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GLEAM
Glasgow Linkage Exclusion Analysis Method
Su Stenhouse, Daniel Ellis, Ayesha
Ahmed and Vicky Murday
The Problem
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Diagnostic molecular genetics has traditionally
been concerned with single gene disorders
Fragile X, cystic fibrosis, Duchenne muscular
dystrophy
As more disease associations are identified and
whole gene sequencing has become routine in
diagnostic laboratories there is more demand for
analysis of disorders which may be caused by
one of several genes
‘Problem’ disorders
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Hypertrophic cardiomyopathy
Long QT
APKD
Dominant retinitis pigmentosa
Tuberous sclerosis
Ehlers Danlos
Dilated cardiomyopathy
The list goes on…..
The Solutions?
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For BRCA 1 &2 in familial breast cancer it is possible
to sequence both genes to search for a mutation
Disorders with more genes such as cardiomyopathy are
harder to deal with
Next generation sequencing technologies promise very
rapid sequencing but still result in massive quantities of
data to analyse
Can the NHS afford the expensive equipment required?
Sequence only the commonest genes but fewer families
benefit
The Old Idea
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Linkage analysis can be used to indicate which
of a number of genes might be involved in a
particular family
However this requires a good family structure
and samples from affected and unaffected
individuals in several generations
Samples are rarely available from the required
number of individuals
CMGS 2006
The Eureka moment
The New Idea
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Conventional linkage analysis uses a large number of
individuals and a few markers
If it were possible to use a very large number of
markers then fewer family members would be required
DNA CHIP technology allows thousands of markers
to be analysed at once
If two affected family members were oppositely
homozygous for any marker within a gene that gene
would be excluded from causing the disease in that
family
Phase
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The crux of this method is that it eliminates the
need to establish phase which is required for
conventional linkage analysis
Single nucleotide polymorphisms (SNPs) are
chosen with a minor allele frequency of 0.3 or
above
The SNPs are chosen along the length of the
genes of interest and the immediate flanking
regions
A Novel Use of CHIP Technology
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Using SNPs to DISPROVE linkage
Relative 1
If these were two
affected patients in a
pedigree and the two
diagrams represented
their genotypes for 4 SNPs
in a putative gene, being
homozygous for different
alleles in the 3rd SNP would
suggest there was no linkage
between the disease and the gene.
Relative 2
A
B
B
A
A
A
A
B
A
A
B
A
B
B
A
B
Choice of SNP allele frequency
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Chance both parents heterozygote from HardyWeinberg is 2pq x 2pq = 4p2q2
Chance for a SNP to be informative is p2q2
divided by 2
So if p=0.3 and q=0.7 chance informative is
0.022
If p=0.5 and q=0.5 then chance informative is
0.031
Choice of SNP numbers
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Using a binomial distribution, if 100 SNPs with
allele frequencies between 0.3 and 0.5 are used
per gene there is a 90 to 96% chance of finding
at least one informative.
If 150 SNPs of those allele frequencies are used
this rises to 96 to 99%
Evidence Base
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Using families with a known BRCA1 or 2
mutation we aimed to establish whether we
would have accurately predicted which gene was
involved using this novel method.
The Families
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We identified 57 Scottish BRCA 1 or BRCA2
families in which we had samples from more
than one affected individual available and 16
were provided from the South West Thames
genetic service.
Proof of Principle
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SNPs were selected along the length of the
BRCA 1 and 2 genes
Illumina system chosen as the platform as
equipment was available to us
Professor Connor kindly agreed to fund the
initial CHIP manufacture
The initial CHIP carried 214 SNPs for BRCA 1
and 170 SNPs for BRCA 2 all with a MAF of
>0.3
Illumina CHIP
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Random SNPs attached to ‘beads in wells’
ILLUMINA GoldengateTM assay uses a four
colour code to identify which SNP is where
Extension and ligation from genomic DNA
PCR ligated template using universal primers
Hybridise to IllumicodeTM array and identify
SNPs
Analysis takes 3 days and can type 96 patients
Costs
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First CHIP is very expensive as the cost is in
manufacturing the first SNP pool
Subsequent CHIPs are relatively cheap as the
same SNP pool can be used
Averaging out the cost of first and subsequent
CHIPs we estimate a cost of about £50 per
patient
Cf ~£800 to sequence each gene in each patient
or £76,800 for 96 patients
Choosing the samples
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As we are looking for opposite homozygosity
parent/child combinations cannot be used as
they will by definition share an allele
Sibs, cousins, aunt/niece etc
The further removed in the pedigree the more
informative they are likely to be.
The Pairs
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69 siblings
21 cousins
14 aunt/niece
2 great aunt/niece
RESULTS
Excluded Gene (BRCA)
2(SH2)_D17055
2(SI1)_D17825
2(SI2)_D31511
rs4941829
rs2872422
rs9531598
rs4943360
rs1359634
rs4941842
rs7321575
rs2245328
rs492821
rs555163
rs1854427
rs660687
rs277151
rs277154
rs277126
rs535962
rs598700
rs1869799
rs1535532
rs1410810
rs277143
None
2(SH1)_D17276
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
Name
Chr
Position
30842815
30853581
30859924
30866590
30879675
30887435
30894875
30959209
30963121
30970018
30972743
30976343
30985599
30985892
30995631
30999138
30999896
31003351
31012398
31026476
31030567
2
A
B
B
B
B
B
A
B
A
A
B
B
A
B
B
B
A
A
B
B
B
A
A
B
A
A
B
A
A
B
B
A
A
A
B
B
B
A
A
A
B
A
A
A
B
B
RESULTS
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2(SH2)_D17055
rs4941829
rs2872422
rs9531598
rs4943360
rs1359634
rs4941842
rs7321575
rs2245328
rs492821
rs555163
rs1854427
rs660687
rs277151
rs277154
rs277126
rs535962
rs598700
rs1869799
rs1535532
rs1410810
rs277143
2(SH1)_D17276
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
BRCA
Name
Chromosome
Genomic Location
30842815
30853581
30859924
30866590
30879675
30887435
30894875
30959209
30963121
30970018
30972743
30976343
30985599
30985892
30995631
30999138
30999896
31003351
31012398
31026476
31030567
A
B
B
B
B
B
A
B
A
A
B
B
A
B
B
B
A
A
B
A
A
B
Formula for the eventual result
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
1
1
1
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
Result
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
RESULTS
SIBS
69
UNINFORMATIVE
INFORMATIVE
35
33
= 48%
17
= 81%
10
= 71%
2
=100%
(1 FAILED)
COUSINS
21
4
AUNT/NIECE
14
4
OTHER
2
0
TOTAL
106
43
62
=58%
SHIL
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Following the success of the proof of principle we
approached Scottish Health Innovations Ltd who are
charged with supporting suitable projects emerging
from the health service.
They agreed to fund a patent application and further
development work
Patent application is now in process
This is intended as a protective patent rather than a
profitable one!
Cardiomyopathy CHIP now about to be trialled
SHIL Nomination
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Without our knowledge the project was
nominated for the Scottish Healthcare
Innovation of the Year Award
Shortlisted projects had to give a presentation to
the selection committee and answer questions
from the panel (Dragon’s Den scenario)
In Perth!
We Won!
Acknowledgements
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We would like to thank the staff at the
molecular lab in St Georges for kindly looking
out and sending us the samples form Vicky’s
erstwhile patients. We know how time
consuming such activity is!