Familial Hypercholesterolaemia: LIPOchip ® experience

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

2. Image 1. Achilles tendon xanthoma Image 2. Xanthelasma (Image from Pietroleonardo & Ruzicka, 2009) 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

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. p.[=] + [=] p.[Asp589fs] + [Lys311Thr] p.[Asp589fs] + [=] 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

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

6. Bristol Genetics Laboratory Simon Broome Audit Data

7. 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.[=]) • 28 of these variants required UV studies (Nb. Most were previously reported to database but with no functional/family studies) Commonly detected mutations in SW diagnostic patients (n=48)

8. Bristol Genetics Laboratory Assay Sensitivity • Testing strategy not sustainable for disease frequency

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. 1 Amplification 2 Fragmentation 3 Labelling 4 Hybridization 5 Results analysis 0 Extraction PCR mixes 1, 2, 3 and 4 DNAse + Alkaline Phosphatase TdT + Biotin-ddUTP Tecan 4800 HS Pro 3hours and 30 minutes 45 minutes 60 minutes 2 hours 2 hours OVERLAPPING PROCESSES DAY 1 DAY 2 Bristol Genetics Laboratory LIPOchip® Processing

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

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. MLPA result LIPOchip result 16 17 18 Long-range PCR confirmation Ex16_F Ex18_R ~5kb 3.5kb N dup 17 N 3.5kb Bristol Genetics Laboratory Duplication LDLR Exon 17

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

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