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Mutation screening in patients affected Mutation screening in patients affected with autosomal dominant with autosomal dominant
hypercholesterolemia – results from the hypercholesterolemia – results from the Department of Health Pilot ProjectDepartment of Health Pilot Project
Alison TaylorAlison Taylor
Molecular Genetics GOSHMolecular Genetics GOSH
Autosomal Dominant Autosomal Dominant HypercholesterolaemiaHypercholesterolaemia
Characterised by:Characterised by: Increased plasma levels of total cholesterol and low Increased plasma levels of total cholesterol and low
density lipoproteindensity lipoprotein Tendon xanthomaTendon xanthoma
Premature symptoms of coronary heart diseasePremature symptoms of coronary heart disease
Heterozygous form 1/500Heterozygous form 1/500 Homozygous form 1/1,000,000Homozygous form 1/1,000,000
Autosomal Dominant Autosomal Dominant HypercholesterolaemiaHypercholesterolaemia
Mutations in:Mutations in: LDLRLDLR Apolipoprotein B-100 gene (APOB)Apolipoprotein B-100 gene (APOB) Proprotein convertase subtilisin/kexin type 9 gene Proprotein convertase subtilisin/kexin type 9 gene
(PCSK9)(PCSK9)
Majority of mutations in the LDLR gene – >1200 Majority of mutations in the LDLR gene – >1200 identifiedidentified
Cascade testing is cost effective way to identify Cascade testing is cost effective way to identify new patientsnew patients
Simon Broome CriteriaSimon Broome Criteria
Simon Broome FH register criteria:Simon Broome FH register criteria: A) TC > 7.5mmol/l or LDL cholesterol >4.9mmol/lA) TC > 7.5mmol/l or LDL cholesterol >4.9mmol/l B) Tendon xanthoma in patient or first degree relativeB) Tendon xanthoma in patient or first degree relative C) Family history of MI before age 50yrs in 2nd C) Family history of MI before age 50yrs in 2nd
degree relative or 60yrs in 1st degree relativedegree relative or 60yrs in 1st degree relative D) Family history of TC >7.5mmol/l in first/second D) Family history of TC >7.5mmol/l in first/second
degree relativedegree relative
Dept of Health ProjectDept of Health Project
Present data from a cohort of 635 patients with Present data from a cohort of 635 patients with clinical diagnosis of heterozygous FHclinical diagnosis of heterozygous FH
Samples referred from six adult lipid clinics in Samples referred from six adult lipid clinics in UKUK
Patients classified using Simon Broome criteriaPatients classified using Simon Broome criteria 190 definite FH (DFH)190 definite FH (DFH) 394 possible FH (PFH)394 possible FH (PFH) 51 unclassified (UFH)51 unclassified (UFH)
FH20 ARMS KitFH20 ARMS Kit
FH20 kit from Tepnel has 3 mixes (A, B and C) Efficient and cost effective screen for FH testing Doesn’t require specialised equipment, completed in 1-2 days Used as initial screen Multiplex ARMS analysis to detect 13 common mutations in familial
hypercholesterolaemia Taylor et al Clinical Genetics 2007: 71: 561-568
p.Arg3527Gln
p.Asp482His
p.Asp374Tyr
Point mutation screenPoint mutation screen
18 exons and promoter region of LDLR gene18 exons and promoter region of LDLR gene Initially done by SSCP, then dHPLCInitially done by SSCP, then dHPLC Now – direct sequence analysisNow – direct sequence analysis
Beckmann Coulter RobotsBeckmann Coulter Robots 20 fragments20 fragments 6 patients, 1 normal control and blank6 patients, 1 normal control and blank 2 PCR plates – 4 sequence plates2 PCR plates – 4 sequence plates
MLPA analysisMLPA analysis
GeneMarker software (SoftGenetics)GeneMarker software (SoftGenetics)
ResultsResults
Mutations found in 232 patients (36.5%)Mutations found in 232 patients (36.5%) DFH – 106 (55.8%)DFH – 106 (55.8%) PFH – 113 (28.7%)PFH – 113 (28.7%) UFH – 13 (25.5%)UFH – 13 (25.5%)
Of mutations detectedOf mutations detected ARMs – 43%ARMs – 43% Point mutation screen – 52.3%Point mutation screen – 52.3% MLPA – 4.7%MLPA – 4.7%
ResultsResults
107 different mutations107 different mutations 12% APOB p.Arg3527Gln 12% APOB p.Arg3527Gln 5% c.654_656delTGG 5% c.654_656delTGG 5% c.313+1G>A5% c.313+1G>A 5% p.Pro685Leu 5% p.Pro685Leu 1.7% p.Asp374Tyr PCSK91.7% p.Asp374Tyr PCSK9 6.9% novel – all predicted to impact on receptor 6.9% novel – all predicted to impact on receptor
functionfunction
Cascade testingCascade testing
All but one of the sites carried out cascade All but one of the sites carried out cascade testing where a mutation found in probandtesting where a mutation found in proband
Cascade testing in 100 familiesCascade testing in 100 families 290 relatives tested 290 relatives tested Mutation found in 166 relatives (56.1%)Mutation found in 166 relatives (56.1%)
ConclusionConclusion
NICE guidelines recommend that DNA testing is NICE guidelines recommend that DNA testing is offered to all identified probandsoffered to all identified probands
Data presented strongly supports the clinical Data presented strongly supports the clinical utility of DNA testing in patients with ADHutility of DNA testing in patients with ADH
Overall detection rate in 635 patients was 36.5%Overall detection rate in 635 patients was 36.5% FH20 ARMs kit is an extremely efficient test in FH20 ARMs kit is an extremely efficient test in
UK patientsUK patients 49% DFH49% DFH 38% PFH38% PFH
ConclusionConclusion
Mutation screening still required as 6.9% Mutation screening still required as 6.9% mutations novelmutations novel
Even in subjects with low clinical suspicion of FH Even in subjects with low clinical suspicion of FH mutation testing will be usefulmutation testing will be useful
AcknowledgementsAcknowledgements
RMG GOSHRMG GOSH Gail NorburyGail Norbury Darrell WangDarrell Wang Brendan MartinBrendan Martin Kunjan PatelKunjan Patel
London IDEASLondon IDEAS Steve HumphriesSteve Humphries Ros WhittallRos Whittall Gretta WoodGretta Wood Mabella FarrerMabella Farrer JE CooperJE Cooper Gaye HadfieldGaye Hadfield Sarah LeighSarah Leigh
Referring CentresReferring Centres RDG NeelyRDG Neely S FairgrieveS Fairgrieve D NairD Nair M BarbirM Barbir J JonesJ Jones S EganS Egan Y LolinY Lolin E HughesE Hughes
Tepnel DiagnosticsTepnel Diagnostics Dept of HealthDept of Health
I am running the London Marathon in April for I am running the London Marathon in April for Great Ormond Street Children's CharityGreat Ormond Street Children's Charity
www.justgiving.com/aetaylorwww.justgiving.com/aetaylor