Variant classification summary

NM_004333.6:c.1741A>T

BRAF · NP_004324.2:p.(Asn581Tyr) · NM_004333.6

GRCh37: chr7:140453987 T>A · GRCh38: chr7:140754187 T>A

Gene: BRAF Transcript: NM_004333.6

Final call

VUS

PM2 supporting PP2 supporting PP3 supporting

Variant details

Gene

BRAF

Transcript

NM_004333.6

Protein

NP_004324.2:p.(Asn581Tyr)

gnomAD AF

ClinVar

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_004333.6:c.1741A>T (p.Asn581Tyr) is a missense variant in exon 14 of BRAF, encoding a substitution at a highly conserved residue within the kinase domain.

2

This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada, meeting PM2 at Supporting strength per the ClinGen RASopathy VCEP v2.3.0 (PM2_Supporting).

3

BRAF exhibits strong constraint against missense variation in gnomAD (missense Z-score >3.09), and missense variants are a common mechanism of RASopathy, meeting PP2 at Supporting strength.

4

The REVEL in silico prediction score is 0.978, exceeding the VCEP threshold of ≥0.7 for PP3 at Supporting strength. SpliceAI predicts no significant splicing impact (max delta = 0.07).

5

N581Y has been observed as a somatic variant in multiple cancer types: colorectal cancer cell line HT55 (Seth 2009, PMID:19474002), melanoma cell line WM3912 (Hutchinson 2015, PMID:26084293), and in a cfDNA NSCLC cohort where it was identified as a novel activating mutation by Ba/F3 assay (Negrao 2020, PMID:32540409). N581Y is catalogued in COSMIC (COSV56062673, n=7). OncoKB classifies it as Likely Oncogenic with Likely Gain-of-function effect.

6

No germline RASopathy probands, de novo occurrences, or family segregation data are available for this variant. VCEP-approved functional assays (BRAF Kinase Activity, MEK/ERK Activation Assays) have no data for N581Y.

7

The N581 residue lies in a statistically significant hotspot, but is outside the VCEP-specified PM1 domains (P-loop AA 459-474 and CR3 activation segment AA 594-627); PM1 is not met.

Final determination: No criteria-combination rule matched the adjudicated criteria in the ClinGen RASopathy Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for BRAF Version 2.3.0 v2.3.0 framework, so the variant remains a Variant of Uncertain Significance pending human review.

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 for BRAF. This is a missense variant (c.1741A>T, p.Asn581Tyr), not a null variant (nonsense, frameshift, or canonical splice site).
PS1	Not met	PS1 requires the same amino acid change as a previously established pathogenic variant. N581Y (p.Asn581Tyr) has been reported in somatic cancer literature (colorectal cancer, melanoma, NSCLC) but has not been established as a germline pathogenic variant in RASopathy. No ClinVar entry classifies N581Y as pathogenic in a germline context.	PMID:19474002 PMID:26084293 PMID:32540409
PS2	Not met	No de novo occurrence of NM_004333.6:c.1741A>T has been reported in a patient with a RASopathy phenotype and confirmed parentage. No proband case data available.
PS3	Not met	The ClinGen RASopathy VCEP-approved functional assays (BRAF Kinase Activity, MEK Activation Assay, ERK Activation Assay) have no data for N581Y in the approved functional studies spreadsheet. The Ba/F3 cell viability assay identifying N581Y as activating (Negrao 2020, PMID:32540409) is not among the VCEP-approved PS3 assays. IMPACT detection of N581Y with predicted modest kinase activity (Hutchinson 2015, PMID:26084293) does not constitute a validated functional assay.	PMID:32540409 PMID:26084293
PS4	Not met	No case-control data demonstrate an increased prevalence of N581Y in RASopathy-affected individuals versus controls. The variant has been reported in somatic cancers (COSMIC n=7) but not in germline RASopathy cohorts.
PS5	N/A	PS5 is not a criterion in the ClinGen RASopathy VCEP v2.3.0 specification for BRAF.
PM1	Not met	Per the RASopathy VCEP, PM1 is applicable only to specific critical and well-established functional domains: exon 6, exon 11, P-loop (AA 459-474), and CR3 activation segment (AA 594-627). N581 (position 581 in exon 14) lies outside these approved domains. Although the residue lies in a statistically significant hotspot, it does not qualify for PM1 under VCEP specifications.
PM2	Met	The variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada v1.0. Per the RASopathy VCEP, absence from gnomAD meets PM2 at Supporting strength (PM2_Supporting).	gnomad_v2 gnomad_v4 gnomad_canada
PM5	Not met	PM5 requires a different pathogenic/likely pathogenic missense change at the same codon (N581). The pm5_candidates search found no qualifying comparator variants with established pathogenicity at codon 581 in BRAF. N581S and N581I have been reported in somatic cancers but none are established as germline pathogenic in the RASopathy context.	pm5_candidates
PM6	Not met	No assumed de novo occurrence of N581Y has been reported. No proband data with unconfirmed parentage available.
PP1	Not met	No co-segregation data available for N581Y in RASopathy families. No informative meioses reported.
PP2	Met	BRAF has a low rate of benign missense variation in gnomAD (missense Z-score well above 3.09) and missense variants are a common mechanism of disease in RASopathies (gain-of-function). PP2 at Supporting strength is appropriate per VCEP specifications.	gnomad_v4
PP3	Met	REVEL score is 0.978, which meets the VCEP threshold of ≥0.7 for PP3 at Supporting strength. SpliceAI max delta score is 0.07, indicating no splicing impact that would contradict the disease mechanism.	revel spliceai
PP4	N/A	PP4 is not applicable per the ClinGen RASopathy VCEP v2.3.0. The VCEP redirects phenotype specificity evaluation to PS4.
PP5	N/A	PP5 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
BA1	Not met	BA1 requires gnomAD filtering allele frequency ≥0.05%. The variant is absent from all gnomAD populations (v2.1, v4.1, Canada).	gnomad_v2 gnomad_v4
BS1	Not met	BS1 requires gnomAD filtering allele frequency ≥0.025%. The variant is absent from all gnomAD populations.	gnomad_v2 gnomad_v4
BS2	Not met	No evidence of N581Y observed in healthy adult individuals. The variant is absent from gnomAD, precluding any observation in presumed healthy controls.
BS3	N/A	BS3 is marked as not applicable in the ClinGen RASopathy VCEP v2.3.0 criteria for BRAF (all strength tiers listed as not applicable).
BS4	Not met	No segregation data available to demonstrate lack of segregation in affected family members. No informative meioses reported.
BP1	N/A	BP1 per the RASopathy VCEP is applicable only to truncating variants (nonsense, frameshift, canonical splice sites, initiation codon, or exon deletions) in genes where LOF is not an established disease mechanism. c.1741A>T is a missense variant and does not qualify.
BP2	Not met	No evidence of an alternative molecular cause of a RASopathy in the same gene (in cis/trans with a pathogenic variant). No points-based scoring evidence available.
BP4	Not met	BP4 requires REVEL ≤0.3 for missense variants per VCEP. The REVEL score for N581Y is 0.978, far exceeding the benign threshold. Multiple computational lines support a deleterious effect.	revel bayesdel spliceai
BP5	Not met	No evidence of an alternative molecular cause of a RASopathy in a different gene, and no phenotype inconsistent with a RASopathy documented.
BP6	N/A	BP6 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.
BP7	N/A	BP7 applies only to synonymous (silent) variants or intronic positions outside canonical splice sites. c.1741A>T is a missense variant resulting in p.Asn581Tyr and does not qualify.

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