximate resolution of cytogenetic FISH?
3-5Mb

What are the three types of FISH probes?
1)Probes for repetitive sequences (Centromeres, telomeric sequences), 2) Unique sequence probes hybridized to a single copy of DNA sequences in a specific gene or chromosome, 3) Whole chromosome paints (or arms) which are cocktails of probes that are chromosome specific and cover the entire length.

List 7 applications of FISH technology?
1) Microdeletion syndromes, 2) Characterization of chromosomal structural abnormalities, 3) identification of marker chromosomes, 4) Aneuploidy detection, 5) Cancer cytogenetics, 6) Gene mapping, 7)Rapid detection of sex chromosomes and the SRY gene

List 5 microdeletion syndromes.
List of Microdeletion Syndromes

Briefly describe Cri-du Chat Syndrome

Describe 3 mechanisms by which uniparental disomy occurs.
1) Trisomic rescue (loss of a chromosome from a trisomic zygote), 2) monosomic rescue (duplication of a chromosome from a monosomic zygote), 3)Gamete complementation (fertilization of a gamete with two copies of a chromosome with no copies from other parent)

What is imprinting?
Normally we inherit one copy of each gene from each parent, some genes are only expressed when they are inherited paternally, some only when maternally, this differential expression based on inheritance is called imprinting, and changes generation to generation.

Which chromosomes are known to have imprinted genes?
Chromosomes 6,7,11,14,and 15.

Describe Prader-Willi Syndrome.
Features: hypotonia, obesity, developmental delay, hypogonadism, short stature, 70%: del(15q11-13), 25% uniparental disomy, 2%:other, diagnoses by FISH for microdeletion, or DNA methylation; due to absence of paternally derived PWS/AS gene

Briefly describe Williams Syndrome.
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Describe DeGeorge Syndrome.
95% 22q11.2 deletion, 5% FISH negative; AD inherit; 1) Conotruncal heart defects, 2)uropathy, 3)polyhydramnios, 4)increased nuchal translucency, 5) IUGR, 6)thymic hypoplasia, 7) characteristic facies, 8) hypoparathyroidism, 9)MR/DD

What is SKY?
A chromosomal analysis technique that has the ability to paint each pair of chromosomes and the sex chromosomes a different flourescing colour.

What kinds of chromosomal transformations is SKY used for?
1) translocations, 2) insertions, 3)marker chromosome identification, 4) cancer tumour genetics

What are thelimitations of SKY?
1) cannot detect del,dup,inv, 2) interpretation difficult if colours too similar

Explain the basic principle of Comparative Genomic Hybridization.
References genomes and the index genome are mixed, if the index genome substantially differs from the reference genome then there will be a neg signal loss or gain for that probe's flourescence, this can be used to determine if there is one allele in the index case that is missing or in excess compared to the reference genome.

How do CGH arrays work?
CGH arrays allow hundreds-thousands of probes to be used to compare the index and the reference genome, giving a complete chromosomal analysis that depends on the resolution of the probe.

Lecture 4

List 3 solid tumours for which cancer cytogenetics are currently used in prognosis and treatment.
1. Lymphoma 2. Breast cancer 3. Bladder carcinoma

What is a chromosomal instability syndrome?
There are several rare single gene syndromes in which there is a characteristic cytogenetic abnormality; affected individuals exhibit elevated rates of chromosome instability, leading to chromosomal rearrangements.

What are the features of ataxia telangiectasia?
1) AR inhertiance 1/40,000, ATM:11q22.3-q23.1 2) Cerebellar ataxia 3) Telangiectasia 4) Growth retardation 5) Immunodeficiency 6) Radiosensitivity *tx with conventional radiation doses, could be fatal 7) Cytogenetics: Chromosomal breakages, telomere instability, radiation sensitivity t(7;14)

What is Nijmegen Breakage Syndrome?
1. microcephaly 2. Bird like face 3. Radiosensitivity 4. rearrangements between 7 and 14, AR, rare NBS1(8q21.3) 5. sensitive to x-rays and bleomycin 6. Growth and mental retardation 7. Ovarian failure 8. Prone to b-cell lymphomas

What is Bloom syndrome?
AR inheritance, rare (1/160,000, BLM:15q26.1, SCE and quadrils growth retartdation/ short stature sun sensitivity / facial lesions Ashkenazi jews

What is Xeroderma pigmentosum?

What is Fanconi Anemia?

What is ICF Syndrome?

What is Roberts Syndrome?

What karytype is most at risk of gonadoblastoma?

What cancer are Kleinfelters patients at increased risk of?

What lymphoproliferative disorders are associated with Down's Syndrome?

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Miscellaneous

What are the steps in preparing a cytogenetics tissue specimen?

1. Specimen received in flow medium and accessioned asap. 2. Specimen cut-up (+/- treated with collagenase), filtered /18G needle 3. Seeded into flask 4. Cultured at 37C 5% CO2 x 48hours 5. Flask flooded with 2ml of media 6. Cultured at 37C 5% CO@ x 2-10days 7. Trypsinize to coverslip when flask growth is confluent 8. Colcemid added to the coverslip x 30min (1/12 dilution) 9. Aspirate off colcemid 10. Add hypo (1/2 0.54 KCl, 1/2 0.75 NaCitrate) x 30 min 11. Add 2mL of fix (1/3 Methanol, 1/3

What is Allerdice or Sandy Point Syndrome?
It is a chromosomal disorder discovered in Sandy Point, NL by Dr. Penny Allderdice, inv(3)(p25q21) characterized by affected offspring with multiple congenital anomalies with surviving children exhibiting severe growth and developmental delays.

What is the most common robertsonian translocation?
Translocation between the long arms of 13 and 14.

What is the most common non-robertsonian translocation?
t(11;22)(q23;q11)

What is a marker chromosome?
A structurally abnormal chromosome in which no part can be identified cytogenetically.

What is the most common chromosomal abnormality in humans?
Aneuploidy - about 5% of pregnancies.

What is the most common cause of triploidy?
Dispermy in 60%

What is the recurrence risk for parents of Down's syndrome child with a "free chromosome"?
1%

List 5 features of Trisomy 8.
1. Prominent cup-shaped ears 2. Prominent lips 3. Somewhat prominent forehead 4. IQ usually 45 to 75 but some have near normal intelligence 5. Deep, longitudinal grooves on soles of feet 6. Joint abnormalities 7. Usually mosaic

List 5 features of Trisomy 9.
1. Growth deficiency (prenatal) 2. Deep-set eyes 3. Low-set, misshapen ears 4. Small lower jaw causes upper lip to overlap lower lip 5. Joint abnormalities 6. Heart and kidney abnormalities 7. Small genitalia (males) 8. Most have severe mental retardation 9. Usually mosaic

List 5 features of Trisomy 13.
1. Cleft lip and/or palate 2. Low set, misshapen ears 3. Small eyes 4. Hemangioma(s) on the face/forehead 5. Defective lateral differentiation of the brain (some have holoprosencephaly) 6. Sixth finger on ulnar side of hand 7. Heart and kidney abnormalities 8. Cryptorchidism in male; bicornate uterus in females 9. Severe mental retardation

List 5 features of Trisomy 14.
1. Normal weight but short length at birth 2. Narrow deep-set eyes 3. Short, bulbous nose 4. Small lower jaw 5. Low-set, misshapen ears 6. Heart abnormalities 7. Severe mental retardation 8. Usually mosaic

List 5 features of Trisomy 18.
1. Growth deficiency (prenatal) 2. Prominent occiput 3. Small mouth and jaw 4. Low-set, misshapen ears 5. Short sternum 6. Clenched hand 7. Short big toe, often flexed upward 8. Rocker-bottom" feet 9. Small pelvis with limited hip movement 10. Heart abnormalities 11. Severe mental retardation

List 5 features of Trisomy 21.
1. Flat face 2. Brushfield spots present when eyes are blue 3. Upslanting eyes 4. Small ears 5. Small mouth (with tongue often protruding) 6. short fingers (especially fifth 7. Heart abnormalities in some cases 8. IQ usually 25-50 but occasionally higher

What is the most common outcome of a pregnancy when the parent has a balanced translocation?
Misscarriage