Classification rationale

PM2 BP4 VUS

NOTCH3 c.5946G>T

NM_000435.2:c.5946G>T (p.Glu1982Asp) is a missense variant in NOTCH3 exon 33 that is absent from all population databases (gnomAD v2.1, v4.1, gnomAD-Canada), meeting PM2 at moderate strength.¹ Multiple computational predictors (REVEL 0.282, BayesDel -0.291, SpliceAI max delta 0.03) uniformly support a benign effect, meeting BP4 at supporting benign strength.² No other ACMG/AMP criteria are met. The variant is absent from ClinVar, has no reported functional data, and is not located in a known mutational hotspot or critical functional domain. Evidence is insufficient for any classification beyond Variant of Uncertain Significance.³ Applying generic ACMG/AMP 2015 combination rules: one moderate pathogenic criterion (PM2) and one supporting benign criterion (BP4) are in conflict. Neither the pathogenic nor benign evidence thresholds are reached, resulting in a classification of Uncertain Significance.⁴

PM2 + BP4 → VUS

1 gnomad_v2 ↗gnomad_v4 ↗gnomad_canada ↗

2 revelbayesdelspliceai ↗

3 clinvar ↗oncokb ↗

4 generic_acmg_combination_rules

Gene diagram · NM_000435.2 · variants mapped to exon structure

NOTCH3 NM_000435.2

Fetching transcript structure from UCSC…

Applied criteria · 2 applied · 21 assessed

Applied · 2

Strength Supporting Moderate Strong Very strong

✓

PM2 moderate Pathogenic

NM_000435.2:c.5946G>T is absent from gnomAD v2.1, v4.1, and gnomAD-Canada. Population frequency is 0%, well below the 0.1% PM2 threshold for a rare missense variant in a gene associated with autosomal dominant disease.

Absent from gnomAD v2.1 (exomes).Absent from gnomAD v4.1 (exomes).Absent from gnomAD-Canada v1.0 (genomes).

gnomad_v2 ↗gnomad_v4 ↗gnomad_canada ↗

✓

BP4 supporting Benign

Multiple lines of computational evidence suggest no deleterious impact on the gene or gene product. REVEL score is 0.282 (below the typical pathogenic threshold of 0.5), BayesDel score is -0.291 (negative direction, consistent with benign), and SpliceAI predicts no splice alteration (max delta score 0.03). Three independent in silico algorithms using different methodologies all support a benign interpretation.

REVEL 0.282 supports benign.BayesDel -0.291 supports benign.SpliceAI max delta 0.03 predicts no splice impact.

spliceai ↗

Assessed · not applied

Pathogenic

PS1 No same amino acid change (p.Glu1982Asp) has been previously reported as pathogenic in ClinVar or the literature.

PS2 No de novo observation with confirmed paternity and maternity has been reported for NM_000435.2:c.5946G>T in any literature source.

PS3 No well-established in vitro or in vivo functional studies have been performed on NM_000435.2:c.5946G>T (p.Glu1982Asp).

PS4 No case-control or cohort prevalence data are available for NM_000435.2:c.5946G>T.

PM1 NM_000435.2:c.5946G>T (p.Glu1982Asp) does not lie in a statistically significant mutational hotspot per cancerhotspots.org.

PM5 No same-residue comparator variant at p.Glu1982 with a different amino acid change has been classified as pathogenic in ClinVar.

PM6 No de novo observation (without confirmed paternity and maternity) has been reported for NM_000435.2:c.5946G>T in any literature source.

PP1 No co-segregation data are available for NM_000435.2:c.5946G>T.

PP2 PP2 requires a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.

PP3 Multiple lines of computational evidence do not support a deleterious effect.

PP4 No patient-specific phenotype or family history information is available for this case.

PP5 NM_000435.2:c.5946G>T is absent from ClinVar.

Benign

BA1 NM_000435.2:c.5946G>T is absent from all population databases (gnomAD v2.1, v4.1, and gnomAD-Canada).

BS1 Population frequency is 0% in gnomAD, below the 0.3% BS1 threshold.

BS2 NM_000435.2:c.5946G>T has not been observed in any healthy adult individual in gnomAD or the literature.

BS3 No well-established in vitro or in vivo functional studies have demonstrated no damaging effect for NM_000435.2:c.5946G>T.

BS4 No non-segregation data are available for NM_000435.2:c.5946G>T.

BP1 BP1 applies when a missense variant occurs in a gene where primarily truncating variants are known to cause disease.

BP2 No observation of NM_000435.2:c.5946G>T in trans with a known pathogenic dominant variant in NOTCH3 has been reported.

BP5 NM_000435.2:c.5946G>T has not been observed in a case where an alternative molecular basis for disease was identified.

BP6 NM_000435.2:c.5946G>T is absent from ClinVar.

N/A · 2 PVS1 · BP7

Research & evidence

Population frequency

gnomAD v4.1

gnomAD v2.1

v4.1

Absent from gnomAD v4.1.

v2.1

Absent from gnomAD v2.1.

🇨🇦 CA

Absent from gnomAD-Canada v1.0.

Allele frequency by ancestry

three datasets · side by side

gnomAD v4.1

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v2.1

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD Canada 🇨🇦

Absent · 0 / ?
0 hom

Not observed in any ancestry group.

gnomAD v4 ↗ gnomAD v2 ↗ gnomAD 🇨🇦 ↗

ClinVar

This variant is absent from ClinVar.

ClinVar ↗

In silico

SpliceAI predicts no significant splice impact for this variant (max delta score = 0.03). REVEL score = 0.282. BayesDel score = -0.291335.

SpliceAI ↗

Functional Unknown Oncogenic Effect

OncoKB did not identify variant-specific reviewed functional evidence for this variant; gene-level curated context is available for reviewer follow-up. NOTCH3 encodes a Type I transmembrane protein of the Notch family. Missense and nonsense mutations in NOTCH3 have been identified in various cancers.

OncoKB ↗

COSMIC

Cancer hotspots

Somatic evidence Not in COSMIC / hotspots

COSMIC

This variant does not lie in a statistically significant hotspot. This variant has not previously been reported in somatic cancers (COSMIC).

Hotspots

This variant does not lie in a statistically significant hotspot.

COSMIC ↗ Hotspots ↗

Sources & reference links

8Sources

ClinVar

gnomAD v2.1

gnomAD v4.1

gnomAD-Canada

SpliceAI

OncoKB

COSMIC

Cancer hotspots