Chromosomal Disorders Flashcards
How is a karyotype prepared?
0.5 mL blood in 5mL culture medium
add phytoemagglutinin (stimulates lymphocytes to divide)
culture 48-72 hours
add colcemid (arrest cells in metaphase)
culture briefly
add hypotonic KCl to swell cells
fix in 3:1 methanol : acetic acid
drop on to microscope slide
brief digestion with trypsin, stain with Giemsa
What does a normal karyotype look likes
2n (diploid)
46 chromosomes
23 pairs
What is G banding?
= pattern characteristic in each chromsome
= heterochromatin = dark bands = AT rich gene pore = stains better with Giemsa
= euchromatin = GC rich = stains poorly
(also have T bands + R bands)
EXTRA READING
What is the structure of a chromosome?
short arm = p (1,2)
long arm = q (1,2,3)
(splits further e.g. 11.21)
What are some possible variations in chromosome number?
Polyploidy
= triploidy = 3N
= tetraploidy = 4N
(common in plants, frogs, fish)
(e.g in humans if egg fertilised by 2 sperm - most die early in embryonic development)
Aneuploidy
= monosomy = 2N-1
= trisomy = 2N+1
(more common in humans)
(caused by non-disjunction)
What is nondisjunction?
EXTRA READING
= chromosomes fail to separate (in meiosis 1/2)
What are some viable autosomal aneuploides?
Trisomy 21 = Down Syndrome
Trisomy 13 = Patau Syndrome
Trisomy 18 = Edward Sydnrome
(monosomy - not viable)
(estimated 1/2 all conceptions have aneuploidy BUT die early on - not aware of the pregnancy)
What is Down Syndrome?
= Trisomy 21
= 1 in 700 live births
= wide skull , flattened at back
= tongue may be furrowed + protruding
= “simian” creases on palms of hands, soles of feet
= epicanthic fold above eyes
= brushfield spots on the iris
= physical and mental retardation (severity is variable)
= increased likelihood of congenital heart defects
=15x increased chance of leukaemia
= susceptibility to Alzheimer’s disease
What is Patau Syndrome?
= Trisomy 13
= 1 in 20,000 live births (most die at birth / miscarried)
= cleft lip and palate
= physical and mental retardation
= defects in multiple organ systems
= most die within first year
What is Edwards Syndrome?
= Trisomy 18
= 1 in 6,000 live births
= clenched fist with first and fourth fingers overlapping the middle two
= rocker bottom feet (extended heel , flat soles)
= heart, kidney + other internal abnormalities
= median lifespan 5-15 days
What are the possible sex chromsome aneuploidies?
= more common
= have survival to adulthood
syndromes:
XO = Turner Syndrome (only viable human monosome)
XXY = Klinfelter Syndrome (sometimes >2 Xs = superklinefelter)
not syndromes:
XXX = Metfemale
XYY = no name (or >2 Ys) ?Jacob’s syndrome
= less severe:
Y chromosome has few genes, they are involved in fertility rather than survival
Multiple chromosomes in females, extra are inactivated
= have reduced fertility
EXTRA READING - XYY
= Jacob’s syndrome - classed as males
= most have no significant physical or developmental differences
= taller than average, increased muscle mass, slight differences in facial structure
= increased risk of learning and behavioural difficulties
What is Turner Syndrome?
= XO (monosomy)
= 1 in 2,500 live births (more lost in miscarriage)
= poorly developed secondary sexual characteristics (e.g. breast development)
= short stature, broad chest, webbed neck, low muscle tone
= rudimentary ovaries - sterile (little oestrogen / no follicles)
= puffy hands and feet at birth
= develop as female
= hormone therapy can give secondary sex characteristics
= can survive into adulthood
What is Klinefelter Syndrome?
= XXY
= 1-2 in 1000 live births
= develop as males BUT have female characteristics
= male genitalia
= breast development
= female distribution of fat and pubic hair
= decrease in male characteristics (less body hair, less deepening of voice)
= testes small and underdeveloped (low fertility - less testosterone produced)
EXTRA READING
= taller than average
= increased risk of ADHD and autism
= increased risk of osteoporosis , breast cancer, autoimmune
= treatment: testosterone replacement
What are examples of chromosome rearrangements?
Deletion
Duplication
Inversion
Nonreciprocal translocation
Reciprocal translocation
(can sometimes have no effect)
What are some examples of congenital (present from birth) disorders associated with chromosome deletions?
Cri-du-Chat Syndrome
= 5q15
Prader Willi Syndrome
= 15q11-13
Angelmann Syndrome
= 15q11-13
Wolf-Hirschhorn Syndrome
= 4q16
= Miller-Dieker Syndrome
= 17q13
Di George Syndrome
= 22q11
What is Cri-du-chat Syndrome?
=5q15
= 1 in 50,000 live births
= babies have cat-like cry
(due to defects in glottis and larynx)
= wide face with saddle nose
= physical and mental retardation
= range of severity, depending on extent (size) of deletion
= low mean survival time
= adolescents have normal puberty and are fertile
(most die before child-bearing age - most cases are spontaneous)
How does crossing-over between repetitive DNA cause chromosome rearrangements?
EXTRA READING
= because repetitive sequences prone to misalignments (forming a loop) and usual crossing-over during meiosis
= can lead to exchange of genetic material between non-homologous chromosomes / between non-allelic regions of same chromosomes
= leads to gene disruptions or alterations in gene dosage (e.g. duplication or deletion)
What is balanced (reciprocal) translocation?
EXTRA READING
= chromosomal abnormality when 2 chromosomes exchange segments of genetic material without any loss or gain of genetic material (rearrangement)
= produces chromosomes with different genes in a different order
= can lead to problems during cell division / or in offspring (or fertility issues)
What are microdeletions?
= can’t be seen on karyotype = too small
= usually occur spontaneously
(because of recombination between repeated sequences e.g. transposable elements)
What is the FISH method?
Spread chromosomes on slide for karyotype
Denature DNA of chromosome in situ
Hybridise with fluorescently labelled oligoprobe corresponding with 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:
= small deletions / duplications cannot be detected
= can only test region corresponding to probe (need for prior knowledge)
What is a CGH array (Comparative Genomic Hybridisation)?
Microarray consisting of thousands of short DNA sequencing spanning 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 = less green fluorescence in several spots
Duplication = excess of green fluorescence in several spots
How are SNP arrays (GWAS) used?
Microarray consisting of thousands of oligonucleotides 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
(2 signals - 2 chromosomes)
Line up with genome and identify regions where signal is equivalent to single variant (if heterozygous)
What is non-invasive pre-natal testing / diagnosis (NIPT/NIPD)?
Up to 10% of free DNA in maternal serum is foetal DNA (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 of pregnancy
Minimum of 5% foetal DNA required
May be used to detect aneuploidies, sex of foetus , some single gene mutations
EXTRA READING
= less invasive (unlike amniocentesis or chorionic villus sampling)
= safer
= cffDNA = cell-free fetal DNA
= can detect the trisomy syndromes
= high detection rate (>99%)
= is a screening test not a diagnostic test (results have to be confirmed with diagnostics)
= cannot detect all chromsomal abnormalities (false negatives and false positives)
