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

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

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%) FH is a genetically heterogeneous disorder, making mutation detection testing problematic

LDL-receptor pathway LDL-receptor pathway maintains intracellular cholesterol homeostasis Cholesterol synthesis LDL-c LDLR ApoB-100 protein Lipid core of cholesterol esters ? PCSK9 LDLR synthesis The products of these three genes all have important roles in the LDL-receptor pathway, which helps to maintain intracellular cholesterol homeostasis - as shown in this simplified figure LDL-c is composed of a lipid core of cholesterol esters encased by ApoB protein The LDL-c complex binds to LDLR, a transmembrane receptor present on most cells, through an interaction with ApoB protein Mutations in the APOB gene affect the strength of this interaction Receptor-ligand internalisation occurs by endocytosis LDL dissociates from its receptor - which is recycled to the cell membrane LDL is degraded releasing cholesterol, which inhibits further cholesterol biosynthesis PCSK9 has been shown to regulate LDLR levels by a post-translational mechanism which is currently not well defined. However, mutations decrease the uptake of LDL into the cell

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

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

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 Laser Light Cy3-streptavidin Biotin Fragmented PCR product On the surface of each LIPOchip® slide, allele-specific oligonucleotides have been immobilised Biotin-labelled, fragmented PCR products are applied to the surface of a slide and allowed to hybridize with these complimentary probes Hybridization is performed using the Tecan Pro station The fluorochrome Cy3-Streptavidin is added and this binds to biotin present on the PCR products An Agilent scanner is used to quantify the signal intensity emitted from each probe following laser excitation Customised software interprets the signals generated and relates this to the genotype for each mutation

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 Signal intensity ratios relating to wild-type and mutant probes are calculated by the software

Graphical display of results generated by LIPOchip® software Normal Homozygous mutant Heterozygous WT/mutant One probe set for several patients Example – actually a probe set for an LDLR SNP – a few included on LIPOchip…..make good internal controls…can distinguish between the different genotypes (normal, heterozygote and homozygote)

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

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

Problem 1: The M064 probe set Normal 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

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

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

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®

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

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

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

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

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