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.

1. 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 Meeting
Paris, 22-05-2014
INTERNATIONAL SOCIETYINTERNATIONAL SOCIETY
FOR GASTROINTESTINALFOR GASTROINTESTINAL
HEREDITARY TUMOURSHEREDITARY TUMOURS
DEPT. MEDICAL GENETICSDEPT. MEDICAL GENETICS
CATHOLIC UNIVERSITYCATHOLIC UNIVERSITY
ROMEROME

4. Human Mutation (2008) 29:1282–1291.

5. 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 Somatic
7 EPCAM
132 Synthetic
2,360 unique
constitutional MMR
gene variants
(MSH2, MLH1, MSH6, PMS2)

6. 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

7. 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

8. QUALITATIVE RULES:
POINTS OF EVIDENCE
a. Co-segregation
b. Tumor molecular characteristics: MSI, IHC (BRAF)
c. Population frequency
d. Risk estimated from case-control studies
e. Presence of the variant on different haplotypes across LS
families
f. Type of sequence variation
g. Functional protein assays
h. mRNA assays
i. Co-occurrence of the variant with a clearly pathogenic
variant in the same gene and CMMRD phenotype

9. 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

10. • 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

11. 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/frameshi
ft
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

12. Nature Genetics 46, 107–115 (2014)
Rationale underlying Class 5
criteria

13. 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

14. Transparent presentation of summary
data for classifications
www.insight-group.org/classifications

15. Future Perspectives
• Rule revision (ie, de novo mutations)
• Revise classifications
• Gene-specific rules
• Intermediate penetrance variants
• Further genes (APC, MUTYH,…)

16. 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