KRAS c.38G>A, p.Gly13Asp

Reasoning

PS3 is met at Supporting strength. The VCEP specifies that well-established functional studies support PS3 and that one approved assay supports PS3_Supporting, while two or more different approved assays support PS3_Moderate. Its approved-assay documentation defines KRAS RAS activation, MEK activation, and ERK activation assays as pathway-specific assays with WT positive controls, negative controls, pathogenic validation controls, and abnormal readouts consisting of increased RAS activation or constitutive/increased/prolonged phosphorylation. The named functional-studies file contains no exact entry for c.38G>A, p.Gly13Asp, or p.G13D; therefore no variant-specific pre-assigned code is available from that file. PMID:26037647 nevertheless directly evaluates KRAS G13D in purified-protein biochemical assays and reports 14-fold faster GDP exchange, 9-fold faster GTP exchange, and approximately 7-fold lower intrinsic GTP hydrolysis than wild type, supporting a damaging gain-of-function effect. Because these are not one of the VCEP-listed approved assay classes and the file states that unlisted assays may only support PS3 at Supporting strength, the evidence is not upgraded to Moderate.

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. ... One approved assay.

Evidence considered

• ClinGen RASopathy VCEP KRAS v2.3 CSPEC PS3 rule: one approved assay supports PS3 at Supporting strength; two or more different approved assays support PS3 at Moderate strength.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, sheet READ ME: the VCEP-listed pathway-specific assays include RAS activation, MEK activation, and ERK activation; two or more unique approved assay types provide sufficient evidence to upgrade PS3 to Moderate, whereas assays not listed may only support PS3_Supporting or BS3_Supporting.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, sheets RAS Activation Assay, MEK Activation Assay, and ERK Activation Assay: KRAS assays are approved and use WT/normal controls, pathogenic validation controls, and abnormal activation or phosphorylation readouts; no exact KRAS c.38G>A/p.Gly13Asp/p.G13D entry is present.
• PMID:26037647, Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations: purified KRAS G13D showed 14-fold faster GDP exchange, 9-fold faster GTP exchange, and approximately 7-fold lower intrinsic GTP hydrolysis than wild-type KRAS.

Still missing

• No exact c.38G>A, p.Gly13Asp, or p.G13D entry was found in SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx.txt.
• The available variant-specific biochemical study is not one of the VCEP-listed approved RAS activation, MEK activation, or ERK activation assay classes; a variant-specific result from an approved assay would be needed to support a stronger calibration.
• No second distinct approved assay result for KRAS G13D was identified.

Sources

Reasoning

The KRAS VCEP limits PM1 to the approved critical functional domains P-loop 10-17, SW1 25-40, SW2 57-64, and SAK 145-156, and excludes specific amino-acid-residue evidence as a PM5 route. p.Gly13Asp is at residue 13, which lies within the approved P-loop domain 10-17. The governing PM1 alignment file confirms the P-loop region as HRAS 10-17 and states that KRAS shares identical residue numbering across the shaded regions.

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

• KRAS VCEP PM1 rule: applicable only to critical and well-established functional domains in the supplementary table, including P-loop AA 10-17, SW1 AA 25-40, SW2 AA 57-64, and SAK AA 145-156; not applicable to specific amino acid residues.
• Alignment-with-PM1-domains.pptx.pdf, page 1 transcription: P-loop (HRAS 10-17); HRAS, KRAS, and NRAS share identical residue numbering across the shaded regions, so the same ranges apply.
• The variant is NP_004976.2:p.(Gly13Asp), placing the altered residue at position 13 within the approved P-loop range 10-17.

Sources

Reasoning

The KRAS VCEP assigns PM5 moderate strength when one likely pathogenic or pathogenic residue change is observed at the same codon. The approved functional-studies table lists KRAS G13C as P in the validation controls for the RAS Activation Assay and ERK Activation Assay. G13C is a different missense residue change at the same codon 13 as the case variant p.Gly13Asp, satisfying the VCEP PM5 moderate rule.

VCEP rule

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

Evidence considered

• KRAS VCEP PM5 moderate rule: 1 [likely] pathogenic residue change at the same codon.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, RAS Activation Assay validation-control row for KRAS: G13C - P.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, ERK Activation Assay validation-control row for KRAS: G13C - P.
• The case variant is p.Gly13Asp (p.G13D), so G13C is an alternate missense change at the same codon 13.

Sources

Reasoning

The variant is a missense substitution, NM_004985.5:c.38G>A (NP_004976.2:p.(Gly13Asp)). The KRAS VCEP rule for missense variants specifies REVEL >= 0.7 for PP3. The case-specific REVEL score is 0.832, which meets that threshold. The SpliceAI result was not used for this missense PP3 path because the VCEP names REVEL for missense variants; no prediction is double-counted across computational criteria.

VCEP rule

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

Evidence considered

• KRAS VCEP v2.3 PP3 missense rule: REVEL >= 0.7.
• Local REVEL predictor lookup for NM_004985.5:c.38G>A / NP_004976.2:p.(Gly13Asp): score 0.832.
• Variant consequence: missense, NM_004985.5:c.38G>A (NP_004976.2:p.(Gly13Asp)).

Sources

Reasoning

The VCEP makes PS4 applicable to autosomal-dominant cases and requires phenotype-specific point scoring, with Supporting at >=1 point, Moderate at >=3 points, and Strong at >=5 points. The literature was checked for the PS4-indexed and high-priority papers. PMID:21063026 reports two pediatric cases with somatic KRAS G13D associated with RALD, but it provides no unaffected-control comparison, no case-control counts for this variant, and no VCEP PS4 point assignment. PMID:15696205 reports a different cohort-level KRAS codon-12/13 association (9/38 refractory versus 0/21 sensitive tumors), but does not mention G13D and is not a germline RASopathy case-control comparison. PMID:19681119 reports no KRAS mutations in 31 embryonal rhabdomyosarcoma tumors and does not mention G13D. The available evidence therefore does not establish a qualifying PS4 point total or a variant-specific affected-versus-unaffected comparison under the KRAS VCEP.

VCEP rule

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 RASopathy Expert Panel KRAS v2.3 CSPEC: PS4 instructions specify point-based strength adjustment for autosomal-dominant cases with phenotypic specifications; thresholds are Supporting >=1 point, Moderate >=3 points, and Strong >=5 points.
• PMID:21063026, Results and discussion, Case 1 and Case 2: two pediatric somatic KRAS G13D RALD cases; the paper reports hematopoietic-lineage distribution and functional assays, not unaffected controls or a case-control count comparison.
• PMID:15696205, Results; Table 2: 9/38 refractory lung adenocarcinomas versus 0/21 drug-sensitive tumors carried KRAS mutations, but the paper does not report KRAS G13D; this is not usable as variant-specific PS4 evidence.
• PMID:19681119, Results, prevalence section; Table 1: no KRAS mutation was observed among 31 primary embryonal rhabdomyosarcoma tumors, and KRAS G13D was not mentioned.
• gnomAD v4.1: 5/1,612,796 alleles overall and 5/1,179,366 European non-Finnish alleles; this population frequency alone does not supply the VCEP's required PS4 case-control comparison or point assignment.

Still missing

• A VCEP-defined PS4 point assignment for the reported cases is unavailable.
• A variant-specific affected-versus-unaffected or population-control comparison with counts is unavailable.

Sources

Reasoning

The governing KRAS VCEP rule requires the variant to be absent from gnomAD controls. Although NM_004985.5:c.38G>A is absent from gnomAD v2.1 non-cancer exomes (0/235,058), it is present in gnomAD v3.1 non-cancer genomes at 3/147,924 alleles and in gnomAD v4.1 all-comers at 5/1,612,796 alleles. Therefore the variant is not absent from gnomAD controls and PM2 is not met.

VCEP rule

The variant must be absent from controls (gnomAD).

Evidence considered

• ClinGen KRAS CSPEC v2.3: PM2 requires 'The variant must be absent from controls (gnomAD).'
• GNOMAD_V2_1_NON_CANCER, exomes-only: absent, 0/235058 alleles, homozygotes 0.
• GNOMAD_V3_1_NON_CANCER, genomes-only: present, AF 2.02807e-05 (0.00203%), 3/147924 alleles, homozygotes 0.
• GNOMAD_V4_1, all-comers: present, AF 3.10021e-06 (0.00031%), 5/1612796 alleles, homozygotes 0.

Sources

Reasoning

The governing KRAS VCEP rule requires a gnomAD filtering allele frequency of at least 0.05%. The highest available relevant frequency is 0.00463% in gnomAD v3.1 non-cancer genomes (3/64,810 alleles; total non-cancer frequency 0.00203%); the gnomAD v4.1 all-comers group maximum is 0.000124%, and the gnomAD v2.1 non-cancer exome frequency is 0% (0/235,058). All are below 0.05%, so BA1 is not met.

VCEP rule

GnomAD filtering allele frequency ≥0.05%.

Evidence considered

• ClinGen KRAS CSPEC v2.3: BA1 requires 'GnomAD filtering allele frequency ≥0.05%.'
• GNOMAD_V3_1_NON_CANCER, genomes-only: total AF 2.02807e-05 (0.00203%; 3/147924 alleles), highest population AF 4.62892e-05 (0.00463%; 3/64810 European non-Finnish alleles), homozygotes 0.
• GNOMAD_V4_1, all-comers: total AF 3.10021e-06 (0.00031%; 5/1612796 alleles), grpmax FAF 1.24e-06 (0.000124%), homozygotes 0.
• GNOMAD_V2_1_NON_CANCER, exomes-only: AF 0 (0/235058 alleles), homozygotes 0.

Sources

Reasoning

The governing KRAS VCEP rule requires a gnomAD filtering allele frequency of at least 0.025%. The highest available relevant frequency is 0.00463% in gnomAD v3.1 non-cancer genomes (3/64,810 alleles; total non-cancer frequency 0.00203%); the gnomAD v4.1 all-comers group maximum is 0.000124%, and the gnomAD v2.1 non-cancer exome frequency is 0% (0/235,058). All are below 0.025%, so BS1 is not met.

VCEP rule

GnomAD filtering allele frequency ≥0.025%.

Evidence considered

• ClinGen KRAS CSPEC v2.3: BS1 requires 'GnomAD filtering allele frequency ≥0.025%.'
• GNOMAD_V3_1_NON_CANCER, genomes-only: total AF 2.02807e-05 (0.00203%; 3/147924 alleles), highest population AF 4.62892e-05 (0.00463%; 3/64810 European non-Finnish alleles), homozygotes 0.
• GNOMAD_V4_1, all-comers: total AF 3.10021e-06 (0.00031%; 5/1612796 alleles), grpmax FAF 1.24e-06 (0.000124%), homozygotes 0.
• GNOMAD_V2_1_NON_CANCER, exomes-only: AF 0 (0/235058 alleles), homozygotes 0.

Sources

Reasoning

The variant is a missense substitution, NM_004985.5:c.38G>A (NP_004976.2:p.(Gly13Asp)). The KRAS VCEP rule for missense variants specifies REVEL <=0.3 for BP4. The case-specific REVEL score is 0.832, which does not meet the BP4 threshold. The SpliceAI result was not substituted for the VCEP's named missense predictor, and the same REVEL result is not counted as independent evidence for another computational criterion.

VCEP rule

For missense variants: REVEL <=0.3.

Evidence considered

• KRAS VCEP v2.3 BP4 missense rule: REVEL <=0.3.
• Local REVEL predictor lookup for NM_004985.5:c.38G>A / NP_004976.2:p.(Gly13Asp): score 0.832.
• Variant consequence: missense, NM_004985.5:c.38G>A (NP_004976.2:p.(Gly13Asp)).

Sources

Reasoning

The KRAS VCEP requires the same amino acid change as a previously established pathogenic variant regardless of nucleotide change, and limits this route to observed analogous residue positions in HRAS, KRAS, MRAS, NRAS, RIT1, and RRAS2. The available VCEP functional-study table lists KRAS G13D as a pathogenic validation control, but it does not establish a different nucleotide change producing p.Gly13Asp; the exact case variant is not a valid self-comparator. The PM1 alignment file was searched for c.38G>A, p.Gly13Asp, and p.G13D, with no entry found. Therefore the alternate-nucleotide same-amino-acid comparator required for PS1 cannot be confirmed from the available evidence.

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

• KRAS VCEP PS1 rule: same amino acid change as a previously established pathogenic variant regardless of nucleotide change, applicable at analogous residue positions in HRAS, KRAS, MRAS, NRAS, RIT1, and RRAS2.
• SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx, RAS Activation Assay and ERK Activation Assay validation-control rows: KRAS G13D is listed as P, but the table does not provide a distinct nucleotide change for the same p.Gly13Asp amino-acid substitution.
• Alignment-with-PM1-domains.pptx.pdf: searches for c.38G>A, p.Gly13Asp, and p.G13D returned no entry.

Still missing

• No alternate-nucleotide HRAS, KRAS, MRAS, NRAS, RIT1, or RRAS2 pathogenic comparator for p.Gly13Asp was identified.
• Alignment-with-PM1-domains.pptx.pdf was searched for c.38G>A, p.Gly13Asp, and p.G13D; no entry for this variant was found.

Sources

Reasoning

The VCEP assigns PS2 according to de novo evidence quantified as points, but the available case evidence does not document a proband with confirmed parental testing, maternity/paternity, or a point assignment. PMID:22722830 concerns acquired/somatic KRAS G13D and explicitly provides no germline or segregation data, so PS2 cannot be assessed.

VCEP rule

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

Evidence considered

• No proband-level parental testing, maternity/paternity confirmation, or VCEP PS2 point assignment is documented in the available case evidence.
• PMID:22722830 reports KRAS G13D as an acquired alteration in cancer models and states that the paper does not provide segregation or germline data for this variant.

Still missing

• Proband-level confirmation that the variant is de novo
• Parental maternity/paternity and genotype results
• VCEP PS2 point assignment

Sources

Reasoning

The VCEP assigns PM6 according to de novo evidence quantified as 2, 1, or 0.5 points, but no proband-level de novo observation without parental testing, parental genotypes, or VCEP point assignment is documented. PMID:22722830 provides no germline or segregation data, so PM6 cannot be assessed.

VCEP rule

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

Evidence considered

• No proband-level de novo observation, parental-testing status, or VCEP PM6 point assignment is documented in the available case evidence.
• PMID:22722830 reports acquired KRAS G13D in cancer samples and states that the paper does not provide germline or segregation data for this variant.

Still missing

• Proband-level de novo observation
• Evidence that parental testing was unavailable or not performed
• VCEP PM6 point assignment

Sources

Reasoning

The VCEP requires at least 3 informative meioses for PP1 Supporting, 5 for Moderate, or 7 for Strong. No affected-relative segregation, informative meioses, or VCEP-countable family observations are documented; PMID:22722830 provides no segregation data.

VCEP rule

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

Evidence considered

• No affected-relative testing, unaffected-relative testing, informative meioses, or VCEP-countable segregation observations are documented in the available case evidence.
• PMID:22722830 reports acquired KRAS G13D in cancer models and provides no segregation data for this variant.

Still missing

• Affected and unaffected relatives with variant testing
• Count of informative meioses
• Phenotype consistency within informative families

Sources

Reasoning

The governing KRAS VCEP entry provides only the point outcomes '-4 Points' and '-1 Point' and does not specify how homozygote or other population observations map to those point values. The available gnomAD data show zero homozygotes in gnomAD v2.1 non-cancer exomes, gnomAD v3.1 non-cancer genomes, and gnomAD v4.1 all-comers, but the VCEP rule cannot be evaluated from the stated evidence-to-point mapping without importing a non-VCEP rule.

VCEP rule

-4 Points. ... -1 Point.

Evidence considered

• ClinGen KRAS CSPEC v2.3 lists BS2 outcomes as '-4 Points.' and '-1 Point.' without an evidence-to-point mapping.
• GNOMAD_V2_1_NON_CANCER, exomes-only: homozygotes 0; allele count 0/235058.
• GNOMAD_V3_1_NON_CANCER, genomes-only: homozygotes 0; allele count 3/147924.
• GNOMAD_V4_1, all-comers: homozygotes 0; allele count 5/1612796.

Still missing

• The VCEP BS2 specification does not define which observed population finding earns -4 or -1 point(s).

Sources

Reasoning

The VCEP requires one informative meiosis for BS4. No tested affected or unaffected relatives, non-segregation observation, or informative meiosis is documented; PMID:22722830 provides no segregation data, so BS4 cannot be assessed.

VCEP rule

Requires only one informative meiosis.

Evidence considered

• No tested affected or unaffected relatives, non-segregation observation, or informative meiosis is documented in the available case evidence.
• PMID:22722830 reports acquired KRAS G13D in cancer models and provides no segregation data for this variant.

Still missing

• A tested informative family meiosis
• Relative phenotype and genotype results demonstrating non-segregation

Sources

Reasoning

The KRAS VCEP specifies BP2 only through its point thresholds, including supporting strength at ≥ (-1) Point. The available case evidence contains no documented observation of this variant in cis or trans with another pathogenic variant, no phase information, and no BP2 point assignment; consequently the VCEP rule cannot be evaluated.

VCEP rule

≥ (-1) Point.

Evidence considered

• ClinGen RASopathy Expert Panel KRAS Version 2.3 CSPEC criterion entry for BP2: Supporting = ≥ (-1) Point; no case evidence documents cis/trans phase with another pathogenic variant or assigns BP2 points.

Still missing

• Documented phase and a VCEP-defined BP2 point assignment for any pathogenic variant observed in cis or trans are unavailable.

Sources

Reasoning

BP5 is applicable under the KRAS VCEP when an alternative molecular cause in a different gene is documented and the phenotype is consistent with the expected RASopathy severity, or when a phenotype inconsistent with RASopathy is fully explained by a different causative variant. The available case materials do not document an alternative causative variant or a VCEP BP5 point assignment for this case, so the criterion cannot be evaluated without substituting a generic rule.

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).

Evidence considered

• KRAS VCEP v2.3 CSPEC BP5: points are awarded for an alternative molecular cause of a RASopathy in a different gene, or for phenotypes inconsistent with a RASopathy fully explained by a different causative variant; strength thresholds are Strong >=(-4) points, Moderate >=(-2) points, and Supporting >=(-1) point.
• No case evidence supplied for this group documents an alternate molecular diagnosis or assigns BP5 points.

Still missing

• Documented alternate molecular cause in a different gene and the relevant phenotype relationship are unavailable.
• A VCEP-defined BP5 point assignment is unavailable.

Sources