Variant classification summary

NM_000251.2:c.366+1G>A

MSH2 · NP_000242.1:p.? · NM_000251.2

GRCh37: chr2:47635695 G>A · GRCh38: chr2:47408556 G>A

Gene: MSH2 Transcript: NM_000251.2

Final call

VUS

PVS1 very strong PM2 supporting

Variant details

Gene

MSH2

Transcript

NM_000251.2

Protein

NP_000242.1:p.?

gnomAD AF

ClinVar

Pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000251.2:c.366+1G>A is a canonical +1 splice donor variant in MSH2 exon 2. Exon 2 skipping is predicted to disrupt the reading frame (155 bp, not divisible by 3) and trigger nonsense-mediated decay, qualifying for PVS1 at very_strong strength under InSiGHT MSH2 VCEP v2.0.

2

The variant is absent from all queried population databases (gnomAD v2.1, v4.1, and gnomAD-Canada v1.0), meeting PM2_Supporting under the VCEP threshold of <0.00002 allele frequency.

3

The same genomic position with a different nucleotide change (g.47408556G>C, equivalent to c.366+1G>C) was classified as Pathogenic by the InSiGHT VCEP pilot with PVS1 and PM2_Supporting, providing strong precedent for the pathogenicity of splice disruption at this position.

4

The variant has been reported in ClinVar as Pathogenic by 6 clinical laboratories and Likely Pathogenic by 2 laboratories (ClinVar ID 245947). It has been observed in COSMIC in 3 somatic cancer specimens (COSV51878772).

5

No literature evidence was found mentioning the exact variant (c.366+1G>A). Thirteen publications were triaged and reviewed; none contained variant-specific clinical, functional, segregation, or de novo data.

6

Applying InSiGHT MSH2 VCEP v2.0 classification rules: PVS1 (very_strong) + PM2_Supporting (supporting) meets Rule 4 (1 Very Strong + ≥2 Supporting = Pathogenic) or Rule 1 (1 Very Strong = Pathogenic). The overall classification is Pathogenic.

Final determination: No criteria-combination rule matched the adjudicated criteria in the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MSH2 Version 2.0 v2.0 framework, so the variant remains a Variant of Uncertain Significance pending human review.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	Met	NM_000251.2:c.366+1G>A is a canonical splice donor variant (IVS+1) in MSH2 exon 2. Exon 2 spans 155 nucleotides (c.212 to c.366), which is not divisible by 3; skipping or cryptic splice activation is predicted to disrupt the reading frame, introduce a premature termination codon, and trigger nonsense-mediated decay. Under InSiGHT MSH2 VCEP v2.0, IVS±1 variants where exon skipping disrupts the reading frame and is predicted to undergo NMD qualify for PVS1 at very_strong strength. The same genomic position with a G>C substitution (g.47408556G>C) was classified by the VCEP pilot as PVS1, confirming precedent.	cspec vcep_vcep_pilot_variants_mmr vcep_pvs1_decisiontree_mmr pvs1_gene_context pvs1_variant_assessment
PS1	N/A	InSiGHT MSH2 VCEP v2.0 PS1 applies to (a) predicted missense substitutions encoding the same amino acid change as a previously established P/LP variant, or (b) variants affecting the same non-canonical splice nucleotide as a confirmed P/LP splice variant. NM_000251.2:c.366+1G>A is a canonical (+1) splice variant, not a missense variant and not at a non-canonical splice nucleotide. PS1 is not applicable.	cspec
PS2	Not met	No de novo observations were identified in the reviewed literature. No parental testing or de novo reports are available for NM_000251.2:c.366+1G>A.
PS3	Not met	No calibrated functional assay data or variant-specific functional studies were identified for NM_000251.2:c.366+1G>A in the reviewed literature. The InSiGHT MMR VCEP functional assay SVI documentation does not contain data for this specific variant. While the variant is at a canonical splice donor and is predicted null by mechanism, PS3 requires experimental functional evidence (calibrated assays with functional odds for pathogenicity), which is not available.
PS4	N/A	InSiGHT MSH2 VCEP v2.0 states PS4 is not utilized due to availability of tumor IHC data (PP4) for variant classification. VCEP explicitly marks PS4 as Not Applicable.	cspec
PS5	N/A	PS5 is not included in the InSiGHT MSH2 VCEP v2.0 criteria ruleset. The same-residue different amino acid change concept is covered by PM5 in this framework, and this variant is a splice variant, not a missense substitution.	cspec
PM1	N/A	InSiGHT MSH2 VCEP v2.0 explicitly states PM1 is not applicable. The VCEP rationale: 'There are no recognized mutational hot spots that could be used for classification purposes. While there are functional domains in the MMR genes, the distribution of pathogenic variants is generalized over all the domains.'	cspec
PM2	Met	NM_000251.2:c.366+1G>A is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. Under InSiGHT MSH2 VCEP v2.0, PM2_Supporting is met when the variant is absent or has an allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4. The VCEP pilot variants spreadsheet also applied PM2_supporting to the same-position variant g.47408556G>C.	gnomad_v2 gnomad_v4 gnomad_canada cspec vcep_vcep_pilot_variants_mmr
PM5	N/A	InSiGHT MSH2 VCEP v2.0 PM5 applies to missense changes at an amino acid residue where a different missense change was classified as P/LP. NM_000251.2:c.366+1G>A is a canonical splice site variant, not a missense substitution, and does not map to an amino acid residue in a way that permits PM5 application.	cspec pm5_candidates
PM6	N/A	InSiGHT MSH2 VCEP v2.0 explicitly marks PM6 as Not Applicable.	cspec
PP1	Not met	No co-segregation data is available for NM_000251.2:c.366+1G>A. None of the reviewed papers report pedigree-level segregation analysis for this specific variant.
PP2	N/A	InSiGHT MSH2 VCEP v2.0 explicitly marks PP2 as Not Applicable. The VCEP rationale: 'Missense variant in a gene with low rate of benign missense changes does not apply.'	cspec
PP3	N/A	InSiGHT MSH2 VCEP v2.0 PP3 for splice prediction applies only to non-canonical splicing nucleotides with SpliceAI delta score >=0.2. NM_000251.2:c.366+1G>A is at a canonical (+1) splice donor position. The VCEP PVS1 rule explicitly states: 'Not to be combined with PP3.' The splice effect is captured by PVS1. HCI prior is not applicable (variant not in missense HCI table).	cspec spliceai
PP4	Not met	No patient-specific tumor phenotype data (MSI status or IHC for MMR proteins) is available for carriers of NM_000251.2:c.366+1G>A in the case materials. The variant has been observed in COSMIC (3 somatic occurrences) but this does not constitute patient constitutional tumor phenotype evidence meeting PP4 thresholds under the VCEP.
PP5	N/A	InSiGHT MSH2 VCEP v2.0 explicitly marks PP5 as Not Applicable. 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.'	cspec
BA1	Not met	NM_000251.2:c.366+1G>A is absent from all queried gnomAD datasets. Under InSiGHT MSH2 VCEP v2.0, BA1 requires a gnomAD v4 Grpmax filtering allele frequency >=0.001 (0.1%). The variant does not meet this threshold.	gnomad_v2 gnomad_v4 gnomad_canada cspec
BS1	Not met	NM_000251.2:c.366+1G>A is absent from gnomAD v4. Under InSiGHT MSH2 VCEP v2.0, BS1 requires a gnomAD v4 Grpmax filtering allele frequency >=0.0001 and <0.001. The variant is absent and does not meet this threshold.	gnomad_v4 cspec
BS2	Not met	No evidence of co-occurrence in trans with a known pathogenic MSH2 variant was identified in the reviewed literature. No healthy adult carriers were identified to satisfy BS2.
BS3	Not met	No functional studies demonstrating a benign effect were identified for NM_000251.2:c.366+1G>A. The InSiGHT MMR VCEP calibrated functional assays do not contain data for this variant. The VCEP BS3_Strong rule for intronic variants showing no mRNA aberration is also not met — no mRNA splicing assay data (with NMD inhibition) is available.
BS4	Not met	No segregation data demonstrating lack of co-segregation with disease was identified for NM_000251.2:c.366+1G>A.
BP1	N/A	InSiGHT MSH2 VCEP v2.0 explicitly marks BP1 as Not Applicable. 'Missense variant in a gene where only loss of function causes disease is not applicable.'	cspec
BP2	N/A	InSiGHT MSH2 VCEP v2.0 explicitly marks BP2 as Not Applicable. The VCEP uses BS2 instead for co-occurrence evidence.	cspec
BP4	N/A	NM_000251.2:c.366+1G>A is a canonical (+1) splice donor variant. Although SpliceAI returns a max delta score of 0.00 (which is <=0.1), SpliceAI is not designed to accurately assess canonical ±1,2 splice site disruption. Applying BP4 would directly contradict the VCEP PVS1 assignment. The VCEP PVS1 rule captures the splice defect; BP4 is not applied when PVS1 is met for the same mechanism.	spliceai cspec
BP5	Not met	No tumor phenotype data (MSS tumors, retained MMR protein expression, or BRAF V600E/MLH1 methylation) is available to suggest a benign interpretation for NM_000251.2:c.366+1G>A.
BP6	N/A	InSiGHT MSH2 VCEP v2.0 explicitly marks BP6 as Not Applicable. 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.'	cspec
BP7	N/A	InSiGHT MSH2 VCEP v2.0 BP7 applies to synonymous (silent) or intronic variants at or beyond -21/+7 that are predicted to have no splicing impact. Although c.366+1 is numerically within this range, it is the canonical +1 splice donor — the most critical nucleotide for 5' splice site recognition. BP7 is intended for deep intronic or exonic synonymous variants that are predicted benign, not for variants disrupting the consensus splice donor sequence. PVS1 captures the splicing impact of this variant.	cspec

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