Classification rationale

PM2 VUS

NOTCH3 c.3257A>C

NM_000435.2:c.3257A>C (p.Asp1086Ala) is a missense variant in NOTCH3 exon 20. This variant is absent from large population databases (gnomAD v2.1, v4.1, and gnomAD-Canada; PM2_supporting). No pathogenic or benign criteria beyond PM2_supporting are met. The variant is not cysteine-altering and lies outside known mutational hotspots. No functional, segregation, de novo, case-control, or clinical classification data are available. Based on ACMG/AMP 2015 generic classification rules (PMID:25741868), a single supporting pathogenic criterion (PM2_supporting) is insufficient to reach Likely Pathogenic or any other classification tier. This variant is classified as a Variant of Uncertain Significance (VUS).¹

PM2 → VUS

1 gnomad_v2 ↗gnomad_v4 ↗gnomad_canada ↗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 · 1 applied · 21 assessed

Applied · 1

Strength Supporting Moderate Strong Very strong

✓

PM2 supporting Pathogenic

This variant is absent from large population databases including gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0 (allele frequency 0.0%), meeting PM2 threshold of <0.1% allele frequency.

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 ↗

Assessed · not applied

Pathogenic

PS1 No previously established pathogenic variant at amino acid residue 1086 has been identified in ClinVar or the literature to support PS1.

PS2 No de novo evidence with both maternity and paternity confirmed is available in ClinVar or the literature for this variant.

PS3 No variant-specific functional evidence (in vitro or in vivo) demonstrating a damaging effect is available in the literature, ClinVar, or OncoKB curated sources.

PS4 No case-control or prevalence data comparing affected vs.

PM1 Residue Asp1086 is not located in a statistically significant mutational hotspot (Cancer Hotspots: not significant).

PM6 No de novo evidence without confirmed paternity/maternity is available in ClinVar or the literature for this variant.

PP1 No cosegregation data with disease in multiple affected family members are available for this variant.

PP2 The variant is a non-cysteine missense change (Asp1086Ala) in NOTCH3.

PP3 REVEL score of 0.744 is borderline but not supported by BayesDel (0.07, below deleterious threshold).

PP4 No phenotype or family history data specific to this variant are available to assess whether the patient's presentation is highly specific for NOTCH3-related disease.

PP5 This variant is absent from ClinVar; no reputable source has classified it as pathogenic.

Benign

BA1 This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada (allele frequency 0.0%).

BS1 This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada (allele frequency 0.0%).

BS2 No evidence of observation in a healthy adult individual in trans with a known pathogenic NOTCH3 variant is available.

BS3 No variant-specific functional studies (in vitro or in vivo) demonstrating no damaging effect are available in the literature, ClinVar, or OncoKB.

BS4 No family segregation data are available to assess lack of segregation with disease.

BP1 NOTCH3-associated disease (CADASIL) is primarily caused by missense variants, not truncating variants.

BP2 No evidence of observation in trans with a known dominant pathogenic NOTCH3 variant is available.

BP4 REVEL score of 0.744 is above 0.5, indicating a prediction of deleterious effect.

BP5 No evidence of an alternate molecular basis for disease in a case carrying this variant is available.

BP6 This variant is absent from ClinVar; no reputable source has classified it as benign.

N/A · 3 PVS1 · PM5 · 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 = 0.05). REVEL score = 0.744. BayesDel score = 0.0697656.

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

Re-run this variant?