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