GLEAM GLEAM Glasgow Linkage Exclusion Analysis Glasgow Linkage Exclusion Analysis Method Method Su Stenhouse, Daniel Su Stenhouse, Daniel Ellis, Ayesha Ahmed and Ellis, Ayesha Ahmed and Vicky Murday Vicky Murday
Jan 12, 2016
GLEAMGLEAMGlasgow Linkage Exclusion Analysis Glasgow Linkage Exclusion Analysis
MethodMethod
Su Stenhouse, Daniel Ellis, Su Stenhouse, Daniel Ellis, Ayesha Ahmed and Vicky Ayesha Ahmed and Vicky
MurdayMurday
The ProblemThe Problem Diagnostic molecular genetics has Diagnostic molecular genetics has
traditionally been concerned with single traditionally been concerned with single gene disordersgene disorders
Fragile X, cystic fibrosis, Duchenne Fragile X, cystic fibrosis, Duchenne muscular dystrophymuscular dystrophy
As more disease associations are As more disease associations are identified and whole gene sequencing identified and whole gene sequencing has become routine in diagnostic has become routine in diagnostic laboratories there is more demand for laboratories there is more demand for analysis of disorders which may be analysis of disorders which may be caused by one of several genes caused by one of several genes
‘‘Problem’ disordersProblem’ disorders
Hypertrophic cardiomyopathyHypertrophic cardiomyopathy Long QTLong QT APKDAPKD Dominant retinitis pigmentosaDominant retinitis pigmentosa Tuberous sclerosisTuberous sclerosis Ehlers DanlosEhlers Danlos Dilated cardiomyopathyDilated cardiomyopathy The list goes on…..The list goes on…..
The Solutions?The Solutions? For BRCA 1 &2 in familial breast cancer it is For BRCA 1 &2 in familial breast cancer it is
possible to sequence both genes to search for possible to sequence both genes to search for a mutationa mutation
Disorders with more genes such as Disorders with more genes such as cardiomyopathy are harder to deal withcardiomyopathy are harder to deal with
Next generation sequencing technologies Next generation sequencing technologies promise very rapid sequencing but still result promise very rapid sequencing but still result in massive quantities of data to analysein massive quantities of data to analyse
Can the NHS afford the expensive equipment Can the NHS afford the expensive equipment required?required?
Sequence only the commonest genes but Sequence only the commonest genes but fewer families benefitfewer families benefit
The Old IdeaThe Old Idea
Linkage analysis can be used to Linkage analysis can be used to indicate which of a number of genes indicate which of a number of genes might be involved in a particular familymight be involved in a particular family
However this requires a good family However this requires a good family structure and samples from affected structure and samples from affected and unaffected individuals in several and unaffected individuals in several generationsgenerations
Samples are rarely available from the Samples are rarely available from the required number of individualsrequired number of individuals
CMGS 2006CMGS 2006
The Eureka momentThe Eureka moment
The New IdeaThe New Idea Conventional linkage analysis uses a large Conventional linkage analysis uses a large
number of individuals and a few markersnumber of individuals and a few markers If it were possible to use a very large If it were possible to use a very large
number of markers then fewer family number of markers then fewer family members would be requiredmembers would be required
DNA CHIP technology allows thousands of DNA CHIP technology allows thousands of markers to be analysed at oncemarkers to be analysed at once
If two affected family members were If two affected family members were oppositely homozygous for any marker oppositely homozygous for any marker within a gene that gene would be excluded within a gene that gene would be excluded from causing the disease in that familyfrom causing the disease in that family
PhasePhase
The crux of this method is that it The crux of this method is that it eliminates the need to establish eliminates the need to establish phase which is required for phase which is required for conventional linkage analysisconventional linkage analysis
Single nucleotide polymorphisms Single nucleotide polymorphisms (SNPs) are chosen with a minor (SNPs) are chosen with a minor allele frequency of 0.3 or aboveallele frequency of 0.3 or above
The SNPs are chosen along the The SNPs are chosen along the length of the genes of interest and length of the genes of interest and the immediate flanking regionsthe immediate flanking regions
A Novel Use of CHIP A Novel Use of CHIP
TechnologyTechnology Using SNPs to DISPROVE linkageUsing SNPs to DISPROVE linkage
If these were two If these were two affected patients in a affected patients in a pedigree and the two pedigree and the two diagrams represented diagrams represented their genotypes for 4 SNPstheir genotypes for 4 SNPsin a putative gene, beingin a putative gene, beinghomozygous for differenthomozygous for differentalleles in the 3alleles in the 3rdrd SNP would SNP would suggest there was no linkage suggest there was no linkage between the disease and the gene.between the disease and the gene.
A
A
A
A
A
A A
A
A
B
B
B
B
B B
B
Relative 1 Relative 2
Choice of SNP allele Choice of SNP allele frequencyfrequency
Chance both parents heterozygote Chance both parents heterozygote from Hardy-Weinberg is 2pq x 2pq = from Hardy-Weinberg is 2pq x 2pq = 4p4p22qq22
Chance for a SNP to be informative is Chance for a SNP to be informative is pp22qq2 2 divided by 2divided by 2
So if p=0.3 and q=0.7 chance So if p=0.3 and q=0.7 chance informative is 0.022informative is 0.022
If p=0.5 and q=0.5 then chance If p=0.5 and q=0.5 then chance informative is 0.031informative is 0.031
Choice of SNP numbersChoice of SNP numbers
Using a binomial distribution, if 100 Using a binomial distribution, if 100 SNPs with allele frequencies SNPs with allele frequencies between 0.3 and 0.5 are used per between 0.3 and 0.5 are used per gene there is a 90 to 96% chance of gene there is a 90 to 96% chance of finding at least one informative.finding at least one informative.
If 150 SNPs of those allele If 150 SNPs of those allele frequencies are used this rises to 96 frequencies are used this rises to 96 to 99%to 99%
Evidence BaseEvidence Base
Using families with a known BRCA1 Using families with a known BRCA1 or 2 mutation we aimed to establish or 2 mutation we aimed to establish whether we would have accurately whether we would have accurately predicted which gene was involved predicted which gene was involved using this novel method.using this novel method.
The FamiliesThe Families
We identified 57 Scottish BRCA 1 or We identified 57 Scottish BRCA 1 or BRCA2 families in which we had BRCA2 families in which we had samples from more than one samples from more than one affected individual available and 16 affected individual available and 16 were provided from the South West were provided from the South West Thames genetic service.Thames genetic service.
Proof of PrincipleProof of Principle
SNPs were selected along the length SNPs were selected along the length of the BRCA 1 and 2 genesof the BRCA 1 and 2 genes
Illumina system chosen as the Illumina system chosen as the platform as equipment was available platform as equipment was available to usto us
Professor Connor kindly agreed to Professor Connor kindly agreed to fund the initial CHIP manufacturefund the initial CHIP manufacture
The initial CHIP carried 214 SNPs for The initial CHIP carried 214 SNPs for BRCA 1 and 170 SNPs for BRCA 2 all BRCA 1 and 170 SNPs for BRCA 2 all with a MAF of >0.3with a MAF of >0.3
Illumina CHIPIllumina CHIP
Random SNPs attached to ‘beads in wells’Random SNPs attached to ‘beads in wells’ ILLUMINA GoldengateILLUMINA GoldengateTMTM assay uses a four assay uses a four
colour code to identify which SNP is wherecolour code to identify which SNP is where Extension and ligation from genomic DNAExtension and ligation from genomic DNA PCR ligated template using universal PCR ligated template using universal
primersprimers Hybridise to IllumicodeHybridise to IllumicodeTMTM array and identify array and identify
SNPsSNPs Analysis takes 3 days and can type 96 Analysis takes 3 days and can type 96
patientspatients
CostsCosts First CHIP is very expensive as the cost First CHIP is very expensive as the cost
is in manufacturing the first SNP poolis in manufacturing the first SNP pool Subsequent CHIPs are relatively cheap Subsequent CHIPs are relatively cheap
as the same SNP pool can be usedas the same SNP pool can be used Averaging out the cost of first and Averaging out the cost of first and
subsequent CHIPs we estimate a cost of subsequent CHIPs we estimate a cost of about £50 per patientabout £50 per patient
Cf ~£800 to sequence each gene in each Cf ~£800 to sequence each gene in each patient or £76,800 for 96 patientspatient or £76,800 for 96 patients
Choosing the samplesChoosing the samples
As we are looking for opposite As we are looking for opposite homozygosity parent/child homozygosity parent/child combinations cannot be used as they combinations cannot be used as they will by definition share an allelewill by definition share an allele
Sibs, cousins, aunt/niece etcSibs, cousins, aunt/niece etc The further removed in the pedigree The further removed in the pedigree
the more informative they are likely the more informative they are likely to be.to be.
The PairsThe Pairs
69 siblings69 siblings 21 cousins21 cousins 14 aunt/niece14 aunt/niece 2 great aunt/niece2 great aunt/niece
RESULTSRESULTS
Po
sition
Ch
r
Nam
e
13545_196-1351
30842815 13 rs494182930853581 13 rs287242230859924 13 rs953159830866590 13 rs494336030879675 13 rs135963430887435 13 rs494184230894875 13 rs732157530959209 13 rs224532830963121 13 rs49282130970018 13 rs55516330972743 13 rs185442730976343 13 rs66068730985599 13 rs27715130985892 13 rs27715430995631 13 rs27712630999138 13 rs53596230999896 13 rs59870031003351 13 rs186979931012398 13 rs153553231026476 13 rs141081031030567 13 rs277143
Excluded Gene (BRCA) 2 None
2(SH
1)_D17276
2(SH
2)_D17055
2(SI1)_D
17825
2(SI2)_D
31511
A AB BB BB B B BB AB A A AA BB BA AA AB BB B
AAA
B BA A
A A AA A AB B B
B B
RESULTSRESULTS
Ge
no
mic
Lo
ca
tion
Ch
rom
os
om
e
Na
me
BR
CA
2(SH
1)_D17276
2(SH
2)_D17055
Result30842815 13 rs4941829 2 A A 1 1 TRUE TRUE TRUE30853581 13 rs2872422 2 B B 1 1 TRUE TRUE TRUE30859924 13 rs9531598 2 B B 1 1 TRUE TRUE TRUE30866590 13 rs4943360 2 B B 1 1 TRUE TRUE TRUE30879675 13 rs1359634 2 B A 1 1 TRUE FALSE FALSE30887435 13 rs4941842 2 B A 1 1 TRUE FALSE FALSE30894875 13 rs7321575 2 A B 1 1 TRUE FALSE FALSE30959209 13 rs2245328 2 B 1 0 FALSE FALSE TRUE30963121 13 rs492821 2 A 1 0 FALSE FALSE TRUE30970018 13 rs555163 2 A 1 0 FALSE FALSE TRUE30972743 13 rs1854427 2 B 1 0 FALSE FALSE TRUE30976343 13 rs660687 2 B 1 0 FALSE FALSE TRUE30985599 13 rs277151 2 0 0 TRUE TRUE TRUE30985892 13 rs277154 2 0 0 TRUE TRUE TRUE30995631 13 rs277126 2 0 0 TRUE TRUE TRUE30999138 13 rs535962 2 0 0 TRUE TRUE TRUE30999896 13 rs598700 2 0 0 TRUE TRUE TRUE31003351 13 rs1869799 2 A 1 0 FALSE FALSE TRUE31012398 13 rs1535532 2 A 1 0 FALSE FALSE TRUE31026476 13 rs1410810 2 B 1 0 FALSE FALSE TRUE31030567 13 rs277143 2 0 0 TRUE TRUE TRUE
Formula for the eventual result
RESULTSRESULTSUNINFORMATIVUNINFORMATIVEE
INFORMATIVINFORMATIVEE
SIBSSIBS 6969(1 FAILED)(1 FAILED)
3535 3333
= 48%= 48%
COUSINSCOUSINS 2121 44 1717
= 81%= 81%
AUNT/AUNT/NIECENIECE
1414 44 1010
= 71%= 71%
OTHEROTHER 22 00 22
=100%=100%
TOTALTOTAL 106106 4343 6262
=58%=58%
SHILSHIL Following the success of the proof of principle Following the success of the proof of principle
we approached Scottish Health Innovations we approached Scottish Health Innovations Ltd who are charged with supporting suitable Ltd who are charged with supporting suitable projects emerging from the health service.projects emerging from the health service.
They agreed to fund a patent application and They agreed to fund a patent application and further development workfurther development work
Patent application is now in processPatent application is now in process This is intended as a protective patent rather This is intended as a protective patent rather
than a profitable one!than a profitable one! Cardiomyopathy CHIP now about to be Cardiomyopathy CHIP now about to be
trialledtrialled
SHIL NominationSHIL Nomination
Without our knowledge the project was Without our knowledge the project was nominated for the Scottish Healthcare nominated for the Scottish Healthcare Innovation of the Year AwardInnovation of the Year Award
Shortlisted projects had to give a Shortlisted projects had to give a presentation to the selection committee presentation to the selection committee and answer questions from the panel and answer questions from the panel (Dragon’s Den scenario)(Dragon’s Den scenario)
In Perth!In Perth!
We Won!We Won!
AcknowledgementsAcknowledgements
We would like to thank the staff at We would like to thank the staff at the molecular lab in St Georges for the molecular lab in St Georges for kindly looking out and sending us kindly looking out and sending us the samples form Vicky’s erstwhile the samples form Vicky’s erstwhile patients. We know how time patients. We know how time consuming such activity is!consuming such activity is!