1. 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 Glasgow

2. Introduction Associated with STS deficiency in fibroblasts and ↑ plasma cholesterol sulphate Associated with STS deficiency in fibroblasts and ↑ plasma cholesterol sulphate X-linked recessive inheritance X-linked recessive inheritance 1 in 2000-6000 males 1 in 2000-6000 males STS gene - Xp22.3 STS gene - Xp22.3 10 exons 10 exons

3. STS enzyme STS enzyme Responsible for hydrolysis of cholesterol sulfate (CS) to cholesterol in epidermis 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 XLI – accumulation of CS in epidermis leads to barrier instability and inhibits desmosomal degradation Phenotype Phenotype Scaly skin on scalp, trunk and limbs Scaly skin on scalp, trunk and limbs Corneal opacities Corneal opacities

4. Placental sulphatase deficiency Placenta – STS-rich tissue Placenta – STS-rich tissue STS is involved in steroid conversion pathway: cholesterol  estriol STS is involved in steroid conversion pathway: cholesterol  estriol Deficiency associated with: Deficiency associated with: longer gestation and poor cervical dilatation longer gestation and poor cervical dilatation Results in slowing of delivery + indicates need for C-section or instrumental delivery Results in slowing of delivery + indicates need for C-section or instrumental delivery ↑ perinatal morbidity + mortality ↑ perinatal morbidity + mortality

5. Associated Conditions Approx. 90% of XLI individuals – complete deletion of the STS gene Approx. 90% of XLI individuals – complete deletion of the STS gene More extensive deletions - contiguous gene deletion syndromes More extensive deletions - contiguous gene deletion syndromes Kallmann syndrome Kallmann syndrome Short stature Short stature X-linked chondrodysplasia punctata X-linked chondrodysplasia punctata X-linked ocular albinism X-linked ocular albinism ADHD ADHD

6. Biochemical Analysis STS activity is measured on white cells or cultured fibroblasts STS activity is measured on white cells or cultured fibroblasts Radiolabelled assay with 3H Dehydroepiandrosterone sulphate as a substrate Radiolabelled assay with 3H Dehydroepiandrosterone sulphate as a substrate Affected males are tested for presence or absence of STS gene by PCR Affected males are tested for presence or absence of STS gene by PCR No info on any intragenic deletions or point mutations No info on any intragenic deletions or point mutations

7. Mutations Several point mutations in STS gene identified Several point mutations in STS gene identified No evidence of genotype-phenotype correlation, regardless of the location or type of the STS mutation No evidence of genotype-phenotype correlation, regardless of the location or type of the STS mutation production of a catalytically inactive STS enzyme 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 both the N-terminal region and the C-terminal region of the STS protein are important for enzyme activity

8. Initial referral Patient NH clinically affected with XLI Patient NH clinically affected with XLI No enzyme activity detected No enzyme activity detected But, normal result for gene deletion analysis But, normal result for gene deletion analysis Request from Dundee for full seq screen of STS coding exons (1-10), including intron/exon boundaries Request from Dundee for full seq screen of STS coding exons (1-10), including intron/exon boundaries Primers designed for sequencing Primers designed for sequencing

9. Y chr Pseudogene Transcriptionally inactive at the promoter Transcriptionally inactive at the promoter Several exons deleted Several exons deleted Significant sequence homology between X-STS and Y-STS genes Significant sequence homology between X-STS and Y-STS genes

10. Results from Temperature Gradient PCR 55°C - 65°C 55°C - 65°C Example gel for exons 1-8 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

11. NH c.583delG p.Val195SerfsX19

12. Further Testing Screening of NH’s mother confirmed her as a carrier. Screening of NH’s mother confirmed her as a carrier. Second referral – Edinburgh Second referral – Edinburgh Patient JM clinically affected, no STS activity and normal result on gene deletion analysis Patient JM clinically affected, no STS activity and normal result on gene deletion analysis

13. JM c.387_391dupAGCAC p.Leu131GlnfsX3

14. Extended Testing A further 10 samples were received from Dr Graham A further 10 samples were received from Dr Graham Dosage analysis carried out to confirm presence of STS gene Dosage analysis carried out to confirm presence of STS gene Full sequencing screen carried out on all 10 exons Full sequencing screen carried out on all 10 exons Four additional mutations detected = high pickup rate Four additional mutations detected = high pickup rate

15. STS dosage analysis DMD 53 STS 5’ DMD 17 STS 3’ DMD 51

16. MD c.1046_1048delAGG p.Glu349del

17. TT c.1649G>A p.Trp550X

18. AE c.1360C>T p.Arg454Cys

19. JA c.494C>T p.Thr165Ile

20. MLPA kit P160 Probes for each of 10 exons Probes for each of 10 exons Other probes include KAL1 and NLGN4X Other probes include KAL1 and NLGN4X In female heterozygotes, 35-50% reduced relative peak area of amplified product expected 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 Deletion of one exon – needs to be confirmed by sequencing to rule out mutation/ polymorphism close to probe ligation site

21. Carrier testing in females

22. Current testing strategy for XLI in Glasgow Enzyme activity measured and gene deletion PCR carried out in Biochemical Genetics Enzyme activity measured and gene deletion PCR carried out in Biochemical Genetics Dosage assay available in Molecular Genetics Lab to identify female carriers 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 MLPA better suited for carrier testing – detects single (or multiple) exon deletions/ duplications as well as deletions of entire gene

23. Sample is received by Biochemical Genetics at Yorkhill Hospital for XLI diagnostic analysis Steroid sulphatase enzyme analysis carried out on white cells Report patient as negative for XLI -ve + ve Dosage analysis to identify partial/ full STS gene deletions + ve Report patient is affected with XLI due to a STS gene deletion Offer mother, and other family members, MLPA testing for carrier status -ve Full screen sequencing of 10 coding exons of STS gene to identify point mutations -ve Offer mother, and other family members, STS sequence testing for identified point mutation Confirm XLI diagnosed biochemically however, genetic basis is unknown

24. Summary Service offered for males affected with XLI – dosage analysis + full screen sequencing for point mutations Service offered for males affected with XLI – dosage analysis + full screen sequencing for point mutations Carrier testing for mothers Carrier testing for mothers Important for genetic counselling for future pregnancies and for predicting risk of difficult labour Important for genetic counselling for future pregnancies and for predicting risk of difficult labour

25. Acknowledgements Molecular Genetics Lab, Yorkhill, Glasgow Molecular Genetics Lab, Yorkhill, Glasgow Su Stenhouse, Sandy Cooke Su Stenhouse, Sandy Cooke Biochemical Genetics Lab, Yorkhill, Glasgow Biochemical Genetics Lab, Yorkhill, Glasgow Gordon Graham Gordon Graham