Cf

Question 1

Location of CFTR? And size?

Answer 1

7q31.2

27 exons

Question 2

Mutations in what other gene can give similar phenotype?

Answer 2

SCN1

Question 3

Incidence of CF

Answer 3

1 in 2000 to 1 in 3000

Carrier frequency 1/22 -1/27

Question 4

Role of CFTR in salmonelli typhi defence?

Answer 4

CFTR membrane chloride channel required for salmonelli typhi to enter intestinal epithelial cells

also hets resistant to typhoid fever and or cholera

Question 5

How many known different mutations?

What % are loss of function ?

Answer 5

> 1,500

2%

Question 6

Example of intronic mutation?

Answer 6

3849+10kb c>t

Question 7

Most common mutation, what percentage?

Answer 7

Delta F508. 80%

Question 8

Example of pancreatic sufficiency mutations? (Can’t digest food properly)

Answer 8

R117H and A455E

Question 9

True or false: strong correlation between genotype and pulmonary function

Answer 9

False: poor correlation

Question 10

Where is the poly T tract and what are the alleles?

Answer 10

Intron 8 adjacent to exon 9 splice acceptor

5, 7, 9 T’s

Question 11

Which poly T is least efficient at splicing and affects expression of R117H? Which is most common?

Answer 11

5T highest level of mRNA lacking exon 9. 5% of alleles

9T is most common

Question 12

Phenotype of R117H and 5t, t7 or 9t with pathogenic variant in trans

Answer 12

5t= variable- could be severe
7t= variable- cf related disease or benign 
9t= v.rare- variable, benign of cf-related

Question 13

Where is Poly TG? What is most common and what increases exon skipping?

Answer 13

Upstream of poly T tract in intron 8
Common = 11
12 or 13 in cis with 5T increases exon 9 skipping

Question 14

Non molecular testing for CF?

Answer 14

Sweat test (gold standard)>60 EQ/L means positive in 98% of CF cases

Immunoreactive trpsinogen (IRT) - IRT serum levels raised in new borns

Question 15

Molecular methods to test for CF?

Answer 15

Amplification refractory multiplex system (ARMS)

• elucigene CF29- gel electrophoresis
• elucigene cfeu2 - capillary electrophoresis

And oligonucleotide ligation assay

Question 16

What is the principle of ARMS?

Answer 16

Primer will only bind if complementary at 3’ end - mutant and wild type primers

Question 17

Cons of CF ARMS 29

Answer 17

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

Question 18

Difference with CF-EU2 kit

Answer 18

WT and mutation specific primers for all mutations except S459R
Includes primers for polyT
STR markers present

Question 19

Problems with CF-EU2 kit

Answer 19

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

Question 20

Explain OLA

Answer 20

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

Question 21

Problems with OLA?

Answer 21

Cross reactivity - 508 on one allele can result in no amplification on second allele. Miss homozygotes

Question 22

Classical cf referrals reasons

Answer 22

Meconium ileus, failure to thrive, respiratory infections

Question 23

Non classical cf referrals

Answer 23

Respiratory problems, broncheatitis, idiopathic chronic pancreatitis, obstructive azoospermia

Question 24

Testing workflow

Answer 24

Identify 2 mutations confirms diagnosis
Then test parents(in trans?)
Then carrier testing offered to family members

Question 25

What %of cf patients will test negative

Answer 25

1%

Question 26

True or false f508 almost always in trans with 9t?

Answer 26

True

Question 27

Tat for carrier testing? Tat for prenatal?

Answer 27

10 days , 3 days prenatal

Question 28

What is exhogenic bowl? What percentage have cf?

Answer 28

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

Question 29

Occurrence of congenital bilateral absence of vas deferens (CBAVD) in men with azoospermia ? What % have CFTR mutations?

Answer 29

Occurs in 1-2% of men with azoospermia of which 50-70% have CFTR

Question 30

Neonatal testing

Answer 30

One of 5 diseases tested for
Blood spots tested for IRT and sequencing of 
F508
G551D- Celtic 
G542X- mediteranian  mutation
621+1 G>t

Question 31

What is the common Ashkenazic Jew mutation?

Answer 31

W1282X- 48%

Question 32

What % of pregnancies have echogenic bowel?

Answer 32

0.6% of which 4% have cf (can use for risk analysis

Question 33

Baby is homoszygous for 508 but dad is not carriers - possible reasons?

Answer 33

Non paternity
Sample mix up
SNP under primer
Deletion of whole allele with 508
Maternal uniparental disomy for Chromosome 7 (microsatellite)

Question 34

What is IRT?

Answer 34

Immunoreactive trypsinogen- pancreatic enzyme precursor elevated in CF