ROS1 c.6076G>A, p.Gly2026Arg

Reasoning

PM2 is met at supporting strength under the generic ClinGen SVI threshold supplied for this case. The variant is absent from gnomAD v2.1 and gnomAD v4.1 all-comers datasets and is also reported absent from the available gnomAD v2.1 non-cancer and v3.1 non-cancer searches. Because no VCEP governs ROS1, the all-comers datasets are the default; the observed frequency is therefore 0, which is at or below the generic PM2 supporting threshold of 0.0001.

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 all-comers: variant absent.
• gnomAD v4.1 all-comers: variant absent.
• gnomAD v2.1 non-cancer and v3.1 non-cancer searches also reported the variant absent; no governing VCEP specifies these subsets because no VCEP covers ROS1.
• Generic PM2 calibration supplied for this case: <=0.0001, SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020; PMID:25741868).

Sources

Reasoning

The variant is a missense substitution, NM_001378902.1:c.6076G>A (p.Gly2026Arg), so the governing generic rule selects the REVEL path only; SpliceAI is not used for missense PP3/BP4. REVEL is 0.862, meeting the published generic PP3 supporting threshold of >=0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997). BayesDel is not used because no generic ACMG-strength calibration is established. The result supports PP3 at supporting strength without double-counting a splice prediction.

Rule

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

Evidence considered

• Case normalization identifies a missense variant: NM_001378902.1:c.6076G>A, NP_001365831.1:p.(Gly2026Arg), in exon 39.
• Local REVEL lookup reports score 0.862 for GRCh38 chr6:117317184 C>T.
• Under the generic one-path rule, a missense variant uses REVEL only; SpliceAI is not evidence for missense PP3/BP4.
• REVEL 0.862 meets the published generic PP3 supporting cutoff >=0.644 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997).
• BayesDel score 0.39197 was not used because the governing generic rule provides no ACMG-strength calibration for BayesDel.

Sources

Reasoning

The governing ACMG/AMP definition is: "The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls." The generic note requires an RR or OR >5.0 with a confidence interval that does not include 1.0. The literature_pass packet listed PMID:24627688 as a PS4 candidate, but its full-text extraction verified that it does not mention ROS1, c.6076G>A, or p.(G2026R) and contains no case-control counts, affected-carrier frequency, control-carrier frequency, OR, RR, or confidence interval. Therefore no qualifying case-control enrichment evidence is present; no OR or CI can be computed.

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:24627688 full-text extraction: the paper is an ALK rearrangement testing guideline and does not mention ROS1 c.6076G>A or p.(G2026R); no variant-specific case-control data were reported.
• The required PS4 comparison was not computable because no affected-carrier count, control-carrier count, OR/RR, or confidence interval was available.

Still missing

• A variant-specific case-control study or affected-versus-control carrier counts are needed to calculate an OR or RR and its confidence interval.

Sources

Reasoning

The governing ACMG/AMP definition is: "Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation." The exact variant has one ClinVar submission classified as Pathogenic, but it is an ordinary clinical-laboratory submission with no assertion criteria, no expert-panel status, and no validated PMID. The literature_pass candidate PMID:24627688 was verified from its full text and does not mention the exact ROS1 variant. Therefore the required reputable-source pathogenic report for 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 exact-variant record NM_001378902.1(ROS1):c.6076G>A (p.Gly2026Arg): Pathogenic, one clinical laboratory, no assertion criteria provided, zero expert-panel submissions, and zero validated PMIDs.
• PMID:24627688 full-text extraction: no mention of ROS1 c.6076G>A or p.(G2026R), so it does not independently provide a reputable pathogenic report for this variant.

Sources

Reasoning

Generic ACMG/AMP BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1 all-comers datasets, and also absent from the available gnomAD v2.1 non-cancer and v3.1 non-cancer searches; therefore no qualifying allele frequency or grpmax FAF at or above the generic 0.05 threshold is present. The ClinVar record is a single somatic, zero-star Pathogenic submission for lung adenocarcinoma and is not a germline pathogenic consensus that would create the discordance caveat described for a stand-alone benign call.

Rule

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

Evidence considered

• gnomAD v2.1 all-comers: variant absent; no allele frequency or grpmax FAF reported.
• gnomAD v4.1 all-comers: variant absent; no allele frequency or grpmax FAF reported.
• gnomAD v2.1 non-cancer and v3.1 non-cancer searches also reported the variant absent; no governing VCEP specifies these subsets because no VCEP covers ROS1.
• Generic BA1 calibration supplied for this case: >=0.05, ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Sources

Reasoning

Generic ACMG/AMP BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1 all-comers datasets, with no filtering allele frequency or grpmax FAF exceeding the generic 0.01 default. No ancestry-specific elevated frequency, prevalence/penetrance-based maximum credible allele frequency calculation, or founder-allele exception is needed because no population frequency was observed.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 all-comers: variant absent; no filtering allele frequency or grpmax FAF reported.
• gnomAD v4.1 all-comers: variant absent; no filtering allele frequency or grpmax FAF reported.
• gnomAD v2.1 non-cancer and v3.1 non-cancer searches also reported the variant absent; no governing VCEP specifies these subsets because no VCEP covers ROS1.
• The generic BS1 default is 0.01; the case provides no observed frequency requiring a maximum-credible-allele-frequency calculation or ancestry-specific penetrance adjustment (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only; SpliceAI cannot substitute for an indeterminate or non-supportive REVEL result in this situation. REVEL is 0.862, which is above the generic BP4 supporting upper threshold of <=0.29 and therefore does not support a benign computational effect. The SpliceAI max delta of 0.03 is not used for missense BP4, and 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)

Evidence considered

• Case normalization identifies a missense variant: NM_001378902.1:c.6076G>A, NP_001365831.1:p.(Gly2026Arg), in exon 39.
• Local REVEL lookup reports score 0.862 for GRCh38 chr6:117317184 C>T.
• Under the generic one-path rule, a missense variant uses REVEL only; SpliceAI max delta 0.03 is not evidence for missense BP4.
• REVEL 0.862 does not meet the published generic BP4 supporting cutoff <=0.29 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997).
• BayesDel score 0.39197 was not used because the governing generic rule provides no ACMG-strength calibration for BayesDel.

Sources

Reasoning

The governing ACMG/AMP definition is: "Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation." The exact-variant ClinVar record is Pathogenic, not Benign or Likely benign, and has one ordinary laboratory submission with no expert-panel classification. No reputable benign report was identified. Therefore BP6 is 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 exact-variant record: Pathogenic from one clinical laboratory, no assertion criteria provided, and zero expert-panel submissions; no Benign or Likely benign classification is present.

Sources

Reasoning

The variant is NM_001378902.1:c.6076G>A, p.(Gly2026Arg), and the supplied evidence does not identify a previously established pathogenic variant causing the same amino-acid change through a different nucleotide change. The single ClinVar submission is a somatic, zero-star Pathogenic assertion without assertion criteria or validated literature support and cannot establish PS1. The extracted papers do not mention this variant or an appropriate same-amino-acid comparator. Therefore PS1 cannot be assessed from the available evidence.

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

• ClinVar variation 376139: exact variant NM_001378902.1(ROS1):c.6076G>A (p.Gly2026Arg), one somatic clinical-laboratory submission, Pathogenic, no assertion criteria, zero review stars; not sufficient to establish PS1.
• The available paper extracts do not report p.Gly2026Arg or a same-amino-acid comparator.

Still missing

• A validated pathogenic comparator producing p.Gly2026Arg by a different nucleotide change is needed.

Sources

Reasoning

No germline proband-level de novo observation, parental genotyping, maternity confirmation, paternity confirmation, or family-history assessment is documented. The only exact-variant ClinVar submission is explicitly of somatic origin for lung adenocarcinoma and therefore does not establish PS2. The criterion cannot be assessed without documented disease in the proband, absence of family history, and confirmation of both maternity and paternity.

Rule

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

Evidence considered

• The exact-variant ClinVar record (NM_001378902.1(ROS1):c.6076G>A, p.Gly2026Arg; Variation ID 376139) contains one clinical-testing submission classified as pathogenic, but its reported origin is somatic and its condition is lung adenocarcinoma; it provides no germline parental testing or de novo evidence.

Still missing

• Documented germline variant result in the proband
• Documented disease phenotype in the proband
• Confirmation of both maternity and paternity
• Documented absence of family history

Sources

Reasoning

The available evidence does not provide a validated, reproducible in vitro or in vivo functional assay for ROS1 NM_001378902.1:c.6076G>A (p.Gly2026Arg). The reviewed publications are lung-cancer testing guidelines and do not report this variant or variant-specific functional results. Therefore, the evidence is insufficient to assess PS3 rather than supporting a damaging functional effect.

Rule

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

Evidence considered

• Lindeman et al. 2013 (PMID:23562183): the reviewed full text does not mention ROS1, c.6076G>A, or p.Gly2026Arg and provides no variant-specific functional evidence.
• Kim et al. 2014 (PMID:24627688): the reviewed paper concerns ALK rearrangement testing and does not mention ROS1 p.Gly2026Arg or provide a ROS1 functional assay.
• Lindeman et al. 2018 (PMID:29355391): the paper discusses ROS1 testing generally but does not report this variant or variant-specific functional data.
• Lindeman et al. 2018 (PMID:29398453): the paper discusses ROS1 structural rearrangements generally but does not report this substitution or variant-specific functional data.

Still missing

• A validated and reproducible in vitro or in vivo assay directly evaluating ROS1 p.Gly2026Arg is needed.
• Assay controls, biological relevance, and quantitative results for the exact variant are unavailable.

Sources

Reasoning

The variant is a missense substitution at ROS1 residue 2026. No VCEP domain table is present for ROS1, and the supplied evidence does not establish that residue 2026 lies in a qualifying mutational hotspot or a critical, well-established functional domain without benign variation. Cancer Hotspots and COSMIC returned no result for this variant, and no generic hotspot/domain source with the required benign-variation context was supplied. Therefore PM1 cannot be assessed.

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 query ROS1 G2026: no statistically significant hotspot result.
• COSMIC query ROS1 c.6076G>A: no result found.
• vcep_materials.json reports no VCEP materials and an empty domain_tables object for ROS1.

Still missing

• A validated ROS1 domain or hotspot source covering residue 2026 and demonstrating absence of benign variation is needed.

Sources

Reasoning

PM3 cannot be assessed from the available evidence. The case identifies NM_001378902.1:c.6076G>A (p.Gly2026Arg), but provides no affected-proband observation with a second pathogenic allele, no pathogenic variant in the same individual, and no parental or offspring testing establishing trans phase. The inheritance mode for a ROS1-related disorder is also not established in the case evidence. Therefore, the required trans configuration and recessive-disease context are unresolved.

Rule

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 the tested variant as NM_001378902.1:c.6076G>A (NP_001365831.1:p.(Gly2026Arg)) but contains no documented affected proband with a second pathogenic allele.
• No parental or offspring genotyping, phase result, cis/trans result, or segregation observation is provided for this variant.
• The case evidence does not establish a relevant recessive inheritance model for ROS1.
• The available ClinVar record is a single zero-star somatic Lung adenocarcinoma submission with no assertion criteria and does not provide germline phase or allelic evidence.

Still missing

• Documented affected-proband observation involving this variant and a second pathogenic or likely pathogenic variant is unavailable.
• Parental or offspring testing establishing trans phase is unavailable.
• The relevant ROS1 disorder inheritance mode is not established in the case evidence.

Reasoning

The variant is a novel missense substitution, p.Gly2026Arg, but no different missense change at residue 2026 with a validated pathogenic determination is identified in the supplied evidence. The PM5 candidate file contains zero candidates and explicitly states that classic same-residue PM5 semantics could not be confirmed safely. The ClinVar record is for the observed variant itself and is an unreviewed somatic assertion, not a different same-residue pathogenic comparator. Therefore PM5 cannot be assessed.

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

• pm5_candidates.json: total_candidates_seen = 0, same_residue_candidates = 0, and no classic PM5 candidate could be confirmed.
• ClinVar variation 376139 concerns the observed p.Gly2026Arg variant itself and is not a different same-residue comparator.

Still missing

• A validated pathogenic missense variant different from p.Gly2026Arg at residue 2026 is needed.

Sources

Reasoning

No germline proband-level de novo observation or clinical documentation supports an assumed de novo event. The exact-variant ClinVar submission is explicitly somatic and does not provide evidence for PM6. No parental testing status is documented, so the criterion cannot be assessed rather than applied.

Rule

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

Evidence considered

• The exact-variant ClinVar record reports a single clinical-testing submission with origin somatic for lung adenocarcinoma; it does not document a germline de novo event or parental testing.

Still missing

• Documented germline variant result in the proband
• Evidence that the variant is de novo or assumed de novo
• Parental testing status

Sources

Reasoning

No affected family members, informative meioses, genotype results, phenotype concordance, or segregation counts are documented for this variant. The available literature extracts concern lung-cancer molecular-testing guidelines and contain no variant-specific family segregation data. PP1 therefore cannot be assessed.

Rule

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

Still missing

• Pedigree and affected-family-member information
• Variant genotypes for affected and unaffected relatives
• Phenotype consistency and age-related assessment
• Informative meioses or segregation count

Reasoning

The supplied evidence does not establish both required PP2 components for ROS1: a low rate of benign missense variation and missense variants as a common disease mechanism. Absence of the observed variant from gnomAD does not establish either gene-level property, and the available literature concerns ROS1 rearrangements or testing guidance rather than a validated germline missense mechanism. Therefore PP2 cannot be 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 observed variant is absent from gnomAD v2.1 and v4.1, but single-variant absence does not establish the PP2 gene-level requirements.
• The supplied literature extracts do not provide a validated ROS1 germline missense disease mechanism or a gene-wide benign missense assessment.

Still missing

• Gene-level benign missense constraint and evidence that missense variants commonly cause disease are needed.

Sources

Reasoning

The governing ACMG/AMP definition is: "Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology." The available case evidence identifies the ClinVar condition as lung adenocarcinoma, but provides no detailed patient phenotype, family history, or evidence that the phenotype is highly specific for a disease with a single genetic etiology. The generic PP4 requirement therefore cannot be assessed from the available evidence.

Rule

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

Evidence considered

• ClinVar records the condition as Lung adenocarcinoma, but the available record contains no detailed phenotype or family-history description and does not establish a single genetic etiology.

Still missing

• Detailed phenotype and family-history information is needed to determine whether the presentation is highly specific for a disease with a single genetic etiology.

Sources

Reasoning

BS2 cannot be assessed from the available population evidence. gnomAD v2.1 and v4.1, including the available non-cancer searches, report the variant as absent, but they provide no observed healthy adult carrier or homozygote/hemizygote observation. Absence from population databases neither demonstrates the healthy-adult observation required by BS2 nor establishes the disorder's age-related penetrance.

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, so neither database supplies an observed healthy adult genotype for BS2.
• The available gnomAD v2.1 non-cancer and v3.1 non-cancer searches also report absence and do not provide a healthy-adult genotype observation.

Still missing

• No documented healthy adult heterozygote, homozygote, or hemizygote observation with relevant age and phenotype information was available.

Sources

Reasoning

The available evidence does not provide a validated, reproducible in vitro or in vivo functional assay showing that ROS1 NM_001378902.1:c.6076G>A (p.Gly2026Arg) has no damaging effect on protein function or splicing. The reviewed publications do not report this variant or variant-specific benign functional results. Therefore, the evidence is insufficient to assess BS3 rather than supporting a normal functional effect.

Rule

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

Evidence considered

• Lindeman et al. 2013 (PMID:23562183): the reviewed full text does not mention ROS1, c.6076G>A, or p.Gly2026Arg and provides no variant-specific benign functional evidence.
• Kim et al. 2014 (PMID:24627688): the reviewed paper concerns ALK rearrangement testing and does not mention ROS1 p.Gly2026Arg or provide a ROS1 assay demonstrating no damaging effect.
• Lindeman et al. 2018 (PMID:29355391): the paper discusses ROS1 testing generally but does not report this variant or variant-specific evidence excluding functional or splicing effects.
• Lindeman et al. 2018 (PMID:29398453): the paper discusses ROS1 structural rearrangements generally but does not report this substitution or variant-specific evidence excluding functional effects.

Still missing

• A validated and reproducible in vitro or in vivo assay directly evaluating ROS1 p.Gly2026Arg is needed.
• Assay controls, biological relevance, and quantitative evidence excluding effects on protein function or splicing are unavailable.

Sources

Reasoning

No affected family members tested for the variant, non-segregating genotypes, pedigree, or phenotype data are documented. Consequently, there is no evidence on which to evaluate lack of segregation or the possibility of phenocopies, and BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family. ... Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals.

Still missing

• Pedigree identifying affected relatives
• Variant testing results in affected relatives
• Clinical phenotype data for affected relatives
• Assessment of phenocopies and alternative familial variants

Reasoning

The supplied evidence does not establish that ROS1 is a gene for which primarily truncating variants are known to cause disease. The available gene-level material describes ROS1 as a receptor tyrosine kinase and discusses oncogenic rearrangements, while the extracted papers do not provide a validated germline variant spectrum demonstrating a primarily truncating disease mechanism. Therefore BP1 cannot be assessed.

Rule

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

Evidence considered

• The supplied evidence does not provide a validated ROS1 germline disease variant spectrum establishing predominance of truncating pathogenic variants.
• Available ROS1 material concerns receptor-tyrosine-kinase biology and oncogenic rearrangements, which does not by itself satisfy BP1.

Still missing

• A validated disease-specific ROS1 variant spectrum demonstrating primarily truncating pathogenic variants is needed.

Reasoning

BP2 cannot be assessed from the available evidence. The case provides no affected-proband or other individual-level observation showing NM_001378902.1:c.6076G>A (p.Gly2026Arg) in trans with a pathogenic variant in a fully penetrant dominant disorder, and no observation showing it in cis with a pathogenic variant. No parental or offspring testing, haplotype data, or other phase evidence is available. The inheritance and penetrance requirements for the trans pathway are therefore unresolved.

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 evidence identifies the tested variant as NM_001378902.1:c.6076G>A (NP_001365831.1:p.(Gly2026Arg)) but reports no cis or trans observation with another pathogenic variant.
• No affected-proband genotype, parental or offspring testing, haplotype result, or phase determination is provided.
• The case evidence does not establish a fully penetrant dominant ROS1 disorder for applying the trans pathway.
• The available ClinVar record is a single zero-star somatic Lung adenocarcinoma submission with no assertion criteria and does not provide germline phase or allelic evidence.

Still missing

• A documented observation of this variant in trans with a pathogenic variant in a fully penetrant dominant disorder is unavailable.
• A documented observation of this variant in cis with a pathogenic variant is unavailable.
• Parental or offspring testing, haplotype data, or another reliable phase determination is unavailable.
• The relevant disorder's penetrance and inheritance model are not established in the case evidence.

Reasoning

The governing ACMG/AMP definition is: "Variant found in a case with an alternate molecular basis for disease." The available evidence does not document an alternate molecular diagnosis, a second pathogenic variant, or another molecular basis for the patient's disease. Because the case-level information needed to evaluate this criterion is absent, BP5 cannot be assessed.

Rule

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

Evidence considered

• No alternate molecular diagnosis or second disease-causing molecular basis is reported in the available case evidence.

Still missing

• A case-level alternate molecular diagnosis or documented second molecular basis for disease is needed.