L6, Chromosomal Disorders Flashcards
1
Q
Describe the preparation of a karyotype:
A
- 0.5ml blood in 5ml culture medium
- Add phytohemagglutinin (stimulates lymphocytes to divide)
- Culture 48-72 hrs
- Add colcemid (arrests cells in metaphase)
- Culture briefly; add hypotonic KCl to swell cells; fix in 3:1 methanol:acetic acid; drop onto microscopic slide
- Brief digestion with trypsin, stain with Giemsa
2
Q
Features of a normal karyotype:
A
- 23 chromosomes
- xx in females, xy in males
3
Q
What is G-banding:
A
- Distinctive pattern produced by Giemsa staining
- G-bands occur in heterochromatin (AT rich so stains well)
4
Q
Labelling conventions for chromosome bands:
A
- Short arm: p
- Long arm: q
- Domains 1, 2 etc. radiate out from centromeres
5
Q
Polyploidy in humans vs other species:
A
- Extra complete sets of chromosomes
- Very rare in humans
- Triploid can arise when an egg is fertilised by 2 sperms; rarely survive to term, usually have severe defects and live only minutes if born
- Tetraploidy virtually not observed in live births
- Salamanders, frogs and leaches are all polyploid
6
Q
Aneuploidy: Examples, relevance to human births
A
- 2N-1: Monosomy
- 2N+1: Trisomy
- Estimated half of human conceptions are aneuploid but most die early on before development
7
Q
What is non-disjunction
A
- Chromosome fails to separate, either in meiosis I or II
- Demonstrate: FCs
8
Q
List the 3 viable autosomal aneuploidies:
A
- Trisomy 21 (Downs syndrome) -> Survival to adulthood
- Trisomy 13 (Patau syndrome)
- Trisomy 18 (Edward syndrome)
- All monosomies are non-viable
- 13 and 18 -> severe, multi-system effects; survival to adulthood prohibited
9
Q
Visible characteristics of Downs syndrome:
A
- Wide skull, flattened at back
- Tongue may be furrowed and protruding
- ‘Simian’ crease on palms of hands and soles of feet
- Epicanthic folds above eyes
- Brushfield spots on the iris
10
Q
Clinical characteristics of DS:
A
- Physical and mental retardation
- Increased likelihood of congenital heart defects
- 15X increased chance of leukaemia
- Susceptibility to AD (50% prevalence in DS populations by age 60)
11
Q
Patau syndrome: Overview
A
- aka trisomy 13
- 1 in 20,000 live births
- Cleft lip and palate
- Physical and mental retardation
- Defects in multiple organ systems
- Most die within first year
12
Q
Edwards syndrome: Overview
A
- 1 in 6,000 live births
- Clenched fist with first and fourth fingers overlapping the middle two
- Rocker bottom feet
- Heart kidney and other internal abnormalities
- Median lifespan 5-15 days
13
Q
List the sex chromosome aneuploidies:
A
- XO: Turner syndrome -> Viable
- XXY: Klinefelter
- XXX: Metafemale
- XYY
14
Q
Typical outcomes in sex chromosome aneuploidies:
A
- Generally more concerned with male infertility than survival
- Extra X’s tend to be inactivated
- XXX and XXY relatively mild to the point of going undiagnosed -> often reduced fertility, compounded by number of extra X’s
- DSD: Proper term for disorders of sex development
15
Q
Turner syndrome: Overview
A
- 1 in 2,500 live births
- Poorly developed secondary sexual characteristics -> develop as girls but sterile
- Hormone therapy can generally be used to overcome secondary sex issues (rudimentary ovaries) but not infertility itself
- Short stature, bone malformations and webbed neck
- Puffy hands and feet at birth
- Structural heart abnormalities reported
16
Q
Klinefelter syndrome overview:
A
- Occurs in around 1 per 1000 live male births
- Male genitalia
- Excessive height at puberty
- Tendency to some female secondary sex characteristics (breasts, fat and pubic hair distribution)
- Testes small and underdeveloped -> low fertility (few to no sperm)
17
Q
Chromosome rearrangements: List the 5 types
A
- Deletion
- Duplication
- Inversion
- Nonreciprocal translocation (one way)
- Reciprocal translocation (both ways)
18
Q
Congenital disorders associated with Chromosome deletions (with chr. bands)
A
- Cri-du-chat: 5p15
- Prader willi: 15q11-13
- Angelmann: 15q11-13
- Wolf-Hirschorn: 4p16
- Miller-Dieker: 17p13
- Di-george: 22q11
19
Q
Cri-du-chat: Overview
A
- 1 in 50,000 live births
- Distinctive cat cry in babies
- Defects in glottis and larynx, wide face and saddle nose
- Physical and mental retardation
- Range of severity which depends on extent of deletion
- Low mean survival time
- Adolescents have normal puberty and are fertile
20
Q
How do chromosome rearrangements come about?
A
- Crossing over between repetitive regions of DNA
- Adjacent sites on same strand -> looping out -> deletion
- Sites on neighbouring strands -> non reciprocal translocation-> duplication in one strand and deletion in other
- Adjacent sites on same strand -> inversion
- Sites on neighbouring strands -> reciprocal translocation
21
Q
How does translocation come about? Types?
A
- Crossing over -> tetravalent
- Both balanced and unbalanced translocations can come about
- Unbalanced are highly unlikely to be viable whereas balanced can often go unnoticed
- If a terminated foetus is found to have this unbalanced karyotype, checking the parent for balanced translocation can be informative of their fertility
22
Q
When is giemsa staining useful in diagnosing chromosomal disorders?
A
- Best for aneuploidies and large deletions or duplications
23
Q
Drawbacks of giemsa staining for diagnosing chromosomal disorders:
A
- Time consuming
- Low sensitivity -> can’t detect rearrangements smaller than 5Mbp
24
Q
Alternatives to giemsa staining in diagnosing chromosomal disorders:
A
- Fluorescent in situ hybridisation (FISH)
- Array comparitive genomic hybridisation (Array CGH)
- Single nucleotide polymorphism (SNP) profiling
- Whole genome sequencing (non invasive pre-natal diagnosis)
25
Q
+ Two Further examples of microdeletions:
So many witchy boils
A
- Smith-Magenis
- Williams-Beuren
- Occur spontaneously because of recombination between repeated sequences (e.g. transposable elements)
26
Q
Describe the FISH method:
A
- Spread chromosomes on slide
- Denature DNA in situ
- Hybridise with fluorescently labelled probe corresponding to region of interest
- Wash off unbound probe
- Stain with DAPI (DNA intercalating stain)
- View under fluorescent microscope
- Need to include control probe for unaffected region of same chromosome (different colour)
27
Q
Probes commonly used in routine FISH testing:
A
- 13, 18, 21 -> Trisomies
- X and Y -> Sex chromosome aneuploidies
27
Q
2 key limitations of FISH:
A
- Small deletions or duplications cannot be detected
- Can only detect the region corresponding to the probe - requires prior knowledge of structures etc
28
Q
Array CGH: Protocol
A
- DNA from patient and control extracted and labelled with different fluorescent dyes
- DNAs mixed together in equal quantities and hybridised to the microarray
- Slide washed and scanned
- Most spots will have equal red and green fluorescence
- Deletions or duplications represented by diminished or excessive green fluorescence across several spots (quantified using software -> plot)
29
Q
SNP arrays: Overview
A
- Microarray consisting of thousands of oligonucleotide pairs spanning genome, each pair differing at a single base
- Hybridise with fluorescently labelled DNA from patient -> exact match required
- Signal from both variants added together
- Identifying regions in genome where signal is equivalent to single variant
- Quite common and cheap, doesn’t necessitate control DNA although they are still often used 9e.g. comparison to parents SNP array profiles)
30
Q
NIPT/NIPD: Overview
What is it used for, when can it be carried out from?
A
- Up to 10% of free DNA in maternal serum is foetal
- Free DNA (minimum 5% foetal DNA required) is amplified and sequenced by NGS -> relative number of sequence reads is used to measure chromosome number
- Can be carried out from 9 weeks
- May be used to detect aneuploidies, sex of foetus and some single gene mutations
31
Q
+ Chromosomal abnormalities occur in … % of births
A
- About 1%
32
Q
+ Example of congenital condition which is not genetic in origin or a de novo mutation
A
- Teratogenic effects of thalidomide
- Causes limb abnormalities and other issues
- Widely prescribed in 1950s and early 1960s to treat morning sickness in pregnancy
33
Q
+ Triple X syndrome
A
- Around 1 in 1000 female live births
- Often go unnoticed; few physical abnormalities
- Often shorter than average
- High prevalence in mental institutions (1 per 250 as of 1994)
- Early menopause is almost universal for the condition
- At risk for speech delays, learning disabilities, neuromotor impairment and mental illness
- Extra X typically originates from errors in maternal meiosis (particularly meiosis I) -> correlated with advanced maternal age
34
Q
+ 46, XX testicular DSD: How are these patients male with XX chromosomes?
A
- Generally have Y-chromosome sequence somewhere in their genome -> usually the SRY gene (male-determining factor)
- Commonly, this gene would be translocated onto the paternal X chromosome (SRY on an autosome is quite rare)
35
Q
+ What about chromosomes 21, 13 and 18 might make them viable as trisomies?
A
- They contain some of the lowest gene densities
- An addition in these chromosomes is thus conferring the minimal amount of extra genes
