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The International Society for Gastrointestinal Hereditary Tumors (InSIGHT) has established a committee (Variant Interpretation Committee; VIC) for the interpretation of sequence variants in the mismatch repair (MMR) genes associated with Lynch syndrome (LS). One of the major steps involved in this process has been the establishment of qualitative specific classification rules for the MMR genes, with the aim to improve the clinical utility of MMR gene testing. The 5-class variant classification system proposed by the International Agency for Research on Cancer (IARC) was used to this purpose, since it links all classes to specific clinical recommendations. Multiple lines of evidence were required for class assignment and in order to classify a variant as pathogenic or likely pathogenic, (Classes 5 and 4, respectively), or as not pathogenic or likely not pathogenic (Classes 1 and 2, respectively), concordant evidence derived from both clinical and functional datasets had to be available. Variants with discordant information or with lack of either clinical or functional information, were considered of uncertain significance (Class 3). The following specific points of evidence were considered: 1. Type of sequence variation; 2) functional protein assays; 3) mRNA assays; 4) phenotype associated in compound heterozygotes for the variant under scrutiny and a clearly pathogenic variant in the same gene; 5) presence of the variant on different haplotypes across LS families; 6) co-segregation data and clinical phenotype; 7) tumor molecular characteristics; 8) population frequency; 9) risk estimated from case-control studies. Since interpretation of functional assays proved to be difficult and variable across committee members, specific supporting information and flowcharts were developed. In addition, whenever available, quantitative multifactorial analysis2 was used and the outcome compared to that of qualitative assessment. The classification scheme was modified by consensus to accommodate new data and inconsistencies over multiple classification teleconferences and face-to-face meetings. Overall, the rules were successfully applied to classify 2,360 variants lodged onto the InSiGHT database. These criteria provide a baseline for standardized clinical classification of MMR gene sequence variation that may be linked to patient and family management in the genetic counseling arena according to published guidelines.
Citation preview
Development of rules for the interpretation of mismatch repair
gene variants based on the 5-tiered IARC classification system
On behalf of the InSiGHT Variant Interpretation Committee
5° Biennial Human Variome Project MeetingParis, 22-05-2014
INTERNATIONAL SOCIETYINTERNATIONAL SOCIETYFOR GASTROINTESTINALFOR GASTROINTESTINALHEREDITARY TUMOURSHEREDITARY TUMOURS
DEPT. MEDICAL GENETICSDEPT. MEDICAL GENETICSCATHOLIC UNIVERSITYCATHOLIC UNIVERSITY
ROMEROME
Human Mutation (2008) 29:1282–1291.
InSiGHT Mutation Database:data cleaning, nomenclature standardization and systematic data review
12,645 MMR gene submissions
10 Source unknown / non-
existant
3,468 nomenclature alterations
(12 not resolvable)
Duplicate entries resolved
230 Somatic7 EPCAM
132 Synthetic
2,360 unique constitutional MMR
gene variants
(MSH2, MLH1, MSH6, PMS2)
Criteria development process
• Used modified Delphi approach– http://en.wikipedia.org/wiki/Delphi_method
• Evolution of criteria – Started with the 117 most commonly cited variants with
discordant interpretation– Iterative amendments & clarifications over 12 meetings
• Quantitative (multifactorial) or qualitative evidence considered
• Work towards fully quantitative (Bayesian), recognising limits– Calibration of in silico algorithms
Current Multifactorial Likelihood Model
Quantitative classification
MMR
Sequence
Variant+ =
Thompson et al Hum Mutat, 2013. 34(1): p.200-09.
Require ≥2 data points to promote robust classification
QUALITATIVE RULES:POINTS OF EVIDENCE
① Co-segregation
② Tumor molecular characteristics: MSI, IHC (BRAF)
③ Population frequency
④ Risk estimated from case-control studies
⑤ Presence of the variant on different haplotypes across LS families
⑥ Type of sequence variation
⑦ Functional protein assays
⑧ mRNA assays
⑨ Co-occurrence of the variant with a clearly pathogenic variant in the same gene and CMMRD phenotype
Qualitative points of evidence
5’…TCT CAA AAA TTT ACG…3’ S Q K F T
5’…TCT CAA TAA TTT ACG…3’ S Q *
Sequence-based
Segregation data Tumour data Frequency data Co-occurrence in trans
In vitro data
Presence/absence of haematological
malignancies, childhood cancers – CMMR-D phenotype
Clinical/molecular Functional
• Major issue in classification process was conflicting data from functional assays
• Functional assay subcommittee formed to tackle the issue
• Flowchart developed to assist assay interpretation
Functional assay interpretation
Class 5 Pathogenic:
Class 4 Likely pathogenic:
Class 3 Uncertain:
Class 2 Likely not pathogenic:
4 points of evidence:
Abrogated function or CMMRD or
different background haplotypes
Co-segregation with disease (~LR
10:1)≥2 tumors with LS
molecular phenotype
Absence in 1000 genomes
PP >0.99
or
Nonsense/frameshift
Full inactivation of variant allele by
splicing aberration
Large deletion
Large duplication confirmed to
encode a frameshift leading to NMD
or
or
or
or
2 points of evidence:
PP 0.95-0.99
Canonical splice site, untested for
splicing
Co-segregation with disease
(~LR 5:1)
Or
≥2 tumors with LS molecular phenotype
Abrogated function or CMMRD or
different background haplotypes
or
or
Synonymous or intronic variant with no mRNA
aberration
AF ≥1% in specific ethnic group
2 points of evidence if proficient function,
otherwise 3 points of evidence required:
Proficient function or co-occurrence with no CMMRD
AF 0.01-1%
No co-segregation with disease (~LR 0.01:1)
≥3 MSS CRC or inconsistent IHC
tumors
Odds Ratio with upper 95% CI <5 in
case-control studies
PP 0.001-0.049
or
or
or
Class 1 Not pathogenic:
3 points of evidence if proficient function,
otherwise 4 points of evidence required:
AF ≥1% in control reference groups
Proficient function or co-occurrence with no CMMRD
AF 0.01-1%
No co-segregation with disease
(~LR 0.01:1)
≥3 MSS CRC or inconsistent IHC
tumors
Odds Ratio with upper 95% CI <4 in
case-control studies
PP <0.001
or
or
Insufficient evidence to
classify
PP 0.05-0.949
or
Nature Genetics 46, 107–115 (2014)
Rationale underlying Class 5 criteria
Class 5: evidence from tumor molecular pathology
Criterion
• > 2 tumors with MSI-H and/or appropriate IHC loss
Rationale
• Provides evidence that the variant is associated with the clinical phenotype. Assumed conservative LR > 5:1 for tumor data
Transparent presentation of summary data for classifications
www.insight-group.org/classifications
Future Perspectives
• Rule revision (ie, de novo mutations)
• Revise classifications
• Gene-specific rules
• Intermediate penetrance variants
• Further genes (APC, MUTYH,…)
InSiGHT Variant Interpretation Committee (VIC)
Bryony A. Thompson
Amanda B. Spurdle
Marc Greenblatt
John-Paul Plazzer
Kiwamu Akagi
Fahd Al-Mulla
Bharati Bapat
Inge Bernstein
Gabriel Capella
Johan T den Dunnen
Desiree du Sart
Mark Farrell
Susan Farrington
Ian Frayling
Established Yokohama, 2007
Ming Qi
Rajkumar Ramesar
Brigitte Royer-Pokora
Rodney Scott
Rolf Sijmons
Carli Tops
Thomas Weber
Juul Wijnen
Michael Woods
Lene Rasmussen
David Goldgar
Sean Tavtigian
Finlay Macrae
Maurizio Genuardi
Thierry Frebourg
Chris Heinen
Elke Holinski-Feder
Maija Kohonen-Corish
Suet Yi Leung
Annika Lindblom
Kristina Lagerstedt
Alexandra Martins
Pal Moller
Monika Morak
Minna Nystrom
Aurelie Fabre
Paivi Peltomaki
Marta Pineda