Lecture 16 Flashcards
Human Karyotype
46 chromosomes
23 pairs of chromosomes
22 pairs autosomes, 2 sex chromosomes
Arranged in order of Reducing size
Human Karyotype clinically
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))
Chromosome Nomenclature
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
Chromosomal Abnormalities
Numerical or structural
Constitutional(downsyndrome) vs acquired( 50% lukemia)
Numerical Chromosomal Abnormalities
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)
How common are numerical abnormalities?
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
Clinical conditions Congenital Down syndrome
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
Clinical conditions Congenital Aneuploidy of the sex chromosomes
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
Acquired abnormalities in case 39 year old male
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
Mosaicism
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)
Structural chromosome abnormalities
Reciprocal translocations Robertsonian Translocations Inversions Deletions -all of these can be either congenital or acquired abnormalities
Roberstonian Translocation
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)
A reciprocal or balanced translocation is:
“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
Unbalanced translocation
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
How do carrier of reciprocal (balanced) translocations present?
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
Inversion
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
Chromsome deletions
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
Clinical application chromosome tests
Karyotype analysis
Fluorescent in Situ Hybridisation (FISH)- use fluorescent label probes to look at genetic areas on chromosomes
Chromosome Analysis
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
Mutation screening
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
Conventional G-banding Karyotype analysis
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
FISH
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
Clinical significance of HER-2 amplification
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
