CTNNB1 c.115_135del, p.Ala39_Ser45del

Reasoning

PM2 Supporting is met: NM_001904.4:c.115_135del is absent from the default all-comers gnomAD v2.1 and v4.1 datasets. No governing VCEP specifies a different population source, and the generic ClinGen SVI-based threshold supplied for PM2 Supporting is <=0.0001. The variant is an in-frame deletion, so the ACMG/AMP indel-calling caveat limits confidence but does not provide evidence of a nonzero population frequency.

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 and v4.1 both report the exact normalized variant NM_001904.4:c.115_135del as absent.
• The generic ClinGen SVI-based PM2 Supporting threshold supplied for this case is <=0.0001 (PMID:25741868).
• The ACMG/AMP caveat notes that population data for indels may be poorly called by next-generation sequencing.

Sources

Reasoning

PP5 requires a reputable source to report the exact variant as pathogenic when the underlying evidence is unavailable for independent evaluation. The exact variant has no ClinVar record or expert-panel classification in the case evidence, and the reviewed literature does not report the exact variant. OncoKB's variant-specific oncogenic context and its cited paper do not establish an exact germline Pathogenic/Likely pathogenic ClinVar expert-panel classification. PP5 is not met.

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 exact-variant query for NM_001904.4:c.115_135del returned no record and therefore no expert-panel Pathogenic/Likely pathogenic classification.
• OncoKB reported a variant-specific Likely Oncogenic somatic context, not an exact germline ClinVar expert-panel Pathogenic/Likely pathogenic classification; its cited PMID:29316426 paper did not report this exact variant.

Sources

Reasoning

BA1 is not met: NM_001904.4:c.115_135del is absent from gnomAD v2.1 and gnomAD v4.1, so its observed allele frequency is below the generic stand-alone threshold of >=0.05. The variant is an in-frame deletion, for which population indel calling may be imperfect; therefore absence is not treated as definitive evidence of a frequency below every possible threshold. No pathogenic ClinVar consensus or other disease association requiring reconciliation was identified in the case evidence.

Rule

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

Evidence considered

• gnomAD v2.1 and v4.1 both report the exact normalized variant NM_001904.4:c.115_135del as absent.
• The generic BA1 stand-alone calibration supplied for this case is >=0.05 (ACMG/AMP 2015, PMID:25741868).
• The ACMG/AMP caveat notes that population data for indels may be poorly called by next-generation sequencing.

Sources

Reasoning

BS1 is not met on the available population data: the variant is absent from gnomAD v2.1 and v4.1, so no allele frequency greater than expected for CTNNB1-related disease is observed. The generic 0.01 default is not reached, and there is no elevated ancestry-specific frequency requiring a maximum-credible-frequency analysis. Because this is an in-frame deletion, indel calling limitations temper confidence in a negative population observation.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 and v4.1 both report the exact normalized variant NM_001904.4:c.115_135del as absent.
• The generic BS1 default supplied for this case is >=0.01; Whiffin et al. 2017 (PMID:28518168) is the cited framework for disorder-specific maximum credible allele-frequency assessment.
• No ancestry-specific elevated frequency, homozygote observation, prevalence estimate, or penetrance estimate was available that would support a different BS1 conclusion.

Sources

Reasoning

NM_001904.4:c.115_135del is an in-frame deletion predicted to remove Ala39-Ser45 in CTNNB1 exon 3. BP3 requires an in-frame deletion in a repetitive region without a known function. The reviewed CTNNB1 exon 3 deletion evidence describes this region as containing regulatory phosphorylation sites involved in beta-catenin degradation, so the required absence of known function is not satisfied. BP3 is therefore not met.

Rule

BP3: In frame-deletions/insertions in a repetitive region without a known function.

Evidence considered

• Variant normalization identifies NM_001904.4:c.115_135del as the in-frame deletion NP_001895.1:p.(Ala39_Ser45del), removing seven amino acids in exon 3.
• Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer, Results/WNT Pathway alterations and Discussion: related CTNNB1 exon 3 deletions remove regulatory phosphorylation sites involved in beta-catenin protein degradation and are associated with WNT pathway activation.
• The exact c.115_135del variant is not explicitly reported, but the documented regulatory function of the affected exon 3 region contradicts BP3's required absence of known function.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The available evidence does not document this exact variant in a clinical case with an independently established alternate molecular cause, so BP5 is not met.

Rule

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

Reasoning

BP6 requires a reputable source to report the exact variant as benign when the underlying evidence is unavailable for independent evaluation. The exact variant is absent from ClinVar, with no expert-panel Benign/Likely benign classification, and no reviewed publication reports the exact variant as benign. BP6 is not met.

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 exact-variant query for NM_001904.4:c.115_135del returned no record and therefore no expert-panel Benign/Likely benign classification.

Sources

Reasoning

The available case evidence does not document a proband with the relevant disease phenotype, confirmed maternity and paternity, or absence of family history for NM_001904.4:c.115_135del. Therefore, the required de novo evidence cannot be assessed.

Rule

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

Evidence considered

• The case materials identify NM_001904.4:c.115_135del in CTNNB1 but provide no proband-level phenotype, parental segregation results, maternity or paternity confirmation, or family-history assessment.

Still missing

• Document the proband's relevant disease phenotype.
• Document parental testing showing the variant is absent in both biological parents.
• Confirm maternity and paternity.
• Document absence of a relevant family history.

Reasoning

The available literature reports recurrent CTNNB1 exon 3 in-frame deletions in colorectal tumors and nuclear beta-catenin staining, consistent with WNT activation, but it does not explicitly report NM_001904.4:c.115_135del or NP_001895.1:p.(Ala39_Ser45del). The reported tumor sequencing and immunohistochemistry are not a validated, variant-specific functional assay establishing the effect of this deletion, and no assay controls or reproducibility data for this variant are available. Therefore, the evidence is insufficient to assess PS3 under the generic definition.

Rule

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

Evidence considered

• Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer, Results/WNT Pathway alterations and Discussion: MSK-IMPACT sequencing of 1,134 colorectal adenocarcinomas identified recurrent CTNNB1 exon 3 in-frame deletions; tumor beta-catenin immunohistochemistry showed nuclear staining consistent with WNT activation, but the paper did not explicitly report c.115_135del or p.(Ala39_S45del).
• Because the reported finding concerns related exon 3 deletions rather than this exact variant, it cannot establish the variant-specific functional evidence required for PS3.

Still missing

• A validated, variant-specific in vitro or in vivo assay is needed to establish whether p.(Ala39_Ser45del) causes a damaging effect.
• Assay controls, reproducibility, and quantitative results for this exact variant are unavailable.

Sources

Reasoning

The governing PS4 definition requires a significantly increased prevalence in affected individuals compared with controls, and ACMG/AMP Note 1 specifies an OR or RR >5.0 with a confidence interval that does not include 1.0. The reviewed PMID:29316426 paper describes 29 CTNNB1 exon 3 deletion cases among 1,134 metastatic colorectal cancers, but it does not report this exact variant, does not provide affected-versus-unaffected or population-control counts for it, and therefore does not permit calculation of an OR/RR or 95% CI. The available evidence is insufficient to assess PS4.

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:29316426, Results/WNT Pathway alterations and Discussion: MSK-IMPACT targeted sequencing of 1,134 colorectal adenocarcinomas reported 29 recurrent CTNNB1 exon 3 deletion cases, but the paper did not report this exact c.115_135del/p.(Ala39_S45del) variant or a case-control comparison for it.
• No affected-versus-control counts, OR/RR, or 95% confidence interval for the exact variant were available; no calculation could validly be performed.

Still missing

• A case-control study or matched-control frequency comparison reporting NM_001904.4:c.115_135del or NP_001895.1:p.(Ala39_Ser45del) is needed.
• Exact affected and control counts, or frequencies sufficient to calculate an OR/RR and 95% confidence interval, are unavailable.

Sources

Reasoning

NM_001904.4:c.115_135del is an in-frame deletion predicted to remove seven amino acids, NP_001895.1:p.(Ala39_Ser45del), in exon 3 of the MANE transcript. PM4 requires the deleted segment to be in a non-repeat region, but the available evidence does not establish whether Ala39-Ser45 is a repeat region or a non-repeat region. Therefore PM4 cannot be assessed reliably.

Rule

PM4: Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.

Evidence considered

• Variant normalization identifies NM_001904.4:c.115_135del as the in-frame protein deletion NP_001895.1:p.(Ala39_Ser45del), removing seven amino acids in exon 3.
• The available evidence does not provide a repeat-region annotation for the exact Ala39-Ser45 segment.
• Because the governing PM4 definition requires a non-repeat region and that location property is unresolved, PM4 is not assessed.

Still missing

• A reliable sequence or domain annotation is needed to determine whether the deleted Ala39-Ser45 segment is a repeat region or a non-repeat region.

Reasoning

The available case evidence does not document an assumed de novo occurrence of NM_001904.4:c.115_135del in an affected patient, nor does it provide parental testing or other evidence establishing that the variant arose de novo. Therefore, PM6 cannot be assessed.

Rule

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

Evidence considered

• The case materials provide no proband-parent genotype comparison or other documented basis for assuming a de novo event.

Still missing

• Document the affected patient's phenotype.
• Document parental testing or a documented basis for assuming de novo status.
• Clarify whether maternity and paternity were confirmed.

Reasoning

The available case evidence contains no documented affected family members carrying NM_001904.4:c.115_135del and no meiosis or segregation observations. Consequently, co-segregation and any stronger application based on increasing segregation data cannot be assessed.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• No affected relatives, family pedigree, genotype results, or informative meioses are documented in the available case evidence.

Still missing

• Provide a pedigree and identify affected relatives.
• Provide genotype and phenotype data for each informative family member.
• Provide the number of informative meioses and segregation results.

Reasoning

PP4 requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available case materials identify CTNNB1 and the molecular variant but do not provide the patient's phenotype, family history, or a documented phenotype-to-disease specificity assessment. PP4 cannot be assessed from the available evidence.

Rule

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

Still missing

• Patient phenotype and relevant family history are needed.
• A clinical assessment explaining whether the phenotype is highly specific for a disease with a single genetic etiology is needed.

Reasoning

BS2 is not assessed because the available evidence contains no documented healthy adult who carries NM_001904.4:c.115_135del in the heterozygous state, no homozygote observation, and no hemizygote observation. Population absence cannot establish the presence or absence of a healthy-adult carrier, and the required full-penetrance early-onset disease context is not independently documented for this specific variant.

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

• The case evidence reports the variant as absent from gnomAD v2.1 and v4.1, but provides no individual-level healthy-carrier observation.
• No reliable homozygote or hemizygote observation was identified in the supplied evidence.

Still missing

• A verified healthy-adult observation with genotype, zygosity, age, phenotype ascertainment, and disease-inheritance context is required to assess BS2.

Reasoning

No well-established in vitro or in vivo study showing that NM_001904.4:c.115_135del or NP_001895.1:p.(Ala39_Ser45del) has no damaging effect on protein function or splicing was identified. The available paper reports related CTNNB1 exon 3 deletions with nuclear beta-catenin staining, not a benign functional result for this exact deletion, so BS3 cannot be assessed.

Rule

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

Evidence considered

• Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer, Results/WNT Pathway alterations and Discussion: the study describes related CTNNB1 exon 3 deletions and nuclear beta-catenin staining, but provides no benign functional assay result for c.115_135del or p.(Ala39_Ser45del).
• The available evidence does not show no damaging effect on protein function or splicing for this exact variant.

Still missing

• A validated, variant-specific assay demonstrating preserved protein function or normal splicing is needed.
• Assay controls, reproducibility, and quantitative benign-effect results for this exact variant are unavailable.

Sources

Reasoning

The available case evidence does not identify affected family members who were tested for NM_001904.4:c.115_135del or document any affected non-carriers. Without an informative family and phenotype-genotype results, lack of segregation cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The case materials contain no affected relative testing results and no documented affected non-carrier.

Still missing

• Provide a pedigree with affected relatives and their clinical diagnoses.
• Provide variant testing results for affected relatives.
• Address possible phenocopies and other pathogenic variants if non-segregation is observed.

Reasoning

BP2 cannot be assessed because the available evidence does not document a pathogenic variant in trans or cis, affected-proband observations establishing such a relationship, or parental/offspring testing to determine phase.

Rule

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.

Evidence considered

• The available case evidence contains no documented pathogenic partner variant in cis or trans.
• No parental or offspring testing, phase determination, or affected-proband observation relevant to BP2 is documented.

Still missing

• Documented pathogenic partner variant and laboratory-supported phase information are needed to evaluate BP2.