CTNNB1 c.121A>G, p.Thr41Ala

Reasoning

The queried variant is CTNNB1 p.Thr41Ala (T41A). The identical amino-acid change was previously reported in multiple independent tumor studies, including adrenocortical carcinoma, primary pigmented nodular adrenocortical disease, and hepatocellular carcinoma. A functional reporter assay also tested T41A and found activation of beta-catenin signaling comparable to recurrent exon 3 activating mutants. These are exact same-amino-acid observations rather than merely alternate substitutions at the residue, and the available reports support a pathogenic/oncogenic effect rather than a splice-mediated effect.

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

• Mutations of beta-catenin in adrenocortical tumors, Table 2, patient 38: CTNNB1 T41A was reported with diffuse beta-catenin accumulation; tumor and leukocyte sequencing supported a somatic alteration.
• Detection of somatic beta-catenin mutations in primary pigmented nodular adrenocortical disease, Results Patient 12/Table 1: an ACC-to-GCC missense change produced T41A in an adrenal adenoma and was confirmed by cDNA sequencing.
• Beta-catenin mutations in hepatocellular carcinoma, Table 1: T41A was reported in three hepatocellular carcinoma tumors, with somatic status assessed using matched non-tumoral tissue.
• Genomic profiling of hepatocellular adenomas, Figure 4D and legend: T41A was directly tested in a CTNNB1-driven TOP-FLASH luciferase assay and activated beta-catenin signaling more strongly than K335I and N387K.

Sources

Reasoning

PS3 is met at supporting strength. PMID:24735922 directly tested CTNNB1 p.T41A (the queried c.121A>G variant) in Huh7 cells using a CTNNB1-driven TOP-FLASH luciferase reporter. The study reports that T41A activated beta-catenin more strongly than the comparator HCA-associated mutants K335I and N387K; mutants were tested in triplicate cotransfections. This is a biologically relevant cell-based functional assay with an appropriate reporter and comparator variants, but the available evidence does not establish clinical diagnostic-laboratory validation, reproducibility across independent assay systems, or a quantitative assay threshold supporting moderate or strong calibration.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Genomic profiling of hepatocellular adenomas reveals recurrent FRK-activating mutations and the mechanisms of malignant transformation, Results: Figure 4D and legend; CTNNB1 T41A was tested in Huh7 cells with a CTNNB1-driven TOP-FLASH luciferase reporter.
• The study states: "We further showed that K335I and N387K mutants were able to activate b-catenin in a hepatocellular cell line, with a lower activity than the S33Y, T41A, and S45P mutants located within exon 3." The extracted source text identifies the comparator as N387K in the finding and assay description; the direct quote is preserved verbatim from the extract.
• The assay context reports triplicate cotransfections, supporting reproducibility within the experiment, but no clinical-laboratory validation or cross-platform replication is documented.

Still missing

• No evidence of validation in a clinical diagnostic laboratory setting.
• No independent assay system or quantitative result threshold was provided for strength escalation.

Sources

Reasoning

PM2 is met at supporting strength. NM_001904.4:c.121A>G (CTNNB1 p.Thr41Ala) is reported absent from gnomAD v2.1 and gnomAD v4.1. Under the supplied generic ClinGen SVI default, absence from these control datasets satisfies the PM2 population observation and is assigned supporting strength; the non-cancer subsets are also reported absent, but no governing VCEP requires their use.

Rule

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v2.1 (exome) reports the variant as absent.
• gnomAD v4.1 (exome) reports the variant as absent.
• The supplied generic PM2 calibration is <=0.0001 and downgrades PM2 to supporting strength; absence is below this threshold.
• The available non-cancer entries also report absence, but generic ACMG/AMP rules govern and do not require substitution of non-cancer subsets for the default all-comers datasets.

Sources

Reasoning

The exact p.T41A variant is reported in somatic adrenocortical, PPNAD-associated adrenal, and hepatocellular carcinoma tumors, but the reviewed reports are tumor case series without unaffected or population control carriers for this exact variant. Leukocyte or adjacent-tissue testing in these studies establishes somatic status, not a case-control comparison. Therefore no valid case/control counts, odds ratio, or 95% confidence interval can be computed, and the ACMG PS4 requirement of OR/RR >5.0 with a confidence interval excluding 1.0 is not demonstrated.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: 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

• PMID:16140927, Table 2: p.T41A was reported in patient 38 with adrenocortical carcinoma; tumor/leukocyte sequencing showed the alteration was somatic, not a comparison with unaffected controls.
• PMID:18419788, Results Patient 12/Table 1: p.T41A occurred in one adrenal adenoma among 18 PPNAD patients and was absent from adjacent tissue; this is a somatic case observation, not a case-control prevalence comparison.
• PMID:10435629, Table 1: p.T41A was reported in three of 119 hepatocellular carcinoma tumors, with matched non-tumor tissue used to confirm somatic status; no unaffected-control carrier count was reported.
• PMID:24735922, Figure 4D/legend: p.T41A was included in a functional assay, but no exact-variant case-control or population prevalence data were reported.
• gnomAD v2.1 and v4.1 report the variant as absent, but no affected-case prevalence or matched case-control counts are available, so an OR and 95% CI were not computed.
• The literature_pass PS4 index was checked; ai_classified_papers is empty, and the indexed PS4 leads do not supply a qualifying exact-variant affected-versus-unaffected comparison in the available evidence.

Still missing

• A matched case-control dataset reporting exact p.T41A carrier counts in affected individuals and unaffected or population controls is unavailable.

Sources

Reasoning

The queried change is p.Thr41Ala. The reviewed literature reports T41A itself and other CTNNB1 exon 3 changes, but no different missense substitution at residue 41 was established as pathogenic in the available evidence. Therefore the required same-residue alternate pathogenic missense comparator for PM5 is absent.

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

• The reviewed reports identify CTNNB1 p.T41A at residue 41, not a different missense amino-acid substitution at residue 41.
• The PM5 comparator collection contains zero same-residue candidates and zero qualifying pathogenic comparator variants.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI is not used for PP3 in this situation. REVEL is 0.403, below the generic PP3 supporting threshold of >= 0.644 calibrated by Pejaver et al. 2022 (PMID:36413997), so PP3 is not met. BayesDel has no established generic ACMG-strength calibration and is not used.

Rule

PP3 (supporting): >= 0.644 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• NM_001904.4:c.121A>G is a missense variant, NP_001895.1:p.(Thr41Ala), so only REVEL applies under the generic missense path.
• Local REVEL lookup reports 0.403 for NM_001904.4:c.121A>G; this is below the PP3 supporting threshold of >= 0.644 from the supplied ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• The available BayesDel score is not used because no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

ClinVar contains exact-variant pathogenic or likely pathogenic submissions, but the submission audit reports zero expert-panel submissions. The available assertions are ordinary laboratory, OMIM literature-only, or other non-expert assertions; therefore no qualifying exact-variant ClinVar expert-panel pathogenic/likely pathogenic classification is present for 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 17580 exactly matches NM_001904.4:c.121A>G (p.Thr41Ala), but the submission audit reports expert_panel_submissions = 0.
• The pathogenic OMIM submissions have no assertion criteria and are not expert-panel classifications; the likely pathogenic submissions are single-laboratory clinical-testing assertions. These do not trigger PP5 under the specified rule.

Sources

Reasoning

BA1 is not met. NM_001904.4:c.121A>G (CTNNB1 p.Thr41Ala) is absent from gnomAD v2.1 and gnomAD v4.1, so no allele frequency reaches the generic stand-alone benign threshold of >=0.05. There is also no reported grpmax FAF or qualifying population with at least 2000 observed alleles that could support a BA1 call. The ClinVar record is not used to override the population result because its submissions include mixed somatic and germline contexts and no expert-panel consensus.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 (exome) reports the variant as absent.
• gnomAD v4.1 (exome) reports the variant as absent.
• No grpmax FAF or qualifying population denominator of at least 2000 observed alleles is available for this variant.

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with no grpmax FAF above the generic default threshold of >=0.01. No ancestry-specific elevated frequency, non-cancer-only frequency, or maximum-credible-allele-frequency calculation is available that would support the criterion. The available evidence therefore does not show an allele frequency greater than expected for the CTNNB1-related disorder.

Rule

BS1: Allele frequency is greater than expected for the disorder. ... BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v2.1 (exome) reports the variant as absent.
• gnomAD v4.1 (exome) reports the variant as absent.
• No filtering allele frequency (grpmax FAF) is reported, so no frequency exceeds the generic BS1 threshold of 0.01.
• No prevalence, penetrance, allelic-heterogeneity inputs, or ancestry-specific frequency are available for a Whiffin maximum-credible-frequency calculation.

Sources

Reasoning

BS2 is not met. The case evidence contains no observation of NM_001904.4:c.121A>G in a healthy adult individual as a heterozygote, homozygote, or hemizygote. gnomAD absence is population-frequency evidence and does not itself document a healthy adult carrier or establish the full-penetrance, early-onset applicability required by BS2.

Rule

BS2: 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

• The available gnomAD records report the variant as absent and provide no healthy-adult carrier observation.
• No case-level healthy-adult genotype, zygosity, age, or penetrance observation is available.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; the SpliceAI score cannot be substituted for an indeterminate missense REVEL result. REVEL is 0.403, which is above the generic BP4 supporting cutoff of <= 0.29 and therefore does not support BP4. The result is in the REVEL gray zone, so BP4 is not met rather than being supported by the SpliceAI max delta of 0.002.

Rule

BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• NM_001904.4:c.121A>G is a missense variant, so BP4 uses REVEL only; SpliceAI is not an alternative path for this variant.
• Local REVEL lookup reports 0.403 for NM_001904.4:c.121A>G; this exceeds the BP4 supporting cutoff of <= 0.29 from the supplied ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• SpliceAI max delta is 0.002, but it is not used for BP4 because the variant is missense and the applicable REVEL result is indeterminate.

Sources

Reasoning

ClinVar contains no qualifying exact-variant expert-panel benign or likely benign classification. The exact-variant record has zero expert-panel submissions, and the available ordinary submissions are pathogenic, likely pathogenic, uncertain, other, or somatic oncogenic assertions; none triggers 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 17580 exactly matches NM_001904.4:c.121A>G (p.Thr41Ala), but the submission audit reports expert_panel_submissions = 0.
• No exact-variant expert-panel benign or likely benign assertion is present; ordinary laboratory, literature-only, uncertain, other, and somatic assertions are excluded by the specified BP6 rule.

Sources

Reasoning

The available evidence does not provide a proband-parent genotype comparison, confirmed maternity and paternity, phenotype-confirmed disease status, or documentation of no family history. Therefore, de novo status cannot be assessed under PS2.

Rule

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

Evidence considered

• The case evidence contains no proband-parent segregation results or documented maternity and paternity confirmation.
• The available variant-specific literature concerns somatic CTNNB1 findings and does not establish de novo occurrence in a patient with the relevant disease.

Still missing

• Proband and parental variant testing results
• Confirmed maternity and paternity
• Phenotype-confirmed disease diagnosis
• Family-history documentation

Reasoning

CTNNB1 residue Thr41 lies in the exon 3 phosphorylation/degradation-regulatory region and is repeatedly described as an oncogenic codon; the queried variant is absent from gnomAD v2.1 and v4.1. However, the generic PM1 requirement also specifies absence of benign variation, and the available evidence does not establish that the relevant region lacks benign missense variation. The cancer-hotspot and COSMIC observations cannot independently close that gap under the governing instruction because no VCEP hotspot route applies.

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

• Functional correlates of mutations in beta-catenin exon 3 phosphorylation sites, abstract: mutations in exon 3 phosphorylation sites activate beta-catenin signaling, with Thr41 among the residues occurring more frequently in malignant tumors.
• Beta-catenin mutations in hepatocellular carcinoma, Table 1 and discussion: T41A occurs in the exon 3 GSK3 phosphorylation-regulatory region and is classified among activating/stabilizing exon 3 mutations.
• Genomic profiling of hepatocellular adenomas, Results Hot Spots of Mutations in CTNNB1 and Figure 4D: exon 3 mutants including T41A produced strong beta-catenin activation.
• gnomAD v2.1 and gnomAD v4.1: the queried CTNNB1 c.121A>G/p.Thr41Ala variant is absent; this does not by itself demonstrate absence of benign variation throughout the region.
• The case has no CTNNB1 VCEP domain table; therefore no authoritative VCEP domain list or VCEP-defined hotspot route is available.

Still missing

• A reliable domain-level benign-variation assessment for the CTNNB1 exon 3 phosphorylation/degradation-regulatory region is unavailable.

Sources

Reasoning

The available evidence does not document an assumed de novo occurrence, parental testing, or a patient with the relevant disease. The variant's somatic tumor reports cannot establish PM6 for a germline interpretation.

Rule

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

Evidence considered

• No parental testing or assumed de novo statement is present in the available case evidence.
• Variant-specific publications reviewed in the evidence bundle describe somatic tumor observations rather than germline de novo cases.

Still missing

• Documentation that the variant is de novo or presumed de novo
• Parental testing results or explicit absence of parental testing
• Phenotype-confirmed disease diagnosis

Reasoning

No affected family members, familial variant testing, co-segregation results, or meioses are reported. Although CTNNB1 is associated with germline disease in the case context, the required family-level segregation evidence is absent.

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 case evidence contains no family pedigree, affected-relative descriptions, familial genotypes, or segregation analysis for NM_001904.4:c.121A>G.
• The cited variant-specific literature reports somatic tumor cohorts or functional data, not co-segregation in affected family members.

Still missing

• Affected relatives and their clinical phenotypes
• Variant testing results for affected and unaffected relatives
• Number of informative meioses
• Assessment of phenocopies or multiple pathogenic variants

Reasoning

The available case material does not provide a validated gene-level benign missense rate for CTNNB1 together with evidence that missense variants are a common mechanism of the disease context being assessed. CTNNB1 evidence in the bundle instead documents a loss-of-function germline disease context, while the queried T41A reports are somatic oncogenic observations; those facts do not establish the two required PP2 conditions.

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

• The case material does not supply a validated CTNNB1 gene-level benign-missense rate or a disease-specific demonstration that missense variants are a common disease mechanism.
• The available CTNNB1 germline context describes loss-of-function disease, whereas the p.T41A literature concerns somatic oncogenic tumors; neither establishes PP2.

Still missing

• A validated gene-level assessment of benign missense variation and disease-mechanism prevalence for PP2 is unavailable.

Reasoning

The available evidence concerns a somatic CTNNB1 p.T41A tumor alteration and does not provide an evaluated patient phenotype or family history that is highly specific for a single genetic etiology. The available tumor types are heterogeneous and do not establish the PP4 phenotype-specificity requirement.

Rule

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

Evidence considered

• The reviewed publications describe p.T41A in somatic tumor specimens from adrenocortical carcinoma, PPNAD-associated adrenal adenoma, and hepatocellular carcinoma, but do not provide a phenotype/family-history assessment meeting PP4.
• The case record identifies p.T41A as a missense CTNNB1 variant and contains no patient-specific highly specific phenotype or family-history evidence for this criterion.

Still missing

• A clinical phenotype and family-history record for the evaluated patient, with a disease having a single highly specific genetic etiology, is unavailable.

Sources

Reasoning

BS3 is not assessed because no well-established functional study in the available evidence demonstrates that CTNNB1 p.T41A has no damaging effect on protein function or splicing. The available T41A reporter assay instead supports increased beta-catenin activation and therefore cannot support BS3.

Rule

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

Evidence considered

• The available PMID:24735922 assay tested CTNNB1 T41A in a TOP-FLASH reporter system and reported activating activity, not preserved benign function or normal splicing.
• No functional study demonstrating absence of a damaging effect for this variant was available in the reviewed functional evidence.

Still missing

• A well-established assay demonstrating no damaging effect on CTNNB1 p.T41A protein function or splicing is absent.

Sources

Reasoning

No affected family members carrying or lacking the variant are described, so lack of segregation cannot be evaluated. There is also no family-level information to assess phenocopies or the possibility of more than one pathogenic variant.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The available case evidence does not report affected relatives who were tested for the variant or any non-segregation observation.
• No family-level data are available to evaluate the BS4 caveat concerning phenocopies or multiple pathogenic variants.

Still missing

• Affected family members and their genotypes
• Clinical phenotypes of affected relatives
• Family structure and informative meioses
• Assessment of phenocopies or multiple pathogenic variants

Reasoning

The bundle indicates a CTNNB1 germline loss-of-function disease context, but it does not provide a source-registry-citable, disease-specific assessment demonstrating that primarily truncating variants are known to cause the disease. The available p.Thr41Ala reports are somatic tumor studies and cannot alone establish the gene-level BP1 premise for the relevant inherited disease context.

Rule

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

Evidence considered

• The case material describes a CTNNB1 germline loss-of-function context but does not provide a source-registry key for the underlying disease cohort or review.
• The cited p.Thr41Ala publications are somatic cancer studies and do not establish the inherited-disease variant spectrum required for BP1.

Still missing

• A source-registry-citable CTNNB1 disease review or cohort quantifying the predominance of truncating disease-causing variants is required.

Reasoning

BP2 cannot be assessed because the available evidence does not document this germline variant in trans with a pathogenic variant in an affected proband or in cis with a pathogenic variant. The retrieved reports of p.T41A are somatic tumor studies and provide no germline family testing, affected-proband phase, or cis/trans determination.

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

• PMID:16140927, Table 2, patient 38: reports tumor p.T41A with tumor/leukocyte sequencing and describes the alteration as somatic; no germline phase or second pathogenic allele is reported.
• PMID:18419788, Results for patient 12 and Table 1: reports heterozygous p.T41A in an adrenal adenoma and describes it as somatic; adjacent tissue findings do not establish cis/trans phase with another pathogenic variant.
• PMID:10435629, Table 1 and methods: reports p.T41A in hepatocellular carcinoma and confirms somatic status using matched non-tumoral tissue; no germline phase or second pathogenic allele is reported.
• PMID:24735922, Results and Figure 4D: tests T41A in a beta-catenin activation assay but reports no clinical family, affected-proband, or allelic-phase observation.

Still missing

• Germline parental or offspring testing and explicit phase data are unavailable.
• No affected-proband report documents a second pathogenic variant in cis or trans with NM_001904.4:c.121A>G.

Sources

Reasoning

The reviewed reports show p.T41A as a somatic alteration within tumors and establish somatic status using leukocyte or adjacent-tissue comparisons, but they do not document a case in which an alternate molecular basis explains the disease instead of this variant. The available evidence therefore cannot establish BP5.

Rule

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

Evidence considered

• PMID:16140927 reports p.T41A in an adrenocortical carcinoma and absence from leukocyte DNA, supporting somatic origin but not an alternate molecular basis for disease.
• PMID:18419788 reports p.T41A confined to an adrenal adenoma and absent from adjacent tissues, supporting somatic localization but not BP5.
• PMID:10435629 reports p.T41A in hepatocellular carcinoma with matched non-tumor tissue used for somatic confirmation; no independent alternate disease-causing molecular basis is reported.

Still missing

• A documented p.T41A-positive case with an independent alternate molecular basis explaining the disease is unavailable.

Sources