Transcript Research Discoveries to Diagnostic Panels

Research discoveries to diagnostic
panels – an update
TPMI Research Day
October 8, 2015
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
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 2015.
Nucleotide Change
c.67-1delG (IVS2)
c.455G>A
c.634C>T
c.1522C>T
c.2564G>A
c.2588G>C
c.3064G>A
c.3322C>T
c.3323G>A
c.4139C>T
c.4163T>C
c.4222T>C
c.4469G>A
c.4537delC
c.4577C>T
c.4918C>T
c.5461-10T>C (IVS38)
c.5714+5G>A (IVS40)
c.6089G>A
c.6449G>A
Amino acid change
# of alleles
Exon
rs#
splice site
p.Arg152Gln
P.Arg212Cys
p.Arg508Cys
p.Trp855X
p.Gly863Ala
p.Glu1022Lys
p.Arg1108Cys
p.Arg1108His
p.Pro1308Leu
p.Leu1388Pro
p.Trp1408Arg
p.Cys1490Tyr
p.Gln1513Argfs*13
p.Thr1526Met
p.Arg1640Trp
splice site?
splice site
p.Arg2030Gln
p.Cys2150Tyr
3
3/11
1
2
5
2/4
1
3
1
4
8
1
2
1
3
1
12
31
1
1
2
5
6
11
16
17
21
22
22
28
28
28
30
30
31
35
38
40
44
47
rs67-1delG
rs82646862
rs61750200
rs138157885
rs6175406
rs76157638
rs61749459
rs61750120
rs61750121
rs61750130
rs61750131
rs61750135
rs61751402
rs4537delC
rs61750152
rs61751404
rs1800728
rs61751407
rs61750641
rs61751384
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
2016.
FAP
APC
IVS3-1G>A (Twillingate mut'n)
FAP
APC
694C>T (R236X) exon 6 ?694 or 649
FAP
APC
3067-3068insA
FAP
APC
exon 12 1622-1623insCA
FAP
APC
(1309)delAAAGA
FAP
APC
(1061)delACAAA
HNPCC
MSH2
Fam C
HNPCC
MSH2
Exon 8 deletion
HNPCC
MSH2
Exons 4-16 deletion
HNPCC
MSH2
c.1661G>C
HNPCC
MSH2
c.488T>G (p.V163G)
HNPCC
MLH1
V49E (c.146T>A)
HNPCC
MLH1
C77R / c.229T>C
HNPCC
MLH1
p.Ala128LysfsX22 (dupl exons 3&4)
HNPCC
MSH6
c.3557-1G>A
HNPCC
MSH6
c.3514dupA
HNPCC
MSH6
c. 3640G>T / p.E1214X
HNPCC
PMS2
c.736_741del6ins12
HNPCC
PMS2
c.593_593insT
HNPCC
PMS2
c.903+1G>A
HNPCC
PMS2
Exon 6-8 deletion
HNPCC
PMS2
exon 10 deletion
MYH
MYH
MYH
MYH
Y165C/G382D
891+3A->C
MYH
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, NR24 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