Locus-specific databases (LSDBs) which accept submissions of variants and their interpretation from diagnostic and research laboratories are subject to problems of duplicate entries, limited annotation of relevant clinical and experimental data, and variable quality of in silico analyses. In addition, ad hoc or generic criteria for interpretation lack refinement for gene specific interpretation, and do not draw on the specific expertise of clinicians and scientists directly working in the field relevant to the genes in question. As a result, interpretation of the same variant submitted from different sources can be discordant, leaving the database and the clinical field open to uncertainty, detracting from its utility for clinical application. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) has fostered the integration of all major mismatch repair databases into a single LSDB. InSiGHT has developed a coordinated approach to the authoritative interpretation of mismatch repair gene variants, engaging a range of gene specific experts, and drawing on comprehensive clinical and experimental information sourced from the published literature and from its own members. The InSiGHT Variant Interpretation Committee comprised of 40 experts in the mismatch repair field was tasked with variant review and classification on a pro bono basis. InSiGHT developed governance and support through incorporation, to protect the committee from possible legal challenge related to clinical use of its conclusions. The Committee applied a standardized classification scheme to constitutional variants in the Lynch Syndrome genes MLH1, MSH2, MSH6 and PMS2, reviewing all clinical and functional data available for the variants [3]. All 2,360 unique sequence alterations were considered and classified. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinically important misclassifications were identified for unique variants initially submitted as not pathogenic (54 unique variants reclassified as pathogenic and 25 reclassified as likely pathogenic) and unique variants submitted as pathogenic (28 unique variants reclassified as not pathogenic, 16 reclassified as likely not pathogenic and 218 reclassified as uncertain clinical significance). Importantly, the consensus results have been disseminated online through the InSiGHT database and ClinVar. Consistent clinical management based on transparent evaluation is now possible for the 1,370 variants that are not obviously protein truncating (e.g. missense, single amino acid indels) based on their nomenclature. This large-scale endeavour demonstrates the value of multidisciplinary collaboration for curation and classification of variants in public locus-specific databases.

1. Progressive of the InSiGHT mismatch repair gene database
and variant interpretation committee – application of
classification rules and dissemination of results.
Finlay Macrae
Secretary, International Society for Gastrointestinal Hereditary
Tumours
For the InSiGHT Variant Interpretation Committee
Head, Colorectal Medicine and Genetics, The Royal Melbourne Hospital
Professor of Medicine, Depts of Medicine, Melbourne and Monash
Universities, Victoria, Australia
HVP meeting, May 2014
UNESCO Building, Paris

3. What is InSiGHT
• The International Society for Gastrointestinal
Hereditary Tumours formed with the merger
of the Leeds Castle Polyposis Group and
ICG HNPCC in 2005
• Incorporated in 2009
• Maintains a database of MisMatch Repair
and other genes responsible for GI cancer
DNA variants through its website
www.insight-group.org

4. InSiGHT and the
Human Variome Project
Consensus
InSiGHT meeting Yokohama,
March 2007

5. InSiGHT Database:
A lead LSDB for the HVP
• Curator: JohnPaul Plazzer (Colorectal Medicine and
Genetics, RMH, and Human Variome Project)

6. MMR mutation
Identified
(DNA Lab)
IHC &/or MSI data added
(Histopathology)
Interpretation of
genotype
(Clinicians & Lab)
Virtual pathology added
(Histopathology)
Functional data added
(Research Labs)
Clinical phenotype
added
(Clinicians)
Identified dataset stored
locally
(Family Cancer Clinic)
De-identified data stored with InSiGHT
Variant Interpretation Committee
(InSiGHT)
DbGaP (NCBI)
ClinVar
UCSC
Data from other
centres can be
submitted to update
information

7. InSiGHT/HVP database
Achievements
• Merge of MMR (Mike Woods), functional assay (R Sijmons) with InSiGHT databases – 2008
• Full time InSiGHT curator appointed - 2010 in Melb JohnPaul Plazzer
• Data submission..data submission…, Germany, Sweden, Denmark, Australia, US CFR,
China, UK, Quest Diags
• InSiGHT Variant Interpretation Committee : Gene specific criteria for pathogenicity – 2011
• Nature Genetics Publication on Variant Interpretation accepted(B Thompson et al) – 2013
• Strongly endorsing Nature Genetics Editorial
• Microattribution: Nature Genetics accepted (Thompson B et al)
• “Follow a Variant”
• MMR Functional assay calibration grant: US RO1 grant(S Tavtigian,M Greenblatt)
• -2013
• International Mismatch Repair Consortium: NHMRC funded 2013
• Clin Var: NCBI engaged 2013 US

9. The InSiGHT Database
• As of the end of December 2012 there were 12,635
submissions to the InSiGHT database of 2,730
unique MMR gene variants lodged in the InSiGHT
database.
• Now (April 2014) there are 14,304 submissions of
2,888 unique variants
• Briefly, multiple lines of evidence were required for
classification, and evidence for each variant had
to include data associating the variant with both
clinical and functional consequences (Online
Methods).

10. InSiGHT Database Visitor
count
Database ‘hits’ in 2012 per month
Can infer from these numbers that
visitors use the InSiGHT database
multiple times per month

11. Should I request
colonoscopy?
Donna-Marie Ian
25 24
Positive FH
MSI high tumour
MLH1 leu559arg
Missense
Not inherited
Robert Hofstra
Rolf Sijmons
Western:no expression
Pulldown: no interaction
MAPP-MMR and SIFT v2
Suggest pathogenic
Polyphen inconclusive
Reported Belvederesi EJHG
2006

12. August 2010 China National Highway 110
traffic jam, China, world’s worst traffic jam
ever….
more than 100 kilometres from August 14 - 26,
inc. at least 11 days of total gridlock.

13. InSiGHT Variant Interpretation Committee
Modus operandi
• Chair appointed by Council: Maurizio Genuardi (Italy)
• Invitations for core and extended committee memberships
• Qualitative approach to classification discussed, circulated, and agreed
• Initial set of missense variants with 3 lines of evidence classified according
to approach agreed across the committee
• Variant list for discussion is circulated to extended committee – any
additional available data canvassed
• All data is then assembled by curator for Core Committee plus rolling
other membership to classify at international teleconferences
(sponsored)
• One line entry to be included on InSiGHT database describing outcomes
• Later, preliminary approach to all InSiGHT members to seek all available
information of variants under consideration at each meeting
• InSIGHT members encouraged to submit data at each contact, and
signal variants of particular clinical concern for Committee consensus
•

14. InSiGHT Variant Interpretation Committee
• M Genuardi (Italy, Chair)
• JP Plazzer (Curator)
• F Macrae (Sec, InSiGHT)
• A Spurdle (Australia)
• B Thomson (Australia)
• M Woods (Canada)
• R Sijmons (Netherlands)
• P Peltomaaki (Finland)
• M Greenblatt (USA)
• I Frayling (UK)
• J Burn (UK)
• M Dunlop (UK)
• S Farrington (UK)
• B Royer Pokora (Germany)
• E Holinski Feder (Germany)
• G Moeslein (Germany)
• I Blanco (Spain)
• G Capella (Spain)
• D Du Sart (Australia)
• M Kohonen Corish (Australia)
• R Scott (Australia)
• B Talseth (Australia)
• R Ramesar (South Africa)
• M Qi (China)
• R Hofstra (Netherlands)
• M Vihinen (Finland)
• M Nystrom (Finland)
• T Weber (USA)
• S Tavtigian (USA)
• D Golgar (USA)
• C Heinen (USA)
• S Lipkin (USA)
• A Lindblom (Sweden)
• K Akagi (Japan)
• F Al-Mulla (Kuwait)
• I Bernstein (Denmark)
• F Wikman (Denmark)
• T Frebourg (France)
• S. Olschwang (France)
• S Leung (Hong Kong)
• T Liu (Sweden)
• P Moller (Norway)
• B Bapat (Canada)
• L Rasmussen (Denmark)

15. Simplified guidelines describing the levels and types of evidence
required for each tier of the classification
Thompson, B. A., Spurdle, A. B., Plazzer, J. P., Greenblatt, M. S., Akagi, K., Al-Mulla, F., ... & Genuardi, M. (2013).
Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the
InSiGHT locus-specific database. Nature genetics.

16. Microsatellite Instability analysis -
Electropherograms
Electropherograms of the fluoresceinated amplification products
for the loci BAT26 and BAT25 from colorectal cancer tissue.
Hoang et. al. 1997; Zhou et. al. 1998

17. Immunohistochemical detection of Loss of Expression
MLH1
MSH2
MSH6
PMS2

18. Altered Classifications
• Proportion of five-tiered classifications for all documented constitutional variants in the
database against the original LOVD database classifications assigned by submitters
• Class 5b includes variants that are not obviously truncating but are considered to be pathogenic on the
basis of combined evidence
Thompson, B. A., Spurdle, A. B., Plazzer, J. P., Greenblatt, M. S., Akagi, K., Al-Mulla, F., ... & Genuardi, M. (2013).
Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the
InSiGHT locus-specific database. Nature genetics.

19. Final Classifications
• Class 5b includes variants that are not obviously truncating but are
considered to be pathogenic on the basis of combined evidence
• In addition to the 990 assumed pathogenic truncating or large-deletion
variants (class 5a), consistent medical management is now also possible for
the remaining 1,370 not obviously truncating variants; these include 167 class 5
(pathogenic) variants (class 5b) (12%), 183 class 4 (likely pathogenic)
variants (14%), 86 class 2 (likely not pathogenic) variants (6%) and 169 class
1 (not pathogenic) variants (12%)
Thompson, B. A., Spurdle, A. B., Plazzer, J. P., Greenblatt, M. S., Akagi, K., Al-Mulla, F., ... & Genuardi, M. (2013).
Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the
InSiGHT locus-specific database. Nature genetics.

20. • Presentation of evidence used for InSiGHT
Classification
• http://www.insight-group.org/variants/classifications/

21. www.insight-group.org/variants/classifications/
Follow a variant

22. Summary• Genetic Medicine is moving centre stage
• Genetic testing will move into the routine domain
• Failure to apply genetic knowledge kills people
• Ability to intervene will drive diagnostics
• We must focus on variant analysis

23. InSiGHT Variant Interpretation Committee
• M Genuardi (Italy, Chair)
• JP Plazzer (Curator)
• F Macrae (Sec, InSiGHT)
• A Spurdle (Australia)
• B Thomson (Australia)
• M Woods (Canada)
• R Sijmons (Netherlands)
• P Peltomaaki (Finland)
• M Greenblatt (USA)
• I Frayling (UK)
• J Burn (UK)
• M Dunlop (UK)
• S Farrington (UK)
• B Royer Pokora (Germany)
• E Holinski Feder (Germany)
• G Moeslein (Germany)
• I Blanco (Spain)
• G Capella (Spain)
• D Du Sart (Australia)
• M Kohonen Corish (Australia)
• R Scott (Australia)
• B Talseth (Australia)
• R Ramesar (South Africa)
• M Qi (China)
• R Hofstra (Netherlands)
• M Vihinen (Finland)
• M Nystrom (Finland)
• T Weber (USA)
• S Tavtigian (USA)
• D Golgar (USA)
• C Heinen (USA)
• S Lipkin (USA)
• A Lindblom (Sweden)
• K Akagi (Japan)
• F Al-Mulla (Kuwait)
• I Bernstein (Denmark)
• F Wikman (Denmark)
• T Frebourg (France)
• S. Olschwang (France)
• S Leung (Hong Kong)
• T Liu (Sweden)
• P Moller (Norway)
• B Bapat (Canada)
• L Rasmussen (Denmark)

24. International Society for Gastrointestinal Hereditary Tumors
2015
June 18-20th
Prof. Benedito Mauro Rossi
Chairman
6th. InSiGHT Meeting - 2015 - Sao Paulo