The BRAF NM_004333.6:c.1781A>G (NP_004324.2:p.(Asp594Gly)) missense variant has previously been reported in somatic cancers (COSMIC; COSV56065695, n = 169 times), and lies in a statistically significant hotspot (cancerhotspots.org). Experimental evidence: Two variant-specific studies show impaired or absent BRAF kinase activity with abnormal RAS-dependent ERK signaling, but the VCEP table does not list this variant. Based on available guidelines, this variant is considered to be a Variant of Uncertain Significance (VUS).
Five-step scale from benign to pathogenic. This variant is uncertain. Select a step to read what it means.
Uncertain.The recorded classification is uncertain significance.
Gene-level background from the recorded interpretation.
Words with a dotted underline have a short definition. Hover or tap them.BRAF encodes a protein kinase that helps control the MAPK/ERK signaling pathway, which regulates cell growth, division, and specialization. Changes in BRAF are associated with cardiofaciocutaneous, Noonan, and Costello syndromes. BRAF is an oncogene, and abnormal activation of its signaling can drive cancers including melanoma, thyroid cancer, colorectal cancer, lung cancer, lymphoma, and hairy cell leukemia.
The recorded framework is ClinGen BRAF VCEP v2.3. The criteria and strengths shown below come from the saved assessment; this page does not recalculate the classification.
The applied criteria and their strengths are shown as assessed under the stated framework.
Met, Supporting: two variant-specific studies show impaired or absent BRAF kinase activity with abnormal RAS-dependent ERK signaling, but the VCEP table does not list this variant.
Met, moderate: residue 594 is the lower boundary of the VCEP-approved CR3 activation segment spanning amino acids 594-627.
Met at Supporting: the variant is absent from both available all-comers gnomAD v2.1 and v4.1 control records.
Met: REVEL 0.989 meets the VCEP PP3 threshold of ≥0.7 for missense variants.
No benign criterion marked as applied in the recorded assessment.
VUS: PS3 (supporting), PM1 (moderate), PM2 (supporting), and PP3 (supporting) do not satisfy any BRAF VCEP pathogenic or benign combination rule.
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1 allele counts were not recorded for this variant.
Absent from gnomAD v2.1.
Not available in gnomAD-Canada v1.0.
Recorded REVEL score: 0.989. Refer to the recorded PP3/BP4 rationale for interpretation.
2 Pathogenic · 1 Likely pathogenic · 1 Uncertain significance. ClinVar API review status: criteria provided, conflicting classifications.
OncoKB identified variant-specific curated literature and context relevant to functional review; biological-effect context: Gain-of-function; curated oncogenicity label: Oncogenic.
This variant has previously been reported in somatic cancers (COSMIC; COSV56065695, n = 169 times).
This variant lies in a statistically significant hotspot.
ACMG/AMP criteria under ClinGen BRAF VCEP v2.3, with the data and sources behind each one.
28 criteria with recorded statuses: 4 met · 3 not met · 11 not assessed · 10 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
Only the five requested phenotype/prevalence criteria were adjudicated under the ClinGen RASopathy Expert Panel BRAF Version 2.3 specification.
PS4 and BP5 were not assessed because the available evidence lacks the VCEP-specific phenotype point assignments and required alternative-cause or case-control data; PP4, PP5, and BP6 are explicitly not applicable.
No criteria match.
The VCEP specifies PS3 Supporting for one approved assay and PS3 Moderate only for two or more different approved assays. The case-specific literature directly evaluates BRAF D594G in cellular functional studies: PMID:28783719 reports no detectable kinase activity and RAS-dependent ERK signaling using RAS-loss, immunoblotting, active-RAS pull-down, co-immunoprecipitation, and dimerization assays; PMID:23352452 reports impaired catalytic activity and dimerization-dependent focus formation. These studies support a damaging/abnormal BRAF functional effect, but the named VCEP functional-studies file contains no row for c.1781A>G, p.Asp594Gly, or p.D594G, and neither paper is the specifically listed BRAF kinase-activity publication in that table. Therefore the evidence supports PS3 at Supporting, but not Moderate under the VCEP's two-different-approved-assays 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.
Residue 594 is within the BRAF VCEP-defined CR3 activation segment (amino acids 594-627), an approved critical and well-established functional domain. The variant is p.Asp594Gly at residue 594, so it meets the VCEP's PM1 domain route. The required RAF-Alignment.xlsx text was searched for c.1781A>G, p.Asp594Gly, and p.D594G; no variant-specific entry was found there, but PM1 is supported by the VCEP's explicitly defined CR3 interval.
Applicable only to critical and well-established functional domains available in the supplementary table (exon 6, exon 11, P-loop [AA 459-474], CR3 activation segment [AA 594-627]). Not applicable to specific amino acid residues (see PM5).
The governing BRAF VCEP permits PM2 at Supporting strength when the variant is absent from controls in gnomAD. The variant is absent from both available all-comers gnomAD v2.1 and gnomAD v4.1 records, satisfying the VCEP's absence requirement.
The variant must be absent from controls (gnomAD).
The VCEP rule applies to missense variants when REVEL is at least 0.7. This variant is a missense substitution, NM_004333.6:c.1781A>G (p.Asp594Gly), and the recorded REVEL score is 0.989, meeting the VCEP threshold. The splice-impact path is not additionally counted because the missense path already supplies PP3 evidence.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The governing BRAF VCEP rule requires a gnomAD filtering allele frequency of at least 0.05% for BA1. The variant is absent from the available all-comers gnomAD v2.1 and gnomAD v4.1 records, so no allele frequency reaches the threshold.
GnomAD filtering allele frequency ≥0.05%.
The governing BRAF VCEP rule requires a gnomAD filtering allele frequency of at least 0.025% for BS1. The variant is absent from the available all-comers gnomAD v2.1 and gnomAD v4.1 records, so no allele frequency reaches the threshold.
GnomAD filtering allele frequency ≥0.025%.
The VCEP rule applies to missense variants using REVEL ≤0.3, and this variant is missense. Its recorded REVEL score is 0.989, which is above the benign threshold, so BP4 is not met. The VCEP's separate splicing-variant path is not used for this missense variant, and the same REVEL prediction is not double-counted with PP3.
For missense variants: REVEL ≤0.3.
The VCEP rule requires the same amino-acid change as a previously established pathogenic variant and limits applicability to observed analogous residue positions in BRAF and RAF1. The searched evidence reports BRAF D594G/c.1781A>G and corresponding D593G numbering variants, but it does not provide a VCEP-established pathogenic comparator classification that can be used without inferring from another source's final classification.
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _BRAF_ and _RAF1_.
The VCEP requires a de novo variant with both maternity and paternity confirmed, in a patient with the disease and no family history, and assigns strength by its point values. The available case evidence does not document a germline proband result, parental testing, confirmed maternity and paternity, or a disease phenotype. The reviewed publications describe somatic tumor observations with matched normal tissue, not germline de novo transmission or parental testing; therefore PS2 cannot be assessed.
De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. ... 4 Points. ... 2 Points. ... 1 Point.
The BRAF VCEP requires point-based scoring for autosomal-dominant cases with RASopathy phenotypic specifications and assigns Supporting, Moderate, or Strong evidence at ≥1, ≥3, or ≥5 points. The reviewed exact-variant literature documents somatic cancer observations, including one D593G tumor in 164 non-Hodgkin lymphoma cases, one colorectal adenoma among 130 tumors, and eight tumors in a cancer cohort, but provides no RASopathy case-control comparison, unaffected-control count, or VCEP phenotypic point assignments. Therefore the governing point score cannot be evaluated; no PS4 strength is assigned. The PS4 rule's case-control note specifies RR/OR >5.0 with a confidence interval excluding 1.0, but the available studies do not provide the required matched case-control counts or estimates.
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 points.
The VCEP provides Moderate PM5 for one likely pathogenic residue change at the same codon and Strong PM5 for at least two likely pathogenic residue changes observed in at least five probands. The reviewed materials report alternate codon-594 substitutions, including D594N, D594A, and D594V, but do not establish that any comparator is likely pathogenic under this VCEP or document the required proband count for the Strong route.
≥2 different [likely] pathogenic residues changes at the same codon observed in ≥5 probands. ... 1 [likely] pathogenic residue change at the same codon.
The VCEP defines PM6 as an assumed de novo variant without confirmation of paternity and maternity, with 2, 1, or 0.5 points according to the applicable strength. The available case evidence does not establish a germline proband observation, a disease phenotype, or that either parent was tested and negative. The reviewed publications are somatic tumor reports rather than unconfirmed germline de novo cases, so PM6 cannot be assessed.
Assumed de novo, but without confirmation of paternity and maternity. ... 2 Points. ... 1 Point. ... 0.5 Points.
The VCEP applies PP1 to co-segregation with disease in affected family members and assigns supporting strength at 3 or more informative meioses, moderate at 5 or more, and strong at 7 or more; segregation in more than one family is recommended. No affected relatives, informative meioses, pedigrees, or family-level phenotype and genotype data are documented. The available publications are somatic cancer reports and do not provide germline familial segregation, so PP1 cannot be assessed.
Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... ≥7 informative meioses. ... ≥5 informative meioses. ... ≥3 informative meioses.
The VCEP requires a missense z score greater than 3.09 in gnomAD. The case evidence provides gnomAD presence/absence statements but no BRAF missense z score, so the criterion cannot be evaluated using the VCEP rule.
Missense z score is >3.09 in gnomAD.
The governing BRAF VCEP evaluates BS2 using point-based scoring for healthy homozygous or heterozygous individuals, with reduced points for apparently unaffected heterozygotes. The available evidence contains no qualifying healthy individual, genotype zygosity, phenotype, age, penetrance, or family-evaluation data from which the required points can be assigned.
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.
The VCEP defines BS4 as lack of segregation in affected members of a family and states that strong strength requires only one informative meiosis. No affected family member, informative meiosis, or family genotype-phenotype comparison is documented. The reviewed publications concern somatic tumors and do not establish non-segregation in an affected family, so BS4 cannot be assessed.
Lack of segregation in affected members of a family. ... Requires only one informative meiosis.
The BRAF VCEP allows BP2 when an alternative molecular cause of a RASopathy is present in the same gene and the phenotype has the expected RASopathy severity; its original summary requires the variant to be observed in trans with a pathogenic variant for a fully penetrant dominant disorder or in cis with a pathogenic variant in any inheritance pattern. The VCEP's supporting threshold is ≥ (-1) Point. The available literature reports BRAF p.D594G with KRAS p.A59E in one colorectal tumor and similar mutant allele frequencies consistent with the same tumor-cell population, but it does not establish germline cis/trans phase with a pathogenic variant or a RASopathy phenotype, so the BP2 points and threshold cannot be evaluated.
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. ... Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern. ... ≥ (-1) Point.
The BRAF VCEP BP5 rule requires an alternative molecular cause in a different gene, or an alternative molecular cause in the same gene with BP2, with a phenotype consistent with the expected RASopathy severity; it also permits phenotypes inconsistent with RASopathy that are fully explained by a different causative variant. The available material documents a somatic KRAS p.A59E co-occurrence in one colorectal cancer specimen, but does not establish an alternative germline molecular cause for a RASopathy phenotype or provide the VCEP BP5 point assignment. The applicable point operators are ≥(-1) for Supporting, ≥(-2) for Moderate, and ≥(-4) for Strong; no BP5 point value is available, so the required substituted inequalities cannot be evaluated (point value ≥ -1, point value ≥ -2, and point value ≥ -4). BP5 is therefore not assessed.
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). ... ≥ (-1) Point. ... ≥ (-2) Points. ... ≥ (-4) Points.
The governing BRAF VCEP specification explicitly marks PVS1 as not applicable. The variant is a missense substitution, NM_004333.6:c.1781A>G, p.(Asp594Gly), rather than a loss-of-function consequence for which PVS1 would be evaluated.
Not Applicable
The BRAF VCEP explicitly designates PM3 as not applicable. Its governing text states "Not applicable." PM3 therefore cannot be applied, regardless of any available phase or allelic observations.
Not applicable.
The VCEP rule states, "No known repetitive areas in gene. Use as described." This variant is a missense substitution, p.(Asp594Gly), with no insertion, deletion, stop-loss event, or resulting protein-length change; therefore the PM4 protein-length-change criterion does not apply.
No known repetitive areas in gene. Use as described.
The BRAF VCEP explicitly marks PP4 as not applicable and directs users to PS4 instead. No independent PP4 assertion is permitted under this governing specification.
Not applicable, see PS4.
The BRAF VCEP marks PP5 not applicable for this VCEP and states that the criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Variant-specific pathogenic reports therefore cannot be converted into PP5 under this framework.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
The BRAF VCEP v2.3 marks BS3 as Not applicable. The functional-studies table likewise proposes BS3 not applicable for the documented BRAF assay class, so benign functional evidence cannot be assigned under this VCEP criterion.
Not applicable
The queried variant is a missense substitution, p.Asp594Gly. The BRAF VCEP BP1 rule applies to truncating variants—nonsense, frameshift, canonical splice-site, initiation-codon, whole-gene, or multi-exon deletions—in genes without established loss-of-function correlation; it does not apply to this missense variant.
This rule has contraindications for use with RASopathies. Given the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon, entire gene or multi exon deletion) in genes without established LOF correlation to disease. See the supplemental material regarding dosage sensitivity information for each individual gene and potential association to disorders associated with LOF variants.
The governing BRAF VCEP specification explicitly marks BP3 as not applicable. The variant is a missense substitution rather than an in-frame insertion or deletion in a repetitive region.
Not Applicable
The BRAF VCEP marks BP6 not applicable for this VCEP and states that the criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Benign reports from reputable sources therefore cannot be converted into BP6 under this framework.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
The VCEP BP7 rule covers synonymous variants and also intronic positions except canonical splice sites or non-coding variants. NM_004333.6:c.1781A>G is an exonic missense variant producing p.Asp594Gly, so it lies outside BP7's stated scope.
A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4.
Every interpreted variant in BRAF, mapped onto the exon structure of NM_004333.6. Select a row to find it on the plot.
| Protein | Location | Classification | Link |
|---|
Absent from gnomAD v4.1.
Absent from gnomAD v2.1.
Not available in gnomAD-Canada v1.0.
The VCEP rule applies to missense variants when REVEL is at least 0.7. This variant is a missense substitution, NM_004333.6:c.1781A>G (p.Asp594Gly), and the recorded REVEL score is 0.989, meeting the VCEP threshold. The splice-impact path is not additionally counted because the missense path already supplies PP3 evidence.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The VCEP rule applies to missense variants using REVEL ≤0.3, and this variant is missense. Its recorded REVEL score is 0.989, which is above the benign threshold, so BP4 is not met. The VCEP's separate splicing-variant path is not used for this missense variant, and the same REVEL prediction is not double-counted with PP3.
For missense variants: REVEL ≤0.3.
The VCEP BP7 rule covers synonymous variants and also intronic positions except canonical splice sites or non-coding variants. NM_004333.6:c.1781A>G is an exonic missense variant producing p.Asp594Gly, so it lies outside BP7's stated scope.
A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved. This rule is also applicable for intronic positions (except canonical splice sites) or non-coding variants and should be used in conjunction with BP4.
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.04). REVEL score = 0.989. BayesDel score = 0.542287.
8 paper records listed. 3 cited in the classification.
into oncogenic B-Raf mutants with high (V600E and G469A), moderate (G464V and L597V), low (G466A), and impaired (D594G) catalytic activity
Location Results, ‘Effect of Dimer Interface Mutations on Disease-Associated Raf Mutants,’ and Figure 3B
Context Engineered B-Raf mutants were assessed using B-Raf-C-Raf heterodimerization assays in cycling HeLa cells and focus-formation assays in NIH3T3 cells.
The most common residue involved by the kinase-impaired mutant was codon 594 (15 of 20 or 75 % of kinase-impaired mutants), specifically, p.D594G (c.1781A > G) in 8 tumors and p.D594N (c.1780G > A) in 5 tumors.
Location Results, Clinical detection of BRAF mutations in different tumors according to kinase activity; Table 2; Table 3, case P5
Context Retrospective cohort of FFPE lung cancer, colorectal cancer, and melanoma specimens analyzed by targeted Ion AmpliSeq Cancer Hotspot Panel v2 NGS; 135 BRAF-mutated tumors were identified.
D594G | None | Yes | Yes | Insensitive
Location Table 1, Classification of cancer-associated BRAF mutants; Figure 1d; Figure 2a-b; Figure 3c
Context Cell-based functional assays using NIH3T3, conditional RAS-knockout cells, and 293H cells; immunoblotting, active-RAS pull-down, co-immunoprecipitation, and vemurafenib sensitivity assays.
Two other kinase-dead BRAF mutants, the classical catalytic lysine mutant (K483MBRAF), and D594VBRAF, a mutant found in human cancer (Wan et al., 2004), also activate MEK in D04 cells.
Results, section ‘BRAF Inhibition Activates CRAF,’ paragraph 3; Results, section ‘Oncogenic Ras and Kinase-Dead Braf Cooperate to Induce Melanoma in Mice’
The remaining one was found at codon 593 (D593G) in the activation segment domain (Table 1, Figure 1).
Results, paragraph 2; Figure 1 legend; Table 1
We also identified one previously detected mutation (10) in codon 593, the GAT!GGT (D593G) mutation (Figure 1), which was found in an adenoma of the sigmoid colon.
Results, ‘Mutation screening of the MAPK pathway’; Table II; Figure 1; Discussion
It should be understood that the current copy-number genomic microarray technologies are not designed to detect point mutations, gene expression levels, methylation anomalies, and microRNA anomalies, all of which may contribute to tumorigenesis.
Page 485, section DNA microarray platforms, under limitations of DNA microarray analysis
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The evidence statements recorded by the interpretation pipeline. They have not been reinterpreted by the page.
Absent from gnomAD v4.1.
Absent from gnomAD v2.1.
Not available in gnomAD-Canada v1.0.
This variant has been reported in ClinVar as Pathogenic (2 clinical laboratories) and as Likely pathogenic (1 clinical laboratory) and as Uncertain significance (1 clinical laboratory). (ClinVarID = 13972)
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.04). REVEL score = 0.989. BayesDel score = 0.542287.
OncoKB identified variant-specific curated literature and context relevant to functional review; biological-effect context: Gain-of-function; curated oncogenicity label: Oncogenic.
This variant lies in a statistically significant hotspot. This variant has previously been reported in somatic cancers (COSMIC; COSV56065695, n = 169 times).
This variant lies in a statistically significant hotspot.