1. Implementation of the National CF Newborn Screening programme: a review of the first year of testing nationwide. Ottie O’Brien (Northern Genetics Service, Newcastle Hospitals NHS Foundation Trust) and all CMGS laboratories providing CF newborn screening.

2. Introduction Biochemical screening of newborns in some parts of UK since 1980 Biochemical screening of newborns in some parts of UK since 1980 Northern Ireland since 1983, Wales 1997, Scotland 2003. Northern Ireland since 1983, Wales 1997, Scotland 2003. Parent-led support group driven towards extension of screening for all newborns in UK Parent-led support group driven towards extension of screening for all newborns in UK National Screening committee not convinced of benefits of screen but supported it because of inequity in UK National Screening committee not convinced of benefits of screen but supported it because of inequity in UK Evidence that early treatment leads to better outcome is accumulating Evidence that early treatment leads to better outcome is accumulating Screening in all areas of England from 1 October 2006 Screening in all areas of England from 1 October 2006

3. Screening protocol for England IRT–DNA–IRT (5-8 days. Raised >99.5 th centile) IRT–DNA–IRT (5-8 days. Raised >99.5 th centile) Maximise diagnosis of affecteds with preventable / treatable disease Maximise diagnosis of affecteds with preventable / treatable disease Minimise pick up of carriers and mild forms (late onset, unpreventable) Minimise pick up of carriers and mild forms (late onset, unpreventable) Stage 1: 4 mutations Stage 1: 4 mutations Stage 2: 29/30 mutations Stage 2: 29/30 mutations (detects 85-90% mutations in Caucasians) 2 nd IRT intended to increase detection of CF 2 nd IRT intended to increase detection of CF Disadvantages – pick up healthy carriers, non- paternity Disadvantages – pick up healthy carriers, non- paternity

5. UK regional laboratories England:1st screen, 4 mutations England:1st screen, 4 mutations c.489+1G>T, p.Phe508del, p.Gly542X, p.Gly551Asp 2 nd screen, 29 mutations 2 nd screen, 29 mutations Wales (Cardiff):1 screen, 8 mutations Wales (Cardiff):1 screen, 8 mutations c.489+1G>T, p.Phe508del, p.Gly542X, p.Gly551Asp p.Gly85Glu, p.Arg553X, c.1766+1G>A, p.Arg1283Met Scotland (Glasgow): 1 screen, 28 mutations Scotland (Glasgow): 1 screen, 28 mutations Birmingham – 1 st screen West Midlands Newborn Screening Centre 2 nd screen West Midlands Regional Genetics Service

6. Cystic Fibrosis Regional Genetics Screening Laboratories Laboratories North West Thames Bristol Wessex Oxford Cambridge Birmingham Trent Manchester Liverpool Leeds Newcastle

7. Methods and reporting Extraction: EZ1, boiled prep, OLA CF33, Extraction: EZ1, boiled prep, OLA CF33, QIA Amp ® DNA micro kit Methods: Tepnel ® CF4, CF29, CF-HT30, Methods: Tepnel ® CF4, CF29, CF-HT30, CF-HT28, Abbot ® CF OLA33, in-house Taqman, PCR + PAGE, sequencing exons 4 & 11 Sample: whole card (7), punches (3), 1 spot, 1 square. Storage issues (2). Sample: whole card (7), punches (3), 1 spot, 1 square. Storage issues (2). Reporting method: variable Reporting method: variable Turnaround: almost all report >97% in 4 days Turnaround: almost all report >97% in 4 days

8. A blood spot card

9. CF screening numbers NW Thames Bristol Wessex Oxford Birmingham Trent Manchester Liverpool Leeds Newcastle Wales Scotland

10. CF screening numbers 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

11. CF screening numbers MonthsTotal screened NW Thames 583579 Bristol 619822 Wessex 8Not known Oxford 12 29915 Birmingham 1269105 Trent 12 73274 Manchester 4Not known Liverpool 1228814 Leeds 1237634 Newcastle 1234447 Wales 1233404 Scotland 1255000

12. CF screening numbers MonthsTotalIRT>99.5 th C screened NW Thames 583579461 Bristol 619822144 Wessex 8Not known 67 Oxford 12 29915289 Birmingham 1269105383 Trent 12 73274413 Manchester 4Not known 75 Liverpool 1228814123 Leeds 1237634281 Newcastle 1234447257 Wales 1233404223 Scotland 1255000406

13. CF screening numbers MonthsTotalIRT>99.5 th C% screened Expected 0.5 NW Thames 5835794610.55 Bristol 6198221440.73 Wessex 8Not known 67 Oxford 1229915 2890.97 Birmingham 12691053830.55 Trent 12 732744130.56 Manchester 4Not known 75 Liverpool 12288141230.43 Leeds 12376342810.75 Newcastle 12344472570.75 Wales 12334042230.67 Scotland 12550004060.74

14. Babies presumed affected NWTh Bristol Wessex Oxford Bham Trent Manch Liverpool Leeds Ncle Wales Scot

15. Babies presumed affected 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

16. Babies presumed affected 2 mut on1 mut panel 1 panel 12nd mut panel 2 NWTh 94 Bristol 23 Wessex 61 Oxford 40 Bham 103 Trent 144 Manch 31 Liverpool 94 Leeds 50 Ncle 61 Wales 6N/A Scot 15N/A

17. Babies presumed affected 2 mut on1 mut panel 10 mut & repeat panel 12nd mut panel 2 raised IRT NWTh 94 not reported Bristol 231 Wessex 61 not reported Oxford 40 not reported Bham 1038 Trent 144 Manch 31 not reported Liverpool 944 Leeds 504 Ncle 611 Wales 6N/A not reported Scot 15N/A not reported

18. Babies presumed affected 2 mut on1 mut panel 10 mut & repeatTotal presumed panel 12nd mut panel 2 raised IRTAffected NWTh 94 not reported 13 Bristol 2316 Wessex 61 not reported 7 Oxford 40 not reported 4 Bham 103821 Trent 144 32 Manch 31 not reported 4 Liverpool 94417 Leeds 5049 Ncle 6118 Wales 6N/A not reported 6 Scot 15N/A not reported 15

19. Babies presumed affected (% 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 91.9540.9 not reported 132.8 Bristol 21.432.110.764.2 Wessex 6911.5 not reported 710.4 Oxford 41.400 not reported 41.4 Bham 102.630.882.1215.5 Trent 143.44114 3.4327.7 Manch 3411.3 not reported 45.3 Liverpool 97.343.34 1713.8 Leeds 51.80041.493.2 Ncle 62.310.41 83.1 Wales 62.7N/A not reported 62.7 Scot 153.7N/A not reported 153.7

20. Babies with 1 mutation NW Thames Bristol Wessex Oxford Birmingham Trent Manchester Liverpool Leeds Newcastle Wales Scotland

21. Babies with 1 mutation Total IRT>99.5 th 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

22. Babies with 1 mutation Total 1 mut on panel 1 IRT>99.5 th 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

23. Babies with 1 mutation Total 1 mut on panel 1% IRT>99.5 th C 0 mut on panel 2 Expected 11 NW Thames 461 255.4 Bristol 144 64.2 Wessex 67 811.9 Oxford 289 186.2 Birmingham 383 225.7 Trent 413 266.3 Manchester 75 45.3 Liverpool 123 97.3 Leeds 281 113.9 Newcastle 257 197.4 Wales 223 209 Scotland 406 4410.8

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

25. Meconium ileus cases Echogenic screen showed pregnancy to be F508del/F508del – family told predicted CF affected. Echogenic screen showed pregnancy to be F508del/F508del – family told predicted CF affected. Baby included in CFNS and had normal IRT - reported as “CF not suspected”. Checked Guthrie for F508del and confirmed original results. Biochem then lowered their cut off levels slightly to try and prevent this happening again. Baby with meconium ileus was missed on blood spots as no raised IRT – had F508del and R117H/5T Baby with meconium ileus was missed on blood spots as no raised IRT – had F508del and R117H/5T Urgent direct referral from a neonatal unit for a child with meconium peritonitis. Screened for the full panel of mutations and identified a c.3528delC heterozygote. Guthrie analysis reported this patient as ‘no mutation detected’ for four mutations. (Received in time frame) Urgent direct referral from a neonatal unit for a child with meconium peritonitis. Screened for the full panel of mutations and identified a c.3528delC heterozygote. Guthrie analysis reported this patient as ‘no mutation detected’ for four mutations. (Received in time frame)

26. R117H 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 99.5 th C so not sent for DNA. Sweat test and subsequent management not yet known.

27. F508del carrier picked up on CFNS. Cascade testing of family revealed 2 yr old cousin was F508del/R117H(7T) and had borderline sweat test. Being treated for CF. F508del carrier picked up on CFNS. Cascade testing of family revealed 2 yr old cousin was F508del/R117H(7T) 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 examples

28. Technical 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.

29. Problems / 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. 2 nd IRT data, 2 nd rare mutation Lack of follow-up e.g. 2 nd IRT data, 2 nd rare mutation Need to have a system for prenatal tests to pre- empt CFNS to avoid conflicting reports. Need to have a system for prenatal tests to pre- empt CFNS to avoid conflicting reports. Regions with significant non-Caucasian population – 4 mutation panel not ideal e.g. NW Thames would like W1282X as large Ashkenazi Regions with significant non-Caucasian population – 4 mutation panel not ideal e.g. NW Thames would like W1282X as large Ashkenazi Use the same protocol as CFNS for all diagnostic CF referrals Use the same protocol as CFNS for all diagnostic CF referrals

30. Summary National CF Newborn Screening programme going well after first year National CF Newborn Screening programme going well after first year Good communication with Biochemistry Good communication with Biochemistry Higher workload than expected Higher workload than expected Lower pick up rate than expected (affecteds and carriers) Lower pick up rate than expected (affecteds and carriers) More babies with 0 mutations and 2 nd high IRT than in algorithm. May not get full screen. More babies with 0 mutations and 2 nd high IRT than in algorithm. May not get full screen. Need to have system for prenatal results Need to have system for prenatal results Would like more feedback about fate of cases with unclear outcome. Would like more feedback about fate of cases with unclear outcome.

31. Acknowledgements GlasgowCardiff North West Thames West Midlands BristolLeedsLiverpoolManchesterOxfordTrentNewcastleWessex Angela Iley Rachel Butler Stuart Payne Sarah Ball, Fiona MacDonald Claire Faulkner, Maggie Williams Sarah Shepherd, David Cockburn, Teresa Patrick, Helen Lindsay Emma McCarthy, Kevin Southern Martin Schwarz Somai Man Richard Kirk Ruth Sutton, Steve Turner Stacey Sandell