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