1. 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

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

4. 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

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. 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

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

9. 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)

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. 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

13. 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

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. 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 ?

18. Implementation of LIPOchip®?
Sensitivity : Cost Ratio
Full screen
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: Progenika:
Oliver Wood
Esta Cross
Alison Skinner
Dr John Harvey
Progenika:
Dr Xabier Abad Lloret
Maximiliano Crosetti