Lecture 16

Question 1

Human Karyotype

Answer 1

46 chromosomes
23 pairs of chromosomes
22 pairs autosomes, 2 sex chromosomes
Arranged in order of Reducing size

Question 2

Human Karyotype clinically

Answer 2

Performed on any dividing cells(can only see in metaphase)
-bone marrow(has actively dividing cells), cancer, Fetal tissue) amniotic cells, chorionic villus, peripheral blood lymphocytes (constitutional chromosome abnomormality)
Gross (rather than specific genes) screen of human genetic material
-numerical and structural changes chromosomes
Congenital disorders - diagnostic information (trisomy)
Cancer- diagnostic and prognostic information (acquired genetic disorders- blood cancers, solid cancers(non-blood))

Question 3

Chromosome Nomenclature

Answer 3

p arm= short arm
q arm= long arm
Telomere= distal end
centromere= central area
9p34- abnormality being on the short arm of chromosome 9 at locus 34

Question 4

Chromosomal Abnormalities

Answer 4

Numerical or structural

Constitutional(downsyndrome) vs acquired( 50% lukemia)

Question 5

Numerical Chromosomal Abnormalities

Answer 5

Aneuploidy

• chromosome number that is not a multiple of the normal haploid number (=23)
• loss of 1 chromosomes = Monosomy (5 or 17)
• gain of 1 chromosome = trisomy
• caused by non-disjunction during meiosis= segregation (Zygote 2n+1 = extra copy of chromosomes)

Question 6

How common are numerical abnormalities?

Answer 6

Up to 20% of pregnancies spontaneously abort
Estimated that 50% of 1st trimester abortions due to chromosomal abnormalities (particularilies aneuploidy)
Most of these are numerical abnormalities - aneuploidy mostly trisomies incompatible for normal fetal development
-get a chromosome analysis

Question 7

Clinical conditions Congenital Down syndrome

Answer 7

Trisomy 21 down Syndrome
-most common congential chromosome disorders
-increase with maternal age
-clinical features:
Mental Retardation
Characteristic facial features
Other abnormalities e.g. cardiac, increase in leukaemia, GI
Congenital heart abnormalities, intestinal abnormalities (strictures within) , flatten nasal fold, prominent epicanthlic folds in corner of the eyes, shortened neck with thickened soft tissue
-200-300x fold increased risk of leukemia

Question 8

Clinical conditions Congenital Aneuploidy of the sex chromosomes

Answer 8

Aneuploidy of the sex chromosomes
Males: Klinefelter syndrome 47, XXY Gain chromosome. Poor beard growth, breast development, under-developed testes. Delayed development of puberty, early gynaecomastia.
Females: Turner Syndrome 45, X0 lost chromosomes. Characteristic facial features, web of skin, constriction of aorta, Poor breast development, under-developed ovaries. Delayed pubertal development

Question 9

Acquired abnormalities in case 39 year old male

Answer 9

High leukocyte count
splenomegaly
high white cell count
suspected chronic myeloid leukaemia
-characteristic acquired chromosomal abnormality: Philadelphia chromosome 22 very small. reciprocal translocation between chromosome 9 and 22. causes leukemia
\+ Alot of changes, C6
Treated with targeted tyrosine kinase inhibitor, with good molecular response
Noted to be developing gynecomastia
Additional laboratory studies 
-low testosterone level
-azoospermia
Acquired c.a.= leukemia
Constitutional abnormality= Klienfelters syndrome

Question 10

Mosaicism

Answer 10

somatic mosaicism
-structural or numerical
(most anaeploudies caused by non-dysjunction in gamete formation
-occurs past zygote formation (post fertilisation= mixture of cells. some trisomy, others monosomy and others normal)
-mitotic non-disjunction
-3% Downs cases
–less evident (phenotype milder)

Question 11

Structural chromosome abnormalities

Answer 11

Reciprocal translocations
Robertsonian Translocations
Inversions
Deletions
-all of these can be either congenital or acquired abnormalities

Question 12

Roberstonian Translocation

Answer 12

predispose (chromosome translocation) development of downsyndrome
Results from the fusion of two acrocentric chromosomes (centromere distal, v small short arm having minimal/no significant coding material(loss of them is no clinical significance)) (chromosomes 13, 14, 15, 21 or 22) to form one chromosome)
A phenotypically normal individual will have 45 chromosomes, not 46
Carrier frequency 1:1000
The 2x most common Robertsonian translocations are:
-der(13;14)
-der (14;21)
-an unbalanced form of der(14;21) is responsible for 4% of all children with downsyndrome (3% moasic)

Question 13

A reciprocal or balanced translocation is:

Answer 13

“t”
A 2x way exchange of material between two non-homologous chromosomes
(at a congenital level) A balanced translocation results in a phenotypically normal individual as no genetic material has been lost or gained

Question 14

Unbalanced translocation

Answer 14

Parent with a balanced translocation, children at risk of unbalanced translocation- monosomy or trisomy
chance of having additional material (3x copies)
-can occur in recurrent miscarriages

Question 15

How do carrier of reciprocal (balanced) translocations present?

Answer 15

Children/offspring are at risk of having a unbalanced translocation

• recurrent miscarriages
• chromosomes examination of products of conception
• birth of a dysmorphic baby who is an unbalanced carrier (abnoraml phenotype)
• Oligospermia in male carriers

Question 16

Inversion

Answer 16

an intra-chromosomal structural re-arrangement, which involves two breaks on the same chromosomes - the resulting chromosome segment rotates by 180 degrees and reinserts itself

• pericentric inversion: (around centromere)
• paracentric inversion: (one side of the centromere)
• no loss of genetic material therefore:
• Inversion carriers are usually phenotypically normal
• 1 in 10,000 pregnancies
• the risk for an inversion carrier of producing unbalanced offspring is very low
• some of the more common inversions e.g. inv(9), inv(2), inv(y) exist as normal variants in the population

Question 17

Chromsome deletions

Answer 17

A terminal deletion is caused by a single break at the terminal region of a chromosomes and loss of that fragment (end)
An interstitial deletion is caused by 2x breaks in the same chromosome and loss of the intervening fragment (middle)
-congenital level results in loss of chromosome and essential genes
-Cri-du-chat syndrome:
severe Mr (IQ

Question 18

Clinical application chromosome tests

Answer 18

Karyotype analysis

Fluorescent in Situ Hybridisation (FISH)- use fluorescent label probes to look at genetic areas on chromosomes

Question 19

Chromosome Analysis

Answer 19

G-banding
-can look at all of the genetic material at once
-but must have large changes (deletions or translocations) - megabases before can see at a chromosome level
-number and structural abnormalities
FISH
-look at individual parts of chromsomes(10kilobases in size)- looks down better at specific genetic areas/genes. still cant look at small base pair deletions/small point mutations

Question 20

Mutation screening

Answer 20

Can now go from looking at large areas of whole chromosome
–> to looking at small base pair deletions/small point mutations
SSCP DGGE PTT cHPLC RFLPs

Question 21

Conventional G-banding Karyotype analysis

Answer 21

on divided cells
-take amniotic fluids, culture, and when dividing arest in metaphase, add nursta cell and put on slide, analyse 20-30 cells, identify individual chromosomes and see if there are any numerical or structural abnormalities
-lots of skill
-done on dividing cells
Suspected congenital disorders:
-e.g. Down syndrome, dysmorphic baby
-products of conception
-recurrent miscarriages
-parents with unrecognised robersonian or unbalanced translocation that could predispose fetus to unbalanced translocation
-prenatal diagnosis
Cancer/Leukaemia
-diagnostic and prognostic marker
-in malignant cells
-acute myeloid leukaemia - low risk(good chance of remission) cancer of early haemotopoetic cells. Translocation.
Chromosomes from leukemia put people in different diagnositic groups (low, intermediate and high risk) AML cytogenetic risk groups- guides treatment options

Question 22

FISH

Answer 22

Fluorescence In Situ Hybridization
This technique exploits the ability of a given DNA molecule to bind specifically - or hybridize - to the DNA template from which it originated
=an adjunct to chromosome analysis; looks at specific genes/parts of genes only
1. Probe. Label with Floursecent dye.
2. Denature
3. Hybridize to complementary area in cells being analysed
ADv: can be done on both dividing and non-dividing cells. Information about particular areas
Karyotype analysis: requires dividing cells and is a whole genome scan.
Specimens suitable for FISH:
Any type of tissue with a viable nucleus
-blood, skin, bone marrow, tumours, amniocytes, paraffin embedded tissues
-dividing and non-dividing cells
Breast cancer pathology Red signal+ probe Her2
green probe= Telomeric Probe
amplification of Her-2 gene

Question 23

Clinical significance of HER-2 amplification

Answer 23

HER-2 oncogenic gene (cancer causing)
-breast cancer pathology
-use FISH to look for these additional copies/HER-2 amplification
over expression = increased copies of of Her-2 protein/epidermal growth factor receptor = target for monoclonal Herceptin