26 - Variant detection in Inherited Disease Flashcards
1
Q
Steps of PCR
A
- Denaturation (95ºC): dsDNA to ssDNA
- Annealing (55-60ºC): Primer binds to target DNA
- Extension (72ºC): Complementary nucleotide bases are added from primer (5’ –> 3’)
2
Q
Varient level diseases
A
- HFE-Associated Hereditary Haemochromatosis
- Huntington Disease
3
Q
HFE-Associated Hereditary Haemochromatosis
A
- Increased iron absorption causes iron overload, resulting in tissue damage
- Autosomal recessive
- Real Time PCR used to diagnose
4
Q
Real Time PCR
A
- Probes are used, which have a fluorescent molecule and a quencher
- Intact probes do not emit fluorescence
- Probes only anneal to the DNA if there is an exact sequence match
- PCR performed
- DNA polymerase cleaves the probe, allowing fluorescence to be emitted
5
Q
Huntington Disease
A
- Progressive neurodegenerative condition
- Autosomal dominant inheritance
- Triplet repeat expansion in HTT gene
- Gain of function
- Fragment analysis used to diagnose
6
Q
Gene level diseases
A
- Cystic Fibrosis
- Duchenne Muscular Dystrophy
7
Q
Cystic Fibrosis
A
- Loss-of-function pathogenic variants in the CFTR gene (encodes
chloride channel) - Impaired transport of chloride ions and water across the cell membrane results in a thickening
of secretions in lungs, pancreas and other organs - Autosomal recessive inheritance
- Sanger sequencing used to diagnose
8
Q
Sanger sequencing
A
Detection of single nucleotide variants or deletions/duplications/ insertions of a small number of nucleotide
9
Q
Duchenne Muscular Dystrophy (DMD)
A
- Progressive muscle disease
- Childhood onset
- Muscle loss firstly occurs in the upper legs and pelvis, leading to wheelchair dependence
- Few survive beyond the third decade (respiratory
complications and cardiomyopathy) - MLPA used to diagnose
10
Q
What causes DMD
A
- X-linked disorder, males are affected
- Females are usually unaffected carriers, but can be at risk of developing cardiomyopathy
- Pathogenic variants in the DMD gene (encoding dystrophin)
11
Q
MLPA
A
- Multiplex Ligation Dependent Probe Amplification
- Method for detecting deletions or duplications of one or more exons, or a whole gene
- If genetic varient not detected, sequence used to detect sequence variants
12
Q
How does MLPA work
A
- Each kit contains multiple sets of probe pairs matching a specific sequence of part of each exon
- The probes are hybridised to patient
DNA - If exon present, PCR product is produced, if not, no product produced
13
Q
Genome level diseases
A
Down Syndrome
14
Q
Down syndrome
A
- Trisomy 21
- Most common chromosomal abnormality
- Karyotype used to diagnose
15
Q
Features of down syndrome
A
- Learning problems and developmental delay
- Dysmorphic features
- Cardiac defects
- Increased risk of leukaemia
- Reduced life expectancy
