Module 2 - Cystic Fibrosis Flashcards Preview

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Flashcards in Module 2 - Cystic Fibrosis Deck (96)
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When was cystic fibrosis first identified and how?

Identified mucous plugs in pancreas in babies dying of malnutrition.
Condition called "cystic fibrosis of the pancreas."
In 1940s it became apparent that CF was of genetic inheritance.


What was the early characterisation of CF? (post 1938)

Fat and protein malabsorption
Failure to thrive
Lung disease
Abnormal electrolyte composition in sweat


How was the gene responsible for CF identified?

1983 - abnormal cAMP-mediated regulation of chloride transport (sweat ducts)
1985 - gene associated with CF mapped to chromosome 7q31.2 by linkage in families
1989 - gene identified by positional cloning


Describe the CFTR gene

"Cystic Fibrosis Transmembrane conductance Regulator"
90kb DNA
27 exons coding for 1480 amino acids
Codes for large integral glycosylated membrane-spanning protein


Describe the CFTR protein. What type of protein is it and how is it regulated?

Member of ABC (ATP-binding cassette) superfamily
Chloride channel protein. It is different from other ABC transporters because the passage of ions is via passive diffusion.
Regulated by cAMP-dependent phosphorylation
Expressed in epithelial cells (apical membranes)


What are the domains of the CFTR protein?

5 domains.
2 membrane spanning domains (MSD) - forma channel for passage of chloride ions
2 nucleotide-binding domains (NBD) - bind and hydrolyse ATP
1 regulatory domain (R) - phosphorylation by cAMP-dependent protein kinase)


How does CFTR interact with other proteins?

Carboxy-terminal is anchored to cytoskeleton and kept close to other proteins.
These proteins influence CFTR functions such as:
- conductance
- regulation of other channels (e.g. eNAC: epithelial sodium channel)
- signal transduction
- localisation at apical plasma membrane


Describe CFTR function in the airways and how a can mutation affect its function in a patient with CF.

In a normal lung: CFTR assists in Cl- secretion. Cl- ions move out of the cell down a concentration gradient, while Na+ ions and water move into the cell. CFTR inhibits ENac so that there is less absorption of Na+.
In a CF lung: Cl- ions build up within the cell resulting in an even greater movement of Na+ ions and water into the cell. --> dehydrated mucous in airways.


Describe CFTR function in the sweat duct and how a mutation can affect its function in a CF patient.

In normal sweat ducts: Cl- ions move into the cell, down a concentration gradient, together with Na+ ions and water. CFTR activates ENac in the sweat duct. Sweat is secreted into the duct by the gland and both Na+ and Cl- are normally reabsorbed by the duct cells before reaching the skin surface.
In CF sweat ducts: Cl- ions cannot enter the cell and Na+ and water remain in the sweat duct. --> elevated Na+ and Cl- levels in the sweat.


How many known mutations of CF are there? What can they cause? In which exons are the majority of mutations found?

More than 1900 known mutations.
These mutations are a variety of CF-causing, non CF-causing, varied penetrance and unknown significance.
Most mutations are found in exons 4, 8, 14 and 20.


What is a Class I mutation? Provide an example of one.

No protein production.

E.g. Spice mutation intron 4 donor site (G -> T) in MSD1. Absent protein, normal mRNA abundance.


What is a Class II mutation? Provide an example of one.

Defective processing (maturation, premature degradation)

E.g. F508del in NBD 1.


What is a Class III mutation? Provide an example of one.

Defective regulation (e.g. decreased ATP binding and hydrolysis).

E.g. S1255P in NBD2.


What is a Class IV mutation? Provide an example of one.

Defective/reduced ion conductance or channel gating.

E.g. R117H in MSD1. Alteration of Cl- channel.


What is a Class V mutation? Provide an example of one.

Reduced protein production (e.g. promoter or splicing abnormality)

E.g. Splice mutation intron 4 donor site (G -> T) in MSD 1. Reduced number of transcripts.


What is a Class VI mutation? Provide an example of one.

Accelerated turnover from cell surface.

E.g. Q1412X. Instability at cell surface.


What is the most common mutation in CF?

F508del. (Class II)
Accounts for 70-75% of mutations in people from Northern European descent. Homozygosity in about 50% of patients with CF.
Misfolded protein retained in ER and targeted for degradation.


What are some class-specific therapies?

Class I: Nonsense mutations corrected by compounds that allow 'read through' of mRNA. (e.g. aminoglycoside antibiotics)
Class II: 'correctors' to improve processing
Class III: 'potentiators' to activate protein
Class IV: flavonoid compounds to augment channel function (increase open probability
Class V: often splicing mutations. Increase levels of correctly spliced mRNA.


What diagnostic test can be used for a F508del mutation?

PCR + gel electrophoresis.
Homozygotes will show a shorter band due to the deleterious nature of the mutation.


How are mutation panels used in testing?

PCR +/- restriction enzymes.
Number of mutations in testing panels differs throughout the world.
Some mutations are specific to particular ethnic groups.
Previously, in VIC a total of 12 mutations were tested, together accounting for 80% of mutations. A further 7 mutations were added.


Testing of mutations by SNP analysis

Multiplex PCR - single reaction, multiple primers, fluorescent tags.
Assay involves primer extension of each hybridised primer so that each product incorporates a coloured fluorescent tag (dNTP) and the colour of the peak depends on which dNTP has been added.
Analysis of each mutation performed in 2 batches on capillary gel electrophoresis.


Most recent VIC CFTR mutation panels and testing

New diagnostic panel includes 38-40 mutations, accounting for 91% of mutations in VIC population.
SNP genotyping + measuring of mass of allele-specific products following PCR.
MALDI-TOF MS - Matrix assisted laser desorption/ionization-time of flight mass spectrometry.
Can genotype a large number of samples (100s-1000s) and a medium number of SNPs (10s-100s)


What is the pattern of inheritance for CF?

Autosomal recessive
Carrier frequency of 1 in 25.
Often no family history.


What ethnicity does it most commonly affect? What is the incidence rate?

Caucasian descent (especially Northern European).
Incidence of approx. 1/2500-3000 live births.


Why are there different rates of prevalence around the world?

Ethnic-specific mutations.


What are the genotype-phenotype correlations in terms of class and lung disease/pancreatic function?

Weak genotype-phenotype correlations for lung disease, more for pancreatic disease.

Classes I, II, and III (and probably VI) - severe lung disease; pancreatic insufficiency.
Classes IV and V - milder lung disease; pancreatic sufficiency.


What is one challenge of mutations with uncertain clinical significance?

Penetrance of mutation may also depend on another intragenic polymorphism.

E.g. R117H and poly T tract in intron 8 in cis.


Environmental and genetic modifiers both play a role in the resultant phenotype of a CF patient. What phenotypes are not well correlated with CFTR mutations?

Lung function, neonatal intestinal obstruction, diabetes and anthropometry (e.g. weight and height).


It is important to identify genetic modifiers because:

- Identifies new targets for therapies
- Increases understanding of disease variability
- Expect these genes/variants to be minimally penetrant in healthy people but effects unmasked in CF patients.
- These gene modifiers may contribute to development or progression of common diseases in general population


What are 3 types of association studies that can be carried out to identify genetic modifiers of CF?

1. Linkage: Track genes/markers associated with specific phenotype in families with CF
2. Candidate gene expression: Genes with known function; correlate variations in gene with presence of features/phenotype in CF patients.
3. Genome wide association studies (GWAS): Examine DNA markers at many positions on multiple chromosomes in populations with and without CF phenotype of interest. Look for SNPs that are shared with much greater frequency in those with same phenotype.