1. Nick Parkin Trainee Clinical Scientist Wessex Regional Genetics Laboratory Cystic Fibrosis rare mutation screen

2. Cystic Fibrosis Autosomal recessive disorder Caused by homozygous or compound heterozygous defects in the CFTR gene (7q31.2) Carrier frequency of ~ 1 in 24 Incidence of ~ 1 in 2400 live births Most prevalent mutation is ΔF508, accounts for ~ 75% of carriers 3 levels of testing offered: CF 4 (4 mutations) CF OLA (32 mutations, 90% of mutations found in people of NW European origin) CF rare screen (whole gene point mutation screen and dosage analysis)

3. Proband GW, aged 13 days, referred as a blood spot No clinical information is given on a Guthrie card They are referred for molecular testing if the patient has an IRT value above the 95th centile The bloodspot was extracted and run on the CF4 kit Case 1

4. CF4 showed that GW is heterozygous for ΔF508 CF4 Results:

5. CF4 result is then confirmed using the CF OLA panel of 32 mutations and checked for the presence of any other mutations. No other mutation found in GW, reported as a carrier of ΔF508. Results:

6. Approximately 2 weeks later a family were referred to us who had 2 affected children GD, neonate, again clinically affected, sample marked as urgent. JD, 2 years old, clinically affected with classic CF (although referred as having CF there had been no molecular testing performed) Case 2

7. On booking in of GD’s sample it became apparent that GD was the previously received GW (confirmed by medical records). GDJD This meant that we now had a more detailed clinical image of the original case

8. Having already genotyped GD (aka GW) there was no need to test him again JD was tested on the CF OLA and as expected showed the same genotype. ΔF508 heterozygous

9. JD was reported as a ΔF508 heterozygote After speaking with the clinicians on the neonatal intensive care unit they were sure that diagnosis of cystic fibrosis was clear cut in both GD and JD It was requested that both brothers be put forward for rare mutation screening

10. Testing strategy for CF rare screen: pre-screen all fragments on dHPLC then direct sequencing MLPA also done to check dosage CF rare screening

11. Results of the pre-screen: multiple exons needed sequencing, 2 in particular looked interesting: Exon 3 Exon 10

12. Exon 10 was expected to be variant, it is the site of the ΔF508 mutation also carried in this family The ΔF508 mutation is a deletion of CTT

13. Exon 3 was problematic Although the fragment worked well for dHPLC we could not get it to sequence New primers were ordered in a hope to resolve these issues With the new primers working the sequencing reaction showed a variant

14. 297-3 C>T (165-3 C>T) Single base substitution in intron 2 3 bases away from the start of exon 3 ? affect splicing activity

15. Previously studied by Beinvenu et al. in 1994 RNA studies showed skipping of exon 3 Reported as a mutation 297-3 C>T (165-3 C>T) However another paper, written in 1995 mentioned 297-3 C>T as a polymorphism Unfortunately the paper gave no references

16. 297-3 C>T (165-3 C>T) Contacted Martin Schwarz in Manchester about this mutation. “I think its safe to say that it may be pathogenic, but avoid making any phenotypic predictions… … If its classical CF rather than CBAVD I’m not so sure”

17. This mutation does not have enough of a negative effect to cause classical CF. The CFTR gene to protein pathway is very inefficient ~70% of the wild type translated protein is broken down before it gets to the epithelial membrane Therefore to cause classic CF you need massivley reduced or complete knock out of CFTR function The mutation only skips exon 3 ~50% of the time, combined with ΔF508 this would still leave 25% of functional CFTR protein. 297-3 C>T (165-3 C>T)

18. There was still one other possibility, exon 23 had shown a slight shift but it did not look like it very convincing.

19. Normal GD JD

20. 4279insA (c.4147dupA) Exon 23 sequencing results:

21. 4279insA (c.4147dupA) Duplication of A in exon 23 of the CFTR gene Mutation creates a stop codon 5bp down stream Premature termination loses 97 amino acids from the C-terminal of the protein Although this premature stop codon removes the end of the protein Haardt et al. 1999, showed that the protein still retains function The mature protein however is degraded 5-6 fold quicker than the wild type protein when it reaches the epithelial membrane

22. 4279insA (c.4147dupA) After speaking to Martin Schwarz again he said that they have found this mutation before However they have only ever been found it with the 297-3 variant (so far) Due to the nature of the 2 mutations (neither being a complete knock out) it is possible that the 2 mutations have an additive effect creating the classical CF presentation. Intersetingly the 297-3 variant had been reported as a polymorphism because it was found in cis with 4279insA

23. This case has now been reported Only the 4279insA was reported as it is non-sense and therefore presumed to be causative. We are awaiting the parental samples to confirm the phase of the mutations. Conclusion

24. Acknowledgements Anne-Marie Reuther (WRGL) Esta Cross (WRGL) Martin Schwartz (Manchester DNA Lab)