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Flashcards in Cf Deck (34):
1

Location of CFTR? And size?

7q31.2
27 exons

2

Mutations in what other gene can give similar phenotype?

SCN1

3

Incidence of CF

1 in 2000 to 1 in 3000

Carrier frequency 1/22 -1/27

4

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*

5

How many known different mutations?
What % are loss of function ?

>1,500
2%

6

Example of intronic mutation?

3849+10kb c>t

7

Most common mutation, what percentage?

Delta F508. 80%

8

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

R117H and A455E

9

True or false: strong correlation between genotype and pulmonary function

False: poor correlation

10

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

Intron 8 adjacent to exon 9 splice acceptor

5, 7, 9 T's

11

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

12

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

13

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

14

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

15

Molecular methods to test for CF?

Amplification refractory multiplex system (ARMS)
-elucigene CF29- gel electrophoresis
-elucigene cfeu2 - capillary electrophoresis

And oligonucleotide ligation assay

16

What is the principle of ARMS?

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

17

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

18

Difference with CF-EU2 kit

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

19

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

20

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

21

Problems with OLA?

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

22

Classical cf referrals reasons

Meconium ileus, failure to thrive, respiratory infections

23

Non classical cf referrals

Respiratory problems, broncheatitis, idiopathic chronic pancreatitis, obstructive azoospermia

24

Testing workflow

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

25

What %of cf patients will test negative

1%

26

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

True

27

Tat for carrier testing? Tat for prenatal?

10 days , 3 days prenatal

28

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

29

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

30

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

31

What is the common Ashkenazic Jew mutation?

W1282X- 48%

32

What % of pregnancies have echogenic bowel?

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

33

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)

34

What is IRT?

Immunoreactive trypsinogen- pancreatic enzyme precursor elevated in CF