The BRAF NM_004333.6:c.1406G>C (NP_004324.2:p.(Gly469Ala)) missense variant has previously been reported in somatic cancers (COSMIC; COSV56061424, n = 139 times), and lies in a statistically significant hotspot (cancerhotspots.org). Experimental evidence: Two different approved assays, MEK and ERK activation, directly support damaging BRAF G469A gain-of-function signaling. Based on available guidelines, this variant is considered to be Likely Pathogenic.
Five-step scale from benign to pathogenic. This variant is likely pathogenic. Select a step to read what it means.
Likely pathogenic.A likely pathogenic classification under the recorded framework.
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, Strong: ClinVar records the exact BRAF p.Gly469Ala variant as Pathogenic, supported by variant-specific functional reports, but review is warranted because submission status is single-submitter.
Met at Moderate: two different approved assays, MEK and ERK activation, directly support damaging BRAF G469A gain-of-function signaling.
Met, Moderate: residue 469 lies within the VCEP-approved P-loop domain spanning amino acids 459-474, so the domain-based PM1 rule applies.
Met at Supporting: the missense REVEL score is 0.724, meeting the VCEP threshold of ≥0.7.
No benign criterion marked as applied in the recorded assessment.
Likely Pathogenic: PS1 (strong) plus PM1 (moderate) satisfy BRAF VCEP Rule11, with additional PS3 (moderate) and PP3 (supporting) evidence.
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1: 3 / 1,614,022 alleles. Recorded AF 1.85871e-06. Recorded homozygote count 0.
This variant is present in gnomAD v2.1 (AF= 3.97741e-06; MAF= 0.00040%, 1/251420 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 2.89134e-05; MAF= 0.00289%, 1/34586 alleles, homozygotes = 0).
Absent from gnomAD-Canada v1.0.
Recorded REVEL score: 0.724. Refer to the recorded PP3/BP4 rationale for interpretation.
1 Pathogenic. ClinVar API review status: criteria provided, single submitter.
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; COSV56061424, n = 139 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 · 6 not met · 9 not assessed · 9 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
PVS1 is not applicable because the VCEP explicitly excludes it in the BRAF v2.3 CSPEC entry and the variant is missense.
PM4 is not met because the variant changes one amino acid without changing protein length.
BP3 is not applicable because the VCEP explicitly marks BP3 Not Applicable.
PS1 Strong and PM1 Moderate are supported by the exact pathogenic p.Gly469Ala record and the VCEP-approved P-loop domain; PM5 is not met, PP2 is not assessed without the required z score, and BP1 is not applicable to this missense variant.
PS4 cannot be assessed because the exact-variant evidence lacks qualifying germline RASopathy cases and affected-control counts; no odds ratio or confidence interval can be calculated.
PP4, PP5, and BP6 are explicitly not applicable under the BRAF RASopathy VCEP.
BP5 cannot be assessed because no alternative molecular cause or fully explained phenotype is documented.
PP3 is met at Supporting from the VCEP missense-specific REVEL threshold.
BP4 is not met because the VCEP missense-specific REVEL threshold is not satisfied.
BP7 is not applicable because the variant is missense coding and outside the VCEP BP7 scope.
BA1 and BS1 are not met because the observed gnomAD allele frequencies are below their VCEP thresholds.
PM2 is not met because the variant is observed in both gnomAD v2.1 and gnomAD v4.1.
BS2 is not assessed because the VCEP point-based rule does not define how the available zero-homozygote observation maps to a point value.
No criteria match.
PS1 is met at Strong strength because the exact p.Gly469Ala (p.G469A; c.1406G>C) variant is recorded as Pathogenic in ClinVar, and variant-specific literature reports the same BRAF G469A change with constitutive signaling and stable homodimerization. The ClinVar record is single-submitter rather than expert-panel reviewed, so human review is warranted for the 'previously established pathogenic' requirement.
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _BRAF_ and _RAF1_.
PS3 is met at Moderate strength. The VCEP CSPEC rule requires two or more different approved assays for Moderate PS3. The governing functional-studies file lists MEK Activation Assay and ERK Activation Assay as approved pathway-specific assays, with MEK activation defined by the phosphorylated-to-unphosphorylated MEK ratio and ERK activation defined by the phosphorylated-to-unphosphorylated ERK ratio. PMID:22510884 directly studied BRAF G469A and found altered MEK signaling and stable homodimerization in transiently transfected cells. PMID:22773810 directly studied BRAF G469A and found constitutive ERK activation during ectopic-expression and EGFR-inhibition experiments. These are two different approved assay types supporting a damaging gain-of-function effect. The named functional-studies table contains no exact c.1406G>C, p.Gly469Ala, or p.G469A variant-specific row; the strength is therefore based on the VCEP-approved assay classes and the directly extracted variant-specific study findings, not on a pre-assigned variant code.
Two or more different approved assays.
PM1 is met at Moderate strength. The variant is a missense change in exon 11 at residue 469, which lies within the VCEP-defined P-loop at amino acids 459-474, an approved critical and well-established functional domain. The VCEP rule explicitly limits PM1 to the listed domains and says it is not for specific amino acid residues; residue 469 is within the approved domain. The authoritative vcep_materials.json domain_tables object has no BRAF entry, so the CSPEC domain definition governs. The required RAF-Alignment.xlsx search found no c.1406G>C, p.Gly469Ala, or p.G469A entry.
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 VCEP PP3 rule has a missense-specific path requiring REVEL ≥0.7; NM_004333.6:c.1406G>C is a missense variant causing p.(Gly469Ala), and the recorded REVEL score is 0.724. The separate splicing-impact clause is not required for this missense path, and the SpliceAI result is not counted because the governing missense path names REVEL.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The governing BRAF VCEP requires the variant to be absent from gnomAD for PM2 at Supporting strength. This variant is present in gnomAD v2.1 at 1/251,420 alleles (AF 3.97741e-06) and in gnomAD v4.1 at 3/1,614,022 alleles (AF 1.85871e-06), so the VCEP's absence requirement is not satisfied.
The variant must be absent from controls (gnomAD).
The VCEP PM4 rule states, "No known repetitive areas in gene. Use as described." The variant is a missense substitution, c.1406G>C, producing p.(Gly469Ala), so it does not create a protein-length change; the PM4 criterion is therefore not met under the VCEP's as-described application.
No known repetitive areas in gene. Use as described.
PM5 is not met because the available variant-specific literature and comparator collection provide no different likely pathogenic or pathogenic missense change at BRAF codon 469. Reports of nearby BRAF G468A/G468R changes concern codon 468, not the same codon, and therefore do not satisfy the VCEP PM5 rule. No VCEP full-text file searched contained a matching p.Gly469Ala, p.G469A, or c.1406G>C entry that would identify a same-codon comparator.
1 [likely] pathogenic residue change at the same codon.
The governing BRAF VCEP specifies BA1 at a gnomAD filtering allele frequency of at least 0.05%. The highest observed gnomAD frequency is 0.00289% in the gnomAD v2.1 Admixed American subgroup (1/34,586 alleles); the combined gnomAD v4.1 frequency is 0.00019% (3/1,614,022 alleles), so the BA1 threshold is not reached.
GnomAD filtering allele frequency ≥0.05%.
The governing BRAF VCEP specifies BS1 at a gnomAD filtering allele frequency of at least 0.025%. The highest observed gnomAD frequency is 0.00289% in the gnomAD v2.1 Admixed American subgroup (1/34,586 alleles); the combined gnomAD v4.1 frequency is 0.00019% (3/1,614,022 alleles), so the BS1 threshold is not reached.
GnomAD filtering allele frequency ≥0.025%.
The VCEP BP4 rule applies to missense variants when REVEL ≤0.3. NM_004333.6:c.1406G>C is missense, but its REVEL score is 0.724, above the governing cutoff; therefore BP4 is not met. SpliceAI is not used to rescue BP4 because the governing missense path names REVEL.
For missense variants: REVEL ≤0.3.
The VCEP PS2 criterion requires a documented de novo occurrence and assigns evidence by point score, with full points for RASopathy phenotypes and reduced points when phenotypic information is minimal. The available case evidence does not document a proband with a RASopathy phenotype, confirmed maternity and paternity, parental testing results, or absence of family history; therefore no PS2 points can be assigned.
Follow SVI recommendations for point-based scoring in conjunction with PM6 (see Reference 1) and 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 PS4 rule requires significantly increased prevalence in affected individuals versus controls and specifies OR or RR >5.0 with a confidence interval excluding 1.0; for very rare variants it permits multiple unrelated patients with the same phenotype plus absence in controls as moderate evidence. The exact-variant literature provides no qualifying germline RASopathy case series or affected-control counts. PMID:22773810 reports BRAF G469A in 1 of 195 tumors with acquired EGFR-inhibitor resistance, but this is a somatic oncology cohort rather than an autosomal-dominant RASopathy comparison; PMID:14612909 reports other variants, not G469A; PMID:22510884 is functional only; and PMID:24033266 is a general methods paper. No qualifying OR or 95% CI can therefore be computed, and no PS4 point total can be assigned.
The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: 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. See manuscript for detailed guidance. ... Note 2: 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.
The VCEP PM6 criterion concerns an assumed de novo variant without confirmation of maternity and paternity and assigns strength by point score. The available case evidence does not document an assumed de novo occurrence, the proband's RASopathy phenotype, or any parental testing or maternity/paternity information; therefore PM6 cannot be scored.
Follow SVI recommendations for point-based scoring in conjunction with PS2 (see Reference 1) and 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 PP1 criterion evaluates co-segregation using informative meioses: at least 3 for Supporting, at least 5 for Moderate, and at least 7 for Strong. No affected family members, genotype-phenotype segregation observations, or informative meioses are documented for this variant, so PP1 cannot be assigned.
Segregation in more than one family is recommended.
PP2 cannot be assessed because the VCEP requires a missense z score greater than 3.09 in gnomAD, and the available case evidence contains allele counts and frequencies but no BRAF missense z score.
Missense z score is >3.09 in gnomAD.
The governing BRAF VCEP gives BS2 only as point-based alternatives (−4 Points. or −1 Point.) and does not provide, in the criterion entry, a mapping from the available population observation to either point value. gnomAD reports zero homozygotes, but assigning a BS2 point value would require an unstated rule.
\-4 Points. ... \-1 Point.
The VCEP BS4 criterion addresses lack of segregation in affected family members and requires only one informative meiosis for Strong evidence. The available case evidence documents no affected relatives, no family genotype-phenotype comparison, and no informative meiosis; absence of such documentation cannot establish non-segregation.
Lack of segregation in affected members of a family.
The VCEP provides BP2 point thresholds but the available evidence contains no documented phase observation showing this variant in trans with a pathogenic variant or in cis with a pathogenic variant. Because the required allelic relationship cannot be evaluated, BP2 is not assessed.
≥ (-4) Points. ... ≥ (-2) Points. ... ≥ (-1) Point.
The VCEP BP5 rule requires an alternative molecular cause of a RASopathy in a different gene, or a phenotype inconsistent with RASopathy fully explained by a different causative variant, with point-based strength adjustment. The available case materials do not identify an alternative causative variant or a qualifying phenotype fully explained by another variant, so BP5 cannot be evaluated. The VCEP's own operator is >=: 0 points >= (-1) point? no; 0 points >= (-2) points? no; 0 points >= (-4) points? no, but this does not substitute for the missing alternate-cause evidence.
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.
The ClinGen RASopathy VCEP BRAF v2.3 CSPEC entry explicitly marks PVS1 as Not Applicable. The case variant is NM_004333.6:c.1406G>C, a missense substitution producing NP_004324.2:p.(Gly469Ala) in exon 11, not a truncating, splice-site, or other loss-of-function consequence within the VCEP's PVS1 scope.
Not Applicable
PM3 is explicitly designated Not Applicable in the ClinGen RASopathy VCEP BRAF Version 2.3 criterion specification. No recessive inheritance or phase assessment is therefore used.
Not Applicable
The BRAF RASopathy VCEP explicitly marks PP4 as not applicable and directs phenotype-specific evidence to PS4.
Not applicable, see PS4.
The BRAF RASopathy VCEP explicitly marks PP5 as not applicable and prohibits its use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
BS3 is not applicable under the BRAF VCEP specification. The CSPEC entry explicitly marks BS3 as not applicable, and the governing functional-studies file records the approved functional assay examples as PS3-supporting with BS3 not applied. The available variant-specific studies show damaging signaling rather than a no-damage result.
Not applicable
BP1 is not applicable because the VCEP limits this rule to truncating variants, including nonsense, frameshift, canonical splice-site, initiation-codon, whole-gene, or multi-exon deletion variants. This variant is a missense substitution, c.1406G>C (p.Gly469Ala).
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 ClinGen RASopathy VCEP BRAF v2.3 CSPEC entry explicitly marks BP3 as Not Applicable. No BP3 evidence can be applied under this governing framework.
Not Applicable
The BRAF RASopathy VCEP explicitly marks BP6 as not applicable and prohibits its use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
The VCEP BP7 rule covers synonymous variants, intronic positions except canonical splice sites, and non-coding variants. NM_004333.6:c.1406G>C is a missense coding variant, so it lies outside BP7 scope; BP7 is not applicable without evaluating splice prediction or conservation evidence.
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 |
|---|
| Admixed American1 / 60,012 | 1.7e-05 |
| South Asian1 / 91,080 | 1.1e-05 |
| European (non-Finnish)1 / 1,179,934 | 8.5e-07 |
| Admixed American1 / 34,586 | 2.9e-05 |
This variant is present in gnomAD v4.1 (AF= 1.85871e-06; MAF= 0.00019%, 3/1614022 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 1.66633e-05; MAF= 0.00167%, 1/60012 alleles, homozygotes = 0).
This variant is present in gnomAD v2.1 (AF= 3.97741e-06; MAF= 0.00040%, 1/251420 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 2.89134e-05; MAF= 0.00289%, 1/34586 alleles, homozygotes = 0).
Absent from gnomAD-Canada v1.0.
The VCEP PP3 rule has a missense-specific path requiring REVEL ≥0.7; NM_004333.6:c.1406G>C is a missense variant causing p.(Gly469Ala), and the recorded REVEL score is 0.724. The separate splicing-impact clause is not required for this missense path, and the SpliceAI result is not counted because the governing missense path names REVEL.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The VCEP BP4 rule applies to missense variants when REVEL ≤0.3. NM_004333.6:c.1406G>C is missense, but its REVEL score is 0.724, above the governing cutoff; therefore BP4 is not met. SpliceAI is not used to rescue BP4 because the governing missense path names REVEL.
For missense variants: REVEL ≤0.3.
The VCEP BP7 rule covers synonymous variants, intronic positions except canonical splice sites, and non-coding variants. NM_004333.6:c.1406G>C is a missense coding variant, so it lies outside BP7 scope; BP7 is not applicable without evaluating splice prediction or conservation evidence.
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 = 0.00). REVEL score = 0.724. BayesDel score = 0.38392.
7 paper records listed. 2 cited in the classification.
This analysis revealed that the MEK phosphorylation potential of this oncoprotein is reduced by o40% by the R509H substitution (Figure 1D and G).
Location Results, subsection “B-Raf V600E, B-Raf insT and B-Raf G469A do not require an intact DIF for efficient signalling,” Figure 1D/G; subsection “B-Raf V600E and B-Raf G469A form more stable homodimers than B-Raf wt,” Figure 3C
Context Human B-RafG469A constructs were transiently expressed in Plat-E cells; MEK/ERK signaling was assessed by western blotting and homodimerization by co-immunoprecipitation of epitope-tagged B-Raf proteins.
another tumor harbored EGFR exon 19 deletion and BRAF G469A
Location Results, “RAS Signaling Pathway Gene Mutations in Tumors from Patients with Acquired Resistance”; functional studies in “Ectopic Expression of NRAS Q61K, BRAF V600E, or BRAF G469A Confers Resistance...”
Context Tumor mutation screening in 195 patient samples with EGFR-mutant lung cancer and acquired resistance to EGFR TKIs; ectopic-expression and drug-response assays in EGFR-mutant PC-9 cells.
All four mutants had elevated basal kinase activity compared with wild-type BRAF
Results, paragraph beginning “To characterize the biological effects of these mutations”; Table 2; Figure 3
Six distinct ATP-competitive RAF inhibitors induced ERK activation in cells with wild-type BRAF, but inhibited signaling in mutant BRAFV600E cells.
Results, first paragraph
Additionally, three (two G468A and one G468R) of the four BRAF mutations in this study involved the same amino acid (G468) that is located in the GXGXXG motif within the G-loop of the kinase domain.
Results and Discussion; Table 1
Once the variant call is validated, literature, variant databases, and population control studies should be evaluated.
Section: Collecting evidence to determine the likelihood of pathogenicity
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= 1.85871e-06; MAF= 0.00019%, 3/1614022 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 1.66633e-05; MAF= 0.00167%, 1/60012 alleles, homozygotes = 0).
This variant is present in gnomAD v2.1 (AF= 3.97741e-06; MAF= 0.00040%, 1/251420 alleles, homozygotes = 0) and has highest observed frequency in the Admixed American population (AF= 2.89134e-05; MAF= 0.00289%, 1/34586 alleles, homozygotes = 0).
Absent from gnomAD-Canada v1.0.
This variant has been reported in ClinVar as Pathogenic (1 clinical laboratory). (ClinVarID = 13971)
SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00). REVEL score = 0.724. BayesDel score = 0.38392.
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; COSV56061424, n = 139 times).
This variant lies in a statistically significant hotspot.