KRAS c.35G>T, p.(Gly12Val)

Reasoning

The KRAS RASopathy VCEP (v2.3) defines PS3 using approved functional assays: one approved assay = Supporting, two or more different approved assays = Moderate (no Strong tier specified in this cspec rule). The VCEP-approved functional-studies table lists PMID 20949621 as a validated source for both the RAS Activation (GTPase biochemistry) Assay and the MEK/ERK Activation Assay for KRAS, and lists p.G12V among the pathogenic validation controls used to establish those assays. Full text of PMID 20949621 (Denayer et al.) confirms direct biochemical characterization of purified RAS-G12V protein: it was used as the 'GTPase deficient mutant' reference, showing markedly decreased intrinsic and GAP(NF1)-stimulated GTP hydrolysis (locking RAS in the active GTP-bound state), and elevated downstream pMEK1/2, pERK1/2, and pAKT relative to RAS-wt in cellular assays. This reproduces abnormal function consistent with the RASopathy/oncogenic gain-of-function disease mechanism across two distinct VCEP-approved assay categories (RAS Activation/GTPase assay and MEK/ERK Activation assay) reported within the same validated source, meeting the VCEP threshold for PS3_Moderate.

Rule

KRAS RASopathy VCEP v2.3 PS3: Moderate for >=2 different approved assays; Supporting for 1 approved assay.

Evidence considered

• KRAS RASopathy VCEP v2.3 cspec PS3 rule: 'Two or more different approved assays' = Moderate; 'One approved assay' = Supporting.
• VCEP SVI-RASopathy-VCEP-V2-Approved-Functional-Studies table lists PMID 20949621 (with 23059812) as validated sources for the KRAS 'RAS Activation Assay' and separately for the KRAS 'MEK Activation Assay' / 'ERK Activation Assay', with G12V listed among pathogenic validation controls for these assays.
• PMID 20949621 full text: 'we used RAS wt, a GTPase deficient mutant (RAS G12V) and a self-activating (fast-cycling) mutant (RAS F28L)' as controls in purified-protein biochemical characterization; all data summarized in Table 1 and Supp. Table S4.
• PMID 20949621 full text: intrinsic and GAP(NF1)-stimulated GTP hydrolysis were markedly reduced for oncogenic p.G12V relative to wt, comparable to or exceeding the reduction seen for several germline RASopathy mutants, consistent with G12V being locked in the GTP-bound active state.
• PMID 20949621 full text: '...pMEK1/2, pERK1/2, and AKT (pAKT) compared to RAS wt but less than RAS G12V' — indicating RAS-G12V produced the highest downstream MAPK pathway activation among the tested proteins, i.e. two distinct assay readouts (GTPase biochemistry and downstream pathway phosphorylation) both showing abnormal, pathway-activating function for G12V.

Still missing

• The second PMID listed for the RAS Activation Assay (23059812) is not present in this case's source registry/full-text set and could not be independently verified to further support assay count.
• G12V functional data in PMID 20949621 appears as a well-established validation control rather than the primary germline variant under study in that paper; no additional independent wet-lab assay paper specific to this exact variant was reviewed beyond the VCEP-approved-assay table entry.

Sources

Reasoning

The KRAS RASopathy VCEP PM1 specification restricts this criterion to variants falling within specific critical, well-established functional domains: P-loop (AA 10-17), Switch I/SW1 (AA 25-40), Switch II/SW2 (AA 57-64), and SAK (AA 145-156), applied at Moderate strength (not applicable to individual residue-specific analysis, which is covered by PM5). Codon 12 (Gly12, altered to Val12) falls within the P-loop domain (AA 10-17) as defined in the VCEP's PM1-domain alignment material, satisfying this rule.

Rule

Applicable only to critical and well-established functional domains: P-loop (AA 10-17), SW1 (AA 25-40), SW2 (AA 57-64), SAK (AA 145-156).

Evidence considered

• cspec (KRAS RASopathy VCEP v2.3) PM1 rule: domain table lists P-loop as AA 10-17 (Moderate strength), applicable only to the listed critical/well-established functional domains.
• VCEP alignment material (Alignment-with-PM1-domains.pptx) confirms the P-loop spans HRAS/KRAS residues 10-17, encompassing codon 12.

Sources

Reasoning

Variant NM_033360.3:c.35G>T (p.Gly12Val) is a missense variant. The ClinGen RASopathy VCEP specification (KRAS-specific cspec v2.3) defines PP3 explicitly as: 'For missense variants: REVEL >= 0.7' at Supporting strength. The local REVEL lookup for this variant returns a score of 0.91, which exceeds the VCEP-specified threshold of 0.7. PP3 is met at Supporting strength per the VCEP's own pre-assigned code/threshold for this criterion.

Rule

For missense variants: REVEL >= 0.7 (ClinGen RASopathy VCEP KRAS specification v2.3).

Evidence considered

• ClinGen RASopathy VCEP cspec v2.3 specifies PP3 (Supporting) rule: 'For missense variants: REVEL >= 0.7.'
• Local REVEL predictor lookup returns score 0.91 for NM_033360.3:c.35G>T (p.Gly12Val), which is >= 0.7.

Sources

Reasoning

PM2 is not met under the KRAS VCEP specification because the variant is not absent from gnomAD: gnomAD v4.1 reports one allele (AF 6.20014e-07) and zero homozygotes. gnomAD v2.1 and gnomAD-Canada report absence, but the VCEP rule requires absence from gnomAD and the v4.1 observation prevents satisfying that requirement.

Rule

The variant must be absent from controls (gnomAD).

Evidence considered

• The ClinGen RASopathy VCEP KRAS Version 2.3 specification states that PM2 Supporting requires the variant to be absent from controls in gnomAD.
• gnomAD v4.1 reports the exact variant at 1/1,612,866 alleles (AF 6.20014e-07) with zero homozygotes, so the variant is not absent from gnomAD.
• The variant is absent from gnomAD v2.1 (0/249,328 alleles) and gnomAD-Canada v1.0, but these negative datasets do not override the observed gnomAD v4.1 allele for the VCEP PM2 rule.

Sources

Reasoning

PM4 addresses protein length changes from in-frame deletions/insertions or stop-loss variants in a non-repetitive region. NM_033360.3:c.35G>T is a single-nucleotide missense substitution (p.Gly12Val) that does not alter protein length (no insertion, deletion, or stop-loss). The KRAS RASopathy VCEP CSPEC defines PM4 with the rule 'no known repetitive areas in gene; use as described' but this rule engages only for variants that change protein length, which this substitution does not. PM4 is therefore not met.

Rule

PM4: Moderate — no known repetitive areas in KRAS; applies only to protein length-altering variants (ClinGen RASopathy VCEP CSPEC v2.3)

Evidence considered

• case_summary.json compact_evidence.cspec PM4 rule: 'No known repetitive areas in gene. Use as described,' defaultStrength Pathogenic Moderate; applies to protein-length-altering variants (in-frame indels, stop-loss), not single-nucleotide substitutions.
• Variant p.Gly12Val is a missense change with no change in protein length, so the PM4 length-change trigger is not met.

Sources

Reasoning

BA1 is not met. The KRAS VCEP Version 2.3 threshold is a gnomAD filtering allele frequency of at least 0.05%. The highest reported frequency is 6.20014e-07 overall in gnomAD v4.1 (1/1,612,866 alleles; 0.000062%) and 8.47959e-07 in the highest-frequency population, European non-Finnish (1/1,179,302; 0.000085%), both far below 0.05%. gnomAD v2.1 reported 0/249,328 alleles.

Rule

GnomAD filtering allele frequency >=0.05%.

Evidence considered

• The ClinGen RASopathy VCEP KRAS Version 2.3 specification defines BA1 as gnomAD filtering allele frequency >=0.05%.
• gnomAD v4.1 reports the exact variant at total AF 6.20014e-07 (1/1,612,866 alleles), with the highest population AF 8.47959e-07 in European non-Finnish individuals (1/1,179,302 alleles); no homozygotes were observed.
• gnomAD v2.1 reports 0/249,328 alleles and no homozygotes for the exact variant.

Sources

Reasoning

BS1 is not met. The KRAS VCEP Version 2.3 threshold is a gnomAD filtering allele frequency of at least 0.025%. The gnomAD v4.1 overall AF is 6.20014e-07 (0.000062%), and the highest population AF is 8.47959e-07 (0.000085%), both below the VCEP threshold. gnomAD v2.1 reports no observed alleles.

Rule

GnomAD filtering allele frequency >=0.025%.

Evidence considered

• The ClinGen RASopathy VCEP KRAS Version 2.3 specification defines BS1 as gnomAD filtering allele frequency >=0.025%.
• gnomAD v4.1 reports one allele among 1,612,866 total alleles (AF 6.20014e-07; 0.000062%) and a maximum reported population AF of 8.47959e-07 in European non-Finnish individuals (0.000085%), below 0.025%.
• gnomAD v2.1 reports 0/249,328 alleles for the exact variant.

Sources

Reasoning

The ClinGen RASopathy VCEP specification defines BP4 as: 'For missense variants: REVEL <= 0.3' at Supporting strength. The REVEL score for this variant is 0.91, well above the 0.3 threshold, so BP4 is not met. SpliceAI max delta score of 0.005 (no significant splice impact) is not independently applicable here because the VCEP's BP4 rule for this variant class is scoped to the missense/REVEL path, and the variant's primary molecular consequence is missense (p.Gly12Val), not splicing-relevant.

Rule

For missense variants: REVEL <= 0.3 (ClinGen RASopathy VCEP KRAS specification v2.3).

Evidence considered

• ClinGen RASopathy VCEP cspec v2.3 specifies BP4 (Supporting) rule: 'For missense variants: REVEL <= 0.3.'
• Local REVEL predictor lookup returns score 0.91, which does not meet the REVEL <= 0.3 threshold required for BP4.

Sources

Reasoning

The case-evidence bundle does not provide proband-level parental testing results, confirmation of both maternity and paternity, or an explicit de novo observation for NM_033360.3:c.35G>T (p.Gly12Val). The KRAS RASopathy VCEP requires confirmed de novo status and applies point-based PS2 scoring with phenotype-specific adjustment; therefore PS2 cannot be assigned from the available evidence.

Rule

PS2: follow SVI point-based scoring with phenotype-specific adjustment; 4 points = Very Strong, 2 points = Strong, 1 point = Moderate (ClinGen RASopathy VCEP CSPEC v2.3)

Evidence considered

• The case-evidence bundle identifies the variant as NM_033360.3:c.35G>T (p.Gly12Val) but contains no proband pedigree, parental genotypes, maternity confirmation, paternity confirmation, or explicit de novo finding.
• The KRAS RASopathy VCEP CSPEC v2.3 defines PS2 as de novo in a patient with disease with both maternity and paternity confirmed and specifies point-based strengths of 4 points (Very Strong), 2 points (Strong), or 1 point (Moderate).

Still missing

• Documented variant testing results for both biological parents are missing.
• Confirmation of both maternity and paternity is missing.
• A documented affected proband with a consistent RASopathy phenotype and no relevant family history is missing.

Sources

Reasoning

The KRAS VCEP framework defines PS4 as Supporting at >=1 point, Moderate at >=3 points, and Strong at >=5 points. The reviewed exact-variant publication reports KRAS c.35G>T (p.Gly12Val) in 1 of 65 sebaceous nevi, but this is a postzygotic somatic nevus-sebaceous cohort rather than a case-control or germline RASopathy cohort. The available evidence therefore does not establish qualifying phenotype prevalence or case-control enrichment for PS4 in the applicable KRAS RASopathy context.

Rule

KRAS VCEP v2.3: PS4 Supporting >=1 point, Moderate >=3 points, Strong >=5 points; qualifying disease-relevant case enrichment is required.

Evidence considered

• The KRAS VCEP Version 2.3 framework specifies PS4 Supporting at >=1 point, Moderate at >=3 points, and Strong at >=5 points.
• PMID:22683711 reports KRAS c.35G>T (p.Gly12Val) in 1 of 65 sebaceous nevi (2%) in a study of postzygotic HRAS/KRAS mutations; the cohort is not a germline RASopathy case-control study.

Still missing

• A disease-relevant case-control analysis or a qualifying KRAS RASopathy case series with an auditable proband count and phenotype ascertainment is not available.
• The single exact-variant observation is somatic mosaic nevus-sebaceous evidence and cannot be assumed to represent germline KRAS RASopathy enrichment.

Sources

Reasoning

The KRAS RASopathy VCEP defines PM5 by counting distinct, already-established [likely] pathogenic amino-acid changes at the SAME codon (codon 12) other than the variant under review (p.G12V): 1 such change = Moderate, >=2 changes seen in >=5 probands each = Strong. This case's dedicated PM5 comparator-harvesting step (pm5_candidates.json) failed to return usable candidates (0 same-residue candidates identified; retrieval errored with 'HTTP Error 429: Too Many Requests') and explicitly instructed not to apply PM5 from that artifact alone. No other source in this case's evidence bundle enumerates verified, individually-cited alternate pathogenic codon-12 substitutions (e.g., G12D, G12A, G12C, G12S, G12R) with grounded per-substitution classification evidence, so PM5 cannot be adjudicated without risking an ungrounded citation.

Rule

>=2 different [likely] pathogenic residue changes at the same codon observed in >=5 probands = Strong; 1 [likely] pathogenic residue change at the same codon = Moderate.

Evidence considered

• pm5_candidates.json: 'found': false; 0 same-residue candidates identified; error 'HTTP Error 429: Too Many Requests'; recommendation explicitly states 'do not apply PM5 from this artifact alone.'
• cspec (KRAS RASopathy VCEP v2.3) PM5 rule: 1 other established pathogenic residue change at same codon = Moderate; >=2 changes in >=5 probands each = Strong.

Still missing

• PM5 comparator harvesting (pm5_candidates.json) failed (HTTP 429) and returned zero same-residue candidates; needs a successful re-run or manual ClinVar/COSMIC same-codon search to identify other established pathogenic codon-12 substitutions with citable sources.
• The VCEP-approved functional-studies spreadsheet references codon-12 validation controls for other RAS genes (e.g., HRAS) but the KRAS-specific validation-control list for codon 12 substitutions besides G12V is truncated/incomplete in the retrieved material and cannot be cited with confidence.

Sources

Reasoning

The case-evidence bundle does not document an assumed de novo occurrence, parental genotypes, or a proband phenotype sufficient for PM6 point assignment. The KRAS RASopathy VCEP specifies PM6 for assumed de novo variants without confirmed maternity and paternity and requires SVI point-based scoring with phenotype-specific adjustment; no such case evidence is available here.

Rule

PM6: follow SVI point-based scoring with phenotype-specific adjustment; 2 points = Strong, 1 point = Moderate, 0.5 points = Supporting (ClinGen RASopathy VCEP CSPEC v2.3)

Evidence considered

• The case-evidence bundle contains no parental genotypes, pedigree, de novo statement, or proband phenotype for NM_033360.3:c.35G>T (p.Gly12Val).
• The KRAS RASopathy VCEP CSPEC v2.3 defines PM6 as assumed de novo without confirmation of paternity and maternity and assigns 2 points (Strong), 1 point (Moderate), or 0.5 points (Supporting), with phenotype-specific adjustment.

Still missing

• Evidence that the variant is de novo or assumed de novo is missing.
• Parental testing and/or documented lack of confirmation of maternity or paternity is missing.
• Phenotypic information needed for full or reduced PM6 point assignment is missing.

Sources

Reasoning

No affected relatives, family pedigree, segregation results, or informative meioses are reported in the case-evidence bundle. Because the KRAS RASopathy VCEP requires co-segregation with disease and recommends segregation in more than one family, PP1 cannot be assigned or strength-adjusted.

Rule

PP1: co-segregation with disease; 3 or more informative meioses = Supporting, 5 or more = Moderate, 7 or more = Strong (ClinGen RASopathy VCEP CSPEC v2.3)

Evidence considered

• The case-evidence bundle contains no pedigree, affected-relative testing, co-segregation data, or informative-meiosis count for NM_033360.3:c.35G>T (p.Gly12Val).
• The KRAS RASopathy VCEP CSPEC v2.3 defines PP1 as co-segregation with disease, recommends segregation in more than one family, and specifies Supporting at 3 or more, Moderate at 5 or more, and Strong at 7 or more informative meioses.

Still missing

• Affected-family-member genotypes and phenotypes are missing.
• The number of informative meioses is not reported.
• Independent families and segregation results are not reported.

Sources

Reasoning

BS2 was not assessed as met because the case bundle contains no documented healthy adult heterozygote or homozygote carrying the exact variant with adequate phenotype and penetrance information. gnomAD shows one heterozygous allele in v4.1 and no homozygotes, but gnomAD population data alone do not establish that the carrier was a healthy adult or that the relevant KRAS RASopathy phenotype would have been detectable and fully penetrant by ascertainment.

Rule

Strength adjustment using point-based scoring based on phenotypic specifications; healthy homozygote or heterozygote observations require phenotype context.

Evidence considered

• The KRAS VCEP Version 2.3 BS2 specification uses point-based strength adjustment for healthy homozygous or heterozygous individuals and requires phenotype context.
• gnomAD v4.1 reports one heterozygous allele and zero homozygotes, but provides no case-specific evidence that the carrier was a clinically evaluated healthy adult or that the relevant disorder was expected to be fully penetrant at the time of observation.

Still missing

• No phenotype-annotated healthy adult carrier observation for NM_033360.3:c.35G>T (p.Gly12Val) was identified.
• No evidence establishing the age, clinical evaluation, and penetrance status of the single gnomAD v4.1 heterozygous carrier was identified.

Sources

Reasoning

No affected family members with documented absence of the variant are reported, and no informative non-segregating meiosis is available. The KRAS RASopathy VCEP requires lack of segregation in affected family members and states that one informative meiosis is sufficient for Strong BS4; that prerequisite evidence is absent, so BS4 cannot be assigned.

Rule

BS4: lack of segregation in affected family members; Strong requires one informative meiosis (ClinGen RASopathy VCEP CSPEC v2.3)

Evidence considered

• The case-evidence bundle contains no affected-relative genotypes, negative segregation results, pedigree, or informative-meiosis data for NM_033360.3:c.35G>T (p.Gly12Val).
• The KRAS RASopathy VCEP CSPEC v2.3 defines BS4 as lack of segregation in affected family members and specifies Strong BS4 with one informative meiosis, while cautioning that phenocopies and multiple pathogenic variants can confound apparent non-segregation.

Still missing

• An affected family member who tested negative for the variant is not documented.
• The phenotype and diagnostic validity of any potentially non-segregating relative are not available.
• The number of informative meioses is not reported.

Sources

Reasoning

BP2 is applicable under the KRAS Version 2.3 VCEP framework and requires evidence of an alternative molecular cause of a RASopathy in the same gene, with phenotype consistent with expected RASopathy severity, evaluated through phase and inheritance observations. The consolidated case-evidence bundle contains no documented second pathogenic KRAS variant, no affected-proband observation with an alternative molecular cause, and no cis/trans or parental phase data for this variant. Therefore BP2 cannot be assigned.

Rule

Points are awarded for an alternative molecular cause of a RASopathy in the same gene (and/or in conjunction with BP5) and the phenotype is consistent with expected severity of the RASopathy. Supporting: ≥ (-1) Point; Moderate: ≥ (-2) Points; Strong: ≥ (-4) Points.

Evidence considered

• The KRAS Version 2.3 VCEP specification marks BP2 as applicable and defines it as an alternative molecular cause of a RASopathy in the same gene, with phenotype consistent with expected severity; its point-based strength rules are supporting at at least -1 point, moderate at at least -2 points, and strong at at least -4 points.
• Review of the consolidated case-evidence bundle identified no case-level documentation of a second pathogenic variant, affected-proband alternative molecular cause, cis/trans phase, or inheritance data relevant to BP2.

Still missing

• Documented alternative molecular cause in the same gene or another qualifying molecular cause is absent from the case evidence bundle.
• No affected-proband phenotype linked to an alternative molecular cause is provided for BP2 assessment.
• No parental or other phase testing establishes whether a second pathogenic variant is in cis or trans with NM_033360.3:c.35G>T.

Sources

Reasoning

The KRAS VCEP framework provides BP5 point thresholds but the case bundle does not contain an auditable BP5 point assessment or sufficient evidence that the evaluated individual has an alternative molecular explanation for the relevant phenotype. ClinVar aggregate and laboratory classifications cannot substitute for BP5 evidence.

Rule

KRAS VCEP v2.3: BP5 is point-based (Supporting >=-1, Moderate >=-2, Strong >=-4); case-specific alternative molecular etiology and an auditable point calculation are required.

Evidence considered

• The KRAS VCEP Version 2.3 framework lists BP5 strengths by point threshold (Supporting >=-1 point, Moderate >=-2 points, Strong >=-4 points), but no case-specific point total is supplied.
• The ClinVar record contains multiple non-expert assertions across heterogeneous somatic and germline conditions; these labels do not establish an alternative molecular diagnosis for BP5.

Still missing

• An auditable, disease-relevant alternate molecular diagnosis in the evaluated individual or a documented BP5 point assignment is not available.
• The available somatic cancer and mosaic-lesion records do not establish BP5 for the evaluated KRAS RASopathy context.

Sources