Detecting Chromosomal Abnormalities Flashcards
How are samples sourced pre and postnatally?
Prenatal:
Amniocentesis
Chorionic villus sampling
Cell-free fetal DNA
Postnatal:
Blood
Saliva
Describe chromosome staining
Most common = G-banding. G = Giemsa, a chemical
Banding is used bc diff types of chromatin stain differently
Euchromatin = GC-rich; loosely packed; genes active
Heterochromatin = AT-rich; tightly packed; genes inactive
Done at metaphase when chromosomes are highly condensed, which takes several days
Looks for aneuploidies, translocations & very large deletions
What is FISH?
Fluorescent in situ hybridisation
Hybridisation is when single stranded nucleic acid binds to a new single stranded nucleic acid (DNA-DNA or DNA-RNA)
1) culture cells to metaphase, which takes several days
2)Denature the probe and target DNA
3)Mix probe and target DNA. Probe binds to target
Uses probes for SPECIFIC parts of genome. Looks for aneuploidies, translocations & large deletions
What is a probe?
A single stranded DNA (or RNA) molecule
Typically 20 – 1kb bases long
Labelled with a fluorescent or luminescent molecule
In some techniques thousands of probes are used simultaneously
How does this help diagnose cri du chat syndrome?
In cri-du-chat syndrome, patients have lost parts of their 5p chromosomes, so it’s a telemetric deletion.
A probe has been designed to specifically anneal to that region of chromosome 5p. In the intact chromosome it anneals and glows yellow.
In the other chromosome, 5p is not there, the probe has nothing to anneal to, it doesn’t glow. This =cri-du-chat syndrome, diagnosed by FISH
What is array CGH?
Detects sub-microscopic chromosomal abnormalities
Patient DNA labelled Green. Control DNA labelled Red
If the patient has deletions, habra less green dots. If the patient has duplications habra more green dots.
Also, if the lines are overriding one another, the patient is fine, but if the 2 lines veer away from each other=problem (seen as DNA dosage loss)
Much quicker as you dont need metaphase staining
What is QF-PCR?
Quantitative fluorescence polymerase chain reaction: identifies trisomies 13, 18 and 21 using microsatellites from extracted DNA:
Perform PCR using primers for microsatellite on chromosome 21 (if testing for Down’s)
Should be two copies of microsatellite (one from mother, one from father)
If homozygous, habra a single peak of high signal. If heterozygous, habra 2 lower signal peaks
Much quicker (~48hrs)
What are microsatelites and how do you detect them?
Di-, tri-, tetra- nucleotide sequence with variable numbers of repeats
Total microsatellite sequence length varies between individuals
To detect them, 1st isolate DNA from individual. Design primers specific to flanking sequences
Then do PCR amplification. Genotype size of fragments based on gel electrophoresis
Describe Non-invasive pre-natal testing (NIPT) and NGS
- Cell free fetal DNA from maternal blood sample
- Trisomy testing
- “High chance” indicator for invasive test
- Looks for aneuploidies using next generation sequencing
- Screening not diagnostic
Outline the process of mitosis
I)Chromosomes duplicate and the copies remain attached to each other.
P)Chromosomes condense and become visible. The spindle forms.
The nuclear membrane breaks apart and the spindle interacts w the chromosomes.
M)The copied chromosomes align in the middle of the spindle.
A)Chromosomes separate into 2 genetically identical groups and move to op poles.
T)Nuclear envelope reforms, spindle begins to break down.
Describe and explain meiosis
Before meiosis, interphase occurs (cell growth, DNA replication).
Homologous chromosomes align to form a bivalent structure. Crossing over occurs: all 4 chromatids come into contact.
At these contact points (chiasmata) chromatids break and rejoin, exchanging DNA sections between non-sister chromatids. This makes recombinant chromosomes (variation).
Anaphase I- Chromosomes are pulled away from each other by spindle fibres
Telophase I- nuclei reforms. 2 new cells
Hay another round of cell division to produce 4 haploid gametes
What is karyotype, polyploid and aneuploid?
Humans have 23 pairs of chromosomes. 22 pairs autosomes, 1 pair sex chromosomes XX/ XY
POLYPLOID: multiple of the haploid number (e.g. 4n=92), not compatible with life
ANEUPLOID: chromosome number is not an exact multiple of haploid number - due to extra or missing chromosome(s) (e.g. 2n+1=47) (trisomy, monosomy)
What are the types of Autosomal and sex chromosome aneuploidies?
- Trisomy 13 (Patau’s)
- Trisomy 18 (Edward’s)
- Trisomy 21 (Down’s)
Others happen but do not carry to term
- Turner’s (45,X) (1 in 5000 female births)
- Triple X syndrome (47,XXX) (1 in 1000 female births)
- Klinefelter’s (47,XXY) (1 in 1000 male births)
- 47,XYY (1 in 1000 male births)
What is mosaicism?
The presence of two or more genetically different cell lines derived from a single zygote.
Mitotic Non-disjunction; majority of cells 2n, some 2n+1 = mosaic
Sometimes maternal non disjunction in the gametes result in 3 copies of chromosome 21. However, the trisomic cell can undergo ‘trisomic rescue’, where the cell recognizes that it has the incorrect number of chromosomes and ensures subsequent cells have the correct number. This is a form of mosaicism (see diagram)
Mosaic phenotype thought to be less severe but is difficult to assess
what is the difference between full and partial trisomy/monosomy?
Full monosomy arises by non disjunction as mentioned
Partial monosomy/trisomy (microdeletion/duplication syndromes) far more common and the mechanism is different
What is x linked inheritance?
Offspring of a carrier female have a 50% (1 in 2) chance of inheriting pathogenic variant
Females w the variant=carriers, unless skewed X-inactivation. Males w the variant=affected
X-linked genes are never passed from father to son (only pass on Y chromosome). All daughters of an affected father are obligate carriers (as pass on only X chromosome which has variant)
Affected males may have de novo variant
Describe inheritance of mitochondrial pathogenic variants
What is the Hardy Wineberg principle?
•Allele and genotype frequencies in a population will remain constant in successive generations. There are no other evolutionary influences.
If the carrier frequency of SMA is 1 in 50, and Jamal is a carrier of SMA, what is the chance that he and Elizabeth will have a child with the condition?
