EGFR c.2156G>C, p.Gly719Ala

Reasoning

PS3 is met at strong strength. Two independent, variant-specific functional studies evaluated EGFR p.Gly719Ala. Kobayashi et al. (PMID:26206867) used sequence-confirmed retroviral constructs in Ba/F3 and NIH/3T3 cells, with wild-type/eGFP controls and the established EGFR Del19 comparator. G719A cells grew without IL-3, formed foci in NIH/3T3 cells, and showed reproducible altered EGFR-TKI sensitivity, including approximately 25-fold greater neratinib sensitivity than Del19. The same study confirmed the pharmacologic phenotype by EGFR phosphorylation assays in HEK293 cells. An orthogonal protein-array/functional study (PMID:27294619) measured ligand-independent EGFR activation in NIH3T3 cells: normalized pTyr1068 phosphorylation was 0.89 for Gly719Ala versus 0.21 for wild-type EGFR, and the authors reported gefitinib sensitivity. These concordant transformation, signaling, and drug-response results directly support an abnormal, oncogenic effect of the variant. The evidence is strong functional support, although the studies were research assays rather than validated clinical diagnostic-laboratory assays.

Rule

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

Evidence considered

• Kobayashi et al. 2015 (PMID:26206867): variant-specific retroviral EGFR G719A assays in Ba/F3 and NIH/3T3 cells; G719A cells grew without IL-3 and formed foci, whereas wild-type/eGFP controls did not.
• Kobayashi et al. 2015 (PMID:26206867): G719A showed altered EGFR-TKI sensitivity, with approximately 25-fold greater neratinib sensitivity than EGFR Del19, and the phenotype was confirmed by EGFR phosphorylation assays in HEK293 cells.
• Yuan et al. 2016 (PMID:27294619): NIH3T3 EGFR phosphorylation assay measured normalized pTyr1068 phosphorylation of 0.89 for Gly719Ala versus 0.21 for wild-type EGFR without EGF, interpreted as ligand-independent activation; gefitinib sensitivity was also reported.

Sources

Reasoning

EGFR p.Gly719Ala lies at codon 719 in the receptor tyrosine-kinase domain. Cancer Hotspots reports EGFR G719 as a statistically significant mutational hotspot, and the source record identifies the queried G719 variant as lying in that hotspot. Independent functional literature places Gly719Ala in the EGFR kinase-domain mutation cluster and reports ligand-independent EGFR activation. The variant is absent from gnomAD v2.1 and v4.1; however, the hotspot extraction is flagged as partially parsed, so the hotspot record 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 reports: 'This variant lies in a statistically significant hotspot.' The queried record is EGFR G719, although the extraction notes that the G719 hotspot row was only partially parsed.
• Protein-structure-guided discovery of functional mutations across 19 cancer types, Results/Figure 6b-c discussion: Gly719Ala is in the EGFR kinase-domain cluster and showed normalized EGFR pTyr1068 phosphorylation of 0.89 versus 0.21 for wild type, indicating ligand-independent activation.
• The exact variant is absent from gnomAD v2.1 and gnomAD v4.1.

Still missing

• Human confirmation of the partially parsed Cancer Hotspots G719 record is recommended.

Sources

Reasoning

PM2 is met at supporting strength. NM_005228.5:c.2156G>C (EGFR p.Gly719Ala) was reported absent from all-comers gnomAD v2.1 and gnomAD v4.1, and the available non-cancer subsets likewise reported absence. Thus the observed allele frequency is 0 in the reported datasets, satisfying the generic PM2 supporting threshold of <=0.0001; no VCEP-specific population source requires substitution of a different dataset.

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 (exome), all-comers: variant absent; observed allele frequency 0 in the queried dataset.
• gnomAD v4.1, all-comers: variant absent; observed allele frequency 0 in the queried dataset.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER also reported the variant absent. The generic PM2 calibration supplied for this case is <=0.0001, supporting strength (ClinGen SVI recommendation, PMID:25741868).

Sources

Reasoning

NM_005228.5:c.2156G>C is an exonic missense substitution, p.(Gly719Ala), so the generic missense REVEL path applies and SpliceAI is not used for PP3. REVEL is 0.824, meeting the published PP3 moderate threshold of >= 0.773 but not the strong threshold of >= 0.932. BayesDel is not used because no generic ACMG-strength calibration is established. The calibrated REVEL result supports PP3 at moderate strength.

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. ... PP3 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Local REVEL predictor lookup: NM_005228.5:c.2156G>C, REVEL = 0.824.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997): generic PP3 moderate threshold >= 0.773 and strong threshold >= 0.932; 0.824 meets moderate only.
• Generic path selection: this missense variant is evaluated with REVEL only; SpliceAI is not evidence for missense PP3, and BayesDel has no established generic ACMG-strength calibration.

Sources

Reasoning

PP5 is not met because the exact variant's ClinVar record has Pathogenic submissions from single laboratories with one-star or no-criteria review status and zero expert-panel submissions. Generic PP5 requires a reputable report of pathogenicity, but under the specified application rule it may be applied only to an exact-variant ClinVar expert-panel classification; that qualifying source is absent. Literature reports and ordinary laboratory submissions cannot substitute for the required expert-panel classification.

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 45225 is an exact match for NM_005228.5(EGFR):c.2156G>C (p.Gly719Ala), with Pathogenic laboratory submissions, review status 'criteria provided, single submitter' or 'no assertion criteria provided,' and expert_panel_submissions = 0.
• The available ClinVar record therefore cannot trigger PP5 under the required exact-variant expert-panel-only rule.

Sources

Reasoning

BA1 is not met. NM_005228.5:c.2156G>C (EGFR p.Gly719Ala) was absent from the default all-comers gnomAD v2.1 and gnomAD v4.1 datasets, and was also absent from the available non-cancer subsets; therefore no filtering allele frequency or observed allele count reaches the generic BA1 threshold of >=0.05. No VCEP-specific population source governs this generic interpretation. The pathogenic ClinVar entry is a single-submitter somatic assertion and does not provide a population-frequency basis for BA1.

Rule

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

Evidence considered

• gnomAD v2.1 (exome), all-comers: NM_005228.5:c.2156G>C was reported absent; no allele count, point estimate, or grpmax FAF was available.
• gnomAD v4.1, all-comers: NM_005228.5:c.2156G>C was reported absent; no allele count, point estimate, or grpmax FAF was available.
• The available GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER records also reported the variant absent. The generic BA1 calibration supplied for this case is >=0.05 (ACMG/AMP 2015, PMID:25741868).

Sources

Reasoning

BS1 is not met on the available population data. The variant was absent from all-comers gnomAD v2.1 and gnomAD v4.1 and from the available non-cancer subsets, so there is no filtering allele frequency or grpmax FAF above the generic BS1 default of >=0.01. Because no elevated frequency was observed, a maximum-credible-allele-frequency calculation is not needed to reject BS1 here; prevalence, penetrance, and allelic heterogeneity cannot turn an absent allele into an above-threshold frequency.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 (exome), all-comers: variant absent; no filtering allele frequency, grpmax FAF, or observed allele count was available.
• gnomAD v4.1, all-comers: variant absent; no filtering allele frequency, grpmax FAF, or observed allele count was available.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER also reported absence. The generic BS1 default supplied for this case is >=0.01; the generic rule requires frequency greater than expected for the disorder, with per-gene maximum-credible-frequency analysis preferred (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

BS3 is not met. The available variant-specific assays do not show a lack of damaging effect; instead, they show oncogenic transformation, ligand-independent EGFR activation, and altered inhibitor sensitivity for p.Gly719Ala. No well-established assay demonstrating preserved EGFR function or normal splicing was identified. The available functional evidence therefore supports PS3 and is incompatible with applying BS3.

Rule

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

Evidence considered

• Kobayashi et al. 2015 (PMID:26206867): G719A caused IL-3-independent Ba/F3 growth and NIH/3T3 focus formation, with wild-type/eGFP controls failing to show these effects.
• Yuan et al. 2016 (PMID:27294619): Gly719Ala produced normalized EGFR pTyr1068 phosphorylation of 0.89 versus 0.21 for wild-type EGFR without EGF, consistent with ligand-independent activation.

Still missing

• No variant-specific well-established assay demonstrating preserved EGFR function or normal splicing was identified.

Sources

Reasoning

BP1 is not supported because the available EGFR evidence instead documents activating missense disease biology: EGFR Gly719Ala produced ligand-independent receptor activation, and EGFR G719A was experimentally studied as an oncogenic driver mutation. These data do not characterize EGFR disease as primarily caused by truncating variants.

Rule

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

Evidence considered

• PMID:27294619 reports that Gly719Ala showed increased normalized EGFR pTyr1068 phosphorylation and ligand-independent activation.
• PMID:26206867 directly studied EGFR G719A in Ba/F3 and NIH/3T3 transformation assays and reported oncogenic driver activity.

Sources

Reasoning

NM_005228.5:c.2156G>C is an exonic missense substitution, so the generic missense REVEL path applies and SpliceAI is not used for BP4. REVEL is 0.824, exceeding the generic BP4 supporting cutoff of <= 0.29 and therefore does not support no impact. 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. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Local REVEL predictor lookup: NM_005228.5:c.2156G>C, REVEL = 0.824.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997): generic BP4 supporting threshold <= 0.29; 0.824 does not meet it.
• Generic path selection: this missense variant is evaluated with REVEL only; SpliceAI is not evidence for missense BP4, and BayesDel has no established generic ACMG-strength calibration.

Sources

Reasoning

BP6 is not met because no exact-variant ClinVar expert-panel classification of Benign or Likely benign is present. ClinVar instead shows exact-variant Pathogenic laboratory submissions and zero expert-panel submissions. Ordinary submissions, aggregate labels, and non-expert assertions cannot trigger BP6 under the specified application 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 45225 is an exact match for NM_005228.5(EGFR):c.2156G>C (p.Gly719Ala), with Pathogenic laboratory submissions and expert_panel_submissions = 0; no benign or likely-benign expert-panel classification is recorded.

Sources

Reasoning

The variant is EGFR p.Gly719Ala. The available evidence documents this same amino-acid change, but no previously established pathogenic EGFR variant caused by a different nucleotide substitution at codon 719 was identified. Exact-variant reports do not satisfy PS1, which requires a same-amino-acid comparator with a different nucleotide change.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• The case variant is NM_005228.5:c.2156G>C, encoding NP_005219.2:p.(Gly719Ala).
• ClinVar documents the exact p.Gly719Ala variant, but an exact-variant report is not evidence of a different nucleotide substitution producing the same amino-acid change.

Still missing

• A verified pathogenic EGFR codon-719 nucleotide-alternative comparator is needed.

Reasoning

Not assessed: the available evidence does not document a proband with a relevant disease phenotype, absence of family history, or confirmed de novo occurrence with both maternity and paternity confirmed. The available publications describe somatic NSCLC cohorts or functional studies rather than parental testing or family data.

Rule

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

Evidence considered

• No case evidence or reviewed publication documents parental testing, confirmed maternity and paternity, or a de novo result for NM_005228.5:c.2156G>C.

Still missing

• Documented disease phenotype for the proband
• Documented absence of family history
• Variant testing results for the proband and both biological parents
• Confirmed maternity and paternity

Reasoning

The generic ACMG/AMP PS4 requirement is a statistically significant increase in variant prevalence among affected individuals versus controls, with OR or RR >5.0 and a 95% confidence interval excluding 1.0. PMID:15738541 reports one EGFR G719A tumor among 59 patients with recurrent NSCLC, but it provides no unaffected or population-control count and no case-control comparison. The broader G719X treatment studies (PMID:31825714 and PMID:31931137) are single-arm or pooled affected-patient outcome cohorts, not case-control studies, and do not provide variant-specific G719A control comparisons. The variant is absent from gnomAD v2.1 and v4.1, but absence alone does not supply a matched affected-versus-control table from which a valid OR and confidence interval can be established. The PS4 leads listed in literature_pass.json (PMID 15710947, 20479403, 24627688, and 34131312) were checked as literature leads; the available case materials do not provide extractable variant-specific case/control counts or a verified OR and confidence interval for them. Therefore PS4 cannot be assessed from the available evidence.

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

• Mitsudomi et al. (PMID:15738541), Results/Fig. 1: one G719A mutation was found among 33 EGFR-mutant tumors in a cohort of 59 recurrent NSCLC patients; the study reports no unaffected or population-control comparison.
• Sequist et al. (PMID:31825714), Results/Table 2: the G719X subgroup had 10 objective responses among 19 patients (53%) and was analyzed in a multicenter single-arm phase II trial; G719A-specific and control data were not reported.
• Yang et al. (PMID:31931137), Results: the G719X category in a pooled database of 693 afatinib-treated NSCLC cases had an objective response rate of 63.4%; this is an affected-patient outcome cohort without an exact-G719A control comparison.
• gnomAD v2.1 and v4.1 report the exact variant as absent; this does not by itself provide the case-control counts and confidence interval required for PS4.

Still missing

• A verified affected-versus-control count or frequency for exact EGFR c.2156G>C/p.Gly719Ala is needed to calculate OR or RR and its 95% confidence interval.
• The PS4-indexed papers PMID:15710947, PMID:20479403, PMID:24627688, and PMID:34131312 lack extractable variant-specific case-control data in the available materials.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not establish that EGFR c.2156G>C is associated with a recessive disorder, does not identify a pathogenic second allele, and provides no parental or offspring testing demonstrating trans phase. The reported tumor and treatment literature is not phase or inheritance 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

• The case evidence identifies EGFR c.2156G>C (p.Gly719Ala) and reports somatic cancer and treatment observations, but contains no affected-proband allelic observations, second pathogenic allele, parental testing, offspring testing, or phase result relevant to PM3.

Still missing

• Disease inheritance mode for the relevant EGFR condition is not established.
• No pathogenic variant in the same individual is documented as the second allele.
• No parental or offspring testing establishes trans phase.

Reasoning

The variant is p.Gly719Ala, but the available case evidence does not identify a different missense substitution at EGFR residue 719 that was independently determined to be pathogenic. The exact p.Gly719Ala reports and broader G719X clinical data cannot substitute for a different pathogenic missense comparator at the same residue.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• The comparator search artifact contains zero confirmed same-residue candidates and explicitly provides no safe PM5 candidate.
• Reports of EGFR G719A or the broader G719X group are not different missense substitutions at residue 719 and therefore do not establish PM5.

Still missing

• A verified pathogenic EGFR residue-719 alternative missense comparator is needed.

Sources

Reasoning

Not assessed: the available evidence does not document an assumed de novo occurrence in a patient with the disease, and it provides no parental testing information showing that maternity or paternity was unconfirmed.

Rule

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

Evidence considered

• No case evidence or reviewed publication documents an assumed de novo NM_005228.5:c.2156G>C result or the parental-testing circumstances required for PM6.

Still missing

• Documented disease phenotype for the proband
• Evidence that the variant is de novo or assumed de novo
• Parental testing and the status of maternity and paternity confirmation

Reasoning

Not assessed: no affected family members, informative meioses, variant testing in relatives, or co-segregation results are documented. The available literature does not provide family segregation data for this variant.

Rule

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

Evidence considered

• No case evidence or reviewed publication documents affected relatives, family testing, informative meioses, or segregation of NM_005228.5:c.2156G>C with disease.

Still missing

• Pedigree with affected and unaffected relatives
• Variant genotypes for informative relatives
• Phenotype status for tested relatives
• Number of informative meioses and co-segregation results

Reasoning

The evidence supports a disease-relevant activating missense mechanism for EGFR, including functional activation by Gly719Ala, but it does not provide a gene-level assessment demonstrating both a low rate of benign missense variation and that missense variants are a common disease mechanism under the generic PP2 rule.

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

• PMID:27294619 reports ligand-independent activation by EGFR Gly719Ala, supporting a missense disease mechanism, but does not establish the required gene-wide benign missense rate.

Still missing

• A gene-level analysis establishing low benign missense variation and common disease-causing missense variation is required.

Sources

Reasoning

The available record identifies the variant in the context of NSCLC, but it does not provide a patient-specific phenotype, family history, or a phenotype description that is highly specific for a single-gene etiology. NSCLC is genetically heterogeneous and therefore cannot be treated as a highly specific phenotype for generic PP4. The missing clinical phenotype and family-history data prevent a complete PP4 assessment.

Rule

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

Evidence considered

• ClinVar identifies the exact variant in the context of Non-small cell lung carcinoma, but the available record contains no patient-specific phenotype or family-history description; NSCLC has multiple genetic etiologies.

Still missing

• Patient-specific phenotype, diagnostic features, and family history are needed to determine whether the presentation is highly specific for a single genetic etiology.

Sources

Reasoning

BS2 cannot be assessed from the available evidence. The population records report absence of the variant but provide no observed healthy adult carrier, homozygote, or hemizygote, and no phenotype-verified healthy-individual cohort is supplied. In addition, the case materials do not establish a fully penetrant early-onset Mendelian disorder model for EGFR p.Gly719Ala against which a healthy observation could be interpreted.

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 variant absent and therefore do not document a healthy adult heterozygote, homozygote, or hemizygote.
• No phenotype-verified healthy-individual or segregation cohort for this variant was available in the population evidence.

Still missing

• A verified healthy adult observation with the relevant zygosity is needed.
• The applicable disorder's inheritance, penetrance, and age of onset are not established in the available evidence.

Reasoning

Not assessed: no affected family members with variant testing and phenotype data are documented, so lack of segregation cannot be evaluated. The available evidence contains no family-level non-segregation result.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No case evidence or reviewed publication documents an affected relative who lacks the variant or any other family-level non-segregation observation.

Still missing

• Affected relatives with documented disease status
• Variant test results for affected relatives
• Family-level genotype–phenotype comparison

Reasoning

BP2 cannot be assessed because the available evidence does not document a pathogenic variant in trans in a fully penetrant dominant disorder or a pathogenic variant in cis in any inheritance pattern. Tumor mutation and drug-response reports do not establish allelic phase with another pathogenic variant.

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 case evidence reports EGFR c.2156G>C in somatic cancer and treatment contexts, but provides no allele-level co-occurring pathogenic variant, phase result, or inheritance information relevant to BP2.

Still missing

• No pathogenic variant in trans or cis is documented for this individual.
• No phase testing or allele-level relationship is reported.
• Fully penetrant dominant inheritance, if relevant, is not established.

Reasoning

No case-level evidence in the available materials documents that this exact variant occurred in a patient with an alternate molecular basis for the disease. The cancer literature reviewed reports EGFR-mutant NSCLC cohorts and treatment outcomes, but it does not establish an alternate molecular cause for a specific case carrying EGFR c.2156G>C/p.Gly719Ala. BP5 therefore cannot be assessed.

Rule

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

Evidence considered

• The reviewed studies report affected NSCLC cohorts and EGFR-mutant treatment outcomes, but none documents an alternate molecular basis for disease in a specific exact-variant case.

Still missing

• A case-level report showing the exact variant together with an independent alternate molecular basis for the disease is needed.

Sources