NM_002524.4:c.35G>A (p.Gly12Asp) is the same amino acid change as well-established pathogenic G12D variants in HRAS, KRAS, and other RAS genes, meeting PS1 at Strong strength per RASopathy VCEP rules.1 Gly12 is located in the P-loop domain (amino acids 10-17), a VCEP-specified critical functional domain and mutational hotspot without benign variation, meeting PM1 at Moderate strength.2 REVEL score of 0.783 meets the VCEP PP3 threshold of ≥0.7 for missense variants, providing Supporting in silico evidence of pathogenicity.3 NRAS G12D has been observed in multiple probands with RASopathy-spectrum phenotypes including juvenile myelomonocytic leukemia, meeting PS4 at Supporting strength (≥1 point).4 The variant is present at extremely low frequency in gnomAD (max AF=0.0046%), far below BA1 (0.05%) and BS1 (0.025%) thresholds, but does not meet PM2 which requires complete absence from gnomAD per VCEP rules.5 Extensive functional evidence for NRAS G12D exists in the literature including mouse models and biochemical assays, but VCEP-approved functional assay verification for the specific variant in germline RASopathy context requires human review.6
NRAS
Final classification
Likely Pathogenic
PS1PS4PM1PP3
NRAS
c.35G>A
p.Gly12Asp
This variant
NM_002524.4:c.35G>A (p.Gly12Asp) is the same amino acid change as well-established pathogenic G12D variants in HRAS, KRAS, and other RAS genes, meeting PS1 at Strong strength per RASopathy VCEP rules.
ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for NRAS Version 2.3.0 v2.3.0 criteria-combination framework: matched Rule11 (1 Pathogenic.Strong + 1 Pathogenic.Moderate) with applied criteria: PS1 strong, PS4 supporting, PM1 moderate, PP3 supporting; maps to Likely Pathogenic.
Classification rationale
PS1PS4PM1PP3
Likely Pathogenic
NRAS c.35G>A
PS1 + PS4 + PM1 + PP3
→
Likely Pathogenic
Gene diagram
· NM_002524.4 · variants mapped to exon structure
NRAS
NM_002524.4
Fetching transcript structure from UCSC…
Applied criteria · 4 applied · 13 assessed
MetEvidence satisfies this criterion.
Not metEvaluated against available evidence; threshold not reached.
Not assessedApplies in principle, but no evidence was found to evaluate it.
N/ADoesn't apply to this variant type.
Applied · 4
Strength
Supporting
Moderate
Strong
Very strong
✓
PS1
strong
Pathogenic
NM_002524.4:c.35G>A (p.Gly12Asp) is the same amino acid change as well-established pathogenic G12D variants in HRAS, KRAS, and other RAS genes, satisfying the RASopathy VCEP PS1 rule for analogous pathogenic residue positions. ClinVar classifies NRAS G12D as Pathogenic (11 clinical laboratories) and Likely pathogenic (3 laboratories).
Same amino acid change (G12D) as previously established pathogenic variants in HRASKRASMRAS
✓
PS4
supporting
Pathogenic
NRAS G12D has been observed in multiple probands with RASopathy-spectrum phenotypes. ClinVar reports 14 usable submissions including clinical laboratories classifying as Pathogenic. G12D is reported in juvenile myelomonocytic leukemia (JMML), a RASopathy-associated condition (PMID:17332249). Meeting VCEP PS4 threshold of at least 1 point (Supporting).
ClinVar: Pathogenic by 11 clinical labsLikely pathogenic by 3 (ClinVarID 39648)NRAS codon 12 mutations found in 112/257 NRAS-mutated AML patients (PMID:16434492)
✓
PM1
moderate
Pathogenic
Gly12 is located in the P-loop domain (amino acids 10-17), one of four critical and well-established functional domains specified by the RASopathy VCEP (P-loop, SW1, SW2, SAK). The P-loop is a mutational hotspot without benign variation, satisfying PM1 at Moderate strength.
VCEP PM1: Gly12 falls within P-loop (AA 10-17)a VCEP-approved critical functional domainStatistically significant hotspot (evidence_brief)
✓
PP3
supporting
Pathogenic
REVEL score is 0.783, which meets the RASopathy VCEP PP3 threshold of ≥0.7 for missense variants. SpliceAI predicts no splice impact (max delta=0.00), consistent with a missense mechanism rather than splicing effect. BayesDel score (0.329) is below typical pathogenic thresholds but REVEL is the specified VCEP metric.
REVEL score: 0.783 (meets VCEP threshold of ≥0.7)SpliceAI max delta: 0.00 (no predicted splice impact)BayesDel score: 0.329
Assessed · not applied
· 12 not met · 1 not assessed
Pathogenic
PS2
No de novo occurrence data (with confirmed maternity and paternity) is available for NM_002524.4:c.35G>A in this evidence packet.
PS3
Extensive functional evidence for NRAS G12D as a gain-of-function variant exists in the literature (multiple mouse models, RAS activation assays, MEK/ERK phosphorylation assays, focus formation assays).
PM2
The RASopathy VCEP PM2 rule requires the variant to be absent from gnomAD controls.
PM5
PM5 is designed for novel missense changes at a codon where a different pathogenic missense is already established.
PM6
No de novo data (without confirmation of maternity and paternity) is available for this variant.
PP1
No segregation data is available for NM_002524.4:c.35G>A.
Benign
BA1
The maximum gnomAD filtering allele frequency for NM_002524.4:c.35G>A is 4.62e-5 (0.0046%) in the European (Finnish) subpopulation (v2.1) and grpmax FAF is 7.63e-6 (v4.1).
BS1
The maximum gnomAD allele frequency for NM_002524.4:c.35G>A (0.0046%) is below the VCEP BS1 threshold of ≥0.025%.
BS2
No data on healthy adult individuals carrying NM_002524.4:c.35G>A is available.
BS4
No segregation data is available.
BP2
No evidence of an alternative molecular cause for a RASopathy in the same gene is available.
BP4
REVEL score is 0.783, which does not meet the VCEP BP4 threshold of ≤0.3 for missense variants.
BP5
No evidence of an alternative molecular cause for disease is available.
N/A · 8
PVS1 · PP2 · PP4 · PP5 · BS3 · BP1 · BP6 · BP7
Research & evidence
Population frequency · supports pathogenic
gnomAD v4.1
gnomAD v2.1
v4.1
This variant is present in gnomAD v4.1 (AF= 1.05331e-05; MAF= 0.00105%, 17/1613956 alleles, homozygotes = 0) and has highest observed frequency in the Ashkenazi Jewish population (AF= 3.37861e-05; MAF= 0.00338%, 1/29598 alleles, homozygotes = 0); grpmax FAF= 7.63e-06.
v2.1
This variant is present in gnomAD v2.1 (AF= 7.95279e-06; MAF= 0.00080%, 2/251484 alleles, homozygotes = 0) and has highest observed frequency in the European (Finnish) population (AF= 4.61936e-05; MAF= 0.00462%, 1/21648 alleles, homozygotes = 0).
🇨🇦 CA
Absent from gnomAD-Canada v1.0.
Allele frequency by ancestry
three datasets · side by side
gnomAD v4.1
0.0011%
· 17 / 1,613,956
0 hom · FAF 0.00076%
0 hom · FAF 0.00076%
Ashkenazi Jewish 1 / 29,598 |
0.0034% |
European (non-Finnish) 15 / 1,179,834 |
0.0013% |
South Asian 1 / 91,082 |
0.0011% |
+ 7 not observed (Remaining individuals, Admixed American, European (Finnish), Amish, East Asian, Middle Eastern, African/African American)
gnomAD v2.1
0.0008%
· 2 / 251,484
0 hom
0 hom
European (Finnish) 1 / 21,648 |
0.0046% |
European (non-Finnish) 1 / 113,762 |
0.00088% |
+ 6 not observed (African/African American, Admixed American, Ashkenazi Jewish, East Asian, Remaining individuals, South Asian)
gnomAD Canada 🇨🇦
Absent
· 0 / ?
0 hom
0 hom
Not observed in any ancestry group.
ClinVar
This variant has been reported in ClinVar as Pathogenic (11 clinical laboratories) and as Likely pathogenic (3 clinical laboratories). (ClinVarID = 39648)
In silico
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). REVEL score = 0.783. BayesDel score = 0.328551.
Functional
Oncogenic
OncoKB identified variant-specific curated literature and context relevant to functional review; biological-effect context: Gain-of-function; curated oncogenicity label: Oncogenic.
COSMIC
Cancer hotspots
Somatic evidence
Hotspot
COSMIC
This variant lies in a statistically significant hotspot. This variant has previously been reported in somatic cancers (COSMIC; COSV54736383, n = 1303 times).
Hotspots
This variant lies in a statistically significant hotspot.
Literature · how each cited paper was used
2papers cited
Each card is an audit: what was searched, what was found, whether it names the variant, which criteria it fed, and why. 5 further PMIDs triaged but not cited — see Sources & references.
High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients.
Searched
G12Dc.35G>AGly12Asp
Found
N-Ras G12D was used as a positive control in functional transformation assays (focus formation, colony formation, RAF pull-down) and was the most biochemically active mutant tested. NRAS G12D was also detected as a somatic mutation in 10 AML patients and 3 CMML patients in the screening cohort.
Variant
✓ Names this variant — characterised directly
Applied to
→PS4
met
Why
Variant-specific functional data and patient observations confirmed. N-Ras G12D used as positive control demonstrating strongest transformative capacity. Referenced in PS3 and PS4 assessments.
cells were infected with equivalent multiplicity of infection of retrovirus-expressing empty vector, N-Ras G12D, N-Ras WT
Location Methods (focus formation assay); Results; Table 1 · Context A31 fibroblast focus formation assay, RAF-1 pull-down assay, murine bone marrow colony-forming assay, HEK 293T/17 cells · full text
Rule & framework references · cited for criterion definitions, not variant evidence
16434492 ↗
Implications of NRAS mutations in AML: a study of 2502 patients.
Sources & reference links
9Sources
Triaged references · 5 PMIDs not cited in assessment
21586752 ↗
Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose- and cell type-dependent manner.
ONCOKB
18372904 ↗
Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.
ONCOKB
23687087 ↗
Nras(G12D/+) promotes leukemogenesis by aberrantly regulating hematopoietic stem cell functions.
ONCOKB