Chromosomal Abnormalities Flashcards
How are cells prepared for karyotyping?
0.5ml blood added to 5ml culture medium. Add phytohaemagglutinin to stimulate lymphocytes to divide. Culture for 48-72 hours and add colcemid to arrest cells in metaphase. Culture briefly and add hypotonic KCl to swell cells. Fix in a mixture of methanol: acetic acid in a 3:1 ratio. Drop onto a microscope slide and let it undergo brief digestion with trypsin and stain with Giemsa.
Why is giema staining used?
Chromosomes have some common structural features and Giemsa staining leaves a recognisable pattern of bands:
G-DARK BANDS: tightly bound DNA less accessible to transcription machinery
G-LIGHT BANDS: less tightly bound DNA, more accessible
What are 3 key points about cytogenic nomenclature?
Normal karyotype 46 XX or 46 XY
9p22.2 = chromosome 9, p-arm (short arm), position 22 on arm
Numbers get bigger as you get further away from centrosome and longer arm known as q-arm
What is the purpose and stages of mitosis?
Purpose: growth and repair, create identical cells, replace exhausted cells
Stages:
1. Interphase – G1, S and G2 phases, DNA replication, cell getting ready for mitosis
2. Prophase – chromatin condenses into chromosomes and pair up
3. Metaphase – nuclear envelope disappears, chromosomes line up at equator
4. Anaphase – sister chromatids separate, and centromeres split
5. Telophase and cytokinesis – chromatin expands and cytoplasm splits
What is the purpose and phases of meiosis?
Purpose: achieve reduction from 2n to n, ensure genetic variation, enable random assortment and recombination.
Phases: 2 phases – meiosis 1 and meiosis 2
Phase 1 – prophase, metaphase, anaphase to 2n cells
Phase 2 – no interphase in between, prophase, metaphase, anaphase (each cell has n genetic material – 4 genetically different haploid cells)
Why is female meiosis vulnerable?
Oogenesis is paused in utero until puberty. One primary oocyte yields only one ovum. Finite number of primary oocytes so increased chance for things to go wrong due to broken up process.
Why does aneuploidy occur and when is it more likely?
May get non-disjunction (causing aneuploidy), probably due to degradation of factors holding homologous chromosomes together. Non-disjunction can occur in either meiosis 1 or 2. Risk of maternal non-disjunction increases w/age e.g. trisomy 21 (Down’s syndrome). Can see 3 chromosome 21s in karyotype. Can see amounts of DNA as well using QF-PCR (quantitative fluorescence PCR). Most trisomy arises from maternal non-disjunction.
When is aneuploidy tolerated?
Not all trisomies are compatible w/life – only certain ones are (Chr 13, 16, 21)
Monosomy is poorly tolerated and often fatal.
Sex chromosome imbalance is tolerated because:
- X-inactivation of excess X chromosomes
- Low gene content of Y chromosome
What are reciprocal translocations?
Balanced reciprocal translocations are an exchange of equal amounts of chromosome material between two nonhomologous chromosomes. Usually phenotypically normal and occurs during synapsis at mitosis and meiosis. However, it increases the chances of unbalanced gametes. When unbalanced, severity of phenotype is dependent on gene content of affected segment. Trisomy usually tolerated better than monosomy and it may arise de novo or from a reciprocal parental abnormality.
What are the different types of possible mutations?
Deletion - random deletion of bases causing a frameshift
Substitution - depends on the base substituted and the codon
Translocations - switching of chromosome portions between chromosomes
Insertions - addition of bases causing a frameshift
Inversions - turning chromosome portion upside down changing order of genes
What do reciprocal chromosomal abnormalities have in common?
- Carriers (1 in 100) are usually phenotypically normal
- Chromosomes have to contort into unusual figurations to achieve synapsis at mitosis and meiosis
- Present a reproductive risk as increased chance of unbalanced gametes
How are genetic disorders grouped?
Phenotype first approach initially used where clinical basis of grouping was children with similar developmental delay and/or dysmorphism, then looking for genetic abnormalities. Yielded many ‘classical’ contiguous gene deletion syndromes. Cost of genomic analysis has reduced, can detect smaller imbalances now and hence in genomic era, much easier to find exact genetic cause.
What is William’s syndrome?
Includes a deletion-based syndrome and one caused by duplication. Where genes are deleted at 7q11.23, features include: Long philtrum, Short, upturned nose, Arched eyebrows, Supravalvular aortic stenosis and a ‘cocktail party’ personality. Where genes are duplicated, features include: Short philtrum, Broad nose, Flat eyebrows, Dilatation of the aorta and delayed speech development and autistic behaviours hindering social interaction. Duplications usually have a milder phenotype than deletions.
How is deletion in William’s syndrome detected?
Detect deletion w/a CGH trace and array comparative genome hybridisation used to detect chromosomal copy number changes. Method is:
- Label sample DNA and reference DNA differently
- Co-hybridise genomic array
- Analyse on software to compare and see missing bit of DNA or gain of extra DNA
What are contiguous gene deletion/duplication syndromes?
A contiguous gene syndrome (CGS), also known as a contiguous gene deletion syndrome is a clinical phenotype caused by a chromosomal abnormality, such as a deletion or duplication that removes several genes lying in close proximity to one another on the chromosome.
These disorders have common breakpoints and are mediated by low copy repeats (LCRs). Risk of non-allelic homologous recombination (NAHR) may be increased by parental LCR inversions. Non-allelic homologous recombination (NAHR) is a form of homologous recombination that occurs between two lengths of DNA that have high sequence similarity but are not alleles.
