KRAS c.436G>A, p.Ala146Thr

Reasoning

The ClinGen RASopathy VCEP functional-studies table lists KRAS A146T, the queried p.Ala146Thr/c.436G>A variant, as a P/LP validation control in the approved KRAS RAS Activation Assay and in the approved KRAS ERK Activation Assay. These are two different approved assay types, and the table documents the relevant assay context: RAS activation measures bound RAS immunoprecipitated with RAF1 or RBD, while ERK activation measures the pERK/ERK ratio basally and after stimulation. The table's approved-assay designation and pre-assigned P/LP control code govern this interpretation; no generic assay threshold is substituted. The VCEP strength rule therefore supports PS3 at Moderate strength. Independent literature is concordant: PMID:20147967 reports A146T in the active GTP-bound conformation and significant focus formation, and PMID:20570890 reports elevated RAS-GTP and phosphorylated ERK in A146T models, but the strength assignment rests on the VCEP table's two approved assay entries.

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. ... Two or more different approved assays.

Evidence considered

• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt, RAS Activation Assay sheet, KRAS column: A146T is listed among the P/LP validation controls; the assay is approved (Y) and proposed strength is PS3_Supporting; BS3_NA.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt, ERK Activation Assay sheet, KRAS column: A146T is listed among the P/LP validation controls; the assay is approved (Y) and proposed strength is PS3_Supporting; BS3_NA.
• The approved RAS Activation Assay measures bound RAS protein immunoprecipitated with RAF1 or synthetic RBD; the approved ERK Activation Assay measures the pERK/ERK ratio basally and after stimulation.
• PMID:20147967, Results: the exact codon-146 G>A Ala-to-Thr change was functionally studied; A146T showed significant focus formation and was detected in the active GTP-bound conformation.
• PMID:20570890, Results and Figure 3: A146T KRAS models showed elevated RAS-GTP and phosphorylated ERK relative to wild-type KRAS.

Still missing

• The VCEP functional-studies table does not provide a variant-specific numeric readout for A146T; the VCEP's pre-assigned P/LP validation-control code and approved assay designations are used as governing evidence.

Sources

Reasoning

The variant changes KRAS residue 146. The KRAS VCEP PM1 rule limits PM1 to the approved P-loop 10-17, SW1 25-40, SW2 57-64, and SAK 145-156 domains and excludes specific amino-acid residue evidence. Residue 146 lies within the approved SAK domain spanning amino acids 145-156, so PM1 is met at Moderate strength. The governing Alignment-with-PM1-domains.pptx.pdf was searched for c.436G>A, p.Ala146Thr, and p.A146T; no variant-specific row was found, but the authoritative domain range covers residue 146.

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 defines the approved domains as P-loop 10-17, SW1 25-40, SW2 57-64, and SAK 145-156; residue 146 is within SAK.
• Alignment-with-PM1-domains.pptx.pdf.txt was searched for c.436G>A, p.Ala146Thr, and p.A146T and had no direct variant row; its paralog alignment supports the approved 145-156 domain mapping.

Still missing

• Alignment-with-PM1-domains.pptx.pdf.txt contains no direct c.436G>A, p.Ala146Thr, or p.A146T entry; PM1 is based on its governing domain mapping.

Sources

Reasoning

The KRAS VCEP assigns PM5 at Moderate strength when one likely pathogenic residue change occurs at the same codon. The case evidence reports that PMID:26970110 identified a different KRAS codon-146 change, p.Ala146Val, in lesional tissue from patients with OES/ECCL and describes KRAS codon-146 mutations as known oncogenic KRAS mutations. This is one alternate residue change at codon 146 supporting PM5 Moderate.

VCEP rule

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

Evidence considered

• PMID:26970110 is summarized in the case evidence as reporting KRAS p.Ala146Val and p.Ala146Thr in lesional tissue from three OES patients and one ECCL patient, and describing codon-146 KRAS mutations as known oncogenic mutations.
• The alternate p.Ala146Val change is a different residue change at the same codon and satisfies the VCEP's Moderate PM5 requirement of one likely pathogenic residue change at that codon.
• PMID:26970110 is not a source_registry key in this case, so it is cited in evidence_used rather than evidence_refs.

Sources

Reasoning

The KRAS VCEP rule applies the missense path and specifies REVEL >= 0.7 for PP3. This variant is missense, NP_004976.2:p.(Ala146Thr), and the documented REVEL score is 0.909, which meets the VCEP threshold. The available SpliceAI result is not needed for the missense REVEL path and was not used as an additional independent PP3 prediction.

VCEP rule

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

Evidence considered

• KRAS VCEP CSPEC criterion PP3: missense variants use REVEL >= 0.7.
• Variant consequence: missense, NP_004976.2:p.(Ala146Thr).
• Local REVEL predictor lookup: score 0.909, exceeding the VCEP PP3 cutoff of 0.7.

Sources

Reasoning

The KRAS VCEP requires the same amino-acid change as a previously established pathogenic variant, regardless of nucleotide change, at an observed analogous residue position in HRAS, KRAS, MRAS, NRAS, RIT1, or RRAS2. The reviewed reports document the queried KRAS c.436G>A p.Ala146Thr/p.A146T change itself, not a distinct nucleotide change producing the same amino-acid substitution. Therefore PS1 is not met.

VCEP rule

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._

Evidence considered

• The case variant is KRAS NM_004985.5:c.436G>A, NP_004976.2:p.(Ala146Thr), also written NP_004976.2:p.(A146T).
• PMID:20147967 reports a G-to-A change causing Ala-to-Thr at codon 146, and PMID:20570890 reports KRAS A146T; neither provides a different nucleotide change producing p.Ala146Thr.

Still missing

• No alternate-nucleotide p.Ala146Thr comparator was identified for the VCEP-listed RAS genes.

Sources

Reasoning

The governing VCEP requires significantly increased prevalence in affected individuals versus controls; its rare-variant alternative permits moderate evidence only when the variant is observed in multiple unrelated patients with the same phenotype and absent from controls. The available variant-specific literature supports affected cancer cohorts (PMID:20147967: KRAS A146T in 7/106 colorectal tumors; PMID:20570890: A146T in 19/415 colorectal tumors) but these are somatic colorectal-cancer cohorts, not RASopathy case-control studies, and the extracted evidence does not provide unaffected matched controls for a valid OR/95% CI. The literature packet identifies PMID:26110767, PMID:30289595, PMID:30891959, and PMID:34131312 as PS4 candidates, but the available records do not establish a qualifying case-control comparison for this variant; PMID:30891959 reports KRAS p.Ala146Thr in two OES subjects, while gnomAD v4.1 contains the variant at 3/1,612,834 alleles, so the VCEP's stated absence-from-controls route is not met. No PS4 point total is documented, so none of the VCEP point thresholds can be applied.

VCEP rule

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. ... Strength adjustment using point-based scoring for autosomal dominant cases with 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)). ... ≥5 points. ... ≥3 points. ... ≥1 point.

Evidence considered

• ClinGen KRAS VCEP PS4 rule: affected-individual prevalence must be significantly increased versus controls; rare variants may use multiple unrelated patients with the same phenotype only when absent from controls.
• PMID:20147967 full-text extract: KRAS p.A146T was found in 7 of 106 colorectal tumors (6.5%); the study used matched blood/tumor DNA but did not provide a qualifying RASopathy unaffected-control comparison for PS4.
• PMID:20570890 full-text extract: A146T was identified in 19 of 415 colorectal tumors; this is a somatic colorectal-cancer cohort and does not establish RASopathy case-control enrichment.
• Literature-pass PS4 candidate list includes PMID:26110767, PMID:30289595, PMID:30891959, and PMID:34131312; the available records do not document a qualifying case-control comparison for this variant.
• PMID:30891959 abstract record reports KRAS p.Ala146Thr mosaicism in two OES subjects, but no unaffected-control comparison is available.
• gnomAD v4.1: 3/1,612,834 total alleles, allele frequency 1.86008e-06; therefore the variant is present rather than absent from controls.

Still missing

• A matched RASopathy case-control dataset with affected and unaffected carrier counts, or a documented VCEP PS4 point score, is unavailable.
• Full text or a variant-specific extract for PMID:26110767, PMID:30289595, PMID:30891959, and PMID:34131312 is unavailable in the case files.

Sources

Reasoning

The KRAS Version 2.3 VCEP requires the variant to be absent from gnomAD controls for PM2 at Supporting strength. The governing rule does not specify a non-cancer or exome-only subset, so the default all-comers gnomAD entries apply. The variant is absent from gnomAD v2.1 but present in all-comers gnomAD v4.1 at AF 1.86008e-06 (3/1,612,834 alleles; homozygotes 0), so it is not absent from gnomAD controls and PM2 is not met. The non-cancer data are concordant with this mixed result: absent in gnomAD v2.1 non-cancer and present in gnomAD v3.1 non-cancer at AF 6.76883e-06 (1/147,736 alleles; homozygotes 0).

VCEP rule

The variant must be absent from controls (gnomAD).

Evidence considered

• KRAS Version 2.3 CSPEC PM2 entry: The variant must be absent from controls (gnomAD).
• gnomAD v2.1 all-comers result for 12-25378562-C>T: absent.
• gnomAD v4.1 all-comers result for 12-25225628-C>T: present at AF 1.86008e-06 (0.00019%), 3/1,612,834 alleles, homozygotes 0.
• gnomAD v2.1 non-cancer result: absent.
• gnomAD v3.1 non-cancer genomes-only result: present at AF 6.76883e-06 (0.00068%), 1/147,736 alleles, homozygotes 0; highest observed European non-Finnish AF 1.54526e-05 (0.00155%), 1/64,714 alleles, homozygotes 0.

Sources

Reasoning

The KRAS Version 2.3 VCEP requires a gnomAD filtering allele frequency of at least 0.05% for BA1. The highest reported all-comers gnomAD v4.1 frequency is 0.00160% in Remaining individuals (1/62,444 alleles), while the combined frequency is 0.00019% (3/1,612,834 alleles); both are below 0.05%. gnomAD v2.1 is absent, and the available non-cancer subsets do not provide a frequency at or above the VCEP threshold.

VCEP rule

GnomAD filtering allele frequency ≥0.05%.

Evidence considered

• KRAS Version 2.3 CSPEC BA1 entry: GnomAD filtering allele frequency ≥0.05%.
• gnomAD v4.1 all-comers result for 12-25225628-C>T: total AF 1.86008e-06 (0.00019%), 3/1,612,834 alleles, homozygotes 0; highest observed Remaining individuals AF 1.60143e-05 (0.00160%), 1/62,444 alleles, homozygotes 0.
• gnomAD v2.1 result for 12-25378562-C>T: absent.

Sources

Reasoning

The KRAS Version 2.3 VCEP requires a gnomAD filtering allele frequency of at least 0.025% for BS1. The highest reported all-comers gnomAD v4.1 frequency is 0.00160% in Remaining individuals (1/62,444 alleles), and the combined frequency is 0.00019% (3/1,612,834 alleles), both below 0.025%. gnomAD v2.1 is absent, and the available non-cancer frequencies are also below the threshold.

VCEP rule

GnomAD filtering allele frequency ≥0.025%.

Evidence considered

• KRAS Version 2.3 CSPEC BS1 entry: GnomAD filtering allele frequency ≥0.025%.
• gnomAD v4.1 all-comers result for 12-25225628-C>T: total AF 1.86008e-06 (0.00019%), 3/1,612,834 alleles, homozygotes 0; highest observed Remaining individuals AF 1.60143e-05 (0.00160%), 1/62,444 alleles, homozygotes 0.
• gnomAD v2.1 result for 12-25378562-C>T: absent.

Sources

Reasoning

The KRAS VCEP rule applies BP4 to missense variants using REVEL <= 0.3. This variant is missense, but its documented REVEL score is 0.909, which does not meet the benign threshold. SpliceAI was not used to establish BP4 because the governing VCEP BP4 rule names REVEL for missense variants.

VCEP rule

For missense variants: REVEL <=0.3.

Evidence considered

• KRAS VCEP CSPEC criterion BP4: missense variants use REVEL <= 0.3.
• Variant consequence: missense, NP_004976.2:p.(Ala146Thr).
• Local REVEL predictor lookup: score 0.909, above the VCEP BP4 cutoff of 0.3.

Sources

Reasoning

The VCEP assigns PS2 using de novo evidence scored as 4, 2, or 1 points, but the available case evidence does not document an affected proband with confirmed maternity and paternity, absence of a family history, or phenotype information sufficient to evaluate this criterion.

VCEP rule

4 Points. ... 2 Points. ... 1 Point.

Evidence considered

• The available case evidence contains no documented parental testing, confirmed maternity or paternity, de novo observation, or informative phenotype assessment for NM_004985.5:c.436G>A (NP_004976.2:p.Ala146Thr).

Still missing

• Documented proband with the variant and a compatible RASopathy phenotype
• Confirmed maternity and paternity
• Documented absence of family history

Sources

Reasoning

The VCEP assigns PM6 using 2, 1, or 0.5 points for assumed de novo observations without the PS2-level confirmation, but the available case evidence contains no documented assumed de novo proband observation or parental testing for this variant.

VCEP rule

2 Points. ... 1 Point. ... 0.5 Points.

Evidence considered

• The available case evidence contains no documented parental testing, de novo observation, or proband-level clinical evidence for NM_004985.5:c.436G>A (NP_004976.2:p.Ala146Thr).

Still missing

• Documented proband with the variant and a compatible RASopathy phenotype
• Parental testing or other evidence supporting a de novo origin
• Information distinguishing assumed de novo evidence from confirmed maternity and paternity

Sources

Reasoning

The VCEP assigns PP1 by the number of informative meioses, with supporting strength at 3 or more, moderate strength at 5 or more, and strong strength at 7 or more informative meioses. No affected relatives, parental transmission, family structure, or informative meioses are documented for this variant.

VCEP rule

≥7 informative meioses. ... ≥5 informative meioses. ... ≥3 informative meioses.

Evidence considered

• The available case evidence contains no documented familial segregation, affected relatives, tested relatives, or informative meiosis count for NM_004985.5:c.436G>A (NP_004976.2:p.Ala146Thr).

Still missing

• Affected relatives and their variant status
• Family structure and tested relatives
• Count of informative meioses

Sources

Reasoning

The KRAS Version 2.3 VCEP applies BS2 through point-based phenotypic specifications using healthy homozygous or heterozygous individuals, with reduced points for apparently unaffected heterozygotes and specific applicability to parent or sibling samples during clinical family evaluations. The population evidence shows zero homozygotes in gnomAD v4.1, but it does not identify a healthy individual, establish phenotype and penetrance, document an unaffected heterozygote in a family evaluation, or provide the VCEP point assignment. Therefore BS2 cannot be evaluated from the available evidence.

VCEP rule

Strength adjustment using point-based scoring based on phenotypic specifications. Phenotypic specifications: based on healthy homozygote or heterozygote individuals, reduced points for apparently unaffected heterozygous individuals, applicable to parent or sibling samples during clinical family evaluations.

Evidence considered

• KRAS Version 2.3 CSPEC BS2 instructions: strength adjustment uses point-based scoring based on phenotypic specifications; healthy homozygote or heterozygote individuals and apparently unaffected heterozygotes are specified, with parent or sibling samples applicable during clinical family evaluations.
• KRAS Version 2.3 CSPEC BS2 strength rules: Strong = -4 Points; Supporting = -1 Point.
• gnomAD v4.1 all-comers result: homozygotes = 0 overall and in the highest-frequency Remaining individuals population (1/62,444 alleles); this does not establish the VCEP's healthy-individual phenotype or point score.

Still missing

• A VCEP-defined BS2 point assignment from a documented healthy homozygous or heterozygous individual, including phenotype and family-sample context, is unavailable.

Sources

Reasoning

The VCEP requires only one informative meiosis for BS4, which evaluates lack of segregation in affected family members. No affected family members, tested relatives, or informative meiosis showing non-segregation are documented for this variant.

VCEP rule

Requires only one informative meiosis.

Evidence considered

• The available case evidence contains no documented affected relative, variant-negative affected family member, or informative meiosis demonstrating lack of segregation for NM_004985.5:c.436G>A (NP_004976.2:p.Ala146Thr).

Still missing

• An affected family member with documented variant-negative testing
• A documented informative meiosis showing lack of segregation

Sources

Reasoning

BP2 is applicable in the KRAS VCEP framework, with strength determined by a negative point total. The available evidence does not document a qualifying affected-proband observation, phase relationship, or pathogenic variant in trans or cis, and the VCEP point assignment cannot therefore be evaluated. No generic ACMG/AMP or other external allelic rule is substituted.

VCEP rule

Strong: ≥ (-4) Points; Moderate: ≥ (-2) Points; Supporting: ≥ (-1) Point.

Evidence considered

• The case evidence contains no documented qualifying phase relationship or affected-proband observation establishing a pathogenic variant in trans or cis for BP2.
• The VCEP BP2 entry supplies only negative-point strength thresholds (Strong: ≥ (-4) points; Moderate: ≥ (-2) points; Supporting: ≥ (-1) point) and no qualifying point total is available for this case.

Still missing

• Documented phase and an evaluable VCEP point assignment for any qualifying pathogenic variant in trans or cis are unavailable.

Sources

Reasoning

The KRAS VCEP permits BP5 when an alternative molecular cause explains the RASopathy phenotype, or when a phenotype inconsistent with RASopathy is fully explained by another causative variant, with strength determined by the VCEP point score. The available evidence does not document an alternative causative variant or a BP5 point total, so the criterion cannot be evaluated.

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

• KRAS VCEP BP5 instructions require an alternative molecular cause in a different gene, or a phenotype inconsistent with RASopathy fully explained by another causative variant.
• No case evidence documents such an alternative causative variant or a BP5 point total.

Still missing

• A documented alternative molecular cause and phenotype assessment, or the VCEP BP5 point score, is unavailable.

Sources