PMS2 c.497T>C, p.(Leu166Pro)

Reasoning

c.497T>C (NM_000535.7) is a missense substitution, NP_000526.2:p.(Leu166Pro). The governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 defines PP3 for missense variants on the gene-specific 'MAPP/PP2 Prior P' probability in the HCI/LOVD PMS2 priors table (hci-lovd.hci.utah.edu), not on REVEL: PP3_Moderate when that prior is >0.81, PP3_Supporting when >0.68 and <=0.81. The on-record HCI-PRIORS-PMS2 table contains the exact coding substitution c.497T>C / p.L166P (row PMS2_01071), with 'Variant/MAPP/PP2_Prior' = 0.9595 (MAPP score 35.66; Custom PP2 score 0.998; reference {PMID22949387:Thompson et al., 2013}), which the case evidence independently reports as hci_prior probability 0.9595. 0.9595 exceeds the 0.81 threshold, so the VCEP pre-assigned strength is PP3_Moderate and is applied directly. Both candidate numeric columns in that row (0.9595 and 0.998) exceed 0.81, so the Moderate assignment is robust to which column is treated as the governing prior. The generic REVEL calibration (PMID:36413997) is superseded here by the gene-specific VCEP rule and was not used to set the strength.

Rule

PMS2 VCEP v2.0 PP3 (Moderate): Missense variant with 'MAPP/PP2 Prior P' score >0.81. (Supporting tier, not applied: >0.68 and <=0.81.)

Evidence considered

• ClinGen InSiGHT PMS2 VCEP specification v2.0 (cspec) PP3: missense variant with 'MAPP/PP2 Prior P' score >0.81 = PP3_Moderate; >0.68 and <=0.81 = PP3_Supporting. The VCEP PP3 path for a missense variant is the gene-specific MAPP/PP2 prior, not the generic REVEL calibration.
• On-record VCEP file HCI-PRIORS-PMS2.txt (declared by this gene's VCEP index as governing PP3/BP4) row 1074: c.497T>C, protein p.L166P, Variant/Custom_PP2_score 0.998, Variant/MAPP_score 35.66, Variant/MAPP/PP2_Prior 0.9595, reference {PMID22949387:Thompson et al., 2013}, DBID PMS2_01071.
• Column headers of HCI-PRIORS-PMS2.txt line 2: '{{ Variant/DNA }}', '{{ Variant/Protein }}', '{{ Variant/Custom_PP2_score }}', '{{ Variant/MAPP_score }}', '{{ Variant/MAPP/PP2_Prior }}'. The header 'Variant/MAPP/PP2_Prior' matches the metric named in the VCEP PP3 rule text.
• Case evidence hci_prior: found=true, coding_variant c.497T>C, protein p.L166P, probability 0.9595, mapp_score 35.66, custom_pp2_score 0.998.
• Variant consequence for c.497T>C is missense (VEP most_severe_consequence = missense_variant), placing it within the missense scope of the VCEP PP3 rule.
• Generic REVEL score for reference only: 0.776, which would be PP3_supporting under the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997); this was not used to set strength because the gene-specific VCEP MAPP/PP2 prior rule governs and is more specific.
• The HCI prior table's underlying data reference is PMID22949387 (Thompson et al. 2013); that PMID is not a source_registry key in this case, so it is not listed in evidence_refs.

Sources

Reasoning

Under the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel PMS2 specification v2.0, BS1 is strong benign evidence when the gnomAD v4 Grpmax filtering allele frequency is >= 0.00028 and < 0.0028 (0.028-0.28%) and the variant is excluded as a founder pathogenic variant. The specification names gnomAD v4 Grpmax and does not designate a non-cancer or exome-only source, so the all-comers gnomAD v4.1 entry governs. gnomAD v4.1 grpmax FAF = 0.00242754 (highest population African/African American, AF 0.00273435, 205/74,972 alleles, 0 homozygotes), which is above the 0.00028 floor and below the 0.0028 ceiling, so BS1 is met at the Strong strength specified. The population signal is supported by every other consulted source: gnomAD v2.1 all-comers grpmax FAF 0.00249164 (total AF 0.000269792, 76/281,698; AFR AF 0.002914), gnomAD v3.1 non-cancer grpmax FAF 0.00242938 (total AF 0.000824592, 122/147,952), gnomAD v2.1 non-cancer exomes AF 0.000216278 (51/235,808) and gnomAD-Canada v1.0 AF 0.00021732 (4/18,406). The variant is not reported anywhere as a pathogenic or founder pathogenic allele, so the exclusion condition attached to the BS1 rule is satisfied. Because the frequency sits in the upper part of the BS1 band, only a modest further increase in the African-ancestry estimate would cross into the BA1 stand-alone band.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.00028 and < 0.0028 (0.028-0.28%) and variant is excluded as founder pathogenic variant (ClinGen InSiGHT PMS2 v2.0, BS1 Strong).

Evidence considered

• PMS2 v2.0 specification (cspec): BS1 default strength 'Benign Strong', rule 'GnomAD v4 Grpmax filtering allele frequency >= 0.00028 and < 0.0028 (0.028-0.28%) and variant is excluded as founder pathogenic variant.' No non-cancer or exome-only population source is specified, so gnomAD v4 all-comers is the governing source.
• gnomAD v4.1 grpmax filtering AF = 0.00242754 >= 0.00028 and < 0.0028, satisfying the BS1 band; total AF 0.00013713 (221/1,611,606 alleles), exome AF 0.00006784, genome AF 0.00080088, homozygotes = 0.
• The elevated frequency is ancestry-specific: African/African American AF 0.00273435 (205/74,972 alleles) versus 0.0000391 in European (non-Finnish) and near-absence elsewhere; no homozygotes in any population.
• Concordant frequencies in the alternative sources: gnomAD v2.1 all-comers grpmax FAF 0.00249164 (AFR 0.002914, 72/24,710; total AF 0.000269792, 76/281,698); gnomAD v3.1 non-cancer AF 0.000824592, grpmax FAF 0.00242938 (122/147,952 alleles); gnomAD v2.1 non-cancer exomes AF 0.000216278 (51/235,808 alleles); gnomAD-Canada v1.0 AF 0.00021732 (4/18,406 alleles). The BS1 band is met under all of these.
• Founder-pathogenic exclusion: no consulted source (ClinVar variation 135944, gene-specific literature or population databases) reports c.497T>C (p.Leu166Pro) as a pathogenic or founder pathogenic allele, so the BS1 rule applies as written.
• Disease context considered for threshold plausibility: PMS2 is a Lynch syndrome mismatch-repair gene, and gene-level studies report PMS2 accounting for a minority of LS mutations (PMID:25980754) with atypical, later-onset presentation (PMID:27443514); the VCEP supplies PMS2-specific frequency thresholds, so no separate prevalence-based adjustment was made.

Still missing

• No ancestry-specific founder-effect documentation exists for the AFR-concentrated frequency (grpmax FAF 0.00242754); the BS1 founder exclusion rests on the absence of any pathogenic/founder report for this variant.

Sources

Reasoning

Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 (framework_mode 'vcep'), so the VCEP PVS1 decision tree is used rather than the generic ClinGen SVI PVS1 framework. The variant is NM_000535.7:c.497T>C, NP_000526.2:p.(Leu166Pro) - a single-nucleotide missense substitution. It satisfies none of the VCEP PVS1 branches: it is not a nonsense or frameshift variant introducing a premature termination codon (no truncation is created; the reference and predicted proteins are both 862 aa with position_first 165 and position_last 166 identical, i.e. no protein-length change), it is not a large single- or multi-exon deletion or duplication, it is not an initiation-codon variant, it is not a canonical GT-AG +/-1 or +/-2 splice-site variant (canonical_splice_consensus false), and it is not a variant at IVS+/-1 or IVS+/-2 where exon skipping or cryptic-site use disrupts or preserves the reading frame. There is also no mRNA/RNA assay evidence from patient constitutional material showing a splicing aberration that creates a premature stop codon or an in-frame deletion disrupting a functional domain, which is the remaining VCEP route to PVS1. SpliceAI predicts no splice impact for this variant (max delta score 0.012), far below the >=0.2 threshold the VCEP uses to call a predicted splice defect, so no splicing-based PVS1 path is triggered either. Because PVS1 is not met, no strength tier is assigned (strength null); the VCEP strength tiers (Very Strong <=codon 798 PTC, Strong initiation codon / presumed tandem duplication / reading-frame-preserving critical-region splice change, Moderate PTC between codons 799 and 862) are all null-variant tiers and are therefore not reached by a missense substitution. The variant's consequence is instead handled by the missense-specific rules (PP3/BP4 via the HCI prior, PS1, PM5), not by PVS1.

Rule

VCEP InSiGHT PMS2 v2.0 PVS1 (PVS1_DecisionTree_MMR) - no null-variant branch satisfied by a missense substitution; PVS1 not met.

Evidence considered

• InSiGHT PMS2 v2.0 CSPEC PVS1 rules: Very Strong for a nonsense/frameshift PTC at or before codon 798, multi-exon or single-exon large genomic alteration, or IVS+/-1/2 variant whose exon skipping or cryptic-site use disrupts the reading frame and is predicted to undergo NMD, or a patient-RNA-confirmed splicing aberration producing a premature stop or in-frame functional-domain disruption; Strong for initiation-codon variants, presumed in-tandem duplication of >=1 exon causing a pre-last-splice-junction frameshift, and reading-frame-preserving critical-region splice changes; Moderate for a PTC between codons 799 and 862.
• VCEP PVS1_DecisionTree_MMR: every PVS1-producing branch is entered from nonsense/frameshift, GT-AG 1,2 splice sites, deletion, duplication, or initiation codon. A missense substitution has no entry point in the tree.
• Variant consequence: HGVS c.497T>C gives NP_000526.2:p.(Leu166Pro); VEP most_severe_consequence is 'missense_variant' with variant_class 'SNV'. No premature termination codon, no frameshift, no stop-loss, and no protein-length change (reference and predicted protein both end at residue 166 within an 862-residue protein).
• SpliceAI lookup for NM_000535.7:c.497T>C returns a maximum delta score of 0.012, i.e. no predicted splicing impact; the variant is not at a canonical splice consensus (canonical_splice_consensus false).
• Gene-level gate: PMS2 germline loss of function is an established disease mechanism and the PVS1 gene gate is eligible, so PVS1 was assessed rather than skipped; the criterion fails on variant consequence, not on gene mechanism.
• Literature was checked and no retrieved publication provides a consequence-level (PTC/splice/frameshift) finding for this variant; the only paper naming it reports it as a variant of uncertain significance with no functional or RNA data.

Still missing

• No patient-derived mRNA/RNA assay result is recorded; that branch of the VCEP PVS1 rule is therefore unverified, but it cannot be met here because no splicing aberration is predicted (SpliceAI max delta 0.012) and the variant is not at a canonical splice site.

Sources

Reasoning

The governing specification is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 (CSPEC). Its PS1_Strong rule requires a predicted missense substitution that encodes the same amino acid change as a variant previously established by this VCEP as Pathogenic via a different underlying nucleotide change (the alternative limb concerns non-canonical splice nucleotides and cannot apply to this missense change). NM_000535.7:c.497T>C is p.(Leu166Pro): c.497 is position 2 of codon 166, and the Leu166 codon is a CTN codon - confirmed by the ClinVar records c.496C>T (p.Leu166=, VCV000231594) and c.498A>G (p.Leu166=, VCV001674255), both synonymous, and c.497T>A (p.Leu166Gln, VCV002451615). Because a Pro at this codon requires C at position 2 (CCN), Pro can only be produced by changing that base to C, i.e. by c.497T>C itself; A gives Gln/His, G gives Arg and T gives Leu. No different underlying nucleotide change can encode p.Leu166Pro, so the structural prerequisite of PS1 is unsatisfiable for this variant. This is consistent with the evidence: the VCEP pilot-variant table contains no PMS2 variant at residue 166, and ClinVar variation 135944 (the variant under assessment) is classified conflicting/likely benign with no pathogenic submission, so no residue-166 or p.Leu166Pro variant has been established as Pathogenic by this VCEP. PS1 is therefore not met.

Rule

PS1_Strong (InSiGHT PMS2 v2.0): 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

• CSPEC v2.0 PS1 rule (Strong): a predicted missense substitution encoding 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); (Moderate): same amino-acid change as a previously established Likely Pathogenic missense variant with normal RNA result plus PM2_supporting.
• Codon 166 identity: ClinVar VCV000231594 (c.496C>T, p.Leu166=), VCV001674255 (c.498A>G, p.Leu166=) and VCV002451615 (c.497T>A, p.Leu166Gln) establish that c.497 is codon position 2 of a CTN Leu codon; only C at that position yields Pro (CCN), so c.497T>C is the unique nucleotide change that encodes p.Leu166Pro.
• VCEP pilot-variant table (VCEP-pilot-variants---MMR.xlsx): no PMS2 variant at residue 166; the PMS2 entries are c.1144+1G>A, c.989-2A>G, c.241G>T (p.Glu81Ter), c.1571dup, c.1753C>A (p.Leu585Ile) and c.614A>C (p.Gln205Pro).
• ClinVar variation 135944 (NM_000535.7(PMS2):c.497T>C (p.Leu166Pro)): conflicting classifications - Likely benign (5 laboratories), Uncertain significance (4), Benign (3), likely benign (1); 0 expert-panel submissions; no submitter reports Pathogenic.
• Literature searched for the exact variant/amino-acid change: PMID:24113346 is the only fetched paper that mentions p.L166P, and it reports it once as a laboratory-classified variant of uncertain significance (ColoNext panel); no publication reports p.Leu166Pro arising from a nucleotide change other than c.497T>C.

Sources

Reasoning

The ClinGen InSiGHT PMS2 specification v2.0 defines PM2 at the only applicable strength, Supporting, as 'Absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset'; no non-cancer or exome-only source is designated, so the all-comers gnomAD v4.1 entry governs. gnomAD v4.1 reports this variant at total AF 0.00013713 (221/1,611,606 alleles) and grpmax filtering AF 0.00242754, roughly 6.9-fold and 121-fold above the 0.00002 cut-off respectively. The variant is present in every supplied dataset, including the non-cancer subsets (gnomAD v2.1 non-cancer exomes AF 0.000216278, 51/235,808; gnomAD v3.1 non-cancer AF 0.000824592, 122/147,952), and in gnomAD-Canada v1.0 (AF 0.00021732). Under gnomAD v2.1 all-comers the total AF is 0.000269792 (76/281,698). No population source reports the variant as absent or rare enough to approach the threshold, so PM2 is not met at any strength; this is a threshold verdict driven by a well-powered, multi-dataset presence rather than by a sparse-data artifact.

Rule

Absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset (ClinGen InSiGHT PMS2 v2.0, PM2 Supporting).

Evidence considered

• PMS2 v2.0 specification (cspec): PM2 applicable only at Supporting strength, rule 'Absent/extremely rare allele frequency <0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset'. The rule names the gnomAD v4 dataset and no non-cancer or exome-only subset, so gnomAD v4.1 all-comers is the governing source.
• gnomAD v4.1: total AF = 0.00013713 (221/1,611,606 alleles), grpmax filtering AF = 0.00242754, exome AF 0.00006784 (99/1,459,274), genome AF 0.00080088 (122/152,332); homozygotes = 0. Total AF exceeds the 0.00002 threshold by ~6.9-fold and grpmax FAF by ~121-fold.
• gnomAD v2.1 all-comers: total AF = 0.000269792 (76/281,698 alleles), grpmax FAF = 0.00249164 (AFR AF 0.002914, 72/24,710); also far above 0.00002.
• Sensitivity check with non-cancer sources (checked because the VCEP does not name them): gnomAD v2.1 non-cancer exomes AF = 0.000216278 (51/235,808 alleles); gnomAD v3.1 non-cancer genomes AF = 0.000824592 (122/147,952 alleles), grpmax FAF 0.00242938. Both remain orders of magnitude above the 0.00002 cut-off, so PM2 is not met under any supplied population source.
• gnomAD-Canada v1.0 (HostSeq genomes): AF = 0.00021732 (4/18,406 alleles), homozygotes = 0, i.e. the variant is also present in an independent population cohort at a frequency well above 0.00002.
• Ancestry distribution: frequency is concentrated in African/African American (v4.1 AF 0.00273435) with the variant effectively absent in several other populations; the multi-population AF estimates all exceed the PM2 threshold, so no ancestry-restricted argument supports rarity.
• Homozygote data: zero homozygotes reported across gnomAD v4.1, v2.1, v3.1 non-cancer and gnomAD-Canada, so the variant's presence is exclusively heterozygous at the frequencies above.

Still missing

• The VCEP PM2 wording does not state whether the 0.00002 cut-off is applied to total AF or to grpmax FAF; both metrics (0.00013713 and 0.00242754) exceed it by a wide margin, so the ambiguity does not affect the verdict.

Sources

Reasoning

CSPEC v2.0 PM5 requires a 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 (PM5_Moderate), or as Likely Pathogenic on the protein level (PM5_Supporting); both limbs may only be used if PP3 is supporting for the variant under assessment. That gate is satisfied - the local HCI 'MAPP/PP2 Prior P' probability for c.497T>C is 0.9595, which exceeds the VCEP's PP3_Moderate cut-off of >0.81 - so the failure is specifically the missing comparator. The only other missense reported at PMS2 codon 166 is c.497T>A (p.Leu166Gln, ClinVar VCV002451615), classified Uncertain significance by a single submitter with no expert-panel review, and the VCEP pilot-variant table contains no PMS2 variant at residue 166 at all. No different missense change at residue 166 has been classified Pathogenic or Likely Pathogenic by this VCEP, so PM5 is not met. The automated comparator harvest (pm5_candidates.json) recorded zero same-residue candidates after its ClinVar query hit HTTP 429; that gap was closed by querying ClinVar directly for all PMS2 residue-166 records, which independently confirms that p.Leu166Gln is the only second missense and that it is not pathogenic.

Rule

PM5_Moderate (InSiGHT PMS2 v2.0): missense change at a residue where a different missense change was classified by this VCEP as Pathogenic on the protein level and not due to aberrant splicing; PM5_Supporting if the other variant is Likely Pathogenic; both only when PP3 is supporting for the variant under assessment.

Evidence considered

• CSPEC v2.0 PM5 rule: missense change at an amino acid residue where a different missense change was classified by this VCEP as Pathogenic (PM5_Moderate) or Likely Pathogenic (PM5_Supporting) on the protein level and not due to aberrant splicing; only used if PP3 is supporting for the variant under assessment.
• PP3 gate for PM5: local HCI prior probability for c.497T>C = 0.9595 (>0.81), corresponding to PP3_Moderate under the VCEP's HCI prior thresholds, so the PP3 precondition for using PM5 is met and the criterion fails solely on the absent comparator.
• Residue-166 comparator search: ClinVar returns four PMS2 records containing Leu166 - c.497T>C (p.Leu166Pro, VCV000135944, variant under assessment), c.497T>A (p.Leu166Gln, VCV002451615, Uncertain significance, single submitter), and the two synonymous changes c.496C>T and c.498A>G (p.Leu166=). No Pathogenic or Likely Pathogenic residue-166 missense exists in ClinVar.
• VCEP pilot-variant table: no PMS2 variant at residue 166; therefore no VCEP-classified different missense change at this residue.
• pm5_candidates.json: found=false, 0 same-residue candidates, 0 expert-panel pathogenic candidates, with error 'HTTP Error 429: Too Many Requests' - the harvest was incomplete, so the comparator search was re-run against ClinVar directly as described above.
• PMID:24113346 reports PMS2 p.L166P once (ColoNext panel, laboratory-classified VUS); it provides no second amino-acid change at residue 166 and no pathogenic comparator.

Still missing

• PM5 requires the comparator to have been classified by the InSiGHT VCEP itself; that internal classification database is not public, so the assessment rests on the VCEP pilot-variant table plus ClinVar, neither of which contains a Pathogenic or Likely Pathogenic residue-166 missense.

Sources

Reasoning

PP5 is triggered only when the exact variant already carries an expert-panel (ClinGen/VCEP-level, 3-star) ClinVar classification of Pathogenic or Likely pathogenic. ClinVar VCV000135944 (variation 135944, NM_000535.7(PMS2):c.497T>C p.Leu166Pro) has an empty expert-panel record - the source data field 'expert_panels' is {} and the submission audit counts 0 expert-panel submissions among 14 submissions (11 usable for classification, 5 usable as criterion leads). Its review status is 'criteria provided, conflicting classifications' (1 star), and every one of its 13 logged submissions is a single clinical-laboratory submission: 5 Likely benign, 4 Uncertain significance, 3 Benign, 1 likely benign. Aggregate ClinVar labels and ordinary laboratory submissions must never trigger PP5, and there is no expert-panel assertion here to consider at any strength, so PP5 is not met. CSPEC v2.0 separately lists PP5 as 'Not Applicable' for PMS2; either way no PP5 credit is available.

Rule

PP5 applies only to an exact-variant expert-panel (3-star ClinGen/VCEP) ClinVar classification of Pathogenic or Likely pathogenic; ordinary single-submitter laboratory classifications and aggregate labels do not qualify.

Evidence considered

• ClinVar VCV000135944 source data: 'expert_panels': {} - no expert-panel record exists for this exact variant.
• ClinVar review status 'criteria provided, conflicting classifications', 1 star, date last updated 2026-04-13; 13 submissions from 13 submitters, all 'criteria provided, single submitter' (5 Likely benign, 4 Uncertain significance, 3 Benign, 1 likely benign).
• ClinVar submission audit: expert_panel_submissions = 0 out of 14 submissions; the criterion leads are ordinary clinical laboratories (LabCorp, Ambry Genetics, Quest Diagnostics, Sema4, Sinai Health System).
• CSPEC v2.0 PP5: applicability = Not Applicable for PMS2.

Sources

Reasoning

Governing specification is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel PMS2 specification v2.0, under which BA1 is stand-alone benign when the gnomAD v4 Grpmax filtering allele frequency is >= 0.0028 (0.28%) and the variant is excluded as a founder pathogenic variant. The rule names gnomAD v4 Grpmax explicitly and does not direct a non-cancer or exome-only source, so the all-comers gnomAD v4.1 entry governs. gnomAD v4.1 grpmax FAF = 0.00242754, driven by the African/African American population (AF 0.002734, 205/74,972 alleles); total v4.1 AF = 0.00013713 (221/1,611,606 alleles), exome AF 0.0000678, genome AF 0.0008009, homozygotes = 0. Observed grpmax FAF is 86.7% of the 0.0028 cut-off, i.e. below it, so BA1 is not met. The all-comers v2.1 estimate (grpmax FAF 0.00249164; total AF 0.000269792, 76/281,698; AFR AF 0.002914) is likewise below 0.0028, and the alternative non-cancer sources do not raise the value above threshold (gnomAD v3.1 non-cancer grpmax FAF 0.00242938, total AF 0.000824592; gnomAD v2.1 non-cancer exomes AF 0.000216278; gnomAD-Canada v1.0 AF 0.0002173). The founder-pathogenic exclusion condition is satisfied because no consulted source reports c.497T>C (p.Leu166Pro) as a pathogenic or founder pathogenic allele. The frequency is therefore captured by BS1 rather than BA1; the shortfall is small, so an upward revision of the AFR frequency estimate could move this variant across the stand-alone threshold.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.0028 (0.28%) and variant is excluded as founder pathogenic variant (ClinGen InSiGHT PMS2 v2.0, BA1 Stand Alone).

Evidence considered

• PMS2 v2.0 specification (ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel, cspec): BA1 default strength 'Benign Stand Alone', rule 'GnomAD v4 Grpmax filtering allele frequency >= 0.0028 (0.28%) and variant is excluded as founder pathogenic variant.' The rule specifies gnomAD v4 Grpmax and names no non-cancer or exome-only subset, so the all-comers gnomAD v4.1 entry is the governing population source.
• gnomAD v4.1 (GRCh38 chr7:6002493 A>G): grpmax filtering AF = 0.00242754; total AF = 0.00013713 (221/1,611,606 alleles); exome AF 0.00006784 (99/1,459,274); genome AF 0.00080088 (122/152,332); homozygotes = 0.
• Highest observed population in gnomAD v4.1 is African/African American (AF 0.00273435, 205/74,972 alleles, 0 homozygotes); the grpmax FAF of 0.00242754 is AFR-driven, so the frequency estimate carries an ancestry-specific component.
• Arithmetic against the VCEP threshold: 0.00242754 / 0.0028 = 0.867, i.e. 86.7% of the BA1 stand-alone cut-off; the estimate lies in the BS1 band (>= 0.00028 and < 0.0028) rather than at or above 0.0028.
• gnomAD v2.1 all-comers: grpmax FAF = 0.00249164; total AF = 0.000269792 (76/281,698 alleles); exome AF 0.00021975 (55/250,284), genome AF 0.00066849 (21/31,414); AFR AF 0.002914 (72/24,710); homozygotes = 0. Also below 0.0028.
• Sensitivity check with the supplied non-cancer subsets (not used as the governing source because the VCEP does not name them): gnomAD v3.1 non-cancer genomes AF = 0.000824592 (122/147,952 alleles), grpmax FAF = 0.00242938 (highest 1KG:ESN AF 0.004808, 1/208); gnomAD v2.1 non-cancer exomes AF = 0.000216278 (51/235,808 alleles), grpmax FAF = 0.00248074; gnomAD-Canada v1.0 AF = 0.00021732 (4/18,406 alleles). None reaches 0.0028, so BA1 remains not met under any of the supplied population sources.
• Founder-pathogenic exclusion: the ClinVar record for variation 135944 contains no submission reporting c.497T>C as a pathogenic or founder pathogenic allele, and no consulted source describes an ancestral founder haplotype for this variant, so the BA1 threshold is applicable as written.
• Homozygote data: zero homozygotes are reported in gnomAD v4.1, v2.1, v3.1 non-cancer and gnomAD-Canada, i.e. no recessive-state carrier is documented for this variant.

Still missing

• No founder-effect documentation (haplotype or ancestry-specific genealogical data) for gnomAD v4.1 grpmax FAF 0.00242754; the founder-pathogenic exclusion was assessed only from the absence of any pathogenic/founder report for this variant in the consulted sources.

Sources

Reasoning

In the ClinGen InSiGHT PMS2 specification v2.0 the generic BS2 formulation is replaced: BP2 is 'Not applicable' with the instruction 'BS2 is used instead', and the operative BS2 rule is co-occurrence in trans with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or another LS cancer above its median age of onset in LS) who has no evidence of CMMRD features, with phase confirmed by testing of parents or offspring. That rule requires an affirmative patient-level observation. No consulted source provides one: the ClinVar record for variation 135944 contains no submission reporting trans co-occurrence or phase data for c.497T>C, no fetched publication reports this variant in trans with a pathogenic PMS2 allele, and this variant-level case carries no proband zygosity or phase information. The only full text naming the variant (PMID:24113346) lists PMS2 p.L166P on a ColoNext hereditary cancer panel as a variant of uncertain significance, with no phase, zygosity or co-occurring-variant data. The generic alternative reading of BS2 (a healthy homozygous adult) is likewise unsupported: gnomAD v4.1, v2.1, gnomAD v3.1 non-cancer and gnomAD-Canada all report zero homozygotes, so no biallelic carrier is documented. With no qualifying in-trans observation in any source, BS2 is not met.

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), with no evidence of CMMRD features, and phase confirmed by testing of parents or offspring (ClinGen InSiGHT PMS2 v2.0, BS2 Strong).

Evidence considered

• PMS2 v2.0 specification (cspec): BS2 default strength 'Benign Strong', 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 ... Confirmation of phase requires testing of parents or offspring.'
• PMS2 v2.0 specification (cspec): BP2 is marked 'Not applicable' with instructionsToUse 'BS2 is used instead', so the generic BS2 wording (healthy homozygous/hemizygous adult) is not the operative rule for this gene.
• No consulted source reports c.497T>C (p.Leu166Pro) in trans with a pathogenic or likely pathogenic PMS2 variant; the ClinVar record for variation 135944 lists no submission reporting trans co-occurrence or phase determination for this variant.
• PMID:24113346 is the only consulted full text naming the variant: 'PMS2 ColoNext p.L166P VUS' in Table 2, with no phase, zygosity, co-occurring variant or segregation data; the associated PMS2 case in that paper involves two unspecified PMS2 VUSs and normal MMR immunohistochemistry in the mother's colon tumor.
• Homozygote check across all supplied population datasets: gnomAD v4.1, v2.1, gnomAD v3.1 non-cancer and gnomAD-Canada each report 0 homozygotes for this variant, so no healthy biallelic carrier is documented and the generic BS2 formulation would also be unsupported.
• Disease-mechanism context: PMS2-associated Lynch syndrome is dominantly inherited with reduced, age-dependent penetrance and CMMRD represents the biallelic state; the VCEP BS2 rule is designed to capture benign evidence from an in-trans carrier past the LS age of onset, an observation absent from all sources consulted here.

Still missing

• No proband-level genotype, zygosity or phase data (parental/offspring testing) are available for this variant, so an in-trans co-occurrence with a pathogenic PMS2 allele can be neither confirmed nor excluded.
• ClinVar submissions for variation 135944 do not expose per-criterion evidence for BS2, so any unsubmitted laboratory observation of trans co-occurrence would not be visible here.

Sources

Reasoning

c.497T>C (NM_000535.7) is a missense substitution, NP_000526.2:p.(Leu166Pro), so the missense BP4 path applies. The governing ClinGen InSiGHT PMS2 VCEP v2.0 rule for BP4 in a missense variant is a gene-specific 'MAPP/PP2 Prior P' probability of <0.11; the on-record HCI-PRIORS-PMS2 table gives 0.9595 for the exact substitution c.497T>C / p.L166P, which is far above 0.11, so BP4 is not met. The second branch of the VCEP BP4 rule (SpliceAI delta <= 0.1) is explicitly restricted to intronic and synonymous variants and therefore does not apply to this missense change. For completeness on the missense path, the generic ClinGen SVI REVEL calibration (PMID:36413997) also fails BP4: REVEL 0.776 is well above the BP4_supporting ceiling of 0.29, and above all higher BP4 tiers, so no benign computational evidence is available for this variant on either reading. The SpliceAI max delta of 0.012 is not cited as BP4 support because the variant is missense, and a clean splice delta says nothing about whether the amino-acid substitution is tolerated.

Rule

PMS2 VCEP v2.0 BP4 (Supporting): Missense variant with 'MAPP/PP2 Prior P' score <0.11; OR (intronic/synonymous only) SpliceAI delta score <= 0.1.

Evidence considered

• ClinGen InSiGHT PMS2 VCEP specification v2.0 (cspec) BP4: missense variant with 'MAPP/PP2 Prior P' score <0.11 (Benign Supporting); OR for intronic and synonymous variants only, SpliceAI delta score <= 0.1 (Walker et al. 2023).
• On-record VCEP file HCI-PRIORS-PMS2.txt row 1074: c.497T>C, protein p.L166P, Variant/MAPP/PP2_Prior 0.9595 — above the <0.11 BP4 threshold, so the benign-supporting branch is not satisfied.
• Case evidence hci_prior: found=true, coding_variant c.497T>C, protein p.L166P, probability 0.9595 — consistent with the on-record table.
• Variant consequence is missense (VEP most_severe_consequence = missense_variant), which fixes BP4 to the missense branch; the SpliceAI branch of the VCEP BP4 rule is scoped to intronic/synonymous variants and is out of scope here.
• Generic ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997) thresholds for BP4: strong <=0.016, moderate <=0.183, supporting <=0.29. Observed REVEL is 0.776, exceeding every BP4 tier, so BP4 would not be met on the generic missense path either.
• SpliceAI max delta score 0.012 was noted but deliberately not used as BP4 evidence, because a missense variant's PP3/BP4 must come from the protein-impact path only.

Sources

Reasoning

BP6 is triggered only when the exact variant already carries an expert-panel (ClinGen/VCEP-level, 3-star) ClinVar classification of Benign or Likely benign. ClinVar VCV000135944 (variation 135944) has an empty expert-panel record ('expert_panels': {}, 0 expert-panel submissions of 14 logged submissions); its review status is 'criteria provided, conflicting classifications' (1 star) and all 13 submissions are single clinical-laboratory submissions (5 Likely benign, 4 Uncertain significance, 3 Benign, 1 likely benign). Although several ordinary submitters call the variant Likely benign or Benign, aggregate ClinVar labels and non-expert laboratory submissions must never trigger BP6, so no BP6 credit is available. CSPEC v2.0 separately lists BP6 as 'Not Applicable' for PMS2; either way BP6 is not met.

Rule

BP6 applies only to an exact-variant expert-panel (3-star ClinGen/VCEP) ClinVar classification of Benign or Likely benign; ordinary single-submitter laboratory classifications and aggregate labels do not qualify.

Evidence considered

• ClinVar VCV000135944 source data: 'expert_panels': {} - no expert-panel benign or likely benign classification exists for this exact variant.
• ClinVar review status 'criteria provided, conflicting classifications', 1 star; submissions are all single clinical-laboratory submissions (5 Likely benign, 4 Uncertain significance, 3 Benign, 1 likely benign).
• ClinVar submission audit: expert_panel_submissions = 0.
• CSPEC v2.0 BP6: applicability = Not Applicable for PMS2.

Sources

Reasoning

The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 (CSPEC doc 1578297058), where PS2 is Applicable but scored by de novo points rather than as a single qualitative rule. Points are earned per proband: 2 points for a de novo variant with both maternity and paternity confirmed in a case with an MMR-deficient Lynch-syndrome-spectrum tumor (MSI/IHC consistent with the affected gene, no MLH1 methylation), 1 point for a confirmed de novo in a case with an LS-spectrum tumor but no MSI/IHC/methylation data, and 0.5 points for an assumed de novo with maternity and/or paternity unconfirmed with an LS-spectrum tumor. Strength follows the point total: >=4 points = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting. This case provides no proband-level data: there is no parental (maternity/paternity) testing, no family history, no affected relative genotypes and no tumor MSI/IHC/methylation information anywhere in the case record, and no source reports c.497T>C (p.Leu166Pro) as a de novo observation in any patient. The only variant-specific publication (PMID:24113346) reports p.L166P as a variant of uncertain significance detected on a single ColoNext panel, with no parental or other family testing described. Zero de novo points can therefore be assigned, and there is no way to reach even the 0.5-point Supporting tier. However, the absence of parental testing is a data gap and not evidence that the variant was inherited, so PS2 is recorded as not assessed rather than not met. Closing the criterion would require parental genotypes for an affected proband plus the proband's tumor MSI/IHC (and MLH1 methylation) status.

Rule

InSiGHT PMS2 v2.0 PS2 (de novo points): >=4 points = Very Strong; 2-3.5 = Strong; 1-1.5 = Moderate; 0.5 = Supporting (2 points = confirmed de novo with MMR-deficient LS-spectrum tumor; 1 point = confirmed de novo with LS-spectrum tumor, no tumor data; 0.5 points = assumed de novo, maternity and/or paternity unconfirmed, with LS-spectrum tumor).

Evidence considered

• cspec (InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0, doc 1578297058) was read first as the governing gene-specific framework; PS2 is Applicable and is scored by de novo points: 2 points per proband for a de novo variant with both maternity and paternity confirmed in a case with an MMR-deficient LS-spectrum tumor, 1 point for confirmed de novo with an LS-spectrum tumor lacking MSI/IHC/methylation data, and 0.5 points for an assumed de novo with maternity and/or paternity unconfirmed with an LS-spectrum tumor; thresholds >=4 points = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting.
• The case evidence bundle contains no proband, no parental genotypes, no family history and no tumor MSI/IHC or MLH1 methylation data for c.497T>C, so no proband can be scored for de novo points.
• PMID:24113346 is the only retrieved full text that names this exact change; it lists PMS2 p.L166P as a VUS detected on one ColoNext panel and provides no parental testing or de novo statement, so it contributes no PS2 points.
• ClinVar submissions for VCV000135944 were inspected for any de novo statement and contain none; per instruction, no classification was inferred from another source's final classification.
• Generic ACMG/AMP PS2 wording (PMID:25741868) was consulted only for the original criterion definition; the VCEP point-based specification takes precedence and is not superseded by a generic de novo assertion.

Still missing

• No proband/tested patient in this case: no affected individual carrying c.497T>C is documented, so no de novo points can be assigned.
• No parental testing (neither maternity nor paternity confirmation) for any carrier, so neither the 2-point nor the 1-point confirmed-de-novo tier can be evaluated.
• No tumor MSI/IHC or MLH1 methylation data for any carrier, which the VCEP requires for the highest (2-point) PS2 tier.
• No source (ClinVar submissions, PMID:24113346, any fetched full text) reports c.497T>C as de novo.

Sources

Reasoning

Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 (framework_mode 'vcep'), in which PS3 is Applicable and strength is set by calibrated functional assay odds rather than by generic ACMG. The VCEP tiers are: Strong for a calibrated functional assay with functional odds for pathogenicity >18.7; Moderate for odds >4.3 and <=18.7, OR an MMR function defect established by the VCEP functional-assay flowchart, OR monoallelic complete loss of expression (<10% of wild-type in cDNA without puromycin, full-length transcript analysed with and without NMD block); Supporting for odds >2.08 and <=4.3. NM_000535.7:c.497T>C is a missense substitution, NP_000526.2:p.(Leu166Pro), so for this variant PS3 can only be reached through a calibrated MMR functional assay (or the missense branch of the flowchart), and the exploratory numeric evidence has been checked against that requirement. (1) Assay documentation: the VCEP calibrated-assay documentation file for MMR genes catalogues the accepted assays and their variant-independent normal/abnormal readout thresholds - Drost 2018/2020 HCT116 repair-capacity assay (normal >=70% for MLH1/MSH2 and >=100% for MSH6/PMS2; abnormal <23% and <18% respectively), Jia 2021 / Scott 2022 LOF-score and OddsPath assays (LOF score <=0 vs >0.4; OddsPath <0.48 vs >2.08), Rath 2022 MLH1 MMR assay, four Thompson 2013 mammalian activity / protein-expression / subcellular-localisation assays (normal >75% and abnormal <25% in two independent assays; nuclear vs cytoplasmic localisation), Morak 2019 cDNA full-length-transcript analysis (biallelic ~50+/-10% vs allelic loss <=10%), and Bouvet 2019 alkylating-agent survival assay - but it reports no assay result, no control comparison and no functional odds for c.497T>C / p.Leu166Pro. (2) No VCEP functional pre-assignment exists for this variant: the VCEP pilot-variants table contains other MMR variants with PS3 applied (e.g. MSH2 c.1807G>A PS3_moderate, MSH6 c.1282A>G PS3, MLH1 c.923A>C PS3, MSH2 c.1012G>A PS3) but does not list PMS2 c.497T>C at all. (3) The splicing route is closed: SpliceAI max delta is 0.012, below the >=0.2 delta at which the VCEP anticipates a splice defect, so the flowchart's splicing branch is not triggered and no splicing-based function defect can be claimed. (4) Literature: no retrieved publication reports a functional assay for this variant; the only paper that names the exact change, PMID:24113346, records PMS2 p.L166P as a variant of uncertain significance identified on a ColoNext panel with no functional work, and the other retrieved papers contain no PMS2 variant-level functional data (the PS3-indexed literature leads were non-retrievable). (5) Database assertions: ClinVar holds no PS3-based assertion for this variant - submitters provided PP3, PP4 and PM1 only, and the University of Chicago submission explicitly states that 'due to insufficient evidences and the lack of functional studies' the variant remains of uncertain significance - and OncoKB reports no variant-specific reviewed functional evidence. (6) The HCI/LOV D 'MAPP/PP2 Prior P' probability of 0.9595 (with REVEL 0.776 and BayesDel 0.369) is computational in-silico evidence that feeds PP3/BP4, not a functional assay readout, and cannot be converted into PS3 at any strength. PS3 is therefore not assessed rather than not met: the criterion applies to this missense variant and was actively pursued across the VCEP assay documentation, the VCEP pilot list, the retrieved literature, ClinVar and OncoKB, but no calibrated functional assay result, positive/negative assay control comparison or functional odds value exists for c.497T>C, so no VCEP PS3 strength tier can be evaluated.

Rule

VCEP InSiGHT PMS2 v2.0 PS3: calibrated functional assay with functional odds for pathogenicity >18.7 (Strong); >4.3 and <=18.7, or MMR function defect per the functional assay flowchart, or monoallelic complete loss of expression <10% of wild-type in cDNA without puromycin (Moderate); >2.08 and <=4.3 (Supporting).

Evidence considered

• ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 (cspec): PS3 is Applicable with gene-specific tiers keyed to calibrated functional assay odds - Strong >18.7, Moderate >4.3 and <=18.7 (or MMR function defect per the functional assay flowchart, or monoallelic complete loss of expression <10% of wild-type in cDNA without puromycin), Supporting >2.08 and <=4.3; the VCEP instruction is to refer to the 'Functional assay SVI documentation (MMR genes)' file for the calibrated assays.
• VCEP calibrated-assay documentation file (Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays'): nine MMR functional assays are catalogued with assay-level normal and abnormal readout thresholds - Drost 2018/2020 repair-capacity assay (normal >=70% MLH1/MSH2 and >=100% MSH6/PMS2; abnormal <23% MLH1/MSH2 and <18% MSH6/PMS2), Jia 2021 / Scott 2022 LOF-score and OddsPath functional assays (LOF score <=0 vs >0.4; OddsPath <0.48 vs >2.08), Rath 2022 MLH1 MMR assay, Thompson 2013 mammalian activity, protein-expression and subcellular-localisation assays (normal >75%, abnormal <25% in two independent assays; nuclear vs cytoplasmic), Morak 2019 cDNA full-length-transcript analysis (biallelic ~50+/-10% vs allelic loss <=10%) and Bouvet 2019 methylating-agent survival assay. The file contains no row, result, control comparison or odds value for PMS2 c.497T>C / p.Leu166Pro.
• VCEP Functional-assay-flowchart: for an in-frame indel or missense variant without predicted splicing impact, the flowchart requires either a calibrated functional assay (convert functional LR to an ACMG/AMP weight) or two independent assays with concordant results (mammalian MMR activity, then protein expression/stability <=25% vs >=75%, then subcellular localisation and cellular-based MMR activity); no branch was entered because no such assay exists for this variant.
• VCEP pilot-variants table (VCEP-pilot-variants---MMR.xlsx): lists MMR variants with VCEP-assigned codes, including PS3 for MSH2 c.1807G>A (PS3_moderate), MSH6 c.1282A>G (PS3), MLH1 c.923A>C (PS3) and MSH2 c.1012G>A (PS3); PMS2 c.497T>C (p.Leu166Pro) is absent, so no VCEP functional call exists for it.
• SpliceAI max delta for this variant is 0.012, well below the >=0.2 delta the VCEP uses to call a predicted splice defect, so no splicing-based functional defect path is triggered (the variant is also not at a canonical splice site).
• ClinVar (variation 135944, 13 submissions): no submitter applied PS3 as a supporting assertion for this variant; the criteria-bearing submissions provided PP3, PP4 and PM1 only, and the University of Chicago submission states that 'due to insufficient evidences and the lack of functional studies' the change remains a variant of uncertain significance, i.e. the absence of functional data is itself documented in the public record.
• OncoKB lookup for PMS2 L166P: 'OncoKB did not identify variant-specific reviewed functional evidence for this variant', returning Unknown Oncogenic Effect with no variant-level citations.
• Literature screening: no retrieved publication reports a functional assay for c.497T>C / p.Leu166Pro; PMID:24113346 names the exact change only as a VUS found on a ColoNext panel, and the remaining retrieved papers (PMID:23788249, PMID:25356965, PMID:25741868, PMID:25980754, PMID:26467025, PMID:27443514) contain no PMS2 variant-level functional data.
• Computational scores were deliberately not converted into PS3: the HCI 'MAPP/PP2 Prior P' probability 0.9595, REVEL 0.776 and BayesDel 0.369188 are in-silico metrics belonging to PP3/BP4 and cannot substitute for a calibrated functional assay readout.

Still missing

• No calibrated MMR functional assay result (e.g. HCT116 repair-capacity assay, OddsPath/LOF-score assay, mammalian MMR activity assay, protein-expression/stability assay, subcellular-localisation assay, cDNA full-length-transcript analysis or alkylating-agent survival assay) has been performed or reported for PMS2 c.497T>C (p.Leu166Pro), so no functional odds for pathogenicity is available and no VCEP PS3 tier (>2.08 Supporting, >4.3 Moderate, >18.7 Strong) can be evaluated.
• No variant-specific positive and negative assay controls (wild-type, known pathogenic and known benign MMR controls, or deficient cell-line controls) are documented for this variant, so assay validity and reproducibility cannot be confirmed.
• No monoallelic-expression or NMD-blocked cDNA result is available, so the VCEP PS3_Moderate route for monoallelic complete loss of expression (<10% of wild-type without puromycin) cannot be evaluated.
• The VCEP pilot-variants table supplied for this gene does not include PMS2 c.497T>C, so there is no VCEP pre-assigned functional interpretation to inherit.
• Two literature leads indexed under PS3 in this case (PMID 2813514 and PMID 28492532) were not retrievable as full text and are not registered sources; PMID 28492532 is a criteria-refinement methodology paper (Sherloc) with no PMS2 variant data, and 2813514 is a malformed seven-digit identifier, so the PS3 literature search could not be completed through those leads.

Sources

Reasoning

Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Guidelines for PMS2, version 2.0, which lists PM3 as applicable with a point-based strength system. The PM3 rule requires co-occurrence of the variant with a known pathogenic or likely pathogenic sequence variant in the same gene (PMS2) in a patient whose clinical features are consistent with constitutional mismatch repair deficiency (CMMRD) per Aronson et al 2022 (referenced to the VCEP 'Table for CMMRD diagnosis'). Points are per case: pathogenic/likely pathogenic in trans = 1.0 point; pathogenic/likely pathogenic with phase unknown = 0.5 points; the sum across cases sets the strength (0.5 point = PM3_Supporting; 1-1.5 points = PM3; 2-3.5 points = PM3_Strong; >=4 points = PM3_VeryStrong). This variant, PMS2 c.497T>C (p.Leu166Pro), is a missense substitution, and the case evidence contains no affected proband observation, no CMMRD-consistent clinical phenotype or CMMRD point tally, no second pathogenic/likely pathogenic PMS2 variant, and no phase information, so no PM3 points can be assigned. The only case-level report of this exact change (PMID:24113346) records it as a single finding classified uncertain significance on a ColoNext panel, with no co-occurring PMS2 variant and no phase described. PM3 is therefore not assessed rather than met or not met: the trans/co-occurrence and affected-proband data on which the criterion depends are absent from this case.

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; pathogenic/likely pathogenic in trans = 1.0 point, pathogenic/likely pathogenic phase unknown = 0.5 points; sum across cases: >=4 points = PM3_VeryStrong, 2-3.5 = PM3_Strong, 1-1.5 = PM3, 0.5 = PM3_Supporting.

Evidence considered

• The PMS2 ClinGen InSiGHT VCEP specification version 2.0 lists PM3 as applicable and defines it as co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD per Aronson et al 2022, with 1.0 point for a pathogenic/likely pathogenic variant in trans and 0.5 points when phase is unknown, summed across cases to set strength (Very Strong >=4, Strong 2-3.5, Moderate 1-1.5, Supporting =0.5 points).
• The case evidence contains no affected proband description, no CMMRD-consistent phenotype or Aronson CMMRD point score, no co-occurring pathogenic/likely pathogenic PMS2 variant, and no phase, parental, or offspring testing information for PMS2 c.497T>C.
• PMID:24113346 (retrospective NGS hereditary cancer panel series) reports the exact change PMS2 p.L166P as a single finding classified as a variant of uncertain significance on one ColoNext panel, and describes no second PMS2 variant, no CMMRD phenotype, and no phase relationship for it.

Still missing

• No affected proband, CMMRD-consistent phenotype, or Aronson et al 2022 CMMRD point tally is documented in the case evidence, so the clinical prerequisite of the PMS2 PM3 rule cannot be satisfied.
• No second pathogenic/likely pathogenic PMS2 sequence variant and no phase (cis/trans) determination — including no parental or offspring testing — is available for this variant, so no in-trans (1.0 point) or phase-unknown (0.5 point) PM3 evidence can be scored.
• No variant-specific PM3 pre-assigned code for c.497T>C (p.Leu166Pro) was found in the PMS2 VCEP materials supplied for this case.

Sources

Reasoning

Under the governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0, PP1 is graded by the combined Bayes likelihood ratio from pedigree co-segregation analysis (the specification recommends COOL/COsegregation OnLine, with multiple pedigrees combined by multiplication, an allele frequency supplied, and the 'overall Bayes Factor' used to set strength): >18.7 in >=2 families = Strong, >4.3 and <=18.7 = Moderate, and >2.08 and <=4.3 = Supporting. No co-segregation analysis exists for c.497T>C. The case record contains no pedigree, no genotypes from relatives of any carrier, no affected/unaffected segregation counts and no likelihood-ratio calculation for any family. The only retrieved publication that names the variant (PMID:24113346) reports p.L166P as a VUS found incidentally on one ColoNext panel and provides no relative testing or segregation data; the family-history discussion in that paper concerns a different patient carrying two unnamed PMS2 variants, one of whom had a mother with a colon tumor showing normal mismatch repair protein expression - it is not linked to c.497T>C and cannot be used as segregation evidence. Because no likelihood ratio can be computed in either direction, PP1 is left not assessed; it cannot be scored as met or not met from the available data. A curator would need a genotyped pedigree (affected carriers plus unaffected relatives) analysed with COOL v3 to close this criterion.

Rule

InSiGHT PMS2 v2.0 PP1: combined Bayes likelihood ratio >18.7 in >=2 families = Strong; >4.3 and <=18.7 = Moderate; >2.08 and <=4.3 = Supporting (pedigrees combined by multiplication; COOL/COsegregation OnLine recommended).

Evidence considered

• cspec (InSiGHT PMS2 v2.0) was read first as the governing framework; PP1 is Applicable and graded by the combined Bayes likelihood ratio from pedigree co-segregation analysis, with COOL/COsegregation OnLine recommended, multiple pedigrees combined by multiplication, and strength thresholds >18.7 = Strong (>=2 families), >4.3 and <=18.7 = Moderate, >2.08 and <=4.3 = Supporting.
• No pedigree, relative genotypes or segregation likelihood-ratio computation for c.497T>C is present anywhere in the case record; the variant is a variant-level curation with no proband-level data.
• PMID:24113346 (only retrieved full text naming p.L166P) reports the change as a VUS on a single ColoNext panel with no relative testing, and its family-history narrative concerns a different patient with two unnamed PMS2 variants, so it provides no co-segregation data for this variant.
• PMID:27443514 and PMID:25980754 were reviewed and provide only gene-level PMS2 observations (carrier frequencies, atypical family-history presentation in unselected endometrial cancer and suspected Lynch syndrome cohorts) with no variant-specific segregation data.
• No co-segregation analysis exists, so no likelihood ratio - and therefore no PP1 strength tier - can be assigned; absence of a segregation study is not evidence against co-segregation.

Still missing

• No pedigree or family structure is recorded for any carrier of c.497T>C.
• No relative genotypes (affected or unaffected) are available, so no co-segregation likelihood ratio can be calculated.
• No COOL/COsegregation OnLine analysis (or equivalent combined Bayes likelihood ratio) has been performed for any family.
• Tumor phenotype for relatives (MSI/IHC, MLH1 methylation) is absent, so phenotype consistency within a pedigree cannot be assessed.

Sources

Reasoning

CSPEC v2.0 defines PP4 for PMS2 as tumour-phenotype evidence and assigns strength by tumour count: >=3 independent CRC/endometrial MSI-H tumours in >=2 families using a standard panel of 5-10 markers or tumour genome and/or loss of MMR protein expression consistent with the variant location = Strong; 2 independent CRC/endometrial MSI-H tumours = Moderate; 1 CRC/Endometrial MSI-H tumour = Supporting; an MSI-H tumour with inconsistent protein expression does not meet PP4 at that level. The case evidence contains no tumour phenotype data for the proband or any relative: there is no MSI result, no MMR immunohistochemistry result, and no tumour inventory anywhere in the assembled sources. The ClinVar record (VCV000135944, variation 135944) lists only broad conditions ('Lynch syndrome 4', 'Mismatch repair cancer syndrome 4', 'Hereditary nonpolyposis colorectal neoplasms') with no MSI or IHC detail, and none of the full-text papers reports a tumour result for p.L166P (PMID:24113346 mentions the variant only as a VUS on a panel; PMID:27443514 provides gene-level endometrial cancer data for PMS2 carriers, with one PMS2 carrier having intact MMR IHC and no MSI, but does not identify that variant as c.497T>C). No tumour-level data exists to score against the 1/2/3-tumour thresholds, so PP4 is not assessed; the missing item is tumour MSI status and/or MMR protein IHC for a tumour from this patient or family.

Rule

CSPEC v2.0 (InSiGHT PMS2 V2.0), PP4: >=3 independent CRC/Endometrial MSI-H tumours in >=2 families = Strong; 2 tumours = Moderate; 1 tumour = Supporting (MSI-H by standard 5-10 marker panel or tumour genome and/or loss of MMR protein expression consistent with the variant location).

Evidence considered

• CSPEC v2.0 PP4 rules: Strong = >=3 independent CRC/Endometrial MSI-H tumours in >=2 families (standard 5-10 marker panel or tumour genome) and/or loss of MMR protein expression consistent with the variant location; Moderate = 2 such tumours; Supporting = 1 such tumour; MSI-H with inconsistent protein expression does not meet PP4.
• No MSI, MMR immunohistochemistry, or tumour list for this case appears in any assembled source (case summary, evidence.json ClinVar/OncoKB entries, literature pass); the ClinVar conditions are broad disease labels without tumour-testing detail.
• PMID:24113346 records p.L166P only as a VUS found on a ColoNext panel, with no tumour phenotype testing for that individual.
• PMID:27443514 describes a PMS2 carrier whose tumour was screen-positive yet had intact MMR immunohistochemistry and no MSI, but the exact PMS2 variant of that patient is not identified in the supplied text, so it cannot be attributed to c.497T>C.

Still missing

• No tumour MSI (5-10 marker panel or tumour genome) result available for the proband or any relative.
• No MMR protein immunohistochemistry result (MLH1/MSH2/MSH6/PMS2 expression) available for any tumour, so consistency with the PMS2 variant location cannot be judged.
• No tumour inventory/CRC or endometrial cancer phenotype data for this case.

Sources

Reasoning

Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0, in which BS3 is Applicable. Its Strong tier is a calibrated functional assay with functional odds for pathogenicity <=0.05, or - for synonymous substitutions and intronic variants only - no mRNA aberration (splicing or allelic imbalance) in laboratory assays conducted with nonsense-mediated decay inhibition; its Supporting tier is calibrated functional odds >0.05 and <=0.48, or variant-specific proficient function in protein- and mRNA-based laboratory assays as per the MMR functional assay flowchart. NM_000535.7:c.497T>C is a missense substitution, NP_000526.2:p.(Leu166Pro), so the synonymous/intronic branch of BS3_Strong is out of scope, and the remaining routes require an actual variant-specific functional result showing proficient (wild-type-like) MMR function - a calibrated assay odds value, or flowchart-consistent concordant assays showing normal repair levels, >=75% relative protein expression, or nuclear localisation with DNA-damage sensitivity and no mutator phenotype. No such result exists for this variant in the case evidence: the VCEP calibrated-assay documentation file lists the accepted assays and their assay-level thresholds but has no entry for c.497T>C / p.Leu166Pro; the VCEP pilot-variants table does not include this variant; SpliceAI max delta 0.012 is below the VCEP >=0.2 splice-defect threshold, so no splicing-based functional claim (benign or pathogenic) is available; and no retrieved publication reports functional work on this change, the only paper naming it (PMID:24113346) classifying it as a VUS without any laboratory assay. ClinVar submissions for this variant did not assert BS3 either - submitters used PP3, PP4 and PM1, and the benign/likely benign classifications were based on other evidence types, from which BS3 may not be inferred (a criterion is never taken from another source's final classification). Computational scores point away from benign function rather than toward it - the HCI 'MAPP/PP2 Prior P' probability is 0.9595 (PP3_Moderate under this VCEP) and REVEL is 0.776 - but in any case these are in-silico metrics (PP3/BP4), not functional assays, and cannot establish BS3. BS3 is therefore not assessed: the criterion was actively evaluated against every functional-evidence source in the case, and no variant-specific functional assay demonstrating proficient function or a benign functional odds value exists to score at any strength.

Rule

VCEP InSiGHT PMS2 v2.0 BS3: calibrated functional assay with functional odds for pathogenicity <=0.05, or synonymous/intronic variant with no mRNA aberration in NMD-inhibited assays (Strong); calibrated odds >0.05 and <=0.48, or variant-specific proficient function in protein- and mRNA-based assays per the MMR functional assay flowchart (Supporting).

Evidence considered

• ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0 (cspec): BS3 is Applicable - Strong for calibrated functional odds for pathogenicity <=0.05, or for synonymous/intronic variants with no associated mRNA aberration (splicing or allelic imbalance) determined in assays with nonsense-mediated decay inhibition; Supporting for calibrated odds >0.05 and <=0.48, or variant-specific proficient function in protein- and mRNA-based laboratory assays per the MMR functional assay flowchart.
• VCEP calibrated-assay documentation file (Functional-assay-SVI-documentation-MMR.xlsx, sheet 'MMR assays'): catalogues the accepted MMR assays and their normal versus abnormal readout thresholds (normal >=70% MLH1/MSH2 and >=100% MSH6/PMS2 repair capacity; normal >75% in two independent mammalian activity, expression or localisation assays; nuclear localisation; biallelic ~50+/-10% transcript) but contains no result for PMS2 c.497T>C / p.Leu166Pro, so no variant-specific proficient-function claim can be made.
• VCEP Functional-assay-flowchart: a 'proficient function' outcome requires concordant results from two independent assays (normal MMR activity relative to wild-type controls, >=75% relative protein expression or wild-type-like subcellular localisation); none of these assays were performed for this variant.
• VCEP pilot-variants table (VCEP-pilot-variants---MMR.xlsx): includes BS3-based VCEP codes for other MMR variants (e.g. MLH1 c.649C>T BS3, MSH2 c.1748A>G BS3_Supporting, MLH1 c.277A>G BS3_Supporting) but does not list PMS2 c.497T>C, so no VCEP benign-functional pre-assignment exists for it.
• ClinVar (variation 135944): no submitter asserted BS3 for this variant; the criteria-bearing submissions used PP3, PP4 and PM1, and the benign/likely benign classifications of other submitters were not accompanied by a functional assay. Per interpretation policy, these final classifications were not used to infer BS3.
• OncoKB lookup for PMS2 L166P reported no variant-specific reviewed functional evidence (Unknown Oncogenic Effect), i.e. no curated benign functional evidence exists.
• SpliceAI max delta 0.012 (< the VCEP >=0.2 splice-defect threshold) removes any splicing-based functional argument, and being a missense substitution the variant is outside the synonymous/intronic branch of BS3_Strong.
• Computational metrics were not used for BS3: the HCI 'MAPP/PP2 Prior P' 0.9595, REVEL 0.776 and BayesDel 0.369188 are in-silico evidence for PP3/BP4 (and here lean non-benign), not functional assay readouts, and cannot support BS3.

Still missing

• No MMR functional assay (repair-activity, protein-expression/stability, subcellular-localisation or cellular-based mutator-phenotype assay) reports proficient, wild-type-like function for PMS2 c.497T>C (p.Leu166Pro), so no variant-specific BS3 evidence exists at any strength.
• No calibrated functional odds value for pathogenicity is available for this variant, so neither the BS3_Strong ceiling (<=0.05) nor the BS3_Supporting band (>0.05 and <=0.48) can be evaluated.
• No assay control comparison (wild-type, known pathogenic and known benign controls) or independent-replicate concordance data are documented for this variant, so assay validity cannot be confirmed even if a result were reported.
• The synonymous/intronic branch of BS3_Strong (no mRNA aberration with NMD inhibition) is inapplicable because the variant is a missense substitution, and no NMD-inhibited mRNA study exists for it.

Sources

Reasoning

Under the governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for PMS2 v2.0, BS4 requires a pedigree-based lack of co-segregation quantified as a combined Bayes likelihood ratio: <0.05 gives BS4_Strong and >0.05 with <=0.48 gives BS4_Supporting, with multiple pedigrees combined by multiplication and COOL/COsegregation OnLine recommended as the analysis tool. No segregation analysis of any kind exists for c.497T>C: the case record has no pedigree, no tested relatives, no affected non-carriers or unaffected carriers, and no Bayes factor. The only retrieved publication naming the variant (PMID:24113346) reports it as a VUS called incidentally on a single ColoNext panel without family testing, so it likewise yields no likelihood ratio. Because absence of a co-segregation analysis is not evidence of non-segregation, BS4 is left not assessed rather than not met - a low likelihood ratio must be demonstrated, not assumed from missing data. Closing the criterion would require a genotyped pedigree including informative non-segregating individuals (for example affected non-carriers or unaffected obligate carriers) analysed through COOL v3.

Rule

InSiGHT PMS2 v2.0 BS4: combined Bayes likelihood ratio <0.05 = Strong; >0.05 and <=0.48 = Supporting (lack of co-segregation; COOL/COsegregation OnLine recommended).

Evidence considered

• cspec (InSiGHT PMS2 v2.0) was read first as the governing framework; BS4 is Applicable and graded by the combined Bayes likelihood ratio for lack of co-segregation: <0.05 = Strong, >0.05 and <=0.48 = Supporting, with pedigrees combined by multiplication and COOL/COsegregation OnLine recommended.
• The case record contains no pedigree, no genotyped relatives and no segregation likelihood ratio for c.497T>C, so BS4 cannot be scored in either direction.
• PMID:24113346 reports p.L166P only as a VUS detected on one ColoNext panel with no family testing and no segregation analysis, providing no likelihood ratio for BS4.
• No source reports a family in which c.497T>C fails to track with disease; missing segregation data is a gap, not benign evidence, so BS4 is left not assessed rather than not met.

Still missing

• No pedigree or family data for c.497T>C, so no combined Bayes likelihood ratio for lack of co-segregation can be produced.
• No genotypes from affected non-carriers or unaffected carriers, which are the informative meioses BS4 depends on.
• No COOL/COsegregation OnLine (or equivalent) analysis has been performed for any family.
• Absence of a segregation study must not be read as demonstrated non-segregation.

Sources

Reasoning

CSPEC v2.0 defines BP5 for PMS2 as tumour-based benign evidence and assigns strength by tumour count and tumour characteristics: >=4 CRC/endometrial tumours with MSS and/or no loss of MMR protein expression and/or LS-spectrum tumours with loss of MMR protein(s) inconsistent with the gene demonstrating genetic variation = Strong (alternatively >=2 BRAF V600E (CRC only) or MLH1 methylation (LS spectrum tumour only) with MSI-H/MLH1 loss); 2 or 3 such tumours = Supporting (alternatively 1 BRAF V600E (colon only) or MLH1 methylation with MSI-H/MLH1 loss). Evaluating this requires tumour-level MSI/MSS status, MMR immunohistochemistry, and BRAF/MLH1-methylation results from the patient's or family's tumours. None of that exists in the assembled evidence: there is no tumour report, no MSI/MSS call, no MMR IHC, and no BRAF or MLH1 methylation result anywhere in the case; the ClinVar record provides only broad disease labels, and no full-text paper reports a tumour result attributable to p.L166P. BP5 is therefore not assessed rather than not met - the tumour-count thresholds cannot be applied without tumour data. (The generic ACMG sense of BP5, a variant found in a case with an alternate cause of disease, is likewise unevaluable because no case-level clinical data are present.)

Rule

CSPEC v2.0 (InSiGHT PMS2 V2.0), BP5: >=4 CRC/Endometrial MSS and/or no-MMR-loss and/or LS-spectrum tumours with MMR loss inconsistent with the variant gene (or >=2 BRAF V600E/MLH1 methylation with MSI-H/MLH1 loss) = Strong; 2-3 tumours (or 1 such marker) = Supporting.

Evidence considered

• CSPEC v2.0 BP5 rules: Strong = >=4 tumours (CRC/Endometrial MSS and/or no loss of MMR protein expression, and/or LS-spectrum tumours with MMR protein loss inconsistent with the gene carrying the variant) OR >=2 BRAF V600E (CRC only)/MLH1 methylation with MSI-H/MLH1 loss; Supporting = 2 or 3 such tumours OR 1 BRAF V600E (colon only)/MLH1 methylation with MSI-H/MLH1 loss.
• No tumour MSI/MSS, MMR IHC, BRAF V600E, or MLH1 methylation data appear in any assembled source for this case.
• ClinVar VCV000135944 lists only broad conditions (Lynch syndrome 4, Mismatch repair cancer syndrome 4, Hereditary nonpolyposis colorectal neoplasms) with no tumour-testing results.
• No full-text paper in the corpus reports MSI/IHC/methylation results attributable to PMS2 p.L166P.

Still missing

• No tumour MSI/MSS status available for any CRC, endometrial, or LS-spectrum tumour in this patient or family.
• No MMR protein immunohistochemistry result available, so 'no loss' or 'inconsistent loss' of MMR protein cannot be established.
• No BRAF V600E or MLH1 promoter methylation result available.
• No case-level clinical history describing an alternate cause of disease, so the generic ACMG BP5 reading is also unevaluable.

Sources