APC c.78C>A, p.(Ser26Arg)

Reasoning

The governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis APC VCEP specification v2.1 (cspec), so generic ACMG frequency defaults do not apply. BA1 is specified as gnomAD Popmax Filtering Allele Frequency >= 0.1% (0.001) at Benign Stand Alone strength, and the specification directs use of the non-cancer dataset from gnomAD (v2.1.1), preferably its Popmax/grpmax filtering AF, with subpopulations usable at the highest minor allele frequency provided >=2000 alleles were tested (not founder populations). In the VCEP-directed non-cancer gnomAD v2.1.1 exomes the variant's popmax filtering AF is 0.02778749 (2.78%), carried by the African/African American subpopulation (AF 3.018%, 448/14844 alleles); the exome-only non-cancer allele frequency is 0.00186391 (249/133590 alleles). That is roughly 28-fold above the 0.1% stand-alone threshold. The VCEP's own derivation of this threshold (prevalence 1:5,000, genetic heterogeneity 0.5, penetrance 0.8, calculated AF >=0.006%, rounded 10-fold to 0.1%) makes an allele frequency of 2.78% incompatible with a pathogenic APC allele. Independent datasets agree: gnomAD v3.1 non-cancer genomes popmax FAF 0.0280573 (2.81%, 1KG:ESN 3.846%); all-comers gnomAD v2.1 grpmax FAF 0.02768 (2.77%) and gnomAD v4.1 grpmax FAF 0.0285595 (2.86%); all-comers total AF 0.31% (v2.1) and 0.17% (v4.1). BA1 is met at stand-alone strength; this criterion alone establishes the variant as Benign.

Rule

BA1 (Benign Stand Alone) - APC VCEP v2.1 / InSiGHT: gnomAD Popmax Filtering Allele Frequency (AF) >= 0.1% (0.001), preferably from the non-cancer dataset from gnomAD (v2.1.1), highest minor allele frequency across subpopulations.

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) BA1 (Benign Stand Alone) rule: 'GnomAD Popmax Filtering Allele Frequency (AF) >= 0.1% (0.001)', with instructions to use the non-cancer dataset from gnomAD (v2.1.1); the supplementary material adds 'preferably based on the Popmax Filtering Allele Frequency of the non-cancer dataset from gnomAD (v2.1.1)' and permits subpopulations with >=2000 tested alleles, considering the highest minor allele frequency.
• Threshold derivation stated in the specification: prevalence 1:5,000, genetic heterogeneity 0.5, penetrance 0.8, allelic heterogeneity 0.06 (Kelly et al. 2018 plus the CardioDB allele frequency calculator) giving a calculated allele frequency of >=0.006%, rounded up 10-fold to >=0.1%.
• gnomAD v2.1 non-cancer exomes (VCEP-directed source, exome-only figures): popmax/exome grpmax filtering AF 0.02778749 (2.78%); AFR 0.03018054 (3.018%, 448/14844 alleles); allele frequency 0.00186391 (249/133590 alleles); SAS 0/22480 alleles.
• gnomAD v3.1 non-cancer genomes: popmax filtering AF 0.0280573 (2.81%), highest subpopulation 1KG:ESN 0.0384615 (3.846%, 8/208 alleles); total allele frequency 0.00856475 (1267/147932 alleles).
• All-comers corroboration: gnomAD v2.1 grpmax filtering AF 0.02768 (2.77%), highest subpopulation African/African American AF 0.03011889 (3.012%, 456/15140 alleles), total AF 0.00310432 (516/166220 alleles); gnomAD v4.1 grpmax filtering AF 0.0285595 (2.86%), highest subpopulation African/African American AF 0.02962306 (2.962%, 2070/69878 alleles), total AF 0.00166343 (2280/1370658 alleles).
• The variant is a missense change p.(Ser26Arg) in an APC region with no recognised pathogenic missense hotspot (the only APC missense positions with (Likely) Pathogenic classification are codons 1026 and 1028), and observation of this allele at 0.31% (all-comers) or 0.19% (non-cancer exomes) is orders of magnitude above the ~0.0008% minor allele frequency of the most frequent pathogenic APC variant, c.3927_3931delAAAGA p.(Glu1309Aspfs*4), as stated in the specification.
• For comparison only: Sherloc (PMID:28492532) treats allele frequencies of 0.5%/'very high', 0.1%/'high' and >8 alleles as benign-range signals for dominant genes; the numeric thresholds applied here are the APC VCEP's own values, not Sherloc's.
• ClinVar consensus (Benign, 10 clinical laboratories) was noted as corroboration only and was not used to infer this criterion.

Sources

Reasoning

The APC VCEP v2.1 specification defines BS1 at Strong strength as a GnomAD Popmax Filtering Allele Frequency >= 0.001% (0.00001), preferably based on the Popmax Filtering Allele Frequency of the non-cancer dataset from gnomAD (v2.1.1); the threshold was derived from prevalence 1:5,000, genetic heterogeneity 1 and penetrance 0.8, giving a calculated allele frequency of >=0.0008% rounded to >=0.001%, which equals the minor allele frequency of the most frequent pathogenic APC variant on gnomAD v2.1.1. The observed popmax filtering AF in the VCEP-directed non-cancer gnomAD v2.1.1 exomes is 0.02778749 (2.78%) and the exome-only allele frequency is 0.00186391; both are more than two orders of magnitude above the 0.001% threshold (the popmax value exceeds it roughly 2,780-fold). The same conclusion holds in the v3.1 non-cancer genomes (popmax FAF 0.0280573; AF 0.00856475) and in the all-comers datasets (v2.1 grpmax FAF 0.02768, total AF 0.00310432; v4.1 grpmax FAF 0.0285595, total AF 0.00166343). BS1 is therefore met at Strong strength, but the same allele-frequency measurements already satisfy BA1 (stand-alone benign), so BS1 is subsumed by BA1 and must not be counted as an additional independent Strong benign line in the combining rules.

Rule

BS1 (Benign Strong) - APC VCEP v2.1 / InSiGHT: gnomAD Popmax Filtering Allele Frequency (AF) >= 0.001% (0.00001), preferably from the non-cancer dataset from gnomAD (v2.1.1).

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) BS1 (Benign Strong) rule: 'GnomAD Popmax Filtering Allele Frequency (AF) >= 0.001% (0.00001)', preferably based on the Popmax Filtering Allele Frequency of the non-cancer dataset from gnomAD (v2.1.1).
• Threshold derivation stated in the specification: prevalence 1:5,000, genetic heterogeneity 1, penetrance 0.8, allelic heterogeneity 0.06, calculated allele frequency >=0.0008%, rounded to >=0.001%; the calculated value equals the minor allele frequency of the most frequent pathogenic APC variant c.3927_3931delAAAGA p.(Glu1309Aspfs*4) on gnomAD v2.1.1.
• gnomAD v2.1 non-cancer exomes (VCEP-directed source, exome-only figures): popmax filtering AF 0.02778749 (2.78%), allele frequency 0.00186391 (249/133590 alleles) - roughly 2,780-fold and 186-fold above the 0.001% threshold respectively.
• gnomAD v3.1 non-cancer genomes: popmax filtering AF 0.0280573, allele frequency 0.00856475 (1267/147932 alleles), both far above the threshold.
• All-comers gnomAD v2.1 (grpmax FAF 0.02768; total AF 0.00310432) and gnomAD v4.1 (grpmax FAF 0.0285595; total AF 0.00166343) independently exceed the BS1 threshold, so the verdict does not depend on the choice between the non-cancer and all-comers sources.
• BS1 and BA1 draw on the identical frequency measurements; because BA1 stands alone as Benign, BS1 is recorded for completeness only and is not counted as a separate benign line of evidence.

Still missing

• BS1 and BA1 are satisfied by the identical allele-frequency measurements; BS1 adds no independent benign weight once BA1 (stand-alone) is met.

Sources

Reasoning

The APC VCEP v2.1 specification defines BS2 at Strong strength as >= 10 points for healthy individuals OR >= 2 observations in the homozygous state (BS2_Supporting is >= 3 points). The specification explicitly bars one route and permits the other: the non-cancer gnomAD (v2.1.1) dataset 'cannot be used for heterozygous healthy individuals' because of limited phenotype information and because it is usually already used for BA1/BS1, but it 'can be used to search for homozygous individuals'. The homozygous-state clause is satisfied. gnomAD v3.1 non-cancer genomes report 18 homozygotes (1267/147932 alleles, AF 0.857%); gnomAD v4.1 reports 33 homozygotes (14 exomes, 19 genomes; 31 of them in the African/African American subpopulation at AF 2.96%); gnomAD v2.1 reports 4 homozygotes (2 exomes, 2 genomes); gnomAD-Canada v1.0 reports 2 homozygotes (36/18400 alleles). Any one of these independent datasets meets the >= 2 homozygous-observation requirement, and the number of homozygotes is below Hardy-Weinberg expectation for an allele at 2.8% frequency in the African subpopulation, arguing against genotyping artefact. The healthy-individual points route cannot be used: gnomAD heterozygous carriers are excluded by the specification, and no reviewed full-text publication reports this variant in individually curated healthy adults or unaffected controls, so no points can be counted from literature. Caveats that reduce confidence to medium and warrant human review: the VCEP-named non-cancer gnomAD v2.1.1 exomes report only 1 homozygote, which is below the >= 2 clause, so the call rests on the newer non-cancer genomes and all-comers datasets; and the specification does not state whether reference-population homozygotes of a variant in a dominantly inherited, early-onset gene should be counted under this clause. The verdict direction (benign) is robust because BA1 independently establishes Benign.

Rule

BS2 (Benign Strong) - APC VCEP v2.1 / InSiGHT: >= 10 points for healthy individuals OR >= 2 times in homozygous state; non-cancer gnomAD (v2.1.1) usable to search homozygotes but not heterozygous healthy individuals.

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) BS2 (Benign Strong) rule: '>= 10 points for healthy individuals OR >= 2 times in homozygous state', with BS2_Supporting at '>= 3 points'; a healthy individual from control/non-cancer/normal/unaffected populations is worth 0.5 points.
• APC VCEP specification v2.1 (InSiGHT) BS2 instructions: the non-cancer dataset from gnomAD (v2.1.1) cannot be used for 'heterozygous healthy individuals' because of limited phenotype information and because it is usually already used for BA1/BS1, but it can be used to search for homozygous individuals.
• gnomAD v3.1 non-cancer genomes: 18 homozygotes (genome homozygote count 18; allele frequency 0.00856475, 1267/147932 alleles) - independently satisfies the >= 2 homozygous-state clause.
• gnomAD v4.1 all-comers: 33 homozygotes (exome 14, genome 19); 31 of these are in the African/African American subpopulation (2070/69878 alleles, AF 2.962%); also 2 homozygotes in 'Remaining individuals'.
• gnomAD v2.1 all-comers: 4 homozygotes (exome 2, genome 2); all 4 in the African/African American subpopulation (456/15140 alleles).
• gnomAD v2.1 non-cancer exomes (VCEP-directed source, exome-only figures): 1 homozygote (249/133590 alleles) - below the >= 2 homozygous-state clause, which is why the call does not rest on this dataset alone.
• gnomAD-Canada v1.0 (HostSeq genomes): 2 homozygotes (36/18400 alleles, AF 0.00195652) - a second, independent population dataset meeting the >= 2 clause.
• Homozygote counts are below Hardy-Weinberg expectation for an allele at ~2.8% frequency in the African subpopulation, so the observations are not explained by a call/genotyping artefact.
• The gnomAD-Canada (HostSeq genomes) resource is a Canadian population genome dataset and carries a homozygote count above the required two; it is used only as corroboration, not as the decisive dataset.

Still missing

• The VCEP-named non-cancer gnomAD v2.1.1 exomes report only 1 homozygote (below the >= 2 clause); the homozygous-state call relies on gnomAD v3.1 non-cancer genomes (18 homozygotes), gnomAD v4.1 (33) and gnomAD v2.1 (4), and this inter-dataset discrepancy should be acknowledged by a reviewer.
• The APC VCEP specification does not explicitly state whether homozygotes of a variant in a dominantly inherited, early-onset condition observed in reference-population datasets should be counted under the '>= 2 times in homozygous state' clause; human confirmation of this application is advised.
• The healthy-individual points route was not enumerable: the specification bars gnomAD heterozygous carriers for this purpose, and no reviewed publication reports this variant in curated healthy or unaffected individuals (PMID:24728327, a 681-healthy-adult whole-genome control cohort, contains no APC variant-level data).

Sources

Reasoning

BP1 applies to 'missense variant in a gene for which primarily truncating variants are known to cause disease', and the governing ClinGen InSiGHT APC VCEP specification keeps BP1 applicable to APC with one explicit carve-out: '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)' (supplementary material: 'APC is a gene for which primarily truncating variants are known to cause disease ... Therefore, for all variants located in the first 15-amino acid repeat of the beta-catenin binding domain (codon 1021-1035) BP1 is not allowed to be used'). The variant under assessment, NP_001120983.2:p.(Ser26Arg), is a missense change at codon 26, which lies well outside codons 1021-1035, so the carve-out does not apply and BP1 is permitted at Supporting strength. The gene-level premise is well supported: APC truncating variants (nonsense, frameshift, splice site) account for >90% of the APC mutational spectrum, with recognised truncating hotspots at codons 1309, 1061, 213 and 1068, and the VCEP's PVS1 rule states that APC is a gene where loss of function is the predominant mechanism of disease; only two missense positions (1026, 1028) have ever reached Likely Pathogenic. The VCEP attaches one procedural caveat to BP1 — 'A number of assumed "missense" variants are in fact splice variants. At least several splice prediction tools should be used' — and that caveat is addressed here to the extent the case data allows: SpliceAI gives a max delta of 0.007 for this variant, with no acceptor/donor gain or loss flagged, so the substitution is not a cryptic splice variant. Only SpliceAI was retrieved of the three VCEP-recommended predictors (SpliceAI, MaxEntScan, VarSeak), which is why confidence is medium rather than high. BP1 is met at Supporting strength.

Rule

APC VCEP BP1 (Supporting): missense variant in a gene where primarily truncating variants cause disease, except missense variants in codons 1021-1035.

Evidence considered

• APC VCEP CSPEC BP1 rule (only Supporting strength is applicable): '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).'
• APC VCEP supplementary material (page 7, BP1 row): 'APC is a gene for which primarily truncating variants are known to cause disease. Based on our knowledge there are only two amino acid positions (1026 and 1028), where reported missense variants can be classified as Likely Pathogenic based on the APC-specific criteria ... Therefore, for all variants located in the first 15-amino acid repeat of the beta-catenin binding domain (codon 1021-1035) BP1 is not allowed to be used. A number of assumed "missense" variants are in fact splice variants. At least several splice prediction tools should be used.'
• The variant under assessment is p.(Ser26Arg) (NM_001127511.2:c.78C>A), a missense substitution at codon 26; codon 26 is far outside the BP1 exclusion window codons 1021-1035, so BP1 is allowed for this variant.
• Gene mechanism: the Clinical Utility Gene Card for FAP/AFAP (PMID:21368914, section 1.6 Mutational spectrum) reports a 'Broad spectrum of point mutations, >90% are truncating (nonsense, del/ins and splice sites). Hot spots: codon 1309 (about 11%, 5-bp del), codon 1061 (7%, 5-bp del), codon 213 (3%, C4T transition), codon 1068 (2%, 4-bp del)', with no codon 26 alteration listed; the APC VCEP PVS1 rule likewise states that APC is a gene where loss of function is the predominant mechanism of disease.
• Splice status of this variant (VCEP BP1 caveat): SpliceAI for NM_001127511.2:c.78C>A gives max delta 0.007, with no acceptor gain/loss or donor gain/loss flagged — far below the VCEP's thresholds for native splice-site loss (SpliceAI 0.8-1.0) or cryptic-site gain (strong 0.8-1.0, moderate 0.2-0.8), and below the generic BP4 supporting boundary of 0.1 (PMID:30661751). The change is therefore not an unrecognised splice variant.
• Corroborating benign context (not part of the BP1 argument itself): gnomAD v4.1 lists this variant at total AF 0.00166 (2280/1,370,658 alleles) with 33 homozygotes and grpmax FAF 0.0286 in African/African American individuals, and ClinVar 133505 classifies it Benign with 10 submissions (2-star).

Still missing

• Only one of the three VCEP-recommended splice predictors was retrieved (SpliceAI, max delta 0.007); MaxEntScan and VarSeak were not run. The VCEP's BP1 instruction asks that at least several splice prediction tools be used to exclude an underlying splice variant. SpliceAI alone is already ~14-fold below the 0.1 no-impact boundary, and the variant is a common benign population polymorphism, so the residual risk is low but not formally excluded by a multi-tool consensus.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for APC v2.1 (CSPEC doc 1529292791), a gene-specific specification that supersedes the generic ACMG/AMP PVS1 definition. In that specification PVS1 is listed as applicable with default strength Pathogenic Very Strong, and all four strength tiers (Very Strong, Strong, Moderate, Supporting) carry the identical rule text 'Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (Figure 1)'. The gene-level gate is satisfied: the VCEP supplementary material states APC is a gene where loss of function is the predominant mechanism of disease, so the failure here is at the variant level, not the gene level. Variant consequence: NM_001127511.2:c.78C>A translates to NP_001120983.2:p.(Ser26Arg), a single-nucleotide missense substitution in exon 1. It creates no premature termination codon, shifts no reading frame, abolishes no start codon, deletes no exon and changes no protein length (no truncation, no elongation, no in-frame length change). The APC modified PVS1 decision tree (Figure 1) distributes only specific canonical splice-site substitutions across List A (PVS1), List B (PVS1_Strong), List C (PVS1_Moderate), List D (PVS1_Supporting) and List E (N/A); c.78C>A appears in none of these lists and codon 26 is not a canonical splice consensus position. Because conversion of a presumed missense change into a splice-null allele is the only residual route to PVS1 for this variant, the splice consequence was checked: SpliceAI reports DS_AG 0.007, DS_AL 0.002, DS_DG 0.005 and DS_DL 0.002 (max delta 0.007; reported summary max delta 0.01), which is far below the 0.2 supporting-impact cutoff for SpliceAI (Jaganathan et al. 2019, PMID:30661751) and shows neither loss of a native splice site nor gain of a cryptic site. NMD, exon-importance and extreme-3'-end considerations are therefore not engaged, because they only bear on variants that create a premature stop codon or disrupt a native splice site, and neither event is present. PVS1 is consequently not met, so no strength tier is assigned and the strength field is left null; no SVI or VCEP downgrade rule was applied or needed, since downgrades only reduce a met, full-strength call and there is nothing here to downgrade. Transcript caveat recorded: the submitted transcript NM_001127511.2 is annotated as APC 'transcript variant 1' but VariantValidator flags mane_select false, refseq_select false and select false, whereas the governing APC VCEP ruleset designates NM_000038.6 as the preferred transcript; the variant is exonic in exon 1 of NM_001127511.2. This transcript discrepancy does not change the verdict, because an N-terminal missense substitution is a non-null allele under any APC transcript, but it is retained as a caveat.

Rule

PVS1 (APC VCEP v2.1): 'Null variant in a gene where LOF is a known mechanism of disease. As per modified decision tree (Figure 1)' - graded Very Strong by default unless a named decision-tree list assigns Strong/Moderate/Supporting or List E declares N/A. Not met here: c.78C>A is a missense substitution with no null consequence, so no strength tier (and therefore no strength string) is emitted.

Evidence considered

• Governing specification (cspec, doc 1529292791): ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for APC Version 2.1. PVS1 is applicable with default strength Pathogenic Very Strong; every 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): List A (PVS1), List B (PVS1_Strong), List C (PVS1_Moderate), List D (PVS1_Supporting) and List E (N/A) contain only named canonical splice-site substitutions (e.g. c.136-1G>A,C,T; c.220G>A,C,T; c.934-1G>A,C,T). c.78C>A is absent from all five lists.
• APC VCEP supplementary material (vcep_apc_specifications_supplementary_material_v2): for PVS1 the expert panel records that APC is a gene where LOF is the predominant mechanism of disease; loss of function is therefore the established disease mechanism and the gene-level PVS1 gate is met, while the variant-level null precondition is not.
• Variant consequence: NM_001127511.2:c.78C>A yields NP_001120983.2:p.(Ser26Arg) - a single-nucleotide missense substitution in exon 1 (variant exonic position start_exon = end_exon = 1), not a nonsense, frameshift, canonical +/-1,2 splice, initiation-codon or exon-deletion variant; no premature stop codon, no frame shift, unchanged protein length.
• SpliceAI (spliceai) for NM_001127511.2:c.78C>A: DS_AG 0.007, DS_AL 0.002, DS_DG 0.005, DS_DL 0.002, max delta 0.007 (reported summary max delta 0.01); no native splice-site loss and no cryptic splice-site gain, and the position is not a canonical splice consensus base.
• SpliceAI interpretive cutoffs applied (Jaganathan et al. 2019, Cell, PMID:30661751): >= 0.5 moderate and >= 0.2 supporting evidence of splice impact; a max delta of 0.007 is far below both.
• Generic ACMG/AMP 2015 guideline (PMID:25741868) supplies the originating PVS1 definition (null variant in a gene where loss of function is a known mechanism of disease) and the very strong strength vocabulary, but for APC the gene-specific VCEP specification governs.
• NMD, exon-importance and extreme-3'-end rules are not applicable here: they are conditional on a premature termination codon or on disruption of a native splice site, and neither is generated by this missense substitution.

Still missing

• No RNA/cDNA, minigene or other functional transcript data exist for this variant; exclusion of a splice-driven null effect rests on in-silico prediction (SpliceAI max delta 0.007) and on the absence of any premature stop codon or reading-frame change.
• The submitted transcript NM_001127511.2 is neither MANE Select nor RefSeq Select and is not the APC VCEP-preferred transcript (NM_000038.6); no independent re-derivation of the equivalent canonical-transcript description of c.78C>A was performed in this group.

Sources

Reasoning

PS1 asks whether the variant under assessment results in the same amino acid change as a previously established (Likely) Pathogenic variant. The governing ClinGen InSiGHT APC VCEP specification (v2.1, CSPEC doc 1529292791) explicitly narrows this for APC: for missense variants, PS1/PS1_Moderate 'can currently only be used for missense variants at these two amino acid positions' 1026 and 1028, i.e. the same amino acid change as c.3077A>G p.(Asn1026Ser) or c.3084T>A p.(Ser1028Arg); the panel further states that no missense variant has been classified as Pathogenic based on current evidence, so the Strong arm has no eligible subject variant at all. The variant under assessment, NM_001127511.2:c.78C>A, is NP_001120983.2:p.(Ser26Arg) — a missense change at codon 26, which is neither 1026 nor 1028, and no differently classed nucleotide change producing p.Ser26Arg carries a (Likely) Pathogenic classification (ClinVar 133505 for exactly this amino-acid change is Benign, 2-star, 10 submitters, 0 expert-panel submissions). The splice arm of PS1 is likewise inapplicable: SpliceAI max delta is 0.007 for this variant, far below any splice-impact threshold, so it is not a variant acting through splicing rather than at the protein level. PS1 is therefore not met.

Rule

APC VCEP PS1: same amino acid change as a previously established (Likely) Pathogenic variant; for missense, currently limited to positions 1026 and 1028.

Evidence considered

• APC VCEP CSPEC rule for PS1 (Strong): 'There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). Other variants leading to the same missense change at these positions meet PS1_Moderate. No missense variant has been classified as Pathogenic based on current evidence.' The splice arm of PS1 requires the variant to affect splicing at the same nucleotide as a previously established (Likely) Pathogenic variant.
• APC VCEP supplementary material (APC_specifications_supplementary-material_V2.pdf, page 1, PS1 row): 'Based on the current knowledge there are only two amino acid positions (1026 and 1028), where reported missense variants can be classified as Likely Pathogenic based on the APC-specific criteria ... Thus, PS1_Moderate can currently only be used for missense variants at these two amino acid positions.'
• Variant under assessment is NP_001120983.2:p.(Ser26Arg) (NM_001127511.2:c.78C>A; NC_000005.9:g.112043492C>A) — a missense substitution at codon 26, not codon 1026 or 1028, and no differently classed nucleotide change yielding p.Ser26Arg was found that is established as (Likely) Pathogenic.
• ClinVar variation 133505 (NM_001127511.3(APC):c.78C>A, p.Ser26Arg) is classified Benign with 10 submissions/10 submitters, 'criteria provided, multiple submitters, no conflicts' (2-star), and zero expert-panel submissions; the only criterion any submitter cited for it was the benign-direction in-silico assessment (conservative change, poorly conserved position).
• SpliceAI lookup for NM_001127511.2:c.78C>A (hg37) returns max delta 0.007 with no acceptor/donor gain or loss flagged, so this variant is not a splice-affected variant and cannot qualify through PS1's splice arm.
• The VCEP's own recommended splice thresholds (SpliceAI native-site loss 0.8-1.0; cryptic-site gain strong 0.8-1.0, moderate 0.2-0.8; MaxEntScan >3 for credible native site and -15% for native site loss, Houdayer et al. 2012) are all far above the observed 0.007.

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, and directs use of the total population from the non-cancer dataset from gnomAD (v2.1.1). The threshold derives from prevalence 1:10,000, allelic heterogeneity 0.06 and penetrance 0.9. The variant is far too common to qualify. In the VCEP-directed non-cancer gnomAD v2.1.1 exomes the allele count is 249 (> 1) and the allele frequency is 0.00186391 (0.186%), roughly 620-fold above the applicable 0.0003% threshold (0.000003). Every other dataset is equally disqualifying: gnomAD v3.1 non-cancer genomes AF 0.00856475 (0.857%, 1267 alleles, ~2,855-fold above threshold), all-comers gnomAD v2.1 AF 0.00310432 (516 alleles) and gnomAD v4.1 AF 0.00166343 (2280 alleles). The generic ClinGen SVI PM2_Supporting threshold of <= 0.0001 is also exceeded, by roughly 5-19 fold depending on dataset. PM2 is not met. Because a common, African-subpopulation-enriched allele such as this could in principle be a population-specific polymorphism rather than a genuinely rare allele in other ancestries, the subpopulation figures were checked explicitly: even the lowest-frequency major subpopulations in gnomAD v2.1 (SAS 0/22480 alleles) do not make the overall frequency rare, and the popmax filtering AF of 2.78% cannot support rarity under any VCEP-permitted reading of the rule.

Rule

PM2 (Pathogenic Supporting) - APC VCEP v2.1 / InSiGHT: allele frequency <= 0.0003% (0.000003) if allele count > 1, OR < 0.001% (0.00001) if allele count <= 1, using the total population from the non-cancer dataset from gnomAD (v2.1.1).

Evidence considered

• APC VCEP specification v2.1 (InSiGHT) PM2 (Pathogenic Supporting) 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'; instructions: 'Use the total population from the non-cancer dataset from gnomAD (v2.1.1)'.
• Threshold derivation stated in the specification: monoallelic inheritance, prevalence 1:10,000 (1:20,000 chromosomes), allelic heterogeneity 0.06, genetic heterogeneity 1, penetrance 0.9, calculated allele frequency <= 0.0003%; the more permissive < 0.001% is allowed only if the allele count is <= 1.
• gnomAD v2.1 non-cancer exomes (VCEP-directed source, exome-only figures): allele count 249 (> 1), allele frequency 0.00186391 (0.186%), i.e. the <= 0.0003% arm applies and is exceeded roughly 620-fold.
• gnomAD v3.1 non-cancer genomes: allele count 1267 (> 1), allele frequency 0.00856475 (0.857%), roughly 2,855-fold above the 0.0003% threshold.
• All-comers gnomAD v2.1 (allele frequency 0.00310432; 516/166220 alleles) and gnomAD v4.1 (allele frequency 0.00166343; 2280/1370658 alleles) are also orders of magnitude above the threshold, so the verdict is unchanged under either the non-cancer or the all-comers source.
• Subpopulation check: the variant is enriched in the African/African American subpopulation (gnomAD v2.1 non-cancer exomes AFR AF 3.018%; v4.1 AFR AF 2.962%) and absent from South Asians in v2.1 non-cancer exomes (0/22480 alleles); the VCEP advises considering subpopulation-specific data, but no subpopulation yields a rarity-compatible frequency for the overall dataset.
• Generic ACMG/ClinGen SVI default for comparison: PM2 (supporting) <= 0.0001 - also not met, since the smallest observed all-comers allele frequency (0.00166343) is about 17-fold higher.

Sources

Reasoning

PM5 requires a different missense change at the same amino acid residue that has been determined to be pathogenic (or, per the APC-specific modification, Likely Pathogenic). The governing ClinGen InSiGHT APC VCEP specification restricts this explicitly: 'Based on the current knowledge this criterion can only be used for missense variants at the two amino acid positions 1026 and 1028, where reported missense variants can be classified as Likely Pathogenic based on the APC-specific criteria', with a different missense at those two residues meeting only PM5_Supporting and a stricter Grantham-distance requirement attached to PM5_Moderate. The variant under assessment is NP_001120983.2:p.(Ser26Arg) at codon 26, which is neither 1026 nor 1028, so no APC-specific PM5 arm can be satisfied regardless of what comparators exist at residue 26. Consistently, the case's same-residue comparator harvest found zero same-residue candidates and zero expert-panel or majority (Likely) Pathogenic comparators, and the ClinVar record for this exact amino-acid change is Benign. The comparator harvest was partial (one query returned HTTP 429), but because the VCEP restricts PM5 to residues 1026 and 1028 as a matter of specification, the conclusion does not depend on a complete comparator search. PM5 is not met.

Rule

APC VCEP PM5: a different missense change determined to be (Likely) Pathogenic at the same residue; currently limited to residues 1026 and 1028, with Grantham distance >= the reported variant.

Evidence considered

• APC VCEP CSPEC PM5_Moderate rule: 'The reported missense variant was determined to be Pathogenic according to the APC-specific modifications. There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). Other different missense variants at these positions meet PM5_supporting. No missense variant has been classified as Pathogenic based on current evidence. Grantham's distance of the variant under assessment must have an equal or higher score than the reported variant.'
• APC VCEP supplementary material (page 4, PM5 row): 'Do not use if the mechanism of pathogenicity for the reported variant is a splicing defect. Multiple in silico splicing predictors should be applied to the reported variant or the splicing defect was confirmed by RNA analysis. In this case check, if PS1 or PS1_Moderate is applicable. Based on the current knowledge this criterion can only be used for missense variants at the two amino acid positions 1026 and 1028.'
• Variant under assessment is p.(Ser26Arg) — residue 26, not 1026 or 1028 — so the APC-specific PM5 condition (a different missense at one of the two recognised residues) cannot be satisfied for this residue at any strength.
• The splicing-defect exclusion in PM5 does not change the outcome: SpliceAI max delta is 0.007 for NM_001127511.2:c.78C>A, i.e. no predicted splicing defect, so the variant is a genuine missense and the pathway would be the missense one (which is residue-restricted).
• Same-residue comparator harvest (pm5_candidates): 6 candidates seen, same_residue_candidates = 0, expert_panel_plp_candidates = 0, non_expert_majority_plp_candidates = 0, conflicted_candidates = 0, recommendation 'no_useful_candidates_found'; the artifact also records an HTTP 429 error and a soft failure warning, so the harvest is incomplete.
• ClinVar variation 133505 covers exactly this amino-acid change (NM_001127511.3(APC):c.78C>A, p.Ser26Arg) and is Benign (10 submissions, 2-star, no conflicts); no submission provides a (Likely) Pathogenic comparer at this residue.

Still missing

• The automated same-residue comparator harvest was incomplete (one ClinVar query returned HTTP 429, and the artifact reports the harvest 'failed softly'); this does not affect the verdict because the APC VCEP restricts PM5 to residues 1026 and 1028 by specification, and no same-residue comparator was found in the queries that did run.

Sources

Reasoning

Scope first: NM_001127511.2:c.78C>A is a coding missense substitution, NP_001120983.2:p.(Ser26Arg) (VEP most_severe_consequence = missense_variant; exon 1 of the coding sequence), so PP3 is in scope and all evidence is read from a single calibrated computational path. The governing specification is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for APC v2.1 (framework_mode = vcep), whose PP3_Supporting rule for presumed missense variants reads: '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', with multiple in silico splicing predictors required to support a deleterious effect. SpliceAI Lookup for this variant returns DS_AG 0.007, DS_AL 0.002, DS_DG 0.005, DS_DL 0.002 - a maximum delta score of 0.007, i.e. no meaningful loss of a native acceptor/donor site and no meaningful gain of a cryptic site. That is far below the supplied supporting cutoff of >= 0.2 and the moderate cutoff of >= 0.5 (Jaganathan et al. 2019, Cell, PMID:30661751), and far below the VCEP's own splice-effect bands (native site loss considered at 0.8-1.0; cryptic-site gain strong at 0.8-1.0, moderate at 0.2-0.8). No splicing predictor supports a deleterious effect, so the VCEP route to PP3 fails. Independently, on the generic missense protein-impact path the local REVEL v1.3 score is 0.383, an indeterminate gray-zone value: it is above the BP4-supporting cutoff (<= 0.29) and below the PP3-supporting cutoff (>= 0.644), and below the moderate (>= 0.773) and strong (>= 0.932) tiers of the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997). BayesDel (-0.0215517) has no established generic ACMG-strength calibration and no APC VCEP cutoff, so it is not_available for PP3. PP3 is therefore not met at any strength on either reading, and the two readings agree.

Rule

APC VCEP v2.1 PP3_Supporting (Applicable): '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.' Applied with the published SpliceAI PP3 cutoffs (supporting >= 0.2, moderate >= 0.5; Jaganathan et al. 2019, PMID:30661751) and, on the generic missense path, the ClinGen SVI REVEL PP3 cutoff (supporting >= 0.644; Pejaver et al. 2022, PMID:36413997).

Evidence considered

• APC/InSiGHT VCEP v2.1 PP3_Supporting rule (cspec): '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.' Applicability = Applicable, default strength = Pathogenic Supporting.
• Consequence type used for path selection: NM_001127511.2:c.78C>A normalizes to NP_001120983.2:p.(Ser26Arg) (protein_1l p.(S26R)); the annotated most severe consequence is missense_variant. The variant is a single coding substitution in exon 1, not a truncating, protein-length-changing, synonymous or intronic change.
• SpliceAI Lookup result (source_registry key 'spliceai'): DS_AG 0.007, DS_AL 0.002, DS_DG 0.005, DS_DL 0.002, max delta score 0.007 (the case sentence rounds this to 0.01). No native donor or acceptor site is lost and no cryptic site is gained to a meaningful degree.
• Thresholds applied verbatim from the supplied calibrations: SpliceAI PP3 supporting >= 0.2 and moderate >= 0.5 (Jaganathan et al. 2019, Cell, PMID:30661751). Observed max delta 0.007 does not reach even the supporting tier.
• VCEP splice-prediction guidance quoted in the specification's PS1 instructions (cspec): for SpliceAI, loss of the native splice site is considered for scores between 0.8 and 1; gain of a cryptic splice site is strong for scores between 0.8 and 1 and moderate for a score between 0.2 and 0.8. The observed 0.007 is far below all of these.
• REVEL v1.3 local predictor lookup (source_registry key 'revel') gave 0.383 for chr5:112707795 C>A (GRCh38) / chr5:112043492 C>A (GRCh37). Against the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997) this is gray-zone: PP3 supporting >= 0.644 is not reached, while BP4 supporting <= 0.29 is also not reached.
• BayesDel score -0.0215517 (source_registry key 'bayesdel') was treated as not_available: no generic ACMG-strength calibration for BayesDel is established and the APC VCEP specifies no BayesDel cutoff.
• MaxEntScan and VarSeak, the other splice programs the VCEP recommends alongside SpliceAI, were not retrieved for this variant; because the VCEP requires multiple splicing predictors to support a deleterious effect, a single non-supportive SpliceAI result already prevents PP3 from being met and the missing programs cannot create support.

Sources

Reasoning

The APC VCEP (v2.1) modifies BP2 to a single supporting-strength rule: the variant must be 'Observed in trans with a (Likely) Pathogenic APC variant OR >= 3 times in an unknown phase with different (Likely) Pathogenic APC variants.' Neither arm of the rule is satisfied for c.78C>A (p.Ser26Arg). The case contains no proband trio, family, or genotype data establishing the phase of this allele, and no second pathogenic/likely pathogenic APC variant is reported alongside it. The ClinVar record (variation 133505) contains ten clinical-laboratory submissions, all Benign, none reporting a trans configuration or a co-occurring pathogenic APC allele. No published paper reviewed for this case reports c.78C>A in trans with a pathogenic APC variant, and none reports it >= 3 times in unknown phase alongside different pathogenic APC variants; the reported literature is either gene/disease-level guidance or a general germline-variation catalogue in which this variant is not named. Because BP2 is defined only by the presence of such an observation, and no such observation was found, BP2 is not met. The benign direction of this variant is supported by other criteria (population frequency, ClinVar concordance), not by BP2.

Rule

Observed _in trans_ with a (Likely) Pathogenic _APC_ variant OR >= 3 times in an unknown phase with different (Likely) Pathogenic _APC_ variants.

Evidence considered

• APC VCEP specification v2.1 defines BP2 (Benign Supporting) as: 'Observed in trans with a (Likely) Pathogenic APC variant OR >= 3 times in an unknown phase with different (Likely) Pathogenic APC variants.'
• ClinVar variation 133505 (NM_001127511.3(APC):c.78C>A, p.Ser26Arg) has 10 submissions from 10 clinical laboratories, all classified Benign; the review status is 'criteria provided, multiple submitters, no conflicts'. No submission reports a compound/trans configuration with a pathogenic APC allele.
• No proband trio, family, or phase data are present in the case; the proband-level genotype includes only this single heterozygous missense allele.
• None of the fetched full texts (PMID:15604628, 21368914, 24728327, 25645574, 25741868, 28492532) name c.78C>A or p.Ser26Arg, and none describes its phase; the papers are gene- or disease-level guidance and a healthy-cohort germline variation catalogue.
• No report was found of c.78C>A occurring >= 3 times in unknown phase with different (Likely) Pathogenic APC variants.

Still missing

• No proband trio, parental testing, or phased genotype data were available, so the phase of c.78C>A relative to any pathogenic APC allele could not be established; this does not change the verdict because no trans or repeated unknown-phase observation was found anywhere.

Sources

Reasoning

Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1, in which PS2 is applicable and is no longer a binary call but a curated de novo score (1-1.5 = Moderate, 2-3.5 = Strong, >=4 = Very Strong). The APC VCEP scores a de novo proband from confirmed maternity and paternity combined with the proband's phenotype points: a de novo with confirmed parental relationships is worth 2 de novo scores at >=1 phenotype point and 1 score at 0.5 phenotype point, so PS2 cannot be reached without both a proband phenotype and parental genotypes from that proband. This case carries no individual-level data of any kind: it is a variant-level lookup of NM_001127511.2:c.78C>A (p.Ser26Arg) with no proband, no phenotype, no parental testing, no trio or duo testing, and no family history. None of the eight papers in the case literature set is indexed under PS2, and none of the six fetched full texts names the variant or reports a de novo occurrence (each records the c.78C>A / p.Ser26Arg search terms as not mentioned in the source; PMID:21368914 supplies only the gene-level statement that 10-40% of FAP cases are de novo, which says nothing about this variant). The ClinVar record (variation 133505) contains ten submissions, all Benign, none reporting a de novo observation or parentage testing. No de novo score can therefore be counted and PS2 cannot be evaluated in either direction. It is recorded not_assessed rather than not_met because the missing element is a documentation gap in this record, not evidence that the variant was inherited: de novo status is independent of the population-frequency and in-silico results that make up the remainder of the case file.

Evidence considered

• ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC v2.1 (cspec doc 1529292791) lists PS2 as applicable, default strength Pathogenic Strong, with strength rules: 1-1.5 de novo scores = Moderate, 2-3.5 = Strong, >=4 = Very Strong; 'For curation of de novo score see Tables 1 and 2'.
• APC VCEP Table 1 and Table 2 (key vcep_table_1_262): a proband is scored 1 phenotype point for a phenotype highly specific for APC and 0.5 for a phenotype consistent with APC; the de novo score per proband is 2 with confirmed parental relationships at >=1 phenotype point (1 at 0.5 point) and 1 with unconfirmed parental relationships at >=1 phenotype point (0.5 at 0.5 point). PS2 therefore requires a de novo proband with confirmed maternity and paternity plus a phenotype, none of which exists here.
• APC VCEP supplementary material annotates the original PS2 text 'De novo (both maternity and paternity confirmed)' with 'Modification based on SVI Recommendation for De Novo' criteria, confirming the VCEP rescaling from the generic ACMG/AMP binary form.
• The consolidated case evidence (review manifest, case summary, source registry, evidence.json, literature pass, full-text map, paper extracts) contains no proband, no individual phenotype, no parental/trio/duo testing and no de novo observation for NM_001127511.2:c.78C>A (p.Ser26Arg); the case record supplies the variant itself and variant-level database/predictor annotations only.
• None of the eight papers in the case literature set is indexed under PS2, and the paper_extracts for the six fetched full texts (PMID:15604628, 21368914, 24728327, 25645574, 25741868, 28492532) all record variant_mentioned = false with searched_for terms including c.78C>A, p.S26R and NP_001120983.2:p.(S26R).
• ClinVar variation 133505 (NM_001127511.3(APC):c.78C>A, p.Ser26Arg) holds 10 submissions from 10 clinical laboratories, all classified Benign, with no de novo observation, parental testing or phenotype detail reported in any submission.
• De novo status is not addressed by any other case source; it is not implied by the variant's observed population frequency or in-silico results, so PS2 is unassessed rather than unmet.

Still missing

• No proband, individual phenotype, parental genotypes or trio/duo data exist anywhere in the case, so the APC VCEP de novo score (Tables 1 and 2) cannot be computed at any strength. Confirming PS2 requires at minimum a proband with both maternity and paternity confirmed plus at least 1 phenotype point (PS2_Moderate at 1-1.5 de novo scores).
• No publication in the case literature set reports this variant as de novo; a targeted literature/database search for a de novo observation of APC c.78C>A would be needed to close this gap.

Sources

Reasoning

Governing specification: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specifications for APC v2.1, where PS3 is defined only through specified functional assays — RNA assays showing (1) a premature stop codon or (2) in-frame skipping of exon 13 or 14, with absence of full-length transcript (Very Strong) or <10% full-length transcript (Strong), or, at Supporting strength, protein assays showing increased beta-catenin-regulated transcription activity and/or decreased binding to beta-catenin by surface plasmon resonance, and that protein-assay route is explicitly restricted to variants within the beta-catenin binding domain (codons 959-2129 of APC). The variant under assessment, NM_001127511.2:c.78C>A p.(Ser26Arg), is a single-nucleotide missense substitution in exon 1 at codon 26 of APC. It lies far outside codons 959-2129, so the VCEP's protein-assay route for PS3 is structurally unavailable for this variant. On the RNA-assay route, no RNA-based study (patient RNA analysis, RT-PCR transcript sizing, minigene, or nonsense-mediated decay assessment) performed on this variant was identified in the ClinVar submission record, in the criterion-lead PMID trail, or in any fetched full text. No functional study showing a damaging effect on APC protein function or splicing therefore exists to satisfy PS3, and none of the case's in-silico scores can substitute for it because PS3 requires experimental assay evidence. PS3 is recorded as not_assessed: the criterion is unevaluated for want of an applicable assay rather than being refuted by a negative assay result.

Rule

APC VCEP v2.1 PS3 — 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); Supporting: in-frame skipping of other exons with reported exon deletion/skipping/loss or insertion of intronic nucleotides, over-expression of an alternative transcript (exons 10, 11 or 15), or protein assays showing increased beta-catenin-regulated transcription activity and/or decreased beta-catenin binding by SPR restricted to variants within codons 959-2129.

Evidence considered

• APC VCEP specification v2.1 (ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel) defines PS3 solely through RNA assays showing a premature stop codon or in-frame skipping of exon 13 or 14 (Very Strong: absence of full-length transcript; Strong: <10% of full-length transcript; Moderate and Supporting tiers require additional splicing reports or alternative-transcript over-expression), and through protein assays at Supporting strength only: 'Protein assays show Increased beta-catenin regulated transcription activity and/or decreased binding to beta-catenin by surface plasmon resonance (only for variants within the beta-catenin binding domain, which refers to codons 959-2129 of APC)'.
• Variant consequence confirmed from the normalized annotation: NM_001127511.2:c.78C>A (NC_000005.10:g.112707795C>A) yields NP_001120983.2:p.(Ser26Arg), a missense substitution at codon 26 in exon 1. Codon 26 is outside the beta-catenin binding domain (codons 959-2129) named in the VCEP PS3 rule, so the VCEP's protein-assay arm is not available for this variant; only an RNA-based assay could in principle support PS3 here.
• ClinVar (variation ID 133505) was examined for criterion-bearing evidence: 11 submissions, 9 usable for classification, only 2 usable as criterion leads (SCV000084139, ITMI; SCV000488267, Counsyl), both citing a single validated PMID (24728327). No submission reports a functional assay; the ClinVar submission audit records no functional-assay criterion lead.
• The fetched full texts were searched for functional-assay content (c.78C>A, p.(S26R)/p.(Ser26Arg), 'codon 26', RNA/protein/functional assay terms): PMID:15604628 (NSGC practice guideline — no assay data), PMID:21368914 (Clinical Utility Gene Card for FAP/AFAP — mutation-spectrum review only), PMID:24728327 (whole-genome sequencing of 681 healthy adults; APC named at gene level only), PMID:25645574 (ACG GI cancer guideline), PMID:25741868 (ACMG/AMP 2015 framework), PMID:28492532 (Sherloc framework). None reports any functional assay on APC c.78C>A, and the exact variant is not named in any of them.
• In-silico predictions available in this case (missense predictor scores and splice prediction) were deliberately not used as PS3 evidence: PS3 requires experimental functional assay data on the gene product, not computational prediction.

Still missing

• No RNA assay (transcript analysis / minigene / NMD assessment) has been performed or published for APC c.78C>A; such an assay is the only VCEP-permitted PS3 route potentially applicable to a missense substitution at codon 26.
• No protein assay (beta-catenin-regulated transcription activity or SPR binding) exists for this variant, and the VCEP restricts that assay type to codons 959-2129, which excludes codon 26 — so that route cannot be generated under the current specification.
• Laboratory-internal or unpublished functional data, if any, were not available to this assessment and should be confirmed by the reviewing laboratory.

Sources

Reasoning

Governing specification: InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel (APC VCEP v2.1), which converts generic PS4 into a phenotype-point scheme rather than a case-control odds-ratio rule. Governing operators, quoted literally from the VCEP text for THIS criterion: ">= 16 phenotype points" (Very Strong), "4-15.5 phenotype points" (Strong), "2-3.5 phenotype points" (Moderate), "1-1.5 phenotype point" (Supporting). All four bands are lower-bound / closed-interval comparisons on a point total that is curated from the proband's phenotype and family history using VCEP Table 1; enrichment strengthens as the point total goes UP, so met requires points >= the band's lower bound (e.g. '4.0 >= 4? yes' would be Strong; the opposite-direction <= likelihood-ratio style comparison does NOT apply to PS4 in this framework). Explicit inequality with numbers substituted: cannot be written, because zero phenotype points could be curated - the case contains no proband phenotype, no polyp/adenoma count, no extra-colonic features (desmoid, medulloblastoma, hepatoblastoma, CHRPE, gastric/duodenal adenomas, osteoma) and no family history, and no variant-specific case observation exists in any reviewed source to score on the proband's behalf. The variant record is a post-normalization substrate only (NM_001127511.2:c.78C>A, NP_001120983.2:p.(Ser26Arg), genomic NC_000005.10:g.112707795C>A). Independent case-control enrichment was also searched: the eight ClinVar-cited references (PMID:15604628, PMID:20301519, PMID:21368914, PMID:24728327, PMID:25645574, PMID:25741868, PMID:26389505, PMID:28492532) contain no odds ratio, relative risk, or affected-vs-control frequency comparison for this variant; the closest observation is a healthy-adult cohort (681 individuals) in which APC is named only as a gene with many rare variants, with no per-variant data for c.78C>A. PS4 is therefore not assessable rather than affirmatively not met: neither input needed to run the VCEP operator (curated phenotype points from a proband, or a case-control enrichment estimate) is present in this case. Gap: proband phenotype and family history (Table 1 features) and/or any case-control study reporting this exact variant.

Rule

APC VCEP v2.1 PS4 (phenotype-point scheme): Very Strong >=16 points; Strong 4-15.5 points; Moderate 2-3.5 points; Supporting 1-1.5 points. Direction: higher point total = stronger; met requires value >= band lower bound.

Evidence considered

• APC VCEP v2.1 (ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specification, doc 1529292791) replaces generic PS4 with phenotype points: '>= 16 phenotype points' (Very Strong), '4-15.5 phenotype points' (Strong), '2-3.5 phenotype points' (Moderate), '1-1.5 phenotype point' (Supporting), all with 'For phenotype points curation see Table 1'.
• VCEP Table 1 scores a phenotype point per proband: 1 point for a phenotype highly specific for APC (typical colorectal phenotype 20-99 colorectal adenomas; desmoid without somatic CTNNB1 variant; WNT-subtype medulloblastoma without somatic CTNNB1 variant; hepatoblastoma without somatic CTNNB1 variant) and 0.5 point for a phenotype merely consistent with APC (other colorectal phenotype >=20 adenomas; unknown CTNNB1 status; multifocal/bilateral CHRPE; >=2 gastric adenomas; >=2 duodenal adenomas; osteoma; typical dominant FAP family history).
• The cspec criteria payload for APC lists PS4 with applicability 'Applicable', defaultStrength 'Pathogenic Strong', and the four phenotype-point bands above; no numeric case-control threshold is specified anywhere for PS4 in this gene framework.
• ClinVar variation 133505 (NM_001127511.3(APC):c.78C>A, p.Ser26Arg) carries 10 clinical-laboratory submissions, all Benign, review status 'criteria provided, multiple submitters, no conflicts' (2 star); none reports a proband phenotype, polyp count, or affected/control frequency that could be turned into phenotype points.
• PMID:24728327 (Bodian et al. 2014, whole-genome sequencing of 681 healthy ancestry-diverse adults) is the only ClinVar-cited paper with a validated PMID trail for this record; it names APC only at gene level ('Among the genes with rare variants in the most individuals are BRCA1, BRCA2, APC, MLL2, and MLL3') and reports no APC per-variant allele frequency, no phenotype, and no c.78C>A observation, so it cannot contribute affected-individual prevalence data.
• Generic ACMG calibration thresholds supplied for this group (case-control odds ratio / REVEL / SpliceAI / population frequency) do not govern PS4 here, because the APC VCEP specification takes precedence and defines PS4 by phenotype points; no odds-ratio cut-off was recalled or substituted.

Still missing

• No proband phenotype, polyp/adenoma count, extra-colonic features, or family history in the case files, so APC VCEP Table 1 phenotype points cannot be curated and no PS4 band (1-1.5 Supporting / 2-3.5 Moderate / 4-15.5 Strong / >=16 Very Strong phenotype points) can be evaluated.
• No case-control study reporting this exact variant (c.78C>A, p.Ser26Arg) - no odds ratio, relative risk, or affected-vs-control comparison was found in any ClinVar-cited reference.

Sources

Reasoning

PM6 in the APC VCEP v2.1 is the assumed-de-novo criterion. It is scored from the same curated de novo table as PS2 but with the reduced requirement that maternity and paternity are not confirmed: a de novo proband with unconfirmed parental relationships scores 1 de novo score (PM6_Moderate, 1-1.5) at >=1 phenotype point and 0.5 score (PM6_Supporting) at 0.5 phenotype point. Even this reduced requirement is unmet on the available data because the case records no assumed-de-novo observation at all: there is no proband, no phenotype point (Table 1), no parentage information and no family history for NM_001127511.2:c.78C>A (p.Ser26Arg). No paper in the case literature set names the variant or reports it as an assumed de novo, and the ClinVar record (variation 133505, ten Benign submissions) likewise reports no de novo or parentage observation. PM6 is therefore not_assessed: without at least an assumed-de-novo proband the criterion cannot be computed at any strength, and the absence of such an observation from a variant-level lookup is a documentation gap, not evidence against a de novo origin. A not_met call would additionally be unsafe, because an unconfirmed de novo event can never be excluded from a case file that contains no family data at all.

Evidence considered

• APC VCEP v2.1 (cspec) lists PM6 as applicable, default Pathogenic Moderate, with strength rules: 0.5 de novo scores = Supporting, 1-1.5 = Moderate, 2-3.5 = Strong; de novo scoring per Tables 1 and 2.
• APC VCEP Table 2 (key vcep_table_1_262) gives the de novo score per proband with unconfirmed parental relationships as 1 at >=1 phenotype point and 0.5 at 0.5 phenotype point, so PM6 still requires an observed de novo proband with at least 0.5 phenotype point; this case has neither.
• APC VCEP supplementary material annotates the original PM6 text 'Assumed de novo, but without confirmation of paternity and maternity' with 'Modification based on SVI Recommendation for De Novo' criteria, consistent with the tabulated scoring system used here.
• The case contains no assumed-de-novo observation, no phenotype points, no parentage information and no family history for NM_001127511.2:c.78C>A (p.Ser26Arg); it is a variant-level lookup with no individual-level data.
• No paper in the case literature set is indexed under PM6, and none of the six fetched full texts names c.78C>A or p.Ser26Arg or reports a de novo occurrence (paper_extracts record variant_mentioned = false for all six).
• ClinVar variation 133505 (10 submissions, all Benign, none with assertion criteria detail) reports no assumed-de-novo observation or parentage information for this variant.

Still missing

• No assumed-de-novo observation with any parentage information is recorded for this variant in the case file or in any source reviewed, so the APC VCEP de novo score for PM6 (0.5 = Supporting, 1-1.5 = Moderate, 2-3.5 = Strong) cannot be computed.
• Closure requires either a proband reported as de novo without confirmed parentage (plus >=0.5 phenotype point) or an explicit statement in a paper/ClinVar submission that the variant arose de novo in an affected individual.

Sources

Reasoning

PP1 under the APC VCEP v2.1 requires co-segregation with disease in families, graded by the number of informative meioses: 3-4 meioses in >=1 family = Supporting, 5-6 meioses in >=1 family = Moderate, >=7 meioses in >=2 families = Strong. Only genotype- and phenotype-positive individuals and obligate carriers with phenotype are counted, and affected individuals must exhibit at least 0.5 phenotype point under Table 1. The case contains no family data whatsoever: no relative of an affected individual, no genotype for any second family member, no phenotype points, and no meiosis count, so the segregation requirement cannot be evaluated at any strength. None of the eight papers in the case literature set is indexed under PP1, and none of the six fetched full texts names c.78C>A or p.Ser26Arg or describes a family segregating the variant (PMID:24728327, the only study built on families, reports healthy-adult germline variation without APC segregation data). The ClinVar record (variation 133505) holds ten single-submitter Benign classifications with no segregation or family data. PP1 is returned not_assessed because zero available meioses is a documentation gap rather than evidence of non-segregation; a not_met call would require affirmative evidence that the variant fails to co-segregate with disease in at least one informative family.

Evidence considered

• APC VCEP v2.1 (cspec) lists PP1 as applicable, default Pathogenic Supporting, with strength rules: 3-4 meioses in >=1 family = Supporting, 5-6 meioses in >=1 family = Moderate, >=7 meioses in >=2 families = Strong.
• APC VCEP instructions for PP1 (cspec) require that affected individuals exhibit at least 0.5 phenotype point under Table 1, that only genotype- and phenotype-positive individuals and obligate carriers with phenotype are counted, and note that carriers who received chemoprevention may also be counted.
• APC VCEP supplementary material annotates PP1 ('Co-segregation with disease in multiple affected family members') with modifications based on Luo et al., 2019, matching the meioses-based thresholds tabulated here.
• The case contains no relative genotypes, no pedigree and no meiosis data; no segregation observation for NM_001127511.2:c.78C>A (p.Ser26Arg) exists in any source consulted.
• No paper in the case literature set is indexed under PP1; none of the six fetched full texts names c.78C>A or p.Ser26Arg. PMID:24728327 studied 681 healthy adults from 352 families by whole-genome sequencing but reports no APC genotype-phenotype segregation, and PMID:21368914 is a gene-level Clinical Utility Gene Card without segregation analysis.
• ClinVar variation 133505 (10 submissions from 10 clinical laboratories, all Benign, review status 'criteria provided, multiple submitters, no conflicts') contains no family or segregation data and no criteria-level detail usable for PP1.

Still missing

• No family members were genotyped and no meioses are documented for this variant, so the APC VCEP meioses thresholds (3-4 = Supporting, 5-6 = Moderate, >=7 in >=2 families = Strong) cannot be applied.
• Evaluating PP1 requires at least one family with 3-4 informative meioses in which affected carriers exhibit >=0.5 phenotype point under Table 1 (genotype- and phenotype-positive individuals and obligate carriers with phenotype only).

Sources

Reasoning

Governing specification: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specifications for APC v2.1, where BS3 is defined only through two evidence routes — (i) an RNA assay of a synonymous or intronic variant in a constitutional patient sample demonstrating no mRNA aberration (Strong when biallelic expression is shown and/or nonsense-mediated decay inhibition is used; Supporting without that confirmation), or (ii) a protein assay showing retention of beta-catenin-regulated transcription activity comparable to wild-type, which is explicitly restricted to variants within the beta-catenin binding domain (codons 959-2129 of APC). The variant under assessment, NM_001127511.2:c.78C>A p.(Ser26Arg), is a missense substitution at codon 26 of exon 1, not a synonymous or intronic variant, and it lies far outside codons 959-2129. Both VCEP-permitted BS3 routes are therefore unavailable for this variant as specified. No functional assay of any kind — RNA-based or protein-based — was identified for this variant in the ClinVar submission record, the criterion-lead PMID trail, or any fetched full text, and the specification adds no generic fallback for missense variants outside the beta-catenin domain. BS3 is recorded as not_assessed because there is no qualifying assay evidence to weigh in either direction; it is not a claim that function was tested and found normal.

Rule

APC VCEP v2.1 BS3 — Strong: RNA assay of a synonymous or intronic variant in constitutional patient sample shows no mRNA aberration AND biallelic expression is shown and/or NMD inhibition was used. Supporting: the same RNA assay without biallelic-expression/NMD confirmation, OR protein assay showing retention of beta-catenin-regulated transcription activity comparable to wild-type, restricted to variants within the beta-catenin binding domain (codons 959-2129 of APC).

Evidence considered

• APC VCEP specification v2.1 defines BS3 only as: Strong — 'RNA assay of a synonymous or intronic variant in constitutional patient sample demonstrates no mRNA aberration AND biallelic expression is shown and/or nonsense-mediated decay inhibition was used'; Supporting — the same RNA assay without biallelic-expression/NMD confirmation, 'OR Protein assay show retention of beta-catenin regulated transcription activity comparable to wild-type (only for variants within the beta-catenin binding domain, which refers to codons 959-2129 of APC)'.
• The VCEP supplementary-material table entry for BS3 records 'No comment' from the expert panel, i.e. no additional relaxation or qualifying statement beyond the rules above; the specification therefore offers no route to BS3 for a missense variant outside codons 959-2129.
• Variant consequence confirmed as a missense substitution at codon 26 of exon 1: NM_001127511.2:c.78C>A, NP_001120983.2:p.(Ser26Arg). It is neither synonymous nor intronic, and codon 26 is outside the beta-catenin binding domain (codons 959-2129).
• ClinVar (variation ID 133505; 11 submissions, 9 usable, 2 criterion-lead submissions citing PMID:24728327) contains no submission reporting functional assay data supporting a benign effect, and no expert-panel submission.
• Fetched full texts (PMID:15604628, PMID:21368914, PMID:24728327, PMID:25645574, PMID:25741868, PMID:28492532) were searched for the variant and for functional/RNA/protein assay content; none reports any functional assay on APC c.78C>A, and the exact variant is not named in any of them.
• No benign functional evidence exists to lower the strength of this criterion; BS3 strength is left unset (null) because the criterion is unevaluated, not met at any tier.

Still missing

• No RNA assay exists for this variant; and the specification limits that route to synonymous or intronic variants, which p.(Ser26Arg) is not.
• No beta-catenin-regulated transcription (or SPR binding) assay exists for this variant, and that route is restricted by the specification to codons 959-2129, excluding codon 26.
• The APC VCEP specification provides no BS3 route for a missense variant outside the beta-catenin binding domain, so BS3 is unlikely to become assessable for this variant even with additional data; a reviewer may confirm whether any non-specified functional evidence should be considered as a gene-level exception.

Sources

Reasoning

BS4 under the APC VCEP v2.1 is the non-segregation criterion and is graded from phenotype points rather than meioses: Strong when an affected member without the variant scores at least 1 phenotype point, or when at least two affected members without the variant each score at least 0.5 point; Supporting when an affected member without the variant scores at least 0.5 point (Table 1). Applying it therefore requires at least one affected, genotyped relative who does not carry NM_001127511.2:c.78C>A (p.Ser26Arg), together with Table 1 phenotype-point data for that individual. The case provides no relatives, no genotyping of any family member, no phenotype points for any individual and no family history, so no affected non-carrier can be identified and no phenotype-point total can be assembled. The ClinVar record (variation 133505, ten Benign submissions, none with assertion-criteria detail) reports no affected non-carrier, and no paper in the case literature set names the variant or describes a family in which an affected individual lacks it. BS4 is therefore not_assessed: without an affected non-carrier the criterion can be neither met nor refuted from this record, and the absence of such a relative is a documentation gap rather than evidence of segregation. The benign direction of this variant in other parts of the case file is supported by other criteria (population frequency, ClinVar concordance), not by BS4.

Evidence considered

• APC VCEP v2.1 (cspec) lists BS4 as applicable, default Benign Strong, with strength rules: 'Affected member without the variant must score at least 1 phenotype point or at least two affected members without the variant must each score at least 0.5 phenotype points' (Strong) and 'Affected member without the variant must score at least 0.5 phenotype points' (Supporting), both referring to Table 1.
• APC VCEP Table 1 (key vcep_table_1_262) defines the phenotype-point scale used by BS4 (1 point for a phenotype highly specific for APC, 0.5 point for a phenotype consistent with APC, 0.5 point for a typical FAP family history), but no individual phenotype or family history is recorded for this case, so no point total can be constructed.
• The case contains no affected relatives, no genotypes for any second individual and no family history, so no affected member lacking c.78C>A exists in the record to evaluate.
• ClinVar variation 133505 (10 submissions from 10 clinical laboratories, all Benign) provides no affected non-carrier, no segregation data and no criteria-level detail; no inference is drawn here from those classifications.
• No paper in the case literature set is indexed under BS4, and none of the six fetched full texts names c.78C>A or p.Ser26Arg or reports a family in which an affected individual lacks the variant.
• The VCEP caveat for BS4 (phenocopies of common phenotypes such as cancer can mimic lack of segregation, and a family may carry more than one pathogenic APC variant) is noted for any future segregation analysis of this variant.

Still missing

• No affected relative without the variant was reported or genotyped, and no Table 1 phenotype-point data exist for any individual, so the APC VCEP BS4 thresholds (>=0.5 point = Supporting; >=1 point in one affected member or >=0.5 in each of two = Strong) cannot be applied.
• Closing this gap requires genotype-plus-phenotype information from at least one affected family member who does not carry c.78C>A; note the VCEP caveats that phenocopies for common phenotypes and a second pathogenic APC variant in the family can mimic or mask lack of segregation.

Sources

Reasoning

Governing specification: APC VCEP v2.1 keeps BP5 Applicable at Benign Supporting, with the rule text 'Only applicable for an alternate genetic basis of the colorectal polyposis phenotype' and the defining instruction '(Likely) Pathogenic variant in another adenomatous polyposis gene (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 rule is qualitative: no numeric threshold, odds ratio, or likelihood-ratio value is defined for BP5 by this VCEP, so no >= or <= comparison and no inequality substitution is required (or permissible) for this criterion. Two conjunctive prerequisites therefore exist: (a) a colorectal polyposis phenotype must be present in the case, and (b) a (likely) pathogenic variant in one of the listed alternate polyposis genes must be documented in that case. Neither is available: the case files carry no proband phenotype, no polyp/adenoma findings, and no results from testing of MUTYH, NTHL1, MSH3, POLD1, POLE or the mismatch-repair genes; the case is a single-variant interpretation artifact for APC c.78C>A (p.Ser26Arg). No reviewed source supplies an alternate molecular basis either (the ACMG/AMP 2015 guideline, the NSGC and ACG practice guidelines, the FAP Clinical Utility Gene Card, the Sherloc methods paper, and the healthy-cohort study are all gene-level or framework-level and name no accompanying alternate-basis variant). BP5 is thus not assessable rather than affirmatively not met: without a documented polyposis phenotype and without other-gene results, the absence of an alternate basis cannot be distinguished from untested status. Gap: proband colorectal phenotype plus multi-gene/panel results covering the alternate adenomatous polyposis genes.

Rule

APC VCEP v2.1 BP5 (Benign Supporting): only applicable for an alternate genetic basis of the colorectal polyposis phenotype - a (Likely) Pathogenic variant in another adenomatous polyposis gene, and only when a colorectal polyposis phenotype is present.

Evidence considered

• cspec APC BP5 entry: applicability 'Applicable', defaultStrength 'Benign Supporting'; supporting-strength rule 'Only applicable for an alternate genetic basis of the colorectal polyposis phenotype.'
• cspec APC BP5 instructionsToUse (defining the alternate genetic bases accepted): '(Likely) Pathogenic variant in another adenomatous polyposis gene (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.'
• No numeric threshold, odds ratio, or likelihood ratio is defined for BP5 by the APC VCEP, so no comparison operator or inequality applies to this criterion; the group's <= direction warning is therefore moot for BP5.
• Case files contain no proband phenotype, polyp/adenoma count, age at onset, or results from other adenomatous polyposis genes; the only case-level variant data are for the APC substitution under assessment (NM_001127511.2:c.78C>A, p.Ser26Arg, NC_000005.10:g.112707795C>A).
• None of the reviewed sources documents an alternate molecular basis for this case: the APC VCEP specification itself, the ACMG/AMP 2015 guideline, and the ClinVar-cited gene-level references (NSGC practice guideline, ACG clinical guideline, FAP Clinical Utility Gene Card, Sherloc methods paper) contain no accompanying alternate-basis variant or multi-gene panel finding.

Still missing

• No colorectal polyposis phenotype documented anywhere in the case, so the VCEP precondition 'only applicable when a colorectal polyposis phenotype is present' cannot be evaluated.
• No results from testing MUTYH, NTHL1, MSH3, POLD1, POLE, MLH1, MSH2, MSH6 or PMS2 in this case, so an alternate (likely) pathogenic variant in another adenomatous polyposis gene cannot be confirmed or excluded.

Sources