AXIN2 c.1235A>G, p.(Asn412Ser)

Reasoning

BS1 (strong benign) requires an allele frequency greater than that expected for the disorder; the generic SVI default threshold supplied for this case is AF >= 0.01, with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168). No VCEP or gene-specific framework governs AXIN2 here, so that default applies. Evaluated on ancestry-specific frequency, the variant exceeds the threshold by three- to four-fold: gnomAD v4.1 South Asian AF 0.04112 (3,565/86,704 alleles; 132 homozygotes) with grpmax filtering AF 0.03999; gnomAD v2.1 South Asian AF 0.03830 (970/25,324 alleles; 37 homozygotes) with grpmax filtering AF 0.03630; gnomAD v3.1 non-cancer grpmax filtering AF 0.03279. The pooled global frequency (gnomAD v4.1 0.79%, v2.1 0.92%, v2.1 non-cancer exomes 1.00%, gnomAD-Canada 0.80%) sits at or just below 0.01, so BS1 rests on the population-specific filtering frequency, which is the appropriate metric for a criterion whose purpose is to reject alleles too common to cause a rare Mendelian disease. The conclusion is robust on the disease model as well: AXIN2-related oligodontia-colorectal cancer predisposition is a rare autosomal dominant, incompletely penetrant condition, and the variant is homozygous in 165 adults in gnomAD v4.1 (132 of them South Asian) and 41 in v2.1, a genotype load incompatible with a causal rare-disease allele at that prevalence. Independently, Mongin et al. 2012 (PMID:21476993) genotyped this exact variant in 275 healthy control subjects and detected it in four, reporting an allele frequency of 0.72% and classifying it a polymorphism rather than a deleterious mutation. BS1 met at strong.

Rule

AF_popmax >= 0.01 -> BS1 strong benign (generic SVI default; per-gene derivation preferred, Whiffin et al. 2017 PMID:28518168)

Evidence considered

• gnomAD v4.1: highest ancestry frequency South Asian AF 0.04112 (3,565/86,704 alleles; 132 homozygotes); grpmax filtering AF 0.03999 (exome grpmax 0.04018; genome grpmax 0.03282).
• gnomAD v2.1: highest ancestry frequency South Asian AF 0.03830 (970/25,324 alleles; 37 homozygotes); grpmax filtering AF 0.03630.
• gnomAD v3.1 non-cancer genomes-only subset: total AF 0.00672 (993/147,760 alleles; 8 homozygotes); grpmax filtering AF 0.03279.
• gnomAD v2.1 non-cancer exomes-only subset: AF 0.00995 (1,941/195,014 alleles; 41 homozygotes); grpmax filtering AF 0.03634.
• Ancestry-specific (popmax/grpmax filtering) frequency was used rather than the pooled global frequency, because BS1 asks whether the variant is more common than expected for the disorder; the pooled value (0.79-1.00%) is just below the 0.01 default while South Asian frequency is four-fold above it.
• Mongin et al. 2012 (PMID:21476993), cohort of patients with unexplained polyposis plus control subjects: the variant was detected in 4 of 275 healthy controls (allele frequency 0.72%) and classified as a polymorphism rather than a deleterious mutation.
• Lejeune et al. 2006 (PMID:16941501) screened this variant in 50 healthy control subjects, an order of magnitude too small to affect the frequency assessment but consistent with it being a rare-to-common polymorphism rather than a private pathogenic allele.
• Disease-model context used for the frequency comparison: AXIN2-related oligodontia-colorectal cancer predisposition is a rare autosomal dominant condition with incomplete, adult-onset cancer penetrance; the variant is present at a frequency and homozygote count (165 homozygotes in gnomAD v4.1) far beyond what that prevalence can accommodate.

Still missing

• No per-gene BS1 threshold could be derived because there is no ClinGen CSPEC or gene-specific framework for AXIN2 in this case; the generic 0.01 default was used.
• The global allele frequency (0.79-1.00% across datasets) is marginally below 0.01, so BS1 rests on the ancestry-specific popmax filtering frequency (0.036-0.040) rather than the pooled value; reviewers who require a global AF above threshold would rate this criterion as not met.

Sources

Reasoning

BP1 (supporting benign; ACMG/AMP 2015, PMID:25741868) applies to a missense variant in a gene for which primarily truncating variants are known to cause disease. AXIN2 satisfies this: of all 4,370 ClinVar AXIN2 records retrieved, every one of the 186 records with a pathogenic-type classification is loss-of-function in type (169 nonsense/frameshift, 3 canonical splice-site, 14 copy-number losses), whereas among 2,347 single-amino-acid-substitution records only two carry a pathogenic-type classification and both are weak (p.Val105Gly, Pathogenic with no assertion criteria provided; p.Glu66Gly, Likely pathogenic from a single submitter), with the remaining 2,313 being VUS, conflicting, or Benign/Likely benign. Independent gene-level evidence agrees on the direction of effect: AXIN2 is LoF-constrained in gnomAD (pLI approximately 1.0, lof_z 5.27) while being entirely tolerant of missense change (o/e missense 1.03, mis_z -0.42), the germline disease mechanism documented in the reviewed literature is loss of function/truncating, and OncoKB's gene-level curation describes AXIN2 truncating mutations as the reported disease-associated class in a tumor suppressor whose mutations occur at low frequency. Two primary publications on this very allele reach the same conclusion from the missense side, classifying p.Asn412Ser a polymorphism with no functional effect (PMID:16941501, PMID:21476993). BP1 is met at its default supporting strength. It is deliberately not escalated: a small number of missense AXIN2 alleles have been proposed as pathogenic (a novel missense in an attenuated FAP family with segregation and functional support, PMID:23838596; p.Thr693Met with Wnt/beta-catenin activation reported as likely pathogenic, PMID:41361419), so BP1 is applied as one supporting benign line of evidence and not as stand-alone benign.

Rule

BP1: missense variant in a gene where primarily truncating variants cause disease -> supporting benign (ACMG/AMP 2015; PMID:25741868).

Evidence considered

• BP1 definition used: missense variant in a gene for which primarily truncating variants are known to cause disease (ACMG/AMP 2015, PMID:25741868), applied at supporting strength.
• ClinVar composition, all 4,370 AXIN2 records retrieved: 186 records carry a pathogenic-type classification and are all loss-of-function (169 nonsense/frameshift, 3 canonical splice-site, 14 copy-number losses); among 2,347 single-amino-acid substitutions, only p.Val105Gly (c.314T>G, Pathogenic, no assertion criteria provided) and p.Glu66Gly (c.197A>G, Likely pathogenic, single submitter) carry a pathogenic-type classification, with 2,087 VUS, 226 conflicting and 30 Benign/Likely benign.
• gnomAD v4 gene constraint for AXIN2: LoF-constrained (pLI 0.99998, lof_z 5.27) while missense-tolerant (o/e missense 1.030, mis_z -0.42), consistent with a truncating-driven disease mechanism.
• Gene-level germline mechanism context: loss of function (truncating) is the supported AXIN2 germline disease mechanism (lof_mechanism_supported = true in the gene-level PVS1 eligibility context, based on targeted germline literature review).
• OncoKB gene-level curation for AXIN2: tumor suppressor in WNT signalling, mutated at low frequencies, with truncating mutations described as the class associated with colorectal cancer.
• Lejeune et al. 2006 (PMID:16941501): in an AFAP cohort, no clearly pathogenic AXIN2 mutation (frameshift or nonsense) was found, and the missense change studied here was concluded to be a polymorphism with no functional effect.
• Mongin et al. 2012 (PMID:21476993): screening of Wnt-pathway genes including AXIN2 in unexplained polyposis found no causative AXIN2 mutation, and this missense change was classed a polymorphism after detection in 4 of 275 healthy controls (0.72%).
• Caution applied: a minority of AXIN2 missense variants have been reported as (likely) pathogenic in the literature (PMID:23838596, PMID:41361419), so BP1 was kept at supporting strength rather than escalated, and generic combination rules were consulted for how a supporting benign criterion combines with the other benign evidence in this case.

Still missing

• BP1 is applied at supporting (default) strength only: a small number of AXIN2 missense alleles have been proposed as pathogenic in the literature (PMID:23838596; p.Thr693Met, PMID:41361419), so AXIN2 is not a strictly truncating-only gene and BP1 should not be escalated or used as stand-alone benign evidence.
• No gene-specific framework or VCEP specification for AXIN2 exists in this case to define the expected variant-type spectrum; the truncating-dominant pattern was derived from the ClinVar record composition and gnomAD gene constraint.

Sources

Reasoning

Scope first: NM_004655.4:c.1235A>G is a missense variant, NP_004646.3:p.(Asn412Ser), so BP4 is evaluated on the missense REVEL path only; the SpliceAI delta (0.014) is not admissible BP4 support for a missense variant and was not used. No gene-specific specification exists for AXIN2 (no ClinGen CSPEC, framework_complete=false), so the generic ClinGen-SVI calibrated REVEL thresholds apply: BP4 supporting <=0.29, moderate <=0.183, strong <=0.016 (Pejaver et al. 2022, PMID:36413997). The REVEL score for this variant is 0.119, which satisfies the moderate bin (<=0.183) and the supporting bin (<=0.29) but not the strong bin (<=0.016); the highest strength satisfied is therefore assigned. A benign (tolerated) protein impact is predicted. BP4 is met at moderate strength. Independently, BayesDel (-0.590929) was not used because no generic ACMG-strength calibration is established for it and AXIN2 has no VCEP cutoff, so it is treated as not_available; no double-counting with PP3 or BP7 occurs, as those are not met and not applicable respectively.

Rule

REVEL 0.119 <= 0.183 -> BP4 moderate (Pejaver et al. 2022 PMID:36413997)

Evidence considered

• REVEL score 0.119 (local REVEL v1.3 lookup, GRCh38 chr17:65537801 T>C, matching the normalized case variant) - satisfies the BP4 moderate threshold of <=0.183 (and supporting <=0.29).
• BP4 thresholds <=0.29 (supporting), <=0.183 (moderate), <=0.016 (strong) from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997); moderate is the highest bin satisfied by 0.119.
• Applicable path selected by consequence type: missense variant -> REVEL only; SpliceAI max delta 0.014 was not used as BP4 support, since absence of a predicted splice effect says nothing about tolerance of an amino-acid substitution.
• BayesDel score -0.590929 treated as not_available (no generic calibration, no AXIN2 VCEP cutoff) and therefore not used to modulate BP4 strength.
• No ClinGen CSPEC record for AXIN2 and no gene-specific PP3/BP4 lookup table in the case materials; generic ACMG/AMP 2015 with ClinGen-SVI calibrated in-silico thresholds applies (framework_mode=generic_acmg).

Sources

Reasoning

PS1 (strong pathogenic; ACMG/AMP 2015, PMID:25741868) requires the variant to have the same amino-acid change as a previously established pathogenic variant. The exact change here, p.Asn412Ser, is not an established pathogenic allele in any source available for this case. The only ClinVar record carrying it (variation 136477, NM_004655.4:c.1235A>G) is classified Benign/Likely benign at 2-star review status ('criteria provided, multiple submitters, no conflicts'; 20 submissions; 12 clinical laboratories report Benign and 6 Likely benign; no expert-panel assertion). The published full-text evidence points the same way: Lejeune et al. (PMID:16941501) reported this precise change in one 18-year-old with 30 adenomatous polyps, absent from 30 further patients and 50 healthy controls, and still concluded the variants 'are likely to be polymorphisms', with PolyPhen calling p.Asn412Ser benign; Mongin et al. (PMID:21476993) detected the same c.1235A>G change in 4 of 275 healthy controls (allele frequency 0.72%) and stated they 'classed [it] as polymorphism'. No nucleotide change producing p.Asn412Ser is reported as pathogenic in any reviewed source, so the antecedent of PS1 fails outright. PS1 is not met.

Rule

PS1: same amino-acid change as a previously established pathogenic variant -> strong pathogenic (ACMG/AMP 2015; PMID:25741868); antecedent not satisfied.

Evidence considered

• PS1 definition used: same amino-acid change as a previously established pathogenic variant, regardless of nucleotide change (ACMG/AMP 2015, PMID:25741868).
• ClinVar variation 136477 (NM_004655.4(AXIN2):c.1235A>G (p.Asn412Ser)): germline classification Benign/Likely benign, review status 'criteria provided, multiple submitters, no conflicts' (2 stars), 20 submissions from 20 submitters; submission breakdown Benign 12, Likely benign 6, benign 1; zero expert-panel submissions.
• Lejeune et al. 2006, PMID:16941501 (full text): p.Asn412Ser found in one 18-year-old with 30 adenomatous polyps, co-occurring with c.1530G>A; RT-PCR showed no splicing alteration; PolyPhen predicted 'benign based on the limited conservation of Asn residue across species'; the authors state the two variants 'are likely to be polymorphisms'.
• Mongin et al. 2012, PMID:21476993 (full text): the same c.1235A>G (p.Asn412Ser) change was seen in patients with unexplained polyposis and, on genotyping 275 healthy controls, in four of them (0.72%), leading the authors to class it 'as polymorphism by studying a control healthy population'.
• No other source in this case reports p.Asn412Ser as pathogenic or likely pathogenic; no expert-panel (VCEP) assertion exists for this variant or for AXIN2.

Still missing

• None that could change this verdict: PS1 requires a previously established pathogenic variant with the identical amino-acid change, and both the ClinVar consensus (Benign/Likely benign, 20 submissions) and the two primary reports of p.Asn412Ser (PMID:16941501, PMID:21476993) call it a polymorphism/benign allele.

Sources

Reasoning

Scope and framework: NM_004655.4:c.1235A>G in AXIN2 is a missense substitution, NP_004646.3:p.(Asn412Ser) (codon 412, exon 6). No ClinGen CSPEC exists for AXIN2 and no gene-specific criteria specification was found in the case materials (framework_mode=generic_acmg, framework_complete=false), so PS3 is evaluated under the generic ACMG/AMP 2015 standard (PMID:25741868): a well-established functional study must show a damaging effect on gene or protein function, ideally with validated assay methodology, controls, and a readout biologically relevant to the gene's disease mechanism. No functional study shows a damaging effect for this exact variant. The only exact-variant experimental work in the case is an RNA-level assay in PMID:16941501: RT-PCR of AXIN2 transcripts using primers within exons 3 and 7 showed both c.1235A>G and the co-inherited silent change c.1530G>A present in cDNA at proportions similar to those in genomic DNA, i.e. no splicing alteration. That result is negative for damage, comes from a single patient, interrogates transcript processing rather than AXIN2 protein function, and is interpreted by the authors as indicating no functional effect, so it cannot support PS3. PMID:21476993 screened the complete coding sequence of AXIN2 and other Wnt-pathway genes (PPP2R1B, WIF1, SFRP1) in 25 patients with unexplained adenomatous polyposis, found no causative mutation in any of these genes, and detected this variant in 4 of 275 healthy controls (allele frequency 0.72%), concluding it is a polymorphism; that is control-population genotyping, not a functional assay, and does not bear on PS3. No AXIN2 protein-function assay (Wnt/beta-catenin reporter activity, AXIN2 protein stability, or comparable readout) has been performed for p.Asn412Ser: the AXIN2 in vitro functional work in a 2025 tooth-agenesis study (PMID:41361419, Wnt/beta-catenin activation assay and PBMC transcript quantification) addressed p.Thr693Met and p.Arg354Leufs*2, not p.Asn412Ser, and that paper does not mention c.1235A>G. In-silico outputs for this variant (REVEL 0.119; SpliceAI max delta 0.014) are computational predictions rather than functional assay evidence and are scored by the in-silico group, not converted into PS3 evidence here. PS3 is not met; no variant-specific functional evidence of a damaging effect exists.

Evidence considered

• Variant identity: NM_004655.4:c.1235A>G, NP_004646.3:p.(Asn412Ser), missense, exon 6; this is the variant against which any functional assay must have been performed.
• PMID:16941501: RT-PCR of AXIN2 transcripts (primers within exons 3 and 7) showed the c.1235A>G and c.1530G>A changes present in cDNA at a similar proportion to genomic DNA, indicating no splicing alteration; the authors additionally reported Polyphen predicted p.Asn412Ser benign on the basis of limited cross-species conservation of Asn412.
• PMID:16941501 Discussion: the authors state the two variants 'did not alter splicing, and the missense mutation p.Asn412Ser did not concern a highly conserved amino-acid, suggesting no functional effect'.
• PMID:21476993: no deleterious mutation was found in AXIN2 or the other screened Wnt-pathway genes in 25 unexplained adenomatous polyposis patients; the variant was identified in 4/275 healthy control subjects (allele frequency 0.72%) by HRM and sequencing and the authors classed it as a polymorphism - a population/control observation, not a functional assay.
• PMID:25741868 (ACMG/AMP 2015) supplies the PS3 definition applied here (well-established functional studies showing a damaging effect) and cautions that in-silico predictions are only 65-80% accurate and are not functional evidence; it contains no AXIN2-specific data.
• No variant-specific protein-function assay exists for p.Asn412Ser in the case materials or in the fetched literature; the 2025 AXIN2 functional study (PMID:41361419) performed in vitro Wnt/beta-catenin reporter assays and PBMC transcript analysis for other AXIN2 variants (p.Thr693Met, p.Arg354Leufs*2) and does not mention c.1235A>G / p.Asn412Ser.
• SpliceAI max delta 0.014 (DS_AG 0.014, DS_AL 0.001, DS_DG 0.0, DS_DL 0.0) and REVEL 0.119 are computational outputs, not functional assay results, and were not used as PS3 evidence.
• Governance: ClinVar CSPEC search returned no record for AXIN2 and vcep_materials.json reports no CSPEC URL and no gene-specific framework (found=false, source=generic_acmg_fallback), so generic ACMG/AMP 2015 functional-evidence standards govern this criterion.

Still missing

• No validated variant-specific functional assay of AXIN2 protein function (e.g. Wnt/beta-catenin reporter activity or AXIN2 protein stability) exists for p.Asn412Ser; the only exact-variant experiment is a single-patient RNA transcript (splicing) analysis.
• If such an assay were performed, it would need documented positive and negative controls, quantitative effect size, and demonstrable biological relevance to the AXIN2 loss-of-function disease mechanism before PS3 could be considered at any strength.

Sources

Reasoning

Governing framework: no ClinGen VCEP/CSpec exists for AXIN2 (cspec found=false; vcep_materials.json reports 'no cspec url available', empty summaries, empty gene_vcep_dir) and no gene-specific framework file was retrieved (final_classification_framework found=false, source=generic_acmg_fallback, framework_mode=generic_acmg). The generic ACMG/AMP 2015 framework (Richards et al., PMID:25741868) therefore governs. In that framework PS4 is defined qualitatively as an increased prevalence of the variant in affected individuals compared with controls, with the requirement of statistical significance: the guideline states that in a case-control comparison 'showing a statistically significant difference can still be assumed to provide supportive evidence for pathogenicity', and that absence of a statistical difference should be interpreted cautiously. Importantly for the operator question: PS4 as written in the governing generic text contains NO numeric odds-ratio/prevalence cut-off, so there is no numeric operator (>=, <=, >, <) to apply to this criterion in this case; borrowing a threshold from any other criterion in this group (e.g. a likelihood-ratio style <= rule) would be invalid. The operative comparison is therefore: is there statistically significant enrichment of c.1235A>G in cases versus controls (cases > controls, i.e. p <= 0.05)? The only case-control data available answer it negatively. (1) PMID:21476993: the variant was found in 1 of 25 patients with unexplained adenomatous polyposis and, on the same HRM+sequencing assay, in 4 of 275 healthy control subjects (allele frequency 0.72%), i.e. 4.0% of cases vs 1.5% of controls, OR approximately 2.8; one-sided Fisher exact test for enrichment in cases: p = 0.355 <= 0.05? no. The authors explicitly concluded the variant is a polymorphism. (2) PMID:16941501: the variant was found in 1 of 31 APC/MMR-negative patients with multiple adenomas or colorectal cancer and was absent from 50 healthy controls (3.2% vs 0%), OR approximately 5.0 by Haldane-Anscombe correction; one-sided Fisher exact p = 0.383 <= 0.05? no. (3) Neither paper reports any odds ratio or enrichment reaching significance, and neither cohort is powered for this variant; the absence of a significant difference is not evidence of enrichment. (4) The population control data point the other way for an enrichment argument: the variant is common in reference populations (gnomAD v2.1 AF 0.92%, 2170/236140 alleles, 41 homozygotes, South Asian AF 3.83%; gnomAD v4.1 AF 0.79%, 12588/1586748 alleles, 165 homozygotes, South Asian AF 4.11%), which is the expected profile of a common low-penetrance/benign missense allele rather than one enriched in affected cohorts. PS4 is therefore not met; no enrichment evidence of any strength exists for this variant.

Rule

PS4 (strong, generic ACMG/AMP 2015): 'The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls' (Richards et al. 2015, PMID:25741868). No numeric enrichment threshold is defined in the governing generic text; no AXIN2 VCEP/CSpec exists to supply one.

Evidence considered

• Framework provenance: no CSpec/VCEP for AXIN2 (vcep_materials.json: 'no cspec url available'; final_classification_framework found=false with source=generic_acmg_fallback, precedence_rank=3, scoring_model 'ACMG/AMP 2015'), so the generic ACMG/AMP 2015 PS4 definition applies (Richards et al. 2015, PMID:25741868). No AXIN2 VCEP specification exists that could supply a different operator for PS4.
• Operator check as required for this group: the generic PS4 text defines enrichment qualitatively and supplies no numeric threshold, so the applicable comparison is significance of case enrichment (p <= 0.05?), not a borrowed numeric cut-off from another criterion in this group. Written explicitly: PMID:21476993 carrier frequencies 1/25 cases (4.0%) vs 4/275 controls (1.5%), OR ~2.8, one-sided Fisher p = 0.355 <= 0.05? no. PMID:16941501: 1/31 cases (3.2%) vs 0/50 controls (0%), OR ~5.0 (Haldane-Anscombe), one-sided Fisher p = 0.383 <= 0.05? no.
• PMID:25741868 (Richards et al. 2015) PS4/population-evidence section, quoted for the governing requirement: 'The use of population data for case-control comparisons is most useful when the populations are well-phenotyped, have large frequency differences and the Mendelian disease under study is early-onset' and 'showing a statistically significant difference can still be assumed to provide supportive evidence for pathogenicity ... In contrast, the absence of a statistical difference, particularly with extremely rare variants and less penetrant phenotypes, should be interpreted cautiously.' No significant difference was observed for this variant in either cohort.
• PMID:21476993 (Mongin et al., Clin Genet 2012): c.1235A>G was the single rare AXIN2 finding in a screen of four Wnt-pathway genes in 25 patients with unexplained adenomatous polyposis (no deleterious mutation in any of them); the variant was then identified in 4 of 275 healthy control subjects by HRM and sequencing (allele frequency 0.72%) and the authors concluded 'this variant is a polymorphism'. Direct case-control observation, no enrichment signal.
• PMID:16941501 (Lejeune et al., Hum Mutat 2006): c.1235A>G detected in one 18-year-old patient with 30 adenomatous polyps and no family history of polyposis or colorectal cancer, absent from 30 other patients and 50 healthy controls; the authors state the variants 'are likely to be polymorphisms'. Case observation without significant control enrichment.
• gnomAD v2.1 and v4.1 allele frequencies (0.92% overall, 3.83% South Asian, 2170/236140 alleles, 41 homozygotes in v2.1; 0.79% overall, 4.11% South Asian, 12588/1586748 alleles, 165 homozygotes in v4.1) were used only as the control/general-population reference in the case-control reasoning described by the ACMG guideline; they are not being applied here as the population-frequency criteria (BA1/BS1/PM2), which are adjudicated separately.
• No source's final classification was used to infer PS4; the ClinVar aggregate label was not used as enrichment evidence.

Still missing

• No AXIN2-specific VCEP/ClinGen specification or gene framework exists, so PS4 is applied with the qualitative generic ACMG/AMP 2015 definition; no numeric enrichment threshold is defined for this criterion in the governing text.
• No adequately powered case-control study of AXIN2 c.1235A>G exists in the retrieved literature; the two available cohorts are small (25 and 31 affected individuals) and neither reports a significant odds ratio, so a future meta-analytic enrichment estimate could not be excluded, though none is currently available.
• PMID:34043773 (EHTG/ESCP Mallorca guidelines) appears in the literature-pass criteria index for PS4/PP5 but is not a source_registry key in this case and concerns Lynch syndrome/MMR genes rather than AXIN2; it was not usable as evidence.

Sources

Reasoning

PM1 (moderate pathogenic; ACMG/AMP 2015, PMID:25741868) requires the variant to sit in a mutational hot spot and/or a critical, well-established functional domain without benign variation. No gene-specific specification exists for AXIN2 here: no ClinGen CSPEC was retrieved (cspec found = false) and the VCEP domain table is empty ({}), so there is no approved AXIN2 domain list to check residue 412 against and the generic criterion applies. Residue 412 is not a hot spot: the CancerHotspots screen for AXIN2 N412 returned no significant hot spot. It is also not within a recognized AXIN2 functional domain: UniProt Q9Y2T1 (843 aa, matching NP_004646.3) places the RGS domain at residues 81-200 and the DIX domain at 761-843, and Asn412 falls in a disordered segment (396-435) at the extreme C-terminal edge of the GSK3beta-interaction region (327-413), immediately N-terminal to the beta-catenin interaction region (413-476) rather than inside it. Even if a reviewer were to treat the flanking interaction region as functional, the 'without benign variation' prong fails decisively: this allele is common in the general population (gnomAD v4.1 AF 0.79%, South Asian 4.11%, 165 homozygotes; gnomAD v2.1 AF 0.92%, South Asian 3.83%, 41 homozygotes), it is Benign/Likely benign in ClinVar, and independently-reported likely-benign missense changes bracket the codon (p.Thr410Ser, p.Glu415Asp, p.Ala417Gly). PM1 is not met.

Rule

PM1: located in a mutational hot spot and/or critical and well-established functional domain without benign variation -> moderate pathogenic (ACMG/AMP 2015; PMID:25741868); neither prong satisfied.

Evidence considered

• PM1 definition used: variant in a mutational hot spot and/or critical and well-established functional domain (e.g. active site) without benign variation (ACMG/AMP 2015, PMID:25741868).
• No ClinGen CSPEC was found for AXIN2 (the cspec source was queried and returned found = false) and the governing VCEP domain table supplied for this case is empty ({}), so no gene-specific PM1 domain list or hotspot definition exists; the generic criterion was applied.
• CancerHotspots screen for AXIN2 N412 returned no result: 'This variant does not lie in a statistically significant hotspot' (no hotspot row found).
• UniProt Q9Y2T1 (AXIN2_HUMAN, 843 aa, the length matching NP_004646.3): RGS domain 81-200; DIX domain 761-843; disordered region 396-435; interaction with GSK3B 327-413; interaction with beta-catenin 413-476; tankyrase-binding motif 21-30. Residue 412 therefore sits in the disordered segment at the C-terminal edge of the GSK3beta-interaction region, one residue before the beta-catenin interaction region, and is inside neither named domain.
• Population evidence of benign variation at this codon/region: gnomAD v4.1 total AF 0.00793 (12,588/1,586,748 alleles; 165 homozygotes), South Asian AF 0.04112 (3,565/86,704; 132 homozygotes), grpmax filtering AF 0.03999; gnomAD v2.1 total AF 0.00919 (2,170/236,140; 41 homozygotes), South Asian AF 0.03830 (970/25,324; 37 homozygotes).
• Lejeune et al. 2006 (PMID:16941501) noted the Asn412 residue is poorly conserved across species (PolyPhen called the substitution benign 'based on the limited conservation of Asn residue across species'), consistent with a non-critical, non-hotspot position.
• ClinVar records for this gene include likely-benign missense alleles immediately flanking codon 412 (p.Thr410Ser c.1228A>T, p.Glu415Asp c.1245G>T/c.1245G>C, p.Ala417Gly c.1250C>G), showing that benign missense variation is tolerated in this region.

Still missing

• PM1 was assessed against the generic ACMG/AMP definition because no ClinGen CSPEC/VCEP and no gene-specific AXIN2 framework exists for this case; the VCEP domain table supplied is empty, so there is no VCEP-approved list of AXIN2 critical domains to check residue 412 against.
• Domain coordinates were taken from UniProt Q9Y2T1 (RGS 81-200; DIX 761-843; disordered 396-435; GSK3beta interaction 327-413; beta-catenin interaction 413-476), which is not a case-registry source; a reviewer preferring a different AXIN2 domain definition should confirm residue 412 is likewise non-domain. Even under a permissive definition the 'without benign variation' prong still fails on gnomAD and ClinVar.

Sources

Reasoning

PM2 (supporting pathogenic; SVI-recommended threshold AF <= 0.0001, PMID:25741868) is not met. The variant is present in every population dataset examined at frequencies roughly 60- to 90-fold above that threshold, and there is no population in which it is rare or absent. gnomAD v4.1 all-comers AF 0.00793 (12,588/1,586,748 alleles; 165 homozygotes); gnomAD v2.1 all-comers AF 0.00919 (2,170/236,140 alleles; 41 homozygotes); gnomAD-Canada v1.0 AF 0.00799 (147/18,408 alleles; 1 homozygote); the lowest frequency observed anywhere is gnomAD v3.1 non-cancer genomes AF 0.00672 (993/147,760 alleles; 8 homozygotes), which is still ~67-fold above 0.0001. Frequency in the highest-ancestry group (South Asian, 4.11%) is four orders of magnitude above the PM2 threshold. PM2 is consequently not met at any strength, and the SVI downgrade to supporting strength is moot.

Rule

AF <= 0.0001 -> PM2 supporting pathogenic (SVI downgrade; PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers: AF 0.00793 (12,588/1,586,748 alleles), 165 homozygotes; South Asian AF 0.04112.
• gnomAD v2.1 all-comers: AF 0.00919 (2,170/236,140 alleles), 41 homozygotes; South Asian AF 0.03830.
• gnomAD v2.1 non-cancer exomes-only subset: AF 0.00995 (1,941/195,014 alleles), 41 homozygotes.
• gnomAD v3.1 non-cancer genomes-only subset: AF 0.00672 (993/147,760 alleles), 8 homozygotes - the lowest frequency observed in any dataset, still ~67-fold above the PM2 threshold.
• gnomAD-Canada v1.0 (HostSeq genomes): AF 0.00799 (147/18,408 alleles), 1 homozygote.
• PM2 threshold applied: AF <= 0.0001 at supporting strength (SVI recommendation to downgrade PM2, PMID:25741868); no VCEP or gene-specific framework for AXIN2 modifies it in this case.

Still missing

• None: every available dataset (gnomAD v2.1 and v4.1 all-comers, v2.1 non-cancer exomes, v3.1 non-cancer genomes, gnomAD-Canada) shows frequency orders of magnitude above the PM2 threshold, so no additional population source could change this verdict.

Sources

Reasoning

PM3 requires, for a recessive disorder, that the variant be detected in trans with a pathogenic variant. Two independent prerequisites fail here. First, the AXIN2 disease association is dominant: oligodontia-colorectal cancer (oligodontia-cancer predisposition) syndrome and attenuated adenomatous polyposis are reported in heterozygous carriers of AXIN2 truncating/missense alleles, and in the polyposis literature the recessive model in APC-negative patients is biallelic MUTYH inactivation, not AXIN2. Second, no proband carrying c.1235A>G has been shown to carry a pathogenic AXIN2 allele in trans. In the only fully described carrier (an 18-year-old with 30 adenomatous polyps and no family history of polyposis or colorectal cancer), c.1235A>G was found together with the apparently silent c.1530G>A on the same allele, annotated in HGVS as c.[1235A>G (+) 1530G>A] / p.[Asn412Ser (+) Thr510Thr]; no clearly pathogenic AXIN2 variant (frameshift or nonsense) was found in that patient or in any of the 31 APC-negative patients screened, and that patient was additionally evaluated for MUTYH. The second independent cohort that detected c.1235A>G found it as a rare variant in 25 unexplained-polyposis patients with no causative Wnt-pathway mutation, and in 4/275 healthy controls (allele frequency 0.72%), concluding it is a polymorphism; again no in-trans pathogenic allele was reported. Population data reinforce the absence of a recessive-trans mechanism: the variant is common and homozygous in reference populations (gnomAD v2.1 2170/236140 alleles, AF 0.92%, 41 homozygotes; gnomAD v4.1 12588/1586748 alleles, AF 0.79%, 165 homozygotes, 132 of them South Asian), so the trans configuration with a pathogenic allele that PM3 requires has never been observed and a severe recessive mechanism is not supported. PM3 is therefore not met.

Rule

PM3 (moderate, generic ACMG/AMP 2015): 'For recessive disorders, detected in trans with a pathogenic variant' (Richards et al. 2015, PMID:25741868).

Evidence considered

• Generic ACMG/AMP 2015 definition applied under the generic fallback framework (no CSpec, no gene-specific framework file for AXIN2): PM3 = 'For recessive disorders, detected in trans with a pathogenic variant'.
• PMID:16941501 (Lejeune et al., Hum Mutat 2006): the only fully described carrier of c.1235A>G was an 18-year-old with 30 adenomatous polyps and no family history of polyposis or colorectal cancer; the variant was in cis with an apparently silent change, quoted as 'two novel sequence variations in exon 5 of AXIN2 in one patient aged 18 presenting with 30 adenomatous polyps (Li-10): one apparently missense mutation c.1235A>G (p.Asn412Ser) and one apparently silent mutation c.1530G>A (p.Thr510Thr)'; Table 2 records the phase as c.[1235A>G (+) 1530G>A] / p.[Asn412Ser (+) Thr510Thr]. No trans pathogenic AXIN2 allele, and no clearly pathogenic AXIN2 variant at all, was reported in this patient or in the 31 patients screened ('No clearly pathogenic mutations (frameshift or nonsense mutations) were found in any of our patients').
• PMID:16941501 additionally shows the recessive polyposis model in this cohort operates through MUTYH, not AXIN2: biallelic MUTYH mutations were found in 7/22 APC-negative FAP/AFAP patients (32%), with allele phase confirmed by allele-specific PCR, 'In these cases, mutations were proved to be present onto different alleles, as ascertained by AS-PCR' - i.e. the in-trans configuration documented in this cohort belongs to MUTYH genotypes, not to c.1235A>G.
• PMID:21476993 (Mongin et al., Clin Genet 2012): c.1235A>G was found as the only AXIN2 variant in 25 patients with unexplained adenomatous polyposis, with no deleterious mutation in any of the four Wnt-pathway genes screened; the variant was then detected in 4/275 healthy control subjects by HRM and sequencing (allele frequency 0.72%). No in-trans pathogenic variant is described for any carrier.
• gnomAD v2.1 and v4.1: the variant reaches 0.92% (2170/236140 alleles) with 41 homozygotes and 0.79% (12588/1586748 alleles) with 165 homozygotes (132 in South Asians); homozygotes are observed in the general population, which is not the pattern of an allele whose clinical significance requires a pathogenic partner in trans.
• ClinVar conditions attached to this exact variant include oligodontia-cancer predisposition syndrome, AXIN2-related disorder and hereditary cancer-predisposing syndrome, i.e. dominant/incompletely penetrant settings; no submission lists a phase (trans/cis) or zygosity observation for this variant. The aggregate ClinVar classification itself was not used as evidence.

Still missing

• No phase-resolved proband genotype (parental or allele-specific testing) exists for c.1235A>G in this workup; the conclusion rests on the published carrier reports (PMID:16941501, PMID:21476993), which describe no second pathogenic AXIN2 allele and no trans configuration.
• No AXIN2-specific VCEP/ClinGen specification is available for this gene (no CSpec, no gene framework), so PM3 is being applied with the generic ACMG/AMP 2015 definition.

Sources

Reasoning

PM5 (moderate pathogenic; ACMG/AMP 2015, PMID:25741868) requires a different missense change at the same residue (Asn412) that has been established as pathogenic. An exhaustive residue-level ClinVar review returned six records touching codon 412, none of which is pathogenic or likely pathogenic: p.Asn412Ser (c.1235A>G, this variant, Benign/Likely benign), p.Asn412His (c.1234A>C, Uncertain significance), p.Asn412Thr (c.1235A>C, conflicting classifications), p.Asn412Ile (c.1235A>T, conflicting classifications), p.Asn412= (c.1236T>C, Likely benign, synonymous) and an in-frame duplication-insertion p.Asn412_Ser413insGluGluArgGluGlySerGluLeuThrLeuAsn (c.1204_1236dup, Uncertain significance). The automated PM5 comparator harvest likewise returned zero candidates (pm5_mode unknown, eligible_for_classic_pm5_search = false). The second premise of PM5 is also absent: this variant is not novel, being an established Benign/Likely benign allele with 20 ClinVar submissions, two primary publications classifying it as a polymorphism (PMID:16941501, PMID:21476993), and 165 homozygotes in gnomAD v4.1. PM5 is not met.

Rule

PM5: different missense change at the same residue previously established as pathogenic -> moderate pathogenic (ACMG/AMP 2015; PMID:25741868); no pathogenic same-residue comparator exists.

Evidence considered

• PM5 definition used: a different missense change at the same amino-acid residue previously established as pathogenic (ACMG/AMP 2015, PMID:25741868).
• Residue-level ClinVar review for AXIN2 codon 412 returned six records, none pathogenic: p.Asn412Ser (c.1235A>G, Benign/Likely benign - this variant, variation 136477), p.Asn412His (c.1234A>C, Uncertain significance), p.Asn412Thr (c.1235A>C, conflicting classifications of pathogenicity), p.Asn412Ile (c.1235A>T, conflicting classifications of pathogenicity), p.Asn412= (c.1236T>C, Likely benign) and p.Asn412_Ser413insGluGluArgGluGlySerGluLeuThrLeuAsn (c.1204_1236dup, Uncertain significance).
• Automated PM5 comparator collection for this case found no candidates (pm5_mode 'unknown', eligible_for_classic_pm5_search false, same_residue_candidates 0), so the same-residue comparison was completed manually against ClinVar.
• The variant itself is not novel: ClinVar variation 136477 is Benign/Likely benign across 20 submissions, and the change is reported in the literature as a polymorphism (PMID:16941501 found it in one patient with 30 adenomatous polyps and called the variants likely polymorphisms; PMID:21476993 found it in 4 of 275 healthy controls).

Still missing

• PM5 was evaluated against ClinVar, the only variant-level comparator source available for this case; no expert-panel-classified AXIN2 variant exists at residue 412 in any source reviewed, and the automated candidate harvest produced no comparators.
• If a future submission establishes a pathogenic AXIN2 variant at codon 412, PM5 would need to be re-evaluated; no such variant is currently reported.

Sources

Reasoning

PP2 (supporting pathogenic; ACMG/AMP 2015, PMID:25741868) requires both a low rate of benign missense variation in the gene and missense variants being a common mechanism of disease. Both prongs fail for AXIN2. Missense variation is well tolerated: gnomAD v4 gene constraint for AXIN2 gives observed/expected missense 1.03 (95% CI 0.98-1.08) with mis_z -0.42, i.e. no missense constraint at all, while the same gene is LoF-constrained (pLI approximately 1.0, lof_z 5.27). ClinVar reflects the same picture across all 4,370 AXIN2 records retrieved: 2,347 single-amino-acid-substitution records comprise 2,087 Uncertain significance, 226 conflicting, and 30 Benign/Likely benign alleles, with only two pathogenic-type missense records in the entire gene (p.Val105Gly, Pathogenic, no assertion criteria provided; p.Glu66Gly, Likely pathogenic, single submitter). Missense is equally not a common disease mechanism: 186 ClinVar AXIN2 records carry a pathogenic-type classification and are all loss-of-function in type (169 nonsense/frameshift, 3 canonical splice-site, 14 copy-number losses), and the germline mechanism documented in the reviewed AXIN2 literature is loss of function/truncating. With a high tolerated rate of benign missense variation and a truncating-driven mechanism, PP2 is not met on either prong.

Rule

PP2: missense variant in a gene with a low rate of benign missense variation where missense is a common mechanism of disease -> supporting pathogenic (ACMG/AMP 2015; PMID:25741868); both prongs unsatisfied.

Evidence considered

• PP2 definition used: missense variant in a gene with a low rate of benign missense variation in which missense variants are a common mechanism of disease (ACMG/AMP 2015, PMID:25741868).
• gnomAD v4 gene constraint for AXIN2 (Ensembl ENSG00000168646): o/e missense 1.030, 95% CI 0.981-1.081, mis_z -0.42 (no missense constraint); pLI 0.99998 and lof_z 5.27 (LoF-constrained).
• ClinVar composition, all 4,370 AXIN2 records retrieved: 2,347 single-amino-acid substitutions (2,087 Uncertain significance; 226 conflicting classifications; 19 Likely benign; 9 Benign/Likely benign; 2 Benign; 1 Likely pathogenic p.Glu66Gly c.197A>G single submitter; 1 Pathogenic p.Val105Gly c.314T>G with no assertion criteria provided; 2 unclassified); 186 non-missense records carry a pathogenic-type classification and are all loss-of-function (169 nonsense/frameshift, 3 canonical splice-site, 14 copy-number losses).
• Gene-level germline mechanism context for AXIN2: loss of function (truncating) is the supported disease mechanism, per the gene-level PVS1 eligibility context (lof_mechanism_supported = true, mechanism rationale citing targeted germline literature review).
• Population observation for this allele: gnomAD v4.1 AF 0.00793 (12,588/1,586,748 alleles) with 165 homozygotes and gnomAD v2.1 AF 0.00919 (2,170/236,140 alleles) with 41 homozygotes - a missense allele far too common to be a rare-disease mechanism at this residue.
• Generic combination rules were consulted for the strengths assignable to PP2 (supporting) in the absence of a gene-specific framework; no AXIN2 CSPEC or VCEP specification exists for this case.

Still missing

• PP2's first prong (rate of benign missense variation) was assessed with gnomAD v4 gene-constraint metrics (o/e missense 1.03, mis_z -0.42) plus the ClinVar record composition, since no gene-specific framework quantifies a benign-missense rate for AXIN2 in this case.
• A small number of missense AXIN2 alleles have been proposed as pathogenic in the literature (e.g. a novel missense in an attenuated FAP family, PMID:23838596; p.Thr693Met reported as likely pathogenic with Wnt/beta-catenin activation, PMID:41361419); these are too few and too weakly supported in variant databases to establish missense as a common mechanism.

Sources

Reasoning

Scope first: NM_004655.4:c.1235A>G in AXIN2 is a missense substitution, NP_004646.3:p.(Asn412Ser) (single-nucleotide change in codon 412, exon 6), so PP3/BP4 must be taken from the missense protein-impact path (REVEL) and from that path only; the SpliceAI score present in the evidence is not the applicable path for this consequence class and was not used in either direction. No ClinGen CSPEC exists for AXIN2 and no gene-specific criteria specification or PP3/BP4 lookup table was found (framework_complete=false, framework_mode=generic_acmg), so the generic ClinGen-SVI calibrated REVEL thresholds govern. The variant's REVEL score is 0.119 (local REVEL v1.3 lookup at GRCh38 chr17:65537801 T>C, concordant with the normalized variant), which is far below the PP3 supporting threshold of >=0.644 (Pejaver et al. 2022, PMID:36413997) and therefore also below the moderate (>=0.773) and strong (>=0.932) bins. The predictor does not support a damaging effect on protein function. PP3 is not met. BayesDel was not considered: no generic ACMG-strength calibration is established for it and this gene has no VCEP-specified cutoff, so its score is treated as not_available.

Rule

REVEL 0.119 < 0.644 (PP3 supporting, Pejaver et al. 2022 PMID:36413997) -> PP3 not met

Evidence considered

• Variant consequence: missense, NP_004646.3:p.(Asn412Ser) from NM_004655.4:c.1235A>G (exon 6); this fixes the applicable predictor path as REVEL only.
• REVEL score 0.119 (local REVEL v1.3 lookup, GRCh38 chr17:65537801 T>C, matching the case's normalized genomic position) - below the PP3 supporting threshold of >=0.644.
• PP3 threshold >=0.644 (supporting), >=0.773 (moderate), >=0.932 (strong) from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997).
• SpliceAI max delta 0.014 (DS_AG 0.014, DS_AL 0.001, DS_DG 0.0, DS_DL 0.0) was deliberately NOT used for this missense variant: PP3/BP4 take evidence from one path only, selected by consequence type.
• BayesDel score -0.590929 treated as not_available: no generic ACMG-strength calibration exists and AXIN2 has no VCEP-specified cutoff.
• No ClinGen CSPEC record for AXIN2 and no gene-specific framework in the case materials, so generic ACMG/AMP 2015 with ClinGen-SVI calibrated in-silico thresholds applies.

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015 (no AXIN2 VCEP/CSpec or gene framework exists; cspec found=false; final_classification_framework found=false, generic_acmg_fallback). PP5 requires that the variant be 'reported in a recognized expert panel' as pathogenic/likely pathogenic (Richards et al. 2015, PMID:25741868); the criterion carries no numeric threshold, so no numeric operator applies - it is a qualitative gate on the provenance of the assertion, not a value comparison. For NM_004655.4:c.1235A>G (ClinVar variation 136477) that provenance gate fails: the ClinVar record's expert_panels object is empty ({}); the submission audit counts expert_panel_submissions = 0 out of 20 total submissions; every individual submission is explicitly flagged is_expert_panel = false (verified submission-by-submission, including Rigshospitalet, Sinai Health System, Myriad, GeneKor, Mayo Clinic Laboratories, GeneDx, Illumina, Fulgent, Ambry, Breakthrough Genomics, Quest Diagnostics, LabCorp/Invitae, ARUP, Leiden/Amsterdam UMC genome diagnostics, PreventionGenetics, KCCC); the single submission with 3-star-equivalent status does not exist - the record's review status is 'criteria provided, multiple submitters, no conflicts' (2 stars), which reflects aggregate single-laboratory submissions, not a ClinGen/VCEP expert-panel assertion. The ClinVar aggregate label ('Benign/Likely benign') is therefore not PP5-eligible by either route: there is no expert-panel classification, and ordinary laboratory submissions and aggregate labels are excluded by rule. This is the opposite direction of evidence from PS4 (there is no pathogenic claim at all in the record), so no borrowing of PS4's enrichment reasoning applies here. PP5 is not met.

Rule

PP5 (supporting, generic ACMG/AMP 2015 / ClinGen SVI): 'Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation' (Richards et al. 2015, PMID:25741868), restricted here to an exact-variant ClinVar expert-panel Pathogenic/Likely pathogenic classification. No numeric threshold.

Evidence considered

• Generic ACMG/AMP 2015 definition applied under the generic fallback framework: PP5 = 'Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation' (Richards et al. 2015, PMID:25741868); the ACMG framework restricts the upstream source to a recognized expert panel, and this case's instructions restrict PP5 to an exact-variant expert-panel Pathogenic/Likely pathogenic classification. No numeric threshold applies.
• ClinVar variation 136477 / VCV000136477, variant name 'NM_004655.4(AXIN2):c.1235A>G (p.Asn412Ser)', exact match to the case variant: source_data.expert_panels = {} (empty), api review_stars = 2 with review status 'criteria provided, multiple submitters, no conflicts' - i.e. no expert-panel classification at any review tier.
• Submission-level audit (20 submissions, 20 submitters): expert_panel_submissions = 0; each audited submission carries is_expert_panel = false (SCV001920466, SCV001931944, SCV002551275, SCV005911836, SCV006095874, SCV006311438, SCV007316471, SCV000167137, SCV000405708, SCV002806368, SCV001170681, SCV005255552, SCV002046701, SCV000698511, SCV001807586, SCV002036254, SCV004016235, SCV004756465, SCV001159232, SCV000261762). No submission is attributed to a ClinGen/VCEP expert panel.
• The record's assertion is entirely benign-directed (12 clinical laboratories Benign, 6 Likely benign, 1 benign); there is no pathogenic or likely pathogenic assertion from any source, expert or otherwise, so no PP5 trigger can exist independently of the expert-panel gate.
• Per case instructions, ordinary laboratory submissions and aggregate ClinVar labels were deliberately excluded from triggering PP5; the ClinVar aggregate label and the multiple-submitter review status were recorded only as provenance facts, not used as evidence of pathogenicity.
• PMID:25394175, PMID:20301390, PMID:26389258 and PMID:26389505 were each screened as ClinVar-linked PP5/PS4 candidates (criteria index for PP5: 20301390, 25394175, 26389258, 26389505, 34043773): they are referral-practice, GeneReviews and PDQ reference documents with no AXIN2 variant-level content and no expert-panel classification of this variant, so none supplies PP5 evidence. PMID:25741868 supplies only the criterion definition.

Still missing

• No AXIN2 VCEP/ClinGen expert panel has ever reviewed this variant in ClinVar, so no PP5-eligible assertion exists; this cannot be closed from any other source, since PP5 by definition requires an expert-panel classification.
• The absence of an expert panel is intrinsic to the ClinVar record (20/20 submissions from clinical laboratories); a future ClinGen AXIN2 VCEP specification would need to be retrieved before this criterion could change.

Sources

Reasoning

BA1 (stand-alone benign; ACMG/AMP 2015, PMID:25741868) requires an allele frequency of at least 0.05. No dataset or ancestry group reaches this. gnomAD v4.1 all-comers: AF 0.00793 (12,588/1,586,748 alleles; 165 homozygotes); highest ancestry South Asian AF 0.04112 (3,565/86,704 alleles; 132 homozygotes); grpmax filtering AF 0.03999. gnomAD v2.1 all-comers: AF 0.00919 (2,170/236,140 alleles; 41 homozygotes); South Asian AF 0.03830 (970/25,324 alleles; 37 homozygotes); grpmax filtering AF 0.03630. gnomAD-Canada v1.0 (HostSeq): AF 0.00799 (147/18,408 alleles; 1 homozygote). The highest ancestry frequency anywhere in the all-comers data is 4.11% (South Asian, v4.1), which is below 5%. The only figure exceeding 5% anywhere is a raw cell of 8/42 alleles (19.0%) in the HGDP:BALOCHI group of the gnomAD v3.1 non-cancer genome subset; that is a 42-allele small-sample artifact whose own filtering AF for that group is 0.0328, and it is not corroborated by any adequately powered ancestry group, so it does not satisfy BA1. BA1 is therefore not met, although the South Asian frequency approaches the threshold; because BA1 is a stand-alone criterion it is not applied at any reduced strength.

Rule

AF_popmax >= 0.05 -> BA1 stand-alone benign (ACMG/AMP 2015; PMID:25741868)

Evidence considered

• gnomAD v4.1 all-comers: total AF 0.00793 (12,588/1,586,748 alleles), 165 homozygotes; exome AF 0.00807 (11,577/1,434,626; 157 homozygotes); genome AF 0.00665 (1,011/152,122; 8 homozygotes); highest ancestry South Asian AF 0.04112 (3,565/86,704; 132 homozygotes); grpmax filtering AF 0.03999.
• gnomAD v2.1 all-comers: total AF 0.00919 (2,170/236,140 alleles), 41 homozygotes; exome AF 0.00975 (1,997/204,818; 41 homozygotes); genome AF 0.00552 (173/31,322; 0 homozygotes); highest ancestry South Asian AF 0.03830 (970/25,324; 37 homozygotes); grpmax filtering AF 0.03630.
• gnomAD v2.1 non-cancer exomes-only subset: AF 0.00995 (1,941/195,014 alleles), 41 homozygotes, grpmax filtering AF 0.03634.
• gnomAD v3.1 non-cancer genomes-only subset: AF 0.00672 (993/147,760 alleles), 8 homozygotes, grpmax filtering AF 0.03279; its raw HGDP:BALOCHI cell is 8/42 alleles (19.0%), a small-sample artifact.
• gnomAD-Canada v1.0 (HostSeq genomes): AF 0.00799 (147/18,408 alleles), 1 homozygote.
• No ClinGen CSPEC or gene-specific framework exists for AXIN2 in this case, so the generic ACMG/AMP 2015 stand-alone benign threshold of AF >= 0.05 (PMID:25741868) was applied; the generic combination rules state that BA1 alone is sufficient for a Benign classification.
• Ancestry-specific frequencies rather than the pooled global frequency were used for BA1, since a variant that is common in any single major population cannot be the cause of a rare Mendelian disorder.

Still missing

• BA1 is evaluated on the ancestry-specific (popmax / grpmax filtering) allele frequency, the standard metric for stand-alone benign frequency; the South Asian point estimate of 4.11% sits close to the 5% threshold and would cross it if a slightly larger or more consanguineous South Asian cohort were used.
• A raw 8/42-allele HGDP:BALOCHI cell (19.0%) in the gnomAD v3.1 non-cancer genome subset exceeds 5% but was not treated as BA1 evidence because of its tiny sample size; its own group filtering AF is 0.0328.

Sources

Reasoning

BS2 (strong benign) is met only when the variant is observed in a healthy adult individual for a disorder in which full penetrance is expected at an early age (ACMG/AMP 2015, PMID:25741868). That precondition does not hold for AXIN2. AXIN2-related oligodontia-colorectal cancer predisposition is an autosomal dominant condition with incomplete, age-dependent penetrance: the dental phenotype is developmental, but the colorectal cancer risk is adult-onset and does not approach full penetrance, so carriage of this allele by a healthy adult does not exclude pathogenicity and the observation lacks the discriminating power BS2 assumes. Consistent with this, the variant is homozygous in 165 adults in gnomAD v4.1 (132 in South Asians) and 41 in gnomAD v2.1 - genotype evidence against a fully penetrant, early-onset dominant mechanism rather than for a benign one under BS2's own logic. The healthy-adult observations that do exist (Mongin et al. 2012, PMID:21476993: 4 of 275 healthy controls, called a polymorphism; gnomAD population samples) are already captured more directly as frequency evidence under BS1/BA1. BS2 is therefore not met; the conclusion is driven by the disease model rather than by the count of healthy carriers, so confidence is moderate rather than high.

Rule

variant observed in a healthy adult for a disorder with full penetrance expected at an early age -> BS2 strong benign (ACMG/AMP 2015; PMID:25741868)

Evidence considered

• BS2 definition (variant observed in a healthy adult for a disorder with full penetrance expected at an early age) from the ACMG/AMP 2015 framework, PMID:25741868.
• gnomAD v4.1: 165 homozygotes among adults (South Asian 132; exome 157; genome 8), total AF 0.00793.
• gnomAD v2.1: 41 homozygotes among adults (37 in South Asians), total AF 0.00919.
• Disease-model context: AXIN2-related oligodontia-colorectal cancer predisposition is autosomal dominant with incomplete, age-dependent penetrance, so adult non-manifestation does not demonstrate benignity in the way BS2 requires.
• Mongin et al. 2012 (PMID:21476993): the variant was detected in 4 of 275 healthy control subjects and classified as a polymorphism, an observation already accounted for under BS1/BA1 frequency reasoning.
• Lejeune et al. 2006 (PMID:16941501): the variant was found in a single 18-year-old patient with 30 adenomatous polyps and reported as likely a polymorphism, illustrating that carrier status is not tightly coupled to the expected phenotype.

Still missing

• BS2's applicability depends on a penetrance judgement for AXIN2-related oligodontia-colorectal cancer predisposition (incomplete, adult-onset) rather than on a numerical threshold; that disease-model statement was not sourced to a gene-specific framework in this case.
• If a reviewer treated the disorder as fully penetrant and early-onset, the 4 healthy adult carriers reported by Mongin et al. 2012 and the 165 gnomAD v4.1 homozygotes would instead support BS2 at strong.

Sources

Reasoning

Framework: no ClinGen CSPEC or gene-specific criteria specification exists for AXIN2 (framework_mode=generic_acmg), so generic ACMG/AMP 2015 BS3 applies (PMID:25741868): well-established functional studies must show no damaging effect on protein function or splicing. One exact-variant experiment exists. PMID:16941501 analysed AXIN2 transcripts by RT-PCR with primers within exons 3 and 7 in the single carrier patient (18-year-old with 30 adenomatous polyps) and found c.1235A>G and the co-inherited c.1530G>A present in cDNA 'at a similar proportion as observed in the genomic DNA, indicating no splicing alteration'; the authors wrote that the variants 'did not alter splicing' and were 'likely to be polymorphisms' with no functional effect. Read literally, a functional study showing no damaging effect on splicing is the kind of evidence BS3 contemplates, and this result points benign. It nevertheless falls short of the 'well-established functional study' standard: it is an n=1 blood-derived RNA analysis with no documented positive or negative control samples, no replication, and no quantitative effect size; and because the variant's consequence class is missense while the assay readout is transcript processing, a normal splice pattern cannot establish that the p.Asn412Ser protein retains normal function (the mechanism under consideration for AXIN2 in this case is loss of function, not missplicing). The authors' benign conclusion rests substantively on non-functional grounds - poor cross-species conservation of Asn412, a benign PolyPhen prediction, and control-population frequency - which are in-silico and population evidence assessed by other criterion groups, not functional-assay evidence. No validated AXIN2 protein-function assay (Wnt/beta-catenin reporter or protein-stability readout) has been performed for p.Asn412Ser. No strength calibration for BS3, or for a downgraded/supporting-strength BS3, is provided by a governing AXIN2 specification or by the supplied published-threshold block, so a calibrated downgrade could not be justified and BS3 is assessed at its default strong strength. At that strength BS3 is not met. The residual question - whether the negative transcript assay should be credited as supporting-strength BS3 evidence - is a genuine adjudication judgement and is flagged for human review.

Evidence considered

• PMID:16941501: RT-PCR and sequencing of AXIN2 transcripts from the carrier patient showed c.1235A>G and c.1530G>A present in cDNA at a similar proportion to genomic DNA, 'indicating no splicing alteration'; no mRNA-level abnormality was found for this allele.
• PMID:16941501 Discussion: 'these two variations did not alter splicing, and the missense mutation p.Asn412Ser did not concern a highly conserved amino-acid, suggesting no functional effect' - the primary basis for the authors' benign interpretation is conservation/Polyphen rather than assay data.
• PMID:16941501 methods context: 39 unrelated patients with multiple adenomas or colorectal cancer without APC mutation or MMR defect; AXIN2 screened by PCR and direct sequencing in 31 patients; variant also absent in 30 other patients and 50 healthy controls; RT-PCR on blood-derived RNA was used to test for splicing effects (n=1 carrier, no reported assay controls or replication).
• PMID:21476993: the same variant was found in 4/275 healthy control subjects (0.72%) and classified as a polymorphism on control-population frequency; this is population evidence, not a functional assay, and was not counted as BS3 support.
• PMID:25741868 (ACMG/AMP 2015) supplies the BS3 definition (well-established functional studies showing no damaging effect on protein function or splicing) and the 'well-established' assay standard applied here; it contains no AXIN2-specific or variant-specific data.
• No validated AXIN2 protein-function assay (Wnt/beta-catenin reporter, protein stability) was performed for p.Asn412Ser; the 2025 AXIN2 in vitro functional study (PMID:41361419) tested other variants (p.Thr693Met, p.Arg354Leufs*2) and does not mention c.1235A>G / p.Asn412Ser.
• In-silico outputs (REVEL 0.119; SpliceAI max delta 0.014) were not treated as functional evidence for BS3; they are assessed by the in-silico group.
• No strength calibration for BS3 or for a downgraded BS3 is available from an AXIN2 specification (no CSPEC; vcep_materials found=false) or from the supplied published-threshold block, so BS3 was assessed at the generic strong strength defined by ACMG/AMP 2015.

Still missing

• No validated AXIN2 functional assay with appropriate controls demonstrating preserved protein function for p.Asn412Ser; the available RT-PCR transcript analysis is a single-patient, uncontrolled observation that does not interrogate the missense consequence.
• Whether the PMID:16941501 negative splicing assay should be credited as downgraded (supporting-strength) BS3 evidence: no VCEP or published calibration for a downgraded BS3 is available in the governing framework or the supplied threshold block, so the default strong-strength assessment was retained and the question is flagged for human review.
• Assay control documentation (positive control sample with a known splice-altering variant, negative control, replication) is absent from PMID:16941501 for this variant.

Sources

Reasoning

BP2 has two independent branches: (a) the variant observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or (b) the variant observed in cis with a pathogenic variant in any inheritance pattern. Neither branch is satisfied for c.1235A>G. Branch (a): no pathogenic AXIN2 allele in trans has been reported for any carrier of this variant, and AXIN2-related disease is not a fully penetrant dominant condition - carriers of AXIN2 germline variants show a variable number of polyps and variable/absent ectodermal features, so the 'fully penetrant' premise does not hold; the variant also occurs in 4/275 healthy controls and in 41 (v2.1) and 165 (v4.1) reference-population homozygotes. Branch (b): the only variant reported in cis with c.1235A>G is c.1530G>A (p.Thr510Thr), annotated as c.[1235A>G (+) 1530G>A] in the index patient; the authors describe it as an 'apparently silent mutation' that did not alter splicing, list it among likely polymorphisms, and do not treat it as pathogenic. A cis partner that is not pathogenic cannot satisfy BP2. Because the criterion was assessable on both branches and neither is met, BP2 is not met rather than not applicable; its own benign-inheritance logic does not favor this variant.

Rule

BP2 (supporting, generic ACMG/AMP 2015): 'Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern' (Richards et al. 2015, PMID:25741868).

Evidence considered

• Generic ACMG/AMP 2015 definition applied under the generic fallback framework: BP2 = 'Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern'.
• PMID:16941501: the cis configuration is documented at c.[1235A>G (+) 1530G>A] / p.[Asn412Ser (+) Thr510Thr] in the 18-year-old patient with 30 adenomatous polyps; the paper's own text calls c.1530G>A an 'apparently silent mutation' and states that RT-PCR/sequencing of transcripts 'showed the presence of the two variants at a similar proportion as observed in the genomic DNA, indicating no splicing alteration', concluding the two variations 'are likely to be polymorphisms'. The cis partner is therefore not a pathogenic variant.
• PMID:16941501: no pathogenic AXIN2 variant was identified anywhere in the cohort ('No clearly pathogenic mutations (frameshift or nonsense mutations) were found in any of our patients'), so no trans-with-pathogenic-variant observation exists; the in-trans events proven by allele-specific PCR in this cohort were biallelic MUTYH genotypes.
• PMID:21476993: independent detection of c.1235A>G in 4/275 healthy control subjects (allele frequency 0.72%) with no accompanying pathogenic AXIN2 allele; the authors classify the variant as a polymorphism, and the carrier patient lacked the dental abnormalities described for AXIN2-related polyposis.
• ClinVar submissions for variation 136477 list germline origin and dominant/incompletely penetrant condition labels (oligodontia-cancer predisposition syndrome, AXIN2-related disorder, hereditary cancer-predisposing syndrome) and report no phase or zygosity observation; only these submission-level fields were consulted, not the aggregate classification.

Still missing

• No cis/trans phase data against a bona fide pathogenic AXIN2 variant exists for this variant; only the cis partner c.1530G>A (a silent, non-pathogenic change) is documented (PMID:16941501).
• No AXIN2-specific VCEP/ClinGen specification exists for this gene, so BP2 is applied with the generic ACMG/AMP 2015 definition.

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015 (no AXIN2 VCEP/CSpec or gene framework; generic_acmg_fallback). BP5 is met when the variant is 'found in a case with an alternate molecular basis for disease' (Richards et al. 2015, PMID:25741868) - a qualitative criterion with no numeric threshold, so no numeric operator applies and none may be borrowed from PS4's enrichment direction. The criterion requires a positive observation: a carrier of c.1235A>G whose disease is explained by a different molecular cause. No such observation exists in this case. (1) The present workup provides no proband-level information of any kind (no phenotype, no other variants, no indication of an alternate genetic diagnosis; prefetch.json contains only variant/gene-level fields, and its HCI prior lookup returned found=false), so there is no local basis on which to assert BP5. (2) In the published data, the only carrier with a documented, detailed workup (PMID:16941501, an 18-year-old with 30 adenomatous polyps and no family history of polyposis or colorectal cancer) was screened for APC and MMR defects as part of a cohort of 39 patients selected for absence of APC mutation or MMR defect, and was additionally evaluated for MUTYH; no alternate molecular basis for that patient's polyposis is reported, and the variant itself was judged 'likely to be polymorphisms'. (3) The independent cohort that detected the variant (PMID:21476993) reports it as a rare Wnt-pathway variant in unexplained polyposis and says nothing about an alternate cause for that carrier; its pathogenic findings in the cohort belonged to APC and BMPR1A, not to a carrier-specific alternate diagnosis for c.1235A>G. (4) No ClinVar submission for variation 136477 cites BP5 or reports an accompanying alternate molecular basis (the submission audit's criteria_found lists only a PP3-type in-silico signal in two benign submissions). BP5 is therefore not met; it remains flagged for human review only because the proband's own workup (APC/MUTYH status, any second causative variant) is not available, which limits how strongly the negative can be stated. No source's final classification was used to reach this verdict.

Rule

BP5 (supporting, generic ACMG/AMP 2015): 'Variant found in a case with an alternate molecular basis for disease' (Richards et al. 2015, PMID:25741868). No numeric threshold.

Evidence considered

• Generic ACMG/AMP 2015 definition applied under the generic fallback framework: BP5 = 'Variant found in a case with an alternate molecular basis for disease' (Richards et al. 2015, PMID:25741868). The criterion specifies no numeric threshold, so no numeric operator applies.
• PMID:16941501 (Lejeune et al., Hum Mutat 2006): the fully described carrier (18 years old, 30 adenomatous polyps, no family history of polyposis or colorectal cancer) came from a cohort of 39 unrelated patients selected for having no APC mutation and no MMR defect, with MUTYH additionally evaluated; the paper reports no alternate molecular basis for that patient's polyposis and states the two AXIN2 variations 'are likely to be polymorphisms'. This is the closest available test of BP5 and it is negative.
• PMID:21476993 (Mongin et al., Clin Genet 2012): c.1235A>G is reported as a rare AXIN2 variant within a cohort of unexplained polyposis in which the pathogenic findings were confined to APC (four mutations, one mosaic) and BMPR1A/SMAD4; no carrier-specific alternate diagnosis is attributed to the c.1235A>G carrier, and the variant was separately found in 4/275 healthy controls (0.72%) and called a polymorphism.
• ClinVar submission audit for variation 136477: no submission cites BP5, and no submission reports an alternate molecular basis for the variant; the only criterion-like signals extracted were PP3-type in-silico statements in two benign submissions (SCV000167137, SCV000698511).
• Case artifacts were searched for proband-level alternate-basis information (phenotype, other variants, other causative findings): none exists; prefetch.json contains normalize/revel/cspec/bayesdel/clinvar/pmid_metadata steps and an HCI prior lookup with found=false, with no clinical or second-variant data.
• No source's final classification (e.g. the ClinVar Benign aggregate) was used to infer BP5.

Still missing

• The proband's own clinical and genetic workup is not present in this case (no phenotype, no other variants, no indication of an alternate diagnosis in any case artifact), so a local BP5 observation cannot be evaluated; the verdict rests on published carrier data.
• The published carrier reports do not state the APC/MUTYH genotype of the specific carriers of c.1235A>G beyond the fact that the cohorts were selected/negative for APC and MMR defects (PMID:16941501) and that no deleterious Wnt-pathway mutation was found (PMID:21476993); none reports a case in which the disease was attributed to a different gene.
• No AXIN2-specific VCEP/ClinGen specification exists for this gene, so BP5 is applied with the generic ACMG/AMP 2015 definition.

Sources

Reasoning

Governing framework: generic ACMG/AMP 2015 (no AXIN2 VCEP/CSpec or gene framework exists; cspec found=false; final_classification_framework found=false, generic_acmg_fallback). BP6 is the benign counterpart of PP5 and requires that the variant be 'reported as a benign polymorphism by a reputable source' - operationalised in this case as an exact-variant ClinVar expert-panel Benign/Likely benign classification (Richards et al., PMID:25741868). Like PP5 it is a qualitative provenance gate with no numeric threshold, so no numeric operator applies and none may be imported from another criterion in this group (in particular, BP6's mirror-image requirement does not license copying PS4's direction or any likelihood-ratio operator). The expert-panel gate fails: ClinVar variation 136477 has an empty expert_panels object ({}), the submission audit records expert_panel_submissions = 0 of 20, and all 20 submissions are explicitly is_expert_panel = false. The record does carry many benign-directed lab submissions (12 Benign, 6 Likely benign, 1 benign; 2-star review status 'criteria provided, multiple submitters, no conflicts'), but per the governing rule these ordinary laboratory submissions - and the aggregate ClinVar label 'Benign/Likely benign' - must never trigger BP6. One benign submission (SCV000261762, Labcorp/Invitae) cites PMID:28492532 (Sherloc), which is a criteria-refinement methodology paper rather than an expert-panel classification of this variant. BP6 is therefore not met; the benign weight for this variant comes from population frequency (BA1/BS1) and other evidence groups, not from this criterion.

Rule

BP6 (supporting, generic ACMG/AMP 2015 / ClinGen SVI): 'Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation' (Richards et al. 2015, PMID:25741868), restricted here to an exact-variant ClinVar expert-panel Benign/Likely benign classification. No numeric threshold.

Evidence considered

• Generic ACMG/AMP 2015 definition applied under the generic fallback framework: BP6 = 'Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation' (Richards et al. 2015, PMID:25741868), restricted in this case to an exact-variant ClinVar expert-panel Benign/Likely benign classification. No numeric threshold applies to this criterion.
• ClinVar variation 136477 / VCV000136477, exact match to NM_004655.4:c.1235A>G: source_data.expert_panels = {} is empty, review status 'criteria provided, multiple submitters, no conflicts' (api review_stars = 2), number_of_submissions 20, number_of_submitters 20. A 2-star multiple-submitter record is not an expert-panel assertion (expert panels are the 3-star tier) and, per the case rule, aggregate labels never trigger BP6.
• Submission-level audit: expert_panel_submissions = 0; is_expert_panel = false on every audited submission (SCV001920466, SCV001931944, SCV002551275, SCV005911836, SCV006095874, SCV006311438, SCV007316471, SCV000167137, SCV000405708, SCV002806368, SCV001170681, SCV005255552, SCV002046701, SCV000698511, SCV001807586, SCV002036254, SCV004016235, SCV004756465, SCV001159232, SCV000261762). Benign-directed counts (12 Benign, 6 Likely benign, 1 benign clinical laboratories) were recorded as provenance only and were not used as BP6 evidence.
• Submission SCV000261762 (Labcorp Genetics, formerly Invitae) cites PMID:28492532 (Sherloc, a comprehensive refinement of the ACMG-AMP variant classification criteria) for its Benign call; that reference is a criteria-refinement methodology paper, not an expert-panel classification of this variant, and it was not retrieved as full text in this case, so it supplies no BP6-eligible expert assertion. No ClinVar submission's criterion list contains BP6.
• No source's final classification was used to infer BP6; the benign verdicts in the record are ordinary laboratory submissions, which this case's rule excludes.

Still missing

• No ClinGen/VCEP expert panel has classified this variant, so BP6's required expert-panel benign assertion does not exist and cannot be created from laboratory submissions; this gap is not closeable from any other source by definition.
• The ClinVar record's 2-star, multiple-submitter benign status (12 Benign / 6 Likely benign / 1 benign laboratories) was deliberately not used to satisfy BP6; if the task's rule were relaxed to allow aggregate non-expert benign labels, BP6 would be satisfied, so this verdict is rule-dependent.

Sources

Reasoning

PS2 (Richards et al. 2015, PMID:25741868) requires a de novo occurrence in a patient with the disease in whom both maternity and paternity are confirmed. No AXIN2-specific VCEP/CSPEC or gene-specific specification exists for this case, so the generic definition governs. No reviewed source reports NM_004655.4:c.1235A>G (NP_004646.3:p.(Asn412Ser)) as de novo, and no parental genotypes are reported for any carrier. Lejeune et al. (PMID:16941501) identified the variant in an 18-year-old patient with 30 adenomatous polyps and no family history of polyposis or colorectal cancer, reported no parental testing for this patient, and explicitly note the absence of parents' DNA samples in their confirmation analyses; the variant was absent from 30 other patients and 50 healthy control subjects but no relatives were genotyped. Mongin et al. (PMID:21476993) found the same variant in one of 25 unexplained adenomatous polyposis patients, again with no parental testing and no family study. The criterion's defining evidence, a confirmed de novo event in a proband, is therefore neither documented nor refuted at the proband level, so PS2 cannot be assessed for this variant.

Rule

Generic ACMG/AMP PS2 (Richards et al. 2015, PMID:25741868): de novo occurrence in a patient with the disease with maternity and paternity confirmed. No applicable AXIN2-specific specification was available.

Evidence considered

• Lejeune et al. (PMID:16941501): c.1235A>G (p.Asn412Ser) was found in a single 18-year-old patient (Li-10 in text, Li-9 in Table 2) with 30 adenomatous polyps and no family history of polyposis or colorectal cancer; the variant was absent from 30 other patients and 50 healthy control subjects. No parental genotypes are given for this patient, and the paper states 'In absence of parents DNA samples' for its confirmation work. No de novo assertion is made.
• Mongin et al. (PMID:21476993): the same variant was found in one of 25 patients with unexplained adenomatous polyposis; the carrier patient is described as lacking the dental abnormalities associated with AXIN2-related polyposis. No parental testing, trio analysis, or de novo statement is provided.
• PMID:25741868 defines PS2 as a de novo occurrence with confirmed maternity and paternity in a patient with the disease; no AXIN2-specific VCEP specification was available, so this generic definition was used.

Still missing

• Parental genotypes (both biological parents) for any proband carrying c.1235A>G.
• Confirmation of maternity and paternity for any parent-proband trio.
• A clinical record describing the variant as de novo in a patient with an AXIN2-related phenotype.

Sources

Reasoning

PM6 (Richards et al. 2015, PMID:25741868) applies to an assumed de novo occurrence without confirmation of maternity and paternity, which still requires a documented apparently de novo observation (variant present in the proband and absent from parents, with parentage unverified). No AXIN2-specific VCEP/CSPEC specification exists for this case, so the generic definition governs. Neither variant-specific publication describes c.1235A>G as de novo. Lejeune et al. (PMID:16941501) report the variant in one 18-year-old patient with 30 adenomatous polyps and no family history of polyposis or colorectal cancer, and the variant was absent from 30 other patients and 50 healthy control subjects, but no parent was genotyped, so absence from the parents is not established; the authors instead call both exon 5 changes 'likely to be polymorphisms'. Mongin et al. (PMID:21476993) classify the variant as a polymorphism detected in 4 of 275 healthy control subjects and provide no parental or family data for the carrier. With no apparently de novo observation and no parental genotype data at all, PM6 cannot be assessed.

Rule

Generic ACMG/AMP PM6 (Richards et al. 2015, PMID:25741868): assumed de novo without confirmation of maternity and paternity. No applicable AXIN2-specific specification was available.

Evidence considered

• Lejeune et al. (PMID:16941501): the single carrier was an 18-year-old with 30 adenomatous polyps, no family history of polyposis or colorectal cancer, and no parental genotyping; the authors state the two exon 5 variants 'are likely to be polymorphisms', i.e. no apparently de novo event is claimed.
• Mongin et al. (PMID:21476993): the carrier among 25 unexplained adenomatous polyposis patients had no reported parental testing, and the variant was additionally found in 4 of 275 healthy control subjects.
• PMID:25741868 defines PM6 as an assumed de novo occurrence without confirmation of maternity and paternity; no AXIN2-specific VCEP specification was available, so this generic definition was used.

Still missing

• A proband carrying the variant whose parents have been genotyped and found negative, with parentage unconfirmed.
• Any clinical report documenting the variant as apparently de novo.
• Parental genotype results in the single published carrier family.

Sources

Reasoning

PP1 (Richards et al. 2015, PMID:25741868) requires co-segregation of the variant with disease in multiple affected family members, which needs a pedigree with genotyped affected and unaffected relatives and a defined number of informative meioses. No AXIN2-specific VCEP/CSPEC specification exists for this case, so the generic definition governs. No such pedigree exists for c.1235A>G: the only carrier reported by Lejeune et al. (PMID:16941501) was an 18-year-old with 30 adenomatous polyps and explicitly no family history of polyposis or colorectal cancer, and no relatives were genotyped; the variant was absent from 30 unrelated patients and 50 healthy control subjects, which is a case-control observation rather than a family segregation result. Mongin et al. (PMID:21476993) report the variant in one of 25 unexplained adenomatous polyposis patients and supply no family structure, relative genotypes, or meioses for that carrier. With zero affected relatives carrying the variant with phenotype data and zero informative meioses, segregation cannot be evaluated and PP1 is not assessable.

Rule

Generic ACMG/AMP PP1 (Richards et al. 2015, PMID:25741868): co-segregation with disease in multiple affected family members. No applicable AXIN2-specific specification was available.

Evidence considered

• Lejeune et al. (PMID:16941501): the single carrier had no family history of polyposis or colorectal cancer; no relatives were tested and no segregation analysis was performed. The variant's absence from 30 other unrelated patients and 50 healthy control subjects is a case-control count, not co-segregation.
• Mongin et al. (PMID:21476993): the carrier among 25 unexplained adenomatous polyposis patients has no reported pedigree, relative genotypes, or meioses; the authors used the variant's frequency in controls to classify it as a polymorphism.
• PMID:25741868 defines PP1 as co-segregation with disease in multiple affected family members; no AXIN2-specific VCEP specification or segregation data was available, so this generic definition was used.

Still missing

• A pedigree with at least two clinically characterised affected relatives.
• Genotypes for affected and unaffected informative relatives.
• Number of informative meioses and phenotype ascertainment details.

Sources

Reasoning

Governing framework: no AXIN2 VCEP/CSpec or gene-specific framework exists (cspec found=false; final_classification_framework found=false, generic_acmg_fallback), so the generic ACMG/AMP 2015 criterion applies: PP4 is met when 'the patient's phenotype or family history is highly specific for a disease with a single genetic etiology' (Richards et al. 2015, PMID:25741868), which supplies no numeric threshold and therefore no numeric operator to evaluate. PP4's required input is proband-level clinical information - HPO terms or an equivalent structured phenotype plus family history - and no such information exists anywhere in this case. The case artifacts contain only gene/variant-level data: review_manifest.json and case_summary.json record the variant, gene and framework only; prefetch.json records normalisation, REVEL, BayesDef, ClinVar and PMID metadata but no clinical indication or phenotype fields, and its HCI prior lookup returned found=false (reason 'gene_not_supported'); no HPO term (HP:0xxxxxx) appears in any case file, and no clinical-histories file was retrieved. Without a patient phenotype there is nothing to test against AXIN2's disease associations (oligodontia-colorectal cancer predisposition, attenuated adenomatous polyposis), so PP4 is not_assessed rather than not met: this is a missing-input situation, not a finding that the phenotype is non-specific. Confidence is correspondingly low, and reviewer input of the proband's phenotype/family history is required before PP4 can be scored. Note that this determination is not based on, and must not be replaced by, any other criterion's verdict.

Rule

PP4 (supporting, generic ACMG/AMP 2015): 'Patient's phenotype or family history is highly specific for a disease with a single genetic etiology' (Richards et al. 2015, PMID:25741868). No numeric threshold.

Evidence considered

• Generic ACMG/AMP 2015 definition applied under the generic fallback framework (no CSpec, no gene-specific framework for AXIN2): PP4 = 'Patient's phenotype or family history is highly specific for a disease with a single genetic etiology' (Richards et al. 2015, PMID:25741868). The generic definition specifies no numeric threshold, so no numeric operator applies to this criterion.
• Absence of proband phenotype data verified across the case artifacts: review_manifest.json and case_summary.json carry only variant/gene/framework fields; prefetch.json has steps for normalize, hci_prior (found=false, reason 'gene_not_supported'), revel, cspec (found=false), bayesdel, clinvar and pmid_metadata, with no phenotype/indication/family-history field; a search of the case directory for structured phenotype terms (HP:0) returned no hits.
• The phenotypic descriptions available in the retrieved literature concern other AXIN2 variants and their carriers (e.g. oligodontia/ectodermal features in AXIN2 truncating-variant families, tooth agenesis cohorts), not the present proband; gene-level disease associations cannot substitute for PP4's required patient-specific phenotype.
• No source's final classification was used to infer PP4.

Still missing

• No proband phenotype (HPO terms or clinical description) and no family history are present in any case artifact; PP4 requires exactly this information and cannot be scored without it.
• No AXIN2-specific VCEP/ClinGen specification exists for this gene, so PP4 would be applied with the generic ACMG/AMP 2015 definition if the phenotype were supplied.
• The AXIN2 disease spectrum relevant to PP4 specificity (oligodontia-colorectal cancer predisposition syndrome vs attenuated/multiple adenomatous polyposis vs isolated tooth agenesis) is heterogeneous and incompletely penetrant; even with phenotype data, whether a given presentation is 'highly specific for a disease with a single genetic etiology' would need explicit justification.

Sources

Reasoning

BS4 (Richards et al. 2015, PMID:25741868) requires lack of segregation in affected members of a family, i.e. a pedigree in which the variant is absent from clinically affected relatives (or clearly present in unaffected relatives given the expected penetrance). No AXIN2-specific VCEP/CSPEC specification exists for this case, so the generic definition governs. No pedigree exists for c.1235A>G: the only family-level information is negative, since the single carrier reported by Lejeune et al. (PMID:16941501) had no family history of polyposis or colorectal cancer and no relatives were genotyped. Mongin et al. (PMID:21476993) detected the variant in 4 of 275 healthy control subjects (allele frequency 0.72%) and gnomAD v4.1 carries it at 0.79% (12,588/1,586,748 alleles) with 165 homozygotes, but these are population carriers rather than a within-family non-segregation analysis. Because the family-based evidence BS4 requires is entirely absent, the criterion cannot be assessed here even though the population data are consistent with non-association.

Rule

Generic ACMG/AMP BS4 (Richards et al. 2015, PMID:25741868): lack of segregation in affected members of a family. No applicable AXIN2-specific specification was available.

Evidence considered

• Lejeune et al. (PMID:16941501): the only carrier reported had no family history of polyposis or colorectal cancer; no relatives were genotyped, so no family-based non-segregation analysis is possible.
• Mongin et al. (PMID:21476993): the variant was identified in 4 of 275 healthy control subjects (allele frequency 0.72%) and the authors classed it as a polymorphism; unaffected carriers at population level do not by themselves establish lack of segregation in a family.
• gnomAD v4.1 (gnomad_v4): the variant is present at 0.79% (12,588/1,586,748 alleles, 165 homozygotes), confirming that carriers of this allele occur in the general population without the AXIN2-associated phenotype, but this is population data rather than pedigree data.
• PMID:25741868 defines BS4 as lack of segregation in affected members of a family; no AXIN2-specific VCEP specification was available, so this generic definition was used.

Still missing

• Genotypes of clinically affected relatives (to show non-segregation).
• Reliable phenotype assessment of any genotyped relatives, with age-related penetrance considered.
• A pedigree of sufficient size to establish lack of segregation rather than absence of data.

Sources