1. A review of the Wilson disease service over the past 15 years Miranda Durkie Sheffield Diagnostic Genetics Service miranda.durkie@sch.nhs.uk

2. Introduction Wilson disease (WD; OMIM#277900) is an autosomal recessive disorder of copper metabolism Wilson disease (WD; OMIM#277900) is an autosomal recessive disorder of copper metabolism Hepatic and/or neurological presentation Hepatic and/or neurological presentation Treatable if diagnosed early Treatable if diagnosed early Over 500 mutations reported in all 21 exons of the ATP7B gene (Cu transporting ATPase) Over 500 mutations reported in all 21 exons of the ATP7B gene (Cu transporting ATPase) Significant numbers of reported cases where both mutations cannot be identified (10-30%) Significant numbers of reported cases where both mutations cannot be identified (10-30%) Lots of postulation about second WD gene but no mutations identified in candidate genes so far Lots of postulation about second WD gene but no mutations identified in candidate genes so far

3. WD service at SDGS WD service available since 1995 (part research) WD service available since 1995 (part research) National (and international) referrals National (and international) referrals Published results from SSCP & confirmation DNA sequencing of 52 UK patients in 1999 with detection rate of 70% Published results from SSCP & confirmation DNA sequencing of 52 UK patients in 1999 with detection rate of 70% SSCP/seq of 3 hotspot exons identified 60% of mutations in British cohort SSCP/seq of 3 hotspot exons identified 60% of mutations in British cohort DNA sequencing replaced SSCP in 1999 and stage 1 screen increased to 7 exons to pick-up 80% mutations DNA sequencing replaced SSCP in 1999 and stage 1 screen increased to 7 exons to pick-up 80% mutations Stage 2 screen = sequencing of all remaining 14 exons Stage 2 screen = sequencing of all remaining 14 exons

4. Service Evaluation Aims Determine mutation spectrum by sequencing Determine mutation spectrum by sequencing Determine mutation detection frequency in British referrals Determine mutation detection frequency in British referrals Determine if further testing for deletions/duplications and promoter variants is warranted Determine if further testing for deletions/duplications and promoter variants is warranted Assess utility of 2 stage screening approach Assess utility of 2 stage screening approach

5. A B C D Cohort 1

6. Cohort 2 Long time lag between start of project in 2004 and completion of MLPA & promoter work in 2009 (limited resources) Long time lag between start of project in 2004 and completion of MLPA & promoter work in 2009 (limited resources) Therefore decided to look at 2 nd cohort of referrals received between November 2004 and April 2009 Therefore decided to look at 2 nd cohort of referrals received between November 2004 and April 2009 Only included cases where 2 mutations had been detected and/or full sequencing had been carried out Only included cases where 2 mutations had been detected and/or full sequencing had been carried out

7. Cohort 2 Wilson disease diagnostic referrals = 68 1 mutation = 4 Confirm clinical diagnosis Promoter & MLPA One variant detected c.- 442G>A Excluded WD = 3 2 mutations = 51 No mutations = 10 Confirm clinical diagnosis Excluded WD = 10 3 mutations = 3 E F

8. Interesting Cohort 1 case D One patient homozygous for common p.His1069Gln, c.3207C>A mutation One patient homozygous for common p.His1069Gln, c.3207C>A mutation Routine family studies in 1999 showed that Dad was heterozygous for this mutation but Mum did not have it Routine family studies in 1999 showed that Dad was heterozygous for this mutation but Mum did not have it Reported as a possible whole exon deletion or primer SNP. Reported as a possible whole exon deletion or primer SNP. Alternative primers showed no primer SNPs Alternative primers showed no primer SNPs In 2007 MLPA kit available for ATP7B (P098 MRC Holland) In 2007 MLPA kit available for ATP7B (P098 MRC Holland) MLPA showed no deletion in Mum or index case MLPA showed no deletion in Mum or index case Used Promega Powerplex kit to check for sample mix- up Used Promega Powerplex kit to check for sample mix- up

9. Powerplex results Index case Mum Dad 189 177;193 D13S317

10. Interesting case D cont. Used fluorescent microsatellite markers along length of chromosome 13 Used fluorescent microsatellite markers along length of chromosome 13 Found 7 markers between 13q11 and 13q14.2 show biparental inheritance Found 7 markers between 13q11 and 13q14.2 show biparental inheritance 6 markers between 13q14.2 to 13q34 (inc ATP7B at 13q14.3) show non-maternal inheritance & inheritance of single paternal allele 6 markers between 13q14.2 to 13q34 (inc ATP7B at 13q14.3) show non-maternal inheritance & inheritance of single paternal allele Paternal segmental UPD confirmed resulting in autozygosity for p.His1069Gln mutation Paternal segmental UPD confirmed resulting in autozygosity for p.His1069Gln mutation First reported case of UPD13 with WD First reported case of UPD13 with WD

11. Putative promoter mutation - Case E Cullen et al Clin Genet 2003 Sardinian mutation c.-441_-427del15 +1 ATG start c.-442G>A

12. Alibaba analysis c.-442G>A Wild-type c.-442G>A YY1 = transcriptional repressor

13. TFsearch results c.-442G>A Wild-type c.-442G>A

14. c.-442G>A further studies Family studies showed that it was inherited from Mum concordant with familial segregation Family studies showed that it was inherited from Mum concordant with familial segregation Not found on 188 normal chromosomes Not found on 188 normal chromosomes Putative new binding of YY1/NF- muE1/GATA affecting normal TF binding?? Putative new binding of YY1/NF- muE1/GATA affecting normal TF binding?? Needs further work Needs further work

15. Interesting cases F 3 individuals found with 3 putative mutations each 3 individuals found with 3 putative mutations each 2 mutations in cis are all different & all missense 2 mutations in cis are all different & all missense 1 is predicted to affect splicing 1 is predicted to affect splicing 1 previously reported case with 3 mutations in literature from isolated mountainous region of Crete with very high WD incidence 1 previously reported case with 3 mutations in literature from isolated mountainous region of Crete with very high WD incidence Important implications for staged screening & presymptomatic testing Important implications for staged screening & presymptomatic testing

16. Mutation distribution Previous SSCP study found mutations in exons 2, 8, 13-15 & 18-19 most prevalent (80%) = Stage 1 screen Previous SSCP study found mutations in exons 2, 8, 13-15 & 18-19 most prevalent (80%) = Stage 1 screen However new data shows exons 2, 5, 8, 13-14, 18- 20 most prevalent However new data shows exons 2, 5, 8, 13-14, 18- 20 most prevalent Old stage 1 New stage 1

17. Summary 117 different mutations, 36 novel, found in 191 patients 117 different mutations, 36 novel, found in 191 patients No deletions/duplications detected therefore MLPA is not warranted for routine diagnosis No deletions/duplications detected therefore MLPA is not warranted for routine diagnosis One promoter variant found therefore promoter analysis is not warranted for routine diagnosis One promoter variant found therefore promoter analysis is not warranted for routine diagnosis 1 st case of UPD13 found in WD 1 st case of UPD13 found in WD 2 missense mutations can occur in cis 2 missense mutations can occur in cis 60% of negative stage 1 screens said “WD not likely” therefore staged screening warranted but stage 1 exons need to be adjusted 60% of negative stage 1 screens said “WD not likely” therefore staged screening warranted but stage 1 exons need to be adjusted Of 186 patients with confirmed diagnosis of WD the mutation detection frequency is 98% Of 186 patients with confirmed diagnosis of WD the mutation detection frequency is 98% Second WD locus is unlikely Second WD locus is unlikely

18. Thank you! Sheffield Diagnostic Genetics Service James Blackburn Ann Dalton Anne Goodeve Ann Lee Maria Panayi Everyone at SDGS past & present who worked on WD Royal Hallamshire Hospital Oliver Bandmann Stefanie Klass Sheffield Children’s NHS Foundation Trust Stuart Tanner Other EuroWilson for supporting EMQN WD scheme