Cystic Fibrosis Newborn Screening in the Bristol Genetics Laboratory : Development & One Year’s Experience

1. Cystic Fibrosis Newborn Screening in the Bristol Genetics Laboratory:Development & One Year’s Experience Dr Claire Faulkner Trainee Project

2. CF Newborn Screening • National Protocol designed to pick up 95% CF cases • Enables early diagnosis and treatment • Multi-stage protocol involving IRT and DNA analysis on bloodspot • IRT (Immunoreactive trypsinogen) • - serum levels elevated in newborns with CF • - normal passage of trypsinogen from pancreas to duodenum blocked • - not 100% specific • - samples raised IRT >99.5th centile (70ng/L) referred to DNA • DNA analysis • - CF4: p.Phe508del, p.Gly551Asp, p.Gly542X, c.489+1G>T • 80% coverage • - CF29: 90% coverage

3. Aims of Project To set up and run Molecular CF Newborn Screening Service for the South West • Develop and validate a method to extract DNA from bloodspots • Optimise and validate 4 mutation assay using real-time PCR • - quick (setup-run-analysis in 3hrs) • - cost-effective (£1 per mutation per sample) • - equipment in-house • - validated by Cardiff laboratory • Write protocols, assess H&S, train technicians, liaise with Biochemistry, day-to-day management of service • Go-live Jan 07 • Compile audit data to monitor and improve service

4. DNA Extraction from Bloodspots

5. Example Real-Time PCR Results Mut/Mut N/Mut N/N Negative controls TaqMan allelic discrimination (AD) for p.Phe508del

6. Real-Time PCR Validation • All samples (136 in duplicate) correctly genotyped except: i. p.Gly551Asp/p.Arg553X sample called as a p.Gly551Asp homozygote ii. p.Phe508del homozygous archived bloodspot called as a p.Phe508del homozygote and a c.489+1G>T heterozygote • 86 stored DNA samples • 34 anti-coagulated bloodspot samples • Blind trial on 8 stored DNA samples • Blind trial 16 archived coagulated newborn screening bloodspots 88 +ve controls

7. p.Gly551Asp/p.Arg553X p.Gly551Asp/p.Arg553X amplification curve change in fluorescence Blue = mutant Pink = normal cycle no. p.Gly551Asp/ p.Gly551Asp N/p.Gly551Asp N/p.Arg553X N/N

8. p.Gly551Asp/p.Arg553X p.Arg553X p.Arg553X N N • Probe incorporates the p.Arg553 site, presence of a mutation at this site likely to decrease binding of the normal probe Mut p.Gly551Asp • Any apparent p.Gly551Asp homozygote verified with another assay e.g. CF29

9. p.Phe508del homozygote also positive for c.489+1G>T Abnormal sample amplification curve change in fluorescence Orange = normal Purple = mutant cycle no. N/N N/c.489+1G>T Normal bloodspot Abnormal sample

10. Real-Time PCR Thresholds • Both AD and amplification curves should be checked for each sample • Ensures contamination or unusual amplification patterns are detected • Introduced threshold levels as a non-subjective method to check amplification curves N/N N/p.Gly551Asp p.Gly551Asp/p.Gly551Asp Blue = mutant Pink = normal

11. One year Audit

12. One year Audit

13. One year Audit

14. One year Audit * 5/6 non-British: Pakistani, African, Bangladeshi, mixed, other 5/6 congenital abnormalities, 1/6 hypoxia at birth

15. Case Study: p.Phe508del/p.Arg117His • Uncertain outcome: PS-CF / late-onset CF / CBAVD / asymptomatic • PolyT testing as reflex for p.Arg117His? • Report issued with interpretative comments and recommending further molecular testing following genetic counselling • PolyT testing later requested: 9T/7T • Clinical follow-up: • - PS, normal sweat test • - respiratory pathogens isolated, antibiotics • - difficult to counsel parents • - consultant paediatrician will continue to monitor clinically but label him as atypical or non-classical CF rather than CF

16. Summary • Real-time PCR is an effective method for mutation detection and an excellent tool for CF Newborn Screening • Essential that both AD and amplification curves checked for each sample, using threshold levels • In one year of running service, over 40,000 cases screened and 17 babies with two CFTR mutations detected • It is important that babies with mild or variable mutations (e.g. p.Arg117His) that are well are not labelled with a diagnosis of CF but are closely monitored for signs of disease

17. Thank you! Molecular Maggie Williams Hilary Sawyer Anne Gardner Mark Greenslade Thais Simmons Rose Salamanca Jean Worgan Jenny Coles Biochemistry Helena Kemp Anny Brown Mark deHora Cardiff Laboratory Sarah Maund Linda Meredith