KRAS c.183A>C, p.Gln61His

Reasoning

The KRAS p.Q61H variant was tested in an isogenic MCF10A human mammary epithelial-cell system using a RAF1-RBD pulldown RAS-activation assay after EGF stimulation. The study explicitly reports that Q61H produced the highest KRAS-GTP levels, with a 5- to 6-fold increase over empty-vector cells, and also observed slightly increased anchorage-independent colony formation. The VCEP functional-studies spreadsheet lists the RAS Activation Assay for KRAS as approved and specifies PS3_Supporting for one approved assay; no variant-specific pre-assigned table code was found for c.183A>C, p.Gln61His, or p.Q61H. Therefore, PS3 is met at Supporting strength from one approved RAS-activation assay.

VCEP rule

One approved assay.

Evidence considered

• ClinGen RASopathy VCEP KRAS v2.3 CSPEC PS3 strength rule: "One approved assay."
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, sheet RAS Activation Assay, KRAS column: the assay measures RAS protein bound to RAF1 or synthetic RBD; the KRAS assay is approved (Y) and its proposed strength is PS3_Supporting; BS3_NA.
• PMID:25705018, Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations using human mammary epithelial cells: isogenic MCF10A cells expressing KRAS-Q61H showed the highest KRAS-GTP levels after EGF stimulation, with a 5- to 6-fold increase versus empty-vector cells, using RAF1-RBD pulldown.
• The VCEP functional-studies file was searched for c.183A>C, p.Gln61His, and p.Q61H; no variant-specific pre-assigned code was found, so the VCEP PS3 strength rule was applied.

Still missing

• No c.183A>C, p.Gln61His, or p.Q61H entry was found in SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt.

Sources

Reasoning

The variant changes KRAS residue 61. The governing KRAS VCEP PM1 rule limits PM1 to the approved supplementary functional domains and lists SW2 as amino acids 57-64. Residue 61 is within SW2, so PM1 is met at Moderate strength. The governing alignment file was searched under c.183A>C, p.Gln61His, and p.Q61H; no variant-specific row was present, so the domain-table range, rather than a pre-assigned variant code, is 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

• The KRAS VCEP supplementary PM1 domain list defines P-loop as AA 10-17, SW1 as AA 25-40, SW2 as AA 57-64, and SAK as AA 145-156.
• The normalized protein consequence is NP_004976.2:p.(Gln61His), so residue 61 maps inside the approved SW2 interval 57-64.
• Alignment-with-PM1-domains.pptx.pdf.txt was searched for c.183A>C, p.Gln61His, and p.Q61H; no exact variant entry was found, while the VCEP domain rule independently places residue 61 in SW2.

Still missing

• No c.183A>C, p.Gln61His, or p.Q61H entry was found when Alignment-with-PM1-domains.pptx.pdf.txt was searched; PM1 was determined from the complete approved domain ranges.

Sources

Reasoning

The KRAS VCEP makes PS4 applicable to autosomal-dominant RASopathy cases and permits strong, moderate, or supporting evidence at ≥5, ≥3, or ≥1 points, respectively. Its governing rule states that affected-case prevalence must be significantly increased versus controls; a case-control RR or OR must be >5.0 with a confidence interval excluding 1.0, while the rare-variant fallback requires the variant in multiple unrelated patients with the same RASopathy phenotype and absence from controls. The reviewed literature packet contains no AI-classified PS4 paper; the candidate papers reviewed here concern somatic lung adenocarcinoma or pilocytic astrocytoma rather than germline RASopathy and do not provide a qualifying case-control comparison or multiple unrelated RASopathy patients with this exact variant. Therefore PS4 is not met. The numeric point thresholds are not reached because no VCEP PS4 point score is available; the >5.0 OR requirement and its confidence-interval condition are likewise not demonstrated.

VCEP rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... 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. ... 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. ... ≥5 points. ... ≥3 points. ... ≥1 point.

Evidence considered

• CSPEC PS4 rule: affected prevalence must be significantly increased compared with controls; RR or OR must be >5.0 with a confidence interval that does not include 1.0, and a rare-variant fallback may use multiple unrelated patients with the same phenotype plus absence from controls.
• CSPEC PS4 strength rules: ≥5 points for Strong, ≥3 points for Moderate, and ≥1 point for Supporting; no VCEP PS4 point score is available in this case, so no substituted inequality can be formed.
• PMID:21610151, Table 1 and Results: the study examined 52 sporadic pilocytic astrocytomas and found KRAS mutations in 2 samples; it was a somatic tumor cohort and did not establish this exact variant as a germline RASopathy case-control signal.
• PMID:23619274, page 485, Limitations of DNA microarray analysis: the paper states that copy-number microarrays are not designed to detect point mutations; it provides no case-control or phenotype-specific evidence for KRAS c.183A>C.
• Literature-pass packet: no paper appears in ai_classified_papers as PS4; the sole AI-classified paper is PMID:25705018 for PS3, not PS4.

Still missing

• A RASopathy-specific case series with phenotype details and exact-variant counts in affected individuals and matched controls would be needed for a stronger PS4 assessment.
• PMID:15696205 could not be text-extracted because the available PDF requires OCR; its candidate status was not treated as evidence.

Sources

Reasoning

The governing KRAS VCEP rule requires that the variant be absent from gnomAD controls. The default all-comers gnomAD v2.1 record contains the variant at 1/251,308 alleles (AF 3.979180925398316e-06), although there are zero homozygotes; gnomAD v4.1 reports it as absent. Because the variant is present in the all-comers gnomAD v2.1 control dataset, the VCEP's complete-absence requirement is not satisfied. The available non-cancer subsets also report absence, but the VCEP rule does not specify a non-cancer-only or exome-only source, so those subsets do not replace the default all-comers assessment.

VCEP rule

The variant must be absent from controls (gnomAD).

Evidence considered

• ClinGen RASopathy Expert Panel KRAS v2.3 CSPEC, PM2 entry: "The variant must be absent from controls (gnomAD)."
• gnomAD v2.1 all-comers record for NM_004985.5:c.183A>C / 12-25380275-T-G: exome AC=1, AN=251308, AF=3.979180925398316e-06, homozygotes=0.
• gnomAD v4.1 all-comers record: variant absent.
• The case's GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER entries report absence, but the governing VCEP PM2 text does not require either non-cancer subset or an exome-only source; therefore the default all-comers gnomAD result governs.

Sources

Reasoning

The variant is a missense variant, so the VCEP's applicable PP3 path is REVEL >= 0.7. The available REVEL score is 0.644, which does not meet that threshold. The VCEP also describes a splicing-impact path requiring the predicted outcome to match the disease mechanism, but this exonic missense variant is not a splice-site variant and SpliceAI predicts no significant splice impact (max delta score 0.119); that result does not provide pathogenic computational evidence and is not counted separately.

VCEP rule

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

Evidence considered

• VCEP CSPEC PP3 rule: for missense variants, REVEL >= 0.7; for splicing impact, the predicted outcome must match the disease mechanism.
• Local REVEL predictor lookup for NM_004985.5:c.183A>C / KRAS p.(Gln61His): REVEL score 0.644.
• SpliceAI Lookup for NM_004985.5:c.183A>C: max delta score 0.119, reported as no significant splice impact; not used as an additional PP3 evidence unit.

Sources

Reasoning

The governing KRAS VCEP rule requires a gnomAD filtering allele frequency of at least 0.05%. The variant's all-comers gnomAD v2.1 frequency is 1/251,308 alleles (AF 3.979180925398316e-06; 0.000397918%), and it is absent from gnomAD v4.1. The highest reported gnomAD v2.1 population frequency is Ashkenazi Jewish, 1/10,076 alleles (AF 9.924573243350536e-05; 0.00992457%), still below 0.05%; therefore BA1 is not met.

VCEP rule

GnomAD filtering allele frequency ≥0.05%.

Evidence considered

• ClinGen RASopathy Expert Panel KRAS v2.3 CSPEC, BA1 entry: "GnomAD filtering allele frequency ≥0.05%."
• gnomAD v2.1 all-comers record for NM_004985.5:c.183A>C / 12-25380275-T-G: total AC=1, AN=251308, AF=3.979180925398316e-06, homozygotes=0; highest population AF was Ashkenazi Jewish AC=1, AN=10076, AF=9.924573243350536e-05, homozygotes=0.
• gnomAD v4.1 all-comers record: variant absent.

Sources

Reasoning

The governing KRAS VCEP rule requires a gnomAD filtering allele frequency of at least 0.025%. The variant's all-comers gnomAD v2.1 frequency is 1/251,308 alleles (AF 3.979180925398316e-06; 0.000397918%), and the highest reported population frequency is Ashkenazi Jewish, 1/10,076 alleles (AF 9.924573243350536e-05; 0.00992457%). Both are below 0.025%, and gnomAD v4.1 reports the variant as absent; therefore BS1 is not met.

VCEP rule

GnomAD filtering allele frequency ≥0.025%.

Evidence considered

• ClinGen RASopathy Expert Panel KRAS v2.3 CSPEC, BS1 entry: "GnomAD filtering allele frequency ≥0.025%."
• gnomAD v2.1 all-comers record for NM_004985.5:c.183A>C / 12-25380275-T-G: total AC=1, AN=251308, AF=3.979180925398316e-06; highest population AF was Ashkenazi Jewish AC=1, AN=10076, AF=9.924573243350536e-05.
• gnomAD v4.1 all-comers record: variant absent.

Sources

Reasoning

The variant is a missense variant, so the VCEP's applicable BP4 path is REVEL <= 0.3. The available REVEL score is 0.644, which is above the VCEP benign computational threshold; therefore BP4 is not met. SpliceAI's no-significant-impact result is not used to establish BP4 because the VCEP's BP4 rule for this situation names REVEL only, and the variant is not a truncating variant for which absence of splice impact could be misinterpreted as absence of protein impact.

VCEP rule

For missense variants: REVEL ≤0.3.

Evidence considered

• VCEP CSPEC BP4 rule: for missense variants, REVEL <= 0.3.
• Local REVEL predictor lookup for NM_004985.5:c.183A>C / KRAS p.(Gln61His): REVEL score 0.644.

Sources

Reasoning

The KRAS VCEP requires the same amino-acid change as a previously established pathogenic variant, with applicability to analogous positions in the listed RASopathy genes. The available extracted literature documents KRAS p.Q61H itself, but no alternate nucleotide change producing the same p.Q61H amino-acid change is documented, and no governing VCEP-table entry for c.183A>C, p.Gln61His, or p.Q61H was found. The evidence is therefore insufficient to establish the VCEP's PS1 comparator requirement without using a non-VCEP source or inferring from the variant's own classification.

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 VCEP rule requires the same amino-acid change as a previously established pathogenic variant regardless of nucleotide change, but the available variant-specific papers report p.Q61H without documenting an alternate nucleotide change producing p.Q61H.
• PMID:20147967 studied KRAS Q61H and reported transforming activity; PMID:25705018 studied KRAS-Q61H and reported a 5- to 6-fold increase in KRAS-GTP after EGF stimulation. These findings concern the queried variant itself and do not establish the required alternate-nucleotide comparator.

Still missing

• No c.183A>C, p.Gln61His, or p.Q61H entry was found when Alignment-with-PM1-domains.pptx.pdf.txt was searched; that file does not establish a PS1 comparator.

Sources

Reasoning

The available case evidence does not document a proband-level de novo observation, confirmed maternity and paternity, parental genotypes, or phenotype consistency sufficient to assign PS2 under the VCEP point-based rule. No qualifying de novo points can therefore be determined.

VCEP rule

4 Points. 2 Points. 1 Point.

Evidence considered

• The available case materials contain no documented proband-level de novo result, parental genotypes, confirmed maternity or paternity, or phenotype-based de novo assessment for NM_004985.5:c.183A>C (p.Gln61His).

Still missing

• Documented proband-level observation of NM_004985.5:c.183A>C (p.Gln61His).
• Confirmed maternity and paternity and validated parental testing results demonstrating absence of the variant in both parents.
• Phenotype consistency for the proband with the relevant RASopathy presentation.

Sources

Reasoning

The KRAS VCEP permits PM5 at Moderate strength for one likely pathogenic residue change at the same codon, or Strong strength for at least two different likely pathogenic residue changes at the same codon observed in at least five probands. The extracted evidence documents KRAS p.Q61H itself and general Q61 mechanism, but does not document a different pathogenic or likely pathogenic amino-acid substitution at codon 61 with the required VCEP support. The collected PM5 candidate summary also reports zero same-residue candidates; PM5 is therefore not assessed rather than inferred from the queried variant's own classification.

VCEP rule

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

Evidence considered

• The VCEP PM5 thresholds are one likely pathogenic residue change at the same codon for Moderate, or at least two different likely pathogenic residue changes at the same codon observed in at least five probands for Strong.
• PMID:20147967 and PMID:25705018 document KRAS Q61H, not a different codon-61 amino-acid substitution with an independently established likely pathogenic classification and the required proband count.
• The collected PM5 candidate summary records zero same-residue candidates; it is consistent with, but does not replace, the governing VCEP evidence requirement.

Still missing

• No c.183A>C, p.Gln61His, or p.Q61H entry was found when Alignment-with-PM1-domains.pptx.pdf.txt was searched; no alternate codon-61 residue entry was identified there.

Sources

Reasoning

The available case evidence does not document a presumed de novo observation with sufficient proband, parental-testing, maternity, paternity, or phenotype information to assign any PM6 points under the VCEP rule. PM6 cannot therefore be assessed.

VCEP rule

2 Points. 1 Point. 0.5 Points.

Evidence considered

• The available case materials contain no documented presumed de novo observation, parental testing, confirmed maternity or paternity, or phenotype-consistency evidence for NM_004985.5:c.183A>C (p.Gln61His).

Still missing

• Documented presumed de novo proband observation of NM_004985.5:c.183A>C (p.Gln61His).
• Parental testing and confirmation of maternity and paternity.
• Phenotype consistency and clinical context supporting a presumed de novo interpretation.

Sources

Reasoning

No informative familial segregation observations or meioses are documented in the available case evidence. The VCEP thresholds of at least 3, 5, or 7 informative meioses therefore cannot be evaluated.

VCEP rule

>=7 informative meioses. ... >=5 informative meioses. ... >=3 informative meioses.

Evidence considered

• The available case materials contain no family segregation table, affected-relative genotypes, unaffected-relative genotypes, or reported informative-meiosis count for NM_004985.5:c.183A>C (p.Gln61His).

Still missing

• Family-level genotypes and phenotypes for the variant and relevant relatives.
• A count of informative meioses, with phase and segregation direction documented for each meiosis.

Sources

Reasoning

The governing KRAS VCEP CSPEC lists BS2 as either "-4 Points." or "-1 Point." but does not state the population or clinical observations that assign either point value. The available gnomAD data show zero homozygotes in gnomAD v2.1 and no gnomAD v4.1 record, but the VCEP text provided does not define how these observations map to the BS2 point options, and no unaffected-individual observation with an applicable point assignment is available. BS2 therefore cannot be evaluated without substituting a non-VCEP rule.

VCEP rule

-4 Points. ... -1 Point.

Evidence considered

• ClinGen RASopathy Expert Panel KRAS v2.3 CSPEC, BS2 entry: "-4 Points." and "-1 Point."
• gnomAD v2.1 all-comers record: homozygotes=0; gnomAD v4.1 all-comers record: variant absent.
• The available case evidence contains no unaffected-individual clinical observation and no VCEP-defined conversion of the observed population data to either BS2 point value.

Still missing

• The VCEP BS2 text does not specify the evidence-to-point mapping for −4 or −1 points.
• No VCEP-defined BS2 point assignment for this variant is available from the case evidence.

Sources

Reasoning

The available case evidence does not document an unaffected relative who carries NM_004985.5:c.183A>C (p.Gln61His) or any informative non-segregating meiosis. The VCEP requirement for one informative meiosis therefore cannot be evaluated.

VCEP rule

Requires only one informative meiosis.

Evidence considered

• The available case materials contain no documented unaffected carrier, familial non-segregation result, or informative-meiosis count for NM_004985.5:c.183A>C (p.Gln61His).

Still missing

• An unaffected relative carrying the variant with phenotype and genotype documented.
• Confirmation that the observation constitutes an informative non-segregating meiosis.

Sources

Reasoning

The KRAS VCEP provides BP2 point-based thresholds of Supporting at ≥ (-1) Point, Moderate at ≥ (-2) Points, and Strong at ≥ (-4) Points. The available case evidence contains no documented phase, allelic partner, or observation establishing the relevant cis/trans relationship with a pathogenic variant, so the required BP2 point total cannot be determined.

VCEP rule

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

Evidence considered

• ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for KRAS Version 2.3, CSPEC criterion entry BP2: Strong at ≥ (-4) Points, Moderate at ≥ (-2) Points, Supporting at ≥ (-1) Point.
• The case evidence reviewed contains no affected-proband observation, family segregation record, or molecular phase result documenting a cis/trans relationship with a pathogenic variant for NM_004985.5:c.183A>C (NP_004976.2:p.(Gln61His)).

Still missing

• Documented cis/trans phase and the identity and classification of any allelic pathogenic variant are unavailable, so BP2 points cannot be assigned.

Sources

Reasoning

BP5 is applicable when an alternative molecular cause of a RASopathy in a different gene is documented, or when the phenotype is inconsistent with RASopathy and fully explained by a different causative variant. The available case evidence does not document a patient phenotype, a different-gene causative variant, or a VCEP BP5 point total. The governing thresholds are Supporting ≥ (-1) point, Moderate ≥ (-2) points, and Strong ≥ (-4) points; because no observed point value is available, no valid substituted inequality can be formed and BP5 is not assessed.

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

• CSPEC BP5 applicability: points are awarded for an alternative molecular cause of a RASopathy in a different gene, or for phenotypes inconsistent with RASopathy that are fully explained by a different causative variant.
• CSPEC BP5 thresholds: Strong ≥ (-4) points, Moderate ≥ (-2) points, Supporting ≥ (-1) point; no observed BP5 point value is available, so no numeric inequality can be evaluated.
• The available case evidence contains no documented alternative causative variant or phenotype-specific BP5 score.

Still missing

• Document the patient's RASopathy phenotype and any confirmed alternative molecular diagnosis, including the VCEP-assigned BP5 point total.

Sources