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Bristol Genetics Laboratory Familial Hypercholesterolaemia: LIPOchip ® experience Laura Yarram Bristol Genetics Laboratory.

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Presentation on theme: "Bristol Genetics Laboratory Familial Hypercholesterolaemia: LIPOchip ® experience Laura Yarram Bristol Genetics Laboratory."— Presentation transcript:

1 Bristol Genetics Laboratory Familial Hypercholesterolaemia: LIPOchip ® experience Laura Yarram Bristol Genetics Laboratory

2 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 2. Xanthelasma (Image from Pietroleonardo & Ruzicka, 2009) Image 1. Achilles tendon xanthoma Definite  Total cholesterol >7.5 mmol/L  Tendon xanthomas Possible  Total cholesterol >7.5 mmol/L  Family history of myocardial infarction

3 Bristol Genetics Laboratory 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

4 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 Exercise ECG positive Genetic diagnosis allows consideration of LDL apheresis p.[Asp589fs] + [Lys311Thr] p.[Asp589fs] + [=] p.[=] + [=]

5 Bristol Genetics Laboratory Current Testing Method ARMS – 20 common mutations +ve -ve Mutation confirmation Sequencing REPORT REPORT Offer sequencing/ MLPA Sequencing +ve-ve REPORT Proceed to MLPA REPORT +ve-ve CASCADE Validated on known positive control samples Tested 104 samples to date Primers designed for all 18 LDLR exons + promoter Sequenced in 17 fragments Validated using 4 known positives MLPA (MRC-Holland: P062B) validated using 4 reported samples, obtained from a Norwegian lab

6 Bristol Genetics Laboratory Simon Broome Audit Data Simon Broome Criteria No. Patients Tested No. +ve dFH1515 (100%) pFH5323 (43%) Unclassified176 (35%) Criteria not met194 (21%) Total Diagnostic10448 (46%) Cascade2715 (56%)

7 Bristol Genetics Laboratory Results to Date  34 pathogenic variants detected to date + 2 variants ‘likely non-pathogenic’ -c G>A and c.969C>T (p.[=])   28 of these variants required UV studies (Nb. Most were previously reported to database but with no functional/family studies) cDNAProteinDiagnostic c.10580G>Ap.Arg3527Gln (APOB)9 (19%) c.1436T>Cp.Leu479Pro4 (8%) c.313+1G>AN/A3 (6%) c.1640T>Cp.Leu547Pro2 (4%) c.662A>Gp.Asp221Gly2 (4%) c.1049G>Cp.Arg350Pro2 (4%) Commonly detected mutations in SW diagnostic patients (n=48)

8 Bristol Genetics Laboratory Assay Sensitivity   Testing strategy not sustainable for disease frequency AssaySensitivity (n=48) ARMS FH2052% Bi-directional sequencing of LDLR (Promoter + 18 exons) 46% MLPA (P062B-C1)2%

9 Bristol Genetics Laboratory LIPOchip ® Background  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

10 Bristol Genetics Laboratory LIPOchip ® Processing 5 Results analysis 1 Amplification 2 Fragmentation 3 Labelling 4 Hybridization PCR mixes 1, 2, 3 and 4 DNAse + Alkaline Phosphatase TdT + Biotin-ddUTP 2 hours 45 minutes60 minutes 3hours and 30 minutes OVERLAPPING PROCESSES Tecan 4800 HS Pro 0 Extraction 2 hours DAY 1DAY 2

11 Bristol Genetics Laboratory Mutations Detected c.429C>A, p.Cys143X c.1432G>A, p.Gly478Arg Mutations not present in FH20 ARMS

12 Bristol Genetics Laboratory c.2093G>A (p.Cys698Tyr) Patient has c.2093G>T (p.Cys698Phe) Slight displacement from the ‘Normal’ group

13 Bristol Genetics Laboratory Del ex7-3’UTR

14 Bristol Genetics Laboratory Duplication LDLR Exon 17 LIPOchip result MLPA result 3.5kb ~5kb N dup 17 N Long-range PCR confirmation Ex16_F Ex18_R 3.5kb

15 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  AssaySensitivity (%) (n=48) Pick up (% of all diagnostic cases) (n=104) ARMS (20 common mutations) 5224 Current LIPOchip (251 mutations) 5827 British LIPOchip (Personal Communication) 7736

16 Bristol Genetics Laboratory Proposed Method of Testing  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)

17 Bristol Genetics Laboratory Conclusion  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

18 Bristol Genetics Laboratory Acknowledgments  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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