Variant classification summary

NM_001354609.1:c.736G>C

BRAF · NP_001341538.1:p.(Ala246Pro) · NM_001354609.1

GRCh37: chr7:140501336 C>G · GRCh38: chr7:140801536 C>G

Gene: BRAF Transcript: NM_001354609.1

Final call

Likely Pathogenic

PS4 supporting PM1 moderate PM2 supporting PM6 supporting PP2 supporting PP3 supporting PP5 supporting

Variant details

Gene

BRAF

Transcript

NM_001354609.1

Protein

NP_001341538.1:p.(Ala246Pro)

gnomAD AF

ClinVar

Pathogenic

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

1

The BRAF c.736G>C (p.Ala246Pro) missense variant is absent from gnomAD population databases (PM2_Supporting).

2

The variant lies within exon 6, a critical functional domain defined by the ClinGen RASopathy VCEP v2.3.0 (PM1_Moderate).

3

The variant has been observed in multiple unrelated probands with cardio-facio-cutaneous syndrome, including the original CFC discovery cohort (Niihori et al. 2006), and is classified as Pathogenic by the ClinGen RASopathy VCEP in ClinVar with submissions from 9 clinical laboratories (PS4_Supporting).

4

A de novo occurrence was reported in a CFC proband with both parents negative, though without molecular confirmation of parentage (PM6_Supporting).

5

In silico predictions support a deleterious effect: REVEL score is 0.928, exceeding the VCEP threshold of 0.7 (PP3).

6

BRAF exhibits a high gnomAD missense z-score (>3.09), indicating constraint against missense variation, and missense variants are a common disease mechanism for RASopathies (PP2).

7

Functional studies by Wen et al. 2013 demonstrated that A246P increases Ras binding affinity relative to wild-type, consistent with a gain-of-function mechanism, though the assay is not in the VCEP-approved functional studies list (PS3 not assessed).

8

Under the ClinGen RASopathy VCEP v2.3.0 combination rules (Rule15: 1 moderate + ≥4 supporting → Likely Pathogenic), the variant meets PM1 (moderate) plus PM2_Supporting, PM6_Supporting, PS4_Supporting, PP2, and PP3 (5 supporting criteria), resulting in a classification of Likely Pathogenic.

Final determination: Rule15 in the ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BRAF Version 2.3.0 v2.3.0 criteria-combination framework is satisfied by the adjudicated criteria, so the variant is classified as Likely Pathogenic.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

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

Criterion	Status	Rationale	Evidence used
PVS1	N/A	PVS1 is not applicable per the ClinGen RASopathy VCEP v2.3.0 specifications for BRAF; this criterion is reserved for null variants (nonsense, frameshift, canonical splice) and is not intended for missense variants in this framework.	cspec
PS1	Not met	PS1 requires the same amino acid change to have been previously established as pathogenic through a different nucleotide change. The c.736G>C (p.Ala246Pro) variant is itself the variant that established A246P as pathogenic; no evidence of a different nucleotide change producing A246P was identified. The CSPEC rule also allows cross-gene analogy with RAF1, but no data on an analogous RAF1 variant was available.
PS2	Not met	PS2 requires de novo occurrence with both maternity and paternity confirmed. Niihori et al. 2006 (PMID:16474404) reported a de novo observation of BRAF c.736G>C (p.Ala246Pro) in a CFC proband with both parents negative, but formal maternity/paternity confirmation was not performed. Without molecular confirmation of parentage, PS2 cannot be applied.	PMID:16474404
PS3	Not assessed	Functional evidence for this variant exists in the literature. Wen et al. 2013 (PMID:24409384) demonstrated that BRAF A246P exhibits increased Ras binding affinity relative to wild-type using optical tweezers single-molecule force measurements. However, this assay (optical tweezers / Ras binding kinetics) is not among the VCEP-approved functional assays (RAS Activation, MEK Activation, ERK Activation, BRAF Kinase Activity, RAF1 Kinase Activity, SHP-2 Phosphatase, LZTR1 Stability). Without testing in a VCEP-approved assay, PS3 cannot be applied under the RASopathy VCEP framework.	PMID:24409384
PS4	Met	This variant has been observed in multiple unrelated probands with cardio-facio-cutaneous (CFC) syndrome / RASopathy phenotype. It was identified among 8 BRAF mutations in 16 individuals from the original CFC cohort (Niihori et al. 2006). Nine independent clinical laboratories have submitted this variant as Pathogenic to ClinVar, and the ClinGen RASopathy VCEP has classified it as Pathogenic (VariationID 13965). The aggregate observation across multiple independent probands supports PS4 at a supporting level.	clinvar PMID:16474404 PMID:24409384
PS5	N/A	PS5 is not defined in the ClinGen RASopathy VCEP v2.3.0 specifications for BRAF. The VCEP framework does not include this criterion.	cspec
PM1	Met	The variant c.736G>C lies within exon 6 (c.712-860), which is one of the critical and well-established functional domains defined by the ClinGen RASopathy VCEP v2.3.0 (along with exon 11, P-loop AA 459-474, and CR3 activation segment AA 594-627). Exon 6 encodes part of the cysteine-rich domain (CRD) critical for BRAF function. The variant is absent from gnomAD, meeting the requirement of no benign variation in the domain.	cspec gnomad_v2 gnomad_v4
PM2	Met	The variant is absent from gnomAD v2.1 and v4.1 population databases. Per the RASopathy VCEP v2.3.0, PM2 is applied at supporting strength when the variant is absent from gnomAD controls.	gnomad_v2 gnomad_v4 cspec
PM5	Not assessed	PM5 requires at least one different [likely] pathogenic residue change at the same codon (Ala246). The PM5 candidate screen identified zero comparator variants at residue 246 in ClinVar or other sources. The CSPEC also allows cross-gene analogy with RAF1, but no data on RAF1 residue 246 comparators was available. Without a comparator variant, PM5 cannot be applied.
PM6	Met	Niihori et al. 2006 (PMID:16474404) reported a de novo occurrence of BRAF c.736G>C (p.Ala246Pro) in a proband with CFC syndrome where both parents tested negative for the variant. However, formal maternity/paternity confirmation was not performed, satisfying PM6 (assumed de novo without confirmed parentage) at supporting level (0.5 points) per the RASopathy VCEP point-based scoring system.	PMID:16474404
PP1	Not assessed	No cosegregation data were identified for this variant. Most RASopathy cases with BRAF mutations are sporadic, and multigenerational families with this variant are rare. PP1 requires at least 3 informative meioses for supporting level.
PP2	Met	BRAF has a high gnomAD missense z-score (well above 3.09), indicating strong constraint against missense variation in the general population. Missense variants are a common mechanism of disease for BRAF (gain-of-function in RASopathies). Per the RASopathy VCEP v2.3.0, PP2 is met at supporting strength when the gene's missense z-score exceeds 3.09 in gnomAD.	gnomad_v2 cspec
PP3	Met	The REVEL score for this missense variant is 0.928, which exceeds the threshold of ≥0.7 specified by the RASopathy VCEP v2.3.0 for PP3 at supporting strength. Multiple in silico tools support a deleterious effect on protein function.	revel cspec
PP4	N/A	PP4 is not applicable per the ClinGen RASopathy VCEP v2.3.0. The VCEP directs users to use PS4 instead for phenotype specificity.	cspec
PP5	Met	Expert panel ClinGen RASopathy Variant Curation Expert Panel classified as Pathogenic.	cspec clinvar
BA1	Not met	BA1 requires a gnomAD filtering allele frequency ≥0.05%. This variant is absent from both gnomAD v2.1 and v4.1, so BA1 is not met.	gnomad_v2 gnomad_v4
BS1	Not met	BS1 requires a gnomAD filtering allele frequency ≥0.025% per the RASopathy VCEP v2.3.0. This variant is absent from both gnomAD v2.1 and v4.1, so BS1 is not met.	gnomad_v2 gnomad_v4 cspec
BS2	Not assessed	BS2 requires observation of the variant in a healthy adult individual. No evidence of this variant being found in healthy adults was identified. Given that this variant is associated with an early-onset developmental disorder and is absent from gnomAD, BS2 is unlikely to be met.
BS3	N/A	BS3 is not applicable per the ClinGen RASopathy VCEP v2.3.0 specifications for BRAF.	cspec
BS4	Not assessed	BS4 requires lack of segregation in affected family members. No nonsegregation data were identified. Given that this variant is well-established as pathogenic in CFC syndrome, nonsegregation evidence is unlikely to exist.
BP1	N/A	BP1 applies only to truncating variants (nonsense, frameshift, canonical splice) in genes where the disease mechanism is primarily gain-of-function missense. This variant is a missense substitution (c.736G>C, p.Ala246Pro) and therefore BP1 is not applicable.	cspec
BP2	Not assessed	BP2 requires observation of the variant in trans with a pathogenic variant in the same gene, or an alternative molecular cause of a RASopathy. No evidence of the variant occurring in trans with another BRAF pathogenic variant in an unaffected individual was identified.
BP3	N/A	SKIPPED per user instruction: trivially not applicable. BP3 is also listed as not applicable per the ClinGen RASopathy VCEP v2.3.0.
BP4	Not met	BP4 requires a REVEL score ≤0.3 for missense variants per the RASopathy VCEP v2.3.0. The REVEL score for this variant is 0.928, which is strongly in the pathogenic range and does not meet the BP4 threshold.	revel cspec
BP5	Not assessed	BP5 requires an alternative molecular cause of a RASopathy in a different gene, with phenotype consistent with expected severity. No evidence of an alternative molecular cause was identified for probands carrying this variant.
BP6	N/A	BP6 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee per the RASopathy VCEP v2.3.0.	cspec
BP7	N/A	BP7 applies only to synonymous (silent) variants with no predicted splice impact. This variant is a missense substitution (c.736G>C, p.Ala246Pro) and therefore BP7 is not applicable.
PM3	N/A	SKIPPED per user instruction: trivially not applicable. PM3 is also listed as not applicable per the ClinGen RASopathy VCEP v2.3.0 (recessive disorder criterion; RASopathies are autosomal dominant).
PM4	N/A	SKIPPED per user instruction: trivially not applicable. This variant is a single nucleotide substitution, not an in-frame deletion/insertion or stop-loss variant.

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