PMS2 c.353+7C>A, p.?

Reasoning

The governing PMS2 VCEP PM2 rule requires an absent or extremely rare allele frequency below 0.00002 in gnomAD v4. The variant is present in gnomAD v4.1 at AF 6.74003e-06 (10/1,483,674 alleles), with grpmax FAF 4.7e-06 and zero homozygotes; therefore PM2 Supporting is met. The gnomAD v2.1 non-cancer exome subset also reports AF 4.48523e-06 (1/222,954 exome alleles), but the VCEP rule specifically names gnomAD v4, so the v4 result governs. No governing-table entry for NM_000535.7:c.353+7C>A or its p.? notation was found in VCEP-pilot-variants---MMR.xlsx.txt.

VCEP rule

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

Evidence considered

• gnomAD v4.1: variant 7-6003683-G-T has AF 6.74003e-06 (10/1,483,674 alleles), grpmax FAF 4.7e-06, and zero homozygotes.
• gnomAD v2.1 non-cancer exomes: AF 4.48523e-06 (1/222,954 exome alleles), zero homozygotes; this is concordant but not the VCEP-named dataset for the rule.
• The governing VCEP pilot table was searched for c.353+7C>A and p.?; no matching row was found.

Sources

Reasoning

The variant is intronic, making the VCEP's intronic/synonymous BP4 path applicable. SpliceAI reports a maximum delta score of 0.014, which is <= 0.1; therefore the VCEP no-splicing-impact threshold is satisfied and BP4_Supporting is met. The missense HCI prior path is not applicable, and the HCI-PRIORS-PMS2 table had no exact c.353+7C>A or p.? entry.

VCEP rule

For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023.

Evidence considered

• SpliceAI result for NM_000535.7:c.353+7C>A: maximum delta score 0.014, indicating no predicted splicing impact under the VCEP cutoff.
• The variant is intronic at c.353+7, which is within the VCEP's intronic BP4 scope.
• HCI-PRIORS-PMS2.txt.txt was searched for the exact notations c.353+7C>A and p.?; no matching row was found, and the indexed HCI path is missense-only.

Still missing

• No exact entry for c.353+7C>A or p.? was found in HCI-PRIORS-PMS2.txt.txt; the VCEP intronic SpliceAI path was evaluable independently.

Sources

Reasoning

The variant is intronic at c.353+7, exactly the 3-prime intronic boundary included by the VCEP phrase at or beyond -21/+7 (5-prime/3-prime exonic). It therefore meets BP7_Supporting by location. The VCEP expressly allows BP7 and BP4 to be satisfied together; this does not double-count BP7 as a second splice prediction.

VCEP rule

A synonymous (silent) or intronic variant at or beyond -21/+7 (5′/3′ exonic). Variants may satisfy both BP7 and BP4.

Evidence considered

• Variant normalization identifies NM_000535.7:c.353+7C>A as an intronic variant in intron 4i.
• The VCEP BP7 rule explicitly includes intronic variants at or beyond -21/+7 and states that BP7 and BP4 may both be satisfied.

Sources

Reasoning

The variant is intronic at the non-canonical +7 position, so the applicable PP3 path is the VCEP's SpliceAI splice-defect path; the governing threshold is delta score >= 0.2. SpliceAI reports a maximum delta score of 0.014 (DS_DL 0.014; DS_AG 0.003; DS_AL 0.001; DS_DG 0.000), below the threshold, so PP3 is not met. The HCI-PRIORS-PMS2 table was searched for c.353+7C>A and p.? with no exact entry; its indexed scope is missense, and no missense prior is applicable to this intronic variant.

VCEP rule

Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023.

Evidence considered

• SpliceAI result for NM_000535.7:c.353+7C>A: maximum delta score 0.014, with DS_AG 0.003, DS_AL 0.001, DS_DG 0.000, and DS_DL 0.014.
• The variant is intronic at c.353+7 and has predicted protein consequence NP_000526.2:p.?, so the missense HCI prior path does not apply.
• HCI-PRIORS-PMS2.txt.txt was searched for the exact notations c.353+7C>A and p.?; no matching row was found.

Still missing

• No exact entry for c.353+7C>A or p.? was found in HCI-PRIORS-PMS2.txt.txt; that table is indexed for missense substitutions, not this intronic variant.

Sources

Reasoning

The governing PMS2 VCEP BA1 threshold is a gnomAD v4 Grpmax filtering allele frequency of at least 0.0028, with exclusion as a founder pathogenic variant. The variant's gnomAD v4.1 grpmax FAF is 4.7e-06 (0.00047%), based on 10/1,483,674 total alleles and zero homozygotes, which is below 0.0028. No governing-table entry for NM_000535.7:c.353+7C>A or its p.? notation was found in VCEP-pilot-variants---MMR.xlsx.txt.

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: variant 7-6003683-G-T has total AF 6.74003e-06 (10/1,483,674 alleles), zero homozygotes, and grpmax FAF 4.7e-06.
• The governing VCEP pilot table was searched for c.353+7C>A and p.?; no matching row was found.

Sources

Reasoning

The governing PMS2 VCEP BS1 threshold is a gnomAD v4 Grpmax filtering allele frequency of at least 0.00028 and less than 0.0028, with exclusion as a founder pathogenic variant. The variant's gnomAD v4.1 grpmax FAF is 4.7e-06 (0.00047%), below 0.00028. No governing-table entry for NM_000535.7:c.353+7C>A or its p.? notation was found in VCEP-pilot-variants---MMR.xlsx.txt.

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: variant 7-6003683-G-T has grpmax FAF 4.7e-06, total AF 6.74003e-06, 10/1,483,674 alleles, and zero homozygotes.
• The governing VCEP pilot table was searched for c.353+7C>A and p.?; no matching row was found.

Sources

Reasoning

The variant is intronic, NM_000535.7:c.353+7C>A, with protein consequence p.?; therefore the same-amino-acid route does not apply. The VCEP also permits PS1 for a variant affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant, or a Likely Pathogenic splice variant for the Moderate route, with similar or worse SpliceAI prediction. SpliceAI predicts a maximum delta score of 0.014 for this variant, but the available evidence does not identify a qualifying VCEP-established pathogenic or likely pathogenic comparator at the same splice nucleotide or provide that comparator's SpliceAI score. The criterion cannot be evaluated from the available 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). OR Variants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. OR Variants affecting the same non-canonical splice nucleotide as a Likely Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI.

Evidence considered

• Variant normalization: NM_000535.7:c.353+7C>A; NP_000526.2:p.? (case evidence).
• SpliceAI result: maximum delta score 0.014 (DS_DL 0.014; DS_AG 0.003; DS_AL 0.001; DS_DG 0.000).
• The governing VCEP rule requires a different nucleotide encoding the same amino-acid change, or a comparator at the same non-canonical splice nucleotide classified by the VCEP as Pathogenic/Likely Pathogenic with similar or worse SpliceAI prediction; no qualifying comparator was available in the case evidence.

Still missing

• No VCEP-established Pathogenic or Likely Pathogenic comparator variant at the same non-canonical splice nucleotide, with comparator SpliceAI evidence, is available for evaluation.

Sources

Reasoning

The VCEP PS2 rule assigns de novo points according to confirmed maternity and paternity, LS-spectrum tumor evidence, and tumor MSI/IHC/methylation status. The available case evidence contains no proband-level de novo observation, parental testing, maternity or paternity confirmation, LS-spectrum tumor result, or de novo point assignment, so PS2 cannot be evaluated.

Evidence considered

• The VCEP PS2 specification provides 2 points per proband for a de novo variant with both maternity and paternity confirmed and an MMR-deficient LS-spectrum tumor, 1 point per proband when tumor data are unavailable, and 0.5 points per proband when maternity and/or paternity are unconfirmed with no tumor data.
• No proband, parental-testing, maternity, paternity, LS-spectrum tumor, MSI/IHC/methylation, or de novo-point evidence is present in the reviewed case materials.

Still missing

• Documented proband de novo status with maternity and paternity confirmation or the applicable unconfirmed-parent scenario.
• LS-spectrum tumor diagnosis and MSI/IHC/methylation results, if available.
• VCEP PS2 de novo point assignment.

Sources

Reasoning

The VCEP permits PS3 from calibrated functional assays or an MMR function defect established through the functional-assay flowchart. The case materials contain no variant-specific functional assay result, functional odds, patient-derived RNA result, or other demonstrated MMR defect for PMS2 c.353+7C>A (NP_000526.2:p.?). The on-record Functional-assay-SVI-documentation-MMR.xlsx was searched for c.353+7C>A and p.? with no matching variant entry; its table documents validated assay designs and calibration controls, but does not supply a result for this variant. The on-record Functional-assay-flowchart.pdf was also searched for c.353+7C>A and p.? with no matching variant entry. Therefore PS3 cannot be evaluated from the available variant-specific evidence.

VCEP rule

Calibrated functional assays with functional odds for Pathogenicity > 18.7 OR 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.

Evidence considered

• Case normalization: NM_000535.7:c.353+7C>A; NP_000526.2:p.?; intronic variant.
• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays': validated assay classes, control requirements, calibration thresholds, and proposed PS3 strengths are listed, but no row identifies c.353+7C>A or p.?.
• Functional-assay-flowchart.pdf: splice-site, silent, or intronic variants require a splicing-assay path; no variant-specific result is provided for c.353+7C>A or p.?.

Still missing

• No variant-specific functional assay result or calibrated functional odds were found in the case evidence.
• No patient-derived RNA or other assay result demonstrating an MMR function defect was found.
• No entry for c.353+7C>A or p.? was found in Functional-assay-SVI-documentation-MMR.xlsx.
• No entry for c.353+7C>A or p.? was found in Functional-assay-flowchart.pdf.

Sources

Reasoning

The VCEP PM3 rule requires co-occurrence of this PMS2 variant with a known pathogenic or likely pathogenic variant in the same gene in a patient with clinical features consistent with CMMRD, with 1.0 point for a confirmed trans configuration or 0.5 points when phase is unknown. The available case evidence contains no affected-proband observation establishing CMMRD features, no pathogenic/likely pathogenic PMS2 partner variant, and no phase result; therefore PM3 cannot be evaluated.

VCEP rule

Co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD as per Aronson et al 2022 - Refer to “Table for CMMRD diagnosis.pdf”. ... Pathogenic/Likely Pathogenic _in trans_: 1.0 point; ... Pathogenic/Likely Pathogenic - phase unknown: 0.5 points. ... Sum all cases with the above evidence to determine the PM3 strength.

Evidence considered

• No case evidence documents a pathogenic or likely pathogenic PMS2 partner variant, an affected proband meeting CMMRD-consistent clinical features, or phase between the variants.
• The VCEP PM3 instructions assign 1.0 point for a pathogenic/likely pathogenic variant in trans and 0.5 points when phase is unknown; no qualifying case was identified for scoring.

Still missing

• Documented co-occurrence with a known pathogenic or likely pathogenic PMS2 variant in an affected proband with CMMRD-consistent clinical features.
• Parental or offspring testing establishing trans phase, or an explicit phase-unknown record if applicable.

Sources

Reasoning

The VCEP PP1 rule requires co-segregation in pedigrees with a combined Bayes likelihood ratio, with Supporting >2.08 and <=4.3, Moderate >4.3 and <=18.7, or Strong >18.7 in at least two families. No pedigree, affected or unaffected relative genotypes, meioses, family count, or combined Bayes likelihood ratio is provided, so PP1 cannot be evaluated.

Evidence considered

• The VCEP PP1 thresholds are Supporting for a combined Bayes likelihood ratio >2.08 and <=4.3, Moderate for >4.3 and <=18.7, and Strong for >18.7 in at least two families.
• No family pedigree, segregation observations, informative meioses, family count, or combined Bayes likelihood ratio is present in the reviewed case materials.

Still missing

• Pedigree identifying affected and unaffected relatives and their variant genotypes.
• Number of informative meioses and families.
• Combined Bayes likelihood ratio, preferably calculated using the VCEP-recommended COOL approach.

Sources

Reasoning

PP4 is applicable, but the available case evidence does not provide the required patient-level tumor MSI/IHC findings or the number of independent qualifying tumors and families. The governing VCEP 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; no such patient phenotype evidence is available here. The required Table-for-CMMRD-diagnosis.pdf was searched for c.353+7C>A and p.? with no variant entry, and VCEP-pilot-variants---MMR.xlsx was searched for the same notations with no entry for this variant.

Evidence considered

• The PP4 VCEP rule requires qualifying CRC/endometrial MSI-H tumor evidence or tumor-genome evidence and/or MMR protein loss consistent with the variant location; no such case-level result is present.
• Table-for-CMMRD-diagnosis.pdf.txt was searched for c.353+7C>A and p.?; no variant-specific entry was found.
• VCEP-pilot-variants---MMR.xlsx.txt was searched for c.353+7C>A and p.?; no variant-specific entry was found.

Still missing

• Patient-level tumor MSI/IHC or tumor-genome results are unavailable for NM_000535.7:c.353+7C>A.
• No entry for c.353+7C>A or p.? was found in Table-for-CMMRD-diagnosis.pdf.txt.
• No entry for c.353+7C>A or p.? was found in VCEP-pilot-variants---MMR.xlsx.txt.

Sources

Reasoning

The VCEP requires co-occurrence in trans with a known pathogenic sequence variant in PMS2 in a patient with colorectal cancer after age 45 or another Lynch syndrome cancer above its median age of onset, absence of CMMRD manifestations, and phase confirmation through parents or offspring. The available case evidence contains no qualifying patient, cancer-age, CMMRD, co-occurrence, or phase-confirmation data, so BS2 cannot be evaluated.

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), 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

• The case evidence bundle provides population observations but no qualifying clinical co-occurrence or phase-confirmation record for BS2.

Still missing

• Qualifying in-trans co-occurrence with a known pathogenic PMS2 variant, affected patient's cancer age and type, CMMRD assessment, and parental or offspring phase confirmation are unavailable.

Sources

Reasoning

The VCEP permits BS3 when calibrated functional odds are benign-range or when an intronic variant has no associated mRNA aberration, with laboratory assays conducted using nonsense-mediated decay inhibition; supporting BS3 can also be based on variant-specific proficient protein and mRNA function under the MMR functional-assay flowchart. The case materials contain no variant-specific RNA, splicing, allelic-imbalance, protein-function, or calibrated functional-odds result for PMS2 c.353+7C>A (NP_000526.2:p.?). The on-record Functional-assay-SVI-documentation-MMR.xlsx was searched for c.353+7C>A and p.? with no matching variant entry, and Functional-assay-flowchart.pdf was searched for the same notations with no matching entry. Therefore BS3 cannot be evaluated.

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. OR 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

• Case normalization: NM_000535.7:c.353+7C>A; NP_000526.2:p.?; intronic variant.
• Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays': validated assay classes and normal/abnormal readout thresholds are documented, but no row identifies c.353+7C>A or p.? and no variant result is supplied.
• Functional-assay-flowchart.pdf: no-impact intronic assessment requires an appropriate splicing assay with NMD inhibitors for proficient function; no such variant-specific result is available.

Still missing

• No NMD-inhibited laboratory assay result addressing splicing or allelic imbalance was found.
• No variant-specific proficient protein and mRNA assay result was found.
• No calibrated functional odds for pathogenicity were found.
• No entry for c.353+7C>A or p.? was found in Functional-assay-SVI-documentation-MMR.xlsx.
• No entry for c.353+7C>A or p.? was found in Functional-assay-flowchart.pdf.

Sources

Reasoning

The VCEP BS4 rule requires lack of co-segregation with a combined Bayes likelihood ratio <0.05 for Strong or >0.05 and <=0.48 for Supporting. No pedigree, non-segregating affected relatives, informative meioses, or combined Bayes likelihood ratio is provided, so BS4 cannot be evaluated.

Evidence considered

• The VCEP BS4 thresholds are Strong for lack of co-segregation with a combined Bayes likelihood ratio <0.05 and Supporting for >0.05 and <=0.48.
• No non-segregation pedigree, relative genotype, informative meiosis, family count, or combined Bayes likelihood ratio is present in the reviewed case materials.

Still missing

• Pedigree identifying affected relatives who do not carry the variant and the relevant unaffected relatives.
• Number of informative non-segregating meioses and families.
• Combined Bayes likelihood ratio, preferably calculated using the VCEP-recommended COOL approach.

Sources

Reasoning

BP5 is applicable under the PMS2 VCEP, but the case contains no qualifying tumor series. The VCEP requires 2 or 3 tumors for Supporting or at least 4 tumors for Strong when tumors show MSS and/or no MMR-protein loss, or inconsistent LS-spectrum protein loss, with an alternative BRAF V600E or MLH1-methylation pathway requiring its own qualifying tumor evidence. COSMIC reports two somatic observations, but it does not establish the required patient-level CRC/endometrial tumor MSI/IHC pattern or the specified alternative molecular basis, so BP5 cannot be assigned.

Evidence considered

• The VCEP BP5 Supporting rule requires '2 or 3 tumors' with the specified MSS/MMR-protein inconsistency pattern or '1 BRAF V600E (Colon only)/MLH1 methylation' pathway; the Strong rule requires at least 4 tumors or at least 2 alternative-pathway tumors.
• COSMIC record COSV56220636 reports the variant in somatic cancers 2 times, but provides no qualifying patient-level MSI/IHC or BRAF/MLH1-methylation evidence for BP5.

Still missing

• Qualifying patient-level tumor MSI status, MMR protein expression, and any BRAF V600E or MLH1 methylation results are unavailable.

Sources