ERBB2 c.929C>A, p.Ser310Tyr

Reasoning

PS3 is met at strong strength. The queried variant is ERBB2 p.(Ser310Tyr), and two independent experimental studies directly evaluated this variant. PMID:22908275 reported robust anchorage-independent colony formation by S310Y in NIH 3T3 cells, transformation of AALE human lung epithelial cells, acquisition of IL-3-independent growth in Ba/F3 cells, and sensitivity to multiple ERBB2 inhibitors. PMID:24997986 independently introduced S310Y into gallbladder cancer cell lines and observed significantly increased proliferation in at least one tested line compared with mock-transfected cells or wild-type ERBB2, with additional enhancement in HEK293T cells when ERBB2 mutants were coexpressed with ERBB3. These are variant-specific, biologically relevant, orthogonal gain-of-function assays with comparator controls and concordant results; the evidence supports a damaging effect for the oncogenic ERBB2 mechanism. No claim of clinical diagnostic-laboratory validation is required by the governing ACMG/AMP definition, and the evidence is sufficiently robust for strong PS3 calibration.

Rule

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

Evidence considered

• PMID:22908275, Results, ‘Extracellular Domain Mutations of ERBB2 Found in Cancer are Oncogenic’: ‘In contrast, the G309E, S310F, and S310Y mutants supported robust colony formation in soft agar’; S310Y also supported AALE transformation, Ba/F3 IL-3-independent growth, and responses to neratinib, afatinib, lapatinib, and trastuzumab.
• PMID:24997986, Results and Figure 3c: ERBB2 S310Y was tested by overexpression in GBC-SD, NOZ, and HEK293T cells; ERBB2 mutants including S310Y significantly increased proliferation versus mock-transfected or wild-type ERBB2 controls, with enhancement upon ERBB3 coexpression.
• The assays are variant-specific and concordant across anchorage-independent growth, epithelial transformation, cytokine-independent growth, inhibitor response, and proliferation, supporting ERBB2 gain of function rather than a nonspecific prediction.
• The cited studies are research cell-based assays rather than functional testing validated in a clinical diagnostic laboratory; this limits the type of evidence but does not negate the direct, reproducible, biologically relevant functional findings under the stated PS3 definition.

Sources

Reasoning

PM2 is met at supporting strength. The variant is reported absent from the default all-comers gnomAD v2.1 and v4.1 datasets and from the available gnomAD v2.1 and v3.1 non-cancer subsets. Absence is below the generic PM2 supporting threshold of <=0.0001; no VCEP-specific population source applies because no VCEP covers ERBB2.

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: variant absent from gnomAD v2.1.
• GNOMAD_V4_1: variant absent from gnomAD v4.1.
• GNOMAD_V2_1_NON_CANCER: variant reported absent from the non-cancer exome-only subset.
• GNOMAD_V3_1_NON_CANCER: variant reported absent from the non-cancer genome-only subset.
• The governing framework is generic ACMG/AMP because no VCEP covers ERBB2; therefore the supplied generic PM2 supporting threshold of <=0.0001 applies.

Sources

Reasoning

The variant is ERBB2 p.Ser310Tyr (c.929C>A). Although this exact variant has a single-submitter Pathogenic ClinVar classification and is reported as oncogenic in somatic functional studies, the reviewed evidence does not identify a distinct nucleotide substitution producing the same p.Ser310Tyr amino-acid change that was previously established as pathogenic. The available same-residue literature concerns S310F and other S310 hotspot alleles, not a qualifying same-amino-acid comparator; the variant is not shown to be a splicing-driven change.

Rule

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

Evidence considered

• ClinVar variation 376188 records NM_004448.4:c.929C>A (p.Ser310Tyr) as Pathogenic from one single-submitter clinical laboratory, but this is the queried nucleotide change rather than a distinct same-amino-acid substitution.
• The reviewed papers report ERBB2 S310Y and other S310 alleles, but do not document a separate nucleotide change encoding p.Ser310Tyr with an established pathogenic classification.

Sources

Reasoning

The queried variant is ERBB2 p.Ser310Tyr. PMID:29967253 discusses the different missense variant S310F at the same residue and describes it as a TKI-sensitizing/oncogenic allele, while PMID:22908275 reports S310F among oncogenic mutants. The reviewed evidence does not establish S310F or another different S310 missense variant as pathogenic under the required clinical standard; somatic oncogenicity and hotspot status alone are not a documented pathogenic determination for 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

• PMID:29967253 reports same-residue S310F in functional and drug-sensitivity analyses, but does not establish it as a clinically pathogenic variant.
• PMID:22908275 reports S310F among oncogenic ERBB2 mutants, and PMID:29420467 describes S310 as a HER2 hotspot; neither provides the required established pathogenic classification for a different missense change at residue 310.

Sources

Reasoning

The variant is a missense substitution, p.(Ser310Tyr), so the governing one-path rule requires REVEL only; SpliceAI is not used for missense PP3/BP4. REVEL is 0.54, below the generic PP3 supporting threshold of >= 0.644 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997), and therefore PP3 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

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

• NM_004448.4:c.929C>A is an exonic missense variant, NP_004439.2:p.(Ser310Tyr).
• REVEL score = 0.54; this is below the generic PP3 supporting cutoff of >=0.644 from ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• SpliceAI max delta = 0.014 was not used because the governing path for a missense variant is REVEL only.
• BayesDel score = 0.0783379 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The exact variant has one ClinVar Pathogenic submission from a single clinical laboratory with a one-star criteria-provided review status and no expert-panel submission. Under the required PP5 rule, an ordinary laboratory submission, aggregate label, or non-expert assertion cannot trigger PP5; no qualifying exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification is present.

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 376188 exactly matches NM_004448.4(ERBB2):c.929C>A (p.Ser310Tyr), but its only submission is Pathogenic from CeGaT, criteria provided, single submitter; expert-panel submissions: 0.

Sources

Reasoning

BA1 is not met. The variant is absent from the default all-comers gnomAD v2.1 and v4.1 datasets and is also reported absent from the available non-cancer subsets, so no filtering allele frequency reaches the generic stand-alone threshold of >=0.05. The variant's pathogenic ClinVar submission is single-submitter evidence and does not alter this population-frequency result.

Rule

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• GNOMAD_V2_1: variant absent from gnomAD v2.1; no allele frequency or grpmax FAF was reported.
• GNOMAD_V4_1: variant absent from gnomAD v4.1; no allele frequency or grpmax FAF was reported.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER: variant reported absent from both available non-cancer subsets.
• ClinVar variation 376188: Pathogenic, criteria provided, single submitter; this is not a high-confidence consensus that could justify overriding the absent population data.

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and v4.1 and from the reported non-cancer subsets, so there is no filtering allele frequency or grpmax FAF showing an allele frequency greater than expected for the disorder. Because no elevated ancestry-specific frequency is present, the reduced-penetrance/founder-allele caveat and a maximum-credible-frequency calculation are not triggered.

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: variant absent from gnomAD v2.1; no grpmax FAF or population point estimate was reported.
• GNOMAD_V4_1: variant absent from gnomAD v4.1; no grpmax FAF or population point estimate was reported.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER: variant reported absent from both available non-cancer subsets.
• The generic BS1 default is 0.01, but no frequency value is available that could exceed either the generic default or a disorder-specific maximum credible allele frequency.

Sources

Reasoning

BS3 is not met. The directly tested p.(Ser310Tyr) variant showed gain-of-function phenotypes rather than no damaging effect: PMID:22908275 reported robust soft-agar colony formation, epithelial transformation, and IL-3-independent growth, while PMID:24997986 reported increased proliferation in ERBB2-mutant cell assays. Therefore, the available functional evidence is directionally inconsistent with a benign functional effect and does not support BS3.

Rule

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

Evidence considered

• PMID:22908275, direct S310Y assays showed robust soft-agar colony formation, AALE transformation, and IL-3-independent Ba/F3 growth.
• PMID:24997986, direct S310Y overexpression assays showed significantly increased proliferation relative to mock-transfected or wild-type ERBB2 controls.
• The functional results do not show absence of damaging effect; they provide positive evidence for ERBB2 activation and oncogenic activity.

Sources

Reasoning

The variant is a missense substitution, p.(Ser310Tyr), so the governing one-path rule requires REVEL only; SpliceAI is not used for missense PP3/BP4. REVEL is 0.54, above the generic BP4 supporting threshold of <= 0.29 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997), so BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

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

• NM_004448.4:c.929C>A is an exonic missense variant, NP_004439.2:p.(Ser310Tyr).
• REVEL score = 0.54; this is above the generic BP4 supporting cutoff of <=0.29 from ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• SpliceAI max delta = 0.014 was not used because the governing path for a missense variant is REVEL only.
• BayesDel score = 0.0783379 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The exact variant has one ClinVar Pathogenic submission from a single clinical laboratory and no expert-panel submission; there is no exact-variant ClinVar expert-panel Benign or Likely benign classification. Ordinary laboratory submissions, aggregate labels, and non-expert assertions cannot trigger BP6 under the required rule.

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 376188 exactly matches NM_004448.4(ERBB2):c.929C>A (p.Ser310Tyr); the only submission is Pathogenic, criteria provided, single submitter, with expert-panel submissions: 0.

Sources

Reasoning

No case-level evidence documents a proband with the disease, absence of family history, confirmed maternity, or confirmed paternity for NM_004448.4:c.929C>A (ERBB2 p.Ser310Tyr). The available evidence is limited to somatic cancer studies and does not establish a germline de novo event; 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 evidence documents NM_004448.4:c.929C>A as an ERBB2 missense variant but provides no proband phenotype, family-history statement, parental genotypes, or maternity/paternity confirmation.
• The fetched literature concerns somatic ERBB2 S310Y in cancer and does not provide a germline de novo observation for this case.

Still missing

• Affected proband phenotype and diagnosis
• Family-history status
• Variant testing in the proband and both biological parents
• Confirmed maternity and paternity

Reasoning

No germline case-control study or affected-versus-unaffected comparison for ERBB2 NM_004448.4:c.929C>A (p.Ser310Tyr) was identified. The reviewed reports describe the variant in somatic cancer cohorts and functional assays, but do not provide the affected and control counts, or a matched-ancestry control frequency, needed to calculate an odds ratio and 95% confidence interval. The governing PS4 threshold is >5.0 with a confidence interval that does not include 1.0; because no eligible estimate is available, no threshold inequality can be written and PS4 is not assessed.

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:22908275 reports ERBB2 S310Y in lung cancer samples and functional assays, but no unaffected control comparison or germline prevalence estimate.
• PMID:24997986 reports somatic ERBB2 S310Y among gallbladder carcinoma tumors and functional assays, but no eligible affected-versus-unaffected germline comparison.
• PMID:29420467 reports 30 patients with HER2 S310 hotspot mutations in a somatic cancer treatment study, without variant-specific S310Y case-control counts or population controls.
• PMID:29967253 was checked because it was retrieved for ERBB2 S310 literature; it discusses S310F rather than S310Y and provides no eligible case-control data.

Still missing

• An eligible germline case-control dataset with affected and unaffected counts, or case frequency plus matched-ancestry control frequency, is needed to calculate OR and 95% CI.

Sources

Reasoning

ERBB2 p.Ser310Tyr lies at residue S310, which the reviewed literature describes as a recurrent extracellular-domain hotspot, and the case materials report a statistically significant Cancer Hotspots result. However, generic PM1 requires a mutational hotspot or critical, well-established functional domain without benign variation. The available materials do not establish the required absence of benign variation across the relevant hotspot/domain, and no VCEP-specific hotspot-source route governs this generic assessment.

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

• The case evidence reports that ERBB2 S310 lies in a statistically significant hotspot, but the hotspot extraction is partial and supplies no benign-variation analysis.
• PMID:22908275 describes ERBB2 S310Y among oncogenic extracellular-domain mutations, and PMID:29420467 describes HER2 S310 as a hotspot extracellular-domain mutation; neither establishes the required absence of benign variation across the relevant region.

Still missing

• A validated assessment of benign variation across the S310 hotspot or relevant ERBB2 functional domain is unavailable.

Sources

Reasoning

The generic PM3 definition applies only to a recessive disorder when the variant is detected in trans with a pathogenic variant, and the governing note requires parental or offspring testing to determine phase. The case evidence contains no affected-proband observation, no pathogenic second allele, and no parental or offspring phase testing for NM_004448.4:c.929C>A (p.Ser310Tyr). Therefore, PM3 cannot be assessed from the available evidence.

Rule

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

Evidence considered

• The consolidated case evidence identifies ERBB2 NM_004448.4:c.929C>A (p.Ser310Tyr), but provides no affected-proband observation, second pathogenic allele, or parental/offspring phase testing.
• The available literature findings concern somatic cancer observations and functional assays, not recessive germline allelic phase or affected-proband compound heterozygosity.

Still missing

• Documented recessive ERBB2 disease context and inheritance mode
• A pathogenic or likely pathogenic second allele in an affected proband
• Parental or offspring testing establishing phase as trans

Reasoning

No case-level evidence documents an affected proband with the variant and a de novo occurrence assumed from parental testing or family data. The available evidence does not report parental genotypes, maternity, paternity, or a germline de novo observation; therefore PM6 cannot be assessed.

Rule

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

Evidence considered

• The case evidence provides no parental genotypes, family testing, or germline de novo observation for NM_004448.4:c.929C>A.
• The fetched literature concerns somatic ERBB2 S310Y in cancer and does not establish PM6 evidence.

Still missing

• Affected proband phenotype and diagnosis
• Parental genotypes or other evidence supporting de novo occurrence
• Maternity and paternity information

Reasoning

No affected family members, informative meioses, variant-positive relatives, or phenotype-consistent segregation data are reported for NM_004448.4:c.929C>A. The case bundle also does not establish the required disease diagnosis in a family or provide evidence that ERBB2 is definitively associated with that disease in this context; 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 segregation study, affected-relative genotype, unaffected-relative genotype, or informative meiosis is reported for NM_004448.4:c.929C>A.
• The available publications describe somatic cancer observations and functional assays, not familial germline segregation.

Still missing

• Disease diagnosis and phenotype in the proband and relatives
• Genotypes of affected and unaffected relatives
• Number of informative meioses
• Evidence establishing the relevant ERBB2-disease relationship

Reasoning

The reviewed case materials do not provide a gene-level analysis demonstrating both that ERBB2 has a low rate of benign missense variation and that missense variants are a common mechanism of disease in the applicable germline disease context. Somatic ERBB2 oncogenicity and individual functional assays do not 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 for both PP2 requirements are unavailable.

Reasoning

The case materials do not provide a patient phenotype or family history for this variant, and the reviewed somatic cancer reports do not establish a highly specific phenotype for a single-gene germline disease. Therefore the phenotype-specificity requirement cannot be evaluated.

Rule

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

Evidence considered

• The case summary contains no patient phenotype or family-history description; the literature reviewed concerns somatic ERBB2 cancer observations rather than a specific germline phenotype.

Still missing

• Patient-level phenotype and family-history information, together with a defined single-gene disease phenotype, is needed for PP4 assessment.

Reasoning

BS2 is not assessed. The case evidence contains no observation of this variant in a healthy adult, no homozygote or hemizygote count, and no phenotype- or age-qualified control observation. The available ClinVar record is a single clinical-laboratory submission and does not provide the healthy-adult observation required for 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

• No healthy-adult carrier observation, homozygote count, or hemizygote count is reported in the available population evidence.
• ClinVar variation 376188 contains one Pathogenic single-submitter record but no healthy-control observation or genotype-count evidence.
• GNOMAD_V2_1 and GNOMAD_V4_1 report the variant as absent rather than documenting a healthy carrier.

Still missing

• A documented healthy adult carrying the variant, with inheritance state and sufficient age/phenotype information, is required to assess BS2.

Sources

Reasoning

No affected family members with documented genotype and phenotype are available for comparison, so there is no evidence of either segregation or non-segregation. The case evidence does not provide a family structure, affected-relative testing, or an alternative explanation such as phenocopy; therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected-relative genotypes or phenotype records are reported for NM_004448.4:c.929C>A.
• No family-level non-segregation analysis or phenocopy assessment is available.

Still missing

• Family structure and affected relatives
• Variant testing in affected relatives
• Phenotype documentation for affected relatives
• Assessment of phenocopies or multiple pathogenic variants

Reasoning

The reviewed materials do not establish that ERBB2 is a gene for which primarily truncating variants are known to cause disease in the relevant germline context. The available ERBB2 literature emphasizes somatic activating missense variants and does not provide a sufficiently characterized disease-variant spectrum to apply BP1.

Rule

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

Still missing

• A validated germline ERBB2 disease-variant spectrum showing predominantly truncating pathogenic variants is unavailable.

Reasoning

The generic BP2 definition requires the variant to be observed in trans with a pathogenic variant in a fully penetrant dominant gene/disorder or in cis with a pathogenic variant in any inheritance pattern. The case evidence contains no affected-proband or family observation documenting a pathogenic partner allele, and no phase result showing cis or trans. 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

• The consolidated case evidence identifies ERBB2 NM_004448.4:c.929C>A (p.Ser310Tyr), but provides no pathogenic partner allele or validated cis/trans phase result.
• The available publications report somatic ERBB2 cancer findings and functional assays, not the allelic phase observations required for BP2.

Still missing

• A documented pathogenic partner allele
• Parental, offspring, or other validated testing establishing cis or trans phase
• Evidence that any dominant-disorder trans observation meets the fully penetrant requirement

Reasoning

The available reports describe this variant in somatic cancers, but the case materials do not establish that it was found in a patient with a confirmed alternate molecular basis for the relevant disease. Somatic occurrence alone does not demonstrate the BP5 circumstance, and no patient-level alternate diagnosis or molecular cause is documented.

Rule

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

Evidence considered

• PMID:22908275 and PMID:24997986 report somatic ERBB2 S310Y in cancer specimens, but do not document an alternate molecular diagnosis for a germline disease case.
• PMID:29420467 reports patients with HER2 S310 hotspot cancers in a somatic treatment study, without documenting the BP5 alternate-molecular-basis circumstance for this exact variant.

Still missing

• A patient-level report documenting the variant together with a confirmed alternate molecular basis for the disease is needed.

Sources