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!