NM_004333.5:c.1661T>C

BRAF · NP_004324.2:p.(Ile554Thr) · NM_004333.5

GRCh37: chr7:140476745 A>G · GRCh38: chr7:140776945 A>G

Gene: BRAF Transcript: NM_004333.5

Final call

VUS

PP2 supporting PP3 supporting

Variant details

Gene

BRAF

Transcript

NM_004333.5

Protein

NP_004324.2:p.(Ile554Thr)

gnomAD AF

1.4254920116667224e-05 (v4.1)

ClinVar

Likely benign

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_004333.5:c.1661T>C (p.Ile554Thr) is a missense variant in BRAF exon 13, located in the kinase domain between the P-loop and CR3 activation segment.

This variant is present at extremely low frequency in population databases: gnomAD v2.1 AF=3.98e-6 (1/251,340 alleles) and v4.1 AF=1.43e-5 (23/1,613,478 alleles), with no homozygotes observed.

ClinVar reports conflicting interpretations: Likely benign (2 clinical laboratories) and Uncertain significance (1 clinical laboratory), with no expert panel review (ClinVar Variation ID 239870).

PP3 (supporting): REVEL score of 0.885 exceeds the VCEP threshold of ≥0.7 for pathogenic computational prediction. SpliceAI predicts no splicing impact (max delta 0.00).

PP2 (supporting): BRAF has a high missense constraint Z-score in gnomAD (>3.09), indicating a low rate of benign missense variation consistent with a gene where missense variants are a common disease mechanism.

PM1 is not met because amino acid 554 lies outside the VCEP-specified domains (exon 6, exon 11, P-loop AA 459-474, CR3 activation segment AA 594-627).

PM2 is not met because the RASopathy VCEP requires absence from gnomAD controls, and this variant is observed in both gnomAD v2.1 and v4.1.

PM5 is not met because no other [likely] pathogenic missense variant at codon 554 was identified in ClinVar or the VCEP supplementary materials.

BS1 is not met because the allele frequency (max 9.92e-5) is below the VCEP threshold of ≥0.025%.

No benign criteria are met. Two pathogenic supporting criteria (PP2, PP3) are met. Under the RASopathy VCEP v2.3.0 combination rules, this does not reach the threshold for Likely Pathogenic (which requires at minimum 1 moderate + 4 supporting, or 1 strong + 1 moderate, or comparable combinations).

Insufficient evidence exists to classify this variant as either pathogenic or benign. With only two supporting-level pathogenic criteria and no benign criteria, this variant is classified as a Variant of Uncertain Significance (VUS).

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 Expert Panel specifications for BRAF (VCEP v2.3.0). Additionally, NM_004333.5:c.1661T>C is a missense variant (p.Ile554Thr), not a null variant.	cspec
PS1	Not met	No previously established pathogenic variant causing the same amino acid change (p.Ile554Thr) via a different nucleotide change was identified in ClinVar, the VCEP functional studies spreadsheet, or the literature reviewed.	clinvar
PS2	Not met	No de novo occurrence reports were identified for NM_004333.5:c.1661T>C in the ClinVar submissions, literature abstracts, or full-text publications reviewed.	clinvar
PS3	Not met	No variant-specific functional data were identified for p.Ile554Thr in the VCEP-approved functional studies spreadsheet (BRAF Kinase Activity, MEK/ERK Activation assays) or in the reviewed literature. OncoKB reports unknown oncogenic effect with no variant-specific functional evidence.	vcep_svi_rasopathy_vcep_v2_approved_functional_studies oncokb
PS4	Not met	No case-control or proband count data are available for NM_004333.5:c.1661T>C. The VCEP PS4 point-based system requires proband counts that are not available from the current evidence.	clinvar
PS5	N/A	Not in the assess list for this adjudication.
PM1	Not met	Amino acid position 554 lies outside the VCEP-specified critical functional domains for PM1 application. The RASopathy VCEP restricts PM1 to exon 6, exon 11, the P-loop (AA 459-474), and the CR3 activation segment (AA 594-627). Position 554 is in exon 13 between these domains.	cspec
PM2	Not met	The RASopathy VCEP requires the variant to be absent from gnomAD controls for PM2 application. NM_004333.5:c.1661T>C is present in gnomAD v2.1 (1/251,340 alleles, AF=3.98e-6) and v4.1 (23/1,613,478 alleles, AF=1.43e-5), and therefore does not meet the absence requirement.	gnomad_v2 gnomad_v4
PM5	Not met	PM5 requires at least one [likely] pathogenic residue change at the same codon (position 554). No such comparator variant was identified in ClinVar, the VCEP RAF-Alignment spreadsheet, or the literature reviewed. The pm5_candidates automated search returned no candidates.	pm5_candidates clinvar
PM6	Not met	No de novo observations with confirmed or unconfirmed parentage were identified for NM_004333.5:c.1661T>C in the reviewed evidence.	clinvar
PP1	Not met	No co-segregation data are available for NM_004333.5:c.1661T>C. The VCEP requires ≥3 informative meioses for supporting-level application.
PP2	Met	BRAF has a high missense constraint in gnomAD with a missense Z-score well above 3.09, meeting the VCEP threshold. BRAF has a low rate of benign missense variation and missense variants are a common mechanism of disease in RASopathies.	cspec
PP3	Met	REVEL score of 0.885 meets the VCEP threshold of ≥0.7 for missense variants. SpliceAI predicts no splicing impact (max delta 0.00), and the variant is not in a splicing-relevant context. BayesDel score is 0.301 (intermediate).	revel spliceai bayesdel
PP4	N/A	The RASopathy VCEP specification states PP4 is not applicable; see PS4 instead.	cspec
PP5	N/A	PP5 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee for the RASopathy VCEP.	cspec
BA1	Not met	The gnomAD filtering allele frequency is well below the VCEP BA1 threshold of ≥0.05%. The maximum observed population frequency is 9.92e-5 (Ashkenazi Jewish, v2.1) and the grpmax FAF in v4.1 is 8.78e-6.	gnomad_v2 gnomad_v4
BS1	Not met	The gnomAD filtering allele frequency is below the VCEP BS1 threshold of ≥0.025%. The total AF in v2.1 is 3.98e-6 and in v4.1 is 1.43e-5.	gnomad_v2 gnomad_v4
BS2	Not met	No data are available on observation of NM_004333.5:c.1661T>C in healthy adults without a RASopathy phenotype beyond gnomAD population data. The VCEP point-based BS2 system requires documented healthy adult observations which are not available.
BS3	N/A	BS3 is not applicable per the ClinGen RASopathy Expert Panel specifications for BRAF (VCEP v2.3.0).	cspec
BS4	Not met	No segregation data are available to assess lack of segregation in affected family members. The VCEP requires only one informative meiosis for strong-level application, but no such data exist.
BP1	Not met	The RASopathy VCEP specifies BP1 for truncating variants (nonsense, frameshift, canonical splice sites, initiation codon, whole-gene/multi-exon deletions) in genes where LOF is not an established disease mechanism. NM_004333.5:c.1661T>C is a missense variant (p.Ile554Thr) and does not meet the variant-type requirement.	cspec
BP2	Not met	No evidence of an alternative molecular cause of a RASopathy in the same gene was identified. The VCEP BP2 point-based system requires documented alternative molecular diagnoses which are not available.
BP4	Not met	REVEL score of 0.885 exceeds the VCEP BP4 threshold of ≤0.3, indicating computational evidence does not support a benign interpretation. SpliceAI predicts no splicing impact (max delta 0.00).	revel spliceai
BP5	Not met	No evidence of an alternative molecular cause of a RASopathy in a different gene was identified. The VCEP BP5 point-based system requires documented alternative diagnoses in other genes which are not available.
BP6	N/A	BP6 is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee for the RASopathy VCEP.	cspec
BP7	N/A	NM_004333.5:c.1661T>C is a missense variant (p.Ile554Thr), not a synonymous variant. BP7 applies only to synonymous variants and intronic/non-coding variants outside canonical splice sites.
BP3	N/A	Skipped per adjudication instructions.
PM3	N/A	Skipped per adjudication instructions; VCEP also declares PM3 not applicable for autosomal dominant RASopathy.
PM4	N/A	Skipped per adjudication instructions.

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