PMS2 c.1268C>G, p.Ala423Gly

Reasoning

The variant is a PMS2 missense substitution, and the governing VCEP table directly assigns the applicable HCI-prior path. The exact HCI-PRIORS-PMS2 row for c.1268C>G / p.A423G has a MAPP/PP2 Prior P score of 0.088, which is below the VCEP BP4 cutoff of 0.11; therefore BP4_Supporting is met. The separate SpliceAI alternative is for intronic and synonymous variants and is not needed for this missense path.

VCEP rule

Missense variant with “MAPP/PP2 Prior P” score <0.11 from [http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique](http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique)

Evidence considered

• HCI-PRIORS-PMS2.txt, matching row PMS2_02772: c.1268C>G, p.A423G, MAPP/PP2 Prior P score 0.088; this exact pre-assigned table value meets the VCEP BP4 missense threshold.
• Case normalization identifies NM_000535.7:c.1268C>G as NP_000526.2:p.(Ala423Gly), a missense SNV.

Sources

Reasoning

The PMS2 VCEP PM2 rule requires an absent or extremely rare allele frequency below 0.00002 in the gnomAD v4 dataset. The gnomAD v4.1 total allele frequency for NM_000535.7:c.1268C>G (p.Ala423Gly/p.A423G) is 0.0000396522 (64/1,614,032 alleles), which exceeds 0.00002; therefore PM2 is not met. The specified non-cancer subsets also do not provide a value below the rule threshold: gnomAD v2.1 non-cancer exomes show 0.0000211356 (5/236,568), and gnomAD v3.1 non-cancer genomes show 0.0000878901 (13/147,912). The VCEP pilot-variants file was searched under c.1268C>G, p.Ala423Gly, and p.A423G, with no matching entry found.

VCEP rule

Absent/extremely rare allele frequency \<0.00002 (\<1 in 50,000 alleles ) in gnomAD v4 dataset

Evidence considered

• gnomAD v4.1: total AF 0.0000396522 (64/1,614,032 alleles), with 0 homozygotes; this exceeds the VCEP PM2 cutoff of 0.00002.
• gnomAD v2.1 non-cancer exomes: AF 0.0000211356 (5/236,568 alleles), with 0 homozygotes.
• gnomAD v3.1 non-cancer genomes: AF 0.0000878901 (13/147,912 alleles), with 0 homozygotes.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1268C>G, p.Ala423Gly, and p.A423G; no matching row was found.

Still missing

• No matching c.1268C>G, p.Ala423Gly, or p.A423G entry was found in VCEP-pilot-variants---MMR.xlsx; the VCEP pilot file therefore supplies no pre-assigned PM2 code for this variant.

Sources

Reasoning

The variant is a PMS2 missense substitution, so the governing missense HCI-prior path applies. The exact HCI-PRIORS-PMS2 row for c.1268C>G / p.A423G assigns a MAPP/PP2 Prior P score of 0.088, which is below both PP3 thresholds (>0.81 for Moderate and >0.68 to <=0.81 for Supporting). The alternative PP3 path is limited to predicted splice defects at non-canonical splicing nucleotides and does not apply to this missense substitution; its SpliceAI max delta of 0.024 is therefore not used to support PP3.

VCEP rule

Missense variant with “MAPP/PP2 Prior P” score >0.81 from [http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique](http://hci-lovd.hci.utah.edu/variants.php?select_db=PMS2_priors&action=search_unique)

Evidence considered

• HCI-PRIORS-PMS2.txt, matching row PMS2_02772: c.1268C>G, p.A423G, MAPP/PP2 Prior P score 0.088; the same row also lists MAPP score 3.16 and prior probability 0.002.
• Case normalization identifies NM_000535.7:c.1268C>G as NP_000526.2:p.(Ala423Gly), a missense SNV.
• SpliceAI result for the exact variant: DS_AG 0.024, DS_AL 0.004, DS_DG 0.001, DS_DL 0.009; max delta score 0.024. This is not used for PP3 because the VCEP splice alternative is restricted to non-canonical splicing nucleotides.

Sources

Reasoning

The PMS2 VCEP BA1 rule requires a gnomAD v4 Grpmax filtering allele frequency of at least 0.0028 (0.28%) and exclusion as a founder pathogenic variant. The gnomAD v4.1 Grpmax FAF for NM_000535.7:c.1268C>G (p.Ala423Gly/p.A423G) is 0.00004101 (0.004101%), far below 0.0028; therefore BA1 is not met. The VCEP pilot-variants file was searched under c.1268C>G, p.Ala423Gly, and p.A423G, with no matching entry found.

VCEP rule

GnomAD v4 Grpmax filtering allele frequency ≥ 0.0028 (0.28%) and variant is excluded as founder pathogenic variant.

Evidence considered

• gnomAD v4.1: Grpmax filtering allele frequency 0.00004101 (0.004101%), with 64/1,614,032 total alleles and 0 homozygotes.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1268C>G, p.Ala423Gly, and p.A423G; no matching row was found.

Still missing

• No matching c.1268C>G, p.Ala423Gly, or p.A423G entry was found in VCEP-pilot-variants---MMR.xlsx; the VCEP pilot file therefore supplies no pre-assigned BA1 code for this variant.

Sources

Reasoning

The PMS2 VCEP BS1 rule requires a gnomAD v4 Grpmax filtering allele frequency of at least 0.00028 and below 0.0028, with the variant excluded as a founder pathogenic variant. The gnomAD v4.1 Grpmax FAF is 0.00004101, which is below 0.00028, so the frequency does not meet the BS1 interval. The VCEP pilot-variants file was searched under c.1268C>G, p.Ala423Gly, and p.A423G, with no matching entry found.

VCEP rule

GnomAD v4 Grpmax filtering allele frequency ≥ 0.00028 and \< 0.0028 (0.028-0.28%) and variant is excluded as founder pathogenic variant.

Evidence considered

• gnomAD v4.1: Grpmax filtering allele frequency 0.00004101 (0.004101%), below the BS1 interval lower bound of 0.00028.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1268C>G, p.Ala423Gly, and p.A423G; no matching row was found.

Still missing

• No matching c.1268C>G, p.Ala423Gly, or p.A423G entry was found in VCEP-pilot-variants---MMR.xlsx; the VCEP pilot file therefore supplies no pre-assigned BS1 code for this variant.

Sources

Reasoning

The VCEP PS1 rule requires a different nucleotide change encoding the same amino-acid change, previously established by this VCEP as Pathogenic, or the specified non-canonical splice scenario. The case variant is PMS2 c.1268C>G, p.Ala423Gly (p.A423G). The governing case materials and the required PMS2 full-text search did not identify a different-nucleotide p.Ala423Gly variant established by this VCEP as Pathogenic, and this missense variant is not a non-canonical splice-nucleotide case. PS1 therefore cannot be evaluated as met from the available VCEP evidence.

VCEP rule

A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect).

Evidence considered

• Variant normalization: NM_000535.7:c.1268C>G; NP_000526.2:p.(Ala423Gly), p.(A423G).
• The required PMS2 full-text search found the exact variant notation in HCI-PRIORS-PMS2.txt.txt, but that table is indexed by the VCEP for PP3/BP4 and does not establish a Pathogenic same-amino-acid comparator for PS1.
• No different-nucleotide p.Ala423Gly comparator established by this VCEP was present in the case's PM5 candidate results.

Still missing

• No different-nucleotide p.Ala423Gly comparator established by this VCEP was identified in the available case materials.
• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in MMR-Functional-domains.pdf.txt; that file is a PM1 domain schematic, not a PS1 classification table.

Sources

Reasoning

The VCEP assigns PS2 strength from de novo points, but the available case evidence does not document a proband, confirmed parental testing, maternity/paternity, or any de novo point assignment for NM_000535.7:c.1268C>G. The criterion therefore cannot be evaluated under the VCEP rule.

Evidence considered

• The VCEP PS2 specification provides strength thresholds based on de novo points, but the case materials contain no proband-parent testing result or de novo point assignment for this variant.

Still missing

• Proband phenotype and confirmed maternity/paternity are not documented.
• Parental testing results and de novo status are not documented.
• No VCEP de novo point assignment is available.

Sources

Reasoning

The variant is a missense substitution, so the VCEP functional-assay framework is applicable. The required VCEP assay documentation and flowchart were consulted, but neither Functional-assay-SVI-documentation-MMR.xlsx nor Functional-assay-flowchart.pdf contains a variant-specific entry for PMS2 c.1268C>G, p.Ala423Gly, or p.A423G. The case evidence bundle and available paper extracts also provide no variant-specific functional readout, calibrated functional odds, or concordant assay results for this variant. Therefore, the VCEP thresholds for calibrated odds or the flowchart's deficient-function paths cannot be evaluated, and PS3 is not assessed.

VCEP rule

Calibrated functional assays with functional odds for Pathogenicity > 18.7 ... Calibrated functional assays with functional odds for pathogenicity >4.3 and <= 18.7 ... OR MMR function defect following functional assay flowchart* ... OR Variants with monoallelic expression: complete loss of expression (<10% of wild-type in cDNA without puromycin) of the variant allele. Full-length transcript should be analysed with and without NMD block. ... Calibrated functional odds for pathogenicity >2.08 and <= 4.3

Evidence considered

• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays': lists approved calibrated MMR assays and their control/readout calibration, including PMS2 abnormal readout <18% for the first assay column, but contains no row for c.1268C>G, p.Ala423Gly, or p.A423G.
• Functional-assay-flowchart.pdf: for an in-frame indel or missense variant without predicted splicing impact, directs evaluation through a calibrated functional assay or two independent concordant assays; no variant-specific result is reported for this variant.
• The case evidence bundle and paper_extracts.json contain no variant-specific functional assay result for PMS2 c.1268C>G/p.A423G.

Still missing

• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in Functional-assay-SVI-documentation-MMR.xlsx.txt.
• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in Functional-assay-flowchart.pdf.txt.
• A variant-specific calibrated functional assay result or qualifying concordant functional assay results are needed to evaluate PS3 strength.

Sources

Reasoning

The VCEP assigns PM3 by point totals for evidence that the variant is present in trans with a pathogenic allele in affected individuals, but the available case evidence contains no affected-proband observation, second pathogenic PMS2 allele, phase determination, or PM3 point assignment for NM_000535.7:c.1268C>G (p.Ala423Gly). Therefore PM3 cannot be evaluated.

Evidence considered

• The case evidence identifies NM_000535.7:c.1268C>G (NP_000526.2:p.(Ala423Gly)) but provides no proband-level allelic observations, second pathogenic PMS2 variant, phase information, or PM3 points.
• The reviewed variant-specific literature extracts do not report this variant or qualifying trans-phase observations.

Still missing

• Affected-proband data with a second pathogenic or likely pathogenic PMS2 allele
• Documented trans phase, preferably confirmed by parental or offspring testing
• VCEP PM3 point assignment for each qualifying observation

Reasoning

The VCEP PM5 rule requires a different missense change at the same amino-acid residue that this VCEP classified as Pathogenic or Likely Pathogenic on the protein level and not because of aberrant splicing; the rule also requires PP3 supporting for the assessed missense variant. The available case PM5 search found no same-residue comparator candidates with an applicable VCEP Pathogenic or Likely Pathogenic classification, and no such governing-table entry was identified in the required full-text search. The available evidence is therefore insufficient to assess PM5.

VCEP rule

Missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing. Only use PM5 if PP3 is supporting for the missense change. Use PM5\_Supporting if other variant is Likely Pathogenic due to a missense alteration.

Evidence considered

• The case PM5 candidate result reports zero same-residue candidates, zero expert-panel Pathogenic/Likely Pathogenic candidates, and zero non-expert-majority Pathogenic/Likely Pathogenic candidates.
• The required PMS2 full-text search found the target and other p.A423 substitutions in HCI-PRIORS-PMS2.txt.txt, but that table supplies HCI prior values and not VCEP classifications required by PM5.
• No qualifying same-residue comparator was identified in the available evidence.

Still missing

• A qualifying different missense variant at PMS2 residue 423, classified by this VCEP as Pathogenic or Likely Pathogenic on the protein level, was not identified.
• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in MMR-Functional-domains.pdf.txt; that file is a PM1 domain schematic, not a PM5 comparator table.

Sources

Reasoning

The VCEP requires co-segregation evidence with a combined Bayes Likelihood Ratio and assigns strength according to the specified thresholds. The available evidence documents no informative pedigree, affected or unaffected tested relatives, meioses, or combined Bayes Likelihood Ratio for this variant.

Evidence considered

• The VCEP PP1 specification requires a combined Bayes Likelihood Ratio, with Supporting >2.08 and ≤4.3, Moderate >4.3 and ≤18.7, and Strong >18.7; no such value or underlying pedigree data is present.

Still missing

• No pedigree with phenotype and genotype data is documented.
• No informative affected or unaffected relatives or meioses are documented.
• No combined Bayes Likelihood Ratio is available.

Sources

Reasoning

No case-specific CRC or endometrial MSI-H tumor, tumor-genome, or PMS2-consistent loss-of-expression evidence is available in the supplied evidence. The exact variant was not found in Table-for-CMMRD-diagnosis.pdf or VCEP-pilot-variants---MMR.xlsx; those files therefore provide no pre-assigned PP4 code for NM_000535.7:c.1268C>G.

VCEP rule

1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers^e or tumor genome ​**and/or**​ loss of MMR protein expression consistent with the variant location. MSI-H tumor with inconsistent protein expression does not meet PP4.

Evidence considered

• The PP4 rule requires one or more CRC/endometrial MSI-H tumors or tumor-genome evidence and/or loss of MMR protein expression consistent with the variant location; the case bundle contains no patient-specific result establishing these findings.
• The two governing reference files named for PP4 were searched under c.1268C>G, p.Ala423Gly, and p.A423Gly; neither contained this variant.

Still missing

• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PMS2/fulltext/Table-for-CMMRD-diagnosis.pdf.txt.
• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PMS2/fulltext/VCEP-pilot-variants---MMR.xlsx.txt.
• Case-specific MSI status, tumor-genome results, and PMS2 immunohistochemistry results are unavailable.

Sources

Reasoning

The PMS2 VCEP BS2 rule requires documented co-occurrence in trans with a known pathogenic sequence variant in PMS2 in a patient meeting the specified cancer-age and CMMRD-exclusion conditions, with phase confirmed through parental or offspring testing. The available evidence documents population observations but no qualifying patient-level co-occurrence, phase confirmation, cancer age, or CMMRD assessment; BS2 therefore cannot be evaluated. The VCEP pilot-variants file was searched under c.1268C>G, p.Ala423Gly, and p.A423G, with no matching entry found.

VCEP rule

Co-occurrence _​in trans​_ with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS^d​), and who has no previous or current evidence of clinical manifestations of CMMRD as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring.

Evidence considered

• No qualifying patient-level co-occurrence, phase-confirmation, cancer-age, or CMMRD-exclusion record is present in the reviewed case evidence.
• VCEP-pilot-variants---MMR.xlsx was searched for c.1268C>G, p.Ala423Gly, and p.A423G; no matching row was found.

Still missing

• Patient-level evidence of co-occurrence in trans with a known pathogenic PMS2 variant is unavailable.
• Parental or offspring testing confirming phase is unavailable.
• The required cancer-age and absence-of-CMMRD conditions cannot be verified.
• No matching c.1268C>G, p.Ala423Gly, or p.A423G entry was found in VCEP-pilot-variants---MMR.xlsx.

Sources

Reasoning

The variant is a missense substitution, so the VCEP functional-assay framework is applicable. The required VCEP assay documentation and flowchart were consulted, but neither Functional-assay-SVI-documentation-MMR.xlsx nor Functional-assay-flowchart.pdf contains a variant-specific entry for PMS2 c.1268C>G, p.Ala423Gly, or p.A423G. The case evidence bundle and available paper extracts also provide no variant-specific proficient-function result, calibrated functional odds, or qualifying mRNA/protein assay result. Therefore, the VCEP thresholds for benign functional evidence cannot be evaluated, and BS3 is not assessed.

VCEP rule

Calibrated functional assays with functional odds for Pathogenicity ≤ 0.05 ... OR Synonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition. Whenever abnormal transcripts are identified at similar levels in controls they will be considered naturally occurring isoforms and not mRNA aberrations. ... Calibrated functional assays with functional odds for Pathogenicity >0.05 & ≤0.48 ... OR Variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart.

Evidence considered

• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays': lists approved calibrated assays, validation controls, and normal/abnormal readout thresholds, but contains no row for c.1268C>G, p.Ala423Gly, or p.A423G.
• Functional-assay-flowchart.pdf: defines proficient function for the relevant missense path only after the specified assay sequence and concordant results; no variant-specific result is reported for this variant.
• The case evidence bundle and paper_extracts.json contain no variant-specific proficient-function protein or mRNA assay result for PMS2 c.1268C>G/p.A423G.

Still missing

• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in Functional-assay-SVI-documentation-MMR.xlsx.txt.
• No entry for c.1268C>G, p.Ala423Gly, or p.A423G was found in Functional-assay-flowchart.pdf.txt.
• A variant-specific calibrated functional result or variant-specific proficient-function protein and mRNA assay result is needed to evaluate BS3 strength.

Sources

Reasoning

The VCEP requires lack of co-segregation with a combined Bayes Likelihood Ratio to assign BS4. The available evidence documents no informative non-segregating relatives, pedigree-level phenotype/genotype data, or combined Bayes Likelihood Ratio for this variant.

Evidence considered

• The VCEP BS4 specification requires a combined Bayes Likelihood Ratio <0.05 for Strong or >0.05 and ≤0.48 for Supporting; no such value or underlying non-segregation data is present.

Still missing

• No pedigree with phenotype and genotype data is documented.
• No informative non-segregating relatives or meioses are documented.
• No combined Bayes Likelihood Ratio is available.

Sources

Reasoning

The VCEP BP5 rule requires either 2 or 3 qualifying tumors for supporting evidence, at least 4 qualifying tumors for strong evidence, or a specified BRAF V600E/MLH1-methylation finding. No case-specific MSS status, MMR-protein expression, inconsistent LS-spectrum loss, BRAF V600E result, or MLH1 methylation result is available, so the criterion cannot be evaluated.

VCEP rule

2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors^d with loss of MMR protein(s) that is inconsistent with the gene demonstrating genetic variation. OR 1 BRAF V600E (Colon only)/_MLH1_ methylation (in LS spectrum tumor only) with MSI-H/_MLH1_ loss.

Evidence considered

• The VCEP BP5 supporting rule requires 2 or 3 qualifying tumors or 1 BRAF V600E/MLH1-methylation result; the supplied case evidence does not document either pathway.

Still missing

• Case-specific tumor MSS/MSI status and MMR-protein expression are unavailable.
• Case-specific BRAF V600E and MLH1 methylation results are unavailable.

Sources