CTNNB1 c.134C>T, p.Ser45Phe

Reasoning

The observed missense variant is CTNNB1 p.Ser45Phe (c.134C>T). The same amino-acid change is reported in hepatocellular carcinoma tumors and in desmoid tumor cells, and a functional study reports approximately 22-fold TOP-flash and 17-fold GS-enhancer activation for S45F. The evidence is somatic/cancer-focused rather than a germline clinical assertion, so the pathogenicity context warrants human review; nevertheless, p.S45F is an established disease-associated activating variant and satisfies the generic same-amino-acid-change definition.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• Legoix et al. 1999, PMID:10435629, Table 1 and exon 3 mutation results: p.S45F was identified in four hepatocellular carcinoma tumors.
• Austinat et al. 2008, PMID:18282277, reporter-assay results: S45F produced approximately 22-fold TOP-flash and approximately 17-fold GS-5' enhancer activation.
• Colombo et al. 2017, PMID:26686699, cultured desmoid-cell study: p.S45F cells showed nuclear beta-catenin accumulation and increased Axin2, Cyclin D1, and c-Myc expression.
• ClinVar variation 17588: exact NM_001904.4:c.134C>T (p.Ser45Phe) record includes pathogenic somatic submissions, but the record has no assertion criteria and zero review stars.

Still missing

• The available pathogenicity evidence is primarily somatic/cancer-focused and lacks a strong germline assertion-criteria submission.

Sources

Reasoning

NM_001904.4:c.134C>T (p.Ser45Phe) has variant-specific functional evidence in two independent studies. In cultured desmoid tumor cells, p.Ser45Phe showed strong nuclear beta-catenin accumulation and significantly increased expression of Wnt/beta-catenin target genes Axin2, Cyclin D1, and c-Myc versus controls, with increases of 8.2-fold, 9.5-fold, and 4.9-fold, respectively (PMID:26686699). In transfected HuH7 cells, p.Ser45Phe produced approximately 22-fold activation of the TOP-flash reporter and approximately 17-fold activation of the GS enhancer reporter, the strongest activation among tested mutants (PMID:18282277). These assays are biologically relevant and include comparator controls, but they rely on cultured or transiently transfected systems and are not shown to be validated, reproducible clinical-laboratory assays; the same study also found no increase in endogenous GS expression after p.Ser45Phe transfection. The evidence supports a damaging/gain-of-function effect, but the available information does not justify strong-level calibration, so PS3 is applied at supporting strength.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. ... Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.

Evidence considered

• Characteristics of cultured desmoid cells with different CTNNB1 mutation status (PMID:26686699): cultured p.Ser45Phe-mutant desmoid cells showed strong nuclear beta-catenin accumulation and significantly increased Axin2, Cyclin D1, and c-Myc expression versus human skin fibroblast controls; reported increases were 8.2-fold, 9.5-fold, and 4.9-fold, respectively.
• Correlation between beta-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma (PMID:18282277): transiently transfected p.Ser45Phe produced the strongest reporter activation, approximately 22-fold for TOP-flash and approximately 17-fold for the GS 5-prime enhancer, but did not increase endogenous GS expression in the tested cell systems.
• Strength calibration: the findings are biologically relevant and variant-specific but are not demonstrated to be validated and reproducible in a clinical diagnostic laboratory; supporting rather than strong PS3 is therefore used.

Still missing

• No validated clinical diagnostic-laboratory assay or explicit reproducibility data were identified.
• The functional studies use cultured tumor cells or transient overexpression and include discordant endogenous GS-expression results.

Sources

Reasoning

p.Ser45Phe lies at CTNNB1 residue S45 in the exon 3 degron region, a recurrent mutational hotspot. The Cancer Hotspots result identifies S45 as a statistically significant hotspot, and the literature describes recurrent S45 mutations and altered beta-catenin stability/function. vcep_materials.json contains no CTNNB1 domain-table entry, so there is no authoritative VCEP domain list to apply; the hotspot path supports PM1, although the hotspot evidence is cancer-derived and the parsed result is partial.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• Cancer Hotspots result for CTNNB1 S45: the queried residue is identified as a statistically significant hotspot; the source notes that its row was only partially parsed.
• Liu et al. 2019, PMID:30699286, Table 2 and hotspot discussion: S45F is recurrently represented in cancer datasets and S45F has protein-stability and target-gene differences from wild-type beta-catenin.
• Austinat et al. 2008, PMID:18282277: S45F produced the strongest reporter activation among the tested beta-catenin mutations.

Still missing

• The hotspot extraction is flagged as partial and requires screenshot-level confirmation.
• No explicit benign-variation analysis for the hotspot was available.

Sources

Reasoning

PM2 is met at supporting strength. NM_001904.4:c.134C>T (CTNNB1 p.Ser45Phe) was absent from gnomAD v2.1 and gnomAD v4.1, and the available non-cancer subsets also reported it as absent. This satisfies the supplied generic PM2 supporting threshold of <=0.0001; the variant is a single-nucleotide substitution, so the indel calling caveat does not apply.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 (exome) reported the exact variant as absent.
• gnomAD v4.1 reported the exact variant as absent.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer entries also reported the exact variant as absent.
• Applied the supplied ClinGen SVI PM2 supporting threshold of <=0.0001 (ClinGen SVI 2020; the case supplies PMID:25741868 for this calibration).

Sources

Reasoning

The available CTNNB1 germline disease context states that most pathogenic CTNNB1 syndrome variants are nonsense or frameshift variants generating premature termination codons, supporting a predominantly truncating disease mechanism. Missense CTNNB1 disease variants are also reported, so this supporting benign criterion should be reviewed in the context of the relevant phenotype and disease mechanism.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• pvs1_gene_context.json: the germline CTNNB1 disease review records that most patients carry pathogenic nonsense or frameshift variants generating premature termination codons, while also noting that missense variants can be associated with disease.

Still missing

• A systematic disease-cohort tally comparing truncating and missense pathogenic variants would improve confidence.

Sources

Reasoning

The variant is a missense substitution, so the governing generic path is REVEL only; SpliceAI is not used for PP3/BP4 in this situation. REVEL is 0.353, below the generic PP3 supporting threshold of >= 0.644 calibrated by ClinGen SVI (Pejaver et al. 2022, PMID:36413997), and therefore does not meet PP3 at any specified strength. BayesDel is not used because no generic ACMG-strength calibration is established. The ACMG/AMP definition requires multiple lines of computational evidence supporting a deleterious effect, but the applicable calibrated REVEL result does not provide that support.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• Local REVEL predictor lookup for NM_001904.4:c.134C>T (CTNNB1 p.Ser45Phe): REVEL score 0.353.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997): generic PP3 supporting threshold >= 0.644; the score 0.353 is below threshold.
• SpliceAI result was not counted because this is a missense variant and the governing generic path specifies REVEL only; BayesDel was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

ClinVar variation 17588 is an exact-variant record, but it has no expert-panel submission and its available pathogenic assertion is an ordinary OMIM submission with no assertion criteria, somatic origin, and no clinical-testing status. Therefore the required reputable exact-variant expert-panel classification supporting pathogenicity is absent; the record cannot trigger PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 17588, exact variant NM_001904.4(CTNNB1):c.134C>T (p.Ser45Phe): expert_panel_submissions = 0; the OMIM Pathogenic submission has no assertion criteria and somatic origin.
• The exact-variant ClinVar record is not an expert-panel classification and therefore does not satisfy the criterion's required source type.

Sources

Reasoning

BA1 is not met. NM_001904.4:c.134C>T (CTNNB1 p.Ser45Phe) was absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer subsets, so its observed allele frequency is 0 and does not reach the generic stand-alone threshold of >=0.05. The variant is a single-nucleotide substitution, so the indel calling caveat does not apply.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 (exome) reported the exact variant as absent.
• gnomAD v4.1 reported the exact variant as absent.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer entries also reported the exact variant as absent.
• Applied generic ACMG/AMP stand-alone benign threshold >=0.05, supplied for this case as ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS1 is not met. The exact variant was absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer subsets, so no elevated allele frequency above the disorder-expected frequency was observed. A maximum-credible-frequency calculation is not needed to reject BS1 because the observed frequency is zero; the generic 1% default is likewise not reached.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v2.1 (exome) reported the exact variant as absent.
• gnomAD v4.1 reported the exact variant as absent.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer entries also reported the exact variant as absent.
• Applied the generic BS1 default threshold of >=0.01 supplied for this case; the observed frequency is 0.

Sources

Reasoning

BS2 is not met. The available population evidence reports the exact variant as absent from gnomAD v2.1 and v4.1, and provides no observation of it in a healthy adult as a heterozygote, homozygote, or hemizygote. No healthy-adult carrier observation or documented full-penetrance early-onset context was supplied.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v2.1 (exome) reported the exact variant as absent and therefore provided no healthy-adult carrier observation.
• gnomAD v4.1 reported the exact variant as absent and therefore provided no healthy-adult carrier observation.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer entries also reported the exact variant as absent.

Sources

Reasoning

No well-established functional study shows that p.Ser45Phe has no damaging effect on CTNNB1 protein function or splicing. Instead, the available variant-specific assays report increased beta-catenin nuclear accumulation, increased Wnt-target gene expression, and increased reporter activity, which are consistent with an activating functional effect. The absence of increased endogenous GS expression in one experimental context does not establish preserved normal function and is insufficient to support BS3.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• Characteristics of cultured desmoid cells with different CTNNB1 mutation status (PMID:26686699): p.Ser45Phe-mutant cells showed strong nuclear beta-catenin accumulation and increased expression of Axin2, Cyclin D1, and c-Myc.
• Correlation between beta-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma (PMID:18282277): p.Ser45Phe produced approximately 22-fold TOP-flash and approximately 17-fold GS-enhancer reporter activation; lack of endogenous GS induction in some cells does not demonstrate absence of damaging effect or normal protein function.

Sources

Reasoning

The variant is a missense substitution, so the governing generic path is REVEL only; SpliceAI is not used for PP3/BP4 in this situation. REVEL is 0.353, above the generic BP4 supporting threshold of <= 0.29, so it does not support BP4. The result is a gray-zone REVEL value: it does not meet BP4 and does not permit substituting the SpliceAI score. BayesDel is not used because no generic ACMG-strength calibration is established. The ACMG/AMP definition requires multiple lines of computational evidence suggesting no impact, which is not provided by the applicable REVEL result.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• Local REVEL predictor lookup for NM_001904.4:c.134C>T (CTNNB1 p.Ser45Phe): REVEL score 0.353.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997): generic BP4 supporting threshold <= 0.29; the score 0.353 exceeds threshold.
• SpliceAI max delta 0.003 was not counted because this is a missense variant and the governing generic path specifies REVEL only; BayesDel was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

ClinVar variation 17588 is an exact-variant record, but it has no expert-panel submission and no qualifying expert-panel benign or likely-benign classification. Ordinary submissions, aggregate labels, and non-expert assertions are excluded from BP6, so this record cannot trigger BP6.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 17588, exact variant NM_001904.4(CTNNB1):c.134C>T (p.Ser45Phe): expert_panel_submissions = 0 and no benign or likely-benign expert-panel classification is present.
• Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions were not used for BP6.

Sources

Reasoning

PS2 cannot be assessed. The available case evidence does not document a clinically affected proband with CTNNB1 c.134C>T, absence of family history, or confirmation of both maternity and paternity. The available exact-variant literature and ClinVar trail concern somatic tumors rather than a confirmed germline de novo occurrence, so the PS2 definition is not satisfied or refuted.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• The exact-variant evidence available for CTNNB1 c.134C>T/p.Ser45Phe is from somatic cancer reports and does not document a germline proband-parent de novo observation.
• No source provides both maternity and paternity confirmation for this variant.

Still missing

• Affected proband phenotype and diagnosis are not documented for this variant.
• Family history is not documented.
• Parental testing with confirmed maternity and paternity is not documented.
• Germline origin of the variant is not documented.

Reasoning

The exact CTNNB1 p.Ser45Phe variant is reported in affected tumor cohorts, including 4 of 119 hepatocellular carcinomas in PMID:10435629, but the reviewed studies do not provide an unaffected/population control frequency or a case-control odds ratio and 95% confidence interval. PMID:10435629 used matched non-tumor liver tissue for somatic confirmation, not an independent unaffected control cohort; PMID:18282277 and PMID:26686699 likewise report affected samples without controls. The governing operator is >5.0 for the odds ratio or relative risk, with a confidence interval that does not include 1.0; the required inequality cannot be written because no OR/RR and confidence interval are available. The literature_pass.json packet's ai_classified_papers contains no paper classified as PS4; its PS4 index leads were not supported by extractable case-control data in the available evidence.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• Legoix et al. 1999, PMID:10435629, Table 1 and Results: p.S45F was found in 4 of 119 hepatocellular carcinomas; matched non-tumor liver tissue was used to assess somatic status, not as an independent unaffected control cohort.
• Austinat et al. 2008, PMID:18282277: S45F was identified in a hepatocellular carcinoma cohort, but no independent control frequency, OR/RR, or 95% CI was reported.
• Zhang et al. 2016, PMID:26686699: p.S45F was studied in cultured cells from three desmoid tumors; this is functional evidence, not a case-control prevalence comparison.
• gnomAD v2.1 and v4.1 report the variant absent, but no affected-case frequency or matched-ancestry denominator is available for an OR/RR calculation.
• Exact governing threshold: 'Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.' No numerical substitution is possible because OR/RR and its confidence interval are unavailable.

Still missing

• A case-control cohort or matched-ancestry control frequency for exact CTNNB1 c.134C>T/p.Ser45Phe is required.
• An odds ratio or relative risk with a 95% confidence interval is required; no valid inequality against >5.0 can be formed from the available data.

Sources

Reasoning

PM3 cannot be assessed because the available evidence contains no established recessive-disease context and no parent or offspring testing demonstrating that NM_001904.4:c.134C>T is in trans with a pathogenic variant.

Rule

For recessive disorders, detected in trans with a pathogenic variant

Evidence considered

• The case evidence reports no family-segregation, parent/offspring testing, phase determination, or affected-proband observation relevant to PM3.

Still missing

• Disease inheritance mode is not established for this assessment.
• No parent or offspring testing is reported to determine phase.
• No affected-proband observation with a confirmed pathogenic variant in trans is reported.

Reasoning

The variant is a missense substitution at residue 45, so PM5 is applicable in principle, but the available case evidence does not establish a different missense substitution at Ser45 as pathogenic. The collected comparator file contains no candidates, and no additional same-residue pathogenic comparator could be verified from the available extracted evidence.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• pm5_candidates.json: zero same-residue candidates and zero candidates classified as expert-panel or non-expert-majority pathogenic/likely pathogenic.

Still missing

• A verified pathogenic alternate missense change at residue Ser45 is needed.

Sources

Reasoning

PM6 cannot be assessed. No qualifying assumed-de-novo occurrence is reported for CTNNB1 c.134C>T: the available evidence does not identify an affected proband or parental testing showing that the variant is absent from the parents. The available exact-variant reports are somatic cancer observations and do not establish a germline de novo event.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• The exact-variant reports available in this case describe somatic tumors, not a germline patient with parental testing.
• No parental testing result is available showing absence of the variant in either parent.

Still missing

• Affected proband phenotype and diagnosis are not documented for this variant.
• Parental genotypes are not documented.
• Germline origin and presumed de novo status are not documented.

Reasoning

PP1 cannot be assessed. No affected family members, informative meioses, familial genotypes, or phenotype-consistent segregation data are documented for CTNNB1 c.134C>T. The available literature concerns sporadic somatic tumors and provides no family co-segregation analysis.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... Note: May be used as stronger evidence with increasing segregation data.

Evidence considered

• The available exact-variant literature reports sporadic somatic cancer findings and does not provide family co-segregation data.
• No family pedigree or genotype-phenotype segregation analysis is available for this variant.

Still missing

• No affected relatives are documented.
• No familial genotypes are documented.
• No phenotype-consistent segregation or informative meioses are documented.

Reasoning

CTNNB1 has documented disease-associated missense variation, but the available case evidence does not provide the gene-level low rate of benign missense variation required by PP2, nor a validated quantitative benign-missense assessment. The variant's absence from gnomAD does not establish the gene-level PP2 premise.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• gnomAD v2.1 and v4.1: the observed variant is absent, but this is variant-level evidence and does not establish CTNNB1's gene-level benign missense rate.
• Rossetti et al. 2021, PMID:33350591: the literature packet identifies CTNNB1 missense variants as associated with disease, but it does not provide the required benign-missense-rate metric for PP2.

Still missing

• A validated gene-level demonstration of low benign missense variation is required.

Sources

Reasoning

The available case materials identify the molecular variant but do not provide a patient phenotype or family history that can be judged highly specific for a disease with a single genetic etiology. Cancer-cohort publications involving this somatic variant do not establish a specific germline clinical phenotype for this case.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• Case materials provide variant normalization and somatic cancer literature but no individual-level phenotype or family-history description for this case.

Still missing

• Detailed patient phenotype, diagnosis, and family history are required.

Reasoning

BS4 cannot be assessed. No affected family members carrying or lacking CTNNB1 c.134C>T are documented, so there is no informative family segregation or non-segregation result. The available exact-variant evidence is from somatic tumors and does not constitute a family-based lack-of-segregation observation.

Rule

Lack of segregation in affected members of a family. ... Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals.

Evidence considered

• No family-based non-segregation observation is available for CTNNB1 c.134C>T.
• The available exact-variant reports concern somatic tumors and do not evaluate segregation in affected relatives.

Still missing

• No affected family members are documented.
• No familial variant testing results are documented.
• No assessment of phenocopies or multiple pathogenic variants in the family is possible.

Reasoning

BP2 cannot be assessed because the available evidence contains no affected-proband or family observation showing NM_001904.4:c.134C>T in trans with a pathogenic variant, and no evidence establishes it in cis with a pathogenic variant.

Rule

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.

Evidence considered

• The case evidence reports no phase, family-segregation, parent/offspring testing, or affected-proband observation demonstrating a pathogenic variant in trans or cis with this variant.

Still missing

• No parent or offspring testing is reported to determine phase.
• No affected-proband observation with a confirmed pathogenic variant in trans is reported.
• No observation of this variant in cis with a pathogenic variant is reported.

Reasoning

The available evidence does not document an individual case in which this variant co-occurs with an established alternate molecular basis for the disease. Somatic tumor reports and the ClinVar record do not provide the required case-level alternate molecular diagnosis for this case.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• Reviewed case evidence and variant-specific publications report CTNNB1 p.Ser45Phe in tumors or functional systems but do not document an alternate molecular basis in an individual case relevant to this assessment.

Still missing

• Case-level molecular findings demonstrating an alternate molecular basis for the relevant disease are required.

Sources