bristol genetics laboratory familial hypercholesterolaemia: lipochip ® experience laura yarram...

Bristol Genetics Laboratory

Familial Hypercholesterolaemia:

LIPOchip® experience

Laura Yarram



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 expectancyon statin treatment

NICE guidelines

Image 2. Xanthelasma(Image from Pietroleonardo & Ruzicka, 2009)

Image 1. Achilles tendonxanthomaDefinite

Total cholesterol >7.5 mmol/LTendon xanthomas

PossibleTotal cholesterol >7.5 mmol/LFamily history of myocardial infarction <age 60 in 1st degree relative


Why Perform Genetic Testing? Patient A, aged 8

LDLR mutation confirmed in familyEquivocal cholesterol – 5.6mmol/L

(FH >6.7mmol/L in children, ‘Normal’ <4.0mmol/L)

Family history of extensive cardiovascular diseaseGreat uncle – Myocardial infarction at aged 31

Patient A – mutation identifiedWill start statin treatment at ~ age 10Early treatment gives the maximum health benefitMore likely to adhere to treatment


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 investigationsExercise ECG positiveGenetic diagnosis allows consideration of LDL apheresis

p.[Asp589fs] + [Lys311Thr]

p.[Asp589fs] + [=]

p.[=] + [=]


Current Testing MethodARMS –

20 commonmutations+ve -ve

Mutationconfirmation Sequencing

REPORT

REPORT Offersequencing/

MLPA

Sequencing

+ve -ve

REPORTProceed to

MLPA

REPORT REPORT

+ve -ve

CASCADE

•Validated on known positive control samples• Tested 104 samples to date

• Primers designed for all 18 LDLR exons + promoter• Sequenced in 17 fragments • Validated using 4 known positives

• MLPA (MRC-Holland: P062B) validated using 4 reported samples, obtained from a Norwegian lab


Simon Broome Audit Data

Simon Broome Criteria

No. Patients Tested

No. +ve

dFH 15 15 (100%)

pFH 53 23 (43%)

Unclassified 17 6 (35%)

Criteria not met 19 4 (21%)

Total Diagnostic 104 48 (46%)

Cascade 27 15 (56%)


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)

cDNA Protein Diagnostic

c.10580G>A p.Arg3527Gln (APOB) 9 (19%)

c.1436T>C p.Leu479Pro 4 (8%)

c.313+1G>A N/A 3 (6%)

c.1640T>C p.Leu547Pro 2 (4%)

c.662A>G p.Asp221Gly 2 (4%)

c.1049G>C p.Arg350Pro 2 (4%)

Commonly detected mutations in SW diagnostic patients (n=48)


Assay Sensitivity

Testing strategy not sustainable for disease frequency

Assay Sensitivity (n=48)

ARMS FH20 52%

Bi-directional sequencing of LDLR (Promoter + 18 exons)

46%

MLPA (P062B-C1) 2%


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


LIPOchip® Processing

5Results analysis

1 Amplification

2Fragmentation

3Labelling

4Hybridization

PCR mixes

1, 2, 3 and 4

DNAse+

Alkaline Phosphatase

TdT+

Biotin-ddUTP

2 hours 45 minutes 60 minutes 3hours and 30 minutes

OVERLAPPING PROCESSES

Tecan 4800 HS Pro

0Extraction

2 hours

DAY 1 DAY 2


Mutations Detected

c.429C>A, p.Cys143X

c.1432G>A, p.Gly478Arg

Mutations not present in FH20 ARMS


c.2093G>A (p.Cys698Tyr)

Patient has c.2093G>T(p.Cys698Phe)

Slight displacement fromthe ‘Normal’ group


Del ex7-3’UTR


Duplication LDLR Exon 17LIPOchip resultMLPA result

3.5kb

~5kb

N dup 17 N

Long-range PCR confirmation

16 17 18

Ex16_F Ex18_R

3.5kb


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

Assay Sensitivity (%) (n=48)

Pick up (% of all

diagnostic cases) (n=104)

ARMS (20 common mutations)

52 24

Current LIPOchip (251 mutations)

58 27

British LIPOchip (Personal Communication)

77 36


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 criteriaFull PCSK9 screen by bi-directional sequencing

(Validation near completion, 12 fragments)Sequencing of APOB hotspot regions (exon 26 and 29)


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


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

bristol genetics laboratory familial hypercholesterolaemia: lipochip ® experience laura yarram...

Documents

current lipochip

concordant slide

utr slide

detected mutations

lipochip result mlpa

sequencing mlpa sequencing

fh20 arms slide

common mutations