NRAS c.181C>A, p.Gln61Lys

Reasoning

The NRAS VCEP limits PM1 to critical and well-established functional domains listed in its supplementary table: P-loop residues 10-17, SW1 residues 25-40, SW2 residues 57-64, and SAK residues 145-156, excluding specific amino acid residues as a PM1 route. The variant changes residue 61, which lies within the approved SW2 domain. The governing alignment file independently identifies Switch II as HRAS 57-64 and states that NRAS shares the same residue numbering across the shaded regions. The authoritative domain_tables entry for NRAS is empty, so the explicit VCEP CSPEC domain list and the named governing alignment file were used.

VCEP rule

Applicable only to critical and well-established functional domains available in the supplementary table (P-loop [AA 10-17], SW1 [AA 25-40], SW2 [AA 57-64], SAK [AA 145-156]). Not applicable to specific amino acid residues (see PM5).

Evidence considered

• The VCEP CSPEC PM1 rule lists SW2 (AA 57-64) as an approved critical and well-established functional domain and excludes specific amino acid residues as the PM1 route.
• Alignment-with-PM1-domains.pptx.pdf identifies Switch II as HRAS 57-64 and states that HRAS, KRAS, and NRAS share identical residue numbering across the shaded regions; residue 61 therefore maps inside NRAS SW2.
• The variant is NP_002515.1:p.(Gln61Lys), placing the altered residue at position 61.

Still missing

• The VCEP-governing file Alignment-with-PM1-domains.pptx.pdf was searched for c.181C>A, p.Gln61Lys, and p.Q61K; no variant-specific entry was found, so domain mapping used its stated residue ranges.

Sources

Reasoning

The variant is missense, NM_002524.5:c.181C>A (p.Gln61Lys). The NRAS VCEP PP3 rule for missense variants requires REVEL >= 0.7; the available REVEL score is 0.841, so PP3 is met at Supporting strength. The splice-prediction path was not separately counted because the missense-specific REVEL path already evaluates PP3, and the same computational prediction must not be double-counted across PP3, BP4, and BP7.

VCEP rule

For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 PP3 rule: for missense variants, REVEL >= 0.7.
• Local REVEL predictor lookup for NM_002524.5:c.181C>A / p.(Gln61Lys): REVEL score 0.841.
• Variant consequence is missense: NM_002524.5:c.181C>A, NP_002515.1:p.(Gln61Lys).

Sources

Reasoning

The VCEP PM2 rule requires absence from controls in gnomAD. The variant is absent from gnomAD v2.1 all-comers exomes (0/251,378 alleles) and gnomAD v2.1 non-cancer exomes (0/236,840 alleles), and is reported absent from gnomAD v3.1 non-cancer; however, the VCEP does not specify a non-cancer or exome-only source, so the default all-comers gnomAD result governs. gnomAD v4.1 all-comers reports the variant once (1/1,611,498 alleles; AF 6.205406398270429e-07), so the variant is not absent from gnomAD and PM2 is not met.

VCEP rule

The variant must be absent from controls (gnomAD).

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC, PM2 rule: The variant must be absent from controls (gnomAD).
• gnomAD v2.1 all-comers exomes: 0/251,378 alleles, homozygotes 0.
• GNOMAD_V2_1_NON_CANCER exomes: 0/236,840 alleles, homozygotes 0; this is not the governing source because the VCEP PM2 rule does not specify non-cancer or exome-only data.
• GNOMAD_V3_1_NON_CANCER: reported absent; this is not the governing source because the VCEP PM2 rule does not specify non-cancer data.
• gnomAD v4.1 all-comers: 1/1,611,498 alleles, allele frequency 6.205406398270429e-07, homozygotes 0.

Sources

Reasoning

The NRAS VCEP PM4 rule states, "No known repetitive areas in gene. Use as described." This variant is a single-nucleotide missense substitution, c.181C>A, producing p.(Gln61Lys), with no protein length change or in-frame indel; therefore PM4 is not met.

VCEP rule

No known repetitive areas in gene. Use as described.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC entry for PM4: "No known repetitive areas in gene. Use as described."
• Variant-level consequence facts: NM_002524.5:c.181C>A is a substitution and produces NP_002515.1:p.(Gln61Lys); the predicted and reference proteins have the same terminal position, 61, indicating no protein length change.

Sources

Reasoning

The NRAS c.181C>A (p.Gln61Lys) variant does not meet the VCEP BA1 threshold. Using the available all-comers gnomAD datasets because the VCEP rule does not specify a non-cancer or exome-only source, the highest reported allele frequency is 6.20541e-07 (1/1,611,498 alleles; 0.0000620541%) in gnomAD v4.1, with 0 homozygotes; gnomAD v2.1 exomes reports 0/251,378 alleles. Both are below the VCEP threshold of 0.05%.

VCEP rule

GnomAD filtering allele frequency ≥0.05%.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC, BA1 rule: GnomAD filtering allele frequency ≥0.05%.
• gnomAD v4.1 all-comers: total allele count 1, total allele number 1,611,498, allele frequency 6.205406398270429e-07, homozygotes 0; highest subpopulation frequency 8.485377149558167e-07 in European non-Finnish individuals.
• gnomAD v2.1 all-comers exomes: 0/251,378 alleles, allele frequency 0, homozygotes 0.

Sources

Reasoning

The NRAS c.181C>A (p.Gln61Lys) variant does not meet the VCEP BS1 threshold. Using the available all-comers gnomAD datasets because the VCEP rule does not specify a non-cancer or exome-only source, the highest reported allele frequency is 6.20541e-07 (1/1,611,498 alleles; 0.0000620541%) in gnomAD v4.1, with 0 homozygotes; gnomAD v2.1 exomes reports 0/251,378 alleles. Both are below the VCEP threshold of 0.025%.

VCEP rule

GnomAD filtering allele frequency ≥0.025%.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC, BS1 rule: GnomAD filtering allele frequency ≥0.025%.
• gnomAD v4.1 all-comers: total allele count 1, total allele number 1,611,498, allele frequency 6.205406398270429e-07, homozygotes 0; highest subpopulation frequency 8.485377149558167e-07 in European non-Finnish individuals.
• gnomAD v2.1 all-comers exomes: 0/251,378 alleles, allele frequency 0, homozygotes 0.

Sources

Reasoning

The variant is missense, NM_002524.5:c.181C>A (p.Gln61Lys), so the NRAS VCEP BP4 missense path applies. That rule requires REVEL <= 0.3; the available REVEL score is 0.841, which does not meet the benign threshold. BP4 is therefore not met. The splice-prediction result is not used to establish BP4 for this missense path and is not double-counted with PP3 or BP7.

VCEP rule

For missense variants: REVEL ≤0.3.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 BP4 rule: for missense variants, REVEL <= 0.3.
• Local REVEL predictor lookup for NM_002524.5:c.181C>A / p.(Gln61Lys): REVEL score 0.841.
• Variant consequence is missense: NM_002524.5:c.181C>A, NP_002515.1:p.(Gln61Lys).

Sources

Reasoning

The NRAS VCEP defines PS1 as the same amino acid change as a previously established pathogenic variant regardless of nucleotide change, applicable to observed analogous pathogenic residue positions in HRAS, KRAS, MRAS, NRAS, RIT1, and RRAS2. The available evidence identifies NRAS Q61K itself, but does not establish a distinct nucleotide change producing Q61K as a previously established pathogenic variant; therefore the VCEP PS1 requirement cannot be confirmed.

VCEP rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous pathogenic residue positions in _HRAS, KRAS, MRAS, NRAS, RIT1,_ and _RRAS2._

Evidence considered

• The VCEP PS1 rule requires the same amino acid change as a previously established pathogenic variant regardless of nucleotide change.
• The case normalization identifies NM_002524.5:c.181C>A as NP_002515.1:p.(Q61K); no distinct pathogenic Q61K nucleotide change was documented in the reviewed evidence.
• The VCEP-governing file Alignment-with-PM1-domains.pptx.pdf was searched under c.181C>A, p.Gln61Lys, and p.Q61K and contained no variant-specific entry.

Still missing

• A validated record for a different NRAS nucleotide change encoding p.Q61K, with an established pathogenic classification, was not available.
• The VCEP-governing file Alignment-with-PM1-domains.pptx.pdf was searched for c.181C>A, p.Gln61Lys, and p.Q61K; no variant-specific entry was found.

Sources

Reasoning

The VCEP requires de novo occurrence with confirmed maternity and paternity in a patient with the disease and no family history, with point-based strength determined by the VCEP's PS2 scoring. The available case evidence documents the variant and general database/literature records but provides no proband-specific RASopathy phenotype, parental genotypes, maternity, paternity, or family-history data; therefore de novo status and PS2 points cannot be evaluated.

VCEP rule

Follow SVI recommendations for point-based scoring in conjunction with PM6 (see Reference 1) and phenotypic specifications: full points awarded with RASopathy phenotypes, reduced points applicable to cases with minimal phenotypic information (i.e. prenatal cases, cases with a clinical order of a RASopathy panel without clinical information, and cases with limited clinical information in other global tests (such as WES)). ... De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. ... 4 Points. ... 2 Points. ... 1 Point.

Evidence considered

• The case evidence identifies NM_002524.5:c.181C>A (p.Gln61Lys) but contains no documented parental testing, maternity or paternity confirmation, proband-specific phenotype, or family history.
• No fetched paper extract provides proband-level de novo evidence for this variant; the available extracted papers are functional or somatic cancer studies rather than segregation or parental-testing reports.

Still missing

• Proband-specific RASopathy phenotype and diagnostic context
• Parental genotypes for NM_002524.5:c.181C>A
• Confirmation of maternity and paternity
• Family history relevant to the de novo assessment

Sources

Reasoning

The NRAS VCEP PS3 rule requires well-established functional studies and specifies one approved assay for supporting strength or two or more different approved assays for moderate strength. The governing approved-functional-studies file lists approved NRAS RAS activation, MEK activation, and ERK activation assay classes, with assay-specific examples and proposed PS3_Supporting strength, but it contains no entry for this variant under c.181C>A, p.Gln61Lys, or p.Q61K. PMID:22718121 directly studies NRasQ61K and reports increased melanocyte survival, growth, invasion, and tumor behavior, but the extracted assays are survival/invasion and animal-model studies rather than one of the listed approved NRAS assay classes; the VCEP file states that animal models and variant-specific assays are excluded from the approved assay set. Therefore, the available evidence does not establish that the VCEP-required approved-assay condition is met, and PS3 is not assessed rather than assigned from a non-approved assay.

VCEP rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Approved assays are available in the supplemental materials. ... One approved assay.

Evidence considered

• VCEP CSPEC PS3 entry: "Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Approved assays are available in the supplemental materials."
• VCEP CSPEC PS3 strength rules: "Two or more different approved assays." for Moderate and "One approved assay." for Supporting.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, READ ME: assays not listed are presumed to lack sufficient historical evidence; animal models and variant-specific assays are excluded from the approved assay set; two or more unique assay types for a variant provide sufficient evidence to upgrade PS3 to Moderate.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, RAS Activation Assay / NRAS row: approved assay Y; proposed strength PS3_Supporting; BS3_NA; listed NRAS PMIDs 19966803, 28594414, and 21263000.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, MEK Activation Assay / NRAS row: approved assay Y; proposed strength PS3_Supporting; BS3_NA; listed NRAS PMIDs 19966803, 28594414, and 21263000.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, ERK Activation Assay / NRAS row: approved assay Y; proposed strength PS3_Supporting; BS3_NA; listed NRAS PMIDs 19966803 and 28594414.
• PMID:22718121 paper_extracts entry: NRasQ61K is explicitly studied in transgenic mouse melanocytes, primary melanocytes, soft-agar growth, invasion/invadopodia, and melanoma allograft models; the extracted direct quote reports NRasQ61K-associated survival and invasiveness effects.

Still missing

• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx was searched for c.181C>A, p.Gln61Lys, and p.Q61K; no exact-variant entry was found.
• A report of this variant in an approved NRAS RAS activation, MEK activation, or ERK activation assay with the required controls is not available in the reviewed evidence.

Sources

Reasoning

The NRAS VCEP makes PS4 applicable and defines the underlying evidence as increased prevalence in affected individuals versus controls; its original rule states: "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. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence." The available variant-specific literature includes a 30-case NRAS-mutant NSCLC cohort in which Q61K is listed among Q61 substitutions, but it does not identify Q61K carriers or provide affected-versus-control counts, an OR/RR, a confidence interval, or multiple unrelated RASopathy patients with this germline variant. No paper in literature_pass.json packet.ai_classified_papers was classified as PS4; therefore no PS4 point total or qualifying case-control comparison can be established from the available evidence.

VCEP 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. See manuscript for detailed guidance. ... In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. ... ≥5 points. ... ≥3 points. ... ≥1 point.

Evidence considered

• ClinGen RASopathy VCEP NRAS v2.3 PS4 rule: the original rule requires affected-versus-control enrichment, with RR/OR >5.0 and a confidence interval excluding 1.0, while allowing a moderate rare-variant path for multiple unrelated same-phenotype patients absent in controls.
• PMID:23515407, Results, "NRAS Mutation Genotypes": 30 NRAS-mutant NSCLC cases included Q61H/K/L/R substitutions and Q61 accounted for 80% of mutations, but the paper did not identify Q61K carriers or provide variant-specific case-control statistics.
• literature_pass.json packet.ai_classified_papers contains only duplicate PMID:22718121 entries classified as PS3, not PS4; its classifier reading concerns functional melanoma experiments rather than affected-control prevalence.

Still missing

• Variant-specific case and matched-control counts or allele frequencies are needed to calculate OR/RR and its 95% confidence interval.
• Alternatively, multiple unrelated patients with the same RASopathy phenotype and documented absence of the variant in controls are needed for the VCEP's rare-variant moderate path.

Sources

Reasoning

The NRAS VCEP assigns PM5 moderate strength for one likely pathogenic residue change at the same codon and strong strength only for at least two different likely pathogenic residue changes at the same codon observed in at least five probands. The reviewed literature reports Q61H, Q61K, Q61L, and Q61R at codon 61, but does not establish the non-Q61K changes as likely pathogenic under this VCEP, and the collected comparator record contains no usable same-residue candidates. The PM5 threshold cannot therefore be evaluated from the available VCEP-governed evidence.

VCEP rule

≥2 different [likely] pathogenic residues changes at the same codon observed in ≥5 probands. ... 1 [likely] pathogenic residue change at the same codon.

Evidence considered

• The VCEP PM5 rule provides moderate strength for 1 likely pathogenic residue change at the same codon and strong strength for at least 2 different likely pathogenic residue changes at the same codon observed in at least 5 probands.
• Li et al., PMID:23515407, Results, NRAS Mutation Genotypes: the 30 NRAS mutations included Q61H/K/L/R and codon Q61 accounted for 80% of mutations, but the paper did not establish the individual non-Q61K substitutions as likely pathogenic.
• The collected PM5 comparator record reported zero same-residue candidates, zero expert-panel likely pathogenic/likely benign candidates, and zero non-expert majority likely pathogenic/likely benign candidates.
• The VCEP-governing file Alignment-with-PM1-domains.pptx.pdf was searched under c.181C>A, p.Gln61Lys, and p.Q61K and contained no variant-specific entry.

Still missing

• A VCEP-governed source establishing at least one different likely pathogenic NRAS residue change at codon 61, or two such changes in at least five probands, was not available.
• The VCEP-governing file Alignment-with-PM1-domains.pptx.pdf was searched for c.181C>A, p.Gln61Lys, and p.Q61K; no variant-specific entry was found.

Sources

Reasoning

The VCEP defines PM6 as assumed de novo occurrence without confirmation of paternity and maternity and requires point-based scoring with phenotype-based adjustment in conjunction with PS2. The available case evidence contains no documented proband-specific RASopathy phenotype, parental testing, or clinical observation establishing assumed de novo status; therefore PM6 cannot be scored.

VCEP rule

Follow SVI recommendations for point-based scoring in conjunction with PS2 (see Reference 1) and phenotypic specifications: full points awarded with RASopathy phenotypes, reduced points applicable to cases with minimal phenotypic information (i.e. prenatal cases, cases with a clinical order of a RASopathy panel without clinical information, and cases with limited clinical information in other global tests (such as WES)). ... Assumed de novo, but without confirmation of paternity and maternity. ... 2 Points. ... 1 Point. ... 0.5 Points.

Evidence considered

• The case evidence identifies NM_002524.5:c.181C>A (p.Gln61Lys) but contains no documented parental testing, parental genotype results, or proband-specific clinical observation supporting assumed de novo status.
• The available extracted literature does not provide a segregation or parental-testing report for this variant.

Still missing

• Proband-specific RASopathy phenotype and diagnostic context
• Evidence that the variant is absent from tested parents or otherwise assumed de novo
• Parental testing details and whether maternity or paternity was confirmed

Sources

Reasoning

The VCEP recommends segregation in more than one family and assigns supporting, moderate, or strong PP1 according to at least 3, 5, or 7 informative meioses, respectively. The available case evidence reports no informative meioses, affected relatives, family segregation results, or multi-family segregation data; therefore PP1 cannot be assessed.

VCEP rule

Segregation in more than one family is recommended. ... ≥7 informative meioses. ... ≥5 informative meioses. ... ≥3 informative meioses.

Evidence considered

• No affected-relative genotypes, family pedigrees, informative meioses, or segregation results are documented in the available case evidence.
• No available paper extract reports family segregation or informative meioses for NM_002524.5:c.181C>A.

Still missing

• Number and results of informative meioses
• Affected relatives and their variant genotypes
• Segregation data from additional families

Sources

Reasoning

The VCEP CSPEC entry provides only point labels for BS2 (−4 Points and −1 Point) and does not state the underlying healthy-adult or homozygote evidence conditions needed to assign those points. The available population data show 0 homozygotes in gnomAD v2.1 and v4.1, but absence of homozygotes is not itself the positive BS2 observation, and no VCEP-defined point assignment or qualifying healthy-adult observation is available.

VCEP rule

-4 Points. ... -1 Point.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC, BS2 entries: −4 Points and −1 Point.
• gnomAD v2.1 all-comers exomes: homozygotes 0.
• gnomAD v4.1 all-comers: homozygotes 0.

Still missing

• VCEP-defined BS2 point-assignment conditions and qualifying healthy-adult or homozygote observations are unavailable.

Sources

Reasoning

The VCEP defines BS4 as lack of segregation in affected family members and requires only one informative meiosis for strong evidence, while cautioning that phenocopies and multiple pathogenic variants can confound apparent non-segregation. The available case evidence contains no affected relatives, family genotypes, informative meiosis, or phenotype assessments that could demonstrate non-segregation; therefore BS4 cannot be assessed.

VCEP rule

Lack of segregation in affected members of a family. ... Requires only one informative meiosis.

Evidence considered

• No affected-family-member genotype results, family segregation data, informative meiosis, or phenocopy/dual-variant assessment is documented in the available case evidence.
• No available paper extract reports non-segregation for NM_002524.5:c.181C>A.

Still missing

• At least one informative meiosis
• Affected family members with variant genotype results
• Phenotype assessments and evaluation for phenocopies or another pathogenic variant

Sources

Reasoning

The NRAS Version 2.3 VCEP BP2 entry assigns strength only from a BP2 point total: Strong at ≥ (-4) points, Moderate at ≥ (-2) points, and Supporting at ≥ (-1) point. The available case evidence documents no phase determination, no affected-proband allelic observation, no pathogenic variant demonstrated in trans or cis, and no VCEP BP2 point total. The criterion therefore cannot be evaluated without applying an unstated point-assignment rule.

VCEP rule

≥ (-4) Points. ... ≥ (-2) Points. ... ≥ (-1) Point.

Evidence considered

• ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for NRAS Version 2.3, criteria entry BP2: Strong at ≥ (-4) Points, Moderate at ≥ (-2) Points, and Supporting at ≥ (-1) Point.
• Case evidence contains no documented phase, affected-proband allelic observation, pathogenic variant in trans or cis, or BP2 point total.

Still missing

• Documented observation of the variant in trans or cis with a pathogenic or likely pathogenic allele, with phase evidence.
• The VCEP-defined BP2 point assignment for any qualifying allelic observation.

Sources

Reasoning

The NRAS VCEP makes BP5 applicable when there is an alternative molecular cause of a RASopathy in a different gene, with a phenotype consistent with expected RASopathy severity, or when a phenotype inconsistent with RASopathy is fully explained by a different causative variant. The available case evidence does not document an alternative causative variant, its gene, phenotype concordance or inconsistency, or a VCEP BP5 point assignment. Consequently no BP5 point total can be evaluated against the VCEP's own thresholds: Strong >=(-4) points, Moderate >=(-2) points, or Supporting >=(-1) point.

VCEP rule

Points are awarded for an alternative molecular cause of a RASopathy in a different gene (and/or in conjunction with BP2) and the phenotype is consistent with expected severity of the RASopathy. Points are also awarded for phenotypes inconsistent with a RASopathy and fully explained by a different causative variant (e.g. WES testing). ... ≥ (-4) Points. ... ≥ (-2) Points. ... ≥ (-1) Point.

Evidence considered

• ClinGen RASopathy VCEP NRAS v2.3 BP5 instructions: points require an alternative molecular cause in a different gene with phenotype consistency, or a phenotype inconsistent with RASopathy fully explained by a different causative variant.
• ClinGen RASopathy VCEP NRAS v2.3 BP5 strength rules: Strong >=(-4) points; Moderate >=(-2) points; Supporting >=(-1) point.
• No case evidence supplied a second causative variant or a BP5 point score.

Still missing

• Documented alternate molecular cause, gene, and phenotype relationship are needed.
• A VCEP-defined BP5 point value is needed before comparing it with the selected strength threshold.

Sources