Cystic Fibrosis Mutations and Testing Methods

Question 1

Testing strategy

Answer 1

Allelic heterogeneity but common mutations account for majority of disease (founder effect)

Cost effective to test for common mutations as first test

Question 2

Referral reasons

Answer 2

Newborn screening and cascade testing

Diagnostic (symptoms of CF or CFTR related disease)

Carrier testing (FH, partner of carrier, FEB)

Question 3

Testing methods

Answer 3

Non molecular:

• sweat test (gold standard) >60mEQ/L 98% cases
• Immunodeficiency trypsinogen serum levels elevated in newborns

Molecular:
-mutation specific (ARMS PCR) CFEU2, Devyser (OLA) oligonucleotide ligation assay

• gene screen (NGS)
• linkage analysis for unidentifiable mutation (historical)

Question 4

ARMS PCR

Answer 4

Allele specific PCR

Two forward primers specific for either WT or mutant allele at 3’ end and third common reverse primer

> 50 mutations in kit and poly T

Question 5

PolyT tract

Answer 5

Intron 8 polythymidine tract, the length of which affects splicing of exon 9

Lengths of 5T 7T and 9T typically observed

5T shows least efficient splicing. mRNA lacks exon 9 resulting in reduced production of functional protein

Freq 5.2,83.3, and 11.5 respectively in general pop

Modifies expression of R117H

Question 6

ARMS PCR controls

Answer 6

Positive control (known mutation)
Negative control (no template)
Amplification control (STR markers) also identity check

Question 7

PolyT tract analysis

Answer 7

Only unmasked in certain circumstances:

• always for males with azoospermia as 5T variant observed in ~21% with CBAVD
• if R117H mutation detected (5T modifies severity)
• for CFTR related disease if another mutation identified as 5T in trans with classical mutation can be sufficient to cause disease

Question 8

PolyTG tract

Answer 8

Adjacent to the polyT tract

Further affects splicing of exon 9

Variable length (generally 10-13 repeats) longer repeats less efficient splicing

Higher numbers (12-13) more often associated with CBAVD in those with 5T variant

TG repeat length thought to be predictor of penetrance of 5T variant

Question 9

R117H

Answer 9

Severity of mutation modified by polyT tract in cis:

7T in trans with known mutation = consistent with CFTR related disease, classical CF unlikely, maybe benign

5T in trans with known mutation = consistent with a diagnosis of CF of variable severity

7T in trans with 5T only = maybe associated with CF related disease or maybe benign

Question 10

5T

Answer 10

in trans with known mutation = maybe associated with CF related disease or maybe benign

in trans with 5T = maybe associated with CF related disease, more likely benign

alone, unlikely to be associated with CFTR related disease

Question 11

Testing cross reactivity

Answer 11

Presence of one CF mutation can affect other primers preventing binding e.g presence of R117H results in reduced peak height of R117C in CF EU2 B mix (wt), or peak absent if hom for R117H

Presence of insertions or deletions for with primers not included can be detected by change in expected size of another amplicon in the WT mix

Question 12

PCR problems

Answer 12

1. Identification of extra peaks in B mix but no mutation in A mix (maybe insertion/ deletion) requires further investigation
2. SNP under primer binding site ( false neg or reported hom instead of het) cannot snpcheck as don’t know sequence. Hom would be confirmed by sequencing. Caveat in report.

Question 13

Heterozygous advantage theory

Answer 13

CFTR membrane chloride channel required for Salmonella to enter intestinal epithelial

Het maybe resistant to typhoid fever and/or cholera

Question 14

Oligonucleotide ligation assay

Answer 14

Genomic DNA amplified using primers flanking regions where common mutations may be detected

Each amplicon generated is probed by 3 oligonucleotide probes

• Common probe: hybridises to amplicon at common sequence (fluorescently labelled)
• Mutant and Normal probes: compete for binding of amplicon (complementary probe hybridises)

Probes ligated to common probe in ligase reaction

Normal and mutant probes have varying no.s of mobility modifying tails (combination of electrophoretic mobility and fluorescence permits identification of CF genotype)

Question 15

Problems with OLA

Answer 15

Presence of DeltaF508 on one chr and F508C on the other may result in failure of the normal probe to hybridise and prevent ligation

Also occrs for I507V and I507del

Question 16

Diagnostic referral reasons

Answer 16

Classical CF:

• meconium ileus
• failure to thrive
• recurrent respiratory infections

Non-classical:

• bronchiectasis
• pancreatitis
• CBAVD/azoospermia
• allergic brochopulmonary aspergillosis

Question 17

Carrier testing referral reasons

Answer 17

Partner of a known carrier or diagnosed with CF (risk of affected child)
Family history cascade screening
Parents of affected child
Egg or sperm donor
Undergoing IVF treatment
Consanguinous couple (without family history)
Pregnancy with FEB (test couple)

Patients must be >16y

Question 18

Prenatal diagnosis

Answer 18

TAT 3 days

Amnio/CVS tested if parents are known carriers

Negative result makes risk very low

Carriers will be identified

Must check for MCC

Question 19

FEB

Answer 19

Ultrasound identifies bowel with brightness equal to surrounding bone

Detected in 0.2-1% of pregnancies (ultrasonography subjective)

3% of grade 2 or above FEB have CF

Parental samples tested for carrier status

If negative, prenatal testing is not recommended due to lowered risk

Question 20

Neonatal screening

Answer 20

four most common mutations in white british pop:

DeltaF508
G551D
G542X
621+1G>T

Carried out if IRT levels raised at day 5

Question 21

Advantages and disadvantages of neonatal screening

Answer 21

A:
lowered disease severity due to early treatment
decreased burden of care

D:
false positives (anxiety)
identification of carriers