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