Variant classification summary

NM_033360.4:c.183A>C

KRAS · NP_203524.1:p.(Gln61His) · NM_033360.4

GRCh37: chr12:25380275 T>G · GRCh38: chr12:25227341 T>G

Gene: KRAS Transcript: NM_033360.4

Final call

Likely Pathogenic

PM5 strong PP2 supporting PP3 supporting

Variant details

Gene

KRAS

Transcript

NM_033360.4

Protein

NP_203524.1:p.(Gln61His)

gnomAD AF

3.979180925398316e-06 (v2.1)

ClinVar

Pathogenic

OncoKB

Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_033360.4:c.183A>C (p.Gln61His) is a missense variant in KRAS, a gene in which gain-of-function missense variants cause RASopathies including Noonan syndrome and cardio-facio-cutaneous syndrome.

2

Multiple different pathogenic missense variants have been established at codon 61 in germline RASopathies (p.Gln61Arg and p.Gln61Lys), satisfying PM5 at Strong strength under the RASopathy VCEP. The Gln61His substitution is concordant with the known pathogenic direction of codon 61 changes which impair GTPase activity.

3

PP2 is applied at Supporting strength per VCEP specification, as PP2 is applicable to all RASopathy genes including KRAS.

4

PP3 is applied at Supporting strength based on multiple computational lines of evidence: REVEL score 0.644 predicts a deleterious effect, residue 61 is a statistically significant mutational hotspot, and the variant is classified as Oncogenic (gain-of-function) by OncoKB.

5

Variant-specific functional evidence from PMID:20147967 demonstrates that KRAS Q61H transforms NIH3T3 cells in focus formation assays and is present in the active GTP-bound conformation. PMID:25705018 shows Q61H produces a 5-6 fold increase in GTP-bound KRAS and promotes anchorage-independent growth in MCF10A isogenic cells. These studies provide strong functional support for a gain-of-function effect but do not meet the strict VCEP requirement for PS3 (must use VCEP-approved functional study publications).

6

PM1 could not be applied because residue 61 falls in Switch II (residues 60-76), which is not explicitly listed in the available VCEP PM1 domain supplemental material (only P-loop 10-17 and Switch I 25-40 are listed). Human review is recommended as Switch II is a well-established critical functional domain and residue 61 is a documented mutational hotspot.

7

PM2 is not met because the variant is present in gnomAD v2.1 at extremely low frequency (1/251,308 alleles) rather than being completely absent from all population databases as required by the VCEP.

8

PVS1 is not applicable per VCEP designation and because this is a missense variant rather than a null variant. PS4, PS2, PM6, PP1, BS2, BS4, BP2, and BP5 could not be assessed due to absence of proband-level clinical data, de novo testing, or segregation analysis.

Final determination: Generic ACMG/AMP 2015 fallback rules support a Likely Pathogenic classification based on the observed combination of pathogenic criteria.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	The ClinGen RASopathy VCEP designates PVS1 as Not Applicable. The variant is a missense substitution (c.183A>C, p.Gln61His), not a null variant eligible for PVS1 under the generic ClinGen SVI decision tree.	cspec
PS1	Not met	No evidence that this exact nucleotide change or the same amino acid change arising from a different nucleotide substitution has been previously established as pathogenic under the RASopathy VCEP criteria.
PS2	Not met	No de novo occurrence data reported in the case materials or literature. The RASopathy VCEP requires confirmed paternity for PS2_Strong and ≥2 independent occurrences for PS2_Very_Strong.
PS3	Not assessed	The RASopathy VCEP restricts PS3 to Strong strength using VCEP-approved functional studies (RAS Activation Assay, MEK Activation Assay, ERK Activation Assay). The VCEP-approved KRAS functional studies (PMID: 20949621, 23059812) did not test p.Gln61His. However, substantial variant-specific functional evidence exists from non-VCEP-approved studies: PMID:20147967 demonstrated Q61H transforms NIH3T3 cells in focus formation assays and activates GTP-bound RAS; PMID:25705018 showed Q61H produces a 5-6 fold increase in GTP-bound KRAS and promotes anchorage-independent growth in MCF10A isogenic cells. This functional evidence supports a gain-of-function effect consistent with RASopathy pathogenesis but cannot be assigned PS3 under strict VCEP rules without submission to the expert panel for approval.	PMID:20147967 PMID:25705018 cspec
PS4	Not met	Insufficient independent proband count data to meet VCEP thresholds (≥1 supporting, ≥3 moderate, ≥5 strong). While ClinVar contains submissions with classifications of Pathogenic and Likely pathogenic, the proband-level clinical data required for PS4 proband counting is not available.	clinvar
PS5	N/A	PS5 is not defined in the RASopathy VCEP criteria. Under generic ACMG/AMP, PS5 applies when the same amino acid change results from a different nucleotide change previously established as pathogenic. This variant is being assessed for its own classification; no independent prior pathogenic assertion for Q61H from a different nucleotide change exists.
PM1	Not met	The RASopathy VCEP PM1 rule references supplemental material for approved functional domains and residues. The only supplemental domain material available (Alignment-with-PM1-domains.pptx) lists P-loop (HRAS residues 10-17) and Switch I (HRAS residues 25-40) as approved PM1 domains. Residue 61 is located in the Switch II region (residues 60-76), which is not explicitly listed in the VCEP-approved PM1 domains. Despite residue 61 being a statistically significant mutational hotspot (cancerhotspots.org) and a critical functional residue for GTP hydrolysis, it falls outside the VCEP-defined PM1 boundaries.	cspec vcep_alignment_with_pm1_domains_pptx
PM2	Not met	The RASopathy VCEP requires the variant to be completely absent from all population databases for PM2_Moderate. This variant is present in gnomAD v2.1 at a very low frequency (1/251,308 alleles; AF=3.98e-06) in the Ashkenazi Jewish subpopulation. Because it is not completely absent, PM2 cannot be applied under the VCEP rule.	gnomad_v2
PM5	Met	Multiple different pathogenic missense variants have been previously reported at codon 61 of KRAS in association with germline RASopathies. KRAS c.182A>G (p.Gln61Arg, Q61R) and KRAS c.181C>A (p.Gln61Lys, Q61K) are established pathogenic variants in Noonan syndrome and cardio-facio-cutaneous syndrome. The amino acid change from glutamine (uncharged polar) to histidine (positively charged basic) at position 61 is concordant with the pathogenic direction of known codon 61 substitutions, all of which impair GTPase activity. The VCEP PM5 threshold of ≥2 different pathogenic missense changes at the same residue is satisfied; PM5_Strong also applies to analogous residues in Group 1 genes (HRAS, NRAS, KRAS). Note: the VCEP instructs that PM5 should not be used independently with PM1 if the residue is explicitly designated a mutational hotspot; since residue 61 is not explicitly listed in VCEP PM1 domain material, this co-use restriction does not apply.	clinvar cspec
PM6	Not met	No de novo occurrence data reported. The RASopathy VCEP requires confirmed de novo (without paternity/maternity confirmation for PM6_Moderate) or ≥2 independent PM6 occurrences for PM6_Strong.
PP1	Not met	No segregation data available. The RASopathy VCEP requires at least 3 informative meioses for PP1_Supporting, ≥5 for PP1_Moderate, and ≥7 for PP1_Strong. No family studies or segregation analysis were identified.
PP2	Met	The RASopathy VCEP explicitly states that PP2 is applicable to all RASopathy genes described and curated in the framework, including KRAS. This is a missense variant in a gene where missense variants are a common mechanism of disease and the gene has a low rate of benign missense variation.	cspec
PP3	Met	Multiple lines of computational evidence support a deleterious effect: REVEL score of 0.644 (above typical 0.5 threshold), a statistically significant mutational hotspot at residue 61, and an oncogenic classification by OncoKB. The variant has been recurrently observed in somatic cancers (COSMIC, n=447). SpliceAI predicts no significant splice impact (max delta=0.12). The RASopathy VCEP allows PP3 at Supporting strength when multiple computational lines support a deleterious effect.	revel bayesdel oncokb spliceai
PP4	N/A	The RASopathy VCEP designates PP4 as Not Applicable for this VCEP. The VCEP instructs to use PS4 for proband counting instead.	cspec
PP5	N/A	The RASopathy VCEP designates PP5 as Not Applicable for this VCEP. The VCEP does not permit the use of PP5 (reputable source classification without available evidence) in RASopathy variant interpretation.	cspec
BA1	Not met	The RASopathy VCEP BA1 threshold is an allele frequency ≥0.05%. The variant allele frequency in gnomAD v2.1 is 3.98e-06 (0.0004%), well below the BA1 cutoff. Absent from gnomAD v4.1.	gnomad_v2 gnomad_v4
BS1	Not met	The RASopathy VCEP BS1 threshold is an allele frequency ≥0.025%. The variant allele frequency in gnomAD v2.1 is 3.98e-06 (0.0004%), far below the BS1 cutoff.	gnomad_v2
BS2	Not met	The RASopathy VCEP states that general population data should not be used for BS2 due to variable expressivity and severity of RASopathies. Requires >3 instances of well-phenotyped family members. No such data available.	cspec
BS3	Not met	BS3 requires well-established functional studies showing no damaging effect. The available functional data (PMID:20147967, PMID:25705018) convincingly demonstrate a gain-of-function effect for Q61H (increased GTP-bound RAS, focus formation, anchorage-independent growth), which is opposite to the requirement for BS3. The VCEP-approved functional assays for KRAS are designated PS3_Supporting; BS3 is not applicable for KRAS under the VCEP approved assays.	PMID:20147967 PMID:25705018
BS4	Not met	No segregation data available. The RASopathy VCEP requires only one informative meiosis but no family studies were identified.
BP1	N/A	BP1 is designated for truncating variants (nonsense, frameshift, canonical splice sites, initiation codon, entire gene or multi-exon deletion) in genes without established LOF correlation to disease. This is a missense variant, not a truncating variant. The VCEP further notes the RASopathy disease mechanism is gain-of-function, meaning BP1 would only apply to truncating variants in genes without LOF disease correlation.	cspec
BP2	Not met	No evidence that this variant has been observed in trans with a pathogenic variant (for dominant disorder) or in cis with a pathogenic variant. No such data was identified in the case materials or literature.
BP4	Not met	The RASopathy VCEP requires multiple lines of computational evidence suggesting no impact on gene or gene product. REVEL score of 0.644 indicates a predicted deleterious effect, not absence of impact. While SpliceAI (max delta=0.12) predicts no splice impact, the overall computational evidence points toward a damaging effect rather than no impact. BP4 cannot be applied when computational evidence supports pathogenicity.	revel spliceai bayesdel
BP5	Not met	No evidence that this variant has been observed in a case with an alternate molecular basis for disease. No such data was identified.
BP6	N/A	The RASopathy VCEP designates BP6 as Not Applicable for this VCEP. The criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP7	N/A	BP7 is designated for synonymous (silent) variants with no predicted splice impact and low nucleotide conservation. This variant is a missense substitution (c.183A>C, p.Gln61His), not a synonymous variant.

Disclaimer: The content and results provided by LYFE Sciences are for research and educational purposes only and must not be used as a substitute for professional medical judgment, diagnosis, or treatment. Always consult a qualified healthcare professional before making any clinical decisions.