Research discoveries to diagnostic panels – an update Darren D. O’Rielly, Ph.D., FCCMG Director, Molecular Genetics Laboratory, Eastern Health Director, Translational Genomics Laboratory, Faculty of Medicine, MUN Research Scientist, Faculty of Medicine, MUN Adjunct Professor, School of Pharmacy, MUN TPMI Research Day October 8, 2015

Overview What are diagnostic panels? Translational Genomics Laboratory Personnel Equipment Available genomic services Translational Projects – current & short-term

Diagnostic panels Multi-variant or multi-gene in design Very cost-effective and time-efficient approach Provide more information to clinicians Enhance clinical decision making

Translational lab personnel Director – Darren O’Rielly Ph.D., FCCMG Research Assistant (RA) – Cindy Penny Translational Genomics Laboratory Working Group Darren O’Rielly Ph.D., FCCMG Terry Lynn Young Ph.D. Cindy Penny - RA Jim Houston – Experienced RA in Young Lab Dianne Codner – Experienced RA in Rahman/O’Rielly Lab Sonya Sheaves – Molecular Genetics Technologist

Translational lab equipment Uniplex targeted genotyping ABI 7500 Real-time PCR system x 2 Multiplex targeted genotyping Sequenom MassArray 4 system Luminex 100/200 system

Translational lab equipment Sanger sequencing, microsatellite and fragment analysis ABI 3500XL Copy number detection and gene expression analysis Agilent TapeStation Agilent Microarray system

Translational lab equipment Next-generation sequencing Ion Chef (liquid handler) Ion Torrent PGM (targeted analysis) Ion Torrent Proton (exome, genome)

Genomic services Available now: Targeted uniplex genotyping Targeted multiplex genotyping Sanger sequencing Available in coming months: Targeted NGS panels Whole-exome sequencing Genome-wide CNV analysis

Projects – 2014 to 2016 Stargardt disease recurrent mutation panel HLA-B*27 tagSNP screening assay Hearing loss targeted mutation assays Sudden cardiac death recurrent mutation panel Comprehensive genetic testing for CRC: Recurrent mutation panel Custom NGS panel and custom exon deletion/duplication assay MSI assay for CRC (5 markers) MLH1 hypermethylation & BRAF V600E assay Oncomine Focus assay

Projects – 2014 to 2016 Stargardt disease recurrent mutation panel HLA-B*27 tagSNP screening assay Hearing loss targeted mutation assays Sudden cardiac death recurrent mutation panel Comprehensive genetic testing for CRC: Recurrent mutation panel Custom NGS panel and custom exon deletion/duplication assay MSI assay for CRC (5 markers) MLH1 hypermethylation & BRAF V600E assay Oncomine Focus assay

Stargardt disease mutation panel A diagnostic panel of 20 mutations in ABCA4 previously identified in the NL population and known to cause Stargardt disease is currently being validated. Validation expected to be completed by end of December Nucleotide ChangeAmino acid change# of allelesExonrs# c.67-1delG (IVS2)splice site32rs67-1delG c.455G>Ap.Arg152Gln3/115rs c.634C>TP.Arg212Cys16rs c.1522C>Tp.Arg508Cys211rs c.2564G>Ap.Trp855X516rs c.2588G>Cp.Gly863Ala2/417rs c.3064G>Ap.Glu1022Lys121rs c.3322C>Tp.Arg1108Cys322rs c.3323G>Ap.Arg1108His122rs c.4139C>Tp.Pro1308Leu428rs c.4163T>Cp.Leu1388Pro828rs c.4222T>Cp.Trp1408Arg128rs c.4469G>Ap.Cys1490Tyr230rs c.4537delCp.Gln1513Argfs*13130rs4537delC c.4577C>Tp.Thr1526Met331rs c.4918C>Tp.Arg1640Trp135rs c T>C (IVS38)splice site?1238rs c G>A (IVS40)splice site3140rs c.6089G>Ap.Arg2030Gln144rs c.6449G>Ap.Cys2150Tyr147rs

Cardiomyopathy screen panel A diagnostic panel of 20 mutations previously identified in the NL population and known to cause cardiomyopathies is currently being validated.

Projects – 2014 to 2016 Stargardt disease recurrent mutation panel HLA-B*27 tagSNP screening assay Hearing loss targeted mutation assays Sudden cardiac death recurrent mutation panel Comprehensive genetic testing for CRC: Recurrent mutation panel Custom NGS panel and custom exon deletion/duplication assay MSI assay for CRC (5 markers) MLH1 hypermethylation & BRAF V600E assay Oncomine Focus assay

Recurrent mutation panel A panel of >20 variants strongly associated with hereditary CRC will be developed as a first-pass test for individuals at moderate- or high-risk of CRC. Design of the panel is expected to be complete by the end of November 2015 and validation finished by the end of March FAPAPCIVS3-1G>A (Twillingate mut'n) FAPAPC694C>T (R236X) exon 6 ?694 or 649 FAPAPC insA FAPAPCexon insCA FAPAPC(1309)delAAAGA FAPAPC(1061)delACAAA HNPCCMSH2Fam C HNPCCMSH2Exon 8 deletion HNPCCMSH2Exons 4-16 deletion HNPCCMSH2c.1661G>C HNPCCMSH2c.488T>G (p.V163G) HNPCCMLH1V49E (c.146T>A) HNPCCMLH1C77R / c.229T>C HNPCCMLH1p.Ala128LysfsX22 (dupl exons 3&4) HNPCCMSH6c G>A HNPCCMSH6c.3514dupA HNPCCMSH6c. 3640G>T / p.E1214X HNPCCPMS2c.736_741del6ins12 HNPCCPMS2c.593_593insT HNPCCPMS2c.903+1G>A HNPCCPMS2Exon 6-8 deletion HNPCCPMS2exon 10 deletion MYH Y165C/G382D MYH 891+3A->C MYH c.536A>G homoz

Hereditary cancer NGS panel A panel of ~20 genes strongly associated with hereditary cancer (CRC/BOC) will be developed using NGS as a test for individuals at moderate- or high-risk of CRC who test negative with the targeted Sequenom panel. BRCA1, BRCA2, CDH1, STK11, PTEN, PALB2, TP53, MLH1, MSH2, MSH6, PMS2, EPCAM, APC, MYH, SMAD4, BMPR1A, CHEK2 and ATM (including a few other genes TBD) Design of the NGS panel is expected to be complete by the end of December 2015 and validation finished by the end of May 2016.

Hereditary cancer exon deletion/duplication assay A custom Agilent microarray will be designed covering all exons for all genes included in the custom hereditary cancer NGS panel. The array design is expected to be completed by the end of January 2016 and the validation finished by the end of August 2016.

CRC tumour genetic testing MSI panel 5 microsatellite markers (BAT-25, BAT-26, NR-21, NR- 24 and MONO-27) First somatic test developed and validated by the Translational Genomics Laboratory MLH1 methylation assay & BRAF V600E assay Oncomine Focus NGS panel Targets relevant hotspots & gene fusions Design includes 52 genes targeted by oncology drugs (e.g., KRAS, BRAF, EGFR) Validation expected to be completed by end of 2016.

Projects – 2017 to 2018 Cardiomyopathy targeted multi-gene NGS panel Hematological oncology multi-gene NGS panel Hereditary deafness multi-gene NGS panel Inherited disease multi-gene NGS panel Clinical whole-exome NGS panel

Acknowledgements