Validation of a novel mutation screening strategy for Familial Hypercholesterolaemia: LIPOchip®, a DNA-array based syst

1. Validation of a novel mutation screening strategy for Familial Hypercholesterolaemia: LIPOchip®, a DNA-array based system Jonathan Callaway Wessex Regional Genetics Laboratory

2. Familial Hypercholesterolaemia (FH) • Autosomal dominant disorder of lipid metabolism • Heterozygous prevalence in UK population of 1 in 500 • Characterised by: • Raised serum LDL-c (low density lipoprotein cholesterol) • Tendon and skin xanthomata (due to cholesterol deposits) • Premature coronary heart disease • Early diagnosis is beneficial to patients since treatment with lipid-lowering therapy (e.g. statins) can result in a near-normal life expectancy by lowering the risk of coronary heart disease • Homozygous FH exists but is rare • Prevalence of 1 in a million • Symptoms more severe: appear in childhood and often lead to early death from coronary heart disease

3. Genes implicated in FH • FH is a genetically heterogeneous disorder • Mutations which cosegregate with the disease have been found in at least three genes: • LDLR(low density lipoprotein receptor) • Over 1000 mutations spread throughout gene • Exonic deletions and duplications (5-10% FH cases) • APOB (apolipoprotein B) • 9 mutations • PCSK9 (proprotein convertase subtilisin/kexin type 9) • 6 mutations • Most mutations identified are in LDLR (~79%) with lower proportion in APOB (~5.5%) and PCSK9 (~1.5%)

4. ApoB-100 protein LDL-c Lipid core of cholesterol esters LDLR ?PCSK9 Cholesterol synthesis LDLR synthesis LDL-receptor pathway maintains intracellular cholesterol homeostasis LDL-receptor pathway

5. Current FH testing strategy • FH20 Elucigene ARMS kit (Tepnel Diagnostics) • Identifies 20 most common mutations in UK population • Sensitivity of only 40% • NICE guidelines recommend DNA testing be used to confirm a diagnosis of FH (March 2009) • Need for an increase in testing sensitivity

6. LIPOchip® • A DNA array-based system designed by Progenika • Used as the primary testing strategy for FH in Spain • Detection of 251 common FH point mutations • 242 LDLR, 3 APOB, 6 PCSK9 • Copy number variation detection in LDLR • Currently targeted towards Spanish population within which the manufacturers claim a sensitivity of 80% • 4 FH20 mutations are not detected by Spanish version • FS206, K290RfsX20, Q363X, C656R • British version is under development • June 2010 availability • Sensitivity of 80-85% • Probe sets for the 4 missing FH20 mutations

7. Light Laser Cy3-streptavidin Biotin Fragmented PCR product LIPOchip®: DNA-array technology • Multiplex PCR amplification • Product fragmentation • Labelling with biotin • Hybridization and washing • Using Tecan 4800 HS Pro station • Addition of Cy3-streptavidin (fluorochrome) • Results analysis • Using Agilent scanner and customised software

8. A scanned LIPOchip® slide • 2 pairs of oligonucleotides per mutation: • Each pair consists of a WT probe and a mutant probe • Signal intensity ratios calculated for WT / (WT+Mut) • Controls for hybridization process and for measuring background signal noise • Copy number variation detection controls

9. Normal Heterozygous WT/mutant Homozygous mutant Graphical display of results generated by LIPOchip® software

10. Validation Strategy • 48 LIPOchip® slides were provided by Progenika to validate the technology • Maximum of 12 samples per run - in order to perform copy number detection two of these must be normal male & female controls • Samples selected for validation: • 10 normal controls • 6 FH20 positive controls • 22 FH20 negative patients • Criteria for selection of FH20 negative patients: • ‘Definite FH’ on referral card; or • High cholesterol level (over 8 mmol/L) plus either • Family history of high cholesterol; or • Family history of cardiovascular disease

11. Results from Validation • Normal controls: • 9/10 slides passed quality control • No point mutations were detected • FH20 positive controls: • 6/6 mutations correctly called by LIPOchip® • FH20 negative patients: • 2 pathogenic LDLR missense mutations: • c.1796T>C (p.Leu599Ser) • c.1618G>A (p.Ala540Thr) • 1 unclassified LDLR variant: • c.2177C>T (p.Thr726Ile)…likely non-pathogenic by in-silico analysis • Mutations were confirmed by direct sequencing

12. Normal Problem 1: The M064 probe set • c.91G>T (p.Glu31X) • ‘No Call’ result was frequently obtained: • 7/9 normal controls • 3/6 positive controls • 20/22 FH20 negative patients • Signal intensity values extended beyond the normal distribution parameters although they were still distinct from the mutation range • Progenika are aware of this problem and hope to resolve it in the forthcoming British version

13. Normal LDLR gene dosage Apparent deletion of promoter + exon 1 Problem 2: Copy number variation detection 7/9 normal controls appeared to have a deletion of the LDLR promoter and exon 1

14. Problem 2: Copy number variation detection Also, poor quality dosage data was often generated… These issues raised the question as to whether LIPOchip® could be used in our laboratory for reliable copy number variation detection

15. Traditional Full Screen • Testing strategy • Combination of dHPLC and direct sequencing of LDLR gene • MLPA for dosage analysis of LDLR gene (MRC-Holland kit P062-C1) • Samples: • 10 normals from the validation • 22 FH20 negative patients tested using LIPOchip®

16. Results from Traditional Full Screen • The 2 pathogenic LDLR mutations and the unclassified LDLR variant identified by LIPOchip® were confirmed • 2 further pathogenic LDLR mutations and an additional unclassified variant were detected: • c.1061A>T (p.Asp354Val) • c.1067delA (p.Ala356ValfsX14) • c.2479G>A (p.Val827Ile)…undecided pathogenicity by in-silico analysis • MLPA did not detect any deletions or duplications in the LDLR promoter or exonic regions of patients or controls

17. Full screen with dHPLC and sequencing plus MLPA – 99.9% sensitivity LIPOchip® - possibly 80-85% sensitivity (currently need to use MLPA in addition) ? Full screen with dHPLC and sequencing Possible Stratified Testing Approach for FH FH20 – 40% sensitivity

18. Full screen LIPOchip® Sensitivity : Cost Ratio Implementation of LIPOchip®?

19. Conclusions • LIPOchip® can be reliably used to detect common FH point mutations with an increase in testing sensitivity • Currently MLPA is required as a necessary complement to LIPOchip® testing • Some mutations detected by LIPOchip® require further investigation regarding their pathogenicity using in-silico analysis • Additional validation work is needed on the British version of LIPOchip®, when available • Costing is an issue for LIPOchip® and will influence the decision on whether or not to use the technology in a diagnostic setting

20. Acknowledgements • Wessex Regional Genetics Laboratory: • Oliver Wood • Esta Cross • Alison Skinner • Dr John Harvey • Progenika: • Dr Xabier Abad Lloret • Maximiliano Crosetti