02: Cytogenetics

Question 1

What is cytogenetics?

Answer 1

The study of inheritance in relation to the structure and function of chromosomes.

Must catch cell during active division (metaphase) in order to view chromosomes.

Question 2

What are the technical advances taht have made it easier to study cytogenetics?

Answer 2

• Mitogens, particularly phytohaemagglutinin (PHA), push cells into active division
• Spindle poisons (colchicine) produce metaphase arrest
• Hypotonic solutions rupture nucleus, spreading out the chromosomes
• Differential staining

Question 3

How are chromosomes identified?

Answer 3

Based on their size, banding pattern and the position of the centromere.

Banding resolution: how many visible bands; “stretch” the chromosome to see more

Question 4

Name the parts of a chromosome and the types.

Answer 4

p: short arm

q: long arm

telomere: end of chromosome

centromere: center of chromosome

satellite: secondary, pinched of constriction

stalk: secondary constriction containing genes which code for ribosomal RNA

heterochromatin: tightly packed form of DNA

metacentric: p & q equal size

sub-metacentric: distinctly shorter p

acrocentric: almost no p

Question 5

Which chromosomes have large heterochromatic regions?

Answer 5

1, 9, 16 & Y

(constitutive heterochromatin)

Question 6

Which chromosomes are acrocentric?

Answer 6

13, 14, 15, 21, 22 & Y

Question 7

What are the types of banding methods used?

Answer 7

G-Banding: G bands produced with Trypsin using Giemsa (GTG); routine method in USA.

Q-Banding: Fluorochromes bind DNA and produce distinct banding patterns of bright/dull fluorescence; first banding method developed for human chromosomes.

C-Banding: C bands produced with Barium hydroxide using Giemsa (CBG); stains constitutive heterochromatin (1, 9, 16, Y).

R-Banding: Pattern is opposite of G-Banding

Question 8

How does chromosome nomenclature work?

Answer 8

Each chromosome arm is divided into regions, or cytogenetic bands, that can be seen using a microscope and special stains.

The cytogenetic bands are labeled p1, p2, p3, q1, q2, q3, etc., counting from the centromere out toward the telomeres.

At higher resolutions, sub-bands can be seen within the bands. The sub-bands are also numbered from the centromere out toward the telomere.

For example, the cytogenetic map location of the CFTR gene is 7q31.2, which indicates it is on chromosome 7, q arm, band 3, sub-band 1, and sub-sub-band 2.

Question 9

What is the process for preparing chromosomes for cytogenetics?

Answer 9

1. Add tissue sample
2. Culture in growth medium
3. Add PHA to stimulate mitosis
4. Incubate 2-3 days
5. Add colchicine to stop mitosis in metaphase
6. Transfer cells to tube and centrifuge to concentrate in layers
7. Transfer to tube containing hypotonic fixative
8. Put cells on microscope slide
9. Add Giemsa stain to enhance chromosomes
10. Identify and photograph chromosomes
11. Cut out chromosome pictures and arrange into a karyotype

Question 10

What is the human genome and what are its basic characteristics?

Answer 10

The complete set of genetic information for humans; this information is located as DNA sequences within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual mitochondria.

6 million base pairs of DNA (~3 meters)

15-20K genes encoding 80-100K proteins

Genes spread out over 24 different chromosomes

One chromosome from each parent for a total of 23 pairs (24 different b/c X & Y), or 46 chromosomes per somatic cell.

Question 11

What is a chromosomal imbalance?

Answer 11

Either the gain/loss of a whole chromosome (full/complete aneuploidy) or part of a chromosome (partial aneuploidy).

May occur in the mosaic or non-mosaic state (mosaicism: presence of two or more populations of cells with different genotypes in one individual who has developed from a single fertilized egg).

Monosomy (one missing chromosome) is generally more devastating than trisomy (one extra chromosome).

Question 12

What is a ploidy?

Answer 12

A category of chromosome changes which involve the addition or loss of complete sets of chromosomes.

Triploidy is the posession of one complete extra set of chromosomes (caused by polyspermy, the fertilization of an egg by more than one sperm); embryos usually spontaneously abort. Written as 69, XXX.

Tetraploidy usually results from the failure of the first zygotic divison; also lethal to the embryo. Written as 92, XXXX.

Question 13

What complete non-mosaic aneuploidies can be observed in liveborns (and are thus surviveable)?

Answer 13

• Down syndrome (Trisomy 21)
• Edward syndrome (Trisomy 18)
• Patau syndrome (Trisomy 13)
• Turner syndrome (Monosomy X)

Other chromosomal imbalances will contain much smaller chromosomal regions (partial aneuploidy) that would allow for the organism to survive.

Question 14

What are the common clinical phenotypes of chromosomal abnormalities?

Answer 14

• Developmental delay/intellectual disability (ID)
• Alteration of facial morphogenesis
• Growth delay
• Malformations of the internal organs (especiall cardiac)

MCA (multiple congenital abnormalities) and ID are indications for chromosome analysis

Question 15

For Down Syndrome, describe:

1. Cytogenetics
2. Phenotype
3. Medical complications

Answer 15

Cytogenetics:

1. Trisomy 21 (47,XY,+21)
2. 95% caused by nondisjunction
3. 95% of trisomies from maternal nondisjunction
4. 75% of errors occur during meiosis I

Phenotype:

1. Hypotonia (low muscle tone)
2. Redundant neck fold/flat occiput
3. Low-set ears w/ characteristic pinnae
4. Protruding/large tongue
5. Abnormal dermatoglyphics (simian line, clinodactyly, wide space between 1st and 2nd toes)
6. Brushfield spots (eyes)

Medical complications:

1. Leukemia (15-20x)
2. Moderate to severe ID (100%)
3. Congenital heart defect (40%)
4. GI obstruction (3%)
5. Respiratory infections common

Question 16

For Edward Syndrome, describe:

1. Cytogenetics
2. Incidence
3. Prognosis
4. Phenotype
5. Medical complications

Answer 16

1. Trisomy 18 (47, XY, +18)
2. 1:3333 live births (most common abnormality in stillbirths)
3. 10% alive by 1 year
4. Rocker-Bottom feet, marked ID
5. Prenatal growth deficiency results in small for gestational age infant (SGA), 90% congenital heart defect (ventricular septal defect)

Question 17

For Patau Syndrome, describe:

1. Cytogenetics
2. Incidence
3. Prognosis
4. Phenotype

Answer 17

1. Trisomy 13 (47, XY, +13); 75% due to meiotic nondisjunction; 20% from translocations (25% of which are familial); 5% due to mosaicism (mitotic nondisjunction)
2. 1:5000 live births (95% spontaneously aborted)
3. 10% alive by 1 year
4. Microcephaly with sloping forehead, holoprosencephaly, ophthalmologic abnormalities, cleft lip +/- palate, low set ears with abnormal helices, polydactyly

Question 18

For Turner Syndrome, describe:

1. Cytogenetics
2. Incidence
3. Phenotype
4. Medical complications
5. Treatment

Answer 18

1. 45, X0 (50%; ~10% spontaneously abort), 45, X0/46XX or 45,X0/46,XY (mosaics; 30-40%); structural X abnormalities (10-20%); 80% are due to loss of paternal X chromosome
2. 1:5000 live female births (99% spontaneously aborted)
3. Very short, infertile, broad chest, widely-spaced nipples, webbed neck, spatial perception decreased
4. Structural kidney defects (50%), coarctation of the aorta (20%)
5. Growth hormone and estrogen

Question 19

For Klinefelter Syndrome, describe:

1. Cytogenetics
2. Incidence
3. Phenotype

Answer 19

1. 47, XXY (48,XXXY or 49,XXXXY rare; severe IDs)
2. 1:1000 live male births
3. Testes small, fail to produce normal levels of testosterone, leading to breast growth (gynaecomastia) in 40% of cases; poorly developed secondary sexual characteristics; no spermatogenesis (sterility); taller and thinner; slight reduction in IQ (10-15 points below siblings)

Question 20

For Triple X, describe:

1. Cytogenetics
2. Incidence
3. Phenotype

Answer 20

1. 47, XXX (48,XXXX and 49,XXXXX rare; mild to moderate ID, variable dysmorphic features)
2. 1:1000 female live births
3. No phenotypic abnormalities; may have sterility or learning disabilities (speech/language); IQ reduction (10-15 points below siblings)

Question 21

For XYY Syndrome, describe:

1. Incidence
2. Phenotype

Answer 21

1. 1:1000 live male births
2. May be phenotypically normal; tall but normally proportioned; 10-15 point IQ reduction (?); problems with impulse control (?)

Question 22

Maternal age is well-recognized in all aneuploidies except which two?

Answer 22

45,X (Turner) and XYY Syndrome

Question 23

What is partial aneuploidy?

Answer 23

Deletions resulting in smaller chromosomal regions which allow for the conceptus to survive, albeit with clinical abnormalities in most cases.

Result from unbalanced rearrangements (translocations and inversions), deletions, duplications, isochromosomes, rings and supernumerary marker chromosomes.

Question 24

What are the types of translocations and inversions?

Answer 24

Translocations:

1. Balanced (no material lossed)
2. Reciprocal (equal exchange of material)
3. Unbalanced (leads to partial mono/trisomy)

Inversions:

1. Pericentric (include centromere)
2. Paracentric (arm only)

Question 25

What is a **Robertsonian translocation**?

Answer 25

Translocation between acrocentric chromosomes (13, 14, 15, 21, 22). Short arm are lost and long arms fuse at centromere. Accounts for 5% of Down syndrome.

Question 26

What is the meiotic behavior of a **paracentric inversion**?

Answer 26

* **Dicentric bridge** is pulled in opposite directions, with the chromosome suspended between the daughter nuclei at telophase. * **Acentric fragment** is not attached to a spindle fiber and is lost.

Question 27

Which is worse: Pericentric or paracentric inversion? Why?

Answer 27

* A **pericentric inversion** leads to a crossover within the loop, resulting in recombinant chromosomes with duplications/deletions distal to the inversion breakpoints. * A **paracentric inversion** crossover within the inversion loop results in the formation of an **acentric fragment** and a **dicentric recombinant chromosome**. * Pericentric inversion is worse, becasue it leads to the birth of a child with abnormalities; a paracentric inversion is lethal.

Question 28

What is the origin of **supernumerary marker chromosomes (SMCs)**?

Answer 28

* **Trisomy rescue**; third chromosome chopped and marker chromosome is remnant. * Incidence in consecutive births is 0.3:1000; risk for phenotypic consequence is minimal. * Risk based on size, morphology and origin of SMC; 80% from acrocentric chromosomes, half of which derived from 15. * Maternally-derived duplications of imprinted proximal 15q region strongly correlated with moderate-severe ID. * Duplications of defined regions of proximal q22 have **Cat Eye Syndrome**.

Question 29

What is **Cri du Chat Syndrome**?

Answer 29

* Deletion of 5p * Phenotype: microcephaly, developmental delay, specific cry

Question 30

What is **Wolf-Hirschhorn Syndrome**?

Answer 30

* Deletion of 4p * 87% de novo interstitial deletion of paternal origin; 13% unbalanced product of parental reciprocal translocation

Question 31

What is an **isochromosome**, and what type of aneuploidy can it cause?

Answer 31

Abnormal chromosome with two identical arms, either two short (p) arms or two long (q) arm. Results in partial trisomy/tetrasomy or partial monosomy & partial trisomy

Question 32

What are the different classifications of chromosomal sequences?

Answer 32

1. Beta satellite 2. Alpha satellite (repeated info in centromere) 3. Classical satellite 4. Telomeric sequences (first unique sequences just next to telomere) 5. Unique gene sequences: large, repeated region

Question 33

What are **microdeletion syndromes**?

Answer 33

Deletion of ~1MB of DNA so that they are most often too small to be seen under the microscope

Question 34

What is **Prader-Willi Syndrome**?

Answer 34

Microdeletion of the paternal copy of **15q11.2**. Phenotype: floppy, trouble feeding, insatiable, overweight, small genitalia, physically and emotionally immature, learning disabilities

Question 35

What is **Angelman syndrome**?

Answer 35

Microdeletion of the maternal copy of **15q11.2**. Phenotype: Developmental delay, speech disabilities, movement/balance disorder, excitable, inappropriately happy

Question 36

What is the cytogenetics of **Williams syndrome**?

Answer 36

Microdeletion on chromosome 7.

Question 37

What is the cytogenetics of **DiGeorge syndrome**?

Answer 37

Microdeletion on chromosome 22.

Question 38

What is the benefit of **Prenatal Interphase FISH**? What are its common **indications**? What are its limitations?

Answer 38

Routine chromosome analysis takes 7-10 days; PI-FISH provides a rapid way to screen for common aneuploidies (13, 18, 21 & X) in uncultured amniotic fluid cells in 1-2 days. Common indications: **advanced maternal age (AMA)**, abnormal maternal serum screening, prenatal ultrasound abnormalities Limitations: Reduced sensitivity compared with other cytogenetic analyses; only most frequent abnormalities detected

Question 39

How does **Comparative Genomic Hybridization (CGH)** work?

Answer 39

Identifies chromosomal gains and losses in a single hybridization procedure (thus revealing any DNA sequence copy number changes), mapping these on a normal chromosome. Red = deletion, green = amplification

Question 40

What are the diagnostic limits of **conventional cytogenetic analysis** (karyotyping)?

Answer 40

At absolute best, imbalances in the realm of 3-5Mb may be detected.

Question 41

What are **microarrays** used for?

Answer 41

* Clinical utility in constitutional & cancer cytogentic diagnostics, **preimplantation genetic diagnosis (PGD)**, hematological and solid tumors. * Limitations are **resolution** (tiny deletion) and **spacing** (deletion between probes). * CGH Microarray * Single Nucleotide Polymorphism Oligonucleotide Microarray Analysis (SOMA)