Transcript X-linked ichthyosis - Association for Clinical Genetic Science

Development of a molecular
genetic diagnostic service for
X-linked ichthyosis, with
emphasis on carrier detection
Eleanor Reavey
West of Scotland Regional
Genetics Laboratory
Yorkhill Hospital
Glasgow
Introduction
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Associated with STS deficiency in
fibroblasts and ↑ plasma cholesterol
sulphate
X-linked recessive inheritance
1 in 2000-6000 males
STS gene - Xp22.3
10 exons
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STS enzyme
Responsible for hydrolysis
of cholesterol sulfate (CS)
to cholesterol in epidermis
XLI – accumulation of CS
in epidermis leads to
barrier instability and
inhibits desmosomal
degradation
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Phenotype
Scaly skin on scalp, trunk
and limbs
Corneal opacities
Placental sulphatase deficiency
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Placenta – STS-rich tissue
STS is involved in steroid conversion pathway:
cholesterol  estriol
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Deficiency associated with:
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longer gestation and poor cervical dilatation
Results in slowing of delivery + indicates need
for C-section or instrumental delivery
↑ perinatal morbidity + mortality
Associated Conditions
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Approx. 90% of XLI individuals – complete
deletion of the STS gene
More extensive deletions - contiguous gene
deletion syndromes
Kallmann syndrome
Short stature
X-linked chondrodysplasia punctata
X-linked ocular albinism
ADHD
Biochemical Analysis
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STS activity is measured on white cells or
cultured fibroblasts
Radiolabelled assay with 3H
Dehydroepiandrosterone sulphate as a substrate
Affected males are tested for presence or
absence of STS gene by PCR
No info on any intragenic deletions or point
mutations
Mutations
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Several point mutations in STS gene identified
No evidence of genotype-phenotype correlation,
regardless of the location or type of the STS
mutation
production of a catalytically inactive STS enzyme
both the N-terminal region and the C-terminal
region of the STS protein are important for
enzyme activity
Initial referral
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Patient NH clinically affected with XLI
No enzyme activity detected
But, normal result for gene deletion
analysis
Request from Dundee for full seq screen
of STS coding exons (1-10), including
intron/exon boundaries
Primers designed for sequencing
Y chr Pseudogene
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Transcriptionally inactive at the promoter
Several exons deleted
Significant sequence homology between
X-STS and Y-STS genes
Results from Temperature Gradient PCR
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55°C - 65°C
Example gel for exons 1-8
Exon 1
2
3
4
5
6
7
8
55C
58C
60C
62C
65C
NH
c.583delG
p.Val195SerfsX19
Further Testing
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Screening of NH’s mother confirmed her
as a carrier.
Second referral – Edinburgh
Patient JM clinically affected, no STS
activity and normal result on gene deletion
analysis
JM
c.387_391dupAGCAC
p.Leu131GlnfsX3
Extended Testing
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A further 10 samples were received from
Dr Graham
Dosage analysis carried out to confirm
presence of STS gene
Full sequencing screen carried out on all
10 exons
Four additional mutations detected = high
pickup rate
STS dosage analysis
DMD 53
STS 5’
DMD 17
STS 3’
DMD 51
MD
c.1046_1048delAGG
p.Glu349del
TT
c.1649G>A
p.Trp550X
AE
c.1360C>T
p.Arg454Cys
JA
c.494C>T
p.Thr165Ile
MLPA kit P160
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Probes for each of 10 exons
Other probes include KAL1 and NLGN4X
In female heterozygotes, 35-50% reduced
relative peak area of amplified product expected
Deletion of one exon – needs to be confirmed by
sequencing to rule out mutation/ polymorphism
close to probe ligation site
Carrier testing in females
Current testing strategy for XLI in
Glasgow
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Enzyme activity measured and gene
deletion PCR carried out in Biochemical
Genetics
Dosage assay available in Molecular
Genetics Lab to identify female carriers
MLPA better suited for carrier testing –
detects single (or multiple) exon deletions/
duplications as well as deletions of entire
gene
Sample is received by
Biochemical Genetics at
Yorkhill Hospital for XLI
diagnostic analysis
Steroid sulphatase enzyme
analysis carried out on white
cells
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+ve
Report patient as
negative for XLI
Dosage analysis to
identify partial/ full STS
gene deletions
+ve
Report patient is
affected with XLI
due to a STS gene
deletion
-ve
Full screen sequencing
of 10 coding exons of
STS gene to identify
point mutations
-ve
Confirm XLI
diagnosed
biochemically
however, genetic basis
is unknown
Offer mother, and other
family members, STS
sequence testing for
identified point mutation
Offer mother, and
other family members,
MLPA testing for
carrier status
Summary
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Service offered for males affected with XLI
– dosage analysis + full screen sequencing
for point mutations
Carrier testing for mothers
Important for genetic counselling for
future pregnancies and for predicting risk
of difficult labour
Acknowledgements
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Molecular Genetics Lab, Yorkhill, Glasgow
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Su Stenhouse, Sandy Cooke
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Biochemical Genetics Lab, Yorkhill,
Glasgow
Gordon Graham