implementation of the national cf newborn screening programme: a review of the first year of testing...

Implementation of the National Implementation of the National CF Newborn Screening CF Newborn Screening

programme: programme: a review of the first year of a review of the first year of

testing nationwide.testing nationwide.

Ottie O’Brien (Northern Genetics Service, Ottie O’Brien (Northern Genetics Service, Newcastle Hospitals NHS Foundation Trust) Newcastle Hospitals NHS Foundation Trust)

and all CMGS laboratories providing CF newborn and all CMGS laboratories providing CF newborn screening.screening.

IntroductionIntroduction

Biochemical screening of newborns in some parts of UK Biochemical screening of newborns in some parts of UK since 1980since 1980

Northern Ireland since 1983, Wales 1997, Scotland Northern Ireland since 1983, Wales 1997, Scotland 2003.2003.

Parent-led support group driven towards extension of Parent-led support group driven towards extension of screening for all newborns in UKscreening for all newborns in UK

National Screening committee not convinced of benefits National Screening committee not convinced of benefits of screen but supported it because of inequity in UKof screen but supported it because of inequity in UK

Evidence that early treatment leads to better outcome is Evidence that early treatment leads to better outcome is accumulatingaccumulating

Screening in all areas of England from 1 October 2006Screening in all areas of England from 1 October 2006

Screening protocol for EnglandScreening protocol for England IRT–DNA–IRT (5-8 days. Raised >99.5IRT–DNA–IRT (5-8 days. Raised >99.5 thth centile) centile) Maximise diagnosis of affecteds with Maximise diagnosis of affecteds with

preventable / treatable diseasepreventable / treatable disease Minimise pick up of carriers and mild forms (late Minimise pick up of carriers and mild forms (late

onset, unpreventable)onset, unpreventable) Stage 1: Stage 1: 4 mutations4 mutations Stage 2: Stage 2: 29/30 mutations 29/30 mutations

(detects 85-90% mutations in Caucasians)(detects 85-90% mutations in Caucasians) 22ndnd IRT intended to increase detection of CF IRT intended to increase detection of CF Disadvantages – pick up healthy carriers, non-Disadvantages – pick up healthy carriers, non-

paternitypaternity

UK regional laboratoriesUK regional laboratories England:England: 1st screen, 4 mutations 1st screen, 4 mutations

c.489+1G>T, p.Phe508del, p.Gly542X, p.Gly551Aspc.489+1G>T, p.Phe508del, p.Gly542X, p.Gly551Asp 22ndnd screen, 29 mutations screen, 29 mutations

Wales (Cardiff):Wales (Cardiff): 1 screen, 8 mutations 1 screen, 8 mutations c.489+1G>T, p.Phe508del, p.Gly542X, p.Gly551Aspc.489+1G>T, p.Phe508del, p.Gly542X, p.Gly551Aspp.Gly85Glu, p.Arg553X, c.1766+1G>A, p.Arg1283Metp.Gly85Glu, p.Arg553X, c.1766+1G>A, p.Arg1283Met

Scotland (Glasgow): Scotland (Glasgow): 1 screen, 28 mutations1 screen, 28 mutations

Birmingham – Birmingham – 11stst screen West Midlands Newborn Screening Centre screen West Midlands Newborn Screening Centre 22ndnd screen West Midlands Regional Genetics Service screen West Midlands Regional Genetics Service

Cystic Fibrosis Regional GeneticsCystic Fibrosis Regional GeneticsScreening Laboratories LaboratoriesScreening Laboratories Laboratories

North West ThamesNorth West Thames BristolBristol WessexWessex OxfordOxford CambridgeCambridge BirminghamBirmingham TrentTrent ManchesterManchester LiverpoolLiverpool LeedsLeeds NewcastleNewcastle

Methods and reportingMethods and reporting

Extraction:Extraction: EZ1, boiled prep, OLA CF33, EZ1, boiled prep, OLA CF33,

QIA AmpQIA Amp®® DNA micro kit DNA micro kit Methods:Methods: Tepnel Tepnel®® CF4, CF29, CF-HT30, CF4, CF29, CF-HT30,

CF-HT28, AbbotCF-HT28, Abbot®® CF OLA33, in-house Taqman, CF OLA33, in-house Taqman, PCR + PAGE, sequencing exons 4 & 11PCR + PAGE, sequencing exons 4 & 11

Sample:Sample: whole card (7), punches (3), 1 spot, 1 whole card (7), punches (3), 1 spot, 1 square. Storage issues (2).square. Storage issues (2).

Reporting method:Reporting method: variable variable Turnaround:Turnaround: almost all report >97% in 4 days almost all report >97% in 4 days

A blood spot cardA blood spot card

CF screening numbers CF screening numbers

NW Thames

Bristol

Wessex

Oxford

Birmingham

Trent

Manchester

Liverpool

Leeds

Newcastle

Wales

Scotland


Months

NW Thames 5

Bristol 6

Wessex 8

Oxford 12

Birmingham 12

Trent 12

Manchester 4

Liverpool 12

Leeds 12

Newcastle 12

Wales 12

Scotland 12


Months Total

screened

NW Thames 5 83579

Bristol 6 19822

Wessex 8 Not known

Oxford 12 29915

Birmingham 12 69105

Trent 12 73274

Manchester 4 Not known

Liverpool 12 28814

Leeds 12 37634

Newcastle 12 34447

Wales 12 33404

Scotland 12 55000


Months Total IRT>99.5th C

screened

NW Thames 5 83579 461

Bristol 6 19822 144

Wessex 8 Not known 67

Oxford 12 29915 289

Birmingham 12 69105 383

Trent 12 73274 413

Manchester 4 Not known 75

Liverpool 12 28814 123

Leeds 12 37634 281

Newcastle 12 34447 257

Wales 12 33404 223

Scotland 12 55000 406


Months Total IRT>99.5th C %

screened

Expected 0.5

NW Thames 5 83579 461 0.55

Bristol 6 19822 144 0.73

Wessex 8 Not known 67

Oxford 12 29915 289 0.97

Birmingham 12 69105 383 0.55

Trent 12 73274 413 0.56

Manchester 4 Not known 75

Liverpool 12 28814 123 0.43

Leeds 12 37634 281 0.75

Newcastle 12 34447 257 0.75

Wales 12 33404 223 0.67

Scotland 12 55000 406 0.74

Babies presumed affectedBabies presumed affected

NWTh

Bristol

Wessex

Oxford

Bham

Trent

Manch

Liverpool

Leeds

Ncle

Wales

Scot


2 mut on

panel 1

NWTh 9

Bristol 2

Wessex 6

Oxford 4

Bham 10

Trent 14

Manch 3

Liverpool 9

Leeds 5

Ncle 6

Wales 6

Scot 15


2 mut on 1 mut panel 1

panel 1 2nd mut panel 2

NWTh 9 4

Bristol 2 3

Wessex 6 1

Oxford 4 0

Bham 10 3

Trent 14 4

Manch 3 1

Liverpool 9 4

Leeds 5 0

Ncle 6 1

Wales 6 N/A

Scot 15 N/A


2 mut on 1 mut panel 1 0 mut & repeat

panel 1 2nd mut panel 2 raised IRT

NWTh 9 4 not reported

Bristol 2 3 1

Wessex 6 1 not reported

Oxford 4 0 not reported

Bham 10 3 8

Trent 14 4 14

Manch 3 1 not reported

Liverpool 9 4 4

Leeds 5 0 4

Ncle 6 1 1

Wales 6 N/A not reported

Scot 15 N/A not reported


2 mut on 1 mut panel 1 0 mut & repeat Total presumed

panel 1 2nd mut panel 2 raised IRT Affected

NWTh 9 4 not reported 13

Bristol 2 3 1 6

Wessex 6 1 not reported 7

Oxford 4 0 not reported 4

Bham 10 3 8 21

Trent 14 4 14 32

Manch 3 1 not reported 4

Liverpool 9 4 4 17

Leeds 5 0 4 9

Ncle 6 1 1 8

Wales 6 N/A not reported 6

Scot 15 N/A not reported 15

Babies presumed affectedBabies presumed affected (% of babies with raised IRT)(% of babies with raised IRT)

2 mut on % 1 mut panel 1 % 0 mut & repeat % Total presumed %

panel 1 2nd mut panel 2 raised IRT Affected

Expected 6 1 0.2 7.2

NWTh 9 1.95 4 0.9 not reported 13 2.8

Bristol 2 1.4 3 2.1 1 0.7 6 4.2

Wessex 6 9 1 1.5 not reported 7 10.4

Oxford 4 1.4 0 0 not reported 4 1.4

Bham 10 2.6 3 0.8 8 2.1 21 5.5

Trent 14 3.4 4 1 14 3.4 32 7.7

Manch 3 4 1 1.3 not reported 4 5.3

Liverpool 9 7.3 4 3.3 4 3.3 17 13.8

Leeds 5 1.8 0 0 4 1.4 9 3.2

Ncle 6 2.3 1 0.4 1 0.4 8 3.1

Wales 6 2.7 N/A not reported 6 2.7

Scot 15 3.7 N/A not reported 15 3.7

Babies with 1 mutationBabies with 1 mutation

NW Thames

Bristol

Wessex

Oxford

Birmingham

Trent

Manchester

Liverpool

Leeds

Newcastle

Wales

Scotland


Total

IRT>99.5th C

NW Thames 461

Bristol 144

Wessex 67

Oxford 289

Birmingham 383

Trent 413

Manchester 75

Liverpool 123

Leeds 281

Newcastle 257

Wales 223

Scotland 406


Total 1 mut on panel 1

IRT>99.5th C 0 mut on panel 2

NW Thames 461 25

Bristol 144 6

Wessex 67 8

Oxford 289 18

Birmingham 383 22

Trent 413 26

Manchester 75 4

Liverpool 123 9

Leeds 281 11

Newcastle 257 19

Wales 223 20

Scotland 406 44


Total 1 mut on panel 1 %

IRT>99.5th C 0 mut on panel 2

Expected 11

NW Thames 461 25 5.4

Bristol 144 6 4.2

Wessex 67 8 11.9

Oxford 289 18 6.2

Birmingham 383 22 5.7

Trent 413 26 6.3

Manchester 75 4 5.3

Liverpool 123 9 7.3

Leeds 281 11 3.9

Newcastle 257 19 7.4

Wales 223 20 9

Scotland 406 44 10.8

Summary of mutations found in affected babiesSummary of mutations found in affected babies(panel 1 and panel 1+2)(panel 1 and panel 1+2)

Sc

ot

Wale

s

NW

th

Bri

sto

l

Wessex

Ox

ford

Bh

am

Tre

nt

Man

ch

Liv

erp

oo

l

Lee

ds

Ncle

To

tal

Phe508del/Phe508del9 6 5 2 4 4 8 12 2 7 5 6 70

Phe508del/G551D 2 2 1 1 1 7Phe508del/G542X 1 1 2Phe508del/c.489+1G>A

1 1 2G542X/G542X 1 1G551D/G551D 1 1

Phe508del/D1152H 1 1 2 1 1 6Phe508del/R117H 1 1 1 1 1 5Phe508del/Ile507del

2 2Phe508del/1717G>A

1 1 2Phe508del/1766+1G>A

1 1 2Phe508del/R1162X 1 1 2

Meconium ileus casesMeconium ileus cases

Echogenic screen showed pregnancy to be Echogenic screen showed pregnancy to be F508del/F508del – family told predicted CF affected.F508del/F508del – family told predicted CF affected.Baby included in CFNS and had normal IRT - reported as Baby included in CFNS and had normal IRT - reported as “CF not suspected”. Checked Guthrie for F508del and “CF not suspected”. Checked Guthrie for F508del and confirmed original results. Biochem then lowered their cut confirmed original results. Biochem then lowered their cut off levels slightly to try and prevent this happening again.off levels slightly to try and prevent this happening again.

Baby with meconium ileus was missed on blood spots as Baby with meconium ileus was missed on blood spots as no raised IRT – had F508del and R117H/5Tno raised IRT – had F508del and R117H/5T

Urgent direct referral from a neonatal unit for a child with Urgent direct referral from a neonatal unit for a child with meconium peritonitis. Screened for the full panel of meconium peritonitis. Screened for the full panel of mutations and identified a c.3528delC heterozygote. mutations and identified a c.3528delC heterozygote. Guthrie analysis reported this patient as ‘no mutation Guthrie analysis reported this patient as ‘no mutation detected’ for four mutations. (Received in time frame)detected’ for four mutations. (Received in time frame)

R117H casesR117H cases Communication issue regarding F508del/R117H (7T/9T)

compound heterozygote.

2 cases of F508del/R117H mutations. No guidance in CFNS for polyT analysis so not carried out on Guthrie referrals. Subsequent confirmatory blood samples requested PolyT analysis and both patients proved to be 7/9T. (These cases have been identified since 31/03/07)

Case NOT detected by CFNS: Mother affected: Phe508del / rare mutation, Father carrier: R117H / N. Baby born with R117H / rare mutation. 1st IRT well below 99.5th C so not sent for DNA.

Sweat test and subsequent management not yet known.

F508del carrier picked up on CFNS. Cascade testing of F508del carrier picked up on CFNS. Cascade testing of

family revealed 2 yr old cousin was F508del/R117H(7T) family revealed 2 yr old cousin was F508del/R117H(7T) and had borderline sweat test. Being treated for CF.and had borderline sweat test. Being treated for CF.

F508del / D1152H identified in a CFNS case. Subsequent testing of siblings. Both F508del / D1152H compound heterozygotes. One asymptomatic but one who had presented with a cough and small size and had been discharged.

Infant with F508del / 2789+insA on CFNS. 17 yr old brother (asymptomatic) requested testing. Also F508del / 2789+insA and had positive sweat test.

Cascade testing examplesCascade testing examples

Technical problemsTechnical problems False positive F508del/F508del using the CF30HT kit.

Reported to Tepnel and they subsequently located a polymorphism under normal F508del primer binding site. Now confirm all F508del homozygotes with alternative assay and different primers. All other homozygous mutations are confirmed by requesting parental samples.

One copy of F508del and no second mutation. Sent to Manchester for rare mutation screen. Found R117H. Checked data again – no evidence of blue mutation peak on CFHT, but drop in height of green normal peak. Repeated blood spot and fresh blood sample, still no blue mutation peak. Contacted Tepnel.

Blood spot QA from CDC USA - CFHT kit failed to detect W1282X mutation. Informed Tepnel.

Problems / suggestionsProblems / suggestions

Samples – variable quality, size, timing.Samples – variable quality, size, timing. More referrals than predicted – cut off too low?More referrals than predicted – cut off too low? Lack of follow-up e.g. 2Lack of follow-up e.g. 2ndnd IRT data, 2 IRT data, 2ndnd rare rare

mutationmutation Need to have a system for prenatal tests to pre-Need to have a system for prenatal tests to pre-

empt CFNS to avoid conflicting reports.empt CFNS to avoid conflicting reports. Regions with significant non-Caucasian Regions with significant non-Caucasian

population – 4 mutation panel not ideal e.g. NW population – 4 mutation panel not ideal e.g. NW Thames would like W1282X as large AshkenaziThames would like W1282X as large Ashkenazi

Use the same protocol as CFNS for all Use the same protocol as CFNS for all diagnostic CF referralsdiagnostic CF referrals

SummarySummary

National CF Newborn Screening programme National CF Newborn Screening programme going well after first yeargoing well after first year

Good communication with BiochemistryGood communication with Biochemistry Higher workload than expectedHigher workload than expected Lower pick up rate than expected (affecteds and Lower pick up rate than expected (affecteds and

carriers)carriers) More babies with 0 mutations and 2More babies with 0 mutations and 2ndnd high IRT high IRT

than in algorithm. May not get full screen.than in algorithm. May not get full screen. Need to have system for prenatal resultsNeed to have system for prenatal results Would like more feedback about fate of cases Would like more feedback about fate of cases

with unclear outcome.with unclear outcome.

AcknowledgementsAcknowledgements

GlasgowGlasgowCardiffCardiff

North West ThamesNorth West ThamesWest MidlandsWest Midlands

BristolBristolLeedsLeeds

LiverpoolLiverpoolManchesterManchester

OxfordOxfordTrentTrent

NewcastleNewcastleWessexWessex

Angela IleyAngela IleyRachel ButlerRachel ButlerStuart PayneStuart PayneSarah Ball, Fiona MacDonaldSarah Ball, Fiona MacDonaldClaire Faulkner, Maggie WilliamsClaire Faulkner, Maggie WilliamsSarah Shepherd, David Cockburn,Sarah Shepherd, David Cockburn,Teresa Patrick, Helen LindsayTeresa Patrick, Helen LindsayEmma McCarthy, Kevin SouthernEmma McCarthy, Kevin SouthernMartin SchwarzMartin SchwarzSomai ManSomai ManRichard KirkRichard KirkRuth Sutton, Steve Turner Ruth Sutton, Steve Turner Stacey SandellStacey Sandell

implementation of the national cf newborn screening programme: a review of the first year of testing...

Documents

screening protocol

extension of screening

predicted cf

uknational screening

benefits of screen

affected babiespanel

panel of mutations

abbot cf ola33