Bristol Genetics Laboratory Familial Hypercholesterolaemia: LIPOchip experience Laura Yarram Bristol Genetics Laboratory Bristol Genetics Laboratory What is FH? Autosomal Dominant 1/500 heterozygotes in UK (1/1,000,000 compound heterozygotes) Caused by LDLR, APOB & PCSK9 mutations Raised cholesterol, Xanthomas/Xanthelasma, Risk of CVD Simon Broome Criteria definite or possible

Normal life expectancy on statin treatment NICE guidelines Image 1. Achilles tendon xanthoma Definite Total cholesterol >7.5 mmol/L Tendon xanthomas Possible Total cholesterol >7.5 mmol/L FamilyImage history of myocardial infarction 2. Xanthelasma st (Image & Ruzicka, 2009)

Why Perform Genetic Testing? Patient A, aged 8 LDLR mutation confirmed in family Equivocal cholesterol 5.6mmol/L (FH >6.7mmol/L in children, Normal <4.0mmol/L) Family history of extensive cardiovascular disease Great uncle Myocardial infarction at aged 31 Patient A mutation identified Will start statin treatment at ~ age 10 Early treatment gives the maximum health benefit More likely to adhere to treatment Bristol Genetics Laboratory Why Perform Genetic Testing? Patient B, aged 55 Pre-treatment cholesterol ~20mmol/L (FH >7.5mmol/L in adult) Unexpected compound heterozygote for two unclassified variants - c.[1766delA] + [932A>C] - p.[Asp589fs] + [Lys311Thr]

Pedigree: Instigated further cardiology investigations p.[=] + [=] ECG positivep.[Asp589fs] + [Lys311Thr] Exercise Genetic diagnosis allows consideration of LDL apheresis p.[Asp589fs] + [=] Current Testing Method +ve ARMS 20 common mutations Mutation confirmation Sequencing Validated on known positive control samples MLPA (MRC-Holland: -ve Tested 104 samples date using P062B) to validated

REPORT Offer 4 reported samples, sequencing/ obtained from a MLPA Norwegian lab Sequencing REPORT +ve REPORT CASCADE Bristol Genetics Laboratory -ve Proceed to MLPA Primers designed for all 18 LDLR +ve -ve exons + promoter Sequenced in 17 fragments REPORT REPORT Validated using 4 known positives

Simon Broome Audit Data Simon Broome Criteria No. Patients Tested No. +ve dFH pFH Unclassified 15 53 17 15 (100%) 23 (43%) 6 (35%) Criteria not met 19 4 (21%) Total Diagnostic 104

48 (46%) Cascade 27 15 (56%) Bristol Genetics Laboratory Bristol Genetics Laboratory Results to Date 34 pathogenic variants detected to date + 2 variants likely non-pathogenic - c.2390-16G>A and c.969C>T (p.[=]) cDNA Protein Diagnostic c.10580G>A

p.Arg3527Gln (APOB) 9 (19%) c.1436T>C p.Leu479Pro 4 (8%) c.313+1G>A N/A 3 (6%) c.1640T>C p.Leu547Pro 2 (4%) c.662A>G p.Asp221Gly 2 (4%) c.1049G>C p.Arg350Pro

2 (4%) Commonly detected mutations in SW diagnostic patients (n=48) 28 of these variants required UV studies (Nb. Most were previously reported to database but with no functional/family studies) Bristol Genetics Laboratory Assay Sensitivity Assay Sensitivity (n=48) ARMS FH20 52% Bi-directional sequencing of LDLR (Promoter + 18 exons) 46%

MLPA (P062B-C1) 2% Testing strategy not sustainable for disease frequency LIPOchip Background Bristol Genetics Laboratory LIPOchip has been in development since 2002 to detect the most prevalent Spanish mutations Current Version (8) includes European specific mutations More than 100 hospitals are using LIPOchip throughout Europe Copy number changes also detected

Specific BritChip due to be released June/July 2010 Validation 40 samples used (36 previously tested, 4 new cases) Blind test All results concordant Bristol Genetics Laboratory LIPOchip Processing 0 Extraction 1 Amplification PCR mixes 1, 2, 3 and 4 2 hours 2 hours 2 Fragmentation DNAse + Alkaline Phosphatase

3 Labelling TdT + Biotin-ddUTP 45 minutes 60 minutes 4 Hybridization Tecan 4800 HS Pro 3hours and 30 minutes OVERLAPPING PROCESSES DAY 1 DAY 2 5 Results analysis

Mutations Detected Bristol Genetics Laboratory c.429C>A, p.Cys143X c.1432G>A, p.Gly478Arg Mutations not present in FH20 ARMS c.2093G>A (p.Cys698Tyr) Bristol Genetics Laboratory Patient has c.2093G>T (p.Cys698Phe) Slight displacement from the Normal group Del ex7-3UTR Bristol Genetics Laboratory Bristol Genetics Laboratory Duplication LDLR Exon 17 MLPA result LIPOchip result Long-range PCR confirmation

Ex16_F 16 Ex18_R 17 3.5kb 18 ~5kb 3.5kb N dup 17 N Bristol Genetics Laboratory LIPOchip Trial Results Point mutation analysis is robust Copy number detection results not always reportable MLPA will still be required in a significant proportion of cases

Assay Sensitivity (%) (n=48) Pick up (% of all diagnostic cases) (n=104) ARMS (20 common mutations) 52 24 Current LIPOchip (251 mutations) 58 27 British LIPOchip (Personal Communication) 77 36 Proposed Method of Testing

Bristol Genetics Laboratory Initial screen using LIPOchip platform Current European chip v.8 detects 251 mutations + copy number changes British LIPOchip predicted to detect 80% of UK mutations Followed by full bi-directional sequencing of LDLR (and MLPA where necessary) Negative patients meeting Simon Broome criteria Full PCSK9 screen by bi-directional sequencing (Validation near completion, 12 fragments) Sequencing of APOB hotspot regions (exon 26 and 29) Conclusion Bristol Genetics Laboratory Comprehensive testing service for FH implemented 131 cases tested overall, 48% mutation positive

LIPOchip evaluated further work required to validate British version on release Network links with lipid and cardiac specialists have been established across the SW region Mechanism for robust funding is yet to be established Acknowledgments Bristol Genetics Laboratory Bristol Genetics Lab Maggie Williams Sarah Burton-Jones Thalia Antoniadi Genetic Technologists Teresa Tovey, Jenny Coles, Gemma Dennis, William Cross and Matthew Garner Extraction Lab team and Array team Biochemistry Department, BRI

Graham Bayly Mathangi Balasubramani Bath, Weston-super-Mare and Gloucester Biochemistry teams GOS Lab Alison Taylor Progenika Xabier Abad Maximilliano Crosetti Gen-probe (Tepnel diagnostics) MRC-Holland

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