Chromosomal disorders Flashcards
Preparation of a karytotype
- 0.5ml blood in 5ml of culture medium
- Add phytohemagglutinin
- Culture 48-72 hours
- Add colcemid
- Culture briefly: add hypotonic KCl to swell cells, fix in methanol: acetic acid (3:1), drop on to microscope slide
- Brief digestion with trypsin, stain with Giemsa
Chromosome strucure
short arm - p - sections 1 and 2 - subsections etc
Long arm - q - Sections 1, 2, 3 - subsections etc
Variations in chromosome number
- Polyploidy: e.g. salamanders
3N: triploidy
4N: tetraploidy - Aneuploidy:
2N-1: monosomy
2N+1: trisomy
Triploidy in babies
Triploid babies often die in utero - don’t often live more than a day
~50% of humans have an aneuploid chromosome
Viable autosomal aneuploidies
Trisomy 21 - Down syndrome
Trisomy 13 - Patau syndrome
Trisomy 18 - Edward syndrome
Down syndrome characteristics
- Wide skull, flatterned at back
- Tongue may be furrowed and protruding’
- ‘Simian’ creases on palms of hands and soles of feet
- Epicanthic folds above eyes
- Brushfield spots on iris
- Physical and cognitive difficulties
- Increased likelihood of congenital heart defects
15X increased chance of leukaemia
susceptibility to Alzheimer’s disease
1 in 700 affected
Patau syndrome
1 in 20,000 live births
cleft lip and palate
physical and mental difficulties
defects in multiple organ systems
most die within the first year
Edwards syndrome
1 in 6,000 live births
-clenched fist with the first and fourth fingers overlapping the middle two
- rocker bottom feet
- heart, kidney and other internal abnormalities
- median lifespan 5-15 days
Sex chromosome aneuploidies
XO - Turner Syndrome
XXY - Klinefelter syndrome
XXXY - pseudo Klinefelter syndrome
XXX - Metafemale
XYY
Turner syndrome symptoms
- Webbed neck
- Puffy feet and hands - low muscle tone
- 1 in 2500 births
- Poorly developed secondary sexual characteristics
- Short stature, broad chest, webbed neck
- Rudimentary ovaries - sterile
- Puffy hands and feet at birth
Klinefelter syndrome
- Male genitalia
- Breast development
- Female distribution of fat and public hair
- decrease in male characteristics
- testes small and underdeveloped - low fertility.
Chromosomal rearrangements
What causes chromosomal rearrangements between repetitive DNA
Crossing over
Congenital disorders associated with chromosome deletions
Cri-du-chat syndrome - 5p15
Prader Willi syndrome - 15q11-13
Angelmann syndrome - 15q11-13
Wolf-Hirschhorn syndrome - 4p16
Miller-Dieker syndrome - 17p13
Di Geroge syndrome - 22q11
Cri-du-chat Syndrome characteristics
- 1in 50,000 live births
- Babies have cat-like cry
- Defects in glottis and larynx
- Wide face with saddle nose
- Physical and mental difficulties
- Range of severity, depending on the extent of the deletion
- Low mean survival time
- Adolescents have normal puberty and are fertile
Diagnosing chromosomal disorders
- Giemsa-stained karyotype best for aneuploidies and large deletions/duplications.
- Are time consuming and can’t detect <5Mbp rearrangements
Other alternatives to Giemsa-stained karyotyping
- Fluorescence in situ hybridisation (FISH)
- Array Comparative Genomic Hybridisation (Array CGH)
- Single Nucleotide Polymorphism (SNP) profiling
- Whole genome sequencing (non-invasive pre-natal diagnosis)
FISH method
Spread chromosomes on slide as for karyotype
Denature DNA of chromosomes in situ
Hybridise with fluorescently labelled probe corresponding to the region of interest
Wash off unbound probe
Stain DNA with fluorescent dye (DAPI)
View under a fluorescent microscope
Need to include control probe for unaffected region of same chromosome (different colour
Limitations of FISH
- Small deletions/duplications cannot be detected
- Can only test region corresponding to probe - need for prior knowledge
Array Comparative Genomic Hybridisation (CGH)
- Microarray consisting of thousands of short DNA sequences spanning the entire genome
- DNA from patient and control extracted and labelled with different fluorescent dyes (e.g. red and green)
- DNAs mixed together in equal quantities and hybridised to the slide
- Slide washed and scanned
- Most spots will have equal red and green fluorescence
- Deletion - relatively less green fluorescence in several spots
- Duplication - excess of green fluorescence in several spots.
SNP arrays
- Microarray consisting of thousands of oligonucleotide pairs spanning genome
- Each pair differs at single base
- Hybridise with fluorescently labelled DNA from patient
- Exact match required
- Signal from both variants added together
- Line up with genome and identify regions where signal is equivalent to single variant (if heterozygous)
Non-invasive pre-natal testing/diagnosis (NIPT/NIPD)
Up to 10% of free DNA in maternal serum is foetal (from placenta)
Free DNA amplified and sequenced by NGS
Relative number of sequence reads used to measure chromosome number
Can be carried out from 9 weeks
Minimum of 5% foetal DNA required
May be used to detect aneuploidies, sex of foetus and some single gene mutations
NIPT to be offered for Down syndrome from 2018 on NHS
