Transcript A review of the Wilson disease service over the past 15 years

A review of the Wilson
disease service over the
past 15 years
Miranda Durkie
Sheffield Diagnostic Genetics Service
[email protected]
Introduction
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Wilson disease (WD; OMIM#277900) is an autosomal
recessive disorder of copper metabolism
Hepatic and/or neurological presentation
Treatable if diagnosed early
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%)
Lots of postulation about second WD gene but no
mutations identified in candidate genes so far
WD service at SDGS
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WD service available since 1995 (part research)
National (and international) referrals
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
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
Service Evaluation Aims
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Determine mutation spectrum by sequencing
Determine mutation detection frequency in
British referrals
Determine if further testing for
deletions/duplications and promoter variants is
warranted
Assess utility of 2 stage screening approach
Cohort 1
A
D
B
C
Cohort 2
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Long time lag between start of project in 2004
and completion of MLPA & promoter work in
2009 (limited resources)
Therefore decided to look at 2nd 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
Cohort 2
Wilson disease diagnostic referrals = 68
1 mutation = 4
2 mutations = 51
No mutations = 10
3 mutations = 3
F
Confirm clinical diagnosis
Excluded WD = 3
E
Promoter & MLPA
One variant detected c.-442G>A
Confirm clinical diagnosis
Excluded WD = 10
Interesting Cohort 1 case D
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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
Reported as a possible whole exon deletion or primer
SNP.
Alternative primers showed no primer SNPs
In 2007 MLPA kit available for ATP7B (P098 MRC
Holland)
MLPA showed no deletion in Mum or index case
Used Promega Powerplex kit to check for sample mix-
Powerplex results
D13S317
Index
case
189
Dad
189
Mum
177;193
Interesting case D cont.
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Used fluorescent microsatellite markers along
length of chromosome 13
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
Paternal segmental UPD confirmed resulting in
autozygosity for p.His1069Gln mutation
First reported case of UPD13 with WD
Putative promoter mutation - Case E
Sardinian mutation
c.-441_-427del15
c.-442G>A
+1 ATG start
Cullen et al Clin Genet 2003
Alibaba analysis c.-442G>A
Wild-type
c.-442G>A
YY1 =
transcriptional
repressor
TFsearch results c.-442G>A
Wild-type
c.-442G>A
c.-442G>A further studies
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Family studies showed that it was inherited from
Mum concordant with familial segregation
Not found on 188 normal chromosomes
Putative new binding of YY1/NFmuE1/GATA affecting normal TF binding??
Needs further work
Interesting cases F
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3 individuals found with 3 putative mutations
each
2 mutations in cis are all different & all missense
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
Important implications for staged screening &
presymptomatic testing
Mutation distribution
Old stage 1
New stage 1
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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, 1820 most prevalent
Summary
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117 different mutations, 36 novel, found in 191 patients
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
1st case of UPD13 found in WD
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
Of 186 patients with confirmed diagnosis of WD the
mutation detection frequency is 98%
Second 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