Cystic Fibrosis Newborn Screening in the Bristol Genetics Laboratory:
Development & One Year’s Experience
Dr Claire Faulkner
Trainee Project
CF Newborn ScreeningCF 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
• 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 ProjectAims of ProjectTo set up and run Molecular CF Newborn Screening Service for the
South West
DNA Extraction from BloodspotsDNA Extraction from Bloodspots
Chelex 100 resin EZ1 robot
DNA qualityPoor
260/280 ~1.6
Excellent260/280 ~1.8
Performance on real-time PCR
Poor – differs to control DNA
Good – comparable to controls
Performance on CF29
Requires dilution
> 1/10Good
Reagent cost
(per sample)£0.14 £4.97
Staff time
(per batch of 6)3 hrs 0.5hrs
Total cost
(per sample)£5.09 £5.77
Negative controls
N/N
N/Mut
Mut/Mut
Example Real-Time PCR ResultsExample Real-Time PCR Results
TaqMan allelic discrimination (AD) for p.Phe508del
• 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 ValidationReal-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
N/p.Arg553X
p.Gly551Asp/p.Arg553Xp.Gly551Asp/p.Arg553X
N/N N/p.Gly551Aspp.Gly551Asp/p.Gly551Asp
p.Gly551Asp/p.Arg553X amplification curve
cycle no.
chan
ge in
flu
ores
cenc
e Blue = mutant
Pink = normal
• Probe incorporates the p.Arg553 site, presence of a mutation at this site likely to decrease binding of the normal probe
p.Gly551Asp/p.Arg553Xp.Gly551Asp/p.Arg553X
• Any apparent p.Gly551Asp homozygote verified with another assay e.g. CF29
NN
p.Arg553X
p.Gly551AspMut
p.Arg553X
Abnormal sampleNormal bloodspotN/N N/c.489+1G>T
p.Phe508del homozygote also positive for c.489+1G>Tp.Phe508del homozygote also positive for c.489+1G>T
Abnormal sample amplification curve
cycle no.
chan
ge in
flu
ores
cenc
e Orange = normal
Purple = mutant
N/N N/p.Gly551Asp
p.Gly551Asp/p.Gly551Asp
Real-Time PCR ThresholdsReal-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
One year AuditOne year AuditObserved Expected
Babies screened 40,421
IRT >99.5th 302 202
Observed Expected
Babies screened 40,421
IRT >99.5th 302 202
2 mutations 17 14
2 mutations on CF4 9 12
p.Phe508del/p.Phe508del 8
p.Phe508del/p.Gly551Asp 1
1 mutation on CF4, 2nd CF29 8 2
p.Phe508del/p.Arg117His 5
p.Phe508del/p.Asp1152His 1
p.Phe508del/p.Arg553X 1
p.Phe508del/c.1766+1G>A 1
One year AuditOne year Audit
One year AuditOne year AuditObserved Expected
Babies screened 40,421
IRT >99.5th 302 202
2 mutations 17 14
1 mutation 13 22
Normal 2nd IRT 12 20
N/p.Phe508del 12
Raised 2nd IRT 1 2
p.Phe508del/p.Arg352Gln 1
One year AuditOne year AuditObserved Expected
Babies screened 40,421
IRT >99.5th 302 202
2 mutations 17 14
1 mutation 13 22
No mutations 272 165
IRT >99.9th 34 12
Normal 2nd IRT 26 12
No 2nd sample 2 -
Raised 2nd IRT 6* <1
* 5/6 non-British: Pakistani, African, Bangladeshi, mixed, other
5/6 congenital abnormalities, 1/6 hypoxia at birth
• 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.Arg117HisCase Study: p.Phe508del/p.Arg117His
• 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
SummarySummary
Thank you!Thank you!
Molecular Maggie WilliamsHilary SawyerAnne GardnerMark GreensladeThais SimmonsRose SalamancaJean WorganJenny Coles
BiochemistryHelena KempAnny BrownMark deHora
Cardiff LaboratorySarah MaundLinda Meredith
