Bristol Genetics Laboratory
Familial Hypercholesterolaemia:
LIPOchip® experience
Laura Yarram
Bristol Genetics Laboratory
Bristol Genetics Laboratory
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 expectancy
on statin treatment


NICE guidelines
Image 1. Achilles tendon
xanthoma
Definite
Total cholesterol >7.5 mmol/L
Tendon xanthomas
Possible
Total cholesterol >7.5 mmol/L
Family
history
of myocardial
Image
2. Xanthelasma
(Image from Pietroleonardo st
2009)
infarction
<age 60 in 1& Ruzicka,
degree
relative
Bristol Genetics Laboratory
Why Perform Genetic Testing?

Patient A, aged 8
 LDLR mutation confirmed in family
 Equivocal cholesterol – 5.6mmol/L
(FH >6.7mmol/L in children, ‘Normal’ <4.0mmol/L)
 Family history of extensive cardiovascular disease
 Great uncle – Myocardial infarction at aged 31

Patient A – mutation identified
 Will start statin treatment at ~ age 10
 Early treatment gives the maximum health benefit
 More likely to adhere to treatment
Bristol Genetics Laboratory
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 investigations
p.[=]
Exercise
+ [=] ECG positivep.[Asp589fs] + [Lys311Thr]
 Genetic diagnosis allows consideration of LDL apheresis
p.[Asp589fs] + [=]
Bristol Genetics Laboratory
Current Testing Method
+ve
ARMS –
20 common
mutations
Mutation
confirmation
Sequencing
Sequencing
REPORT
+ve
REPORT
CASCADE
•Validated on known positive
control
samples• MLPA (MRC-Holland:
-ve
• Tested 104 samples
date
P062B)tovalidated
using
REPORT Offer
4 reported samples,
sequencing/
obtained from a
MLPA
Norwegian lab
-ve
Proceed to
MLPA
• Primers designed for all 18 LDLR
+ve
-ve
exons + promoter
• Sequenced in 17 fragments
REPORT
• REPORT
Validated using 4 known positives
Bristol Genetics Laboratory
Simon Broome Audit Data
Simon Broome
Criteria
dFH
pFH
Unclassified
No. Patients
Tested
15
53
17
No. +ve
Criteria not met
19
4 (21%)
Total Diagnostic
104
48 (46%)
Cascade
27
15 (56%)
15 (100%)
23 (43%)
6 (35%)
Bristol Genetics Laboratory
Results to Date

34 pathogenic variants detected to date
 + 2 variants ‘likely non-pathogenic’
- c.2390-16G>A and c.969C>T (p.[=])

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)

28 of these variants required UV studies
(Nb. Most were previously reported to database but with no
functional/family studies)
Bristol Genetics Laboratory
Assay Sensitivity

Assay
Sensitivity
(n=48)

ARMS FH20
52%
Bi-directional sequencing of
LDLR (Promoter + 18 exons)
46%
MLPA (P062B-C1)
2%
Testing strategy not sustainable for
disease frequency
Bristol Genetics Laboratory
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
Bristol Genetics Laboratory
LIPOchip® Processing
0
Extraction
1
Amplification
PCR mixes
1, 2, 3 and 4
2 hours
2 hours
2
Fragmentation
DNAse
+
Alkaline
Phosphatase
3
Labelling
TdT
+
Biotin-ddUTP
45 minutes
60 minutes
4
Hybridization
Tecan 4800
HS Pro
3hours and 30
minutes
OVERLAPPING PROCESSES
DAY 1
DAY 2
5
Results
analysis
Bristol Genetics Laboratory
Mutations Detected
c.429C>A, p.Cys143X
c.1432G>A, p.Gly478Arg
Mutations not present in FH20 ARMS
Bristol Genetics Laboratory
c.2093G>A (p.Cys698Tyr)
Patient has c.2093G>T
(p.Cys698Phe)
Slight displacement from
the ‘Normal’ group
Bristol Genetics Laboratory
Del ex7-3’UTR
Bristol Genetics Laboratory
Duplication LDLR Exon 17
MLPA result
LIPOchip result
Long-range PCR confirmation
Ex16_F
16
Ex18_R
17
3.5kb
18
~5kb
3.5kb
N
dup 17
N
Bristol Genetics Laboratory
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
Bristol Genetics Laboratory
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 criteria
 Full PCSK9 screen by bi-directional sequencing
(Validation near completion, 12 fragments)
 Sequencing of APOB hotspot regions (exon 26 and 29)
Bristol Genetics Laboratory
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
Bristol Genetics Laboratory
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