HRAS c.34G>A, p.(Gly12Ser)

Reasoning

The ClinGen RASopathy VCEP HRAS v2.3 specification uses point-based PS2 scoring: Very Strong for 4 points, Strong for 2 points, and Moderate for 1 point. For the exact HRAS NM_001130442.2:c.34G>A (p.Gly12Ser) variant, Aoki et al. documented parental genotyping in four families and identified the mutations as de novo; two informative c.34G>A cases (COS38 and COS64) had WT/WT parents. Zampino et al. directly genotyped parental DNA, proved paternity in all families, and reported de novo origin for all cases; nine informative families with HRAS mutations, including the recurrent c.34G>A variant, showed paternal origin from unaffected fathers. The directly established de novo observations exceed the VCEP threshold for Strong PS2, with phenotype-consistent Costello syndrome cohorts.

Rule

ClinGen RASopathy VCEP HRAS v2.3: PS2 Very Strong = 4 points; Strong = 2 points; Moderate = 1 point.

Evidence considered

• The HRAS ClinGen RASopathy VCEP v2.3 specification defines PS2 point scoring as Very Strong at 4 points, Strong at 2 points, and Moderate at 1 point.
• Aoki et al. reported four heterozygous de novo HRAS mutations in 12 of 13 affected individuals and stated that parental DNA from four families demonstrated de novo occurrence. In Table 1, the exact 34G>A (p.Gly12Ser) variant was present in COS38 and COS64, with WT/WT parental genotypes.
• Zampino et al. found a de novo heterozygous HRAS change in all subjects diagnosed with Costello syndrome, including eight c.34G>A (p.Gly12Ser) cases; parental DNA genotyping demonstrated de novo origin in all cases, paternity was proved in all families, and nine informative families showed paternal germline origin.
• The cited cohorts describe phenotypically affected individuals with Costello syndrome, including the characteristic phenotype associated with p.Gly12Ser, supporting full VCEP de novo points rather than reduced points for limited clinical information.

Sources

Reasoning

ClinGen RASopathy VCEP v2.3 defines PS3 as applicable with strength tied to the number of distinct VCEP-approved assay categories used (SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx): Supporting for one approved assay, Moderate for two or more. Multiple independent clinical-testing laboratories (Ambry Genetics, GeneDx, Illumina Laboratory Services) captured in ClinVar submissions for this exact variant (HRAS c.34G>A / p.Gly12Ser) converge on functional-study narratives describing (1) a RAS-GTP activation/loading assay showing increased active GTP-bound HRAS and altered GTP/GDP dissociation kinetics, and (2) downstream MEK/ERK/AKT phosphorylation and growth-factor-independent proliferation assays, both of which correspond to VCEP-approved assay categories ('RAS Activation Assay' and 'MEK/ERK Activation Assay') listed for HRAS in the VCEP functional-studies reference table. These findings are biologically consistent with the well-established G12-hotspot mechanism of impaired intrinsic/GAP-stimulated GTPase activity causing constitutive RAS-GTP accumulation and MAPK pathway hyperactivation, which is the accepted RASopathy/oncogenic disease mechanism for this codon. However, the primary functional-study papers referenced by these submitters (e.g., Wey et al. GTP/GDP dissociation kinetics; Gremer et al. MEK/ERK/AKT phosphorylation) and the myopathy functional paper (PMID:17412879, van der Burgt et al.) are not present in this case's source_registry with retrievable full text, so the specific assay methodology, validation controls, and exact quantitative readouts for p.Gly12Ser could not be directly quoted or independently verified against the VCEP's approval criteria for this case. Strength is therefore capped at Supporting (single verifiable category of convergent evidence) rather than Moderate, and flagged for human review to locate/verify the primary assay publications.

Rule

ClinGen RASopathy VCEP v2.3 PS3: Supporting for one approved assay type showing a damaging effect; Moderate for two or more different approved assays. Approved assay categories defined in SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.

Evidence considered

• cspec (ClinGen RASopathy VCEP v2.3): PS3 defaultStrength Pathogenic Strong per rules, but VCEP-specific strength_rules cap it at Moderate for '>=2 different approved assays' and Supporting for 'one approved assay'; approved assays are defined in the VCEP supplemental functional-studies materials.
• ClinVar submitter (Ambry Genetics, SCV accession context): 'functional studies show that this variant promotes enhanced MEK, ERK, and AKT phosphorylation and growth-factor independent proliferation, indicating that this variant impacts protein function.'
• ClinVar submitter (GeneDx, SCV000207842): 'functional studies indicate that the G12S variant alters GTP and GDP dissociation rates resulting in increased active GTP-bound HRAS, which up-regulates the RAS/MAPK pathway.'
• ClinVar submitter (Illumina Laboratory Services, SCV004801624): 'functional studies found that when the p.[Gly12Ser]...' (assay narrative truncated in captured evidence signal, but flags a functional-assay claim for this exact variant).
• VCEP SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx (vcep_materials): defines 'RAS Activation Assay' (measures bound/active RAS via immunoprecipitation) and 'MEK Activation Assay'/'ERK Activation Assay' (phospho-MEK/phospho-ERK ratios) as pathway-specific approved assay categories applicable to HRAS, listing prior HRAS-assay PMIDs (e.g., 19995790, 19035362, 25914166, 23335589, 21850009) as the gene's approved-assay literature base, though none of these are variant-specific full-text sources retrieved in this case.

Still missing

• Primary functional-study publications underlying the ClinVar submitter narratives (RAS-GTP loading/dissociation-kinetics assay; MEK/ERK/AKT phosphorylation assay) are not included in this case's source_registry or fetched full text, preventing direct quotation and verification against VCEP-approved assay validation criteria.
• PMID:17412879 (van der Burgt et al., HRAS myopathy functional study) appears in the ClinVar PMID trail as 'unresolved'/not validated and has no full text available in this case bundle, so its specific functional findings for this variant could not be directly confirmed.

Sources

Reasoning

The HRAS VCEP specifies PS4 Strong for at least 5 points. Exact-variant case-series evidence exceeds this threshold: c.34G>A (p.Gly12Ser) was observed in 7 Costello syndrome patients in the original report, in 30 of 37 HRAS-positive Costello syndrome patients in an independent 43-case series, and in 3 unrelated Dutch Costello syndrome patients. The original report also found the variant in affected Costello syndrome patients and no HRAS mutations in 28 Noonan syndrome controls. Overlap between later reports is possible, but the single 43-case series alone exceeds the VCEP threshold.

Rule

ClinGen RASopathy VCEP HRAS Version 2.3: PS4 Strong at >=5 points; Moderate at >=3 points; Supporting at >=1 point.

Evidence considered

• The HRAS VCEP Version 2.3 rule specifies PS4 Supporting for at least 1 point, Moderate for at least 3 points, and Strong for at least 5 points.
• Aoki et al. reported c.34G>A (p.Gly12Ser) in 7 of 13 individuals with Costello syndrome and reported no mutations in 28 individuals with Noonan syndrome or in one additional individual with Costello syndrome.
• Kerr et al. found HRAS mutations in 37 of 43 clinically diagnosed Costello syndrome patients; c.34G>A (G12S) accounted for 30 of the 37 mutation-positive cases.
• van Steensel et al. identified the exact G12S substitution in all 3 unrelated Dutch patients with Costello syndrome.
• Zampino et al. detected the recurrent c.34G>A change in 8 subjects, with all subjects diagnosed with Costello syndrome carrying a de novo HRAS mutation.

Sources

Reasoning

The HRAS VCEP (v2.3) specifies PM1 (Moderate) as applicable only to critical, well-established functional domains: P-loop (AA 10-17), SW1 (AA 25-40), SW2 (AA 57-64), and SAK (AA 145-156), explicitly stating it is not applicable to specific single residues (reserved for PM5). Residue Gly12 of HRAS falls within the P-loop (AA 10-17), which is corroborated by the VCEP's own Alignment-with-PM1-domains supplemental material listing HRAS 10-17 as the P-loop domain. Therefore PM1 is met at Moderate strength.

Rule

HRAS VCEP v2.3 PM1: Moderate - variant located within P-loop (AA10-17), SW1 (AA25-40), SW2 (AA57-64), or SAK (AA145-156).

Evidence considered

• HRAS VCEP v2.3 cspec: PM1 Moderate applies to P-loop (AA 10-17), SW1 (AA 25-40), SW2 (AA 57-64), SAK (AA 145-156); not applicable to individual residues (see PM5).
• VCEP supplemental slide (Alignment-with-PM1-domains.pptx) confirms P-loop domain boundary as HRAS residues 10-17, which includes codon 12 (Gly12).

Sources

Reasoning

PM2 is met at Supporting strength under the HRAS VCEP specification. The variant is reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. The applicable VCEP rule requires absence from controls in gnomAD and assigns Supporting strength.

Rule

The variant must be absent from controls (gnomAD).

Evidence considered

• The HRAS VCEP specification (version 2.3) states that PM2 requires the variant to be absent from controls in gnomAD and specifies Supporting strength.
• The variant NM_001130442.2:c.34G>A (HRAS p.Gly12Ser) is reported absent from gnomAD v2.1.
• The variant is reported absent from gnomAD v4.1.
• The variant is reported absent from gnomAD-Canada v1.0.

Sources

Reasoning

Expert panel ClinGen RASopathy Variant Curation Expert Panel classified as Pathogenic.

Rule

Requested rule: exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification supports PP5 at Supporting strength; non-expert submissions and aggregate labels do not qualify.

Evidence considered

• ClinVar Variation ID 12602 identifies the exact variant as NM_005343.4(HRAS):c.34G>A (p.Gly12Ser), with Pathogenic classification and review status reviewed by expert panel; the expert panel is the ClinGen RASopathy Variant Curation Expert Panel.
• The PP5 determination uses only the exact-variant expert-panel assertion and does not use the 40 ordinary clinical-laboratory submissions or the aggregate ClinVar label independently.
• ClinVar expert panel classification

Sources

Reasoning

PM6 is reserved for an assumed de novo variant without confirmation of paternity and maternity. The relevant HRAS reports provide parental genotyping and, in the Zampino cohort, explicit paternity verification; therefore this evidence is adjudicated as PS2 rather than PM6.

Rule

ClinGen RASopathy VCEP HRAS v2.3: PM6 requires assumed de novo status without confirmation of paternity and maternity; confirmed parental testing is evaluated under PS2.

Evidence considered

• The HRAS VCEP specification defines PM6 as assumed de novo without confirmation of paternity and maternity, with point-based strengths of Strong at 2 points, Moderate at 1 point, and Supporting at 0.5 points.
• Aoki et al. reported parental DNA testing in four families, including WT/WT parental genotypes for two exact c.34G>A cases.
• Zampino et al. performed parental DNA genotyping and verified paternity in all informative families; this is confirmed de novo evidence and does not meet the PM6 definition.

Sources

Reasoning

NM_001130442.2:c.34G>A is a missense variant (p.Gly12Ser). The governing HRAS RASopathy VCEP cspec (v2.3) PP3 rule requires REVEL >= 0.7 for missense variants. The prefetched local REVEL lookup score is 0.695, which falls just below the VCEP threshold, so PP3 is not met under the gene-specific rule. SpliceAI (max delta score = 0.00) shows no predicted splice impact, so the alternate splicing-impact path for PP3 is also not triggered for this coding missense change. BayesDel score (0.211813) was retrieved but is not used to support PP3 because no verified published calibration threshold for BayesDel is available to this pipeline.

Rule

HRAS RASopathy VCEP cspec v2.3 PP3: missense REVEL >= 0.7 (Supporting)

Evidence considered

• HRAS RASopathy VCEP cspec v2.3 PP3 rule: 'For missense variants: REVEL >= 0.7. For splicing impact, predicted outcome must match disease mechanism.'
• Local REVEL predictor lookup score for NM_001130442.2:c.34G>A = 0.695, below the VCEP's 0.7 PP3 threshold.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00).' -- confirms no splice-based route to PP3.
• BayesDel score 0.211813 was returned by the local lookup but is treated as not_available for PP3/BP4 purposes because no named, citable publication with a verified calibrated threshold for BayesDel is available to this pipeline.

Sources

Reasoning

BA1 is not met. The ClinGen RASopathy VCEP HRAS specification requires a gnomAD filtering allele frequency of at least 0.05% for BA1. The variant is reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; therefore no BA1-level population frequency is present.

Rule

GnomAD filtering allele frequency >=0.05%.

Evidence considered

• The HRAS VCEP specification (version 2.3) defines BA1 as gnomAD filtering allele frequency >=0.05%.
• The variant NM_001130442.2:c.34G>A (HRAS p.Gly12Ser) is reported absent from gnomAD v2.1.
• The variant is reported absent from gnomAD v4.1.
• The variant is reported absent from gnomAD-Canada v1.0.

Sources

Reasoning

BS1 is not met. The HRAS VCEP specification requires a gnomAD filtering allele frequency of at least 0.025% for BS1. The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, so the required increased population frequency is not observed.

Rule

GnomAD filtering allele frequency >=0.025%.

Evidence considered

• The HRAS VCEP specification (version 2.3) defines BS1 as gnomAD filtering allele frequency >=0.025%.
• The variant NM_001130442.2:c.34G>A (HRAS p.Gly12Ser) is reported absent from gnomAD v2.1.
• The variant is reported absent from gnomAD v4.1.
• The variant is reported absent from gnomAD-Canada v1.0.

Sources

Reasoning

The HRAS RASopathy VCEP cspec (v2.3) BP4 rule requires REVEL <= 0.3 for missense variants (with an alternate splicing-negligible-impact path for splice variants). The variant's REVEL score is 0.695, well above the 0.3 BP4 threshold, so BP4 is not met via the missense path. SpliceAI shows no splice impact (max delta = 0.00), but this variant is a missense substitution (p.Gly12Ser), not a splice-region variant, so the splicing-negligible path is not the applicable route and does not independently satisfy BP4 for this coding missense change. BayesDel (0.211813) is not used because it lacks a verified, citable calibration threshold in this pipeline.

Rule

HRAS RASopathy VCEP cspec v2.3 BP4: missense REVEL <= 0.3 (Supporting)

Evidence considered

• HRAS RASopathy VCEP cspec v2.3 BP4 rule: 'For missense variants: REVEL <= 0.3. For splicing variants: predicted outcome is negligible or does not match disease mechanism.'
• Local REVEL predictor lookup score for NM_001130442.2:c.34G>A = 0.695, above the VCEP's 0.3 BP4 threshold, placing it in the intermediate (non-actionable) REVEL zone.
• BayesDel score 0.211813 not applied to BP4 because no verified, named-publication calibration threshold for BayesDel is available to this pipeline.

Sources

Reasoning

No exact-variant ClinVar expert-panel classification of Benign or Likely benign was identified. The exact-variant expert-panel classification present in ClinVar is Pathogenic, so BP6 is not met. Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions were not used to trigger or negate this criterion.

Rule

Requested rule: exact-variant ClinVar expert-panel Benign or Likely benign classification supports BP6; no such classification is present.

Evidence considered

• ClinVar Variation ID 12602 records the exact HRAS c.34G>A (p.Gly12Ser) variant as Pathogenic with review status reviewed by expert panel, attributed to the ClinGen RASopathy Variant Curation Expert Panel; no Benign or Likely benign expert-panel classification is present in the supplied record.

Sources

Reasoning

The HRAS VCEP (v2.3) specifies PS1 (Strong) for a variant producing the same amino acid change as a previously established pathogenic variant regardless of nucleotide change, and extends this to observed analogous residue positions in HRAS, KRAS, MRAS, NRAS, RIT1, and RRAS2. The case-evidence bundle contains no search or comparator data identifying an alternate nucleotide-level variant (e.g., a different codon 12 substitution such as c.34G>T or c.34G>C) that independently produces p.Gly12Ser and has been separately established as pathogenic, nor any analogous-position (e.g., KRAS/NRAS G12S) comparator variant curation. The variant under review (NM_001130442.2:c.34G>A, p.Gly12Ser) is itself the ClinVar expert-panel-classified pathogenic record, so it cannot be used as its own PS1 comparator.

Rule

HRAS VCEP v2.3 PS1: Strong - same amino acid change as a previously established pathogenic variant regardless of nucleotide change; applicable for observed analogous residue positions in HRAS, KRAS, MRAS, NRAS, RIT1, and RRAS2.

Still missing

• No dedicated PS1 comparator search was performed or captured in this case bundle (unlike PM5, which has a pm5_candidates.json artifact); need explicit query for alternate nucleotide changes producing p.Gly12Ser and/or analogous-residue pathogenic variants in KRAS, NRAS, MRAS, RIT1, RRAS2 before this criterion can be adjudicated.

Reasoning

The HRAS VCEP (v2.3) PM5 rule requires 1 or more different established [likely] pathogenic residue changes at the same codon (Moderate for 1 change; Strong for >=2 changes each observed in >=5 probands). This case's dedicated PM5 comparator-harvesting step failed softly (HTTP 429 Too Many Requests) before any same-residue candidate variants (e.g., HRAS p.Gly12Ala, p.Gly12Cys, p.Gly12Asp, p.Gly12Val, p.Gly12Arg) could be retrieved or classified, and no such comparator data appears elsewhere in the case-evidence bundle. Without confirmed pathogenic/likely-pathogenic status and proband counts for alternate codon-12 residue changes, PM5 cannot be adjudicated.

Rule

HRAS VCEP v2.3 PM5: Strong if >=2 different [likely] pathogenic residue changes at the same codon observed in >=5 probands; Moderate if 1 [likely] pathogenic residue change at the same codon.

Evidence considered

• pm5_candidates.json documents a failed automated same-residue comparator search (errors: HTTP Error 429: Too Many Requests) with 0 same-residue candidates identified out of 13 total candidates seen, and explicitly recommends the criterion not be applied from that artifact alone.

Still missing

• Automated same-residue comparator search (pm5_candidates.json) failed due to rate limiting (HTTP 429) and returned zero candidates; a manual ClinVar/literature search for other pathogenic HRAS codon 12 missense changes (e.g., G12A, G12C, G12D, G12V, G12R) with proband counts is required to adjudicate PM5.

Sources

Reasoning

No affected-relative segregation series or informative meiosis count for the clinical case is provided. The available publications establish de novo origin from unaffected parents, which is PS2 evidence and is not PP1 segregation evidence. The HRAS VCEP threshold of at least 3 informative meioses for Supporting, 5 for Moderate, or 7 for Strong therefore cannot be applied.

Rule

ClinGen RASopathy VCEP HRAS v2.3: PP1 Supporting = at least 3 informative meioses; Moderate = at least 5; Strong = at least 7.

Evidence considered

• The HRAS VCEP specification requires at least 3 informative meioses for PP1 Supporting, at least 5 for Moderate, and at least 7 for Strong.
• The reviewed HRAS reports concern de novo variants in affected probands and unaffected parents; they do not provide affected-relative segregation data for this case.

Still missing

• No case-specific affected relatives with genotype and phenotype are reported.
• No informative PP1 meioses are documented.

Sources

Reasoning

BS2 is not assessed. No documented healthy adult carrying this variant in the required disease-relevant genotype or a documented healthy homozygote/heterozygote observation was identified in the consolidated case-evidence bundle. Absence from population databases is not equivalent to evidence of a healthy carrier.

Rule

Point-based scoring based on healthy homozygote or heterozygote individuals; reduced points may apply to apparently unaffected heterozygous individuals during clinical family evaluation.

Evidence considered

• The HRAS VCEP specification describes BS2 as requiring point-based evidence from healthy homozygote or heterozygote individuals, with reduced points for apparently unaffected heterozygous individuals.
• The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; these absence findings provide no healthy-carrier observation for BS2.

Still missing

• A validated observation of the variant in a healthy adult, with genotype and adequate age/phenotype assessment for the expected penetrance of HRAS-related disease, is needed to assess BS2.

Sources

Reasoning

No informative affected or unaffected relatives carrying the variant, other than the tested unaffected parents of de novo probands, are available to evaluate failure to segregate with disease. The VCEP requires one informative meiosis for BS4 Strong, and no such non-segregating family observation is documented for this case.

Rule

ClinGen RASopathy VCEP HRAS v2.3: BS4 Strong requires one informative meiosis showing non-segregation.

Evidence considered

• The HRAS VCEP specification defines BS4 Strong as requiring only one informative meiosis.
• The reviewed reports document de novo occurrence from unaffected parents rather than failure of the variant to segregate with disease in an informative family.

Still missing

• No informative non-segregating family meiosis is reported.
• No genotype-phenotype data for affected or unaffected relatives beyond the de novo parental tests are available.

Sources

Reasoning

The HRAS VCEP framework provides BP2 point-based strength rules, but the case bundle contains no affected-proband or family-level data establishing that NM_001130442.2:c.34G>A (p.Gly12Ser) is in cis with another pathogenic variant, nor does it provide a documented second pathogenic variant in the same individual. The available literature describes this HRAS variant as a heterozygous de novo variant in an autosomal dominant disorder, but does not establish the phase relationship required for BP2. Therefore BP2 cannot be applied or excluded from the available evidence.

Rule

BP2 point-based strength rules per ClinGen RASopathy/HRAS VCEP version 2.3; no qualifying phase observation documented.

Evidence considered

• The HRAS VCEP version 2.3 provides BP2 point-based rules: Strong at >=(-4) points, Moderate at >=(-2) points, and Supporting at >=(-1) point.
• The case bundle does not document a second pathogenic variant or phase information for the evaluated proband.
• Published HRAS Costello syndrome reports reviewed for this group describe heterozygous de novo HRAS variants but do not report the evaluated variant in cis with another pathogenic variant (PMIDs:16170316, 17054105).

Still missing

• No case-specific report of a second pathogenic or likely pathogenic variant in the same individual.
• No parental testing, haplotyping, or other phase data for this case establishing cis or trans configuration with another pathogenic variant.

Sources

Reasoning

No qualifying evidence was identified for the benign alternate-molecular-basis criterion BP5. The available case literature reports Costello syndrome and HRAS c.34G>A (p.Gly12Ser), but does not provide a documented alternate molecular diagnosis for the same affected individuals that would support BP5. No BP5 inference is made from other criteria or classifications.

Rule

Use BP5 only when the variant is found in an affected individual with an alternate molecular basis for the phenotype; no qualifying case was identified.

Evidence considered

• The HRAS VCEP provides BP5 point-based strength rules but the reviewed exact-variant case reports describe HRAS-associated Costello syndrome rather than an alternate molecular etiology in the same affected individuals.
• No qualifying BP5 alternate-diagnosis observation was available in the consolidated case-evidence bundle.

Still missing

• A documented affected individual with the exact variant and an established alternate molecular cause for the phenotype was not identified in the reviewed evidence.

Sources