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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
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
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
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
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