NRAS c.38G>A, p.Gly13Asp

Reasoning

NRAS c.38G>A produces p.Gly13Asp (p.G13D), and the same amino-acid change is reported in prior disease cases, including a constitutional activating NRAS mutation in JMML and an earlier reported germ-line activating NRAS mutation. This satisfies the VCEP PS1 rule for an established pathogenic same-amino-acid change in NRAS, at Strong strength.

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

• PMID:19775298 explicitly reports NRAS c.38G>A (p.G13D) as a heterozygous activating mutation in a male infant with JMML and notes a previously reported germ-line activating NRAS G13D mutation.
• PMID:17332249 reports the same NRAS codon-13 substitution, GGT (Gly) to GAT (Asp), corresponding to p.Gly13Asp/p.G13D, in a JMML case.
• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC PS1 rule applied at Strong strength.

Sources

Reasoning

PMID:19775298 reports NRAS c.38G>A (p.G13D) as a constitutional heterozygous variant in a male infant with JMML. The variant was detected across multiple constitutional DNA sources, including buccal swab, hair bulbs, lymphoblastoid cells, endothelial cells, and cultured skin fibroblasts, and the report states that it was absent in both parents; this is one documented confirmed de novo case. Under the VCEP's PS2 point scale, one documented case supplies 1 point, meeting the Moderate rule.

VCEP rule

1 Point.

Evidence considered

• PMID:19775298 reports NRAS c.38G>A (p.G13D) in a male infant with JMML and describes the variant as a de novo germ-line mutation absent in both parents.
• PMID:19775298 examined peripheral blood, buccal swab, hair bulbs, lymphoblastoid and T-lymphoblastoid cells, endothelial cells, and cultured skin fibroblasts; the variant was consistently present across the examined DNA sources.
• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC, PS2 Moderate row: 1 Point.

Sources

Reasoning

The variant changes NRAS residue 13. The governing NRAS PM1 specification limits PM1 to the P-loop at amino acids 10-17, Switch I at 25-40, Switch II at 57-64, and SAK at 145-156; residue 13 lies within the approved P-loop region. The governing alignment file was searched for c.38G>A, p.Gly13Asp, and p.G13D and contains no variant-specific row, so the domain-range rule and alignment are the applicable evidence.

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

• ClinGen RASopathy Expert Panel NRAS v2.3 PM1 rule defines the approved regions as P-loop AA 10-17, SW1 AA 25-40, SW2 AA 57-64, and SAK AA 145-156, and excludes specific amino-acid residues as a separate PM1 route.
• Alignment-with-PM1-domains.pptx.pdf, page 1 transcription: P-loop (HRAS 10-17); the file states that HRAS, KRAS, and NRAS share identical residue numbering across the shaded regions, so residue 13 is in the NRAS P-loop.
• Alignment-with-PM1-domains.pptx.pdf was searched for c.38G>A, p.Gly13Asp, and p.G13D; no variant-specific entry was present.

Still missing

• Alignment-with-PM1-domains.pptx.pdf was searched for c.38G>A, p.Gly13Asp, and p.G13D; no variant-specific entry was found.

Sources

Reasoning

The VCEP requires the variant to be absent from gnomAD controls for PM2. NRAS c.38G>A (p.Gly13Asp) is present in the default all-comers gnomAD datasets: 1 allele among 251492 in gnomAD v2.1 and 1 allele among 1614022 in gnomAD v4.1. Although no homozygotes are observed and the allele frequency is very low, the variant is not absent, so the VCEP PM2 requirement is not satisfied.

VCEP rule

The variant must be absent from controls (gnomAD).

Evidence considered

• gnomAD v2.1 all-comers: allele count 1, allele number 251492, AF 3.976269622890589e-06, homozygotes 0.
• gnomAD v4.1 all-comers: allele count 1, allele number 1614022, AF 6.195702412978262e-07, homozygotes 0.
• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC entry requires the variant to be absent from controls (gnomAD) for PM2.

Sources

Reasoning

The VCEP rule applies the missense path at REVEL ≥ 0.7, or a splicing-impact path only when the predicted outcome matches the disease mechanism. This variant is missense, and the available REVEL score is 0.699, below the VCEP cutoff. SpliceAI reports a maximum delta score of 0.009, indicating no significant splice impact; therefore no qualifying splicing-impact prediction matching the disease mechanism is present. The criterion is not met.

VCEP rule

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

Evidence considered

• The VCEP CSPEC entry for PP3 states: 'For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.'
• The variant is classified as missense (NM_002524.5:c.38G>A; NP_002515.1:p.(Gly13Asp)).
• Local REVEL lookup reports score 0.699, which is below the VCEP PP3 cutoff of 0.7.
• SpliceAI reports DS_AG 0.002, DS_AL 0.0, DS_DG 0.009, DS_DL 0.0, with maximum delta score 0.009; the source summary describes no significant splice impact.

Sources

Reasoning

The NRAS c.38G>A (p.Gly13Asp) variant does not meet the VCEP BA1 threshold. In the default all-comers gnomAD datasets, the allele frequency is 3.976269622890589e-06 (0.00040%; 1/251492 alleles; 0 homozygotes) in gnomAD v2.1 and 6.195702412978262e-07 (0.00006%; 1/1614022 alleles; 0 homozygotes) in gnomAD v4.1. The highest reported subpopulation frequencies are 9.92063492063492e-05 (0.00992%; Ashkenazi Jewish) in v2.1 and 8.475380714101677e-07 (0.00008%; European non-Finnish) in v4.1; all are below the VCEP cutoff of 0.05%.

VCEP rule

GnomAD filtering allele frequency ≥0.05%.

Evidence considered

• gnomAD v2.1 all-comers: 1/251492 alleles, AF 3.976269622890589e-06 (0.00040%), 0 homozygotes; highest subpopulation AF 9.92063492063492e-05 (0.00992%) in Ashkenazi Jewish individuals.
• gnomAD v4.1 all-comers: 1/1614022 alleles, AF 6.195702412978262e-07 (0.00006%), 0 homozygotes; highest subpopulation AF 8.475380714101677e-07 (0.00008%) in European non-Finnish individuals.
• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC entry sets BA1 at a gnomAD filtering allele frequency of at least 0.05%.

Sources

Reasoning

The NRAS c.38G>A (p.Gly13Asp) variant does not meet the VCEP BS1 threshold. The default all-comers gnomAD frequencies are 3.976269622890589e-06 (0.00040%; 1/251492 alleles; 0 homozygotes) in gnomAD v2.1 and 6.195702412978262e-07 (0.00006%; 1/1614022 alleles; 0 homozygotes) in gnomAD v4.1. The highest observed subpopulation frequency is 9.92063492063492e-05 (0.00992%) in Ashkenazi Jewish individuals in v2.1, which remains below the VCEP BS1 cutoff of 0.025%.

VCEP rule

GnomAD filtering allele frequency ≥0.025%.

Evidence considered

• gnomAD v2.1 all-comers: overall AF 3.976269622890589e-06 (0.00040%), with the highest subpopulation AF 9.92063492063492e-05 (0.00992%) in Ashkenazi Jewish individuals.
• gnomAD v4.1 all-comers: overall AF 6.195702412978262e-07 (0.00006%), with the highest subpopulation AF 8.475380714101677e-07 (0.00008%) in European non-Finnish individuals.
• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC entry sets BS1 at a gnomAD filtering allele frequency of at least 0.025%.

Sources

Reasoning

The VCEP limits BP4 to missense variants with REVEL ≤ 0.3. This variant is missense, but its REVEL score is 0.699, which exceeds the VCEP benign-supporting cutoff. The criterion is not met; SpliceAI is not used to establish BP4 because the VCEP's governing BP4 rule names only REVEL for this situation.

VCEP rule

For missense variants: REVEL ≤0.3.

Evidence considered

• The VCEP CSPEC entry for BP4 states: 'For missense variants: REVEL ≤0.3.'
• The variant is classified as missense (NM_002524.5:c.38G>A; NP_002515.1:p.(Gly13Asp)).
• Local REVEL lookup reports score 0.699, above the VCEP BP4 cutoff of 0.3.

Sources

Reasoning

The NRAS VCEP CSPEC permits PS3 when there is a damaging functional result from one or more approved assays, with Supporting for one approved assay and Moderate for two or more different approved assays. The governing functional-studies spreadsheet identifies RAS, MEK, and ERK activation assays as approved pathway-specific assays, but the exact variant is not documented as an NRAS assay result. The only exact bare-protein notation found in the spreadsheet, G13D, occurs in the HRAS validation-control columns for the RAS Activation and ERK Activation assay sections, not in the NRAS columns; the NRAS validation-control lists contain other variants. The reviewed exact-variant paper extracts report clinical or molecular observations but no variant-specific functional assay result. Therefore the available evidence does not establish an applicable approved functional assay result for NRAS c.38G>A (p.Gly13Asp/p.G13D), so PS3 cannot be assigned at any strength.

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. ... Two or more different approved assays.

Evidence considered

• NRAS 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.
• NRAS CSPEC PS3 strength rules: Supporting = One approved assay; Moderate = Two or more different approved assays.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt, READ ME: RAS Activation Assay, MEK Activation Assay, and ERK Activation Assay are approved pathway-specific assays; the spreadsheet states that two or more unique assay types provide sufficient evidence to upgrade PS3 to Moderate.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt, RAS Activation Assay, validation-controls row 76: the HRAS column lists G13D - P, while the NRAS column lists only G12V - P, I24N - P, T58I - P/LP, and G60E - P.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt, ERK Activation Assay, validation-controls row 135: the HRAS column lists G13D - P, while the NRAS column lists only G12V - P, T50I - P, T58I - P/LP, and G60E - P.
• PMID:17332249 paper extract: NRAS codon 13 GGT (Gly) to GAT (Asp) is reported in JMML case 4, but the extract states that no variant-specific functional result is provided for this NRAS p.G13D case.
• PMID:19775298 paper extract: NRAS c.38G>A (p.G13D) is reported as a constitutional activating mutation in a JMML case, with molecular testing across tissues; no functional assay result for this variant is reported in the extract.

Still missing

• No NRAS c.38G>A, p.Gly13Asp, or p.G13D assay-result entry was found in SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt; the bare G13D entries are in HRAS validation-control columns, so a variant-specific NRAS functional result remains needed.
• Possible protein-numbering alias in the literature: PMID:21079152 (G12D)

Sources

Reasoning

The NRAS VCEP rule requires a scored PS4 point total: Strong at ≥5 points, Moderate at ≥3 points, or Supporting at ≥1 point. The available literature documents the exact variant in individual JMML cases (PMID:17332249; PMID:19775298) and one melanoma treatment cohort (PMID:18375819), but no source supplies the VCEP-defined PS4 point assignments or a case-control comparison with unaffected controls. The VCEP-specific point total therefore cannot be calculated without substituting a non-VCEP rule; PS4 is not assessed.

VCEP rule

≥5 points. ... ≥3 points. ... ≥1 point.

Evidence considered

• ClinGen RASopathy VCEP NRAS v2.3 PS4 table: Strong requires ≥5 points; Moderate requires ≥3 points; Supporting requires ≥1 point.
• Matsuda et al. (PMID:17332249), Table 1 case 4: NRAS codon 13 GGT (Gly) → GAT (Asp), corresponding to c.38G>A/p.G13D, in a JMML cohort.
• Oliveira et al. (PMID:19775298): exact NRAS 38G>A mutation reported in a male infant with JMML as a de novo constitutional mutation.
• PMID:18375819: one of six metastatic melanoma patients had G13D NRAS; this is an affected-treatment cohort observation, not a verified unaffected-control comparison.
• No numerical case-control counts, odds ratio, confidence interval, or VCEP point assignment sufficient to substitute into a PS4 threshold were available.

Still missing

• VCEP-defined PS4 point assignments for the reported cases are unavailable.
• No verified affected-versus-unaffected or matched-population case-control counts were found for this exact variant.
• Possible protein-numbering alias in the literature: PMID:21079152 (G12D)

Sources

Reasoning

The VCEP PM5 rule requires at least one likely pathogenic alternate residue change at the same codon, or at least two different likely pathogenic residue changes observed in at least five probands for Strong PM5. The supplied case evidence and paper extracts document NRAS p.G13D but do not provide a verified alternate amino-acid substitution at codon 13 with a likely pathogenic classification under this VCEP. Therefore PM5 cannot be assessed from the permitted evidence.

VCEP rule

1 [likely] pathogenic residue change at the same codon.

Evidence considered

• The case PM5 candidate summary reports zero same-residue candidates and zero expert-panel or non-expert-majority likely pathogenic candidates; it is not used as a classification by itself.
• The reviewed paper extracts report p.G13D but do not establish an alternate likely pathogenic NRAS codon-13 residue change.
• ClinGen RASopathy Expert Panel NRAS v2.3 PM5 Moderate rule: 1 [likely] pathogenic residue change at the same codon; Strong rule: ≥2 different [likely] pathogenic residue changes at the same codon observed in ≥5 probands.

Still missing

• A same-codon NRAS codon-13 alternate amino-acid change with VCEP-compatible likely pathogenic evidence is not available in the supplied evidence.

Sources

Reasoning

No separate unconfirmed de novo observation is available. The available report documents parental absence and therefore provides confirmed de novo evidence evaluated under PS2 rather than an assumed de novo observation for PM6.

Evidence considered

• PMID:19775298 documents that the variant was absent in both parents, which is confirmed de novo evidence rather than an unconfirmed de novo observation.

Still missing

• No independent case with assumed de novo status but without documented parental confirmation is available.

Sources

Reasoning

No informative meioses or affected and unaffected relatives with segregation data are reported for this variant. The available evidence is a single de novo case and does not establish familial segregation.

Evidence considered

• The available variant-specific report describes a single de novo case and does not provide a count of informative familial meioses.

Still missing

• Informative meiosis count and genotype-phenotype data for affected and unaffected relatives are unavailable.

Sources

Reasoning

The VCEP provides BS2 only as a point-based rule (-4 points for strong or -1 point for supporting), but the available evidence does not document a healthy adult homozygote, heterozygote, or an applicable VCEP phenotypic point assignment for this variant. The gnomAD records show zero homozygotes, but absence of homozygotes does not establish the positive healthy-individual observation required to assign BS2 points. Therefore BS2 cannot be assessed under the VCEP rule.

VCEP rule

-4 Points. ... -1 Point.

Evidence considered

• ClinGen RASopathy Expert Panel NRAS v2.3 CSPEC lists BS2 strengths as -4 Points and -1 Point.
• gnomAD v2.1 reports 0 homozygotes among 251492 alleles; gnomAD v4.1 reports 0 homozygotes among 1614022 alleles.
• No documented healthy adult homozygote or heterozygote observation, penetrance assessment, or VCEP phenotypic point assignment for this variant is present in the available evidence.

Still missing

• A documented healthy adult carrier or homozygote observation, with the VCEP-specified phenotypic point assignment, is unavailable.

Sources

Reasoning

No non-segregation observation is reported. The case provides parental absence in a de novo context but does not report an informative meiosis in which the variant failed to track with the phenotype.

Evidence considered

• The available variant-specific report does not describe an affected or unaffected relative carrying the variant in a non-segregating family.

Still missing

• No informative meiosis demonstrating non-segregation between the variant and phenotype is available.

Sources

Reasoning

The VCEP permits BP2 when an alternative molecular cause of a RASopathy is present in the same gene, with phenotype consistent with the expected severity, and assigns strength from the VCEP point total. The available variant-specific report describes NRAS c.38G>A (p.G13D) as a de novo constitutional heterozygous variant and does not establish a second pathogenic variant in NRAS in cis or trans, nor provide a VCEP-defined BP2 point assignment. Therefore BP2 cannot be evaluated from the available evidence without phase and alternative-cause documentation.

VCEP 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

• ClinGen RASopathy Expert Panel NRAS Version 2.3 CSPEC criteria entry for BP2: points are awarded for an alternative molecular cause of a RASopathy in the same gene, and strength is based on the VCEP thresholds of >= (-1), >= (-2), or >= (-4) points.
• PMID:19775298, case report: NRAS c.38G>A (p.G13D) was reported as a heterozygous de novo germ-line variant; the report does not establish a second pathogenic NRAS variant or cis/trans phase for this individual.

Still missing

• Documented alternative pathogenic NRAS variant in the same individual.
• Phase determination showing the alternative variant in cis or trans, as applicable.
• Phenotype assessment against the VCEP's expected RASopathy severity and the VCEP-defined BP2 point assignment.

Sources

Reasoning

The NRAS VCEP BP5 rule requires a VCEP scoring total: Strong at ≥ (-4) points, Moderate at ≥ (-2) points, or Supporting at ≥ (-1) point. No VCEP BP5 point score or governing BP5 evidence calculation is available for this variant, so the required inequality cannot be written with an evidence value substituted and BP5 cannot be assessed without importing a non-VCEP rule.

VCEP rule

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

Evidence considered

• ClinGen RASopathy VCEP NRAS v2.3 BP5 table: Strong requires ≥ (-4) points; Moderate requires ≥ (-2) points; Supporting requires ≥ (-1) point.
• No BP5-specific VCEP score is present in the available evidence, so no substituted numeric inequality can be evaluated.

Still missing

• VCEP-defined BP5 point score and the underlying evidence calculation are unavailable.

Sources