Chromosomal Abnormalities I

Question 1

Chromosome Structure

Answer 1

Chromosomes usually exists as chromatin. DNA double helix bounds to histones. Octamer of histones form nucleosome.

Question 2

Euchromatin

Answer 2

• Extended state, dispersed through nucleus

- Allows gene expression

Question 3

Heterochromatin

Answer 3

• Highly condensed, genes not expressed

- Tightly bound

Question 4

Genetic locus

Answer 4

The location of a particular gene on a chromosome also can be defined as a genomic region.

Question 5

What is located at the genetic locus?

Answer 5

At each genetic locus, an individual has two alleles, one on each homologous chromosomes.

Question 6

Single chromosome and Double chromosome

Answer 6

In interphase, the chromosome is shown as a single chromatid. In S-phase, there is a doubling of DNA, with two duplicate chromatids which is also called a chromosome.

Question 7

S phase (synthesis)

Answer 7

• Go from single chromatid to double chromatid

- The allele to each loci on the chromatid is replicated identically.

Question 8

Metacentric

Answer 8

• p and q arm even length
• 1-3 and 16-18
• Centromere: In the middle of P+Q; important for telomeres to connect

Question 9

Submetacentric

Answer 9

• p arm shorter than q (p for petite)

Question 10

Acrocentric

Answer 10

• Long q and small p
• P contains no unique DNA
• 13-15, 21-22, Y

Question 11

Karyotyping

Answer 11

• Classic karyotyping is what is expected to be seen

- Easier to visualize as more condensed

Question 12

Which cells don’t have 23 pairs of perfect chromosomes?

Answer 12

Gametes have 23 single chromosomes - Polychromic.

But ideally, every cell apart from the gametes will have 23 perfect pairs

Question 13

What are the two types of chromosomal changes? How are these changes detected?

Answer 13

• Numerical: can be detected through traditional karyotyping, FISH, QF-PCR, NGS
• Structural: Can detect through traditional karyotyping, FISH

Question 14

Haploid

Answer 14

One set of chromosomes (n=23) as in a normal gamete

Question 15

Diploid

Answer 15

The cell contains two sets of chromosomes (2n = 46; normal in humans)

Question 16

Polyploid

Answer 16

Multiple of the haploid number (e.g. 4n = 92)

Question 17

Aneuploid

Answer 17

Chromosome number which is not an exact multiple of haploid number - due to extra or missing chromosome(s) e.g. 2n + 1 = 47 for example trisomies

Question 18

Numerical Abnormalities

Answer 18

• Trisomy: Aneuploidy
• Monosomy: Aneuploidy
• Mosaicism: Mixed group of cells; some may be aneuploidy and some are not.

Question 19

Summarise meiosis 1 and 2

Answer 19

Allelic recombination
Important that homologous chromosomes align correctly Homologous Pairs are pulled apart = disjunction
2 rounds of division to form haploid daughter cells

Question 20

Disjunction

Answer 20

Pulling apart at anaphase

Question 21

Summarise mitosis

Answer 21

• Single round
• No alignment of HP
• Generates diploid daughter cells that are identical to parents unlike meiosis

Question 22

How does aneuploidy arise?

Answer 22

Occur when there is non-disjunction = no separation.
Scenario 1)- this can be in one daughter cell that receives 2 chromosomes in meiosis 1.
- then in meiosis 2, there is a dizomic and the other has none.

Scenario 2) - Meiosis 1 is correct however, non-disjunction at meiosis 2 where there are two chromosomes in one daughter cell.

Question 23

What is formed when nondisjunction occurs?

Answer 23

There is a mixture of trisomy and monosomy whenever non-disjunction occurs. The implications depend on which chromosomes occur.

Question 24

What are some of the common aneuploidy abnormalities? What happens to most trisomies and monosomies?

Answer 24

• Trisomy 13 - Pateau
• Trisomy 18 - Edward’s
• Trisomy 21 - Down’s
• Autosomes are sensitive to most trisomies and monosomies so most will miscarry naturally.

Question 25

What causes mosacisim?

Answer 25

Mitotic non-disjunction - In the daughter cell, one will have 3 copies and the other 1 copy. This is perpetuated in future daughter cells. - Monosomies usually are broken down but some trisomies are maintained. - End up with a mixture of diploid and trisomic = mosaicism.

Question 26

Why is mosaicism important in terms of clinical presentation of diseases?

Answer 26

For example, in down's - the disease is less severe if there are mosaic body cells.

Question 27

Define mosaicism

Answer 27

The presence of two or more genetically different cell lines derived from a single zygote.

Question 28

Apart from mitotic non-disjunction; what else can cause mosaicism?

Answer 28

Anaphase lag

Question 29

What is anaphase lag?

Answer 29

- Same as trisomic rescue - Slow-down anaphase so doesn't work properly - Meiotic NDJ -> every cell is trisomic but then during post-zygotic proliferation -> Anaphase is paused. - One of the chromosomes in the cell is removed in a vesicle. - This results in 2n rather than 2n +1. This causes a mixture again.

Question 30

Clinical relevance of Mosaicism

Answer 30

- Mosaic phenotypes thought to be less severe - Difficult to asses the proportion of the different cells and which tissues/organs are affected - Difficult to predict the mosaicism that are seen in pretty much all chromosomal trisomies. - Examples of mosaic diseases: down, klinefelter and turner - Klinefelter and turners: Not diagnosed till after puberty or children- wanted.

Question 31

Monosomy

Answer 31

- Autosomal are very rare - only one case reported which is thought to be a mistake. - Relatively common sex chromosome monosomy = Turner's - Full monosomy arises by Meiotic NDJ - Partial Monosomy (microdeletion syndromes - can also lead to Turner's) far more common - Pateau's and Edward's less likely to live as long. - Monosomy in autosomy doesn't occur really. Less damaging to have multiple copies that it is to have one copy.

Question 32

How does Turner's syndrome (45, X) arise?

Answer 32

1) Non-disjunction of chromosomes in Meiosis 1 | 2) So extra sex chromosomes in Meiosis 2 in an egg

Question 33

Nullisomic gametes

Answer 33

1. Empty + X chr = XO = Turner's (physically female) | 2. Empty + Y chr = lethal

Question 34

Disomic gametes

Answer 34

XX 1. XX + X chr = XXX = Triple X syndrome 2. XX + Y chr = XXY = Klinefelter's (physically male) XY 1. XY + X chr = XXY = Klinefelter's 2. XY + Y chr = XYY = XYY syndrome - thought to be associated with violence as a lot of male prisoners with violent crimes have this.

Question 35

Summary of numerical abnormalities

Answer 35

- Types (all can be mosaic): - Autosomal: Trisomy 13, 18, 21 - Sex chromosomes: XO, XXY, XYY - Mechanisms: - Nondisjunction - Anaphase lag

Question 36

How to find out a karyotype?

Answer 36

1. Take blood 2. Add Culture medium and phylomagglutinin 3. Culture at 37 degrees for 3 days 4. Add colchicine and hypotonic saline 5. Cells will be fixed 6. Spread cells onto slide by dropping (burst cells) 7. Digest with trypsin and stain with Giernsa 8. Analyse "metaphase spread" 9. Forms a karyotype

Question 37

How to diagnose chromosome abnormalities prenatally?

Answer 37

- Chorionic Villus Sampling - Amniocentesis - then the samples taken can be used to conduct a karyotype

Question 38

Chorionic Villus Sampling

Answer 38

- 11-14 weeks - Miscarriage rate 0.5% to 1% - Maternal contamination - Transverse limb defects - Any invasive test is only done if there are limited risks e.g. age etc - Most chromosomal work is fetal

Question 39

Amniocentesis

Answer 39

- > 16 weeks - Extraction of amniotic fluid - Biochemical diagnosis possible - Miscarriage risk (0.5 - 1%) - Not used as a definitive but cells are cultured

Question 40

G-banding

Answer 40

- Uses a Giemsa stain (G = Giemsa) - Metaphase of chromosomes shown - Classical karyotyping methodology - Line-up based on size, banding, centromere position - The difference in band colour is to do with hetero (dark) and euchromatin (light). - Heterochromatin: AT-rich; tightly packed; genes inactive - Euchromatin: GC-rich; loosley packed; genes active - Both stain differently

Question 41

What is FISH?

Answer 41

Fluorescent in situ hybridization - Cultured cells - Microscopic (5-10Mb) - detects for deletions to this size anything smaller won't be detected.

Question 42

How is FISH carried out?

Answer 42

1. Fluorescent probe 2. Denature probe and target DNA (typically single-stranded DNA probe) 3. Mix probe and target DNA 4. The probe binds to target 5. Using UV light, the probe will glow.

Question 43

What are some uses of fluorescence in chromosome testing?

Answer 43

- Spectral karyotyping = multiple probes looking over the whole chromosome - Target specific FISH

Question 44

What is a quicker test for trisomy?

Answer 44

Quantitative fluorescence PCR | - Using microsatellites

Question 45

Examples of invasive prenatal diagnosis testing

Answer 45

- Amniocentesis (14-20 wks, amniotic fluid) | - Chorionic villus sampling (CVS) (11-14 wks, placental cells)

Question 46

Examples of non-invasive prenatal diagnosis testing

Answer 46

- Cell-free foetal DNA (cffDNA): DNA fragments in maternal plasma (10 wks onwards) -> extract foetal DNA from maternal plasma by extracting and enriching plasma - Actually for trisomies still need confirmation with amnio/CVS

Question 47

How is the DNA from cffDNA used?

Answer 47

1. Can use standard PCR to amplify the gene in the cffDNA from foetal DNA. 2. Can use next-gen sequencing and capture foetal DNA that reads across the entire genome and will produce equal coverage.