W6L9 - Diseases Resulting from Genetic Changes Flashcards
Cystic Fibrosis
Autosomal recessive disease
Symptoms vary and are dependent on the mutation present
Severe cases diagnosed early:
- respiratory distress
- intestinal issues
Less severe cases may present later in life:
- pancreatitis
- respiratory disorders (asthma, pneumonia)
People with CF live to roughly 36 years old
What is CF caused by?
Mutations in the CFTR gene
Encodes for a protein called Cystic Fibrosis Transmembrane Conductance Regulator
- transports chloride ions out of cells
- helps maintain correct volume of mucus on the ciliated epithelia in the lung so that cilia can move
Decreased mucus volume prevents cilia movement and also increased mucus concentration
This leads to bacterial colonisation and growth
CF Diagnosis
New born CF Screen Test - tests for immunoreactive trypsinogen Sweat Test - localised skin sweating is stimulated - sweat collected - and chloride concentration is determined
Oligonucleotide Ligation Assay for CF
Uses 3 different probes for each allele
One common probe which binds to 3’ mutation site
- fluorescently labelled
Probes specific for the normal and mutated allele
- each probe linked to a unique migration modifier
- this gives ligation product specific electrophoretic ability
Temporal Temperature Gradient Electrophoresis for CF
Based on DNA denaturation due to increasing temperature
DNA which contains mutations will melt at different temperatures
If DNA denatured during electrophoresis, its electrophoretic mobility will be affected
CFTR gene amplified by PCR and reaction products electrophoresed with TTGE
α-Thalassaemia Diagnosis
Individuals asymptomatic
Present with a microcytic, hypochromic blood picture
[Hb] normal or mildly decreased
Iron studies normal
- differentiates α-thal from Fe lowered anaemia
Hb electrophoresis and HbA2 are normal
- differentiates α-thal from β-thal
What causes α-Thalassaemia?
Mutation in the α-globin gene
Gene cluster roughly 40kb
Each individual inherits a pair of α-globin genes from each parent, therefore 4
A mutation in one or more α-globin gene(s) = α-thal
Severity depends on number of genes deleted
Written genotypes for α-Thalassaemia and what it means
Indicates the number of functional α-globin genes on an individual’s chromosomes
αα - means two functional genes on a chromosome
α- - one functional gene on a chromosome
– - no functional gene on chromosome
Written in pairs to indicated both chromosomes e.g. α-/αα means one functional gene on one and two on the other chromosome
Principle for Gap PCR - α-Thalassaemia
Most α-thal mutations are deletions of large regions of the α-globin gene cluster
These regions wouldn’t be able to be amplified from a normal α-globin gene cluster
Deletions bring areas of α-globin gene cluster closer together, making them amenable to PCR
α-Thalassaemia Multiplex PCR Steps
- Two probes designed to anneal next to each other on target DNA sequence
- Left hand probe contains universal F primer binding region
- Right probe contains a stuffer sequence and a univeral R primer binding region
- Target DNA denatured and probes allowed to bind
- Once bound, ligase added to ligate adjacent probes together
- Probes amplified by PCR using fluorescently labelled F primer and a standard R primer
- Amplicons analysed by capillary electrophoresis
Increase of decrease in fluorescent signal compared to a normal control used to identify duplications or deletions in target regions
Ratios of Fluorescent Signal and what they mean - α-Thal Multiplex Results
Ratio of roughly 1 - indicates that copy number of target region is the same in both test and normal samples
Ratio above 1 - indicates copy number of target region is higher in test sample
- test has duplication of that area of the gene
Ratio of roughly 0.5 - indicates copy number of target region in test sample is half of the normal
- test has a heterozygous deletion of that area of the gene
Ratio of roughly 0 - indicates target region is absent in test sample
- test has a homozygous deletion of that area of the gene
