The BRAF NM_004333.6:c.1742A>G (NP_004324.2:p.(Asn581Ser)) missense variant has previously been reported in somatic cancers (COSMIC; COSV56058108, n = 48 times), and lies in a statistically significant hotspot (cancerhotspots.org). Experimental evidence: One approved BRAF kinase assay found approximately six-fold higher basal activity for the N581S-equivalent mutant than wild-type. 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 at Supporting: one approved BRAF kinase assay found approximately six-fold higher basal activity for the N581S-equivalent mutant than wild-type.
No benign criterion marked as applied in the recorded assessment.
VUS: PS3 (supporting) was applied, but the BRAF VCEP's criteria-combination rules require additional evidence for a definitive classification.
Evidence summaries and captured source records, where available. Missing entries should not be interpreted as negative findings.
gnomAD v4.1: 2 / 1,573,802 alleles. Recorded AF 1.27081e-06. Recorded homozygote count 0.
Absent from gnomAD v2.1.
Not available in gnomAD-Canada v1.0.
Recorded REVEL score: 0.666. Refer to the recorded PP3/BP4 rationale for interpretation.
2 Likely 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; COSV56058108, n = 48 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: 1 met · 9 not met · 9 not assessed · 9 not applicable. Open any row for the full reasoning, the rule and the evidence behind it.
All four requested segregation and de novo criteria are not assessed because the available case evidence lacks proband-parent testing, family segregation, phenotype-linked relatives, and informative meiosis data.
For this BRAF p.(Asn581Ser) case, the VCEP-specific phenotype criteria resolve to PP4, PP5, and BP6 not applicable; PS4 and BP5 remain not assessed because the available evidence does not supply the required RASopathy phenotype and point-scoring data.
No generic ACMG/AMP phenotype or literature criterion was substituted for a VCEP rule.
The governing framework is the ClinGen RASopathy Expert Panel BRAF Version 2.3 specification.
The variant is missense p.(Asn581Ser), so PP3 and BP4 use their VCEP REVEL missense paths; REVEL 0.666 meets neither threshold.
BP7 is not applicable because the variant is not synonymous, intronic, or non-coding.
No criteria match.
PS3 is met at Supporting strength. The VCEP CSPEC rule states that well-established in vitro or in vivo functional studies supportive of a damaging effect qualify, with Supporting strength assigned for one approved assay. PMID:15035987 reports the variant under the study's B-RAF numbering as N580S-B-RAF, corresponding to BRAF p.N581S in this case, and found approximately six-fold higher basal kinase activity than wild-type in an in vitro kinase assay measuring direct MEK phosphorylation. The governing functional-studies file identifies BRAF kinase activity as an approved gene-specific assay, measuring kinase phosphorylation of MEK and ERK in transfected cells, and assigns the assay PS3_Supporting; BS3_NA. The same file specifies wild-type and vector controls, four pathogenic/likely pathogenic validation controls, two benign/likely benign-labelled validation controls, and increased MBP phosphorylation as the abnormal readout. No entry for c.1742A>G, p.Asn581Ser, or p.N581S was found in SVI-RASopathy-VCEP-V2-Approved-Functional-Studies.xlsx; therefore the assay-level pre-assigned PS3_Supporting code and the variant-specific PMID:15035987 result were used, without upgrading to Moderate because only one approved assay is documented for this variant.
Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. ... One approved assay.
PS1 is not met. The BRAF VCEP requires the same amino acid change as a previously established pathogenic variant, regardless of nucleotide change, and limits use to observed analogous residue positions in BRAF and RAF1. The reviewed evidence identifies this variant as BRAF p.N581S, including the alternate study notation N580S-B-RAF, but does not identify a distinct previously established pathogenic variant producing the same Asn-to-Ser amino acid change. PMID:15035987 provides functional evidence for this variant itself, not a qualifying prior pathogenic same-amino-acid comparator.
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Applicable for observed analogous residue positions in _BRAF_ and _RAF1_.
PM1 is not met. The BRAF VCEP limits PM1 to critical and well-established functional domains in exon 6, exon 11, the P-loop at amino acids 459-474, and the CR3 activation segment at amino acids 594-627, and excludes specific amino acid residues as a PM1 route. The queried residue is BRAF amino acid 581, outside the listed P-loop and CR3 activation segment and not in exon 6 or exon 11. vcep_materials.json has no BRAF domain_tables entry, and the governing RAF-Alignment.xlsx file contains no entry for c.1742A>G, p.Asn581Ser, or p.N581S. No VCEP-named hotspot route is available for this criterion.
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 applies PM2 at supporting strength when the variant is absent from controls in gnomAD. The variant is absent from gnomAD v2.1 and the available non-cancer v2.1 and v3.1 entries, but it is present in the default all-comers gnomAD v4.1 dataset with AC=2, AN=1,573,802, AF=1.270807890700355e-06 (reported MAF 0.00013%) and zero homozygotes. Because the VCEP rule requires absence from gnomAD and the default all-comers source documents presence, PM2 is not met.
The variant must be absent from controls (gnomAD).
PM4 is applicable to protein-length changes caused by in-frame insertions or deletions in a non-repeat region or to stop-loss variants. This variant is a missense substitution producing p.Asn581Ser, with no insertion, deletion, stop-loss event, or protein-length change; therefore the PM4 condition is not met. The VCEP's PM4 strength rule also states that there are no known repetitive areas in the gene and to use PM4 as described, which does not expand it to missense substitutions.
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. ... No known repetitive areas in gene. Use as described.
PM5 is not met. The BRAF VCEP assigns 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 available PM5 comparator evidence reports zero same-residue candidates and no expert-panel or non-expert majority likely pathogenic comparators; no qualifying alternate residue change at codon 581 was identified.
1 \[likely\] pathogenic residue change at the same codon. ... ≥2 different \[likely\] pathogenic residues changes at the same codon observed in ≥5 probands.
NM_004333.6:c.1742A>G is a missense variant producing p.(Asn581Ser). Under the VCEP missense pathway, PP3 requires REVEL >= 0.7. The recorded REVEL score is 0.666, so the PP3 threshold is not met. The separate splicing-impact clause is not used for this missense-pathway assessment because the VCEP specifically names REVEL for missense variants; no additional computational predictor is combined with it.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
The governing BRAF VCEP defines BA1 as a stand-alone benign criterion when the gnomAD filtering allele frequency is at least 0.05%. gnomAD v4.1 reports a total filtering allele frequency of 0.00013% (2 of 1,573,802 alleles), with the highest observed population frequency in European (non-Finnish) individuals at 0.00017%; both are far below 0.05%. gnomAD v2.1 and the non-cancer subsets are absent, but the default all-comers gnomAD v4.1 result is present and remains below the VCEP threshold.
GnomAD filtering allele frequency ≥0.05%.
The governing BRAF VCEP defines BS1 at strong strength when the gnomAD filtering allele frequency is at least 0.025%. gnomAD v4.1 reports a total filtering allele frequency of 0.00013% (2 of 1,573,802 alleles), and the highest population frequency is 0.00017% in the European (non-Finnish) population; both are below 0.025%. The available gnomAD v2.1 and non-cancer entries are absent, but the default all-comers gnomAD v4.1 observation remains below the VCEP threshold.
GnomAD filtering allele frequency ≥0.025%.
NM_004333.6:c.1742A>G is a missense variant producing p.(Asn581Ser), so the VCEP missense BP4 pathway applies and requires REVEL <=0.3. The recorded REVEL score is 0.666, which is above that cutoff; therefore BP4 is not met. No splice prediction is combined with this missense-specific REVEL pathway.
For missense variants: REVEL ≤0.3.
The BRAF VCEP specifies PS2 using a point-based de novo framework, but the available evidence does not document a proband-level de novo observation, confirmed parental testing, or any assignable PS2 points.
The VCEP makes PS4 applicable through point-based scoring for autosomal-dominant RASopathy cases, with full points requiring RASopathy phenotypes and reduced points for minimal phenotype information. The reviewed variant-specific evidence does not provide RASopathy patients, unaffected controls, or a case-control comparison. PMID:26498038 reports p.N581S in a neoplastic-specimen cohort (case M9) with a concomitant PIK3CA variant, but this is a somatic cancer cohort and does not establish the VCEP's RASopathy phenotype evidence. The PS4 source list was checked: PMID:23619274, PMID:24033266, and PMID:24627688 do not mention this BRAF variant, and no ai_classified_papers were present. Therefore no PS4 point total, odds ratio, or confidence interval can be calculated from the available evidence.
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.
The BRAF VCEP specifies PM6 using a point-based presumed-de novo framework, but the available evidence does not document an affected proband, parental testing, or a case-specific point assignment.
The BRAF VCEP requires informative meioses for PP1, but the available evidence contains no affected or unaffected relatives, segregation results, or informative meiosis count.
PP2 is not assessed. The BRAF VCEP requires a missense z score greater than 3.09 in gnomAD. The case evidence provides gnomAD allele observations for the queried variant but does not provide the gene-level missense z score needed to evaluate this rule, and no substitute predictor or generic threshold is permitted.
Missense z score is >3.09 in gnomAD.
The governing BRAF VCEP provides BS2 only as a point-based rule at strong strength for -4 points or supporting strength for -1 point. The available evidence shows zero homozygotes in gnomAD v4.1 and no reported unaffected-individual observations or VCEP point assignment for this variant. A zero homozygote count in a population database does not establish the VCEP's required -1 or -4 point value, so BS2 cannot be evaluated without the VCEP-defined point evidence.
-4 Points. ... -1 Point.
The BRAF VCEP requires one informative meiosis for BS4, but the available evidence contains no non-segregation result or informative meiosis involving an affected individual and relatives.
BP2 is applicable under the BRAF VCEP, but the available case evidence does not document a germline alternative molecular cause of RASopathy in the same gene, its phase relative to NM_004333.6:c.1742A>G, or a phenotype consistent with the expected severity. Therefore no VCEP BP2 point total can be established and no strength can be assigned.
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. ... ≥ (-1) Point.
The VCEP permits BP5 for an alternative molecular cause of a RASopathy in a different gene, or for a phenotype inconsistent with RASopathy that is fully explained by a different causative variant, with points assigned by phenotype consistency and severity. The available case evidence does not document an alternative germline RASopathy cause, a different causative variant explaining the phenotype, or the VCEP's BP5 point value. PMID:26498038 reports a concomitant PIK3CA p.K111E in a somatic cancer specimen, but it does not establish an alternative molecular cause of a germline RASopathy phenotype under this rule. Therefore BP5 cannot be scored.
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.
PVS1 is not applicable under the BRAF VCEP specification. The variant is a missense substitution, NM_004333.6:c.1742A>G (p.Asn581Ser) in exon 15, rather than one of the null variant classes named by the governing criterion, and the VCEP explicitly marks PVS1 as not applicable.
Not applicable.
The BRAF RASopathy VCEP explicitly marks PM3 as not applicable. The framework specifies autosomal dominant inheritance for RASopathy, so the recessive-disorder in-trans observation criterion is not used.
Not applicable.
The BRAF VCEP explicitly marks PP4 as not applicable and directs evaluation through PS4 instead.
Not applicable, see PS4.
The BRAF VCEP explicitly states that PP5 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. Literature and database assertions therefore cannot be used as PP5 under this governing specification.
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 CSPEC specification. The CSPEC entry explicitly marks BS3 as Not applicable, so the available functional result showing increased kinase activity cannot be evaluated under BS3 and no benign functional evidence is assigned.
Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
BP1 is not applicable. The BRAF VCEP states that, despite contraindications for RASopathies, BP1 should be used for truncating variants including nonsense, frameshift, canonical splice-site, initiation-codon, entire-gene, or multi-exon deletions in genes without established loss-of-function correlation. This variant is a missense substitution, BRAF c.1742A>G p.(Asn581Ser), so it is outside the variant types named by the BP1 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 not applicable under the BRAF VCEP specification. The VCEP explicitly marks BP3 as not applicable, despite the general ACMG/AMP description involving in-frame insertions or deletions in a repetitive region without a known function; this variant is instead a missense substitution.
No known benign repetitive areas in RASopathy genes.
The BRAF VCEP explicitly states that BP6 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. A reputable-source benign assertion therefore cannot be applied as BP6 under this governing specification.
This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
BP7 is limited by the VCEP to synonymous variants, intronic positions other than canonical splice sites, or non-coding variants, and requires no predicted splice impact plus lack of high nucleotide conservation. NM_004333.6:c.1742A>G is a missense variant producing p.(Asn581Ser), so it is outside BP7 scope and BP7 is not applicable.
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 (non-Finnish)2 / 1,143,670 | 1.7e-06 |
This variant is present in gnomAD v4.1 (AF= 1.27081e-06; MAF= 0.00013%, 2/1573802 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.74876e-06; MAF= 0.00017%, 2/1143670 alleles, homozygotes = 0); grpmax FAF= 2.9e-07.
Absent from gnomAD v2.1.
Not available in gnomAD-Canada v1.0.
NM_004333.6:c.1742A>G is a missense variant producing p.(Asn581Ser). Under the VCEP missense pathway, PP3 requires REVEL >= 0.7. The recorded REVEL score is 0.666, so the PP3 threshold is not met. The separate splicing-impact clause is not used for this missense-pathway assessment because the VCEP specifically names REVEL for missense variants; no additional computational predictor is combined with it.
For missense variants: REVEL ≥ 0.7. For splicing impact, predicted outcome must match disease mechanism.
NM_004333.6:c.1742A>G is a missense variant producing p.(Asn581Ser), so the VCEP missense BP4 pathway applies and requires REVEL <=0.3. The recorded REVEL score is 0.666, which is above that cutoff; therefore BP4 is not met. No splice prediction is combined with this missense-specific REVEL pathway.
For missense variants: REVEL ≤0.3.
BP7 is limited by the VCEP to synonymous variants, intronic positions other than canonical splice sites, or non-coding variants, and requires no predicted splice impact plus lack of high nucleotide conservation. NM_004333.6:c.1742A>G is a missense variant producing p.(Asn581Ser), so it is outside BP7 scope and BP7 is not applicable.
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 possible splice impact for this variant (max delta score = 0.33). REVEL score = 0.666. BayesDel score = 0.132077.
8 paper records listed. 1 cited in the classification.
Surprisingly the N580SB-RAF mutant is 6-fold more active than WTB-RAF. Although Asn580 is located outside of the P loop and DFG/activation segment, its replacement by Ser is likely to disrupt the inactive conformation of the DFG motif.
Location Results, "Structural Basis for Activating Oncogenic Mutations," paragraph discussing intermediate mutants; Figure 1B
Context Cancer-associated B-RAF mutants were expressed as myc-epitope-tagged proteins in COS cells, and basal B-RAF kinase activity was measured in vitro by direct MEK phosphorylation using physiological ATP and MEK concentrations.
Although substantially less significant, activating mutations in BRAF (q = 1.19 3 103), which is a direct downstream target of KRAS, emerged as an additional predictor of synergy between Chk1- and MK2 inhibitors.
Results, "A Cell-Line-Based Screen Identifies Oncogenic KRAS as a Predictor for Synergistic Interactions between Chk1- and MK2 Inhibitors," paragraph 3
Case M9d melanoma S467L (26 %) PIK3CA/P57S (23 %) N581S (30 %) PIK3CA/K111E (32 %)
Table 1; Table 2; Table 4, case M9
In this work, we define a third class of BRAF mutants consisting of those with low or absent kinase activity. They are RAS-dependent and sensitive to ERK-dependent feedback of RAS. They activate ERK by increasing their binding to RAS and require coexistent mechanisms for RAS activation in the tumour in order to function.
Results/Discussion, paragraph defining class 3 BRAF mutants
Functional evidence that links the variant to disease phenotypes is important to establish causality.
Collecting evidence to determine the likelihood of pathogenicity, paragraph beginning Functional evidence
Herein, we reviewed several publications related with consensus opinions and recommendations for molecular testing in lung cancer, including ALK testing, and propose a guideline recommendation for ALK testing approved by the Cardiopulmonary Pathology Study Group of the Korean Society of Pathologists (Table 1).
Introduction, final paragraph
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= 1.27081e-06; MAF= 0.00013%, 2/1573802 alleles, homozygotes = 0) and has highest observed frequency in the European (non-Finnish) population (AF= 1.74876e-06; MAF= 0.00017%, 2/1143670 alleles, homozygotes = 0); grpmax FAF= 2.9e-07.
Absent from gnomAD v2.1.
Not available in gnomAD-Canada v1.0.
This variant has been reported in ClinVar as Likely pathogenic (2 clinical laboratories). (ClinVarID = 177776)
SpliceAI predicts possible splice impact for this variant (max delta score = 0.33). REVEL score = 0.666. BayesDel score = 0.132077.
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; COSV56058108, n = 48 times).
This variant lies in a statistically significant hotspot.