The BRAF NM_004333.6:c.1397G>T (NP_004324.2:p.(Gly466Val)) missense variant has previously been reported in somatic cancers (COSMIC; COSV56057462, n = 53 times), and lies in a statistically significant hotspot (cancerhotspots.org). Experimental evidence: Two different approved assay types—reduced BRAF kinase activity and cellular MEK/ERK activation—support a damaging functional effect for p.G466V. 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, Moderate: two different approved assay types—reduced BRAF kinase activity and cellular MEK/ERK activation—support a damaging functional effect for p.G466V.
Met, Moderate: residue 466 lies within the VCEP-approved exon 11 P-loop domain spanning amino acids 459-474.
Met: REVEL score 0.781 exceeds the BRAF VCEP PP3 threshold of 0.7 for missense variants.
No benign criterion marked as applied in the recorded assessment.
VUS: PS3 (moderate), PM1 (moderate), and PP3 (supporting) do not satisfy a 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: 1 / 1,614,062 alleles. Recorded AF 6.19555e-07. Recorded homozygote count 0.
This variant is present in gnomAD v2.1 (AF= 3.97731e-06; MAF= 0.00040%, 1/251426 alleles, homozygotes = 0) and has highest observed frequency in the European (Finnish) population (AF= 4.62235e-05; MAF= 0.00462%, 1/21634 alleles, homozygotes = 0).
Not available in gnomAD-Canada v1.0.
Recorded REVEL score: 0.781. Refer to the recorded PP3/BP4 rationale for interpretation.
1 Pathogenic. ClinVar API review status: no assertion criteria provided.
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; COSV56057462, n = 53 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: 3 met · 4 not met · 11 not assessed · 10 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
PVS1, PM4, and BP3 were adjudicated only under the ClinGen RASopathy Expert Panel BRAF Version 2.3 specification; no other criteria were assessed.
PM1 is met at Moderate strength because p.Gly466Val lies in the VCEP-approved exon 11 P-loop interval (amino acids 459-474).
PS1 and PM5 are not met because the available records do not establish qualifying independent pathogenic same-amino-acid or alternate-same-codon comparators under the VCEP rules.
PP2 is not assessed because the required gnomAD missense z score is unavailable, and BP1 is not applicable to this missense variant.
PS3 is met at Moderate strength because the variant has direct evidence from two different approved assay types: BRAF kinase activity and cellular MEK/ERK pathway activation.
BS3 is not applicable under the BRAF VCEP specification.
Only PS4, PP4, PP5, BP5, and BP6 were adjudicated.
PS4 and BP5 remain not_assessed because the VCEP-specific evidence requirements cannot be evaluated with the available phenotype and cohort data.
PP4, PP5, and BP6 are not_applicable under the BRAF VCEP's explicit applicability rules.
The governing framework is the ClinGen RASopathy Expert Panel BRAF specification v2.3.
This is a missense variant, so PP3 and BP4 are evaluated only through the VCEP's named missense predictor, REVEL; PP3 is met and BP4 is not met from the same score, without double-counting it.
BP7 is not applicable because the variant is missense rather than synonymous, intronic, or non-coding.
No criteria match.
The BRAF VCEP defines PS3 as well-established in vitro or in vivo functional studies supportive of a damaging effect, with one approved assay supporting PS3 and two or more different approved assays supporting PS3 at Moderate strength. The governing functional-studies file identifies BRAF kinase activity and MEK/ERK activation assays as approved BRAF-relevant assays, each with proposed PS3_Supporting use. PMID:15035987 directly evaluates the queried substitution under the equivalent G465V notation and reports reduced basal BRAF kinase activity plus cellular MEK/ERK activation through endogenous C-RAF. These are two different approved assay types and therefore meet PS3 at Moderate strength. PMID:22649091 independently reports reduced kinase activity and MEK/ERK/CRAF pathway activation for BRAF G466V, corroborating the functional effect.
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.
The VCEP limits PM1 to critical, well-established functional domains listed in its supplementary table: exon 6, exon 11, the P-loop at amino acids 459-474, and the CR3 activation segment at amino acids 594-627. The queried missense variant is in exon 11 at residue 466, which is within the authoritative P-loop interval 459-474. The VCEP's domain_tables entry is empty for BRAF, so the CSPEC rule's explicit complete domain list governs. PM1 is met at Moderate strength.
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 variant is a missense substitution, NM_004333.6:c.1397G>T, p.(Gly466Val). The BRAF VCEP PP3 rule applies to missense variants when REVEL is at least 0.7; the recorded REVEL score is 0.781, so PP3 is met at Supporting strength. The SpliceAI result was not used for PP3 because the missense REVEL path is sufficient and the same computational prediction must not be double-counted across PP3, BP4, and BP7.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The VCEP requires the same amino-acid change as a previously established pathogenic variant and limits the analogous-residue route to BRAF and RAF1. The reviewed literature reports the queried substitution under offset notation (G465V/G1394T) and reports other G-loop substitutions, but the available records do not establish a distinct previously established pathogenic variant with the same amino-acid change at the applicable residue. The exact variant's ClinVar record is Pathogenic but has no assertion criteria and cannot serve as an independent PS1 comparator. Therefore PS1 is not met.
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 BRAF VCEP requires the variant to be absent from controls in gnomAD for PM2 at Supporting strength. This variant is present in the default all-comers gnomAD datasets: gnomAD v2.1 has 1/251426 alleles (AF 3.9773134043416354e-06; homozygotes 0), and gnomAD v4.1 has 1/1614062 alleles (AF 6.195548869869931e-07; homozygotes 0). Because the variant is observed in gnomAD controls, the VCEP PM2 requirement is not satisfied.
The variant must be absent from controls (gnomAD).
The VCEP assigns PM5 at Moderate strength for one likely pathogenic residue change at the same codon and Strong strength for at least two different likely pathogenic residue changes at the same codon observed in at least five probands. The collected evidence includes nearby G-loop substitutions and offset notation for the queried variant, but does not establish any alternate residue change at codon 466 as likely pathogenic under this VCEP rule. The case PM5 candidate summary records zero same-residue candidates. PM5 is therefore not met.
1 [likely] pathogenic residue change at the same codon.
The variant is a missense substitution, so the BRAF VCEP BP4 missense path applies. That path requires REVEL ≤0.3. The recorded REVEL score is 0.781, which does not satisfy the BP4 cutoff; therefore BP4 is not met. The available SpliceAI score was not substituted for REVEL because the governing VCEP missense rule names REVEL for this situation.
For missense variants: REVEL ≤0.3.
The VCEP specifies PS2 by de novo evidence points, but the available case evidence does not document that NM_004333.6:c.1397G>T (p.Gly466Val) occurred in an affected proband, parental testing results, confirmed maternity and paternity, or any assigned de novo points. The criterion therefore cannot be evaluated under the VCEP rule.
4 Points. ... 2 Points. ... 1 Point.
The BRAF VCEP requires PS4 point-based evidence for autosomal-dominant cases with RASopathy phenotypic specifications. The literature packet contains no ai_classified_papers, and the extracted exact-variant reports are cancer/somatic observations rather than RASopathy case-control cohorts. PMID:12068308 reports the queried amino-acid substitution under the paper's G465V notation in the NCI-H1666 NSCLC cell line and states that cancer samples were compared with 341 normal control DNAs, but it does not provide an eligible RASopathy affected-case count, matched control count for the same phenotype, odds ratio/RR with a confidence interval, or a VCEP PS4 point assignment. Therefore the VCEP PS4 rule cannot be evaluated from the available evidence; no generic case-control or rare-variant substitute was applied.
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)).
The VCEP specifies PM6 by de novo evidence points, but the available case evidence does not document an affected proband with this variant, unconfirmed parental status, or any VCEP-assigned PM6 points. The criterion cannot be evaluated without case-specific de novo evidence under the VCEP rule.
2 Points. ... 1 Point. ... 0.5 Points.
The VCEP requires a specified number of informative meioses for PP1, but the available case evidence contains no affected or unaffected relatives, segregation results, or informative meiosis count for NM_004333.6:c.1397G>T. The criterion cannot be assessed under the VCEP threshold rule.
≥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 allele observations for this variant but does not provide the BRAF missense z score. Because the governing threshold cannot be evaluated from the available evidence, PP2 is not assessed.
Missense z score is >3.09 in gnomAD.
The BRAF VCEP requires the gnomAD filtering allele frequency to be at least 0.05% for BA1. The available gnomAD records report unfiltered total and highest-subpopulation frequencies, but do not provide a filtering allele frequency (grpmax FAF is null): gnomAD v2.1 total AF 3.97731e-06, highest-subpopulation AF 4.62235e-05 (0.00462%), and gnomAD v4.1 total AF 6.19555e-07, highest-subpopulation AF 1.56235e-05 (0.00156%), all with zero homozygotes. Because the required filtering AF is unavailable, these values cannot be substituted to evaluate the VCEP threshold.
GnomAD filtering allele frequency ≥0.05%.
The BRAF VCEP requires the gnomAD filtering allele frequency to be at least 0.025% for BS1. The available gnomAD records report unfiltered total and highest-subpopulation frequencies, but do not provide a filtering allele frequency (grpmax FAF is null): gnomAD v2.1 total AF 3.97731e-06, highest-subpopulation AF 4.62235e-05 (0.00462%), and gnomAD v4.1 total AF 6.19555e-07, highest-subpopulation AF 1.56235e-05 (0.00156%), all with zero homozygotes. Because the required filtering AF is unavailable, these values cannot be substituted to evaluate the VCEP threshold.
GnomAD filtering allele frequency ≥0.025%.
The BRAF VCEP evaluates BS2 by point-based evidence from healthy homozygous or heterozygous individuals, assigning -4 points for Strong or -1 point for Supporting. The available population data show zero homozygotes in gnomAD v2.1 and v4.1, but do not identify a healthy individual carrying the variant or provide a VCEP-assigned point. The variant's presence as one allele in gnomAD cannot establish the required healthy-individual observation, so BS2 cannot be scored.
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. ... \-4 Points. ... \-1 Point.
The VCEP requires one informative meiosis for BS4, but the available case evidence contains no family testing, non-segregation result, unaffected informative relative, or documented informative meiosis for NM_004333.6:c.1397G>T. The criterion cannot be assessed under the VCEP rule.
Requires only one informative meiosis.
The BRAF VCEP permits BP2 when an alternative molecular cause of a RASopathy is present in the same gene, the phenotype has the expected RASopathy severity, and the applicable point threshold is met. The available case evidence does not document a second pathogenic variant in BRAF or another molecular cause in the same affected individual, its phase with NM_004333.6:c.1397G>T (p.Gly466Val), or a phenotype assessment tied to such an alternative cause. Consequently, the BP2 rule cannot be evaluated without substituting an unsupported assumption.
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.
The BRAF VCEP permits BP5 when an alternative molecular cause in a different gene, with a phenotype consistent with the expected RASopathy severity, is documented, or when a phenotype inconsistent with RASopathy is fully explained by a different causative variant. The case evidence identifies BRAF p.Gly466Val in somatic cancer and provides no patient phenotype, alternative pathogenic variant in another gene, or documentation that another variant fully explains the phenotype. The VCEP BP5 point rule therefore cannot be evaluated; no point value or threshold comparison is available.
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).
PVS1 is not applicable under the ClinGen RASopathy Expert Panel BRAF Version 2.3 specification, which explicitly marks PVS1 as not applicable. The variant is a missense substitution, NM_004333.6:c.1397G>T, producing NP_004324.2:p.(Gly466Val), not a loss-of-function consequence.
Not Applicable
The BRAF VCEP explicitly marks PM3 as not applicable and states that it is not applicable. Therefore, phase or affected-proband observations cannot activate PM3 under this framework.
Not applicable.
The VCEP's own PM4 entry states, "No known repetitive areas in gene. Use as described." PM4 therefore does not apply to this variant: NM_004333.6:c.1397G>T is a missense substitution, NP_004324.2:p.(Gly466Val), with no in-frame insertion, deletion, stop-loss event, or protein-length change.
No known repetitive areas in gene. Use as described.
The BRAF VCEP explicitly marks PP4 as not applicable and directs evaluation to PS4 instead. PP4 is not applied independently.
Not applicable, see PS4.
The BRAF VCEP marks PP5 not applicable at every strength and states that the criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. ClinVar and literature assertions therefore cannot activate PP5 under this VCEP.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
The BRAF VCEP CSPEC entry marks BS3 as Not applicable, and the approved functional-studies file lists BS3_NA for the BRAF kinase, MEK activation, and ERK activation assay rows. Therefore BS3 cannot be applied, regardless of the functional findings available for this variant.
Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
The VCEP states that BP1 is contraindicated for RASopathies and should be used for truncating variants, specifically nonsense, frameshift, canonical splice-site, initiation-codon, whole-gene, or multi-exon deletion variants in genes without established loss-of-function correlation. This variant is a missense substitution, c.1397G>T p.Gly466Val, so it is outside the variant types named by the rule.
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.
BP3 is explicitly marked Not Applicable in the ClinGen RASopathy Expert Panel BRAF Version 2.3 specification. The variant is a missense substitution, NM_004333.6:c.1397G>T, producing NP_004324.2:p.(Gly466Val), rather than a variant type for which BP3 could be applied.
Not Applicable
The BRAF VCEP marks BP6 not applicable at every strength and states that the criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Benign assertions from reputable sources therefore cannot activate BP6 under this VCEP.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
The BRAF VCEP BP7 rule covers synonymous variants, intronic positions except canonical splice sites, and non-coding variants. NM_004333.6:c.1397G>T produces the missense protein change p.(Gly466Val), so it is outside BP7's named variant-type scope. BP7 is therefore not applicable, regardless of the available splice-prediction result.
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 |
|---|
| European (Finnish)1 / 64,006 | 1.6e-05 |
| European (Finnish)1 / 21,634 | 4.6e-05 |
This variant is present in gnomAD v4.1 (AF= 6.19555e-07; MAF= 0.00006%, 1/1614062 alleles, homozygotes = 0) and has highest observed frequency in the European (Finnish) population (AF= 1.56235e-05; MAF= 0.00156%, 1/64006 alleles, homozygotes = 0).
This variant is present in gnomAD v2.1 (AF= 3.97731e-06; MAF= 0.00040%, 1/251426 alleles, homozygotes = 0) and has highest observed frequency in the European (Finnish) population (AF= 4.62235e-05; MAF= 0.00462%, 1/21634 alleles, homozygotes = 0).
Not available in gnomAD-Canada v1.0.
The variant is a missense substitution, NM_004333.6:c.1397G>T, p.(Gly466Val). The BRAF VCEP PP3 rule applies to missense variants when REVEL is at least 0.7; the recorded REVEL score is 0.781, so PP3 is met at Supporting strength. The SpliceAI result was not used for PP3 because the missense REVEL path is sufficient and the same computational prediction must not be double-counted across PP3, BP4, and BP7.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The variant is a missense substitution, so the BRAF VCEP BP4 missense path applies. That path requires REVEL ≤0.3. The recorded REVEL score is 0.781, which does not satisfy the BP4 cutoff; therefore BP4 is not met. The available SpliceAI score was not substituted for REVEL because the governing VCEP missense rule names REVEL for this situation.
For missense variants: REVEL ≤0.3.
The BRAF VCEP BP7 rule covers synonymous variants, intronic positions except canonical splice sites, and non-coding variants. NM_004333.6:c.1397G>T produces the missense protein change p.(Gly466Val), so it is outside BP7's named variant-type scope. BP7 is therefore not applicable, regardless of the available splice-prediction result.
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 = 0.01). REVEL score = 0.781. BayesDel score = 0.580097.
8 paper records listed. 2 cited in the classification.
We identified four cancer-associated B-RAF mutants whose basal kinase activities were reduced to between 30% and 80% of the activity of WT B-RAF (Figure 2A). We term these mutants the impaired activity group. Strikingly, however, despite their reduced in vitro kinase activity, three of the mutants still activated endogenous ERK in COS cells, although to lower levels than other mutants (Figures 2B and 2C).
Location Results, “Three B-RAF Cancer Mutants Have Impaired Kinase Activity but Activate ERK in Cells”; “Impaired Activity Mutants Stimulate C-RAF In Vivo”
Context Functional studies of cancer-associated B-RAF mutants using in vitro kinase assays, COS-cell transfection and ERK/MEK activation assays, Xenopus embryo assays, C-RAF depletion by siRNA, and analysis of the H1666 cancer cell line.
To determine the functional significance of Y472CBRAF, we used site-directed mutagenesis to create Y472CBRAF, G466VBRAF (kinase-impaired), and V600EBRAF (constitutively active) in a Flag-tagged wtBRAF construct. We transfected the constructs into COS7 cells, isolated the Flag-tagged proteins and tested for kinase activity. As expected, V600EBRAF had increased kinase activity and G466VBRAF had reduced kinase activity. Y472CBRAF showed severely reduced kinase activity that was less than 10% that of wtBRAF (Figure 1A).
Location Results: Identification of a Novel Inactivating Mutation in BRAF; Lung Cancer Cells Expressing Y472CBRAF Express Activated MEK, ERK, and CRAF
Context Site-directed mutagenesis of Flag-tagged BRAF constructs and kinase assays in COS7 cells; transfection and MEK/ERK and CRAF activity assays in COS7, H226, and H661 cells; dasatinib sensitivity and senescence assays in NSCLC cell lines including cells with endogenous G466V BRAF.
This analysis identified 3 additional mutations, i.e. G463R, G465R and G465E, in 3 individual patients, thereby confirming that mutations in the G-loop of BRaf occur at low frequencies in melanoma.
Results, “Mutations within the BRAF gene”; Results, “Analysis of the BRaf mutants D593V, G463R, G465R and G465E”
G1394T G465V 1 NCl-H1666 NSCLC WT G465V 89 ND A total of 11% of mutations are in the glycine residues (G463, G465 and G468) of the GXGXXG motif within the glycine-rich loop of the kinase domain.
Table 1; Results, paragraph discussing glycine-rich-loop mutations; Table 3
Screenshots represent source records captured during evidence collection. Select one to view the recorded source content.
The evidence statements recorded by the interpretation pipeline. They have not been reinterpreted by the page.
This variant is present in gnomAD v4.1 (AF= 6.19555e-07; MAF= 0.00006%, 1/1614062 alleles, homozygotes = 0) and has highest observed frequency in the European (Finnish) population (AF= 1.56235e-05; MAF= 0.00156%, 1/64006 alleles, homozygotes = 0).
This variant is present in gnomAD v2.1 (AF= 3.97731e-06; MAF= 0.00040%, 1/251426 alleles, homozygotes = 0) and has highest observed frequency in the European (Finnish) population (AF= 4.62235e-05; MAF= 0.00462%, 1/21634 alleles, homozygotes = 0).
Not available in gnomAD-Canada v1.0.
This variant has been reported in ClinVar as Pathogenic (1 clinical laboratory). (ClinVarID = 13967)
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01). REVEL score = 0.781. BayesDel score = 0.580097.
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; COSV56057462, n = 53 times).
This variant lies in a statistically significant hotspot.