ERBB2 c.2329G>T, p.Val777Leu

Reasoning

PS3 is met at strong strength for ERBB2 p.Val777Leu (NM_004448.4:c.2329G>T). Two independent variant-specific functional studies provide concordant evidence of increased ERBB2 function relative to wild type. PMID:23220880 reported greater HER2 kinase-specific activity, increased phosphorylation of signaling proteins, invasive growth in Matrigel, increased soft-agar colony formation, and accelerated xenograft growth; the direct comparison states that V777L had greater tyrosine-kinase specific activity than wild type and strongly increased phosphorylation. PMID:22046346 independently reported enhanced JNK/SAPK and ERK1/2 activation, increased colony formation in soft agar, and retained lapatinib sensitivity. These assays span biochemical activity, downstream signaling, cellular transformation, and in-vivo tumor growth and are biologically relevant to ERBB2 gain of function. Wild-type comparators were used, but the available evidence does not state that the assays were validated and reproduced in a clinical diagnostic laboratory; therefore the evidence supports strong rather than an overstated level. No study in the reviewed material demonstrates absence of damaging effect or normal function for this variant.

Rule

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

Evidence considered

• Bose et al. 2013 (PMID:23220880): recombinant HER2 kinase-domain assays showed greater tyrosine-kinase specific activity for V777L than wild type, with increased activity after in-vitro dimerization; V777L increased phosphorylation of signaling proteins, invasive Matrigel structures, soft-agar colony formation, and xenograft tumor growth.
• Kancha et al. 2011 (PMID:22046346): ERBB2-V777L showed enhanced JNK/SAPK and ERK1/2 activation versus wild type in HEK293 cells and increased soft-agar colony formation; lapatinib sensitivity was also measured in Ba/F3 cells.
• The evidence is concordant across biochemical, signaling, cellular transformation, and in-vivo tumor-growth assays, with wild-type comparisons reported; no clinical-diagnostic-laboratory validation statement was available.

Still missing

• The reviewed studies do not state that the assays were validated and reproduced in a clinical diagnostic laboratory setting.

Sources

Reasoning

ERBB2 p.Val777Leu lies in the ERBB2 tyrosine-kinase domain, a well-established functional domain. Multiple functional studies directly show altered kinase activity, downstream signaling, transforming growth, or tumor growth for V777L, and the variant is absent from gnomAD v2.1 and v4.1. These findings support the critical-functional-domain route of generic PM1 without relying on a VCEP-specific hotspot rule.

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

• Bose et al. locate HER2 V777L in the kinase domain and report greater tyrosine-kinase activity than wild type, increased signaling, invasive cellular phenotypes, and accelerated xenograft growth.
• Kancha et al. characterize ERBB2 V777L among kinase-domain mutations and report enhanced JNK/SAPK and ERK1/2 activation and increased colony formation.
• The inflammatory breast carcinoma report states that V777L is in the HER2 kinase domain and is associated with aberrant and enhanced downstream signaling.
• The SUMMIT study describes V777 as a recurrent HER2 kinase-domain hotspot and reports V777L in four treated patients; this is supportive context, while the PM1 determination rests on the critical-domain route.
• The queried variant is absent from gnomAD v2.1 and gnomAD v4.1, providing the available population-variation check for the generic PM1 assessment.

Sources

Reasoning

PM2 is met at supporting strength. NM_004448.4:c.2329G>T (ERBB2 p.Val777Leu) is absent from gnomAD v2.1 and gnomAD v4.1, and the non-cancer gnomAD v2.1 and v3.1 queries also report absence. Thus the observed allele frequency is 0, satisfying the generic PM2 supporting threshold of <=0.0001; no governing VCEP requires a different population subset 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: NM_004448.4:c.2329G>T absent.
• gnomAD v4.1: NM_004448.4:c.2329G>T absent.
• gnomAD v2.1 non-cancer and v3.1 non-cancer queries also report absence; these are concordant with the default all-comers datasets.
• No VCEP covers ERBB2 in this case, so generic ACMG/AMP rules govern.
• Generic calibration supplied for this case: PM2 supporting threshold <=0.0001 (ClinGen SVI recommendation supplied with PMID:25741868).

Sources

Reasoning

The variant is a missense substitution, NM_004448.4:c.2329G>T (p.Val777Leu), so the governing generic rule selects the REVEL path only; SpliceAI and BayesDel are not used for BP4 in this situation. REVEL is 0.284, which meets the generic BP4 supporting threshold of <=0.29. Therefore BP4 is met at supporting strength.

Rule

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

Evidence considered

• NM_004448.4:c.2329G>T is an exon 20 missense variant producing p.Val777Leu, so only the missense REVEL path applies.
• Local REVEL lookup reports 0.284 for the variant (source_registry key: revel).
• The generic BP4 supporting threshold is REVEL <=0.29, from the ClinGen SVI REVEL calibration by Pejaver et al. 2022 (PMID:36413997); 0.284 meets it.
• SpliceAI max delta 0.076 was not counted because the governing missense path selects REVEL only; BayesDel was not counted because no generic ACMG-strength calibration is established.

Sources

Reasoning

PMID:24516025 reports the exact ERBB2 V777L variant in an inflammatory breast carcinoma together with a second ERBB2 mutation, S310F, each at approximately 47% allele frequency. The paper states that the clinical response occurred during combination HER2-targeted treatment and could not be attributed specifically to V777L because S310F was also present. This documents an alternate molecular basis in the same disease case, satisfying the supplied BP5 definition.

Rule

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

Evidence considered

• PMID:24516025, Results and Table 1: targeted NGS identified ERBB2 V777L and S310F in the same inflammatory breast carcinoma, each at 47% allele frequency.
• PMID:24516025, Discussion: the patient's response to combination HER2-targeted therapy could not be attributed specifically to V777L because the tumor also carried S310F and received combination treatment.
• The co-occurring S310F provides the alternate molecular basis required by the supplied BP5 rule.

Sources

Reasoning

The queried variant is ERBB2 p.Val777Leu (c.2329G>T). The reviewed literature documents this exact amino-acid change and its activating somatic effects, but no previously established pathogenic variant caused the same Val-to-Leu change through a different nucleotide change. The available ClinVar submission classifies the exact variant as uncertain significance, not pathogenic. Therefore PS1 is not met.

Rule

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

Evidence considered

• The case normalization identifies NM_004448.4:c.2329G>T as NP_004439.2:p.(Val777Leu).
• Bose et al. explicitly studied HER2 V777L and reported activating functional effects; this is the same nucleotide/protein variant, not a different nucleotide change producing Val777Leu.
• Kancha et al. explicitly studied ERBB2 V777L; the paper likewise does not establish a distinct pathogenic nucleotide change for the same amino-acid substitution.
• The ClinVar record for variation 44991 reports the exact variant as uncertain significance with criteria provided by a single submitter.

Sources

Reasoning

The PS4-indexed literature was checked against the supplied abstracts. PMID 16988931 reports HER2 mutations in lung tumors but not exact V777L and compares tumor subgroups rather than affected individuals with unaffected or population controls. PMID 31046123 reports V777L in functional pancreatic-cancer experiments without patient/control prevalence data. PMID 32017710 reports grouped ERBB2 mutations in 3/7 sporadic-schwannomatosis-associated tumors versus 0/6 neurofibromatosis-associated tumors and 0/1 nonsyndromic tumor, but not exact-V777L affected-versus-control counts. No qualifying exact-variant case-control counts were available, so an OR and 95% CI could not be computed. The governing operator is literally >: OR > 5.0? not evaluable, because no qualifying OR exists; therefore the required OR threshold and confidence-interval condition are not demonstrated.

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

• PS4-indexed PMID 16988931 abstract: 9/403 lung adenocarcinomas carried HER2 mutations, with subgroup comparison 7/113 versus 2/290; V777L and unaffected/population controls were not reported.
• PS4-indexed PMID 31046123 abstract: V777L was included in a functional pancreatic epithelial-cell and xenograft study; no exact-variant patient/control prevalence data were reported.
• PS4-indexed PMID 32017710 abstract: grouped ERBB2 mutations including p.Val777Leu occurred in 3/7 sporadic-schwannomatosis-associated tumors and in 0/6 neurofibromatosis-associated and 0/1 nonsyndromic tumors; this was not an exact-V777L case-control comparison.
• No qualifying exact-variant affected-versus-unaffected or population-control counts were available, so OR and 95% CI were not computed.
• The governing PS4 operator is > and the threshold is 5.0: OR > 5.0? not evaluable; the confidence interval condition also cannot be evaluated.

Reasoning

The variant affects residue Val777. The reviewed same-residue evidence identifies V777L and recurrent V777 hotspot occurrence, but does not identify a different missense substitution at residue 777 that has been determined pathogenic. Therefore the required PM5 comparator is absent and PM5 is not met.

Rule

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

Evidence considered

• Bose et al. report V777L as the tested ERBB2 substitution and do not report a different pathogenic missense change at residue 777.
• Chang et al. identify ERBB2 V777 as a recurrent somatic hotspot but do not establish a different pathogenic missense substitution at that residue.
• The SUMMIT study reports V777L among V777 hotspot alleles but supplies no different pathogenic Val777 missense comparator.
• The same-residue candidate collection contains zero confirmed candidates and does not establish a classic PM5 comparator.

Sources

Reasoning

The variant is a missense substitution, NM_004448.4:c.2329G>T (p.Val777Leu), so the governing generic rule selects the REVEL path only; SpliceAI and BayesDel are not used for PP3 in this situation. REVEL is 0.284, below the generic PP3 supporting threshold of >=0.644. Therefore 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.). ... As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent

Evidence considered

• NM_004448.4:c.2329G>T is an exon 20 missense variant producing p.Val777Leu, so only the missense REVEL path applies.
• Local REVEL lookup reports 0.284 for the variant (source_registry key: revel).
• The generic PP3 supporting threshold is REVEL >=0.644, from the ClinGen SVI REVEL calibration by Pejaver et al. 2022 (PMID:36413997); 0.284 does not meet it.
• SpliceAI max delta 0.076 and BayesDel 0.00780532 were not counted because the governing missense path selects REVEL only and no generic BayesDel calibration is established.

Sources

Reasoning

The exact-variant ClinVar record is a single-submitter, criteria-provided Uncertain significance classification with zero expert-panel submissions. Other ordinary submissions include an Oncogenic assertion, but no exact-variant expert-panel Pathogenic or Likely pathogenic classification is present. Therefore the required generic PP5 trigger is absent.

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 44991 exactly matches NM_004448.4:c.2329G>T (p.Val777Leu), but its review status is criteria provided, single submitter, with classification Uncertain significance and zero expert-panel submissions.
• The ClinVar submission audit records no exact-variant expert-panel Pathogenic or Likely pathogenic assertion; ordinary laboratory submissions and aggregate or non-expert assertions do not trigger PP5.

Sources

Reasoning

BA1 is not met. NM_004448.4:c.2329G>T (ERBB2 p.Val777Leu) is absent from gnomAD v2.1 and gnomAD v4.1, so its filtering allele frequency is 0 and does not reach the generic stand-alone threshold of >=0.05. The non-cancer gnomAD v2.1 and v3.1 queries also report absence. No population-frequency conflict requiring the BA1 stand-alone caution is present in the available evidence.

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.2329G>T absent; no allele count or frequency reported.
• gnomAD v4.1: NM_004448.4:c.2329G>T absent; no allele count or frequency reported.
• gnomAD v2.1 non-cancer and v3.1 non-cancer queries also report the variant absent.
• Generic calibration supplied for this case: BA1 stand-alone threshold >=0.05 (ACMG/AMP 2015, PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with filtering allele frequency 0, which is not greater than expected for the disorder and is below the generic default threshold of >=0.01. Because no elevated ancestry-specific frequency is present, a maximum-credible-allele-frequency calculation is not needed to reject this criterion.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: NM_004448.4:c.2329G>T absent; filtering allele frequency is therefore 0.
• gnomAD v4.1: NM_004448.4:c.2329G>T absent; filtering allele frequency is therefore 0.
• No elevated ancestry-specific frequency, founder signal, or population threshold artifact is reported for this variant.
• Generic calibration supplied for this case: BS1 default threshold >=0.01; the prompt directs use of filtering allele frequency rather than popmax point estimate (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

BS3 is not met because the available variant-specific functional evidence does not show no damaging effect on ERBB2 function or splicing. Instead, PMID:23220880 and PMID:22046346 report increased kinase activity or downstream signaling and increased transforming phenotypes for p.Val777Leu compared with wild type. The studies therefore provide evidence in the opposite direction from the benign-functional requirement of BS3. No benign functional assay demonstrating normal protein function or splicing was identified.

Rule

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

Evidence considered

• Bose et al. 2013 (PMID:23220880): V777L had greater HER2 tyrosine-kinase specific activity than wild type and strongly increased phosphorylation of signaling proteins, with increased cellular transformation and xenograft growth.
• Kancha et al. 2011 (PMID:22046346): V777L showed enhanced downstream signaling and increased soft-agar colony formation compared with wild-type ERBB2.
• No reviewed source reported a normal-function or normal-splicing result for ERBB2 p.Val777Leu.

Still missing

• No variant-specific benign functional assay demonstrating normal ERBB2 protein function or splicing was identified.

Sources

Reasoning

The exact-variant ClinVar record is a criteria-provided single-submitter Uncertain significance classification with zero expert-panel submissions. No exact-variant expert-panel Benign or Likely benign classification is present, so the required generic BP6 trigger is absent.

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 44991 exactly matches NM_004448.4:c.2329G>T (p.Val777Leu), but the record is single-submitter Uncertain significance with zero expert-panel submissions.
• The ClinVar submission audit shows no exact-variant expert-panel Benign or Likely benign assertion; ordinary laboratory submissions and aggregate or non-expert assertions do not trigger BP6.

Sources

Reasoning

No proband-level germline de novo observation is documented. The available literature and case records concern somatic ERBB2 V777L in tumors and do not establish both maternity and paternity, absence of family history, or a germline patient with the disease; 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 available variant-specific reports describe somatic ERBB2 V777L in tumors and provide no parental testing or germline de novo evidence.
• No proband, maternity, paternity, or family-history data relevant to PS2 are present in the reviewed case records.

Still missing

• Germline variant testing in the proband is not documented.
• Both maternity and paternity confirmation are not documented.
• The proband's disease phenotype and family history are not documented for this variant.

Reasoning

PM3 cannot be assessed because the available evidence does not establish that this ERBB2 variant occurs in a recessive disorder, in an affected proband, or in trans with a pathogenic variant. No parental or offspring testing, phase result, second pathogenic allele, or eligible affected-proband observation was identified. The available literature and ClinVar evidence concern somatic ERBB2 cancer observations rather than germline allelic observations and therefore do not establish PM3.

Rule

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

Evidence considered

• The case materials identify NM_004448.4:c.2329G>T (p.Val777Leu) in ERBB2 but provide no germline affected-proband observation, parental/offspring testing, phase result, or second pathogenic allele.
• The reviewed variant-specific literature and ClinVar records describe somatic ERBB2 cancer findings and do not establish a germline in-trans observation relevant to PM3.

Still missing

• A defined recessive ERBB2 disorder context is not established.
• No affected-proband observation with a pathogenic allele in trans is documented.
• No parental or offspring testing establishes phase.
• No second pathogenic variant is documented in the relevant individual.

Reasoning

No assumed de novo germline occurrence is documented for this variant. The available reports are somatic tumor studies and contain no proband-level de novo observation or parental testing status; therefore PM6 cannot be assessed.

Rule

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

Evidence considered

• The available variant-specific reports describe somatic ERBB2 V777L in tumors and do not report an assumed de novo germline case.
• No parental testing information relevant to PM6 is present in the reviewed case records.

Still missing

• A germline de novo observation in an affected proband is not documented.
• Parental testing status is not documented.

Reasoning

No affected family members, informative meioses, or segregation results are documented for the variant. The available evidence consists of somatic tumor observations and functional studies rather than familial cosegregation; 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

• The available variant-specific reports do not provide family-level segregation, affected-relative genotypes, or informative meioses.
• Somatic tumor observations and functional assay results do not constitute PP1 cosegregation evidence.

Still missing

• Affected relatives and their phenotypes are not documented.
• Variant genotypes and disease status across family members are not documented.
• Informative meioses or segregation results are not documented.

Reasoning

The available evidence shows that ERBB2 p.Val777Leu is absent from gnomAD and has somatic gain-of-function evidence, but it does not establish both required gene-level premises: a low rate of benign missense variation and missense variants as a common mechanism of disease in the applicable germline disease context. The criterion is therefore not assessed.

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 queried variant is absent from gnomAD v2.1 and v4.1, but single-variant absence is not a gene-level benign-missense-rate assessment.
• The functional and OncoKB evidence concerns somatic ERBB2 gain of function and does not by itself establish the generic PP2 germline disease-mechanism premise.

Still missing

• Gene-level evidence quantifying benign missense variation for ERBB2.
• Established applicable disease context showing missense variants are a common mechanism of disease for ERBB2.

Sources

Reasoning

The case materials describe somatic ERBB2 tumor findings but do not provide an individual patient phenotype or family history that is highly specific for a disease with a single genetic etiology. The required clinical phenotype information is absent.

Rule

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

Evidence considered

• The case materials provide somatic tumor and molecular findings but no individual clinical phenotype or family-history description suitable for PP4 assessment.

Still missing

• Patient phenotype and family history matched to a specific disease with a single genetic etiology.

Reasoning

BS2 is not assessed. The available population evidence reports only that the variant is absent from gnomAD v2.1 and v4.1; it does not identify any healthy adult carrier, homozygote, or hemizygote. There is also no case-specific evidence establishing the relevant disorder's inheritance, expected penetrance, or early-age full penetrance needed to interpret a healthy-adult observation under BS2.

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 report the variant absent, not an observed healthy adult genotype.
• The available case evidence does not provide a healthy-adult observation or sufficient disorder-specific penetrance and age-of-onset information.

Still missing

• No documented healthy adult individual carrying this variant in the required zygosity.
• No case-specific disease inheritance, penetrance, and age-of-onset context for applying BS2.

Sources

Reasoning

No family with affected members tested for the variant is documented, so lack of segregation cannot be evaluated. The available somatic tumor literature does not provide family-level non-segregation evidence; therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No family-level variant testing or affected-relative phenotype data relevant to non-segregation are present in the reviewed case records.
• The available literature concerns somatic ERBB2 V777L in tumors and does not establish BS4 evidence.

Still missing

• A family containing affected members is not documented.
• Variant testing and phenotypic status for affected relatives are not documented.
• Potential phenocopies or multiple pathogenic variants cannot be evaluated without family data.

Reasoning

The available case evidence is dominated by somatic ERBB2 missense oncogenicity and does not establish that ERBB2 disease is caused primarily by truncating variants. No applicable gene-level disease series or authoritative mechanism summary was supplied to satisfy the BP1 premise, so BP1 is not assessed.

Rule

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

Evidence considered

• The available literature concerns somatic ERBB2 missense mutations and does not establish the required truncating-variant disease pattern.
• No VCEP or other authoritative gene-level mechanism source is available in this case for BP1.

Still missing

• Authoritative gene-level evidence showing that primarily truncating ERBB2 variants cause the relevant disease.

Reasoning

BP2 cannot be assessed because the available evidence does not document this variant with a pathogenic variant in trans in a fully penetrant dominant disorder or with a pathogenic variant in cis in any inheritance pattern. No affected individual, second pathogenic allele, parental or offspring testing, or cis/trans phase result was identified. Somatic cancer reports and functional studies do not provide the required allelic phase 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 case materials contain no documented cis or trans relationship between this variant and another pathogenic variant, and no parental or offspring phase testing.
• The reviewed somatic cancer observations and functional studies do not establish the allelic configuration required for BP2.

Still missing

• No pathogenic partner variant is documented.
• No parental or offspring testing establishes cis or trans phase.
• No fully penetrant dominant disorder context with an affected individual is documented.