APC c.4893T>C, p.(Ser1631=)

Reasoning

The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis APC VCEP specification v2.1 (cspec), which defines BA1 as a stand-alone benign criterion when the gnomAD Popmax Filtering Allele Frequency is >= 0.1% (0.001). The variant's popmax filtering AF is 0.007257 (0.726%) in gnomAD v2.1 and 0.008497 (0.850%) in gnomAD v4.1, with the highest observed frequency in the African/African American population (0.825% and 0.906% respectively). This is roughly 7-8 fold above the 0.1% stand-alone threshold, and the overall all-comers allele frequency is also elevated (0.000800, 226/282598 alleles in v2.1; 0.000468, 756/1614156 alleles in v4.1). The threshold is unambiguously exceeded in every available dataset, including the non-cancer subsets, so BA1 is met at stand-alone strength. This criterion alone establishes the variant as Benign.

Rule

BA1 (Benign Stand Alone) — APC VCEP v2.1: gnomAD Popmax Filtering Allele Frequency (AF) >= 0.1% (0.001).

Evidence considered

• gnomAD v2.1 all-comers: total AF 0.000799723 (226/282598 alleles, 0 homozygotes); highest population African/African American AF 0.00825387 (0.825%); grpmax filtering AF 0.00725736 (0.726%); exome grpmax filtering AF 0.00682161, genome grpmax filtering AF 0.00725736.
• gnomAD v4.1 all-comers: total AF 0.000468356 (756/1614156 alleles, 9 homozygotes); highest population African/African American AF 0.00906159 (0.906%); grpmax filtering AF 0.00849683 (0.850%); exome grpmax filtering AF 0.00849628, genome grpmax filtering AF 0.00808593.
• APC VCEP specification v2.1 (InSiGHT) BA1 rule: 'GnomAD Popmax Filtering Allele Frequency (AF) >= 0.1% (0.001)', applied at Benign Stand Alone strength.
• The governing APC VCEP specification does not name a non-cancer or exome-only population source for BA1, so the all-comers gnomAD v2.1/v4.1 entries are the default; the non-cancer subsets present in the case were nevertheless checked and give the same verdict (gnomAD v2.1 non-cancer exomes grpmax filtering AF 0.00669606; gnomAD v3.1 non-cancer genomes grpmax filtering AF 0.00790141), both far above 0.1%.
• Population context: the variant is a synonymous change, p.(Ser1631=), observed at a frequency far above expectations for a pathogenic APC allele and at a frequency exceeding the disease prevalence-derived ceiling, which is the mechanistic basis for applying the stand-alone benign criterion.

Still missing

• The registered gnomAD-Canada (HostSeq genomes) source returned no variant record, so it contributes no frequency data; it is not needed for the verdict, which rests on gnomAD v2.1 and v4.1.

Sources

Reasoning

The APC VCEP v2.1 specification defines BS1 at Strong strength when the gnomAD Popmax Filtering Allele Frequency is >= 0.001% (0.00001). The observed popmax filtering AF is 0.007257 (0.726%) in gnomAD v2.1 and 0.008497 (0.850%) in gnomAD v4.1 — about 700-850 fold above the BS1 threshold — and the overall all-comers AF (0.000800 and 0.000468) is likewise orders of magnitude above any frequency expected for a pathogenic APC allele. The threshold is exceeded in all datasets examined, including the non-cancer subsets (non-cancer exome grpmax filtering AF 0.00670; non-cancer v3.1 genome grpmax filtering AF 0.00790). BS1 is therefore met at Strong strength. The same allele-frequency evidence already satisfies BA1 (stand-alone benign), so BS1 is subsumed by BA1 and is not additive; it is recorded here for completeness rather than double-counted in the benign combination.

Rule

BS1 (Benign Strong) — APC VCEP v2.1: gnomAD Popmax Filtering Allele Frequency (AF) >= 0.001% (0.00001).

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) BS1 rule: 'GnomAD Popmax Filtering Allele Frequency (AF) >= 0.001% (0.00001)', applied at Benign Strong strength.
• gnomAD v2.1 all-comers grpmax filtering AF 0.00725736 (0.726%), total AF 0.000799723 (226/282598 alleles).
• gnomAD v4.1 all-comers grpmax filtering AF 0.00849683 (0.850%), total AF 0.000468356 (756/1614156 alleles).
• Non-cancer subsets (checked although the VCEP does not mandate them): gnomAD v2.1 non-cancer exomes AF 0.000574611 (136/236682 alleles, 0 homozygotes), exome grpmax filtering AF 0.00669606; gnomAD v3.1 non-cancer genomes AF 0.00248104 (367/147922 alleles), grpmax filtering AF 0.00790141 — all still above the BS1 threshold.
• BS1 and BA1 draw on the identical frequency measurements; BA1 (stand-alone) already renders the variant Benign, so BS1 adds no independent benign weight.

Still missing

• BA1 and BS1 are both satisfied by the same allele-frequency data; BS1 should not be counted as an additional independent benign line of evidence alongside BA1 in combining rules.

Sources

Reasoning

The APC VCEP v2.1 specification defines BS2 at Strong strength when there are >= 10 points for healthy individuals OR the variant is observed >= 2 times in the homozygous state; a 'healthy individual' from control/non-cancer/normal/unaffected populations is worth 0.5 points. gnomAD v4.1 reports 9 homozygotes in total for this variant (4 in exomes and 5 in genomes), all within the African/African American group (9/75042 alleles, allele frequency 0.906%), which satisfies the >= 2 homozygotes clause at Strong strength. The healthy-individual points route is also satisfied, since gnomAD is a control/non-cancer population reference and the 756 carriers reported in v4.1 far exceed the 10-point (20 individual) threshold at 0.5 points per individual. Two caveats warrant human confirmation: gnomAD v2.1 reports 0 homozygotes for the same variant (so the homozygote evidence rests on the newer v4.1 and v3.1 datasets, where v3.1 non-cancer genomes independently report 4 homozygotes), and the applicability of the homozygous-state clause to reference-population homozygotes of a dominant-condition gene variant is an interpretation the specification does not spell out. Given the consistency of the supporting frequency evidence across datasets and the benign direction of the result, BS2 is called met at Strong, with review advised.

Rule

BS2 (Benign Strong) — APC VCEP v2.1: >= 10 points for healthy individuals OR >= 2 times in homozygous state.

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) BS2_Strong rule: '>= 10 points for healthy individuals OR >= 2 times in homozygous state', with a healthy individual from control/non-cancer/normal/unaffected populations worth 0.5 points; BS2_Supporting is '>= 3 points'.
• gnomAD v4.1 all-comers homozygous state: total_hom 9 (exome_hom 4, genome_hom 5); all 9 homozygotes are in the African/African American group (ac 680, an 75042, hom 9, AF 0.00906159).
• gnomAD v4.1 all-comers carrier count: 756 alleles observed (377 exome, 379 genome), i.e. several hundred control/non-cancer reference individuals, far exceeding the >= 10-point healthy-individual threshold when control-population individuals are scored at 0.5 points each.
• gnomAD v2.1 all-comers: 226 alleles, 0 homozygotes (exome_hom 0, genome_hom 0); gnomAD v2.1 non-cancer exomes: 136 alleles, 0 homozygotes — the v2.1 datasets alone therefore do not meet the homozygous-state clause.
• gnomAD v3.1 non-cancer genomes: total AF 0.00248104 (367/147922 alleles), total_hom 4 — an independent non-cancer dataset that also satisfies the >= 2 homozygotes clause.
• The variant is synonymous (p.(Ser1631=)) and non-loss-of-function, so homozygosity in reference-population individuals is biologically consistent with a benign interpretation rather than with biallelic APC disease.

Still missing

• gnomAD v2.1 reports 0 homozygotes for this variant while gnomAD v4.1 reports 9; the homozygous-state clause is satisfied using the larger, current v4.1 dataset (and independently by the v3.1 non-cancer genomes, which report 4 homozygotes), but the inter-version discrepancy should be acknowledged.
• The specification does not explicitly state whether reference-population homozygotes of a variant in a dominantly inherited gene should be counted under the '>= 2 times in homozygous state' clause; human review is advised to confirm this application.
• Exact per-individual 'healthy individual' point totals from curated control cohorts were not enumerated; the points route was inferred from population-reference status rather than from individually curated control records.

Sources

Reasoning

The governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1 retains BP1 at Benign Supporting strength and states that BP1 is applicable to APC with the exception of missense variants located in the first 15-amino-acid repeat of the beta-catenin binding domain (codon 1021-1035). The APC VCEP supplementary material records the corresponding panel comment that APC is a gene for which primarily truncating variants cause disease; APC loss-of-function through nonsense and frameshift alleles is the established FAP mechanism, so a change that does not alter the protein's amino-acid sequence is consistent with a benign, non-disease-causing allele. The variant NM_001127510.3:c.4893T>C is synonymous, NP_001120982.1:p.(Ser1631=), and lies at codon 1631, far outside the single excluded region (codons 1021-1035); the variant is therefore not the beta-catenin-domain missense exception, and BP1 applies. Independently of the definitional question of whether BP1's original ACMG wording ('missense variant') extends to a synonymous change, the governing VCEP has affirmatively declared the criterion applicable to APC except in one specified missense region, and this variant is outside that region. BP1 is therefore met at Supporting strength. Cancer Hotspots reported no significant hotspot at S1631, and ClinVar classifies the variant Benign/Likely benign across multiple submitters, both consistent with this benign-direction call, though neither was used to infer the criterion.

Rule

BP1 (APC VCEP v2.1): Supporting - applicable to APC except missense variants located in the first 15-amino-acid repeat of the beta-catenin binding domain (codon 1021-1035); APC is a gene for which primarily truncating variants cause disease.

Evidence considered

• The InSiGHT APC VCEP specification (cspec) gives BP1 at Benign Supporting strength with the rule: 'BP1 is applicable to APC with the exception of missense variants located in the first 15-amino-acid repeat of the beta-catenin binding domain (codon 1021-1035).'
• The APC VCEP supplementary material records the panel comment that APC is a gene for which primarily truncating variants cause disease, which is the premise of BP1.
• The variant is synonymous (NP_001120982.1:p.(Ser1631=)) at codon 1631, well outside the excluded codons 1021-1035, so it is not the beta-catenin-domain missense exception.
• PMID:25741868 (Richards et al. 2015) is the source of the BP1 definition - a variant in a gene for which primarily truncating variants are known to cause disease - and supplies the criterion's structure, not variant-level data.
• PMID:26467025 reports that synonymous (amino-acid-preserving) variants were overwhelmingly scored in the benign range (96% of synonymous coding variants studied), supporting the direction of BP1 for this non-amino-acid-altering change.

Still missing

• BP1 is defined in ACMG/AMP for missense variants; the APC VCEP's modification states the criterion is applicable to APC except missense in codons 1021-1035 but does not explicitly address synonymous changes, so application to a synonymous variant rests on the VCEP's affirmative applicability statement rather than an explicit synonymous-variant clause.

Sources

Reasoning

Scope first: BP4 is evaluated on the SpliceAI path because NM_001127510.3:c.4893T>C is a synonymous (silent) change, p.(Ser1631=), not a missense change; the APC VCEP explicitly states BP4 is not applicable to missense variants and is reserved for synonymous (silent) or intronic variants, so this variant is inside the criterion's scope. The governing rule (ClinGen InSiGHT APC v2.1) is satisfied when 'multiple in silico splicing predictors suggest no impact on gene or gene product'. SpliceAI Lookup returns a maximum delta score of 0.00 (DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00), i.e. no native acceptor or donor site is weakened and no cryptic site is created, and the same lookup reports Pangolin SG 0.003 / Pangolin SL -0.007 - two independent algorithms concurring on no impact. Against the published SpliceAI benign threshold of <=0.1 (Jaganathan et al. 2019, Cell, PMID:30661751), the 0.00 max delta is comfortably below the cutoff, and it also sits below the VCEP's own 'moderate cryptic gain' band of 0.2-0.8. BP4 is met at supporting strength. The APC VCEP permits BP7 together with BP4 ('The use of BP7 with BP4 is allowed'), so the shared predictor evidence is not treated as an impermissible double count.

Rule

APC VCEP v2.1 BP4 (supporting): 'Synonymous (silent) or intronic variants: Multiple in silico splicing predictors suggest no impact on gene or gene product.' (Missense variants: BP4 is not applicable.) Generic SpliceAI BP4 supporting threshold: max delta <= 0.1 (Jaganathan et al. 2019, PMID:30661751).

Evidence considered

• APC VCEP v2.1 BP4 (benign supporting) rule: 'Missense variants: BP4 is not applicable. Synonymous (silent) or intronic variants: Multiple in silico splicing predictors suggest no impact on gene or gene product.' Recommended programs are referenced to the panel's PS1 instructions (SpliceAI, MaxEntScan, VarSeak).
• NM_001127510.3:c.4893T>C is a synonymous variant (p.(Ser1631=)), which places it in BP4's synonymous/intronic scope rather than the inapplicable missense scope.
• SpliceAI Lookup result: maximum delta 0.00 (DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00) with Pangolin SG 0.003 and Pangolin SL -0.007 - two independent algorithms agree that the native splice sites are unaffected and no cryptic site is created.
• Generic calibrated SpliceAI BP4 threshold of <=0.1 (Jaganathan et al. 2019, Cell, PMID:30661751) applied because the APC VCEP supplies no separate numeric cutoff; the observed max delta of 0.00 is well below it, so BP4 supporting is met.
• The APC VCEP explicitly allows BP4 to be combined with BP7 ('The use of BP7 with BP4 is allowed'), so the identical predictor set underpinning both codes is applied as the specification intends rather than counted twice against an unstated prohibition.

Still missing

• MaxEntScan and VarSeak (also recommended in the APC VCEP specification) were not retrieved; the two available algorithms both predict no splice impact, so the 'multiple predictors' condition of the VCEP BP4 rule is already satisfied.

Sources

Reasoning

Scope first: NM_001127510.3:c.4893T>C is a synonymous (silent) variant, p.(Ser1631=), which is squarely within BP7's scope. The governing gene-specific rule - the ClinGen InSiGHT APC v2.1 BP7 supporting rule - reads: 'A synonymous (silent) or intronic variant at or beyond +7/-21 for which multiple splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site.' The variant is a synonymous coding change lying well within APC's large coding exon, not at a canonical or exon-proximal splice position, so it is at or beyond the +7/-21 boundary and inside BP7's scope. Two algorithms in the SpliceAI Lookup output agree there is no splice impact: SpliceAI maximum delta 0.00 (DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00 - the splice consensus sequence is unaffected and no new site is created, since both gain scores are 0) and Pangolin SG 0.003 / SL -0.007. This satisfies the 'multiple splicing prediction algorithms predict no impact' requirement, and the result is far below the closest published benign benchmark for splice predictors (SpliceAI <=0.1; Jaganathan et al. 2019, Cell, PMID:30661751) as well as below the VCEP's 0.2-0.8 moderate cryptic-gain band. BP7 is met at supporting strength; the APC VCEP states that use of BP7 with BP4 is allowed, so this does not double-count the shared prediction with BP4.

Rule

APC VCEP v2.1 BP7 (supporting): 'A synonymous (silent) or intronic variant at or beyond +7/-21 for which multiple splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site.'

Evidence considered

• APC VCEP v2.1 BP7 (benign supporting) rule: 'A synonymous (silent) or intronic variant at or beyond +7/-21 for which multiple splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site.' The panel's BP7 instructions add: 'The use of BP7 with BP4 is allowed.'
• NM_001127510.3:c.4893T>C is a synonymous variant (p.(Ser1631=)) within the large APC coding exon, so it sits at/beyond the +7/-21 boundary and inside BP7's scope; it is not a canonical +/-1 or 2 splice site (the APC VCEP's PVS1 decision-tree lists cover only those positions).
• SpliceAI Lookup result for this variant: maximum delta 0.00, with DS_AG 0.00 and DS_DG 0.00 (no new splice site created) and DS_AL 0.00 and DS_DL 0.00 (no native acceptor/donor site lost), plus Pangolin SG 0.003 and Pangolin SL -0.007. Two algorithms predict no impact on the splice consensus sequence and no creation of a cryptic site.
• The published SpliceAI benign benchmark of <=0.1 (Jaganathan et al. 2019, Cell, PMID:30661751) is far exceeded by the observed 0.00, and the VCEP's own guidance (native site loss at 0.8-1.0; strong cryptic gain 0.8-1.0, moderate 0.2-0.8) is likewise not approached.

Still missing

• MaxEntScan and VarSeak were not retrieved, but SpliceAI and Pangolin (two independent algorithms) both predict no impact, satisfying the VCEP requirement for multiple algorithms.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1 (superseding the generic ACMG/AMP PVS1 definition), which grades PVS1 through the APC modified PVS1 decision tree (Figure 1) and requires a null variant - nonsense, frameshift, canonical +/-1 or 2 splice site, initiation codon, or single/multi-exon deletion - in a gene where loss of function is the disease mechanism. The variant under assessment is NM_001127510.3:c.4893T>C, a single-nucleotide substitution in the APC transcript designated by the VCEP (NM_001127510) and it is synonymous: NP_001120982.1:p.(Ser1631=), so residue Ser1631 is unchanged. No premature termination codon is created and no reading frame is altered, therefore no truncated or elongated protein product is produced and no protein-length change occurs. NMD does not enter the analysis: NMD and the extreme-3'-end/exon-skipping cautions only bear on variants that generate a premature stop or disrupt a native splice site, and neither event is present here. Splice consequence was checked because a synonymous change can be loss-of-function through aberrant splicing: SpliceAI reports a maximum delta score of 0.00 (DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00; Pangolin SG 0.003, SL -0.007), i.e. no loss of the native splice site and no gain of a cryptic site, and the position is not a canonical splice consensus base. The variant therefore fails PVS1's null-variant precondition outright, so the criterion is not met and no strength tier is assigned. Because PVS1 is not met, the strength field is left null; no downgrade rule was applied or needed, since downgrading applies only when a criterion is met at full strength and a named SVI/VCEP rule reduces it.

Rule

PVS1 (APC VCEP v2.1): Null variant (nonsense, frameshift, canonical +/-1/2 splice site, initiation codon, single/multi-exon deletion) in a gene where LOF is the known mechanism, graded per the APC modified decision tree (Figure 1) - Very Strong / Strong / Moderate / Supporting. Not met here: the variant is a synonymous substitution with no null consequence, so no strength tier applies.

Evidence considered

• Governing framework: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for APC v2.1 (cspec). PVS1 is listed as applicable with default strength Pathogenic Very Strong, and every strength tier (Very Strong/Strong/Moderate/Supporting) is defined as 'Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (Figure 1)'.
• APC VCEP modified PVS1 decision tree (vcep_fig_1_apc_pvs1_decision_tree_2023_10_20) sorts specific variants into List A (PVS1), List B (PVS1_Strong), List C (PVS1_Moderate), List D (PVS1_Supporting) and List E (N/A); all entries are nonsense/frameshift/canonical splice-site changes. c.4893T>C is not listed, and it is not one of the variant types the tree evaluates.
• APC VCEP supplementary material (vcep_apc_specifications_supplementary_material_v2) comment on PVS1: 'APC is a gene where LOF is the predominant mechanism...' - loss of function is thus the established APC disease mechanism, so the gene-level gate is satisfied; the failure here is at the variant level, not the gene level.
• Variant consequence: NM_001127510.3:c.4893T>C translates to NP_001120982.1:p.(Ser1631=) - a synonymous (silent) substitution that leaves the amino-acid sequence and protein length unchanged (no truncation, no elongation, no in-frame length change).
• SpliceAI (spliceai) for NM_001127510.3:c.4893T>C: max delta score 0.00; component scores DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00; Pangolin SG 0.003 and SL -0.007. This excludes a splice-driven loss-of-function route (no native site loss, no cryptic site gain) that could otherwise have made the synonymous change act as a null allele.
• The variant lies within the large APC coding exon spanning c.1959-8532 in the VCEP supplementary exon table, but that region's exon-skipping consequence (out-of-frame) is irrelevant here because the variant does not alter splicing and does not create a premature stop codon, so neither NMD nor 3'-end truncation considerations arise.
• No RNA/cDNA assay, minigene, or other functional transcript data for this variant exist in the case record; the absence of a loss-of-function transcript effect rests on the silent nature of the change plus in-silico splice prediction.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1, which supersedes the generic ACMG/AMP PS1 definition. Under this specification PS1 has two arms: (1) for missense variants, when the variant under assessment results in the same amino acid change as a previously established Pathogenic (Strong) or Likely Pathogenic (Moderate) APC missense variant; and (2) for splice variants, when the variant affects splicing at the same nucleotide as a previously established Pathogenic/Likely Pathogenic variant, provided the splice prediction is above the thresholds defined in the instructions or is similar to the established variant by multiple in silico predictors. Neither arm can be satisfied here. The variant NM_001127510.3:c.4893T>C is synonymous, NP_001120982.1:p.(Ser1631=) - it produces no amino acid change at all, so it cannot result in the same amino acid change as any established pathogenic missense variant. The VCEP states that only two APC missense variants are currently Likely Pathogenic, c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg), and neither is at codon 1631; the variant does not alter that residue or any other residue. The splice arm also fails because SpliceAI max delta score is 0.00 (DS_AG/DS_AL/DS_DG/DS_DL all 0.0) so the change does not affect splicing at any nucleotide, and no previously established pathogenic APC variant at c.4893 is documented in the collected evidence. PS1 is therefore not met.

Rule

PS1 (APC VCEP v2.1): Strong - same amino acid change as a previously established Pathogenic APC variant (missense) or splice effect at the same nucleotide as a previously established Pathogenic variant above the defined splice-prediction thresholds; Moderate - same, relative to a previously established Likely Pathogenic variant. VCEP notes only two Likely Pathogenic APC missense variants exist: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg).

Evidence considered

• The InSiGHT APC VCEP specification (cspec) defines PS1 for missense variants as the same amino acid change as a previously established Pathogenic (Strong) or Likely Pathogenic (Moderate) APC missense variant, and for splice variants as affecting splicing at the same nucleotide as an established pathogenic variant with splice predictions above threshold.
• The variant is synonymous: normalization gives NP_001120982.1:p.(Ser1631=), i.e. no amino acid substitution, so the missense arm of PS1 cannot be satisfied at any strength.
• The cspec text states that only two APC missense variants are currently Likely Pathogenic - c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg) - neither of which is at residue 1631.
• SpliceAI (spliceai) reports max delta score 0.00 for NM_001127510.3:c.4893T>C (DS_AG 0.0, DS_AL 0.0, DS_DG 0.0, DS_DL 0.0), i.e. no predicted splice effect, so the splice arm of PS1 is not satisfied and no same-nucleotide pathogenic splice variant is evidenced.
• ClinVar reports this variant as Benign/Likely benign with no expert-panel submission and no established pathogenic APC variant at codon 1631; the ClinVar aggregate assertion was not used to infer PS1.

Sources

Reasoning

The APC VCEP v2.1 specification defines PM2 (at Supporting strength) as rare in controls, operationalised as an allele frequency <= 0.0003% (0.000003) when the allele count is > 1, or < 0.001% (0.00001) when the allele count is <= 1. The variant is far too common to qualify: in gnomAD v2.1 the all-comers allele frequency is 0.000800 (226 alleles, count > 1) and in gnomAD v4.1 it is 0.000468 (756 alleles), roughly 150-270 fold above the 0.000003 threshold. The non-cancer subsets are equally disqualifying (gnomAD v2.1 non-cancer exome AF 0.000575; gnomAD v3.1 non-cancer genome AF 0.002481). The variant also fails the generic SVI PM2 Supporting threshold of <= 0.0001 (0.01%), which it exceeds by ~5-8 fold. PM2 is therefore not met, and no intervening or stronger pathogenic-frequency evidence is present.

Rule

PM2 (Pathogenic Supporting) — APC VCEP v2.1: allele frequency <= 0.0003% (0.000003) if allele count > 1, OR < 0.001% (0.00001) if allele count <= 1.

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) PM2 rule: 'Rare in controls, defined by an allele frequency <= 0.0003% (0.000003) if the allele count is > 1 OR by an allele frequency < 0.001% (0.00001) if the allele count is <= 1', at Pathogenic Supporting strength.
• gnomAD v2.1 all-comers: total AF 0.000799723, allele count 226 (> 1), therefore the <= 0.000003 threshold applies and is exceeded ~267 fold.
• gnomAD v4.1 all-comers: total AF 0.000468356, allele count 756 (> 1), exceeding the <= 0.000003 threshold ~156 fold.
• Non-cancer subsets: gnomAD v2.1 non-cancer exomes AF 0.000574611 (136 alleles) and gnomAD v3.1 non-cancer genomes AF 0.00248104 (367 alleles) are likewise far above the PM2 threshold, so the verdict is unchanged under either the all-comers or the non-cancer source.
• Generic ACMG/ClinGen SVI default for comparison: PM2 (supporting) <= 0.0001 — also not met, since the smallest observed all-comers AF (0.000468) is ~5 fold higher; the more permissive VCEP threshold and the stricter generic threshold both fail.

Sources

Reasoning

The governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1 defines PM5 as a missense change at an amino acid residue where a different missense change has previously been established as Pathogenic (Moderate strength) or Likely Pathogenic (Supporting strength), with the additional requirement that the Grantham distance of the variant under assessment be equal to or greater than that of the reported variant; the specification further notes that only two APC missense variants are currently Likely Pathogenic, c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg), and that no APC missense variant has been classified as Pathogenic. The variant NM_001127510.3:c.4893T>C is synonymous, NP_001120982.1:p.(Ser1631=): it does not substitute any amino acid, so there is no 'amino acid residue where a different missense change has been seen' and no Grantham distance can be computed. The case's PM5 candidate collection (pm5_candidates.json) correspondingly found zero same-residue candidates and records the variant as ineligible for the classic same-residue missense PM5 search because its consequence class is synonymous, not missense. PM5 is therefore assessed and not met.

Rule

PM5 (APC VCEP v2.1): Moderate - missense change at an amino acid residue where a different missense change has been established as Pathogenic; Supporting - same, relative to a previously established Likely Pathogenic missense change. Grantham distance of the variant under assessment must be equal to or higher than that of the reported variant. Only p.(Asn1026Ser) and p.(Ser1028Arg) are currently Likely Pathogenic in APC.

Evidence considered

• The InSiGHT APC VCEP specification (cspec) defines PM5 as a missense change at a residue where a different missense change is established as Pathogenic (Moderate) or Likely Pathogenic (Supporting), requiring the variant's Grantham distance to be equal to or higher than the reported variant's.
• The specification lists only two Likely Pathogenic APC missense variants, c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg), and no Pathogenic APC missense variant; neither comparator position is residue 1631.
• Normalization gives NP_001120982.1:p.(Ser1631=), a synonymous change that alters no amino acid residue, so neither a same-residue comparison nor a Grantham distance can be generated.
• The case's PM5 comparator artifact (pm5_candidates) reported zero candidates seen and zero same-residue candidates, with the variant flagged ineligible for the classic same-residue missense PM5 search because its consequence class is synonymous.

Still missing

• No missense change exists at residue 1631 to compare against a previously established pathogenic APC missense variant; the applicability gap is intrinsic to a synonymous variant and cannot be closed by further evidence collection.

Sources

Reasoning

Scope first: NM_001127510.3:c.4893T>C normalizes to NP_001120982.1:p.(Ser1631=), a synonymous (silent) change, not a missense substitution, so the applicable computational path is splice prediction rather than REVEL (which is neither available nor applicable to a non-missense change). The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for APC v2.1, whose PP3 supporting rule requires that 'multiple in silico splicing predictors support a deleterious effect' for a non-canonical splicing variant (and directs that only splicing predictors - not conservation/evolution models - be used). The specification names SpliceAI, MaxEntScan and VarSeak as recommended programs but supplies no separate numeric PP3 cutoff, so the published calibrated SpliceAI thresholds apply: supporting >=0.2 and moderate >=0.5 (Jaganathan et al. 2019, Cell, PMID:30661751). SpliceAI Lookup returns a maximum delta score of 0.00 (DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00) - no loss or gain of an acceptor or donor site - and the same output reports Pangolin SG 0.003 / SL -0.007, likewise indicating no splice effect. Not a single available predictor supports a deleterious splicing effect, and 0.00 is far below the 0.2 supporting threshold and far below the specification's 0.8-1.0 splice-loss / strong-cryptic-gain band. PP3 is not met.

Rule

APC VCEP v2.1 PP3 (supporting): 'Missense variants: Do not use computational prediction models for conservation, evolution, etc. In silico splicing predictors should be used for presumed missense variants to reveal possible splicing effects. Non-canonical splicing variants: Multiple in silico splicing predictors support a deleterious effect.' Generic SpliceAI PP3 supporting threshold: max delta >= 0.2 (Jaganathan et al. 2019, PMID:30661751).

Evidence considered

• APC VCEP v2.1 (ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel, doc 1529292791) PP3 supporting rule: for non-canonical splicing variants, multiple in silico splicing predictors must support a deleterious effect; for presumed missense variants only splicing predictors are to be used. Recommended programs are SpliceAI, MaxEntScan and VarSeak, with numeric guidance given in the panel's PS1 instructions (SpliceAI splice-site loss considered for 0.8-1.0; cryptic-site gain strong for 0.8-1.0 and moderate for 0.2-0.8).
• The case normalization identifies NM_001127510.3:c.4893T>C as NP_001120982.1:p.(Ser1631=), a synonymous variant, which selects the splice-prediction path for PP3 and makes REVEL not applicable.
• SpliceAI Lookup output for this variant: maximum delta score 0.00, with DS_AG 0.00, DS_AL 0.00, DS_DG 0.00 and DS_DL 0.00 (no acceptor or donor site lost or gained); the same lookup reports Pangolin SG 0.003 and Pangolin SL -0.007, also indicating no splice effect.
• Generic calibrated SpliceAI thresholds were applied because the APC VCEP provides no separate numeric PP3 cutoff: PP3 supporting >=0.2 and PP3 moderate >=0.5 (Jaganathan et al. 2019, Cell, PMID:30661751). The observed max delta 0.00 is below both, and no second predictor supports a deleterious splicing effect, so PP3 supporting is not met.
• No variant-specific publication reporting splice-prediction or RNA data for c.4893T>C was identified; the papers extracted for this case (PMID:24996433, PMID:23852704, PMID:26467025, PMID:25741868) do not mention this variant.

Still missing

• MaxEntScan and VarSeak (the other splice programs recommended in the APC VCEP specification) were not retrieved for this variant; both available algorithms (SpliceAI, Pangolin) predict no splice effect, so their absence cannot create PP3 support and does not change the verdict.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1, which supersedes the generic ACMG/AMP PS2 definition. Under this specification PS2 covers a confirmed de novo event (both maternity and paternity confirmed) and is graded by a de novo score curated from Tables 1 and 2: a proband with >=1 phenotype point contributes 2 de novo points and a proband with 0.5 phenotype points contributes 1 de novo point when parental relationships are confirmed. Strength thresholds are Very Strong at >=4 de novo scores, Strong at 2-3.5 and Moderate at 1-1.5. The case record contains no proband, no parental/maternity-paternity testing, no phenotype points and no family data of any kind; the collected evidence is entirely variant-level (ClinVar, gnomAD v2.1/v4.1/v3.1 non-cancer/HostSeq, SpliceAI, OncoKB, COSMIC, Cancer Hotspots) plus generic guidance literature. Because neither the confirmed-parentage requirement nor the phenotype-point component of the de novo score can be evaluated, PS2 cannot be adjudicated and is returned not_assessed rather than not_met; the missing data (a de novo observation with confirmed parentage) was never available to be contradicted.

Rule

PS2 (APC VCEP v2.1): de novo (both maternity and paternity confirmed) - Very Strong >=4 de novo scores; Strong 2-3.5; Moderate 1-1.5. De novo score curated per Tables 1 and 2.

Evidence considered

• The InSiGHT APC VCEP specification (cspec) is the governing framework and defines PS2 as a de novo event with both maternity and paternity confirmed, graded by de novo scores curated from Tables 1 and 2 (Very Strong >=4, Strong 2-3.5, Moderate 1-1.5).
• Table 1 and 2 (vcep_table_1_262) converts each proband's phenotype points into de novo scores: >=1 phenotype point = 2 de novo points (1 if parental relationships unconfirmed); 0.5 phenotype points = 1 de novo point (0.5 if unconfirmed).
• No proband, parental testing result, or phenotype record appears anywhere in the case evidence; the evidence corpus is limited to variant-level annotations.
• ClinVar reports this variant as Benign/Likely benign (multiple submitters, no conflicts), but no ClinVar submission was used to infer PS2 and no de novo claim is asserted by any source.

Still missing

• No proband-level record identifying an affected individual carrying this variant.
• No parental testing (maternity and paternity confirmation) reported for any proband.
• No phenotype-point curation for any proband, so no de novo score (0.5/1/2 per proband, Tables 1 and 2) can be assigned.

Sources

Reasoning

The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for APC v2.1, which replaces the generic PS3 definition with calibrated RNA-assay rules: a premature stop codon or in-frame skipping of exon 13 or 14 with absence of full-length transcript (Very Strong) or with <10% full-length transcript (Strong), other defined exon-skipping / alternative-transcript findings (Moderate or Supporting), and, for variants within the beta-catenin binding domain (codons 959-2129), protein assays showing increased beta-catenin-regulated transcription activity and/or decreased beta-catenin binding by surface plasmon resonance (Supporting). NM_001127510.3:c.4893T>C encodes NP_001120982.1:p.(Ser1631=), a synonymous (silent) change at codon 1631, which lies inside that codon 959-2129 domain, so the protein-assay route is nominally in scope even though the amino-acid sequence is unchanged. However, no variant-specific functional evidence exists in any reviewed source: no RNA transcript analysis (transcript structure, proportion of full-length transcript, alternative-transcript over-expression), no beta-catenin-regulated transcription or surface plasmon resonance measurement, and no assay control or reproducibility data. ClinVar (variation 132688, Benign/Likely benign, 2 stars, 18 submissions) records no functional testing, and the submission-level evidence signals describe only in-silico splicing predictions. OncoKB reports 'Unknown Oncogenic Effect' with no variant-specific functional or biological-effect annotation and no functional PMIDs. The four retrieved full-text publications do not report this variant or any APC functional assay. PS3 therefore cannot be applied at any strength.

Rule

APC VCEP PS3: RNA assays show a premature stop codon or in-frame skipping of exon 13/14 (absence of full-length transcript = Very Strong; <10% full-length transcript = Strong), other defined exon-skipping/alternative-transcript findings (Moderate/Supporting), or protein assays show increased beta-catenin-regulated transcription activity and/or decreased beta-catenin binding by surface plasmon resonance for variants within the beta-catenin binding domain (codons 959-2129), Supporting.

Evidence considered

• APC InSiGHT VCEP v2.1 PS3 specification: RNA assays showing a premature stop codon or in-frame skipping of exon 13 or 14 with absence of full-length transcript (Very Strong) or <10% full-length transcript (Strong); a premature stop codon or exon 13/14 skipping together with reports of exon deletion/skipping/loss or intronic insertion (Moderate); other in-frame skipping with absent or <10% full-length transcript (Moderate); in-frame skipping of exons other than 13/14 or over-expression of an alternative transcript (exons 10, 11 or 15) or increased beta-catenin-regulated transcription activity and/or decreased beta-catenin binding by surface plasmon resonance for variants within the beta-catenin binding domain (codons 959-2129) (Supporting).
• The VCEP supplementary material adds that absence of the full-length transcript or a variant-allele full-length proportion <10% should be demonstrated, for example by Sanger sequencing of the full-length fragment or allele-specific expression, and that alternative-transcript over-expression must be demonstrated comparably to the published exon 15 example.
• The variant is NM_001127510.3:c.4893T>C, NP_001120982.1:p.(Ser1631=): a synonymous change at codon 1631, within the VCEP-specified beta-catenin binding domain (codons 959-2129), although it does not alter the amino-acid sequence.
• ClinVar variation 132688 (Benign/Likely benign, criteria provided, multiple submitters, no conflicts; 2 stars; 18 submissions) contains no functional study; submission evidence signals cite only in-silico splicing predictions, e.g. '5/5 in silico tools via alamut predict no significant effect on normal splicing'.
• OncoKB lists APC p.Ser1631= with oncogenicity label 'Unknown Oncogenic Effect', no variant-specific functional annotation, no biological effect and no cited PMIDs.
• No variant-specific RNA transcript analysis, beta-catenin-regulated transcription assay, or surface plasmon resonance binding measurement was identified in any reviewed source, and none of the retrieved full-text publications reports functional testing of this variant.

Still missing

• A variant-specific RNA assay is required: transcript analysis demonstrating the presence and proportion of full-length transcript (VCEP PS3 thresholds are absence of full-length transcript for Very Strong and <10% full-length transcript for Strong), or defined exon-skipping / alternative-transcript findings at the calibrated strengths.
• For the protein route in scope at this domain position (codons 959-2129), beta-catenin-regulated transcription activity and/or beta-catenin binding by surface plasmon resonance have not been measured.
• No assay validation, wild-type or disease-positive control, or reproducibility data are available for any APC functional assay applied to this variant.

Sources

Reasoning

The governing specification is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel APC specification v2.1, which modifies PS4 from a plain case-control enrichment criterion into a proband phenotype-point system based on Fortuno et al. 2020 (PMID: 33300245) using Table 1 (maximum 1 phenotype point per proband; 1 point for a phenotype highly specific for APC, 0.5 point for a phenotype consistent with but not highly specific for APC). Exact comparison operators as written in the governing source text for THIS criterion: PS4_Very Strong requires phenotype points >= 16; PS4 (Strong) requires 4-15.5 phenotype points; PS4_Moderate requires 2-3.5 phenotype points; PS4_Supporting requires 1-1.5 phenotype points. Every one of these operators is a >= / lower-bound operator (evidence strengthens as the phenotype-point value goes UP), e.g. the minimum qualifying condition for any PS4 weight is 'phenotype points >= 1.00?'. Substituting this case's value: the number of affected probands carrying c.4893T>C for whom Table 1 phenotype points can be scored is 0, so the written inequality is '0 >= 1? no'. The criterion also requires the variant's prevalence in affected individuals to be significantly increased versus controls, and no exact-variant case or control counts, odds ratio, p-value or confidence interval exist for this variant. This case contains no proband-level phenotype, family history or multi-gene panel result for a carrier of this variant (the case is a variant-only submission; prefetch.json carries no clinical/phenotype fields), so no phenotype points can be assigned and the enrichment comparison cannot be constructed. The exact-variant ClinVar record (ClinVarID 132688, 18 single-submitter clinical-laboratory submissions, zero expert-panel submissions) supplies only classifications, not variant-specific case/control counts, and a source's final classification cannot be transferred into PS4. PS4 therefore cannot be adjudicated from the available evidence and is returned not_assessed rather than not_met, because the value needed for its own comparison is missing, not demonstrably below threshold.

Rule

APC VCEP PS4: 'The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls'; modified per Fortuno et al. 2020 (PMID: 33300245) to a phenotype-point system from Table 1: >= 16 phenotype points = Very Strong; 4-15.5 = Strong; 2-3.5 = Moderate; 1-1.5 = Supporting (max 1 point per proband).

Evidence considered

• APC VCEP specification v2.1 PS4 rules (verbatim): '>= 16 phenotype points' = PS4_Very Strong; '4-15.5 phenotype points' = PS4 (Strong); '2-3.5 phenotype points' = PS4_Moderate; '1-1.5 phenotype point' = PS4_Supporting. All four weights are lower-bound (>=) constructions, so any PS4 weight strengthens as the phenotype-point value increases.
• APC VCEP supplementary material, PS4 row: 'The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls' with the modification noted as 'Modifications are based on Fortuno et al. 2020 (PMID: 33300245)'.
• APC VCEP Table 1 (point system relevant to PS2, PS4, PM6, PP1 and BS4, maximum 1 point per proband): 1 phenotype point for a colorectal phenotype highly specific for APC (e.g. 20-99 colorectal adenomas with confirmation and age <= 20 years); 0.5 point for a phenotype consistent with but not highly specific for APC (e.g. >= 20 colorectal adenomas at 20-70 years, or a documented FAP/AFAP diagnosis).
• Written inequality for this case: number of affected probands with scorable Table 1 phenotype points = 0, therefore '0 >= 1?' is no; no PS4 strength is reached.
• This case provides no proband phenotype, family history or carrier multi-gene result; the variant-only input means no carrier exists in the supplied evidence for whom phenotype points could be counted.
• The exact-variant ClinVar record reports Benign (12 laboratories), Likely benign (5 laboratories) and benign (1 laboratory) from single submitters, all 'criteria provided, single submitter' or 'no assertion criteria provided', with zero expert-panel submissions; these are submissions of a classification and carry no variant-specific case/control counts for PS4, so they were not converted into PS4 evidence.
• PMID:20301519 (APC-Associated Polyposis Conditions) and PMID:26389258 (PDQ Cancer Genetics Risk Assessment and Counseling) were flagged by literature triage as candidate sources for PS4/PP5 but no full text was retrieved for either (screenshot only), and both are gene-level clinical resources that would not carry variant-specific case data; PMID:24996433, a RAS-testing guidance document also flagged for PS4/PP5, was extracted and contains no mention of APC c.4893T>C or of APC at all.

Still missing

• No proband-level personal or family phenotype for a carrier of APC c.4893T>C, so no Table 1 phenotype points can be scored (minimum qualifying value for any PS4 weight is >= 1 point per proband)
• No exact-variant case and control counts, odds ratio/relative risk, p-value or 95% confidence interval establishing increased prevalence in affected individuals versus controls

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1, which supersedes the generic ACMG/AMP PM6 definition. Under this specification PM6 covers an assumed de novo event without confirmation of paternity and maternity and is graded by the same de novo score curation as PS2 (Tables 1 and 2), at lower weights: Strong at 2-3.5 de novo scores, Moderate at 1-1.5 and Supporting at 0.5. Even the lowest applicable strength therefore requires at least 0.5 de novo points, i.e. a proband with at least 0.5 phenotype points reported as a de novo occurrence. The case record contains no proband, no phenotype points and no statement that this variant arose de novo in any individual; all collected evidence is variant-level (ClinVar, gnomAD, SpliceAI, OncoKB, COSMIC, Cancer Hotspots) plus generic guidance literature. With no de novo observation and no phenotype-point curation available, PM6 cannot be adjudicated and is returned not_assessed rather than not_met.

Rule

PM6 (APC VCEP v2.1): assumed de novo, without confirmation of paternity and maternity - Strong 2-3.5; Moderate 1-1.5; Supporting 0.5 de novo scores (Tables 1 and 2).

Evidence considered

• The InSiGHT APC VCEP specification (cspec) defines PM6 as assumed de novo without confirmation of paternity/maternity, graded Strong 2-3.5, Moderate 1-1.5, Supporting 0.5 de novo scores using the same Tables 1 and 2 as PS2.
• Table 1 and 2 (vcep_table_1_262) supplies the phenotype-point to de novo-score conversion (proband with >=1 phenotype point = 1 de novo point unconfirmed; 0.5 phenotype points = 0.5 de novo points unconfirmed).
• No source in the case record reports this variant as de novo, and no proband phenotype is documented, so no de novo score of any magnitude can be produced.
• ClinVar's Benign/Likely benign aggregate assertion was noted but not used to derive PM6, per the rule against inferring a criterion from another source's final classification.

Still missing

• No report of a de novo occurrence of this variant in any proband (with or without parental confirmation).
• No phenotype-point curation for any proband, so no de novo score can be assigned.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1, which supersedes the generic ACMG/AMP PP1 definition. Under this specification PP1 depends solely on the number of informative meioses co-segregating with disease: Strong at >=7 meioses in >=2 families, Moderate at 5-6 meioses in >=1 family and Supporting at 3-4 meioses in >=1 family. The lowest applicable strength therefore requires at least 3 informative meioses in a single family. The case record contains no pedigree, no family members, no genotype-phenotype tracing and no meiosis count; every collected source is variant-level (ClinVar, gnomAD, SpliceAI, OncoKB, COSMIC, Cancer Hotspots) or generic guidance literature. With zero informative meioses available for curation, PP1 cannot be adjudicated and is returned not_assessed rather than not_met.

Rule

PP1 (APC VCEP v2.1): Strong >=7 meioses in >=2 families; Moderate 5-6 meioses in >=1 family; Supporting 3-4 meioses in >=1 family.

Evidence considered

• The InSiGHT APC VCEP specification (cspec) defines PP1 by meiosis count: Strong >=7 meioses in >=2 families; Moderate 5-6 meioses in >=1 family; Supporting 3-4 meioses in >=1 family.
• No segregation data, pedigree, relative genotypes or meiosis counts are present anywhere in the case evidence.
• Table 1 and 2 (vcep_table_1_262) provides the phenotype-point definitions that would be used to score affected relatives during a segregation analysis, but no relatives are documented to which they could be applied.
• ClinVar's Benign/Likely benign aggregate assertion was not used to infer PP1.

Still missing

• No pedigree or family data for any proband carrying this variant.
• No genotype-phenotype tracing in relatives, so no informative meiosis count can be derived (Supporting needs >=3 meioses in >=1 family).

Sources

Reasoning

The APC InSiGHT VCEP specification defines BS3_Strong for an RNA assay of a synonymous or intronic variant in a constitutional patient sample demonstrating no mRNA aberration together with demonstrated biallelic expression and/or nonsense-mediated decay inhibition, and BS3_Supporting for the same RNA result without those controls, or for a protein assay showing retention of beta-catenin-regulated transcription activity comparable to wild type (only for variants within the beta-catenin binding domain, codons 959-2129; the VCEP supplementary material otherwise adds 'no comment' for BS3). NM_001127510.3:c.4893T>C is a synonymous variant at codon 1631, inside the beta-catenin binding domain, so both the RNA and the protein-benign routes are nominally in scope. No RNA assay has been performed: there is no transcript analysis in a constitutional sample, no assessment of transcript structure or full-length transcript, and no biallelic-expression or NMD-inhibition data, and no protein assay of beta-catenin-regulated transcription activity showing wild-type-equivalent retention has been reported. The only splicing data available are in silico (SpliceAI max delta 0.00; the ClinVar submitters likewise cite only in-silico predictors), and an in-silico prediction is not the RNA assay the VCEP requires, so it cannot satisfy BS3 at any strength. ClinVar (variation 132688) and OncoKB contain no variant-specific functional assay result, and no reviewed publication reports one. BS3 therefore cannot be applied.

Rule

APC VCEP BS3: RNA assay of a synonymous or intronic variant in a constitutional patient sample demonstrates no mRNA aberration AND biallelic expression is shown and/or nonsense-mediated decay inhibition was used (Strong); OR RNA assay with no mRNA aberration without biallelic-expression/NMD demonstration, OR protein assay showing retention of beta-catenin-regulated transcription activity comparable to wild-type for variants within the beta-catenin binding domain (codons 959-2129) (Supporting).

Evidence considered

• APC InSiGHT VCEP v2.1 BS3 specification: RNA assay of a synonymous or intronic variant in a constitutional patient sample demonstrates no mRNA aberration AND biallelic expression is shown and/or nonsense-mediated decay inhibition was used (Strong); RNA assay of a synonymous or intronic variant showing no mRNA aberration without demonstration of biallelic expression or NMD inhibition, OR protein assay showing retention of beta-catenin-regulated transcription activity comparable to wild type for variants within the beta-catenin binding domain (codons 959-2129) (Supporting).
• The variant is a synonymous change, NP_001120982.1:p.(Ser1631=) at codon 1631, within the beta-catenin binding domain (codons 959-2129) specified for the protein-assay route.
• ClinVar variation 132688 (Benign/Likely benign, 2 stars, 18 submissions) records no RNA or protein functional assay; its submission-level evidence signals cite only in-silico splicing prediction programs (splice site finder, MaxEntScan, NNSPLICE, GeneSplicer).
• OncoKB reports APC p.Ser1631= as 'Unknown Oncogenic Effect' with no variant-specific functional or biological-effect data and no cited PMIDs.
• SpliceAI Lookup reports a maximum delta score of 0.00 for this variant; this is an in-silico prediction and is not the RNA assay required by the VCEP for BS3.

Still missing

• An RNA assay in a constitutional patient sample showing no mRNA aberration is required; demonstrated biallelic expression and/or NMD inhibition would be needed for BS3_Strong rather than Supporting.
• Alternatively, a protein assay demonstrating beta-catenin-regulated transcription activity comparable to wild type would support BS3_Supporting for this domain-localised variant (codons 959-2129); no such assay has been performed.
• No assay controls (wild-type comparator, positive/negative controls) or reproducibility data are available.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1, which supersedes the generic ACMG/AMP BS4 definition. Under this specification BS4 requires a lack of segregation: affected member(s) without the variant must score phenotype points, using Strong when one affected member without the variant scores >=1 phenotype point or at least two affected members without the variant each score >=0.5 points, and Supporting when one affected member without the variant scores >=0.5 phenotype points (Table 1). The case record contains no family members, no genotypes of affected relatives and no phenotype-point curation of anyone; the evidence is entirely variant-level (ClinVar, gnomAD, SpliceAI, OncoKB, COSMIC, Cancer Hotspots) plus generic guidance literature. Without any affected individual tested negative for this variant, absence of segregation cannot be established or refuted, so BS4 is returned not_assessed rather than not_met. The synonymous nature of the change (p.(Ser1631=)) is not itself BS4 evidence, since BS4 is defined solely by phenotype status of untested-for-variant affected relatives.

Rule

BS4 (APC VCEP v2.1): Strong - affected member without the variant scores >=1 phenotype point, or >=2 affected members without the variant each score >=0.5 phenotype points; Supporting - affected member without the variant scores >=0.5 phenotype points (Table 1).

Evidence considered

• The InSiGHT APC VCEP specification (cspec) defines BS4 by absence of segregation: Strong when an affected member without the variant scores >=1 phenotype point (or two such members each score >=0.5), Supporting when an affected member without the variant scores >=0.5 phenotype points.
• Table 1 and 2 (vcep_table_1_262) supplies the phenotype-point assignments (1 for highly specific phenotypes such as typical 20-99 colorectal adenomas, 0.5 for other APC-consistent phenotypes) that BS4 would apply to affected non-carriers.
• No pedigree, affected relative or genotype information is present in the case evidence, so no affected individual lacking the variant can be identified and scored.
• ClinVar's Benign/Likely benign aggregate assertion was not used to derive BS4.

Still missing

• No affected family members documented, so no affected individual without the variant can be scored for phenotype points.
• No genotype results for relatives of any proband, so absence of co-segregation cannot be demonstrated (Supporting needs one affected non-carrier scoring >=0.5 phenotype points).

Sources

Reasoning

Under the APC VCEP Version 2.1, BP2 (Benign Supporting) is satisfied when the variant is observed in trans with a (Likely) Pathogenic APC variant, OR observed at least 3 times in an unknown phase with different (Likely) Pathogenic APC variants. Both arms require case-level allelic evidence: a second (likely) pathogenic APC allele in the same individual, and, for the first arm, a demonstrated phase. NM_001127510.3:c.4893T>C (NP_001120982.1:p.(Ser1631=)) is a synonymous change evaluated here as a variant-level record: it contains no affected-proband genotype, no second APC variant, no experimental or pedigree-based cis/trans phase determination, and no qualifying co-occurrence count against any (likely) pathogenic APC variant. ClinVar variation 132688 is represented by 19 submissions (17 usable for classification, all Benign or Likely benign; zero expert-panel submissions and zero criterion leads), none of which reports a co-occurring pathogenic APC allele or an allelic configuration for this record. Population observations are unphased and therefore cannot establish cis or trans: gnomAD v2.1 reports 226/282,598 alleles with 0 homozygotes, while gnomAD v4.1 reports 756/1,614,156 alleles with 9 homozygotes (all in African/African American individuals). Those 9 population homozygotes are themselves inconsistent with an in-trans partner pathogenic allele in those individuals, but they are anonymous population observations and do not substitute for the case-level phase evidence BP2 requires. BP2 is therefore not assessable on the available evidence rather than affirmatively refuted; the decisive gap is the absence of any second APC variant and any phase determination.

Rule

APC VCEP Version 2.1 BP2 (Benign Supporting): observed in trans with a (Likely) Pathogenic APC variant OR observed at least 3 times in an unknown phase with different (Likely) Pathogenic APC variants.

Evidence considered

• The APC VCEP Version 2.1 lists BP2 as applicable at Supporting strength with the rule: observed in trans with a (Likely) Pathogenic APC variant OR at least 3 times in an unknown phase with different (Likely) Pathogenic APC variants.
• No second APC variant, proband genotype, phase assignment, or qualifying co-occurrence observation is documented anywhere in this record; the evaluation is a single-variant record with database and computational results only.
• ClinVar variation 132688 has 19 submissions (17 usable for classification), all Benign or Likely benign, with zero expert-panel submissions and zero criterion leads; no submission reports an allelic configuration or a co-occurring pathogenic APC variant.
• gnomAD v2.1: 226 alternate alleles in 282,598 with 0 homozygotes (total AF 0.0008; highest subpopulation African/African American AF 0.0083). gnomAD v4.1: 756 alternate alleles in 1,614,156 with 9 homozygotes (total AF 0.00047; African/African American AF 0.0091, 9 homozygotes). These unphased population counts cannot establish cis or trans configuration with a pathogenic variant.
• ClinVar-cited papers checked for this record (PMID:26467025, PMID:23852704, PMID:24996433) contain no phase, in-trans/in-cis, second-variant, proband or inheritance observations for this variant or for APC.

Still missing

• Documented co-occurrence of c.4893T>C with a (Likely) Pathogenic APC variant in the same individual, with cis/trans configuration stated
• Experimental (parental/offspring genotyping) or pedigree-based cis/trans phase determination
• At least three independent observations of this variant in unknown phase alongside different (Likely) Pathogenic APC variants
• Affected-proband genotype and phenotype context, if a case-level record is available

Sources

Reasoning

The governing APC VCEP specification modifies BP5 to a narrow, condition-dependent rule. The supplementary material BP5 row states verbatim: 'Can be used, if a (Likely) Pathogenic variant in another adenomatous polyposis gene was identified (heterozygous variants in POLD1 or POLE; biallelic variants in MUTYH, NTHL1 or MSH3; in patients with onset in childhood / adolescence: biallelic variants in MLH1, MSH2, MSH6 or PMS2). This rule is only applicable when a colorectal polyposis phenotype is present.' The CSPEC criteria list records BP5 as 'Only applicable for an alternate genetic basis of the colorectal polyposis phenotype.' Two conditions must therefore both hold: (1) a (Likely) Pathogenic variant in a named alternate polyposis gene must have been identified in the case, and (2) a colorectal polyposis phenotype must be present. This case supplies neither: the input is a single APC variant with no proband phenotype or family history, and no multi-gene panel result, alternate-gene variant, or polyposis phenotype documentation exists anywhere in the case evidence. No numeric threshold applies to this criterion, so no comparison operator can be quoted. BP5 is returned not_assessed (the criterion's precondition data are absent) rather than not_met, since the absence of an alternate genetic basis in the supplied evidence reflects missing case-level data rather than a demonstrated lack of an alternate molecular basis.

Rule

APC VCEP BP5: usable only if a (Likely) Pathogenic variant in another adenomatous polyposis gene was identified (heterozygous POLD1 or POLE; biallelic MUTYH, NTHL1 or MSH3; biallelic MLH1, MSH2, MSH6 or PMS2 for childhood/adolescence onset) AND a colorectal polyposis phenotype is present.

Evidence considered

• APC VCEP supplementary material, BP5 row (verbatim): 'Can be used, if a (Likely) Pathogenic variant in another adenomatous polyposis gene was identified (heterozygous variants in POLD1 or POLE; biallelic variants in MUTYH, NTHL1 or MSH3; in patients with onset in childhood / adolescence: biallelic variants in MLH1, MSH2, MSH6 or PMS2). This rule is only applicable when a colorectal polyposis phenotype is present.'
• ClinGen InSiGHT APC specification v2.1 criteria list records BP5 as 'Only applicable for an alternate genetic basis of the colorectal polyposis phenotype.'
• The case input is a single APC variant (NM_001127510.3:c.4893T>C) with no proband phenotype, family history, or multi-gene testing result in prefetch.json or any other case file; neither precondition of the APC BP5 rule can be established.

Still missing

• No documentation of a colorectal polyposis phenotype in the case, which is a precondition for BP5 under the APC VCEP rule
• No multi-gene panel or other result identifying a (Likely) Pathogenic variant in an alternate adenomatous polyposis gene (POLD1, POLE, MUTYH, NTHL1, MSH3, or childhood/adolescence-onset MLH1/MSH2/MSH6/PMS2)

Sources