ERBB2 c.2313_2324dup, p.Tyr772_Ala775dup

Reasoning

PS3 is met at strong strength. PMID:16843263 experimentally evaluated the corresponding HER2 exon 20 YVMA insertion in multiple complementary systems, including in vitro kinase assays, phosphorylation and signaling assays, epithelial-cell proliferation, apoptosis, migration, soft-agar transformation, three-dimensional Matrigel invasion and acinar morphogenesis, and an in vivo xenograft assay. Relative to wild-type HER2, the mutant showed markedly increased kinase activity, at least five-fold greater phosphorylation of kinase-dead EGFR in vitro, enhanced ligand-independent signaling, increased transformation and invasion, protection from apoptosis, and tumors in 4/6 mice versus none with wild-type HER2 or vector. Mutant-specific RNA interference reduced mutant HER2 protein by 90%, reduced downstream signaling, increased apoptosis, and decreased proliferation in H1781 cells. The study used appropriate wild-type and vector controls and convergent biochemical, cellular, and in vivo readouts; although it was not a validated clinical diagnostic laboratory assay, the breadth and concordance of the functional evidence support strong PS3. No BS3 evidence is inferred from this result because the assays demonstrate increased activity rather than absence of damaging effect.

Rule

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

Evidence considered

• Wang et al. 2006, Cancer Cell, PMID:16843263: the corresponding HER2 exon 20 YVMA insertion was tested against wild-type HER2 and vector controls in biochemical, cellular, and in vivo assays.
• Wang et al. 2006, Figure 4C and Results: the insertion mutant phosphorylated kinase-dead EGFR at least five-fold more than wild-type HER2 in vitro.
• Wang et al. 2006, Figure 1D and Results: cells expressing the insertion mutant produced established tumors in 4 of 6 athymic nude mice, whereas no tumors were detected with wild-type HER2 or vector.
• Wang et al. 2006, Figure 6 and Results: mutant-specific RNA interference reduced mutant HER2 protein by 90%, reduced phospho-MAPK and phospho-Akt, increased apoptosis, and decreased proliferation in H1781 cells.
• Wang et al. 2006, Summary and Discussion: the insertion mutant showed increased kinase activity, ligand-independent signaling, transformation, invasion, and survival relative to wild-type HER2.

Sources

Reasoning

PM2 is met at supporting strength. The exact ERBB2 NM_004448.4:c.2313_2324dup variant is absent from gnomAD v2.1 and gnomAD v4.1, and the evidence separately reports absence from the gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets. Therefore the variant is absent from the available population controls and is below the supplied generic PM2 supporting threshold of <=0.0001. No governing VCEP specifies an alternate population source because no VCEP covers ERBB2 in this case.

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: exact variant absent; no observed alleles reported, consistent with AF 0 in the queried dataset.
• gnomAD v4.1: exact variant absent; no observed alleles reported, consistent with AF 0 in the queried dataset.
• The evidence also reports exact-variant absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets.
• No VCEP or gene-specific population specification is present; generic ACMG/AMP rules therefore govern.
• Generic PM2 supporting threshold supplied in the prompt: <= 0.0001, identified as the ClinGen SVI recommendation (PMID:25741868).

Sources

Reasoning

NM_004448.4:c.2313_2324dup is an in-frame 12-bp duplication in the MANE Select transcript NM_004448.4, producing NP_004439.2:p.(Tyr772_Ala775dup), a four-amino-acid protein-length increase. The change lies in exon 20 within the ERBB2 tyrosine-kinase domain, a structured functional region rather than a low-complexity repetitive region, and the evidence bundle provides no indication that this site is a repetitive region. It therefore meets generic PM4 for an in-frame protein-length change in a non-repeat region. This is a generic PM4 application; the variant is not a stop-loss change and PVS1 is not being assessed in this group.

Rule

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

Evidence considered

• VariantValidator/Mutalyzer normalization in the case evidence: NM_004448.4:c.2313_2324dup maps to NP_004439.2:p.(Tyr772_Ala775dup), with the variant in exon 20.
• The normalized protein consequence is an in-frame duplication of Tyr772 through Ala775, adding four amino acids and increasing protein length.
• The affected ERBB2 exon-20 segment is within the tyrosine-kinase domain; the supplied protein sequence and case evidence do not identify a low-complexity or repetitive region at this site.

Reasoning

Generic ACMG/AMP PS4 requires a statistically significant increase in affected individuals compared with controls, with OR or RR >5.0 and a 95% confidence interval excluding 1.0. PMID:15457249 reports ERBB2 mutations in 4% of 120 primary lung tumors and 10% of adenocarcinomas, but the abstract provides no unaffected or population-control comparison and does not identify this exact duplication in the abstract. PMID:29420467 reports the exact Y772_A775dup in 12.0% of the SUMMIT cohort versus 2.7% in comparator published genomic datasets, but those comparators are not established unaffected or population controls and no variant-specific OR or 95% CI is reported; therefore no qualifying case-control estimate can be calculated. PMID:24627688 is an ALK testing guideline and provides no relevant ERBB2 case-control evidence.

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:15457249 PubMed abstract: 4% of 120 primary lung tumors had ERBB2 kinase-domain mutations and 10% of lung adenocarcinomas had mutations; no control group or exact-variant count is reported in the abstract.
• PMID:29420467, Results and Discussion: the exact Y772_A775dup was present in 12.0% of the SUMMIT cohort versus 2.7% in comparator published genomic datasets; these are not documented unaffected/population controls, and no variant-specific OR or 95% CI is provided, so no OR/CI was computed.
• PMID:24627688 PubMed abstract: an ALK-rearrangement testing guideline with no ERBB2-specific case-control evidence.

Still missing

• A matched affected-versus-unaffected or population-control dataset for the exact ERBB2 c.2313_2324dup variant, with counts or an OR/RR and 95% CI, is unavailable.

Sources

Reasoning

PP5 is restricted here to an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic. ClinVar variation 13875 matches NM_004448.4:c.2313_2324dup exactly, but the record has review status 'criteria provided, single submitter', reports zero expert-panel submissions, and contains only ordinary laboratory or literature-only submissions; therefore 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 variation 13875 exactly matches NM_004448.4(ERBB2):c.2313_2324dup (p.Tyr772_Ala775dup), but its review status is 'criteria provided, single submitter' and the submission audit reports expert_panel_submissions = 0.
• The ClinVar record contains Pathogenic/Likely pathogenic ordinary submissions, not an exact-variant expert-panel classification; those submissions were not used to trigger PP5.

Sources

Reasoning

BA1 is not met. The exact ERBB2 NM_004448.4:c.2313_2324dup variant is absent from gnomAD v2.1 and gnomAD v4.1, so no allele frequency or filtering allele frequency is available, much less a grpmax FAF of at least 0.05. The absence also means there is no population-frequency basis for a stand-alone benign call; the somatic oncogenic/clinical literature does not substitute for the required population-frequency threshold.

Rule

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

Evidence considered

• gnomAD v2.1: exact variant absent; no allele count, total allele number, AF, or grpmax FAF reported.
• gnomAD v4.1: exact variant absent; no allele count, total allele number, AF, or grpmax FAF reported.
• Generic calibration threshold supplied for BA1: >= 0.05, identified in the prompt as the ACMG/AMP 2015 stand-alone benign allele-frequency threshold (PMID:25741868).

Sources

Reasoning

BS1 is not met. The exact variant is absent from gnomAD v2.1 and v4.1, including the reported non-cancer subsets, so there is no filtering allele frequency showing that the allele is greater than expected for the disorder. Because no elevated frequency is observed, a maximum-credible-allele-frequency calculation is not needed to reject the generic 0.01 trigger in this case. No population-frequency threshold artifact or ancestry-specific elevation is present in the available data.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: exact variant absent; no grpmax FAF or observed allele count is available for comparison with a disease-specific maximum credible allele frequency.
• gnomAD v4.1: exact variant absent; no grpmax FAF or observed allele count is available for comparison with a disease-specific maximum credible allele frequency.
• The evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer subsets; no non-cancer frequency is available.
• Generic BS1 default supplied in the prompt: >= 0.01; the prompt directs that filtering allele frequency (grpmax FAF), not popmax point estimate, must be used.
• The prompt identifies Whiffin et al. 2017 (PMID:28518168) as the source for maximum credible allele-frequency reasoning, but no elevated frequency requiring that calculation is present here.

Sources

Reasoning

BS3 is not met. The reviewed functional study did not show no damaging effect on protein function or splicing; instead, the corresponding HER2 exon 20 insertion demonstrated increased kinase activity, ligand-independent signaling, cellular transformation, invasion, survival, and tumorigenicity relative to wild-type HER2. No well-established benign functional or splicing assay result for this variant was identified. The absence of a benign assay 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

• Wang et al. 2006, PMID:16843263: all variant-directed functional results supported gain of function relative to wild-type HER2, rather than a normal or benign effect; the paper did not provide a benign splicing assay for NM_004448.4:c.2313_2324dup.

Still missing

• No well-established benign functional or splicing assay result for NM_004448.4:c.2313_2324dup was identified.

Sources

Reasoning

The variant is an in-frame duplication, but BP3 requires an in-frame deletion or insertion in a repetitive region without a known function. This ERBB2 duplication affects exon 20 in the tyrosine-kinase domain, a known functional region, so the required absence of known function is not satisfied. The available evidence does not place the variant in a nonfunctional repetitive region.

Rule

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

Evidence considered

• VariantValidator/Mutalyzer normalization in the case evidence places NM_004448.4:c.2313_2324dup (p.Tyr772_Ala775dup) in exon 20 of ERBB2.
• The affected exon-20 region encodes the ERBB2 tyrosine-kinase domain, providing known functional context and excluding the BP3 condition of a repetitive region without known function.

Reasoning

BP6 is restricted here to an exact-variant ClinVar expert-panel classification of Benign or Likely benign. ClinVar variation 13875 has no benign or likely-benign expert-panel classification and reports zero expert-panel submissions; its available submissions are pathogenic, likely pathogenic, or oncogenic single-submitter assertions. BP6 is therefore 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 variation 13875 exactly matches NM_004448.4(ERBB2):c.2313_2324dup (p.Tyr772_Ala775dup), but the audit reports expert_panel_submissions = 0.
• Available ClinVar submissions are Pathogenic, Likely pathogenic, or Oncogenic; no exact-variant benign or likely-benign expert-panel assertion is present.

Sources

Reasoning

No germline proband phenotype record, family-history assessment, or parental testing results are provided. The available case evidence concerns a somatic ERBB2 tumor variant and does not establish confirmed maternity and paternity or a de novo occurrence in a patient with the disease.

Rule

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

Evidence considered

• The available variant-specific publication reports Y772_A775dup in a somatic HER2-mutant cancer treatment cohort and explicitly provides no segregation data; it does not establish germline de novo status.
• No case-level germline parental testing, maternity confirmation, paternity confirmation, or family-history evidence is present in the supplied records.

Still missing

• Germline proband disease phenotype and family-history documentation
• Parental testing confirming both maternity and paternity
• Evidence that the variant is de novo rather than somatic or inherited

Sources

Reasoning

PM3 cannot be assessed because the available record contains no established recessive ERBB2 disorder, no affected-proband observation with a pathogenic allele, and no parental or offspring testing establishing that this variant is in trans with a pathogenic variant. The available ClinVar records describe somatic lung-cancer submissions rather than germline phase evidence.

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

• ClinVar variation 13875 matches NM_004448.4:c.2313_2324dup and lists available submissions as somatic, including lung adenocarcinoma-related records; it does not provide germline phase, a pathogenic allele in trans, or affected-proband segregation data.
• The case evidence identifies no CSPEC/VCEP and provides no recessive ERBB2 inheritance framework or parental/offspring phase testing for this variant.

Still missing

• Confirm the relevant inherited disease and inheritance mode for ERBB2.
• Obtain affected-proband data identifying a pathogenic second allele, if present.
• Obtain parental or offspring testing to establish trans phase.

Sources

Reasoning

No evidence documents an assumed de novo germline variant in the proband. The available variant-specific evidence is from somatic cancer studies and contains no parental or family testing information, so an assumed de novo occurrence cannot be assigned.

Rule

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

Evidence considered

• The variant-specific SUMMIT study evaluates HER2 mutations in tumors and reports no segregation data, so it cannot support assumed germline de novo status.
• No case-level report documenting the proband's germline status or parental genotypes is available in the supplied records.

Still missing

• Germline testing of the proband
• Parental testing or documented absence of parental testing
• Case-level evidence supporting de novo status

Sources

Reasoning

No affected family members, informative meioses, segregation table, or genotype-phenotype observations are documented. The available variant-specific publication concerns somatic tumor responses and does not provide familial segregation evidence.

Rule

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

Evidence considered

• The available variant-specific publication reports tumor-level treatment outcomes and no segregation data; it does not provide affected relatives or informative meioses.
• No familial pedigree, affected-relative testing, or genotype-phenotype co-segregation information is present in the supplied records.

Still missing

• Pedigree with clinically affected relatives
• Genotypes and phenotypes for informative family members
• Number of informative meioses and segregation observations

Sources

Reasoning

The governing PP4 definition requires a patient phenotype or family history that is highly specific for a disease with a single genetic etiology. The available case evidence identifies a somatic lung-cancer context but does not provide an individual phenotype, family history, or a disease-specific clinical presentation sufficient to assess this criterion.

Rule

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

Evidence considered

• Case summary identifies a somatic lung-cancer context but supplies no patient-level phenotype or family-history description for PP4 assessment.

Still missing

• Patient-level phenotype and family-history information, including the disease being evaluated and its etiologic specificity, is unavailable.

Reasoning

BS2 is not assessed. The available case evidence contains no identified healthy adult carrier, no homozygote or hemizygote observation, and no individual-level population genotype data for this exact variant. Absence from gnomAD is relevant to PM2 but cannot establish the BS2 requirement of observation in a healthy adult with the inheritance-specific genotype and an early, fully penetrant disorder.

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 available gnomAD records report the exact variant as absent rather than documenting a healthy adult genotype or an individual-level observation.
• No case-specific publication was identified in the available literature extracts that reports a healthy adult carrying this exact germline variant.

Still missing

• No individual-level healthy-adult observation with the genotype required for the relevant inheritance model.
• No evidence establishing that the relevant disorder is fully penetrant and expected to manifest early for applying BS2.

Reasoning

No affected family members or familial genotypes are documented, so lack of segregation cannot be evaluated. The available somatic cancer publication does not provide a family study and cannot establish non-segregation or address phenocopy and multilocus explanations.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The available variant-specific publication reports tumor responses and no segregation data; it does not document affected relatives who lack the variant.
• No family-level genotype-phenotype dataset is present, so non-segregation cannot be distinguished from phenocopy or additional pathogenic variants.

Still missing

• Affected-family-member phenotypes
• Variant testing in affected relatives
• Family structure sufficient to evaluate segregation and possible phenocopies

Sources

Reasoning

BP2 cannot be assessed because the available record contains no germline evidence that this variant is in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant. The available ClinVar and literature records concern somatic cancer observations and do not establish germline phase or a pathogenic partner allele.

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

• ClinVar variation 13875 matches NM_004448.4:c.2313_2324dup and lists available submissions as somatic; the record does not document a germline pathogenic partner variant or cis/trans phase.
• The available variant-specific paper evidence describes somatic ERBB2/HER2 cancer observations and does not provide germline allelic phase or inheritance data for this variant.

Still missing

• Determine whether a fully penetrant dominant ERBB2 disorder is relevant, or identify the applicable inheritance context.
• Identify any pathogenic partner variant in the proband.
• Obtain molecular phasing to establish cis or trans configuration.

Sources

Reasoning

BP5 requires the variant to be found in a case with an alternate molecular basis for disease. The available evidence documents the ERBB2 variant in somatic cancer cohorts but does not identify an individual case with a separate molecular diagnosis that explains the disease instead of this variant.

Rule

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

Evidence considered

• Available literature reports ERBB2 Y772_A775dup in somatic cancer cohorts and treatment-response analyses, but no individual alternate molecular diagnosis is documented for BP5.

Still missing

• A case-level report demonstrating an alternate molecular basis for the relevant disease, independent of ERBB2 c.2313_2324dup, is unavailable.

Sources