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

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Presentation on theme: "Cystic Fibrosis Newborn Screening in the Bristol Genetics Laboratory : Development & One Year’s Experience Dr Claire Faulkner Trainee Project."— Presentation transcript:

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 >99.5 th 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 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 Aims of Project To set up and run Molecular CF Newborn Screening Service for the South West

4 DNA Extraction from Bloodspots Chelex 100 resinEZ1 robot DNA quality Poor 260/280 ~1.6 Excellent 260/280 ~1.8 Performance on real-time PCR Poor – differs to control DNA Good – comparable to controls Performance on CF29 Requires dilution > 1/10 Good Reagent cost (per sample) £0.14£4.97 Staff time (per batch of 6) 3 hrs0.5hrs Total cost (per sample) £5.09£5.77

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

6 86 stored DNA samples 34 anti-coagulated bloodspot samples Blind trial on 8 stored DNA samples Blind trial 16 archived coagulated newborn screening bloodspots 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 88 +ve controls

7 N/p.Arg553Xp.Gly551Asp/p.Arg553X N/N N/p.Gly551Asp p.Gly551Asp/ p.Gly551Asp p.Gly551Asp/p.Arg553X amplification curve cycle no. change in fluorescence Blue = mutant Pink = normal

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

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

10 N/NN/p.Gly551As p p.Gly551Asp/p.Gly551Asp 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 Blue = mutant Pink = normal

11 One year Audit ObservedExpected Babies screened40,421 IRT >99.5 th

12 ObservedExpected Babies screened40,421 IRT >99.5 th mutations mutations on CF4912 p.Phe508del/p.Phe508del8 p.Phe508del/p.Gly551Asp1 1 mutation on CF4, 2 nd CF2982 p.Phe508del/p.Arg117His5 p.Phe508del/p.Asp1152His1 p.Phe508del/p.Arg553X1 p.Phe508del/c G>A1 One year Audit

13 ObservedExpected Babies screened40,421 IRT >99.5 th mutations mutation1322 Normal 2 nd IRT1220 N/p.Phe508del12 Raised 2 nd IRT12 p.Phe508del/p.Arg352Gln1

14 One year Audit ObservedExpected Babies screened40,421 IRT >99.5 th mutations mutation1322 No mutations IRT >99.9th3412 Normal 2 nd IRT2612 No 2 nd sample2- Raised 2 nd IRT6*<1 * 5/6 non-British: Pakistani, African, Bangladeshi, mixed, other 5/6 congenital abnormalities, 1/6 hypoxia at birth

15 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 Case Study: p.Phe508del/p.Arg117His

16 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 diseaseSummary

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


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