APC c.4360A>G, p.(Lys1454Glu)

Reasoning

The governing APC VCEP v2.1 specification defines BA1 (Benign Stand Alone) as a gnomAD Popmax Filtering Allele Frequency >= 0.1% (0.001) and explicitly directs use of the non-cancer gnomAD dataset (v2.1.1) for this criterion, so the all-comers totals are not the operative source. In the VCEP-directed non-cancer gnomAD v2.1.1 exomes, the variant's popmax filtering AF is 0.00669606 (0.6696%), driven by the African/African American population (exome AFR 117/14,902 = 0.785%; genome-inclusive AFR 194/23,608 = 0.822%; genome popmax filtering AF 0.0072541). This exceeds the 0.1% BA1 threshold by roughly 6.7-fold. The VCEP rule deliberately keys on the popmax filtering AF rather than the global frequency, which matters here: the total non-cancer AF is 0.000582992 (138/236,710 exome alleles) and 0.0008056695 with genomes (216/268,100) - i.e. 0.058% and 0.081%, both below 0.1% - so BA1 is met specifically through ancestry-specific population maximum frequency, not through the cohort-wide rate. That is the biologically correct reading: a pathogenic allele for FAP/attenuated FAP (a dominant, early-onset, high-penetrance disorder with population prevalence on the order of 1 in 10,000-30,000) cannot be carried by ~0.7% of a population group, and an allele at that frequency is orders of magnitude above the maximum credible disease-allele frequency. The determination is robust to dataset choice: the all-comers gnomAD v4.1 release is concordant (total AF 0.000473942, 765/1,614,122 alleles; popmax filtering AF 0.00853602; AFR AF 0.00910205) and also exceeds 0.1% at the popmax level. Two threshold/sampling artifacts were noted and set aside: the 2.63% HGDP:Yoruba figure in gnomAD v3.1 non-cancer is 1/38 alleles and is a small-sample artifact rather than a population estimate, and gnomAD-Canada v1.0 (10/18,410, AF 0.0005431831) is a small cohort that does not change the popmax calculation. BA1 is therefore met at Stand Alone strength; as a stand-alone criterion it is by itself sufficient to support a Benign classification for this variant.

Rule

GnomAD Popmax Filtering Allele Frequency (AF) >= 0.1% (0.001). APC VCEP v2.1 BA1, Stand Alone; dataset directed by the specification: non-cancer gnomAD v2.1.1.

Evidence considered

• Governing rule applied: APC VCEP v2.1 BA1 (Benign Stand Alone) - 'GnomAD Popmax Filtering Allele Frequency (AF) >= 0.1% (0.001)', with the specification's dataset directive to 'Use the non-cancer dataset from gnomAD (v2.1.1)'.
• VCEP-directed source, non-cancer gnomAD v2.1.1 exomes (variant 5-112175651-A-G): popmax filtering AF 0.00669606 (0.6696%); total exome AF 0.000582992 (138/236,710 alleles), 0 homozygotes; genome popmax filtering AF 0.0072541; combined total AF 0.0008056695 (216/268,100 alleles), 0 homozygotes.
• Ancestry breakdown in the directed non-cancer v2.1.1 dataset: African/African American exome AF 117/14,902 = 0.785% and genome-inclusive AFR AF 194/23,608 = 0.822%, which is the population maximum that generates the 0.6696% popmax filtering AF; other populations are far lower (e.g. Admixed American well under 0.1%).
• Concordance check with the all-comers gnomAD v4.1 release (chr5-112839954-A-G): total AF 0.000473942 (765/1,614,122 alleles), popmax filtering AF 0.00853602, African/African American AF 0.00910205 (683/75,038 alleles) - also above the 0.1% popmax BA1 threshold, so the call does not depend on which release is used.
• Disease-prevalence reasoning: FAP/attenuated FAP is an autosomal dominant, early-onset, near-fully-penetrant hereditary cancer syndrome with population prevalence on the order of 1 in 10,000-30,000; an allele frequency of ~0.7% in any ancestry group is orders of magnitude above the maximum credible pathogenic-allele frequency for such a disorder, which is the biological basis of the stand-alone benign call.
• Threshold/artifact handling: the 2.63% HGDP:Yoruba frequency in gnomAD v3.1 non-cancer is 1/38 alleles and was rejected as a small-sample artifact rather than a population estimate; gnomAD-Canada v1.0 (10/18,410 alleles, AF 0.0005431831) is a small cohort and was not used for the popmax calculation.

Still missing

• BA1 rests on the popmax (ancestry-specific maximum) filtering AF of 0.6696% rather than the cohort-wide AF (0.058% exome-only, 0.081% with genomes), which sits below the 0.1% threshold; this is the metric the APC VCEP v2.1 rule specifies, but it means the criterion depends on the African/African American population frequency and on the correct identification of that population maximum.
• The VCEP-specified dataset (non-cancer gnomAD v2.1.1) is exome-dominated and predates gnomAD v4; gnomAD v4.1 all-comers data were used only as a concordance check, and no gnomAD v4 non-cancer subset was available in this case.

Sources

Reasoning

The APC VCEP v2.1 specification defines BS1 (Benign Strong) as a gnomAD Popmax Filtering Allele Frequency >= 0.001% (0.00001) and, like BA1, directs use of the non-cancer gnomAD v2.1.1 dataset. The same VCEP-directed figure that satisfies BA1 also satisfies BS1: the non-cancer v2.1.1 exome popmax filtering AF is 0.00669606 (0.6696%), roughly 670-fold above the 0.001% (0.00001) BS1 ceiling, and every other frequency metric for this variant is above the BS1 threshold as well (non-cancer exome total AF 0.000582992; non-cancer exome+genome total AF 0.0008056695; all-comers v4.1 total AF 0.000473942 with popmax filtering AF 0.00853602). BS1 is therefore factually met. It is recorded here as met/Strong because the criterion's own threshold is exceeded by a wide margin, but it is redundant in this case: because the higher BA1 threshold is also crossed, BA1 operates as the stand-alone criterion and no additional benign weight should be summed from BS1. The same ancestry consideration that drives BA1 applies - the excess frequency is concentrated in the African/African American population - and BS1 is met on both the cohort-wide and the ancestry-specific reading, so no threshold artifact affects this criterion.

Rule

GnomAD Popmax Filtering Allele Frequency (AF) >= 0.001% (0.00001). APC VCEP v2.1 BS1, Strong; dataset directed by the specification: non-cancer gnomAD v2.1.1.

Evidence considered

• Governing rule applied: APC VCEP v2.1 BS1 (Benign Strong) - 'GnomAD Popmax Filtering Allele Frequency (AF) >= 0.001% (0.00001)', with the specification's dataset directive to 'Use the non-cancer dataset from gnomAD (v2.1.1)'.
• VCEP-directed source, non-cancer gnomAD v2.1.1 exomes: popmax filtering AF 0.00669606 (0.6696%) versus the 0.001% (0.00001) BS1 threshold - exceeded ~670-fold.
• Corroborating frequency figures, all above the BS1 threshold: non-cancer exome total AF 0.000582992 (138/236,710); non-cancer exome+genome total AF 0.0008056695 (216/268,100); all-comers gnomAD v4.1 total AF 0.000473942 (765/1,614,122) with popmax filtering AF 0.00853602.
• BA1 (popmax filtering AF >= 0.1%) is also met for this variant, so BS1 is redundant rather than additive in the final combination; it was still adjudicated on its own threshold as required.

Still missing

• BS1 is met but is superseded by BA1 in the combination logic, since the stand-alone BA1 threshold (0.1%) is also exceeded; it should not be counted as additional independent benign evidence on top of BA1.

Sources

Reasoning

Governing specification: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel (APC VCEP) v2.1. The BS3 rule as literally written provides two Supporting options: (i) 'RNA assay of a synonymous or intronic variant in constitutional patient sample demonstrates no mRNA aberration, without demonstration of biallelic expression or use of nonsense-mediated decay inhibition' (Strong is reserved for the same result WITH biallelic expression and/or NMD inhibition), or (ii) '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)'. Option (i) is unavailable: c.4360A>G is a missense change, not synonymous or intronic, and no RNA study exists. Option (ii) is the applicable route, and this variant is eligible because NP_001120982.1:p.(Lys1454Glu) is codon 1454, inside the specified 959-2129 window. The assay evidence satisfies option (ii). PMID:18199528 (Azzopardi et al. 2008, Cancer Res 68:358-63) measured beta-catenin-regulated transcription (CRT) with a luciferase reporter system (pTOPFLASH/pFOPFLASH in APC-deficient SW480 cells, CMV-APC constructs, results averaged over at least 10 independent experiments, Student's t test) and validated the assay with in-run controls: six synonymous variants that all suppressed CRT as effectively as wild type (normal controls), the wild-type APC construct itself, and four truncating mutations (1309D, 1450D, 1517D, 1914D) as mutant controls, of which 1309D failed to suppress CRT (P < 0.0001) and the others suppressed it less effectively than wild type (P < 0.05) - i.e. the assay discriminated loss of function with both positive and negative controls. Sixteen nonsynonymous variants were then tested; K1454E was one of them (Figure 2C x-axis: I1307K, E1317Q, V1352A, M1413V, T1445A, A1446T, K1454E, P1467S, A1474T, I1572T, C1578G, I1579V, R1676G, D1714N, G1921S, P1934L), and only seven (43.8%) showed significantly reduced CRT suppression. K1454E was not among those seven: read from Figure 2C its mean CRT activity is approximately 1.15x the wild-type-normalised value (1.0) with no significance marker, against approximately 1.5-2.2x for the seven compromised variants. Retention of beta-catenin-regulated transcription activity comparable to wild type is therefore demonstrated for this exact variant. Corroboration: two independent clinical-laboratory ClinVar submissions describe this same result for this exact variant - LabCorp SCV000694052 (2016): 'one functional study showed that this variant has a normal effect in an in vitro beta-catenin regulated transcription (crt) assay (azzopardi_2008)'; and Sinai Health System SCV001450573, which cites the same functional-study conclusion. OncoKB independently curates the APC K1454E biological effect as 'Likely Neutral' with PMID:18199528 as its citation. Independent germline observations point the same way: the variant was present in both normal liver and hepatic adenoma in a patient with no colonic polyposis and was interpreted as a polymorphism (PMID:11904335), and it was carried by 2 of 969 healthy controls (PMID:18199528). Strength calibration: the VCEP caps this protein-assay route at Supporting - 'retention of beta-catenin regulated transcription activity comparable to wild-type' is listed only under the Supporting tier, and the Strong tier of BS3 is restricted to RNA-assay evidence with biallelic expression and/or NMD inhibition, which does not exist here. Supporting strength is therefore the maximum defensible and is the strength assigned. Caveats that are recorded rather than hidden: the per-variant value for K1454E appears only in a figure (no numeric value, replicate spread or P-value published for this variant); the CRT read-out is an indirect Wnt-pathway transcription proxy rather than a direct beta-catenin binding measurement (no SPR data exist); and the assay was performed in a somatic-tumour APC-deficient cell line, not in patient-derived material. Explicit comparison with the governing threshold: does the assay show 'retention of beta-catenin regulated transcription activity comparable to wild-type' for a variant within codons 959-2129? Yes - approximately 1.15x the wild-type-normalised value, no significant difference reported and no significance marker, in a variant at codon 1454 (within 959-2129), assayed with validated normal and mutant controls. BS3 is met at Supporting strength.

Rule

APC VCEP v2.1 BS3: RNA assay of a synonymous or intronic variant showing no mRNA aberration (Strong when biallelic expression and/or NMD inhibition is shown; Supporting without) OR protein assay showing retention of beta-catenin regulated transcription activity comparable to wild-type, eligible only for variants within the beta-catenin binding domain (codons 959-2129) - Supporting strength

Evidence considered

• APC VCEP v2.1 BS3_Supporting, quoted verbatim: '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, see PMID: 33348689)'; the BS3_Strong tier is restricted to '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'.
• Variant eligibility for the protein-assay route: NP_001120982.1:p.(Lys1454Glu) = codon 1454, within the VCEP-specified beta-catenin binding domain of codons 959-2129.
• PMID:18199528 (Azzopardi et al. 2008) assay controls and validation, quoted: 'we made and tested six synonymous variants (normal controls) and four truncating mutations (mutant controls) within the beta-catenin down-regulating domain ... the six synonymous variants and the wild-type APC construct all suppressed CRT in an APC-deficient cell line (Fig. 2A)'; '1309D, which is associated with a severe FAP phenotype, failed to suppress CRT (P < 0.0001) whereas 1450D, 1517D, and 1914D suppressed CRT more effectively than 1309D but not as effectively as the wild-type construct (P < 0.05; Fig. 2B)'.
• PMID:18199528 result for the assayed set, quoted: 'Next, we tested the 16 nonsynonymous variants, and in accordance with our hypothesis that some of these rare variants may compromise function, seven (43.8%) had a significantly reduced ability to suppress CRT (Fig. 2C). Both I1307K and E1317Q were found to be functionally compromised.'
• Direct examination of Figure 2C of PMID:18199528 confirms K1454E is one of the 16 assayed variants (x-axis label 'K1454E'), that no significance marker appears over its bar, and that its mean activity is approximately 1.15x the wild-type-normalised value (1.0) versus approximately 1.5-2.2x for the seven significantly compromised variants.
• ClinVar submission SCV000694052 (LabCorp, Likely benign, 2016) evidence text: 'one functional study showed that this variant has a normal effect in an in vitro beta-catenin regulated transcription (crt) assay (azzopardi_2008)' - an independent laboratory reading of the same assay for the same variant.
• ClinVar submission SCV001450573 (Department of Pathology and Laboratory Medicine, Sinai Health System) records the same functional-study conclusion for this variant, giving two independent clinical-laboratory readings of the CRT result.
• OncoKB curation for APC K1454E: classification and biological effect both 'Likely Neutral', with PMID:18199528 as the cited source.
• PMID:11904335 (Torbenson et al. 2002), quoted: 'in Case 9, a base pair change in Codon 1454 did lead to a change in the amino acid sequence, from lysine to glutamine. The amino acid change was present in both the normal liver and adenoma and was interpreted as a polymorphism. The patient had no clinical evidence for a colonic polyposis syndrome.' - an independent germline carrier without polyposis, consistent with the assay result; it is an observation, not an assay, so it is not itself BS3 evidence.
• PMID:18199528 Table 1: K1454E detected once in non-FAP/non-MAP patients with multiple colorectal adenomas and twice in 969 healthy controls, i.e. the variant is present in the general population, consistent with a retained-function allele.
• No computational-prediction score was used for this criterion: the APC VCEP explicitly prohibits conservation/evolution in-silico models for missense variants, and BS3 is an assay-based criterion.

Still missing

• The K1454E CRT result is published only graphically (Figure 2C of PMID:18199528); no numeric activity value, error bars or P-value specific to this variant are reported, so the exact magnitude (approximately 1.15x the wild-type-normalised value) was read from the published bar chart.
• No RNA-based transcript assay exists and no biallelic-expression or NMD-inhibition data exist, so the VCEP BS3_Strong tier (RNA assay with biallelic expression and/or NMD inhibition) is not reached and cannot be reached for a missense variant; Supporting is the ceiling.
• No direct beta-catenin binding measurement (e.g. surface plasmon resonance) exists for K1454E; retention of function is inferred from the indirect CRT transcription read-out rather than from binding affinity.
• The source assay examined a single artificial SW480 (APC-deficient colorectal) cell system; no patient-derived or in vivo functional confirmation of retained APC function for germline K1454E was identified.

Sources

Reasoning

The APC VCEP/InSiGHT specification 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 variant under assessment is a missense change at codon 1454 (p.(Lys1454Glu)), which is outside that excepted 1021-1035 window, so the exception does not apply and BP1 is usable at Supporting strength. APC is a gene in which primarily truncating (loss-of-function) variants cause disease - the VCEP's PVS1 framework treats loss of function as the established mechanism - so a genuine missense substitution satisfies the original BP1 definition. The VCEP's instruction for BP1 warns that a number of assumed missense variants are in fact splice variants and requires splice predictors to be checked; SpliceAI max delta score is 0.001 (DS_AG 0.001, all other deltas 0.0) and Pangolin scores are near zero, so no hidden splice effect is present and the variant is a true amino-acid substitution. BP1 is met at Supporting strength.

Rule

APC VCEP BP1 (Supporting): 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).

Evidence considered

• APC VCEP/InSiGHT specification (v2.1) BP1 rule and applicability: '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 variant is at codon 1454, outside the excepted window.
• APC VCEP BP1 instructionsToUse: a number of assumed missense variants are in fact splice variants, and at least several splice prediction tools should be used before applying BP1.
• pvs1_gene_context.json: loss-of-function mechanism supported (lof_mechanism_supported true), supporting the premise that APC disease is driven primarily by truncating variants.
• SpliceAI lookup for NM_001127510.3:c.4360A>G: max delta score 0.001 (DS_AG 0.001, DS_AL 0.0, DS_DG 0.0, DS_DL 0.0), Pangolin SG 0.022 / SL -0.001 - no predicted splice effect, confirming the change is a genuine missense substitution rather than a cryptic splice variant.

Sources

Reasoning

APC is a gene in which loss of function is the predominant disease mechanism and the APC VCEP framework is applicable (PVS1 default strength Pathogenic Very Strong), so PVS1 was assessed rather than skipped. However, NM_001127510.3:c.4360A>G is a single-nucleotide substitution that changes one amino acid, NP_001120982.1:p.(Lys1454Glu): it is a missense allele, not a null/truncating allele. It is not a nonsense variant, not a frameshift, and not a canonical +/-1 or +/-2 splice-consensus change; Mutalyzer/VariantValidator predict a single-residue substitution with no change in protein length (position_last_original 1454 = position_last_predicted 1454), so no truncated gene product is predicted. Transcript relevance: the transcript of record is NM_001127510.3 (APC transcript variant 2), and the variant lies within exon 17, the large terminal coding exon (c.1959-8532), far from any exon/intron boundary, so canonical splice-site disruption is impossible at this position. The APC VCEP PVS1 decision tree (Fig. 1) restricts PVS1 to null variants, and its Lists A-E consist exclusively of canonical splice-site nucleotide changes; c.4360A>G appears in none of them. SpliceAI (hg37, distance 500) returns a max delta score of 0.001 (DS_AG 0.001, DS_AL 0.000, DS_DG 0.000, DS_DL 0.000), excluding a cryptic splice-site gain or loss that could create a splice-mediated null allele. Because the variant is not a null variant, no specific named SVI or VCEP downgrade rule (NMD escape, non-critical/last exon, removal of <10% of protein) is engaged and no strength tier can be assigned; the strength field is therefore null rather than a strength-tier word, since a strength tier only exists for a met PVS1 call.

Rule

APC VCEP v2.1 PVS1 modified decision tree (Fig. 1): null variants only (nonsense, frameshift, canonical +/-1,2 splice sites); missense substitutions not in Lists A-E.

Evidence considered

• Variant consequence: Mutalyzer/VariantValidator predict NM_001127510.3:c.4360A>G (NC_000005.10:g.112839954A>G) yields NP_001120982.1:p.(Lys1454Glu) - a single amino-acid substitution, not a premature termination codon, frameshift, or splice-consensus change.
• Protein length change: none - Mutalyzer reports position_first 1453, position_last_original 1454, position_last_predicted 1454, so neither truncation nor elongation of APC is predicted.
• NMD: not applicable - no premature termination codon is introduced, so the NMD-escape question never arises and cannot be used as a PVS1 downgrade pathway.
• Transcript relevance/exon importance: NM_001127510.3 (APC transcript variant 2) is the transcript of record; VariantValidator places the change in exon 17 (start_exon = end_exon = 17 in both GRCh37 and GRCh38), the large terminal coding exon spanning c.1959-8532, remote from any exon/intron junction.
• SpliceAI Lookup (hg37, distance 500, basic mask): max delta score 0.001 (DS_AG 0.001, DS_AL 0.000, DS_DG 0.000, DS_DL 0.000; Pangolin SG 0.022, SL -0.001) - no cryptic splice-site gain or loss, excluding a splice-mediated null allele.
• Governing framework: the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for APC v2.1 keeps PVS1 applicable with default strength Pathogenic Very Strong, but limits it to null variants via the modified Fig. 1 decision tree, whose Lists A-E (PVS1, PVS1_Strong, PVS1_Moderate, PVS1_Supporting, N/A) list only canonical +/-1/2 splice-site nucleotide changes; p.Lys1454Glu is not among them.
• APC VCEP supplementary specification comment on PVS1: 'APC is a gene where LOF is the predominant mechanism...' - a gene-level statement of mechanism that does not convert a missense substitution into a null allele.
• PMID:18199528 names this exact allele, K1454E, as a rare (MAF <2%) inherited nonsynonymous APC variant found in 2/969 healthy controls as well as in adenoma patients - a missense change, not a truncating one.
• No SVI/VCEP downgrade rule was applied and no strength tier is recorded because the criterion is not met at all; strength is null, not the criterion code and not a weakened strength word.

Still missing

• No RNA/cDNA study of this allele was available; the missense-not-null determination rests on the predicted consequence plus SpliceAI, which is sufficient because PVS1 requires a null allele.

Sources

Reasoning

PS1 requires the variant under assessment to produce the same amino acid change as a variant already established as Pathogenic (PS1_Strong) or Likely Pathogenic (PS1_Moderate) under the APC-specific modifications. The variable amino-acid change here is p.(Lys1454Glu), and the APC VCEP specification states that only two APC missense variants have ever been classified Likely Pathogenic - c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg) - and that no APC missense variant has been classified Pathogenic. p.(Lys1454Glu) is neither of those amino-acid changes, no established Pathogenic (L)Pathogenic variant with the amino-acid change Lys1454Glu (or any other missense substitution at codon 1454) exists, and the exact variant is classified Benign/Likely benign by multiple clinical laboratories in ClinVar. The patient-cohort label of 'deleterious' applied to this allele in PMID:23970361 is a single-study assertion, not a classification reached under the APC-specific modifications, and does not satisfy the rule. PS1 is therefore not met.

Rule

APC VCEP PS1: The previously established variant was classified as Pathogenic (PS1_Strong) or Likely Pathogenic (PS1_Moderate) according to the APC-specific modifications. Missense variants: when the variant under assessment results in the same amino acid change as previously established (Likely) Pathogenic variant(s). There are currently only two Likely Pathogenic missense variants: c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg). No missense variant has been classified as Pathogenic based on current evidence.

Evidence considered

• APC VCEP/InSiGHT specification (v2.1) PS1 rule: for missense variants PS1 applies only when the variant under assessment results in the same amino acid change as a previously established (L)Pathogenic variant, and the specification names only two Likely Pathogenic APC missense variants, c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg); it states no APC missense variant has been classified Pathogenic.
• ClinVar (VariationID 127295) classifies the exact variant NM_000038.6(APC):c.4360A>G p.(Lys1454Glu) as Benign (11 laboratories), Likely benign (6), benign (1) and Uncertain significance (1); no submitter reports it as Pathogenic or Likely pathogenic, and no expert-panel submission is present.
• PMID:23970361 labels the Lys1454Glu allele 'D' (deleterious) in a single FAP patient (patient 19), an assertion with no functional, segregation or recurrence data, not a classification performed under the APC-specific modifications.
• No other germline codon-1454 missense substitution with an established Pathogenic or Likely Pathogenic classification was identified in any case source.

Sources

Reasoning

Governing specification: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel (APC VCEP) v2.1, which replaces the generic PS3 concept for APC. In the VCEP the PS3 routes are (a) RNA assays of splicing aberrations, tiered from Supporting to Very Strong by the type of transcript aberration and the residual full-length transcript percentage, and (b) for variants lying within the beta-catenin binding domain (codons 959-2129), a PROTEIN assay at Supporting strength, defined literally as '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 under assessment: NM_001127510.3:c.4360A>G, NP_001120982.1:p.(Lys1454Glu), codon 1454 - i.e. inside the 959-2129 window, so the protein-assay route is the only route that could apply. Route (a) is not available: the variant is a missense change with no RNA-based transcript study, so the RNA tiers (premature stop codon, inframe skipping of exon 13 or 14, other inframe skipping, alternative-transcript over-expression) cannot be scored. Route (b) is evaluable and fails. The only variant-specific functional assay identified anywhere in the assembled evidence is PMID:18199528 (Azzopardi et al. 2008, Cancer Res 68:358-63), a beta-catenin-regulated transcription (CRT) luciferase reporter assay (CMV-APC constructs, pTOPFLASH/pFOPFLASH reporters, SW480 APC-deficient cells, at least 10 independent experiments, Student's t test), with an explicitly validated control set: six synonymous variants as normal controls, the wild-type APC construct, and four truncating mutant controls (1309D, 1450D, 1517D, 1914D). In that assay K1454E is one of the 16 nonsynonymous variants tested (Figure 2C x-axis labels), and it is NOT one of the seven variants (43.8%) that showed significantly reduced ability to suppress CRT - the compromised set includes I1307K and E1317Q and is separated by a wide gap from the remainder (the seven significant bars sit at approximately 1.5-2.2x the wild-type-normalised value, whereas K1454E sits at approximately 1.15x). Read directly from Figure 2C, K1454E retains CRT suppression and shows no significant change in beta-catenin-regulated transcription, so there is no demonstrated 'increased beta-catenin regulated transcription activity', and no surface plasmon resonance beta-catenin binding data exist at all. Two clinical-laboratory ClinVar submissions (LabCorp SCV000694052; Sinai Health System SCV001450573) independently characterise this same study as showing a normal effect for this variant in the CRT assay, consistent with the figure. Evidence that must not be counted toward PS3: the somatic K1454E substitution reported in invasive urothelial carcinoma (PMID:18844223; 3 of 70 tumours, absent from matched normal tissue) is a somatic-tumour observation, not a validated in vitro or in vivo functional assay, and it carries no measurement of APC function; likewise the germline carriers reported by PMID:23970361 (one FAP patient, no assay) and PMID:11904335 (variant present in both normal liver and hepatic adenoma, interpreted as a polymorphism) provide no assay read-out. A review of APC missense biology (PMID:21859464) states that the majority of mutation-cluster-region missense variants lie outside the essential beta-catenin repeats and that the mechanism by which they impair APC function is unresolved, and reports no variant-specific assay. Explicit comparison with the governing thresholds: is there an RNA-assay transcript aberration satisfying any PS3 tier (premature stop codon and/or inframe skipping of exon 13 or 14 with absent or <10% full-length transcript, or the Moderate/Supporting combinations)? No - no RNA data exist. Is there a protein assay showing increased beta-catenin regulated transcription activity and/or decreased beta-catenin binding (PS3_Supporting floor)? No - the single available protein/transcription assay shows activity not significantly different from wild type. PS3 is therefore not met at any strength.

Rule

APC VCEP v2.1 PS3: RNA assays of splicing aberrations (Very Strong / Strong / Moderate / Supporting tiers by transcript aberration and residual full-length transcript); protein assay showing increased beta-catenin regulated transcription activity and/or decreased beta-catenin binding by SPR at Supporting strength, eligible only for variants within the beta-catenin binding domain (codons 959-2129)

Evidence considered

• APC VCEP v2.1 (ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel) PS3_Supporting protein-assay route, quoted verbatim: '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) [Reference 2]'; PS3 RNA-assay tiers require, at Very Strong, 'a premature stop codon OR inframe skipping of exon 13 or 14 AND the absence of full-length transcript', declining to Supporting for other inframe skipping plus reports of exon deletion/skipping/loss and for alternative-transcript (exons 10, 11, 15) over-expression.
• Variant position relative to the VCEP protein-assay window: NP_001120982.1:p.(Lys1454Glu) is codon 1454, inside the specified 959-2129 beta-catenin-binding region (domain boundaries referenced by the VCEP to PMID:33348689, Juanes 2020), so the protein-assay route is formally eligible for this variant.
• PMID:18199528 (Azzopardi et al. 2008) Methods, quoted: 'Variants were introduced into CMV-APC constructs using the QuikchangeXL site-directed mutagenesis kit ... The luciferase reporter plasmids pTOPFLASH and pFOPFLASH, which contained wild-type or mutated TCF-response elements, were used to assess beta-catenin-regulated transcription (CRT) ... results averaged from at least 10 independent experiments and compared using Students' t test' - a validated, controlled assay format.
• PMID:18199528 Results, quoted: 'We examined the functional consequences of 16 of the nonsynonymous variants identified within the beta-catenin down-regulating domain using CRT assays ... the six synonymous variants and the wild-type APC construct all suppressed CRT in an APC-deficient cell line (Fig. 2A) ... 1309D, which is associated with a severe FAP phenotype, failed to suppress CRT (P < 0.0001) ... Next, we tested the 16 nonsynonymous variants ... seven (43.8%) had a significantly reduced ability to suppress CRT (Fig. 2C). Both I1307K and E1317Q were found to be functionally compromised.'
• Direct examination of Figure 2C of PMID:18199528: the x-axis of the 16-variant panel is labelled I1307K, E1317Q, V1352A, M1413V, T1445A, A1446T, K1454E, P1467S, A1474T, I1572T, C1578G, I1579V, R1676G, D1714N, G1921S, P1934L - i.e. K1454E was assayed. No significance marker is shown over the K1454E bar, and its mean activity is approximately 1.15x the wild-type-normalised value (1.0), whereas the seven variants reported as significantly compromised form a separate group at approximately 1.5-2.2x.
• ClinVar submission SCV000694052 (Women's Health and Genetics/Laboratory Corporation of America, LabCorp; Likely benign, 2016), submitted evidence text: 'one functional study showed that this variant has a normal effect in an in vitro beta-catenin regulated transcription (crt) assay (azzopardi_2008)'. ClinVar submission SCV001450573 (Department of Pathology and Laboratory Medicine, Sinai Health System) independently reports the same functional-study conclusion for this variant.
• OncoKB record for APC K1454E records the curated biological effect and oncogenicity as 'Likely Neutral', citing PMID:18199528 (the CRT study) - i.e. an independent curated source reads the same assay as neutral rather than damaging.
• PMID:18844223 (Kastritis et al. 2009): the identical AAA>GAA codon-1454 (Lys>Glu) change was found as a somatic mutation in 3 of 70 muscle-invasive urothelial carcinomas and was absent from matched normal urothelium; this is a somatic-tumour sequencing observation with no functional read-out and is not a PS3-eligible assay.
• PMID:21859464 (Minde et al. 2011) review, quoted: 'Remarkably, the majority of reported missense mutations in APC MCR are located outside the essential 15aa and 20aa repeat regions and their flanking N-terminal regions required for binding b-catenin'; the paper reports no variant-specific assay or measurement for Lys1454Glu.
• No REVEL, SpliceAI or BayesDel threshold was used for this criterion: PS3/BS3 are assay-based criteria, and the APC VCEP explicitly forbids computational-prediction models for missense variants (PP3/BP4 pathway, not this group).

Still missing

• The per-variant CRT result for K1454E is published only as a bar in Figure 2C of PMID:18199528; no numeric value, replicate scatter or P-value is reported for this variant, so the 'no significant difference from wild type' reading rests on the published figure (approximately 1.15x the wild-type-normalised value, with no significance marker) plus two clinical-laboratory ClinVar submissions that characterise the study as showing a normal effect.
• No RNA-based transcript study exists for this variant, so the VCEP's RNA-assay PS3 tiers (premature stop codon; inframe skipping of exon 13/14; other inframe skipping; alternative-transcript over-expression) could not be scored at all.
• No surface plasmon resonance (or other direct) measurement of APC beta-catenin binding is available for K1454E; only the indirect CRT transcription read-out exists.
• Somatic recurrence of K1454E in 3/70 invasive urothelial carcinomas (PMID:18844223) is suggestive of a tolerated/late somatic event in a different tissue context but is not a validated assay and cannot be converted into PS3 weight; whether any published study has scored APC protein levels, beta-catenin turnover or Wnt target output in cells carrying germline K1454E specifically was not established from the fetched literature.

Sources

Reasoning

Governing specification: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel (APC VCEP) v2.1. PS4 in APC is not an odds-ratio criterion; it is scored from the Table 1 phenotype-point system. Exact operators as literally written in the governing VCEP text: '>= 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 (for this criterion higher values strengthen; there is no inverse/likelihood-ratio tier). The supporting tier is the relevant one here. Reported index patients carrying this exact variant: PMID:23970361 Table II, patient 19 - 47-year-old female with familial adenomatous polyposis (TNM III, Astler-Coller C2, no extra-colonic manifestations), from a cohort defined by families with >100 colorectal polyps in >=2 successive generations. Table 1 awards 1 point only for a 'typical colorectal phenotype', which requires 20-99 colorectal adenomas at <=20 yr, or >=100 colorectal adenomas at <=30 yr, or >=1000 colorectal adenomas at any age, or an accepted descriptor such as 'innumerable'; at 47 years that 1-point row is not met, and the patient instead meets the 0.5-point 'other colorectal phenotype' row (>=20 colorectal adenomas at 20 to 70 yr, or a documented diagnosis of FAP/AFAP). The family-history 0.5 point cannot simply be added: Table 1 footnote c permits family history only if at least one variant carrier and one additional relative each fulfil >=0.5 point, which is not documented. Score = 0.5 phenotype points from the only carrier with a scoreable APC phenotype. The case itself records no proband phenotype, family history or clinical indication, so it contributes 0 points. Explicit comparison written out: total = 0.5 phenotype points; PS4_Supporting minimum is 1 (range 1-1.5). Is 0.5 >= 1? No. Is 0.5 < 1? Yes. Therefore PS4 is not met at any strength (all thresholds 16, 4, 2 and 1 exceed the observed 0.5). Concordant observations: PMID:18199528 detected K1454E once among non-FAP colorectal-adenoma patients and twice among 969 healthy controls (no enrichment in affected individuals; direction opposite to PS4); PMID:11904335 detected the same change in both normal liver and a hepatic adenoma of a patient with no colonic polyposis and interpreted it as a polymorphism; PMID:21859464 does not name the variant and reports no affected-individual data; PMID:15604628 is a syndrome-level genetic-counseling guideline with no variant-level data.

Rule

APC VCEP v2.1 PS4: >= 16 phenotype points (Very Strong); 4-15.5 (Strong); 2-3.5 (Moderate); 1-1.5 phenotype point (Supporting), using the Table 1 phenotype-point system

Evidence considered

• APC VCEP v2.1 PS4 tiers are defined by phenotype points summed over index patients (max 1 point per proband), with the literal operators '>= 16 phenotype points', '4-15.5 phenotype points', '2-3.5 phenotype points', '1-1.5 phenotype point'.
• VCEP supplementary material states PS4 modifications are based on Fortuno et al. 2020 (PMID:33300245), i.e. prevalence in affected individuals is operationalised through the phenotype-point system rather than a raw odds ratio.
• Table 1 of the APC specifications: 1 point requires a typical colorectal phenotype (20-99 adenomas at <=20 yr, or >=100 adenomas at <=30 yr, or >=1000 adenomas at any age, or an 'innumerable/uncountable/countless/carpeting' descriptor); 0.5 point for other colorectal phenotype (>=20 histologically confirmed adenomas at 20 to 70 yr, or a documented FAP/AFAP diagnosis).
• PMID:23970361 (De Queiroz Rossanese et al. 2013): 20 non-relative Brazilian FAP index patients from families with >=2 successive affected generations and >100 polyps; patient 19 is a 47-year-old female with FAP (TNM III, Astler-Coller C2, no extra-colonic manifestations) carrying Lys1454Glu - the only carrier with a scoreable APC phenotype, worth 0.5 points.
• PMID:18199528 (Azzopardi et al. 2008): K1454E detected once in non-FAP/non-MAP multiple-CRA patients and twice in 969 healthy controls; authors list it among variants found in both patients and controls, so it shows no enrichment in affected individuals.
• PMID:11904335 (Torbenson et al. 2002): the identical codon-1454 AAA>GAA (Lys>Glu) change was present in both normal liver and hepatic adenoma of one patient who had no colonic polyposis, and was interpreted as a polymorphism.
• PMID:21859464 (Minde et al. 2011): review of APC missense biology; does not name c.4360A>G/p.Lys1454Glu and reports no affected-individual phenotype data.
• PMID:15604628 (NSGC guideline): syndrome-level recommendations only; no variant-level or case-count data for this variant.

Still missing

• No proband phenotype or clinical indication is recorded for this case, so no Table 1 phenotype points could be scored for the index patient; only literature-reported carriers were scorable.
• Adenoma count (>=100 documented for the cohort, but not for patient 19 individually) and whether the assessment age qualifies for the 1-point typical-phenotype row are incompletely documented in PMID:23970361; if a proband met the 1-point row, PS4_Supporting would be reached.
• No case-control/cohort enrichment estimate exists for this variant; the only case-control-adjacent observation (PMID:18199528) is not enrichment-positive.

Sources

Reasoning

The APC VCEP v2.1 PM2 rule (Pathogenic Supporting) requires the variant to be rare in controls, defined as an allele frequency <= 0.0003% (0.000003) when the allele count is > 1, or an allele frequency < 0.001% (0.00001) when the allele count is <= 1; the specification directs use of the non-cancer gnomAD v2.1.1 dataset. In that VCEP-directed dataset the variant is neither rare nor even very infrequent in the exomes: it is observed 138 times in 236,710 alleles (AF 0.000582992, 0.0583%), with an allele count of 138, so the applicable ceiling is 0.000003 (0.0003%). The observed AF is roughly 194-fold above that ceiling. Because the allele count is far greater than 1, the more permissive AC <= 1 branch does not apply. The result is unchanged across every frequency source in the case: all-comers gnomAD v2.1 (AF 0.000806731, 228/282,622 alleles), all-comers gnomAD v4.1 (AF 0.000473942, 765/1,614,122 alleles), non-cancer gnomAD v3.1 genomes (AF 0.00250879, 371/147,880 alleles), and gnomAD-Canada v1.0 (AF 0.0005431831, 10/18,410 alleles) - each is orders of magnitude above the PM2 suppressor ceiling. PM2 is designed to capture variants that are absent or at extremely low frequency in population controls; this variant is a recurrent, widely observed population allele, with its highest frequency concentrated in the African/African American population, so the criterion is not met and the correct disposition is not_met rather than not_assessed. No pathogenic weight can be taken from PM2 for this variant.

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. APC VCEP v2.1 PM2, Supporting; dataset directed by the specification: total population of the non-cancer gnomAD v2.1.1 dataset.

Evidence considered

• Governing rule applied: APC VCEP v2.1 PM2 (Pathogenic Supporting) - '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', with the specification's dataset directive to 'Use the total population from the non-cancer dataset from gnomAD (v2.1.1)'.
• VCEP-directed source, non-cancer gnomAD v2.1.1 exomes: AF 0.000582992 (0.0583%), 138/236,710 alleles, 0 homozygotes. Allele count 138 > 1, so the applicable ceiling is 0.000003 (0.0003%); the observed AF is about 194-fold above it.
• All-comers gnomAD v2.1 (228/282,622 alleles, AF 0.000806731) and all-comers gnomAD v4.1 (765/1,614,122 alleles, AF 0.000473942; African/African American AF 0.00910205) are similarly far above the PM2 ceiling, so the call is robust to dataset choice.
• Additional frequency sources reviewed and concordant: non-cancer gnomAD v3.1 genomes (371/147,880 alleles, AF 0.00250879) and gnomAD-Canada v1.0 (10/18,410 alleles, AF 0.0005431831).
• PM2 is intended for variants absent from or at extremely low frequency in controls; this variant is a recurrent population allele present in multiple independent datasets, so it cannot be scored as rare in controls and contributes no pathogenic support.

Still missing

• PM2 is not met in every available frequency source, so no further data could change the determination; no gap remains for this criterion.

Sources

Reasoning

PM5 requires a different missense change at the same amino acid residue (codon 1454) that has been determined Pathogenic or Likely Pathogenic under the APC-specific modifications, with the Grantham distance of the variant under assessment at least equal to that of the reported comparator. The APC VCEP specification states that only two APC missense variants are Likely Pathogenic, c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg), and that no APC missense variant has been classified Pathogenic; neither lies at codon 1454. The ClinVar same-residue comparator harvest for codon 1454 returned 0 same-residue candidates and 0 (L)Pathogenic comparators. No variant at codon 1454 other than p.(Lys1454Glu) itself is reported as pathogenic in any reviewed source: PMID:11904335 found the codon 1454 AAA>GAA change in both normal liver and adenoma of a patient without colonic polyposis, interpreting it as a polymorphism, and PMID:18844223 reports the identical p.(Lys1454Glu) substitution as a somatic tumour change, not as a different pathogenic missense at the residue. There is therefore no qualifying comparator, and the Grantham-distance condition cannot even be evaluated. The variant is also not novel (seen in 2/969 healthy controls, PMID:18199528), which is contrary to the criterion's premise. PM5 is not met.

Rule

APC VCEP PM5 (Moderate): 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. PM5 (Supporting): same rule with the comparator determined Likely Pathogenic.

Evidence considered

• APC VCEP/InSiGHT specification (v2.1) PM5 rule: the reported comparator must have been determined Pathogenic (PM5 moderate) or Likely Pathogenic (PM5 supporting) under the APC-specific modifications, and the Grantham distance of the variant under assessment must be equal to or higher than that of the reported variant; the specification lists only c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg) as Likely Pathogenic APC missense variants.
• pm5_candidates.json (classic_same_residue_missense mode): 6 candidates seen, 0 same-residue candidates, 0 expert-panel (L)Pathogenic candidates, 0 non-expert majority (L)Pathogenic candidates; harvesting failed softly with HTTP 429 and the artifact explicitly instructs not to apply PM5 from it alone.
• ClinVar VariationID 127295 reports the exact p.(Lys1454Glu) variant as Benign/Likely benign, with no Pathogenic or Likely pathogenic submission; no ClinVar record of a different pathogenic missense substitution at codon 1454 was identified.
• PMID:11904335 detected the codon 1454 AAA>GAA (Lys>Glu) change in both the normal liver and the hepatic adenoma of Case 9, with no clinical evidence of a colonic polyposis syndrome, and interpreted it as a polymorphism; the same source notes codon 1454 is only occasionally affected by insertions/deletions and lists no missense mutations there in the APC mutation database.
• PMID:18844223 found the identical codon 1454 AAA>GAA (Lys>Glu) change as a somatic mutation in three patients (5, 7 and 8) with muscle-invasive urothelial carcinoma; it is the same amino-acid change as the variant under assessment, not a different pathogenic missense at the residue, and it is a somatic tumour finding with no germline classification.
• PMID:18199528 explicitly names K1454E and reports it once in a non-FAP non-MAP colorectal adenoma patient and twice among 969 healthy controls, listing it among variants detected in both patients and controls; it is not one of the 16 variants functionally assayed by the beta-catenin-regulated transcription reporter.

Still missing

• ClinVar same-residue comparator harvesting returned HTTP 429 (Too Many Requests) and found 0 same-residue candidates; the negative result was independently corroborated against the VCEP's explicit list of established (L)Pathogenic APC missense variants and the primary literature.

Sources

Reasoning

NM_001127510.3:c.4360A>G encodes the presumed missense substitution NP_001120982.1:p.(Lys1454Glu), so PP3 is in scope. The governing ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis VCEP specification for APC (v2.1) directs that for presumed missense variants PP3 be assessed with in silico splicing predictors rather than conservation/evolution models, and requires multiple in silico splicing predictors to support a deleterious effect. SpliceAI predicts no significant splice impact for this variant: maximum delta score 0.001 (DS_AG 0.001, DS_AL 0.000, DS_DG 0.000, DS_DL 0.000), which is far below the 0.2 supporting threshold (Jaganathan et al. 2019, PMID:30661751). The companion Pangolin outputs are likewise non-supportive (SG 0.022, SL -0.001). No splice prediction supports a deleterious effect, so PP3 is not met. REVEL (0.554) and BayesDel (0.113841) are present but are not usable for PP3 under APC: the VCEP excludes conservation/evolution prediction models for missense variants, and no governing APC BayesDel cutoff is specified.

Rule

APC VCEP PP3: for presumed missense variants, do not use conservation/evolution computational models; use in silico splicing predictors, with multiple predictors supporting a deleterious effect.

Evidence considered

• The case variant is NM_001127510.3:c.4360A>G, encoding the presumed missense change NP_001120982.1:p.(Lys1454Glu); PP3 is therefore in scope.
• The APC VCEP specification states that for missense variants computational prediction models for conservation and evolution should not be used and that in silico splicing predictors should be used to reveal possible splicing effects, requiring multiple in silico splicing predictors to support a deleterious effect.
• SpliceAI reports DS_AG 0.001, DS_AL 0.000, DS_DG 0.000 and DS_DL 0.000, for a maximum delta score of 0.001; this indicates no significant splice impact.
• The supplied generic SpliceAI calibration defines PP3 supporting at maximum delta >= 0.2 (Jaganathan et al. 2019, Cell, PMID:30661751); the observed maximum delta of 0.001 does not reach even the supporting threshold.
• REVEL 0.554 and BayesDel 0.113841 were present in the evidence but are not used for PP3: the governing APC specification excludes conservation/evolution prediction models for missense variants and supplies no BayesDel cutoff.

Sources

Reasoning

PP5 may be triggered only by an exact-variant classification from a ClinVar expert panel: a Pathogenic or Likely pathogenic assertion in a 3-star expert-panel review. ClinVar records for this variant (variant IDs 127295, 537431, 824928, 1035875, 4802496; aggregate label Benign) comprise 21 submissions from clinical laboratories with review status 'criteria provided, single submitter' and an expert-panel submission count of zero. There is therefore no expert-panel Pathogenic/Likely pathogenic assertion for this exact variant, and ordinary laboratory submissions and the aggregate label cannot trigger PP5. The APC VCEP v2.1 specification additionally marks PP5 as 'Not Applicable for this VCEP' - 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee' - citing Biesecker et al. 2018 (PMID:29543229). Both the gene-specific specification and the global expert-panel pathway therefore return no PP5 support.

Rule

PP5 requires an exact-variant ClinVar expert-panel (3-star) Pathogenic/Likely pathogenic assertion; APC VCEP v2.1 marks PP5 Not Applicable (Biesecker et al. 2018, PMID:29543229)

Evidence considered

• ClinVar submission audit for this variant: 21 total submissions, 18 usable for classification, 2 criterion-lead submissions, and 0 expert-panel submissions; aggregate classification Benign with review status 'criteria provided, single submitter' (1 star), so no 3-star expert-panel Pathogenic/Likely pathogenic assertion exists.
• ClinVar per-submitter breakdown: Benign (11 clinical laboratories), Likely benign (6), benign (1), Uncertain significance (1) - all ordinary laboratory submissions, none an expert panel.
• APC VCEP v2.1 PP5 text: 'Not used as recommended by Biesecker et al. 2018 (PMID: 29543229): Expert opinions should only be considered if accompanied by the primary evidence used.'
• PMID:15604628 was screened as a PP5 candidate from the ClinVar submission trail but is an NSGC genetic risk-assessment/counseling guideline containing only syndrome-level APC content and no assertion about this variant.

Still missing

• If an InSiGHT/ClinGen APC VCEP expert-panel classification of this variant were later published, PP5/BP6 would need to be re-evaluated from that assertion.

Sources

Reasoning

The APC VCEP v2.1 BS2 rule has two limbs: '>= 10 points for healthy individuals OR >= 2 times in homozygous state' (Strong), with a Supporting tier at '>= 3 points for healthy individuals'; a healthy individual is worth 1 point if aged >= 50 with < 5 adenomatous polyps and no Table-1 features (or if colorectal cancer/polyposis was not the testing indication), and 0.5 points if identified only by keywords such as control, non-cancer, normal, or unaffected population. Neither limb is satisfied on the evidence available. (1) Homozygous state: the specification directs use of the non-cancer gnomAD v2.1.1 dataset for the homozygote search ('The non-cancer dataset from gnomAD (v2.1.1) can be used to search for homozygous individuals'), and in the VCEP-directed source this variant has 0 homozygotes in exomes (0/236,710 alleles) and 0 in genomes; the all-comers v2.1.1 release likewise reports 0 homozygotes (228/282,622 alleles). With zero homozygotes recorded, the '>= 2 times in homozygous state' limb cannot be met from the specified dataset. (2) Healthy-individual points: the specification prohibits counting heterozygous healthy individuals from the gnomAD non-cancer dataset ('cannot be used for heterozygous healthy individuals, because of the limited phenotype information and since it is usually already used for BA1/BS1'), so gnomAD carriers cannot be scored. The published evidence supplies only a small number of healthy carriers: PMID:18199528 detected the exact K1454E change in 2 of 969 healthy controls (2 x 0.5 = 1 point) and once in a non-FAP/non-MAP colorectal adenoma patient; PMID:11904335 found the codon 1454 AAA>GAA change in both normal and adenomatous liver of a single patient with no colonic polyposis and interpreted it as a polymorphism (that individual is not a scored healthy adult control). The total is at most ~1 point, well below the >= 3 points required even for BS2 Supporting and far below the >= 10 points for Strong. A counter-consideration is recorded: newer non-cancer/current releases not specified by this v2.1.1-calibrated rule do show homozygotes - gnomAD v3.1 non-cancer genomes report 4 homozygotes (371/147,880 alleles) and all-comers gnomAD v4.1 reports 9 homozygotes, all in the African/African American population - which would satisfy the '>= 2 times in homozygous state' limb if BS2 were applied to current releases. Because the governing specification names the non-cancer gnomAD v2.1.1 dataset for this criterion and that dataset shows no homozygotes, BS2 is recorded as not_met, but the discrepancy is flagged for human review; it does not affect the final classification, which is already Benign by BA1.

Rule

>= 10 points for healthy individuals OR >= 2 times in homozygous state (Strong); >= 3 points for healthy individuals (Supporting). APC VCEP v2.1 BS2; homozygote search directed to the non-cancer gnomAD v2.1.1 dataset.

Evidence considered

• Governing rule applied: APC VCEP v2.1 BS2 - '>= 10 points for healthy individuals OR >= 2 times in homozygous state' (Strong) and '>= 3 points for healthy individuals' (Supporting); the specification's dataset directive states the non-cancer gnomAD v2.1.1 dataset cannot be used for heterozygous healthy individuals but can be used to search for homozygous individuals.
• Homozygote search in the VCEP-directed source (non-cancer gnomAD v2.1.1): exome homozygote count 0 (138/236,710 alleles, AF 0.000582992) and genome homozygote count 0; the all-comers v2.1.1 release likewise reports 0 homozygotes (228/282,622 alleles). The '>= 2 times in homozygous state' limb is therefore not met in the specified dataset.
• Homozygote counts in datasets not specified by the VCEP: gnomAD v3.1 non-cancer genomes 4 homozygotes (371/147,880 alleles) and gnomAD v4.1 all-comers 9 homozygotes (765/1,614,122 alleles, all in African/African American); these would meet the >= 2-homozygote limb if current releases are used and are flagged for review.
• PMID:18199528 (Azzopardi et al. 2008): the exact K1454E variant was detected twice among 969 healthy controls and once among non-FAP/non-MAP colorectal adenoma patients; at 0.5 points per keyword-defined healthy individual this contributes about 1 point, below the >= 3-point Supporting floor and the >= 10-point Strong floor.
• PMID:11904335 (Torbenson et al. 2002): the codon 1454 AAA>GAA (Lys>Glu) change was present in both normal liver and adenoma of one hepatic adenoma case and was interpreted as a polymorphism in a patient with no clinical evidence of colonic polyposis; the individual is not a scored healthy adult control under the VCEP point system.
• PMID:23970361 (De Queiroz Rossanese et al. 2013): the only clinical-context report naming this exact variant describes it in a 47-year-old FAP patient (patient 19) with > 100 polyps; an affected proband is the opposite of the healthy-individual evidence BS2 requires and contributes no BS2 points.
• The VCEP prohibits scoring gnomAD non-cancer heterozygous carriers toward BS2, and no other series of healthy adults with the required age/polyp/Table-1 phenotype documentation was available; total healthy-individual points therefore remain below the Supporting threshold.

Still missing

• gnomAD v3.1 non-cancer genomes (4 homozygotes; 371/147,880 alleles) and all-comers gnomAD v4.1 (9 homozygotes, all African/African American) post-date the VCEP's v2.1.1-calibrated BS2 rule; if the '>= 2 times in homozygous state' limb is applied to current releases, BS2 Strong would be met. This discrepancy requires human adjudication, which is why needs_human_review is set.
• The healthy-individual point tally is incomplete by construction: gnomAD non-cancer carriers cannot be scored per the specification, and outside PMID:18199528 (2 healthy controls) no other healthy-adult carrier series with the required age/polyp/Table-1 data was available in this case.
• BS2 status does not change the classification here, because BA1 (Stand Alone) is met and independently supports Benign.

Sources

Reasoning

The governing APC VCEP v2.1 specification defines BP2 at Supporting strength as: 'Observed in trans with a (Likely) Pathogenic APC variant OR >= 3 times in an unknown phase with different (Likely) Pathogenic APC variants.' Neither branch is satisfied. (1) In trans: across the 21 ClinVar submissions catalogued for variation ID 127295 (11 clinical laboratories Benign, 6 Likely benign, 1 benign, 1 Uncertain significance, 0 expert-panel submissions), no submission reports c.4360A>G co-occurring in trans with a pathogenic or likely pathogenic APC allele. The full-text evidence is consistent: PMID:18199528 detected the exact K1454E change once among non-FAP/non-MAP colorectal adenoma patients and twice among 969 healthy controls, with no report of a second APC pathogenic allele in cis or trans; PMID:23970361 reports K1454E in a single FAP patient (patient 19) with no second APC variant and no phase data; PMID:11904335 found the codon 1454 AAA>GAA change in both normal liver and adenoma of one hepatic adenoma case and explicitly interpreted it as a polymorphism in a patient with no colonic polyposis syndrome; PMID:18844223 found the identical AAA>GAA codon 1454 change somatically in three bladder tumours (patients 5, 7, 8) and confirmed its absence from matched normal urothelium, which argues directly against a germline in-trans configuration. (2) >= 3 occurrences in unknown phase with different (Likely) Pathogenic APC variants: the published occurrences of K1454E are with the same change at the same codon, not with three or more different (likely) pathogenic APC variants, and no case has >= 3 independent partner pathogenic alleles recorded. Population data do not supply the missing branch either: gnomAD v2.1 reports allele frequency 0.000806731 with 228/282622 alleles and zero homozygotes, and gnomAD v4 likewise, so there is no homozygous or biallelic carrier genotype reported. Candidate partners for a cis/trans configuration exist in the case only as individual reports (PMID:18844223 somatic events; the benign/likely benign ClinVar aggregate), and none places a pathogenic APC allele on the opposite chromosome. The criterion is therefore not met on the available evidence, with the caveat that no direct phase testing was performed, so this rests on the absence of any reported in-trans observation rather than on a genotype-resolved negative.

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

• Governing rule applied: APC VCEP v2.1 BP2 (Benign Supporting), rule text 'Observed in trans with a (Likely) Pathogenic APC variant OR >= 3 times in an unknown phase with different (Likely) Pathogenic APC variants'; the generic ACMG/AMP BP2 wording ('observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant') was superseded by this gene-specific rule.
• ClinVar variation ID 127295 (APC c.4360A>G, p.Lys1454Glu) aggregates 21 submissions: 11 clinical laboratories Benign, 6 Likely benign, 1 benign, 1 Uncertain significance, with 0 expert-panel submissions; none of the catalogued submissions reports the variant in trans with a pathogenic/likely pathogenic APC allele or reports >= 3 unknown-phase occurrences with different pathogenic APC variants.
• PMID:18199528 (Azzopardi et al. 2008): the exact K1454E variant was found once in a non-FAP/non-MAP colorectal adenoma patient and twice among 969 healthy controls, within the beta-catenin down-regulating domain; the paper reports no second APC pathogenic allele and no phase information for these carriers.
• PMID:23970361 (De Queiroz Rossanese et al. 2013): K1454E (codon 1454) reported in patient 19, a 47-year-old female with FAP; no co-occurring APC pathogenic variant and no phase data are given, so no in-trans configuration is documented.
• PMID:11904335 (Torbenson et al. 2002): the codon 1454 AAA>GAA change was present in both normal liver and adenoma in one hepatic adenoma case and was interpreted as a polymorphism of unknown significance in a patient with no clinical evidence of colonic polyposis; no second APC allele reported.
• PMID:18844223 (Kastritis et al. 2009): the identical AAA>GAA codon 1454 Lys>Glu change was detected as a somatic event in three muscle-invasive urothelial carcinoma patients (5, 7, 8) and confirmed absent from matched normal urothelium, indicating it is not a germline allele co-segregating with a pathogenic APC allele in these individuals.
• PMID:22995991 (Berg et al. 2013): no mention of APC c.4360A>G or p.Lys1454Glu; the paper reports a different APC variant (c.2608C>T, p.P870S) as a likely polymorphism removed from consideration, and its thesis is that HGMD 'disease-causing' annotations in presumed-healthy genomes are frequently benign; it contains no in-trans or phase information relevant to this variant.
• gnomAD v2.1 (5-112175651-A-G): allele frequency 0.000806731, 228/282622 alleles, homozygote count 0; gnomAD v4.1 (chr5-112839954-A-G) reviewed with no biallelic/homozygous carrier reported. This documents that no homozygous carrier genotype exists in population data, which is not itself a BP2 route but confirms the absence of any biallelic configuration.
• No family study, parental testing, offspring testing, or read-backed phasing was available for this case, so the BP2 assessment rests on the absence of any reported in-trans observation across ClinVar submissions, the cited full-text series, and population databases.

Still missing

• No parental, offspring, or read-backed phasing was performed, so phase of c.4360A>G relative to any other APC allele is unestablished in this case; BP2 is not met because no published or submitted observation places it in trans with a (likely) pathogenic APC variant.
• No report of >= 3 independent occurrences of c.4360A>G in unknown phase alongside different (likely) pathogenic APC variants exists in the assembled evidence (ClinVar submissions, cited full texts, or population databases).

Sources

Reasoning

The APC VCEP v2.1 restricts BP5 to a case that has BOTH a colorectal polyposis phenotype AND a (Likely) Pathogenic variant in another adenomatous polyposis gene: heterozygous POLD1 or POLE; biallelic MUTYH, NTHL1 or MSH3; and, for onset in childhood/adolescence, biallelic MLH1, MSH2, MSH6 or PMS2. The specification states the rule 'is only applicable when a colorectal polyposis phenotype is present'. No alternate molecular basis is documented anywhere in this case: there is no case-level genotype information, no record of MUTYH or other polyposis-gene panel testing, and no recorded colorectal polyposis phenotype. The one reported carrier outside an FAP setting (PMID:11904335, a hepatic adenoma patient whose normal liver also carried the change) had no colonic polyposis and no alternative genetic cause identified. BP5 therefore has no evidentiary basis and is not met.

Rule

APC VCEP v2.1 BP5 (Supporting): only applicable for an alternate genetic basis of the colorectal polyposis phenotype - (Likely) Pathogenic variant in another adenomatous polyposis gene (POLD1/POLE heterozygous; MUTYH/NTHL1/MSH3 biallelic; MLH1/MSH2/MSH6/PMS2 biallelic for childhood-adolescent onset)

Evidence considered

• APC VCEP v2.1 BP5 instruction text: '(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.'
• BP5 is a Benign Supporting-only criterion in the APC VCEP specification (stand-alone, very strong, strong and moderate tiers are Not applicable).
• PMID:11904335: the codon-1454 change was found in a hepatic adenoma patient with no colonic polyposis (and in the patient's normal liver), a context in which no alternate polyposis gene defect was identified; this carrier does not satisfy the BP5 precondition of a colorectal polyposis phenotype plus an alternate genetic basis.
• No case-level data in this case document testing of MUTYH, POLD1, POLE, NTHL1, MSH3 or mismatch-repair genes, and no colorectal polyposis phenotype is recorded for the proband.

Still missing

• No case-level phenotype or genetic workup (MUTYH, POLD1/POLE, NTHL1, MSH3, MMR panel) is recorded, so an alternate molecular basis cannot be excluded - only shown to be unreported.

Sources

Reasoning

BP6 may be triggered only by an exact-variant classification from a ClinVar expert panel: a Benign or Likely benign assertion in a 3-star expert-panel review. ClinVar holds no such assertion for this variant (variant IDs 127295, 537431, 824928, 1035875, 4802496). The 21 submissions are from clinical laboratories with review status 'criteria provided, single submitter' and the expert-panel submission count is zero; the aggregate label Benign, however consistently benign across submitters, is an aggregate of ordinary laboratory submissions and must not trigger BP6. The APC VCEP v2.1 specification likewise marks BP6 as 'Not Applicable for this VCEP' - 'This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee' - citing Biesecker et al. 2018 (PMID:29543229). BP6 is therefore not met, and the benign evidence in this case must come from the frequency, segregation, functional and co-occurrence criteria rather than from this criterion.

Rule

BP6 requires an exact-variant ClinVar expert-panel (3-star) Benign/Likely benign assertion; APC VCEP v2.1 marks BP6 Not Applicable (Biesecker et al. 2018, PMID:29543229)

Evidence considered

• ClinVar submission audit for this variant: 21 total submissions, 18 usable for classification, and 0 expert-panel submissions; aggregate classification Benign with review status 'criteria provided, single submitter' (1 star).
• ClinVar per-submitter breakdown: Benign (11 clinical laboratories), Likely benign (6), benign (1), Uncertain significance (1) - ordinary laboratory submissions only.
• APC VCEP v2.1 BP6 text: 'Not used as recommended by Biesecker et al. 2018 (PMID: 29543229): Expert opinions should only be considered if accompanied by the primary evidence used.'

Still missing

• No expert-panel (InSiGHT/ClinGen APC VCEP) classification of this variant is present in ClinVar; if one is published, BP6 must be re-evaluated.

Sources

Reasoning

Governing specification: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for APC, Version 2.1 (APC VCEP), under which PS2 is applicable and is scored in a graded, point-based form. Per the APC VCEP supplementary material, PS2 is modified on the basis of the SVI Recommendation for De Novo Criteria (PS2 & PM6) v1.1 and Fortuno et al. 2020 (PMID:33300245), and the magnitude is set by the de novo score of Tables 1 and 2: >= 4 de novo scores = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate. Table 2 assigns 2 de novo points per proband for a de novo occurrence with confirmed parental relationships when the proband has >= 1 Table 1 phenotype point, and 1 point when the proband has 0.5 phenotype points; the parents must be documented as unaffected (colonoscoped with < 5 colorectal adenomas and no other Table 1 conspicuities, or > 60 years with no gastrointestinal tumour signs and an unremarkable family history). Scoring PS2 therefore requires a proband carrying c.4360A>G p.(Lys1454Glu), a parentally confirmed (maternity and paternity) de novo event, and a Table 1 phenotype score for that proband. This case contains no proband: it is a variant-level record (ClinVar variation 127295, gnomAD queries, in silico predictors), and no parental testing, trio data, or proband clinical description is present in any case file. The seven full-text papers reviewed for variant-level content report no de novo occurrence of c.4360A>G: PMID:18199528 explicitly characterises K1454E as a rare inherited nonsynonymous APC variant detected in the patient cohort and in 2 of 969 healthy controls; PMID:23970361 reports it as a germline allele in a 47-year-old FAP patient drawn from families with >= 2 successive affected generations; PMID:11904335 found the same codon 1454 AAA>GAA change constitutionally in both normal liver and adenoma of a woman with no clinical evidence of colonic polyposis and interpreted it as a polymorphism; PMID:18844223 reports it as a somatic tumour change in urothelial carcinoma. Population data are also incompatible with a recurrent de novo origin: gnomAD v4.1 lists 765 alleles including 9 homozygotes with a grpmax filtering allele frequency of 0.0085. Because the proband-level de novo observation, the parental confirmation and the Table 1 phenotype score that PS2 requires are all absent, no de novo score can be assigned and PS2 cannot be evaluated in either direction; it is recorded as not_assessed rather than met or not_met.

Evidence considered

• APC VCEP v2.1 sets PS2 strengths by de novo score: >= 4 = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, and directs scoring to Tables 1 and 2.
• Table 2 (VCEP Table-1&2 document) scales the de novo score by parental confirmation and proband phenotype points: >= 1 phenotype point gives 2 de novo points when parental relationships are confirmed and 1 when unconfirmed; 0.5 phenotype points give 1 and 0.5 respectively.
• PMID:18199528 names K1454E directly and describes it as a rare inherited nonsynonymous variant in the beta-catenin down-regulating domain, detected in a non-FAP colorectal adenoma patient and in 2 of 969 healthy controls - an inherited, recurrent occurrence rather than a de novo event.
• PMID:23970361 reports the identical germline Lys1454Glu change in a 47-year-old FAP patient, from a cohort of families with >= 2 successive affected generations; the paper does not report the inheritance origin of the allele for that patient and performs no segregation analysis.
• PMID:11904335 detected the same codon 1454 AAA>GAA change in both normal liver and adenoma tissue of a woman with no clinical evidence of colonic polyposis and interpreted it as a polymorphism, i.e. a constitutional rather than a de novo occurrence.
• PMID:18844223 reports the identical codon 1454 Lys>Glu change as a somatic mutation in three urothelial carcinoma patients, confirmed absent from matched normal tissue; a somatic tumour finding is not a germline de novo event.
• gnomAD v4.1 records 765 alleles for this variant including 9 homozygotes, with grpmax filtering allele frequency 0.0085 (0.85%) and African/African American AF 0.0091; recurrent de novo origin is not consistent with this population profile.

Still missing

• No proband with APC c.4360A>G p.(Lys1454Glu) and no clinical/phenotype description for an index case in any case file.
• No parental testing or trio data (maternity and paternity confirmation) to establish or exclude a de novo event.
• No Table 1 phenotype point score for a proband, which Table 2 requires in order to convert a de novo observation into a de novo score.
• No parental phenotype documentation (colonoscopy findings or age/absence of gastrointestinal tumour signs) to confirm the parents were unaffected, as the APC VCEP PS2 modification requires.

Sources

Reasoning

Under the APC VCEP v2.1 specification, PM6 ('assumed de novo, but without confirmation of paternity and maternity') is applicable and is modified on the same basis as PS2, i.e. the SVI Recommendation for De Novo Criteria (PS2 & PM6) v1.1 and Fortuno et al. 2020 (PMID:33300245), with magnitude again set by the de novo score of Tables 1 and 2: 2-3.5 de novo scores = Strong, 1-1.5 = Moderate, 0.5 = Supporting (the specification also cites PM6_Very Strong at >= 4 de novo scores). The relaxation relative to PS2 is only in the parental relationships - the variant must still be an assumed de novo occurrence in a proband, and Table 2 still requires a Table 1 phenotype point score for that proband (1 de novo point for >= 1 phenotype point and 0.5 de novo points for 0.5 phenotype points when parental relationships are unconfirmed). No proband and no parental data exist for this case, and no source reviewed reports an assumed de novo occurrence of APC c.4360A>G p.(Lys1454Glu) in any patient. To the contrary, every variant-level source that names this allele places it in an inherited or constitutional context: PMID:18199528 calls it 'rare inherited' and finds it in healthy controls as well as adenoma patients; PMID:11904335 finds it constitutionally in a woman with no polyposis; PMID:23970361 finds it as a germline allele in an FAP patient from multi-generation affected families; PMID:18844223 finds it somatically in tumours. There is therefore no proband phenotype score and no de novo observation from which a de novo score could be computed, so PM6 cannot be scored and is recorded as not_assessed.

Evidence considered

• APC VCEP v2.1 PM6 strengths are set by de novo score: 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting, with Tables 1 and 2 governing how the score is curated; the specification's instructionsToUse additionally cites PM6_Very Strong at >= 4 de novo scores.
• The PM6 entry in the APC supplementary material reproduces the PS2 parent-phenotype requirements (parents colonoscoped with < 5 colorectal adenomas and no other Table 1 conspicuities, or > 60 years with no gastrointestinal tumour signs and unremarkable family history) and warns about somatic/postzygotic mosaicism.
• Table 2: an assumed de novo event without confirmed parental relationships yields 1 de novo point at >= 1 proband phenotype point and 0.5 de novo points at 0.5 phenotype points, so a phenotype-scored proband is still mandatory for PM6.
• PMID:18199528 describes K1454E as a rare inherited nonsynonymous APC variant found once in a non-FAP colorectal adenoma patient and twice among 969 healthy controls, and lists it among variants detected in both patients and controls - an inherited, recurrent allele rather than an assumed de novo event.
• PMID:23970361 reports germline Lys1454Glu in a 47-year-old FAP patient from families with >= 2 successive affected generations and provides no de novo statement for this variant.
• PMID:11904335 found the codon 1454 AAA>GAA change in both normal liver and adenoma of a woman without colonic polyposis and called it a polymorphism, i.e. a constitutional germline change.
• gnomAD v4.1 shows 765 alleles with 9 homozygotes (grpmax FAF 0.0085), which is not compatible with the variant being a recurrent assumed de novo event.

Still missing

• No proband-level observation of APC c.4360A>G p.(Lys1454Glu) reported as an assumed (unconfirmed) de novo occurrence.
• No parental genotype information of any kind (neither confirmation nor exclusion of maternity/paternity).
• No proband Table 1 phenotype point score, required by Table 2 to convert an assumed de novo event into a de novo score.
• No parental phenotype documentation to establish that the parents were unaffected, as the APC VCEP PM6 modification requires.

Sources

Reasoning

The APC VCEP v2.1 specification applies PP1 for APC with defined meiosis-based thresholds: segregation in >= 7 meioses in >= 2 families = Strong, 5-6 meioses in >= 1 family = Moderate, and 3-4 meioses in >= 1 family = Supporting, with the modification (based on Luo et al. 2019, PMID:31648317 and Jarvik & Browning 2016, PMID:27236918) that affected individuals must exhibit at least 0.5 point of the Table 1 phenotype point system and that only genotype-positive and phenotype-positive individuals and obligate carriers with phenotype are counted (relatives with >= 10 or 'multiple' colorectal adenomas count as 0.5 point). Even the Supporting strength therefore requires a minimum of 3 informative meioses in one family with genotype-phenotype concordance. This case contains no pedigree, no meioses, and no genotyped relatives for APC c.4360A>G p.(Lys1454Glu): the case comprises a variant-level ClinVar record, gnomAD frequency data, in silico prediction scores and a set of full-text papers screened for this allele. Among those papers only PMID:23970361 places the variant in families (FAP kindreds with >= 2 successive affected generations), but it reports no co-segregation analysis, no meiosis count, and no carrier genotype-phenotype table for Lys1454Glu specifically; the other sources report the variant in unrelated individuals (PMIDs 18199528, 11904335) or as a somatic tumour finding (PMID:18844223). With zero meioses documented and no phenotype scoring of variant-positive relatives, PP1 cannot be evaluated; it is recorded as not_assessed, contributing no pathogenic evidence.

Evidence considered

• APC VCEP v2.1 PP1 thresholds: >= 7 meioses in >= 2 families = Strong; 5-6 meioses in >= 1 family = Moderate; 3-4 meioses in >= 1 family = Supporting.
• APC VCEP PP1 modification (Luo et al. 2019, PMID:31648317; Jarvik & Browning 2016, PMID:27236918): affected individuals must show at least 0.5 Table 1 phenotype point, relatives with >= 10 or 'multiple' colorectal adenomas count as 0.5 point, and only genotype-positive/phenotype-positive individuals and obligate carriers with phenotype are counted.
• PMID:23970361 gives germline Lys1454Glu in one FAP patient from a cohort whose families had >= 2 successive affected generations, but reports no co-segregation analysis and no meiosis count for this variant, so no segregation data can be extracted.
• PMID:18199528 reports K1454E in unrelated individuals (one non-FAP adenoma patient and 2/969 healthy controls) with no family-level genotypes, providing no co-segregation information.
• No case file contains a pedigree, meiosis count, or genotyped relative for this variant; the literature screened for the allele reports no family study of it.

Still missing

• No pedigree or family structure for any carrier of APC c.4360A>G p.(Lys1454Glu), and no meiosis count.
• No genotype data for relatives of any carrier, so variant-positive and variant-negative family members cannot be identified.
• No Table 1 phenotype point scoring of relatives, which the APC VCEP PP1 modification requires (affected individuals must have >= 0.5 phenotype point).
• No information on obligate carriers, chemoprevention, or reduced-penetrance carriers, which the specification allows to be counted.

Sources

Reasoning

Under the APC VCEP v2.1 specification, BS4 ('lack of segregation in affected members of a family') is applicable and is graded with the VCEP's Table 1 phenotype point system: an affected member without the variant who scores at least 1 phenotype point, or at least two affected members without the variant who each score at least 0.5 phenotype point, reaches Strong; a single affected member without the variant scoring at least 0.5 phenotype point reaches Supporting. Meeting BS4 therefore requires a family containing an affected, Table 1-scored relative who has been tested and found not to carry APC c.4360A>G p.(Lys1454Glu). No such genotype-phenotype data exist for this case. The case is a variant-level record, and none of the sources naming this allele provides a family in which an affected relative was tested negative: PMID:23970361 reports the variant as a germline finding in one affected FAP patient (phenotype-positive carrier, not a non-carrier); PMID:18199528 reports the variant in an adenoma patient and in 2 of 969 healthy controls, i.e. in unaffected population controls rather than affected relatives, and PMID:11904335 reports it in a woman with no clinical evidence of colonic polyposis, again an unaffected carrier. Observations of a variant in unaffected individuals belong to the BS2/BA1/BS1 logic, not to BS4, which is specifically about affected family members lacking the variant. Because no affected variant-negative relative has been identified or phenotype-scored, BS4 cannot be assessed and is recorded as not_assessed, contributing no benign evidence.

Evidence considered

• APC VCEP v2.1 BS4 grading: affected member without the variant scoring >= 1 Table 1 phenotype point, or >= 2 affected members without the variant each scoring >= 0.5 point, = Strong; a single affected member without the variant scoring >= 0.5 point = Supporting.
• Table 1 of the APC VCEP defines the phenotype points BS4 thresholds reference: 1 point for a phenotype highly specific for APC (e.g. typical colorectal phenotype 20-99 colorectal adenomas at <= 20 years, or >= 1000 adenomas at any age, or a histologically confirmed desmoid without somatic CTNNB1 variant) and 0.5 point for a phenotype consistent with APC but not highly specific (e.g. >= 20 colorectal adenomas at 20-70 years, CHRPE, multiple gastric or duodenal adenomas, osteomas).
• PMID:23970361 reports Lys1454Glu in one affected FAP patient (a variant-positive affected individual), which is the opposite of the BS4 scenario of an affected non-carrier, and reports no genotyping of relatives.
• PMID:18199528 found K1454E in 2 of 969 healthy controls and in one non-FAP adenoma patient; the controls are unaffected population samples, not affected relatives, so they cannot be scored under BS4.
• PMID:11904335 found the same codon 1454 change in a woman with no clinical evidence of colonic polyposis and interpreted it as a polymorphism, again an unaffected carrier rather than an affected non-carrier.

Still missing

• No family of a carrier in which affected relatives were genotyped, so no affected variant-negative individual is available.
• No Table 1 phenotype point scoring of any relative, which BS4's >= 1 (Strong) and >= 0.5 (Supporting) thresholds require.
• No pedigree linking phenotype-positive and phenotype-negative relatives to the variant.
• Note: the variant is documented in unaffected individuals (2/969 healthy controls in PMID:18199528; a woman without colonic polyposis in PMID:11904335), but unaffected carriers support BS2/BA1/BS1-type evidence, not BS4.

Sources