Validation of a novel mutation screening strategy for Familial

Hypercholesterolaemia: LIPOchip®, a DNA-array based

system

Jonathan CallawayWessex 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%)

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

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 technology1. Multiplex PCR amplification2. Product fragmentation3. Labelling with biotin4. Hybridization and washing

- Using Tecan 4800 HS Pro station - Addition of Cy3-streptavidin (fluorochrome)

5. Results analysis- Using Agilent scanner and customised software

Laser

Light

Cy3-streptavidin

Biotin

Fragmented PCR product

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

Graphical display of results generated by LIPOchip® software

Normal

Homozygous mutant

Heterozygous WT/mutant

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

(i) Family history of high cholesterol; or(ii) 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

• 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

Normal

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

Full screen with dHPLC and sequencing plus MLPA – 99.9% sensitivity

Full screen with dHPLC and sequencing

?

LIPOchip® - possibly 80-85% sensitivity (currently need to use MLPA in addition)

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:– Oliver Wood– Esta Cross– Alison Skinner– Dr John Harvey

• Progenika:– Dr Xabier Abad Lloret– Maximiliano Crosetti