ERBB2 c.929C>T, p.Ser310Phe

Reasoning

PMID:29954840 directly examined ERBB2 p.Ser310Phe (S310F) in biologically relevant gallbladder-carcinoma models. The authors expressed S310F and S310Y in GBC-SD and NOZ cells and compared the mutant group with wild-type ERBB2, evaluating proliferation, colony formation, migration, ERBB-pathway signaling, PD-L1 expression, co-culture effects, and inhibitor response. The reported mutant-associated increases in oncogenic cellular phenotypes and pathway signaling support an activating/damaging effect on ERBB2 function. This is direct variant-specific functional evidence with relevant controls and multiple orthogonal readouts, but the paper analyzed S310F together with S310Y and did not provide separately quantified S310F results; it is not a validated, reproducible clinical diagnostic-laboratory assay. Therefore PS3 is met at supporting strength rather than a stronger level.

Rule

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

Evidence considered

• Li et al. 2019, PMID:29954840: ERBB2 S310F and S310Y were expressed in GBC-SD and NOZ cells; the mutant group was evaluated against wild-type ERBB2 using proliferation, colony-formation, migration, ERBB-pathway signaling, PD-L1, co-culture, and inhibitor assays.
• The paper's results support increased oncogenic cellular phenotypes and ERBB2 pathway activity for the S310F/S310Y mutant group, but do not separately quantify the S310F contribution.
• The available evidence is variant-specific and biologically relevant but is not a validated or reproducible clinical diagnostic-laboratory assay.

Still missing

• The study reports S310F together with S310Y rather than providing separately quantified S310F results.
• Independent replication and validated clinical diagnostic-laboratory assay performance were not established in the available evidence.

Sources

Reasoning

ERBB2 p.Ser310Phe lies at residue S310, which the Cancer Hotspots result identifies as a statistically significant hotspot. The variant is absent from gnomAD v2.1 and v4.1, providing no observed population variation for this variant; no VCEP-specific domain table or hotspot restriction applies because no VCEP covers ERBB2. The hotspot extraction is noted as partially parsed and should receive human confirmation.

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 ERBB2 S310: 'This variant lies in a statistically significant hotspot'; the record was flagged as partially parsed.
• gnomAD v2.1: ERBB2 NM_004448.4:c.929C>T is absent.
• gnomAD v4.1: ERBB2 NM_004448.4:c.929C>T is absent.

Still missing

• Human confirmation of the partially parsed Cancer Hotspots S310 record is recommended.

Sources

Reasoning

PM2 is met at supporting strength. NM_004448.4:c.929C>T (ERBB2 p.Ser310Phe) was absent from gnomAD v2.1 and gnomAD v4.1, and the reported gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets were also absent. This is below the supplied generic PM2 supporting threshold of <=0.0001. The variant is a single-nucleotide substitution, so the ACMG/AMP 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: NM_004448.4:c.929C>T was absent; observed allele frequency 0.
• gnomAD v4.1: NM_004448.4:c.929C>T was absent; observed allele frequency 0.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records were also reported absent.
• Applied the supplied ClinGen SVI generic PM2 supporting threshold of <=0.0001 (ClinGen SVI 2020 recommendation, cited in the governing prompt; ACMG/AMP framework PMID:25741868).
• The variant is a C>T substitution, not an indel; the ACMG/AMP indel calling caveat is therefore not applicable.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule requires the missense computational path using REVEL only; SpliceAI and BayesDel are not used for PP3 in this situation. REVEL is 0.533, below the generic ClinGen SVI supporting PP3 threshold of >=0.644 (Pejaver et al. 2022, PMID:36413997), so PP3 is not met.

Rule

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

Evidence considered

• Variant consequence: missense, NM_004448.4:c.929C>T (NP_004439.2:p.(Ser310Phe)); therefore the applicable generic path is REVEL only.
• REVEL score = 0.533 (local REVEL predictor lookup; source_registry key: revel).
• Generic ClinGen SVI REVEL calibration: PP3 supporting requires >=0.644 (Pejaver et al. 2022, PMID:36413997), as supplied in the case rules. SpliceAI and BayesDel were not counted for this missense PP3 assessment.

Sources

Reasoning

PP5 is not met. The exact ClinVar record for NM_004448.4:c.929C>T reports an Oncogenic classification with criteria provided, single submitter, one somatic submission, no expert-panel submission, and no usable submission details supporting an exact germline Pathogenic or Likely pathogenic classification. The available literature-pass PP5 leads were not variant-specific evidence: PMID:22918138 does not mention the variant, and PMID:34131312 is a general technical standard.

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 submission audit for the exact variant: classification Oncogenic; review status criteria provided, single submitter; submissions total 0 in the extracted audit, expert-panel submissions 0, and no validated criterion-lead submissions.
• The ClinVar API submission record, where available, describes a single somatic submission rather than an expert-panel germline Pathogenic or Likely pathogenic assertion.
• PMID:22918138 paper_extracts entry: the paper does not mention ERBB2 c.929C>T or p.S310F and therefore cannot supply a reputable variant-specific PP5 assertion.

Sources

Reasoning

BA1 is not met. NM_004448.4:c.929C>T (ERBB2 p.Ser310Phe) was absent from gnomAD v2.1 and gnomAD v4.1; therefore its observed allele frequency is 0 in the available datasets, far below the generic BA1 threshold of >=0.05. The non-cancer subsets were also reported absent, and no population frequency, ancestry-specific enrichment, or threshold artifact supports BA1.

Rule

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

Evidence considered

• gnomAD v2.1: NM_004448.4:c.929C>T was absent; observed allele frequency 0.
• gnomAD v4.1: NM_004448.4:c.929C>T was absent; observed allele frequency 0.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records were also reported absent; no ancestry-specific elevation was present.
• Applied published generic BA1 calibration: >=0.05, ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant was absent from gnomAD v2.1 and gnomAD v4.1, with observed allele frequency 0, so it is not greater than expected for the disorder and is below the generic BS1 default threshold of >=0.01. The absence of an elevated frequency means the reduced-penetrance, late-onset, founder-allele, prevalence, and allelic-heterogeneity caveats do not create a BS1-positive path here; no maximum-credible-frequency calculation is needed to reject an elevated-frequency criterion.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v2.1: NM_004448.4:c.929C>T was absent; observed allele frequency 0.
• gnomAD v4.1: NM_004448.4:c.929C>T was absent; observed allele frequency 0.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records were also reported absent.
• Applied the generic BS1 default threshold of >=0.01 supplied in the governing rules; Whiffin et al. 2017 (PMID:28518168) was considered for maximum-credible-frequency context, but no elevated frequency requiring that calculation exists.

Sources

Reasoning

BS2 is not met. No healthy adult individual carrying NM_004448.4:c.929C>T was identified in the available population evidence, and the variant was absent from gnomAD v2.1, gnomAD v4.1, and the reported non-cancer subsets. Thus there is no documented homozygous, heterozygous, or hemizygous observation in an unaffected adult to satisfy BS2. The case bundle does not establish the inheritance model or full early-age penetrance of a constitutional ERBB2 disorder, but that uncertainty does not convert the absent observation into BS2 evidence.

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 gnomAD v4.1: the variant was absent, with no observed healthy adult carrier or homozygote.
• The reported gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer records were also absent.
• No case-specific population record documents a healthy adult heterozygote, homozygote, or hemizygote for this variant.

Still missing

• The inheritance model and expected early-age full penetrance of the relevant constitutional ERBB2 disorder are not established in the available case evidence.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule requires the missense computational path using REVEL only; the SpliceAI result cannot substitute for an indeterminate missense REVEL result. REVEL is 0.533, above the generic ClinGen SVI supporting BP4 threshold of <=0.29 (Pejaver et al. 2022, PMID:36413997), so BP4 is not met.

Rule

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

Evidence considered

• Variant consequence: missense, NM_004448.4:c.929C>T (NP_004439.2:p.(Ser310Phe)); therefore the applicable generic path is REVEL only.
• REVEL score = 0.533 (local REVEL predictor lookup; source_registry key: revel).
• Generic ClinGen SVI REVEL calibration: BP4 supporting requires <=0.29 (Pejaver et al. 2022, PMID:36413997), as supplied in the case rules. The SpliceAI max delta of 0.003 was not used because splice prediction is not the applicable path for a missense variant; BayesDel has no established generic ACMG-strength calibration.

Sources

Reasoning

BP6 is not met. The exact ClinVar record is Oncogenic with criteria provided, single submitter, and has no expert-panel submission or exact-variant Benign/Likely benign assertion. No reputable source in the available evidence reports this exact variant as benign or likely benign.

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 submission audit for the exact variant: classification Oncogenic; review status criteria provided, single submitter; expert-panel submissions 0; no validated submissions or criterion leads.
• No exact-variant Benign or Likely benign expert-panel assertion is present in the supplied ClinVar evidence.

Sources

Reasoning

The variant is ERBB2 p.Ser310Phe, but the available evidence does not establish a previously established pathogenic variant producing the same amino-acid change through a different nucleotide change. The available variant-specific paper reports S310F experimentally, not a qualifying independent pathogenic same-amino-acid comparator.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• Li et al. 2019, PMID:29954840: the paper explicitly studied ERBB2 p.S310F, so it is evidence about the index variant rather than a different nucleotide change causing the same amino-acid substitution.

Still missing

• A curated pathogenic comparator with the same amino-acid change and a different nucleotide change is needed.

Sources

Reasoning

The available case evidence does not document a proband with a relevant germline disease phenotype, confirmed maternity and paternity, parental genotypes showing the variant absent in both parents, or absence of a family history. The available variant-specific literature concerns somatic cancer and does not provide de novo germline evidence. Therefore PS2 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 contain no documented germline proband, parental, or family-history evidence for NM_004448.4:c.929C>T (ERBB2 p.Ser310Phe).
• The variant-specific paper extract available for PMID:29954840 describes somatic gallbladder-cancer models and does not report germline de novo testing or family segregation.

Still missing

• Document the proband's disease phenotype and relevant family history.
• Obtain validated parental maternity and paternity confirmation.
• Obtain parental testing demonstrating the variant is absent in both biological parents.

Reasoning

No case-control comparison could be established for NM_004448.4:c.929C>T (NP_004439.2:p.Ser310Phe). The literature-pass PS4 leads were checked: PMID:22918138 does not mention ERBB2 or this variant, and PMID:34131312 is listed only as a general technical-standard lead without variant-specific counts in the available evidence. The available variant-specific literature describes somatic cancer cohorts or a single patient, but provides no affected-case carrier count paired with unaffected/population controls and no RR/OR with a 95% CI. Therefore the ACMG/AMP PS4 threshold cannot be evaluated; no OR or CI was calculable.

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

• PMID:22918138 paper_extracts entry: the full text was checked and does not mention ERBB2, c.929C>T, or p.S310F; it contains no variant-specific case, control, or population-frequency data.
• The literature-pass PS4 index also listed PMID:34131312, but the available record identifies it as a general ACMG microarray technical standard and supplies no variant-specific case-control data.
• Available evidence identifies the variant in somatic cancer contexts and reports absence from gnomAD v2.1, v4.1, and gnomAD-Canada, but absence alone is not a case-control comparison and does not supply the affected-case frequency, OR, or CI required here.

Still missing

• A case-control dataset for the exact variant with affected-case and unaffected or matched-ancestry control counts is required.
• If counts are obtained, calculate the OR and 95% CI and document the explicit comparison: OR > 5.0 and CI excludes 1.0.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not establish a recessive disease context for ERBB2, an affected-proband observation, a pathogenic allele in trans, or parental/offspring testing to determine phase. The case evidence concerns an ERBB2 p.Ser310Phe somatic oncogenicity context and contains no segregation or allelic-phase data. Therefore, the required trans configuration and pathogenic status of the partner allele are unconfirmed.

Rule

PM3: For recessive disorders, detected in trans with a pathogenic variant...Note: This requires testing of parents (or offspring) to determine phase.

Evidence considered

• The case summary identifies ERBB2 NM_004448.4:c.929C>T (p.Ser310Phe) and a somatic oncogenicity context, but provides no affected-proband segregation, partner pathogenic variant, or parental/offspring phase result.
• No allelic-phase or inheritance observations were present in the reviewed case evidence for this criterion.

Still missing

• Establish the relevant inherited disease and inheritance mode for ERBB2.
• Obtain affected-proband data for the relevant disorder.
• Identify and clinically establish a pathogenic partner variant.
• Obtain parental or offspring testing to determine cis/trans phase.

Reasoning

A paper reports ERBB2 S310Y alongside S310F in functional cell assays, but the available evidence does not establish S310Y as a pathogenic variant under generic ACMG/AMP criteria. Therefore, the same-residue comparator does not meet the required 'determined to be pathogenic' condition, and no qualifying comparator is available to apply PM5.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• Li et al. 2019, PMID:29954840, Results: 'We expressed the S310Y and S310F mutants of ERBB2'; the study reports functional effects for the mutants but does not establish S310Y as a generic ACMG/AMP pathogenic comparator.

Still missing

• An alternate missense variant at Ser310 with an established pathogenic classification is needed.

Sources

Reasoning

The available case evidence does not document an affected proband with a relevant germline phenotype or an assumed de novo occurrence. No parental testing, maternity or paternity information, or family-history information is provided. Therefore PM6 cannot be assessed.

Rule

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

Evidence considered

• The case materials contain no documented germline de novo observation or parental testing for NM_004448.4:c.929C>T (ERBB2 p.Ser310Phe).
• The available variant-specific literature concerns somatic cancer and does not provide an assumed de novo germline observation.

Still missing

• Document the proband's disease phenotype and relevant family history.
• Document the variant in the proband and its absence or untested status in both parents.
• Clarify whether maternity and paternity were confirmed.

Reasoning

The available case evidence contains no affected family members, family genotypes, phenotype assessments, or meioses demonstrating co-segregation. It also does not establish a familial disease context for this germline variant. Therefore PP1 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 family-based germline segregation data are documented for NM_004448.4:c.929C>T (ERBB2 p.Ser310Phe).
• The available variant-specific paper extract reports somatic cancer experiments rather than affected family members or familial co-segregation.

Still missing

• Identify affected and unaffected relatives with relevant phenotype information.
• Obtain variant testing for informative family members.
• Document the disease diagnosis and segregation pattern across informative meioses.

Reasoning

The case evidence does not provide a gene-level assessment showing both a low rate of benign missense variation in ERBB2 and that missense variants are a common mechanism of disease. Variant-level absence from gnomAD and functional evidence for S310F cannot establish both PP2 requirements.

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.

Still missing

• Gene-level benign missense constraint and disease-mechanism evidence are required.

Reasoning

The available case materials do not provide a defined patient phenotype or family history that is highly specific for a disease with a single genetic etiology. They identify a somatic ERBB2 p.Ser310Phe cancer variant and cancer-cohort contexts, but that information does 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

• Case summary identifies ERBB2 p.Ser310Phe and somatic cancer context but provides no patient phenotype or family-history description for PP4.

Still missing

• A documented patient phenotype or family history and the associated single-gene disease diagnosis are required.

Reasoning

No well-established functional study in the available evidence demonstrates that ERBB2 p.Ser310Phe has no damaging effect on protein function or splicing. The directly relevant study reports activating cellular effects rather than a benign functional result, and the other reviewed literature does not provide a validated normal-function assay for this variant.

Rule

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

Evidence considered

• PMID:29954840 reports activating rather than benign functional effects for the ERBB2 S310F/S310Y mutant group and therefore does not support BS3.
• No available reviewed source provided a well-established benign functional or splicing result for ERBB2 p.Ser310Phe.

Still missing

• A validated, variant-specific assay showing preserved ERBB2 protein function or normal splicing is unavailable.

Sources

Reasoning

The available case evidence does not identify affected family members who were tested for the variant, nor does it report genotypes and phenotypes sufficient to evaluate non-segregation. No family structure or meiosis data are available. Therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No family-based germline non-segregation data are documented for NM_004448.4:c.929C>T (ERBB2 p.Ser310Phe).
• The available variant-specific literature concerns somatic cancer and does not report affected relatives who lack the variant.

Still missing

• Identify affected relatives and document their disease phenotypes.
• Obtain reliable variant genotypes for affected relatives.
• Document informative meioses and any affected non-carriers, while considering phenocopies and multiple pathogenic variants.

Reasoning

The case evidence does not establish that ERBB2 is a gene in which primarily truncating variants are known to cause disease. The available ERBB2 S310F cancer and functional evidence does not address the required gene-level truncating-versus-missense disease mechanism.

Rule

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

Still missing

• Gene-level disease-mechanism evidence demonstrating predominantly truncating pathogenic variants is required.

Reasoning

BP2 cannot be assessed because the available evidence does not show this variant observed in trans with a pathogenic variant in a fully penetrant dominant disorder, or observed in cis with a pathogenic variant in any inheritance pattern. No affected-proband genotype, pathogenic partner allele, phase result, or relevant inheritance-mode evidence is provided. The available ERBB2 cancer literature and somatic database observations do not establish the allelic relationships required for BP2.

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 observation of ERBB2 p.Ser310Phe in cis or trans with a pathogenic variant and no family-based phase testing.
• The cited ERBB2 literature and databases describe somatic cancer or oncogenicity evidence, not the allelic observations required for BP2.

Still missing

• Obtain an affected-proband genotype including a clinically pathogenic partner variant.
• Determine whether the partner variant is in trans or cis by parental, offspring, or equivalent phase testing.
• Establish whether the relevant disorder is fully penetrant and dominant when evaluating the trans pathway.

Reasoning

No case report or clinical record in the available evidence documents this variant in a case with an alternate molecular basis for the disease. Somatic cancer cohort records and the variant-specific functional study do not establish an alternate molecular diagnosis in an affected case, so BP5 cannot be adjudicated from the supplied evidence.

Rule

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

Evidence considered

• PMID:29954840 paper_extracts entry: the study reports ERBB2 S310F in gallbladder-carcinoma cohort and cell-line experiments, but does not document an alternate molecular basis in a clinical case.

Still missing

• A documented case containing the exact variant and an independently established alternate molecular basis for the relevant disease is required.

Sources