MSH3 c.131C>G, p.(Ala44Gly)

Reasoning

No MSH3-specific VCEP or gene framework was retrieved, so the supplied generic PM2 supporting threshold of allele frequency <=0.0001 was applied. Using the default all-comers population sources, gnomAD v4.1 reports total AF 7.488925751045641e-06, highest subgroup AF 1.021347872787718e-05, and zero homozygotes; gnomAD v2.1 reports total AF 8.907892392659896e-06, highest subgroup AF 1.9632094548167345e-05, and zero homozygotes. All observed frequencies are below 0.0001, so PM2 is met at supporting strength.

Rule

Generic PM2 supporting threshold: allele frequency <=0.0001.

Evidence considered

• gnomAD v4.1 all-comers data report 12/1,602,366 alleles, total AF 7.488925751045641e-06, highest subgroup AF 1.021347872787718e-05, and zero homozygotes.
• gnomAD v2.1 all-comers exome data report 2/224,520 alleles, total AF 8.907892392659896e-06, highest subgroup AF 1.9632094548167345e-05, and zero homozygotes.
• Because no governing VCEP specifies a non-cancer or exome-only source, the default all-comers gnomAD v2.1 and v4.1 entries were used; the available non-cancer subsets do not alter the conclusion.
• Generic PM2 supporting threshold: allele frequency <=0.0001, from the supplied published calibration data based on ClinGen SVI guidance and Richards et al. 2015 (PMID:25741868).

Sources

Reasoning

The variant is a missense substitution, NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)), so BP4 is evaluated through the missense REVEL path only. The REVEL score is 0.138, which is at or below the generic ClinGen SVI moderate BP4 threshold of <=0.183 from the Pejaver et al. 2022 calibration (PMID:36413997); therefore BP4 is met at moderate strength. SpliceAI was not substituted because a splice prediction does not assess missense protein tolerance.

Rule

For a missense variant, apply BP4 at the strongest applicable calibrated strength; REVEL <=0.183 meets the supplied generic moderate BP4 threshold (ClinGen SVI calibration, Pejaver et al. 2022, PMID:36413997).

Evidence considered

• The variant is a missense substitution, NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)), so REVEL is the applicable BP4 path.
• REVEL score is 0.138, meeting the published generic ClinGen SVI moderate BP4 threshold of <=0.183 from Pejaver et al. 2022 (PMID:36413997).
• No governing MSH3 VCEP or gene-specific computational specification was available, so the supplied generic REVEL calibration was applied.
• The available SpliceAI max delta of 0.002 was not used because this missense assessment is restricted to REVEL.

Sources

Reasoning

The variant is a missense substitution, NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)), so PP3 is evaluated through the missense REVEL path only. The REVEL score is 0.138, below the generic ClinGen SVI supporting PP3 threshold of >=0.644 from the Pejaver et al. 2022 calibration (PMID:36413997); therefore PP3 is not met. SpliceAI and BayesDel were not used because they are not the applicable calibrated path for this missense criterion.

Rule

For a missense variant, apply PP3 only when the REVEL score reaches a published calibrated threshold; the supplied generic supporting threshold is >=0.644 (ClinGen SVI calibration, Pejaver et al. 2022, PMID:36413997).

Evidence considered

• The variant is a missense substitution, NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)), so REVEL is the applicable PP3 path.
• REVEL score is 0.138, below the published generic ClinGen SVI supporting PP3 threshold of >=0.644 from Pejaver et al. 2022 (PMID:36413997).
• No governing MSH3 VCEP or gene-specific computational specification was available, so the supplied generic REVEL calibration was applied.
• SpliceAI and BayesDel were not used for PP3; a missense variant must not use splice prediction, and BayesDel has no generic ACMG strength calibration.

Sources

Reasoning

PP5 is restricted here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. The exact-match ClinVar record for NM_002439.5:c.131C>G has zero expert-panel submissions; the available submissions are ordinary single-submitter laboratory assertions, including two Uncertain significance submissions and one Likely benign submission. None is an expert-panel Pathogenic/Likely pathogenic classification. The ClinVar record therefore does not trigger PP5.

Rule

PP5 applies only when an exact-variant ClinVar expert panel classifies the variant as Pathogenic or Likely pathogenic; ordinary laboratory and aggregate labels are excluded.

Evidence considered

• Exact-variant ClinVar variation 655141 has 0 expert-panel submissions.
• The available exact-variant submissions are single-submitter laboratory assertions: two Uncertain significance and one Likely benign; none is an expert-panel Pathogenic or Likely pathogenic classification.

Sources

Reasoning

No MSH3-specific VCEP or gene framework was retrieved, so the generic ACMG/AMP stand-alone BA1 threshold of allele frequency >=0.05 was applied. The variant's highest reported all-comers frequency is 1.021347872787718e-05 in gnomAD v4.1 European non-Finnish individuals, with total AF 7.488925751045641e-06; gnomAD v2.1 total AF is 8.907892392659896e-06. These frequencies are far below 0.05, so BA1 is not met.

Rule

Generic ACMG/AMP BA1 stand-alone threshold: allele frequency >=0.05.

Evidence considered

• gnomAD v4.1 all-comers data report 12/1,602,366 alleles, total AF 7.488925751045641e-06, and the highest observed subgroup AF of 1.021347872787718e-05 in European non-Finnish individuals, with zero homozygotes.
• gnomAD v2.1 all-comers exome data report 2/224,520 alleles, total AF 8.907892392659896e-06, and zero homozygotes.
• Generic ACMG/AMP BA1 threshold: allele frequency >=0.05, from the supplied published calibration data based on Richards et al. 2015 (PMID:25741868).

Sources

Reasoning

No MSH3-specific VCEP or gene framework was retrieved, so the supplied generic BS1 strong benign threshold of allele frequency >=0.01 was applied. The highest reported all-comers frequency is 1.021347872787718e-05 in gnomAD v4.1 European non-Finnish individuals; the total gnomAD v4.1 AF is 7.488925751045641e-06 and the gnomAD v2.1 total AF is 8.907892392659896e-06. The observed frequencies are below 0.01, so BS1 is not met.

Rule

Generic BS1 strong benign threshold: allele frequency >=0.01.

Evidence considered

• gnomAD v4.1 all-comers data report a total AF of 7.488925751045641e-06 and a highest observed subgroup AF of 1.021347872787718e-05 in European non-Finnish individuals, with zero homozygotes.
• gnomAD v2.1 all-comers exome data report a total AF of 8.907892392659896e-06 and zero homozygotes.
• The supplied generic ACMG/ClinGen-SVI calibration specifies BS1 at allele frequency >=0.01; no MSH3-specific threshold was available.

Sources

Reasoning

No MSH3-specific VCEP or gene framework was retrieved. The population datasets show zero homozygotes for this variant in gnomAD v2.1 and gnomAD v4.1, including the largest observed European non-Finnish subgroup counts, so the required observation of the variant in healthy individuals in a genotype configuration inconsistent with disease is absent. BS2 is therefore not met.

Rule

BS2 requires observation of the variant in healthy individuals in a genotype configuration inconsistent with the established disease mechanism.

Evidence considered

• gnomAD v2.1 reports 0 homozygotes among 224,520 alleles overall and 0 in the highest-frequency European non-Finnish subgroup.
• gnomAD v4.1 reports 0 homozygotes among 1,602,366 alleles overall and 0 in the highest-frequency European non-Finnish subgroup.
• No independent healthy-individual cohort or genotype observation meeting BS2 requirements was identified in the available evidence.

Sources

Reasoning

BP6 is restricted here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. The exact-match ClinVar record for NM_002439.5:c.131C>G has zero expert-panel submissions. Although one ordinary laboratory submission is Likely benign, it is a single-submitter clinical laboratory assertion and is explicitly excluded from BP6 under the governing instruction. The record therefore does not trigger BP6.

Rule

BP6 applies only when an exact-variant ClinVar expert panel classifies the variant as Benign or Likely benign; ordinary laboratory and aggregate labels are excluded.

Evidence considered

• Exact-variant ClinVar variation 655141 has 0 expert-panel submissions.
• One ordinary single-submitter laboratory assertion is Likely benign, but ordinary laboratory submissions and non-expert assertions are excluded from BP6.

Sources

Reasoning

PS1 requires a previously established pathogenic variant producing the same amino-acid change. The target is MSH3 NM_002439.5:c.131C>G, NP_002430.3:p.(Ala44Gly), but no validated pathogenic comparator producing p.Ala44Gly was identified. The retrieved ClinVar record is internally inconsistent because its source-data variant title identifies DHFR rather than MSH3, so it cannot establish PS1.

Evidence considered

• The target variant is MSH3 NM_002439.5:c.131C>G, NP_002430.3:p.(Ala44Gly).
• ClinVar variation 655141 has conflicting submissions, with two uncertain-significance submissions and one likely-benign submission, no expert-panel submission, and a source-data variant title for DHFR rather than MSH3.
• The reviewed literature extracts do not mention MSH3 c.131C>G or p.Ala44Gly.

Still missing

• A validated pathogenic comparator producing MSH3 p.Ala44Gly is unavailable.

Sources

Reasoning

PS2 cannot be assessed because the available case evidence documents no affected proband-parent trio, parental genotypes, confirmed absence of the variant in both biological parents, or validated de novo observation. The variant is a missense change in MSH3, but variant consequence and population observations do not establish de novo status. Additional evidence required is testing of both biological parents with reliable assay methods, together with confirmation of maternity/paternity and phenotype compatibility.

Rule

Generic ACMG/AMP PS2 requires a confirmed de novo variant in an affected individual with confirmed parentage; no strength can be assigned without parental testing and appropriate phenotype assessment.

Evidence considered

• The case evidence contains no proband-parent testing results, parental variant status, meiosis information, or confirmed de novo observation.
• The variant is annotated as MSH3 NM_002439.5:c.131C>G, p.(Ala44Gly), but the missense consequence does not itself establish PS2.

Still missing

• Parental genotypes are not documented.
• Maternity and paternity confirmation are not documented.
• A validated de novo call in the proband is not documented.
• Phenotype compatibility for a de novo MSH3 finding is not documented.

Reasoning

No variant-specific functional assay was identified for MSH3 NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)). The available sources provide no validated biochemical, cellular, protein-function, mismatch-repair, or other disease-relevant assay with appropriate controls that could support PS3; therefore PS3 cannot be assessed rather than inferred from computational predictions, population data, somatic occurrence, or clinical assertions.

Evidence considered

• The case evidence contains no variant-specific functional assay result for NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)).
• PMID:25394175 does not mention MSH3 or this variant and provides no functional evidence.
• PMID:25741868 does not mention this variant or provide variant-specific functional data; its discussion of missense interpretation is general guidance, not an assay result.
• PMID:28492532 does not mention MSH3 or this variant and provides no variant-specific functional evidence.

Still missing

• A validated, disease-relevant functional assay directly comparing MSH3 p.(Ala44Gly) with wild-type and appropriate positive and negative controls is unavailable.
• No quantitative assay result or published calibration supports PS3 strength for this variant.

Sources

Reasoning

No applicable MSH3 VCEP or gene-specific framework was available, so the generic ACMG/AMP PS4 concept was used. PS4 requires evidence that the variant is observed significantly more often in affected individuals than in appropriate controls. The available records provide no case-control counts, odds ratio, enrichment statistic, or statistically supported affected-case series for exact variant NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)). The three reviewed publications do not mention this exact variant and do not provide variant-specific clinical prevalence data. Therefore PS4 cannot be assessed from the available evidence.

Rule

Generic PS4: significantly increased prevalence of the exact variant in affected individuals compared with appropriate controls.

Evidence considered

• No MSH3-specific VCEP framework was available; generic ACMG/AMP 2015 was therefore the fallback framework.
• The reviewed exact-variant literature packet contained no paper reporting NM_002439.5:c.131C>G or NP_002430.3:p.(Ala44Gly) in a clinical prevalence or case-control analysis.

Still missing

• Exact-variant affected-case and appropriate-control counts or a validated enrichment statistic are unavailable.

Sources

Reasoning

PM1 requires a mutational hotspot or critical, well-established functional domain without benign variation. No applicable MSH3 VCEP specification was retrieved, vcep_materials.json contains no MSH3 domain-table entry, and the Cancer Hotspots result is negative for residue A44. Therefore, no complete authoritative domain or hotspot basis is available to establish or exclude PM1.

Evidence considered

• No applicable MSH3 VCEP or CSPEC was retrieved.
• The authoritative domain_tables object is empty for MSH3, so no approved critical-domain list is available for residue 44.
• The Cancer Hotspots result reports no statistically significant hotspot for MSH3 A44.

Still missing

• A governing MSH3 critical-domain or hotspot specification is unavailable.
• Independent evidence establishing residue 44 as a critical functional-domain or mutational-hotspot position is unavailable.

Reasoning

No applicable MSH3 VCEP or gene-specific framework was available, so the generic ACMG/AMP rule was considered. PM3 requires the variant to be observed in trans with a pathogenic or likely pathogenic allele in an affected individual, with phase and disease-relevant inheritance established. The available case evidence contains no affected-proband observation, second allele, phase result, segregation data, or documented biallelic MSH3 genotype for NM_002439.5:c.131C>G (NP_002430.3:p.Ala44Gly). Therefore PM3 cannot be assessed.

Rule

Generic ACMG/AMP PM3: evidence of the variant in trans with a pathogenic or likely pathogenic variant in an affected individual, with phase and relevant inheritance established.

Evidence considered

• The case materials identify NM_002439.5:c.131C>G as MSH3 p.Ala44Gly but provide no affected-proband genotype, second MSH3 allele, phase, segregation, or inheritance observation.
• No MSH3-specific VCEP or CSPEC specification was retrieved; generic ACMG/AMP PM3 was therefore the applicable fallback rule.

Still missing

• Affected-proband or affected-family observation involving NM_002439.5:c.131C>G
• A second pathogenic or likely pathogenic MSH3 variant
• Validated trans phase and inheritance information
• Evidence that the observed genotype is relevant to the disease mechanism

Reasoning

PM5 requires a pathogenic missense variant at the same residue. The target is MSH3 p.Ala44Gly, but the comparator collection contains zero same-residue candidates and explicitly does not establish classic PM5 eligibility. The retrieved ClinVar record is internally inconsistent and does not provide a validated pathogenic comparator at residue 44.

Evidence considered

• The target variant is MSH3 p.Ala44Gly at residue 44.
• The collected comparator record contains zero same-residue candidates and does not establish classic PM5 eligibility.
• ClinVar variation 655141 provides conflicting classifications, no expert-panel pathogenic comparator, and source-data identity for DHFR rather than MSH3.

Still missing

• A validated pathogenic missense comparator at MSH3 residue 44 is unavailable.

Sources

Reasoning

PM6 cannot be assessed because the available case evidence provides no credible report of a presumed de novo variant without parental testing. No clinical narrative, family history, parent phenotype, maternity/paternity information, or observation of the variant in the proband is supplied that would support a presumed de novo event. Population presence of the variant is not evidence for or against PM6.

Rule

Generic ACMG/AMP PM6 may be considered for a presumed de novo occurrence when parental testing is unavailable, but requires credible clinical and family evidence; none is available here.

Evidence considered

• No case report, family history, parent phenotype, or presumed de novo observation is documented in the available evidence.
• The available literature extracts do not mention MSH3 NM_002439.5:c.131C>G or provide variant-specific clinical or segregation observations.

Still missing

• A clinical report describing presumed de novo occurrence is not documented.
• Parental testing or a reason it was unavailable is not documented.
• Family history and parent phenotypes are not documented.
• Confirmation that the variant was observed in the affected proband is not documented in a clinical narrative.

Reasoning

PP1 cannot be assessed because no affected relatives, unaffected relatives, pedigrees, familial variant testing, or informative meioses are documented. There is therefore no count of variant-positive affected relatives or segregation events from which supporting, moderate, or strong PP1 could be assigned. The absence of family data is not evidence of non-segregation.

Rule

Generic ACMG/AMP PP1 requires co-segregation of the variant with disease in affected family members; strength depends on the number and informativeness of segregation observations.

Evidence considered

• The case evidence contains no pedigree, affected-relative observations, familial genotypes, segregation results, or informative meioses.
• No variant-specific publication in the extracted literature provides segregation evidence for MSH3 NM_002439.5:c.131C>G.

Still missing

• No pedigree or family structure is documented.
• No affected-relative phenotypes are documented.
• No familial genotypes are documented.
• No informative meioses or segregation-event count is documented.

Reasoning

Generic PP2 requires a gene in which missense variation is a common disease mechanism and benign missense variation is uncommon. The available gene-level context supports MSH3 loss of function, but does not establish that missense variation is a common or predominant disease mechanism, nor provide a validated MSH3 missense-enrichment analysis.

Evidence considered

• The ACMG/AMP framework states that missense interpretation depends on conservation, protein location and context, and biochemical consequence, but does not establish MSH3-specific PP2 eligibility.
• The available gene context supports MSH3 loss of function, not a predominantly missense disease mechanism.
• No validated MSH3-specific missense-enrichment threshold or analysis was provided.

Still missing

• A validated MSH3 missense-enrichment or missense-mechanism analysis is unavailable.
• The proportion of pathogenic MSH3 variants that are missense versus truncating is not established here.

Sources

Reasoning

No applicable MSH3 VCEP or gene-specific phenotype specification was available. PP4 requires a highly specific phenotype or family history compatible with a disease for which the gene is known to cause disease. The case materials identify MSH3 and the missense variant but provide no patient phenotype, disease diagnosis, family history, tumor phenotype, or other clinical presentation that can be evaluated for specificity. General gene-level statements and the absence of exact-variant discussion in the reviewed publications do not establish PP4. Therefore PP4 cannot be assessed.

Rule

Generic PP4: patient phenotype or family history is highly specific for a disease associated with the gene.

Evidence considered

• No MSH3-specific VCEP phenotype definition was available.
• The available case materials contain no individual-level phenotype or family-history description for this variant.

Still missing

• A documented patient phenotype or family history and an applicable MSH3 disease-specific phenotype definition are unavailable.

Reasoning

No variant-specific functional assay was identified for MSH3 NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)). The available sources provide no validated benign-function result from a biologically relevant assay with appropriate controls, so BS3 cannot be assessed; absence of functional evidence is not evidence of a normal effect.

Evidence considered

• The case evidence contains no variant-specific functional assay result demonstrating preserved MSH3 function for NM_002439.5:c.131C>G (NP_002430.3:p.(Ala44Gly)).
• PMID:25394175 does not mention MSH3 or this variant and provides no benign functional evidence.
• PMID:25741868 does not mention this variant or provide variant-specific functional data; its discussion of missense interpretation is general guidance, not a benign assay result.
• PMID:28492532 does not mention MSH3 or this variant and provides no variant-specific functional evidence.

Still missing

• A validated, disease-relevant functional assay directly demonstrating preserved MSH3 function for p.(Ala44Gly), with appropriate controls, is unavailable.
• No quantitative assay result or published calibration supports BS3 strength for this variant.

Sources

Reasoning

BS4 cannot be assessed because no unaffected relatives have documented clinical phenotypes and validated negative familial variant testing. No informative non-segregation observation, age-related risk assessment, or family structure is available. The absence of reported segregation data cannot be treated as evidence that the variant fails to segregate with disease.

Rule

Generic ACMG/AMP BS4 requires lack of segregation in affected families, supported by informative unaffected relatives who have been appropriately assessed and tested; no such evidence is available.

Evidence considered

• The case evidence contains no unaffected-relative phenotypes, familial negative tests, or non-segregation observations.
• No variant-specific publication in the extracted literature provides evidence that the MSH3 variant is present in informative unaffected relatives.

Still missing

• No unaffected relatives with documented phenotypes are identified.
• No negative familial variant-testing results are documented.
• No age-related risk or phenotype ascertainment information is documented.
• No informative non-segregation event is documented.

Reasoning

Generic BP1 requires a missense variant in a gene for which disease is known to be caused primarily by truncating variants and missense variants are not an established mechanism. Although MSH3 loss of function is supported, the available evidence does not establish a primarily truncating disease spectrum or exclude pathogenic missense variation.

Evidence considered

• The ACMG/AMP framework was consulted for missense-variant interpretation principles.
• The available gene context supports loss of function in MSH3 but does not quantify the disease-causing variant spectrum or demonstrate that missense variants are not a disease mechanism.
• No MSH3-specific VCEP rule defining BP1 eligibility was retrieved.

Still missing

• A validated MSH3 variant-spectrum analysis demonstrating predominantly truncating disease variants is unavailable.
• Evidence excluding a pathogenic missense mechanism for MSH3 is unavailable.

Sources

Reasoning

No applicable MSH3 VCEP or gene-specific framework was available, so the generic ACMG/AMP rule was considered. BP2 requires documented allelic configuration, such as the variant occurring in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant, depending on the applicable disease model. The available evidence contains no individual-level genotype, pathogenic comparator allele, phase result, family study, or inheritance information for NM_002439.5:c.131C>G (NP_002430.3:p.Ala44Gly). Population presence and the ClinVar submissions do not establish cis or trans configuration. Therefore BP2 cannot be assessed.

Rule

Generic ACMG/AMP BP2: documented cis/trans allelic configuration with a pathogenic variant in the disease model specified by the applicable framework; phase must be established rather than inferred from population or aggregate database data.

Evidence considered

• The case materials provide population and ClinVar records but no individual-level cis/trans phase, pathogenic comparator allele, segregation, or inheritance data.
• No MSH3-specific VCEP or CSPEC specification was retrieved; generic ACMG/AMP BP2 was therefore the applicable fallback rule.

Still missing

• Individual-level observation containing the target variant and a pathogenic comparator allele
• Validated cis or trans phase
• Relevant inheritance and disease-model information

Reasoning

No MSH3-specific framework was available, so the generic BP5 concept was considered. BP5 requires that a variant be found in a patient with an alternative molecular basis for the disease, such that the variant is unlikely to explain the phenotype. The available materials contain no documented patient phenotype, no confirmed alternative pathogenic molecular diagnosis, and no case-level evidence linking this exact variant to a patient with an alternative molecular basis. The single COSMIC observation is somatic database presence only and does not establish this BP5 scenario. Therefore BP5 cannot be assessed.

Rule

Generic BP5: variant found in a patient with an alternative molecular basis for the disease.

Evidence considered

• ClinVar provides laboratory assertions but no case-level alternative molecular diagnosis for this variant.
• The available COSMIC record reports one somatic occurrence but does not document the clinical BP5 scenario of an affected patient with an alternative molecular basis.

Still missing

• A documented affected case carrying the exact variant with an independent, confirmed molecular basis for the disease is unavailable.

Sources