CTNNB1 c.98C>G, p.Ser33Cys

Reasoning

The exact variant, CTNNB1 p.Ser33Cys (c.98C>G), was tested in a HEK293 TCF-4/cyclin D1 promoter luciferase assay. The S33C construct significantly increased transcriptional activity by 5.4-fold over vector control and 3.6-fold over wild-type CTNNB1. The study used site-directed mutagenesis, sequencing confirmation that only the intended point mutation was introduced, vector and wild-type comparators, an established hotspot mutant positive comparator, triplicate transfections, and an internal pSEAP control. This is variant-specific functional evidence supporting an activating/damaging effect, but it is a single in-vitro reporter study rather than a validated, reproducible clinical diagnostic laboratory assay; therefore PS3 is calibrated to supporting strength.

Rule

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

Evidence considered

• Lee et al. 2009, Results, Fig. 3: the exact p.Ser33Cys variant increased beta-catenin transcriptional activity 5.4-fold versus vector control and 3.6-fold versus wild type in a TCF-4/cyclin D1 promoter luciferase assay.
• Lee et al. 2009, Methods: site-directed S33C mutagenesis was performed on wild-type CTNNB1, intended point mutations were sequence-confirmed, mutant and wild-type constructs were co-transfected with the TCF4 luciferase reporter, measurements used triplicate transfections, and pSEAP served as an internal control.
• The assay included vector and wild-type controls and the known beta-catenin hotspot mutant T41A/S45A as a positive functional comparator; the evidence is variant-specific but comes from one in-vitro reporter system and is not documented as a validated clinical diagnostic assay.

Still missing

• No independent replication in a second assay system or validated clinical diagnostic laboratory setting was identified.

Sources

Reasoning

The variant is p.Ser33Cys in CTNNB1. The Cancer Hotspots result states that CTNNB1 S33 lies in a statistically significant hotspot. Independent paper extracts place Ser33 within the exon 3 phosphorylation/degradation-targeting region and describe recurrent CTNNB1 mutations at phosphorylation sites. The variant is absent from gnomAD v2.1 and v4.1, providing no observed population benign variation for this variant. Under the generic ACMG/AMP PM1 definition, the documented mutational hotspot and critical functional region support PM1; no VCEP-specific restriction applies because no VCEP covers CTNNB1.

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 query CTNNB1 S33 returned 'This variant lies in a statistically significant hotspot' and identified the S33 hotspot row.
• The variant is absent from gnomAD v2.1 and gnomAD v4.1.
• PMID:19582367 reports mutations at CTNNB1 phosphorylation sites S33, S37, S41, and T45 and functionally tested S33C.
• PMID:21761366 describes CTNNB1 exon 3 mutations affecting serine/threonine phosphorylation sites within the beta-catenin degradation-targeting region.
• PMID:18500270 reports Ser33Cys in tumor specimens and evaluates the beta-catenin phosphorylation sequence.

Sources

Reasoning

PM2 is met at supporting strength. NM_001904.4:c.98C>G (CTNNB1 p.Ser33Cys) was absent from gnomAD v2.1 and gnomAD v4.1; the available gnomAD v2.1 non-cancer and v3.1 non-cancer queries also reported absence. Under the generic rules, the variant is therefore below the supplied PM2 supporting threshold of <=0.0001 (0.01%). No alternate VCEP population source governs this case, and the result is a single-nucleotide substitution rather than an indel for which the stated calling caveat would be especially relevant.

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: variant absent; no allele count or allele frequency was reported.
• gnomAD v4.1: variant absent; no allele count or allele frequency was reported.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer queries also reported absence; no VCEP specifies that these subsets must replace the default all-comers datasets.
• Generic calibration supplied for this case: PM2 <= 0.0001 (0.01%), described in the prompt as the ClinGen SVI recommendation to downgrade PM2 to supporting (PMID:25741868).

Sources

Reasoning

The CTNNB1 gene-level disease context supports loss of function as a disease mechanism and states that most CTNNB1 syndrome patients carry pathogenic nonsense or frameshift variants. This establishes that disease-causing variants are primarily truncating in the relevant germline disease context, while the present variant is missense p.Ser33Cys. Therefore BP1 is met at supporting strength.

Rule

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

Evidence considered

• The gene-level CTNNB1 disease context supports loss of function and states that most CTNNB1 syndrome patients carry pathogenic nonsense or frameshift variants.
• The case variant is a missense substitution, NM_001904.4:c.98C>G (p.Ser33Cys).

Sources

Reasoning

The variant is NP_001895.1:p.(Ser33Cys) and is explicitly reported in several somatic tumor studies, but the available evidence does not establish a previously pathogenic variant at the same amino-acid change in the ACMG/AMP sense. ClinVar records the variant as other with no assertion criteria, and the available OncoKB label is somatic oncogenicity rather than an established germline pathogenic classification. Therefore PS1 is not met.

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

• ClinVar variation 376231 identifies NM_001904.4:c.98C>G (p.Ser33Cys) but reports classification 'other' with no assertion criteria.
• OncoKB reports a variant-specific somatic oncogenic label and gain-of-function context, not an ACMG pathogenic germline assertion.
• PMID:18500270, PMID:16685513, PMID:19582367, and PMID:21761366 explicitly report the same p.Ser33Cys change in tumor specimens, but do not provide an established ACMG pathogenic classification for it.

Sources

Reasoning

The available CTNNB1 germline disease context supports loss of function as a disease mechanism and reports that most CTNNB1 syndrome patients carry nonsense or frameshift variants. That evidence does not support both requirements for PP2: a low rate of benign missense variation and missense variants being a common disease mechanism. The gene-level mechanism evidence instead supports a predominantly truncating loss-of-function model, so PP2 is not met.

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 gene-level CTNNB1 disease context supports loss of function and states that most CTNNB1 syndrome patients carry pathogenic nonsense or frameshift variants.
• The reviewed evidence does not provide a validated quantitative or comparative demonstration of both PP2 requirements.

Sources

Reasoning

The variant is an exonic missense substitution, NM_001904.4:c.98C>G (p.Ser33Cys), so the governing generic rule permits the missense REVEL path only; SpliceAI and BayesDel are not used for PP3. REVEL is 0.453, which is below the supplied PP3 supporting threshold of >=0.644 and within the gray zone, so PP3 is not met. The threshold is the supplied ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... Missense variant -> REVEL only.

Evidence considered

• Variant consequence: exonic missense, NM_001904.4:c.98C>G (p.Ser33Cys).
• Local REVEL lookup reports score 0.453 for NC_000003.12:g.41224610C>G.
• The supplied generic calibration defines PP3 supporting as REVEL >=0.644; REVEL 0.453 is indeterminate/gray-zone and does not meet PP3.
• The supplied PP3 threshold is the ClinGen SVI REVEL calibration from Pejaver et al. 2022 (PMID:36413997). SpliceAI max delta 0.002 and BayesDel -0.0309578 were not used because the governing missense path is REVEL only and BayesDel has no established generic calibration.

Sources

Reasoning

ClinVar variation 376231 is an exact match to NM_001904.4:c.98C>G (p.Ser33Cys), but its classification is "other" with no assertion criteria and no expert-panel submission. The record therefore does not meet the required exact-variant ClinVar expert-panel Pathogenic/Likely pathogenic condition for PP5.

Rule

Generic rules for PP5/BP6: apply them only for an exact-variant ClinVar expert-panel classification: Pathogenic/Likely pathogenic supports PP5; Benign/Likely benign supports BP6.

Evidence considered

• ClinVar variation 376231 exactly matches NM_001904.4(CTNNB1):c.98C>G (p.Ser33Cys), but the aggregate classification is "other", review status is "no assertion criteria provided", and expert-panel submissions are absent.
• The available ClinVar record is not an expert-panel Pathogenic/Likely pathogenic assertion and cannot trigger PP5.

Sources

Reasoning

BA1 is not met. NM_001904.4:c.98C>G (CTNNB1 p.Ser33Cys) was absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer subset queries, so there is no allele frequency at or above the generic stand-alone threshold of 0.05 (5%). The variant's somatic cancer reports do not establish a benign population-frequency signal, and no population evidence supports a BA1 stand-alone benign conclusion.

Rule

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

Evidence considered

• gnomAD v2.1: variant absent; no allele count, allele frequency, or grpmax FAF was reported.
• gnomAD v4.1: variant absent; no allele count, allele frequency, or grpmax FAF was reported.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer queries also reported absence; no governing VCEP population-source substitution applies because no VCEP covers CTNNB1.
• Generic calibration supplied for this case: BA1 >= 0.05 (5%), identified in the prompt as the ACMG/AMP 2015 stand-alone benign allele-frequency threshold (PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1, so the filtering allele frequency (grpmax FAF) is not elevated and cannot meet the generic default threshold of 0.01 (1%). A maximum-credible-allele-frequency calculation is not needed to reject BS1 here because the observed population frequency is zero in the reported datasets; no ancestry-specific elevated frequency, founder effect, or threshold artifact is present.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: variant absent; therefore no filtering allele frequency or grpmax FAF was reported.
• gnomAD v4.1: variant absent; therefore no filtering allele frequency or grpmax FAF was reported.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer queries also reported absence; no VCEP specifies an alternate population source.
• The required BS1 frequency measure is the filtering allele frequency (grpmax FAF), not a popmax point estimate; no grpmax FAF was available because the variant was absent.
• Generic calibration supplied for this case: BS1 >= 0.01 (1%), with per-gene maximum-credible-frequency derivation preferred under Whiffin et al. 2017 (PMID:28518168).

Sources

Reasoning

The available variant-specific functional study does not show a lack of damaging effect. Instead, p.Ser33Cys increased TCF-4/cyclin D1 reporter activity relative to both vector control and wild-type CTNNB1. Therefore, the evidence does not support the benign functional criterion BS3.

Rule

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

Evidence considered

• Lee et al. 2009, Results, Fig. 3: the exact p.Ser33Cys construct significantly increased transcriptional activity versus vector control and wild-type CTNNB1, so the assay does not demonstrate preserved function or absence of a damaging effect.

Sources

Reasoning

The variant is an exonic missense substitution, NM_001904.4:c.98C>G (p.Ser33Cys), so the governing generic rule permits the missense REVEL path only; the SpliceAI result cannot substitute for REVEL. REVEL is 0.453, which exceeds the supplied BP4 supporting threshold of <=0.29 and is within the gray zone, so BP4 is not met. The threshold is the supplied ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) ... Missense variant -> REVEL only.

Evidence considered

• Variant consequence: exonic missense, NM_001904.4:c.98C>G (p.Ser33Cys).
• Local REVEL lookup reports score 0.453 for NC_000003.12:g.41224610C>G.
• The supplied generic calibration defines BP4 supporting as REVEL <=0.29; REVEL 0.453 is indeterminate/gray-zone and does not meet BP4.
• The supplied BP4 threshold is the ClinGen SVI REVEL calibration from Pejaver et al. 2022 (PMID:36413997). SpliceAI max delta 0.002 was not used because a missense variant must be assessed through the REVEL path only; BayesDel -0.0309578 was not used because no generic calibration is established.

Sources

Reasoning

ClinVar variation 376231 is an exact match to NM_001904.4:c.98C>G (p.Ser33Cys), but its classification is "other" with no assertion criteria and no expert-panel submission. The record therefore does not meet the required exact-variant ClinVar expert-panel Benign/Likely benign condition for BP6.

Rule

Generic rules for PP5/BP6: apply them only for an exact-variant ClinVar expert-panel classification: Pathogenic/Likely pathogenic supports PP5; Benign/Likely benign supports BP6.

Evidence considered

• ClinVar variation 376231 exactly matches NM_001904.4(CTNNB1):c.98C>G (p.Ser33Cys), but the aggregate classification is "other", review status is "no assertion criteria provided", and expert-panel submissions are absent.
• The available ClinVar record is not an expert-panel Benign/Likely benign assertion and cannot trigger BP6.

Sources

Reasoning

No case-level evidence documents a proband with the disease, absence of family history, or confirmed maternity and paternity for NM_001904.4:c.98C>G. The available variant reports are somatic tumor studies and do not establish germline de novo status.

Rule

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

Evidence considered

• No case-level proband, pedigree, or parental-testing data document confirmed maternity and paternity or absence of family history.
• The available variant-specific literature describes somatic tumor findings rather than germline de novo testing.

Still missing

• Proband disease phenotype and family-history documentation
• Maternity confirmation
• Paternity confirmation
• Evidence that the variant is germline in the proband

Reasoning

The exact p.Ser33Cys variant is reported in tumor cohorts, including 2 of 146 medulloblastomas (PMID:16685513), 1 of 18 brain metastases (PMID:19582367), 3 endometrial carcinomas across study groups (PMID:18500270), and 1 of 16 craniopharyngiomas (PMID:21761366). These reports do not provide a qualifying affected-versus-unaffected/population control comparison for this exact variant, so an odds ratio or relative risk and its confidence interval cannot be calculated. The reviewed PS4 candidate PMID:26822237 does not mention p.Ser33Cys and reports only aggregate CTNNB1 findings. Therefore the PS4 threshold cannot be assessed.

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:16685513, Table 1: p.S33C (TCT>TGT) occurred in 2 of 146 medulloblastomas; the study is a tumor cohort without unaffected/population controls for the exact variant.
• PMID:19582367, Results/Table 1: p.S33C occurred in 1 of 18 brain metastasis specimens and was absent from the other tumor groups; this is not an affected-versus-unaffected control comparison.
• PMID:18500270, Tables 3 and 5: p.S33C was found in two tamoxifen-exposed endometrial carcinomas and one random-group endometrial carcinoma; all reported groups are affected carcinoma cohorts, not unaffected controls.
• PMID:21761366, Table 2: p.S33C occurred in tumor tissue from 1 of 16 patients with adamantinomatous craniopharyngioma; blood and normal-pituitary testing did not establish a case-control prevalence estimate.
• PMID:26822237, Results: aggregate CTNNB1 mutations occurred in 10 of 121 tumor-WES participants (8.3%), but the exact p.Ser33Cys variant was not mentioned and no exact-variant control comparison was provided.

Still missing

• A matched affected-case and unaffected/population-control comparison for CTNNB1 p.Ser33Cys with counts or frequencies is unavailable.

Sources

Reasoning

The available evidence documents p.Ser33Cys itself but does not identify a different missense substitution at Ser33 that has been determined to be pathogenic. The case's comparator collection contains no same-residue candidates, and the available extracts do not provide a qualifying pathogenic alternate missense variant. A validated same-residue pathogenic comparator search or adjudication is still required.

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 PM5 candidate artifact reports zero same-residue candidates and no confirmed pathogenic comparator.
• ClinVar provides no usable expert-panel or criterion-lead pathogenic comparator for the reviewed variant record.

Still missing

• A validated alternate missense variant at CTNNB1 Ser33 determined to be pathogenic is needed for PM5 assessment.

Sources

Reasoning

No case-level evidence documents an assumed de novo occurrence of NM_001904.4:c.98C>G. There is no proband phenotype, parental testing, or family-history information establishing a de novo event without parental confirmation.

Rule

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

Evidence considered

• No case-level parental-testing, pedigree, or explicit assumed-de novo assessment is documented.
• The available variant-specific literature describes somatic tumor findings and does not establish a germline de novo event.

Still missing

• Documented proband disease phenotype
• Parental testing results or an explicit unconfirmed de novo assessment
• Family-history information
• Evidence that the variant is germline in the proband

Reasoning

No affected family members, pedigree, segregation observations, or meioses are documented for NM_001904.4:c.98C>G. The available reports concern somatic tumors and cannot establish cosegregation in a disease family.

Rule

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

Evidence considered

• No family pedigree, affected-relative genotypes, phenotype concordance, or informative meioses are documented.
• Somatic tumor reports do not provide familial cosegregation evidence.

Still missing

• Pedigree with affected relatives
• Genotype and phenotype for each affected family member
• Number of informative meioses
• Confirmation that the gene is definitively associated with the family disease

Reasoning

The case evidence supplied for this group does not provide the patient's phenotype or family history, and no phenotype-specific clinical description is available to determine whether it is highly specific for a disease with a single genetic etiology.

Rule

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

Still missing

• Patient phenotype and family history, including a disease-specific clinical summary, are not available.

Reasoning

BS2 cannot be assessed from the available evidence. No report documents this variant in a healthy adult individual, and the population datasets provide aggregate absence rather than phenotype-confirmed healthy-carrier observations. Therefore, the requirement for an observed healthy adult cannot be established or excluded from these data.

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 and v4.1 reported the variant as absent; these aggregate population records do not document healthy-adult phenotype or zygosity observations.
• No case evidence reviewed for this population group reports the variant in a healthy adult individual.

Still missing

• A phenotype-confirmed healthy adult observation with inheritance-appropriate zygosity is not available.

Sources

Reasoning

No affected family members with negative variant testing, pedigree, phenotype assessment, or other non-segregation evidence are documented. Therefore, lack of segregation cannot be evaluated, and the phenocopy caveat cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-relative genotype data or pedigree-based non-segregation observations are documented.
• No information is available to evaluate phenocopies or the possibility of more than one pathogenic variant in the family.

Still missing

• Affected family members and their clinical phenotypes
• Variant-testing results for affected relatives
• Pedigree and inheritance information
• Assessment of possible phenocopies or multiple pathogenic variants

Reasoning

BP2 requires an affected-proband or other observation showing this variant in trans with a pathogenic variant in a fully penetrant dominant disorder, or in cis with a pathogenic variant in any inheritance pattern. The available reports of NM_001904.4:c.98C>G (p.Ser33Cys) describe somatic tumor observations and provide no germline second-allele result, pathogenic partner variant, or phase determination. Therefore BP2 cannot be assessed from the available evidence.

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

• Stratification of medulloblastoma on the basis of histopathological grading, Table 1 and Results: p.S33C was observed in two medulloblastoma tumors; the study provides tumor sequencing data, not germline phase or a pathogenic partner allele.
• Clinicopathological and molecular analysis of endometrial carcinoma associated with tamoxifen, Tables 3 and 5: p.S33C was reported in endometrial carcinoma specimens; no germline allelic relationship was provided.
• CTNNB1 mutation is rare in brain tumours but involved as a sporadic event in a brain metastasis, Results/Table 1/Figures 2-3: p.S33C was a heterozygous tumor mutation, with no segregation or population-frequency data and no pathogenic allele in cis or trans.
• CTNNB1 gene mutations in adamantinomatous craniopharyngiomas, Table 2 patient 5: c.98C>G/p.S33C was found in tumor DNA and cDNA but not patients' genomic DNA; the authors characterize the mutations as somatic, with no phase or pathogenic partner-variant evidence.

Still missing

• Germline testing for a pathogenic second variant is not documented.
• Parental, offspring, or other phase testing is not documented.
• No affected-proband report documents the required trans or cis allelic relationship.

Sources

Reasoning

The available evidence documents somatic CTNNB1 p.Ser33Cys in several tumor cohorts but does not document this variant in a case with an alternate molecular basis for the disease. The BP5 requirement therefore cannot be assessed from the supplied case evidence.

Rule

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

Still missing

• A case report or cohort record documenting an alternate molecular basis for the relevant disease is unavailable.