5 - Clinical Cytogenetics

Question 1

What are the three functional elements that chromosomes require for stability?

Answer 1

1. Origin of replication
2. Centromere
3. Telomere

Question 2

Describe the origin of replication of a chromosome?

Answer 2

Multiple locations on a chromosome where DNA replication can begin.

Question 3

What is the function of the centromere?

Answer 3

Attachment point for spindle and thus is essential for chrom movement from equator during mitosis and meiosis.

Question 4

Describe centromeres on human chromosomes?

Answer 4

Regional centromeres: their function resides in epigenetic marks provided by CENP histones.

Composed of tandemly repeated 171 bp AT-rich sequences called a-satellite DNA.

Question 5

What does the alpha-satelite DNA contain? What else is at the centrosome?

Answer 5

A 1 7bp binding motif for CENP-B.

CENP-A replaces histone H3 at a small number of nucleosomes at the centromere.

Centromeric DNA supports assembly of kinetochore protein complex for spindle attachment.

Question 6

What are telomeres?

Answer 6

Repeated TTAGGG sequence at the end of chromosomes.

Characteristic terminal loop.

Binds protein complex called shelterin that blocks access to DNA repair machinery, limiting
recombination.

Question 7

What is the Hayflick limit?

Answer 7

The limited capacity that a somatic cell has to divide in its lifetime.

Differs by tissue.

Question 8

How do cells in the germline and cancer cells differ from somatic cells?

Answer 8

They have telomerase, which can lengthen telemeres by adding TTAGGG hexamers.

Question 9

What happens to cells as they approach the Hayflick limit? What else can occur to cells with very short telomeres?

Answer 9

Undergo growth arrest and they become post-mitotic.

Critically short telomeres stimulate p53-mediated arrest. They could also have damage that activates another type of tumor suppressor to undergo apoptosis.

Question 10

What happens when a subset of cells escape the various protective mechanisms that should occur while approaching Hayflicks limit?

Answer 10

Senescence, a post-mitotic state that features resistance to apoptosis.

If they continue to divide, they undergo crisis. If they survive this, they are transformed and have high malignant potential.

Question 11

What happens to acentric fragments of chromosomes during recombination?

Answer 11

Without a centromere, they don’t move from the equatorial plate at anaphase and are lost at high freq.

This leads to deletions of genes carried by them.

Question 12

What happens to dicentric chromosomes during recombination?

Answer 12

They go through a breakage-fusion-bridge cycle.

At anaphase they may migrate to opposite poles, breaking the chrom between them. The free ends are recombinogenic and promote rearrangements. This is a common occurrence at crisis, when telomere insufficiency can initiate formation of a dicentric chrom.

Question 13

What is a paracentric inversion?

Answer 13

An inversion that does not include the centromere and both breaks occur in one arm of the chromosome.

Question 14

What happens if crossing over occurs within a paracentric inversion?

Answer 14

Then the recombinant chromosomes include a dicentric chromosome and an acentric chromosome.

Question 15

What is a pericentric inversion?

Answer 15

An inversion that includes the centromere and there is a break point in each arm.

Question 16

How can translocations be recognized in meiosis?

Answer 16

When the chromosomes fuse, you will see quadrivalents (four chrom fused together) if a translocation has occurred.

Normal karyotypes only form bivalents.

Question 17

What are autosomal aneuploidies? What is the consequence?

Answer 17

An abnormal number of chromosomes in an autosomal cell.

Most autosomal aneuploidies, including autosomal monosomies and trisomies, are lethal in gestation.

Question 18

What is down syndrome caused by? What are the characteristics? What is the incidence of down syndrome?

Answer 18

Most common autosomal aneuploidy, trisomy 21, typically from maternal non-disjunction.

Intellectual impairment, hypotonia, prominent epicanthic folds, simian palm. Increased risk of leukemia and short statues.

~1/700 live births

Question 19

What is Edwards syndrome? What is the incidence? What are the characteristics?

Answer 19

Trisomy 18, incidence ~1/5000 live births (usually die within days of birth).

Microsomia, microcephaly, cardiac defects, microopthalmia (small jaw), overlapping digits.

Question 20

What is Pateau syndrome? What are some associated characteristics? What is the incidence?

Answer 20

Trisomy 13, incidence ~1/15,000. Survival usually less than one year.

Midline defects: holoprosencephaly (undeveloped forebrain), cleft lip/palate, cardiac defects. Polydactyly, prominent heel.

Question 21

How does a variable number of X chromosomes escape the dire consequences that accompany autosomal aneuploidy?

Answer 21

Most X-linked genes are subjected to dosage compensation.

Regardless of the number of X chrom, most X-linked genes are expressed at the same level because all X chrom except one are epigeneticallt silenced.

Question 22

How are all but one X chromosomes silenced?

Answer 22

The chrom has an X inactivation center that includes the Xist locus.

Xist encodes non-coding RNA that can bind the X chromosome from the inactivation center and to the adjacent regions.

Question 23

What does Xist binding to the X chromosome do?

Answer 23

Promotes stable epigenetic silencing of chromosome via specific DNA methylaton/demethylation, histone methylation/demethyl and histone acetylation/deacetylation.

Question 24

What happens to the inactive X chromosome(S)? How does the number of X chromosomes compare to the number of barr bodies?

Answer 24

It stays in a concensed heterochromatin state and manifests as a barr body, a dense structure adjacent to the nuclear envelope.

Number of bass bodies is one less than the number of X chromosomes.

Question 25

What happens to the inactivated X chrom in germ-line cells? How does this occur?

Answer 25

It is reactivated prior to meiosis.

The Xist locus encodes a second non-coding transcript, TsiX, from the opposite strand.

Xist and Tsix RNAs are complementary and bind to eachother to prevent Xist from binding the X chromosome.

This leaves the X chromosome active.

Question 26

Is all of the barr body inactivated?

Answer 26

No, theres a small region that’s still active and expression of the genes within this region is NOT dosage compensated.

Phenotypes observed in pts with X chrom aneuploidy are attributed to this.

Question 27

What is Klinefelter syndrome? What are the corresponding characteristics? What is the incidence?

Answer 27

XXY, but can have 3 or 4X.

Tall stature, hypogonadism, small testes, female fat distribution, gynecomastia.

Sterile secondary to immature sperm.

~1/700 live births.

Question 28

What is Jacob’s syndrome? What are the associated characteristics? What is the incidence?

Answer 28

Caused by extra Y chromosome.

Tall stature, mild cognitive impairment, dyslexia, mild developmental delay.

~1/700 live births.

Question 29

What is turners syndrome? What are the associated characteristics? What is the incidence?

Answer 29

Anueploidy of sex chromosome: 45 X0 karyotype.

Short statue, broad webbed neck, primary amenorrhea, sterility, heart defects.

~1:2000 love born, 99% lost before term.

Question 30

What are the characteristics of a triple X (or more) female? What is the incidence?

Answer 30

Phenotype (if any) is mild. Difficult to diagnose.

Tall statue, hypertelorism, exaggerated epicanthal folds, hypotonia.

~1/100 live female births.

Question 31

How does G-banding for normal karyotypes work?

Answer 31

Prepared from metaphase chromosome preparation.

Stained by C-banding or giemsa. The basic karyotype (visual appearance of the chromosome) is sufficient to detect aneuploidy.

Question 32

What are the advantages of G-banded normal karyotypes? What are the disadvantages?

Answer 32

Low cost, widespread availability.

Disadvantages: technical demands, need to examine many cells (hundreds) from each sample, time consuming.

Question 33

What is single locus FISH and how is it performed? What is an example of a disease that can be detected using this technique?

Answer 33

Fluorescent in situ hybridization - used to detect specific rearrangements such as chronic myelogenous leukemia.

Prepare chromosomes, denature on slide, and hybridize with fluorescent probes.

Question 34

What is an advantage to single locus FISH? What is the disadvantage?

Answer 34

It’s good if you’re seeking a specific hypothesized abnormality.

Not good if you don’t know what you’re looking for. (if this is the case, you should do chrom painting).

Question 35

What is chromosome painting? What is the limitation?

Answer 35

Uses hybridization to repetitive sequences that are unique to each chrom, allowing recognition of rearrangements.

Limitation: requires good metaphase preparations and have limited resolving power.

Question 36

What is comparative genomic hybridization (CGH)?

Answer 36

Same concept of hybridizing a fluorescent probe to DNA, but instead of having DNA presented as a chromosome spread it is bound to a microarray.

Microarray has high density of probes that span the genome.

Question 37

How is the reference DNA used in comparative genomic hybridization?

Answer 37

It is all diploid, so if there’s a copy number variation in the test DNA, then the test DNA will be either more or less abundant than in the reference DNA.

This results in a difference in signal intensity.

Question 38

What is the advantage to using comparative genomic hybridization?

Answer 38

Due to the high density of probes, very small regions of copy number variations can be detected.