A review of the A review of the Wilson disease Wilson disease service over the service over the

past 15 yearspast 15 yearsMiranda DurkieMiranda Durkie

Sheffield Diagnostic Genetics Sheffield Diagnostic Genetics ServiceService

miranda.durkie@sch.nhs.ukmiranda.durkie@sch.nhs.uk

IntroductionIntroduction Wilson disease (WD; OMIM#277900) is an Wilson disease (WD; OMIM#277900) is an

autosomal recessive disorder of copper autosomal recessive disorder of copper metabolismmetabolism

Hepatic and/or neurological presentationHepatic and/or neurological presentation Treatable if diagnosed earlyTreatable if diagnosed early Over 500 mutations reported in all 21 exons of Over 500 mutations reported in all 21 exons of

the the ATP7BATP7B gene (Cu transporting ATPase) gene (Cu transporting ATPase) Significant numbers of reported cases where Significant numbers of reported cases where

both mutations cannot be identified (10-30%)both mutations cannot be identified (10-30%) Lots of postulation about second WD gene but Lots of postulation about second WD gene but

no mutations identified in candidate genes so no mutations identified in candidate genes so farfar

WD service at SDGSWD service at SDGS WD service available since 1995 (part research)WD service available since 1995 (part research) National (and international) referralsNational (and international) referrals Published results from SSCP & confirmation DNA Published results from SSCP & confirmation DNA

sequencing of 52 UK patients in 1999 with detection sequencing of 52 UK patients in 1999 with detection rate of 70%rate of 70%

SSCP/seq of 3 hotspot exons identified 60% of SSCP/seq of 3 hotspot exons identified 60% of mutations in British cohortmutations in British cohort

DNA sequencing replaced SSCP in 1999 and stage 1 DNA sequencing replaced SSCP in 1999 and stage 1 screen increased to 7 exons to pick-up 80% screen increased to 7 exons to pick-up 80% mutationsmutations

Stage 2 screen = sequencing of all remaining 14 Stage 2 screen = sequencing of all remaining 14 exonsexons

Service Evaluation AimsService Evaluation Aims

Determine mutation spectrum by Determine mutation spectrum by sequencingsequencing

Determine mutation detection Determine mutation detection frequency in British referralsfrequency in British referrals

Determine if further testing for Determine if further testing for deletions/duplications and promoter deletions/duplications and promoter variants is warrantedvariants is warranted

Assess utility of 2 stage screening Assess utility of 2 stage screening approachapproach

A

B

C

D

Cohort 1Cohort 1

Cohort 2Cohort 2

Long time lag between start of Long time lag between start of project in 2004 and completion of project in 2004 and completion of MLPA & promoter work in 2009 MLPA & promoter work in 2009 (limited resources) (limited resources)

Therefore decided to look at 2Therefore decided to look at 2ndnd cohort of referrals received between cohort of referrals received between November 2004 and April 2009November 2004 and April 2009

Only included cases where 2 Only included cases where 2 mutations had been detected and/or mutations had been detected and/or full sequencing had been carried outfull sequencing had been carried out

Cohort 2Cohort 2

Wilson disease diagnostic referrals = 68

1 mutation = 4 2 mutations = 51 No mutations = 10 3 mutations = 3

Confirm clinical diagnosis

Promoter & MLPA

Confirm clinical diagnosis

Excluded WD = 3 Excluded WD = 10

One variant detected c.-442G>A

E

F

Interesting Cohort 1 Interesting Cohort 1 casecase DD

One patient homozygous for common One patient homozygous for common p.His1069Gln, c.3207C>A mutationp.His1069Gln, c.3207C>A mutation

Routine family studies in 1999 showed that Dad Routine family studies in 1999 showed that Dad was heterozygous for this mutation but Mum was heterozygous for this mutation but Mum did not have itdid not have it

Reported as a possible whole exon deletion or Reported as a possible whole exon deletion or primer SNP.primer SNP.

Alternative primers showed no primer SNPsAlternative primers showed no primer SNPs In 2007 MLPA kit available for ATP7B (P098 In 2007 MLPA kit available for ATP7B (P098

MRC Holland) MRC Holland) MLPA showed no deletion in Mum or index caseMLPA showed no deletion in Mum or index case Used Promega Powerplex kit to check for Used Promega Powerplex kit to check for

sample mix-upsample mix-up

Powerplex resultsPowerplex resultsIndex case

Mum

Dad

189

189

177;193

D13S317

Interesting Interesting casecase DD cont. cont. Used fluorescent microsatellite markers Used fluorescent microsatellite markers

along length of chromosome 13along length of chromosome 13 Found 7 markers between 13q11 and Found 7 markers between 13q11 and

13q14.2 show biparental inheritance13q14.2 show biparental inheritance 6 markers between 13q14.2 to 13q34 (inc 6 markers between 13q14.2 to 13q34 (inc

ATP7BATP7B at 13q14.3) show non-maternal at 13q14.3) show non-maternal inheritance & inheritance of single paternal inheritance & inheritance of single paternal alleleallele

Paternal segmental UPD confirmed resulting Paternal segmental UPD confirmed resulting in autozygosity for p.His1069Gln mutationin autozygosity for p.His1069Gln mutation

First reported case of UPD13 with WDFirst reported case of UPD13 with WD

Putative promoter mutation Putative promoter mutation - - Case ECase E

Cullen et al Clin Genet 2003

Sardinian mutation c.-441_-427del15

+1 ATG start

c.-442G>A

Alibaba analysis c.-Alibaba analysis c.-442G>A442G>A

Wild-type

c.-442G>A

YY1 = transcriptional repressor

TFsearch results c.-TFsearch results c.-442G>A442G>A

Wild-type

c.-442G>A

c.-442G>A further c.-442G>A further studiesstudies

Family studies showed that it was Family studies showed that it was inherited from Mum concordant with inherited from Mum concordant with familial segregationfamilial segregation

Not found on 188 normal Not found on 188 normal chromosomeschromosomes

Putative new binding of YY1/NF-Putative new binding of YY1/NF-muE1/GATA affecting normal TF muE1/GATA affecting normal TF binding??binding??

Needs further workNeeds further work

Interesting cases Interesting cases FF 3 individuals found with 3 putative 3 individuals found with 3 putative

mutations eachmutations each 2 mutations 2 mutations in cisin cis are all different & all are all different & all

missensemissense 1 is predicted to affect splicing1 is predicted to affect splicing 1 previously reported case with 3 1 previously reported case with 3

mutations in literature from isolated mutations in literature from isolated mountainous region of Crete with very mountainous region of Crete with very high WD incidencehigh WD incidence

Important implications for staged Important implications for staged screening & presymptomatic testingscreening & presymptomatic testing

Mutation distributionMutation distribution

Previous SSCP study found mutations Previous SSCP study found mutations in exons 2, 8, 13-15 & 18-19 most in exons 2, 8, 13-15 & 18-19 most prevalent (80%) = Stage 1 screenprevalent (80%) = Stage 1 screen

However new data shows exons 2, 5, However new data shows exons 2, 5, 8, 13-14, 18-20 most prevalent8, 13-14, 18-20 most prevalent

Old stage 1New stage 1

SummarySummary 117 different mutations, 36 novel, found in 117 different mutations, 36 novel, found in

191 patients191 patients No deletions/duplications detected therefore No deletions/duplications detected therefore

MLPA is not warranted for routine diagnosisMLPA is not warranted for routine diagnosis One promoter variant found therefore One promoter variant found therefore

promoter analysis is not warranted for promoter analysis is not warranted for routine diagnosisroutine diagnosis

11stst case of UPD13 found in WD case of UPD13 found in WD 2 missense mutations can occur 2 missense mutations can occur in cis in cis 60% of negative stage 1 screens said “WD 60% of negative stage 1 screens said “WD

not likely” therefore staged screening not likely” therefore staged screening warranted but stage 1 exons need to be warranted but stage 1 exons need to be adjusted adjusted

Of 186 patients with confirmed diagnosis of Of 186 patients with confirmed diagnosis of WD the mutation detection frequency is 98%WD the mutation detection frequency is 98%

Second WD locus is unlikelySecond WD locus is unlikely

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