1. GLEAM Glasgow Linkage Exclusion Analysis Method Su Stenhouse, Daniel Ellis, Ayesha Ahmed and Vicky Murday

2. The Problem Diagnostic molecular genetics has traditionally been concerned with single gene disorders Diagnostic molecular genetics has traditionally been concerned with single gene disorders Fragile X, cystic fibrosis, Duchenne muscular dystrophy 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 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

3. ‘Problem’ disorders Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy Long QT Long QT APKD APKD Dominant retinitis pigmentosa Dominant retinitis pigmentosa Tuberous sclerosis Tuberous sclerosis Ehlers Danlos Ehlers Danlos Dilated cardiomyopathy Dilated cardiomyopathy The list goes on….. The list goes on…..

4. The Solutions? For BRCA 1 &2 in familial breast cancer it is possible to sequence both genes to search for a mutation 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 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 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? Can the NHS afford the expensive equipment required? Sequence only the commonest genes but fewer families benefit Sequence only the commonest genes but fewer families benefit

5. The Old Idea Linkage analysis can be used to indicate which of a number of genes might be involved in a particular family 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 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 Samples are rarely available from the required number of individuals

6. CMGS 2006 The Eureka moment

7. The New Idea Conventional linkage analysis uses a large number of individuals and a few markers 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 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 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 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

8. Phase The crux of this method is that it eliminates the need to establish phase which is required for conventional linkage analysis 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 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 The SNPs are chosen along the length of the genes of interest and the immediate flanking regions

9. A Novel Use of CHIP Technology Using SNPs to DISPROVE linkage Using SNPs to DISPROVE linkage 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 3 rd SNP would suggest there was no linkage between the disease and the gene. A A A A A AA A A B B B B BB B Relative 1Relative 2

10. Choice of SNP allele frequency Chance both parents heterozygote from Hardy- Weinberg is 2pq x 2pq = 4p 2 q 2 Chance both parents heterozygote from Hardy- Weinberg is 2pq x 2pq = 4p 2 q 2 Chance for a SNP to be informative is p 2 q 2 divided by 2 Chance for a SNP to be informative is p 2 q 2 divided by 2 So if p=0.3 and q=0.7 chance informative is 0.022 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 If p=0.5 and q=0.5 then chance informative is 0.031

11. Choice of SNP numbers 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. 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% If 150 SNPs of those allele frequencies are used this rises to 96 to 99%

12. Evidence Base 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. 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.

13. The Families 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. 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.

14. Proof of Principle SNPs were selected along the length of the BRCA 1 and 2 genes 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 Illumina system chosen as the platform as equipment was available to us Professor Connor kindly agreed to fund the initial CHIP manufacture 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 The initial CHIP carried 214 SNPs for BRCA 1 and 170 SNPs for BRCA 2 all with a MAF of >0.3

15. Illumina CHIP Random SNPs attached to ‘beads in wells’ Random SNPs attached to ‘beads in wells’ ILLUMINA Goldengate TM assay uses a four colour code to identify which SNP is where ILLUMINA Goldengate TM assay uses a four colour code to identify which SNP is where Extension and ligation from genomic DNA Extension and ligation from genomic DNA PCR ligated template using universal primers PCR ligated template using universal primers Hybridise to Illumicode TM array and identify SNPs Hybridise to Illumicode TM array and identify SNPs Analysis takes 3 days and can type 96 patients Analysis takes 3 days and can type 96 patients

16. Costs First CHIP is very expensive as the cost is in manufacturing the first SNP pool 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 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 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 Cf ~£800 to sequence each gene in each patient or £76,800 for 96 patients

17. Choosing the samples As we are looking for opposite homozygosity parent/child combinations cannot be used as they will by definition share an allele 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 Sibs, cousins, aunt/niece etc The further removed in the pedigree the more informative they are likely to be. The further removed in the pedigree the more informative they are likely to be.

18. The Pairs 69 siblings 69 siblings 21 cousins 21 cousins 14 aunt/niece 14 aunt/niece 2 great aunt/niece 2 great aunt/niece

19. RESULTS

20. RESULTS

21. RESULTS UNINFORMATIVEINFORMATIVE SIBS69 (1 FAILED) 3533 = 48% COUSINS21417 = 81% AUNT/NIECE14410 = 71% OTHER202=100% TOTAL1064362=58%

22. SHIL 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. 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 They agreed to fund a patent application and further development work Patent application is now in process Patent application is now in process This is intended as a protective patent rather than a profitable one! This is intended as a protective patent rather than a profitable one! Cardiomyopathy CHIP now about to be trialled Cardiomyopathy CHIP now about to be trialled

23. SHIL Nomination Without our knowledge the project was nominated for the Scottish Healthcare Innovation of the Year Award 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) Shortlisted projects had to give a presentation to the selection committee and answer questions from the panel (Dragon’s Den scenario) In Perth! In Perth!

24. We Won!

25. Acknowledgements 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! 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!