Chromosomal disorders

Question 1

Preparation of a karytotype

Answer 1

1. 0.5ml blood in 5ml of culture medium
2. Add phytohemagglutinin
3. Culture 48-72 hours
4. Add colcemid
5. Culture briefly: add hypotonic KCl to swell cells, fix in methanol: acetic acid (3:1), drop on to microscope slide
6. Brief digestion with trypsin, stain with Giemsa

Question 2

Chromosome strucure

Answer 2

short arm - p - sections 1 and 2 - subsections etc

Long arm - q - Sections 1, 2, 3 - subsections etc

Question 3

Variations in chromosome number

Answer 3

• Polyploidy: e.g. salamanders
  3N: triploidy
  4N: tetraploidy
• Aneuploidy:
  2N-1: monosomy
  2N+1: trisomy

Question 4

Triploidy in babies

Answer 4

Triploid babies often die in utero - don’t often live more than a day

Answer 5

~50% of humans have an aneuploid chromosome

Question 6

Viable autosomal aneuploidies

Answer 6

Trisomy 21 - Down syndrome

Trisomy 13 - Patau syndrome

Trisomy 18 - Edward syndrome

Question 7

Down syndrome characteristics

Answer 7

• 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

Question 8

Patau syndrome

Answer 8

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

Question 9

Edwards syndrome

Answer 9

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

Question 10

Sex chromosome aneuploidies

Answer 10

XO - Turner Syndrome

XXY - Klinefelter syndrome

XXXY - pseudo Klinefelter syndrome

XXX - Metafemale

XYY

Question 11

Turner syndrome symptoms

Answer 11

• 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

Question 12

Klinefelter syndrome

Answer 12

• Male genitalia
• Breast development
• Female distribution of fat and public hair
• decrease in male characteristics
• testes small and underdeveloped - low fertility.

Question 13

Chromosomal rearrangements

Question 14

What causes chromosomal rearrangements between repetitive DNA

Answer 14

Crossing over

Question 15

Congenital disorders associated with chromosome deletions

Answer 15

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

Question 16

Cri-du-chat Syndrome characteristics

Answer 16

• 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

Question 17

Diagnosing chromosomal disorders

Answer 17

• Giemsa-stained karyotype best for aneuploidies and large deletions/duplications.
• Are time consuming and can’t detect <5Mbp rearrangements

Question 18

Other alternatives to Giemsa-stained karyotyping

Answer 18

• Fluorescence in situ hybridisation (FISH)
• Array Comparative Genomic Hybridisation (Array CGH)
• Single Nucleotide Polymorphism (SNP) profiling
• Whole genome sequencing (non-invasive pre-natal diagnosis)

Question 19

FISH method

Answer 19

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

Question 20

Limitations of FISH

Answer 20

• Small deletions/duplications cannot be detected
• Can only test region corresponding to probe - need for prior knowledge

Question 21

Array Comparative Genomic Hybridisation (CGH)

Answer 21

• 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.

Question 22

SNP arrays

Answer 22

• 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)

Question 23

Non-invasive pre-natal testing/diagnosis (NIPT/NIPD)

Answer 23

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