Cf Flashcards
Location of CFTR? And size?
7q31.2
27 exons
Mutations in what other gene can give similar phenotype?
SCN1
Incidence of CF
1 in 2000 to 1 in 3000
Carrier frequency 1/22 -1/27
Role of CFTR in salmonelli typhi defence?
CFTR membrane chloride channel required for salmonelli typhi to enter intestinal epithelial cells
also hets resistant to typhoid fever and or cholera
How many known different mutations?
What % are loss of function ?
> 1,500
2%
Example of intronic mutation?
3849+10kb c>t
Most common mutation, what percentage?
Delta F508. 80%
Example of pancreatic sufficiency mutations? (Can’t digest food properly)
R117H and A455E
True or false: strong correlation between genotype and pulmonary function
False: poor correlation
Where is the poly T tract and what are the alleles?
Intron 8 adjacent to exon 9 splice acceptor
5, 7, 9 T’s
Which poly T is least efficient at splicing and affects expression of R117H? Which is most common?
5T highest level of mRNA lacking exon 9. 5% of alleles
9T is most common
Phenotype of R117H and 5t, t7 or 9t with pathogenic variant in trans
5t= variable- could be severe 7t= variable- cf related disease or benign 9t= v.rare- variable, benign of cf-related
Where is Poly TG? What is most common and what increases exon skipping?
Upstream of poly T tract in intron 8
Common = 11
12 or 13 in cis with 5T increases exon 9 skipping
Non molecular testing for CF?
Sweat test (gold standard)>60 EQ/L means positive in 98% of CF cases
Immunoreactive trpsinogen (IRT) - IRT serum levels raised in new borns
Molecular methods to test for CF?
Amplification refractory multiplex system (ARMS)
- elucigene CF29- gel electrophoresis
- elucigene cfeu2 - capillary electrophoresis
And oligonucleotide ligation assay
What is the principle of ARMS?
Primer will only bind if complementary at 3’ end - mutant and wild type primers
Cons of CF ARMS 29
4 tube reaction - dif combinations f primers products separated on gel- compare t standards
Contains mutant 508 and wild type in different tubes
Can’t determine heterozygosity of their mutations
Can get non specific bands
Separate poly t kit
Difference with CF-EU2 kit
WT and mutation specific primers for all mutations except S459R
Includes primers for polyT
STR markers present
Problems with CF-EU2 kit
Cross reactivity - presence of one mutation can affect another
Eg. R117H will reduce R117C
SNP under primer-binding site. Can’t snp check as commercial kit
Explain OLA
5 primers amplify CFTR regions
Each amplicoj probed by 3 probes.
Common probe hybridises to common sequence (fluoresently labelled) and normal and mutant compete (dif non nucleotide mobility modifiers tails attached )
Ligation of perfect matched probes then run on ABI
Problems with OLA?
Cross reactivity - 508 on one allele can result in no amplification on second allele. Miss homozygotes
Classical cf referrals reasons
Meconium ileus, failure to thrive, respiratory infections
Non classical cf referrals
Respiratory problems, broncheatitis, idiopathic chronic pancreatitis, obstructive azoospermia
Testing workflow
Identify 2 mutations confirms diagnosis
Then test parents(in trans?)
Then carrier testing offered to family members
What %of cf patients will test negative
1%
True or false f508 almost always in trans with 9t?
True
Tat for carrier testing? Tat for prenatal?
10 days , 3 days prenatal
What is exhogenic bowl? What percentage have cf?
Brightness in bowel more than surrounding bone
Detected on ultrasound in 0.2-1.2% of pregnancies
3% of those cases have cr
BPG say test parents not foetus
Occurrence of congenital bilateral absence of vas deferens (CBAVD) in men with azoospermia ? What % have CFTR mutations?
Occurs in 1-2% of men with azoospermia of which 50-70% have CFTR
Neonatal testing
One of 5 diseases tested for Blood spots tested for IRT and sequencing of F508 G551D- Celtic G542X- mediteranian mutation 621+1 G>t
What is the common Ashkenazic Jew mutation?
W1282X- 48%
What % of pregnancies have echogenic bowel?
0.6% of which 4% have cf (can use for risk analysis
Baby is homoszygous for 508 but dad is not carriers - possible reasons?
Non paternity Sample mix up SNP under primer Deletion of whole allele with 508 Maternal uniparental disomy for Chromosome 7 (microsatellite)
What is IRT?
Immunoreactive trypsinogen- pancreatic enzyme precursor elevated in CF
