Development of a Development of a molecular genetic molecular genetic

diagnostic service for X-diagnostic service for X-linked ichthyosis, with linked ichthyosis, with emphasis on carrier emphasis on carrier

detectiondetection

Eleanor ReaveyEleanor ReaveyWest of Scotland Regional West of Scotland Regional

Genetics LaboratoryGenetics LaboratoryYorkhill HospitalYorkhill Hospital

GlasgowGlasgow

IntroductionIntroduction

Associated with STS deficiency in Associated with STS deficiency in fibroblasts and fibroblasts and ↑ plasma cholesterol ↑ plasma cholesterol sulphatesulphate

X-linked recessive inheritanceX-linked recessive inheritance 1 in 2000-6000 males1 in 2000-6000 males STS gene - Xp22.3STS gene - Xp22.3 10 exons10 exons

STS enzymeSTS enzyme

Responsible for Responsible for hydrolysis of hydrolysis of cholesterol sulfate (CS) cholesterol sulfate (CS) to cholesterol in to cholesterol in epidermisepidermis

XLI – accumulation of XLI – accumulation of CS in epidermis leads to CS in epidermis leads to barrier instability and barrier instability and inhibits desmosomal inhibits desmosomal degradationdegradation

PhenotypePhenotype

Scaly skin on scalp, Scaly skin on scalp, trunk and limbstrunk and limbs

Corneal opacitiesCorneal opacities

Placental sulphatase Placental sulphatase deficiencydeficiency

Placenta – STS-rich tissuePlacenta – STS-rich tissue STS is involved in steroid conversion STS is involved in steroid conversion

pathway: cholesterol pathway: cholesterol estriol estriol

Deficiency associated with:Deficiency associated with: longer gestation and poor cervical longer gestation and poor cervical

dilatationdilatation Results in slowing of delivery + indicates Results in slowing of delivery + indicates

need for C-section or instrumental deliveryneed for C-section or instrumental delivery ↑ ↑ perinatal morbidity + mortalityperinatal morbidity + mortality

Associated ConditionsAssociated Conditions

Approx. 90% of XLI individuals – complete Approx. 90% of XLI individuals – complete deletion of the STS genedeletion of the STS gene

More extensive deletions - contiguous More extensive deletions - contiguous gene deletion syndromesgene deletion syndromes

Kallmann syndromeKallmann syndrome Short statureShort stature X-linked chondrodysplasia punctataX-linked chondrodysplasia punctata X-linked ocular albinismX-linked ocular albinism ADHDADHD

Biochemical AnalysisBiochemical Analysis

STS activity is measured on white cells or STS activity is measured on white cells or cultured fibroblastscultured fibroblasts

Radiolabelled assay with 3H Radiolabelled assay with 3H Dehydroepiandrosterone sulphate as a Dehydroepiandrosterone sulphate as a substrate substrate

Affected males are tested for presence or Affected males are tested for presence or absence of STS gene by PCRabsence of STS gene by PCR

No info on any intragenic deletions or No info on any intragenic deletions or point mutationspoint mutations

MutationsMutations

Several point mutations in STS gene identifiedSeveral point mutations in STS gene identified No evidence of genotype-phenotype No evidence of genotype-phenotype

correlation, regardless of the location or type correlation, regardless of the location or type of the STS mutationof the STS mutation

production of a catalytically inactive STS production of a catalytically inactive STS enzymeenzyme

both the N-terminal region and the C-terminal both the N-terminal region and the C-terminal region of the STS protein are important for region of the STS protein are important for enzyme activityenzyme activity

Initial referralInitial referral

Patient NH clinically affected with XLIPatient NH clinically affected with XLI No enzyme activity detectedNo enzyme activity detected But, normal result for gene deletion But, normal result for gene deletion

analysisanalysis Request from Dundee for full seq Request from Dundee for full seq

screen of STS coding exons (1-10), screen of STS coding exons (1-10), including intron/exon boundariesincluding intron/exon boundaries

Primers designed for sequencingPrimers designed for sequencing

Y chr Pseudogene Y chr Pseudogene

Transcriptionally inactive at the Transcriptionally inactive at the promoterpromoter

Several exons deletedSeveral exons deleted Significant sequence homology Significant sequence homology

between X-STS and Y-STS genesbetween X-STS and Y-STS genes

Results from Temperature Gradient Results from Temperature Gradient PCRPCR

5555°C - 65°C°C - 65°C Example gel for exons 1-8Example gel for exons 1-8

Exon 1 2 3 4 5 6 7 8

55C

58C

60C

62C

65C

NHNHc.583delGc.583delG

p.Val195SerfsX19p.Val195SerfsX19

Further TestingFurther Testing

Screening of NH’s mother confirmed Screening of NH’s mother confirmed her as a carrier. her as a carrier.

Second referral – EdinburghSecond referral – Edinburgh Patient JM clinically affected, no STS Patient JM clinically affected, no STS

activity and normal result on gene activity and normal result on gene deletion analysisdeletion analysis

JMJMc.387_391dupAGCACc.387_391dupAGCAC

p.Leu131GlnfsX3p.Leu131GlnfsX3

Extended TestingExtended Testing

A further 10 samples were received A further 10 samples were received from Dr Grahamfrom Dr Graham

Dosage analysis carried out to Dosage analysis carried out to confirm presence of STS geneconfirm presence of STS gene

Full sequencing screen carried out on Full sequencing screen carried out on all 10 exonsall 10 exons

Four additional mutations detected = Four additional mutations detected = high pickup ratehigh pickup rate

STS dosage analysisSTS dosage analysis

DMD 53 STS 5’ DMD 17 STS 3’ DMD 51

MDMDc.1046_1048delAGGc.1046_1048delAGG

p.Glu349delp.Glu349del

TTTTc.1649G>Ac.1649G>Ap.Trp550Xp.Trp550X

AEAEc.1360C>Tc.1360C>T

p.Arg454Cysp.Arg454Cys

JAJAc.494C>Tc.494C>T

p.Thr165Ilep.Thr165Ile

MLPA kit P160MLPA kit P160

Probes for each of 10 exons Probes for each of 10 exons Other probes include KAL1 and NLGN4XOther probes include KAL1 and NLGN4X In female heterozygotes, 35-50% reduced In female heterozygotes, 35-50% reduced

relative peak area of amplified product relative peak area of amplified product expectedexpected

Deletion of one exon – needs to be Deletion of one exon – needs to be confirmed by sequencing to rule out confirmed by sequencing to rule out mutation/ polymorphism close to probe mutation/ polymorphism close to probe ligation siteligation site

Carrier testing in femalesCarrier testing in females

Current testing strategy for XLI Current testing strategy for XLI in Glasgowin Glasgow

Enzyme activity measured and gene Enzyme activity measured and gene deletion PCR carried out in Biochemical deletion PCR carried out in Biochemical GeneticsGenetics

Dosage assay available in Molecular Dosage assay available in Molecular Genetics Lab to identify female carriersGenetics Lab to identify female carriers

MLPA better suited for carrier testing – MLPA better suited for carrier testing – detects single (or multiple) exon detects single (or multiple) exon deletions/ duplications as well as deletions deletions/ duplications as well as deletions of entire geneof entire gene

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

Summary Summary

Service offered for males affected Service offered for males affected with XLI – dosage analysis + full with XLI – dosage analysis + full screen sequencing for point screen sequencing for point mutationsmutations

Carrier testing for mothersCarrier testing for mothers Important for genetic counselling for Important for genetic counselling for

future pregnancies and for predicting future pregnancies and for predicting risk of difficult labourrisk of difficult labour

Acknowledgements Acknowledgements

Molecular Genetics Lab, Yorkhill, Molecular Genetics Lab, Yorkhill, GlasgowGlasgow

Su Stenhouse, Sandy CookeSu Stenhouse, Sandy Cooke

Biochemical Genetics Lab, Yorkhill, Biochemical Genetics Lab, Yorkhill, GlasgowGlasgow

Gordon GrahamGordon Graham