NM_000435.2:c.2299C>A

NOTCH3 · NP_000426.2:p.(Arg767Ser) · NM_000435.2

GRCh37: chr19:15295828 G>T · GRCh38: chr19:15185017 G>T

Gene: NOTCH3 Transcript: NM_000435.2

Final call

VUS

PM2 supporting BP4 supporting benign

Variant details

Gene

NOTCH3

Transcript

NM_000435.2

Protein

NP_000426.2:p.(Arg767Ser)

gnomAD AF

6.779302518239714e-07 (v4.1)

ClinVar

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_000435.2:c.2299C>A (p.Arg767Ser) is a missense variant in NOTCH3 with extremely low population frequency (1/1,475,078 alleles in gnomAD v4.1, AF = 6.78e-7), meeting PM2 at supporting level.

Multiple in silico predictors (REVEL 0.073, BayesDel −0.352, SpliceAI max delta 0.10) concordantly predict a benign effect, satisfying BP4 at supporting level.

The variant is absent from ClinVar, COSMIC, and the published literature. No functional studies, segregation data, de novo reports, or same-residue pathogenic comparators are available.

With one supporting pathogenic criterion (PM2) and one supporting benign criterion (BP4), the evidence is balanced and insufficient to classify this variant as either likely pathogenic or likely benign per generic ACMG/AMP 2015 combination rules (PMID:25741868). The variant is classified as a Variant of Uncertain Significance (VUS).

Final determination: Generic ACMG/AMP 2015 fallback rules do not meet benign, likely benign, likely pathogenic, or pathogenic combination thresholds, so the variant is classified as Variant of Uncertain Significance.

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	NM_000435.2:c.2299C>A is a missense variant (p.Arg767Ser); it does not fall into the default generic PVS1 null-variant buckets of nonsense, frameshift, or canonical ±1,2 splice consensus variants. PVS1 is reserved for loss-of-function null variants.	pvs1_variant_assessment pvs1_gene_context
PS1	Not met	No alternative nucleotide change at NOTCH3 c.2299 has been reported as pathogenic in ClinVar or the literature. This variant is entirely absent from ClinVar, precluding any same-nucleotide pathogenic comparator.	clinvar
PS2	Not met	No de novo observation with confirmed maternity and paternity has been reported for NM_000435.2:c.2299C>A.
PS3	Not met	No functional or experimental studies testing NM_000435.2:c.2299C>A (p.Arg767Ser) were identified. OncoKB reports no variant-specific functional evidence. The literature pass returned zero PMIDs for this variant. In the absence of any direct experimental or systematic-range functional characterization, PS3 cannot be applied.	oncokb
PS4	Not met	No case-control studies demonstrating statistically higher prevalence of NM_000435.2:c.2299C>A in affected individuals compared to controls are available.
PS5	Not met	No family studies demonstrating segregation of NM_000435.2:c.2299C>A with disease have been reported.
PM1	Not met	Residue Arg767 is not located in a statistically significant mutational hotspot per cancerhotspots.org. No variant-specific domain-level functional evidence was identified to support PM1. While this position lies within the NOTCH3 extracellular EGF-like repeat region, the variant is not cysteine-altering and there is no specific literature evidence demonstrating that p.Arg767Ser disrupts a critical functional domain.	oncokb
PM2	Met	NM_000435.2:c.2299C>A is extremely rare in population databases. The variant is present at a single allele in gnomAD v4.1 (AF = 6.78 × 10⁻⁷; 1/1,475,078 alleles, 0 homozygotes), with its highest subpopulation frequency in European (non-Finnish) at 9.19 × 10⁻⁷ (1/1,088,162). It is absent from gnomAD v2.1 and gnomAD-Canada. This frequency is well below the 0.1% (0.001) PM2 threshold for non-VCEP adjudication.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	Not met	No pathogenic missense variant at the same amino acid residue (Arg767) has been identified in ClinVar. The PM5 candidate search returned zero same-residue comparator variants.	pm5_candidates clinvar
PM6	Not met	No de novo observation (maternity and paternity unconfirmed) has been reported for NM_000435.2:c.2299C>A.
PP1	Not met	No cosegregation data with disease in multiple affected family members is available for NM_000435.2:c.2299C>A.
PP2	Not assessed	PP2 requires demonstration that the gene has a low rate of benign missense variation and that missense variants are a common disease mechanism. While NOTCH3 CADASIL is predominantly caused by missense variants (cysteine-altering), no gene-level missense constraint metrics (e.g., Z-score, missense o/e ratio) are available in the evidence brief to support application of PP2.
PP3	Not met	Multiple in silico tools predict a benign effect for NM_000435.2:c.2299C>A (p.Arg767Ser). REVEL score is 0.073 (well below the 0.5 threshold for pathogenicity), BayesDel score is −0.352 (negative, indicating benign), and SpliceAI predicts no splice impact (max delta = 0.10). No computational evidence supports a deleterious effect.	revel bayesdel spliceai
PP4	Not met	No detailed patient phenotype or family history data are available to assess whether the phenotype is highly specific for NOTCH3-related disease.
PP5	Not met	NM_000435.2:c.2299C>A is absent from ClinVar and has not been reported as pathogenic by any reputable clinical diagnostic laboratory or expert panel.	clinvar
BA1	Not met	The allele frequency in gnomAD v4.1 is 6.78 × 10⁻⁷ (0.00007%), far below the 1% BA1 threshold. This variant is not a common polymorphism.	gnomad_v4
BS1	Not met	The allele frequency in gnomAD v4.1 (AF = 6.78 × 10⁻⁷, highest in NFE at 9.19 × 10⁻⁷) is far below the 0.3% non-VCEP BS1 threshold. This rare frequency does not exceed what would be expected for a NOTCH3 disease-causing variant.	gnomad_v4
BS2	Not met	While a single allele is observed in gnomAD v4.1, there is no confirmatory evidence that this carrier is an unaffected healthy adult, and a single observation is insufficient to meet BS2 requirements for a dominant disorder.	gnomad_v4
BS3	Not met	No functional or experimental studies demonstrating that NM_000435.2:c.2299C>A (p.Arg767Ser) has no damaging effect on protein function or splicing have been identified. The in silico benign predictions (REVEL 0.073, BayesDel −0.352) are computational, not experimental, and do not satisfy BS3's requirement for functional assay evidence.	oncokb revel bayesdel
BS4	Not met	No family studies demonstrating lack of segregation of NM_000435.2:c.2299C>A with disease are available.
BP1	Not met	BP1 applies when a missense variant is found in a gene where only truncating variants cause disease. For NOTCH3, CADASIL is predominantly caused by missense variants (cysteine-altering) in the EGF-like repeat domains, not exclusively by truncating variants. Additionally, the PVS1 gene context confirms that NOTCH3 loss-of-function is a supported disease mechanism, not the sole mechanism.	pvs1_gene_context
BP2	Not met	No data are available to determine whether NM_000435.2:c.2299C>A has been observed in trans with a known pathogenic NOTCH3 variant in a case of recessive disease.
BP3	N/A	NM_000435.2:c.2299C>A is a missense substitution; BP3 applies only to in-frame insertions or deletions in repetitive regions.
BP4	Met	Multiple independent in silico tools predict a benign effect for NM_000435.2:c.2299C>A (p.Arg767Ser). REVEL score is 0.073 (well below pathogenic threshold), BayesDel score is −0.352 (negative, consistent with benign), and SpliceAI predicts no splice-altering effect (max delta = 0.10). Three concordant benign predictions from independent algorithms satisfy BP4 at supporting benign level.	revel bayesdel spliceai
BP5	Not met	No data are available demonstrating that NM_000435.2:c.2299C>A is found in a case with an alternate established molecular basis for disease.
BP6	Not met	NM_000435.2:c.2299C>A is absent from ClinVar and has not been reported as benign by any reputable clinical diagnostic laboratory or expert panel.	clinvar
BP7	N/A	NM_000435.2:c.2299C>A is a missense variant (p.Arg767Ser), not a synonymous (silent) variant. BP7 is reserved for synonymous variants with no predicted splice impact.
PM3	N/A	NOTCH3-related CADASIL is an autosomal dominant disorder; PM3 applies only to recessive disorders where a variant is detected in trans with a pathogenic variant.
PM4	N/A	NM_000435.2:c.2299C>A is a single-nucleotide missense substitution; PM4 applies only to in-frame deletions/insertions, stop-loss variants, or initiation codon changes.

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