Variant classification summary

NM_000435.2:c.4688C>T

NOTCH3 · NP_000426.2:p.(Pro1563Leu) · NM_000435.2

GRCh37: chr19:15284927 G>A · GRCh38: chr19:15174116 G>A

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.(Pro1563Leu)

gnomAD AF

6.240872723641674e-07 (v4.1)

ClinVar

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000435.2:c.4688C>T (p.Pro1563Leu) is a missense variant in NOTCH3. It is absent from gnomAD v2.1 (0/242,354 alleles) and ultra-rare in gnomAD v4.1 (1/1,602,340 alleles; AF=6.24e-7), meeting PM2_Supporting.

2

Multiple computational predictors indicate a benign impact: REVEL score 0.08, BayesDel score -0.37, and SpliceAI max delta 0.00. This meets BP4 (supporting benign).

3

The variant is a missense substitution (not a null variant), so PVS1 is not applicable. It alters a non-cysteine residue (Pro1563) in EGF-like repeat 30, where the majority of established pathogenic NOTCH3 variants affect conserved cysteine residues; PM1 is not met.

4

The variant is absent from ClinVar and has no reported functional studies, de novo observations, case-control data, or family segregation data. OncoKB classifies it as 'Unknown Oncogenic Effect.'

5

In the generic ACMG/AMP 2015 framework, the current evidence yields PM2_Supporting and BP4_Supporting_Benign. These criteria are insufficient for a definitive classification; the variant remains a Variant of Uncertain Significance (VUS) pending additional clinical and functional data.

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.4688C>T is a missense variant (p.Pro1563Leu), not a null variant (nonsense, frameshift, or canonical splice site). PVS1 is not applicable to missense variants in the generic ACMG/AMP framework (PMC6185798).	pvs1_variant_assessment pvs1_generic_framework
PS1	N/A	No evidence of another nucleotide change at c.4688 producing the same amino acid change (p.Pro1563Leu) that has been previously established as pathogenic.	clinvar
PS2	Not met	No de novo observation with confirmed maternity and paternity has been identified for this variant.
PS3	Not assessed	No well-established functional studies demonstrating a damaging effect for the exact variant NM_000435.2:c.4688C>T (p.Pro1563Leu) were identified.
PS4	Not assessed	No case-control or cohort data demonstrating statistically significant enrichment of this variant in affected individuals versus controls were identified.
PS5	N/A	PS5 is not a standard criterion in the ACMG/AMP 2015 generic framework. No applicable VCEP-specific PS5 definition exists for NOTCH3.	generic_acmg_combination_rules
PM1	Not met	Pro1563 resides in EGF-like repeat 30 within the extracellular domain of NOTCH3, a region known to harbor CADASIL-causing mutations. However, established pathogenic NOTCH3 missense variants almost exclusively affect conserved cysteine residues within EGF repeats. Pro1563 is not a cysteine residue, and no evidence was identified to establish codon 1563 as a mutational hotspot or critical functional domain for which benign variation is absent.
PM2	Met	NM_000435.2:c.4688C>T is absent from gnomAD v2.1 (0/242,354 alleles) and observed at an ultra-rare frequency in gnomAD v4.1 (1/1,602,340 alleles; AF=6.24e-7), well below the 0.1% threshold for PM2_Supporting in the generic ACMG/AMP framework.	gnomad_v2 gnomad_v4
PM5	N/A	No known pathogenic missense variants at codon 1563 of NOTCH3 (i.e., different amino acid change at Pro1563) were identified to serve as PM5 comparators. The automated PM5 candidate collection found zero same-residue candidates.	pm5_candidates clinvar
PM6	Not met	No de novo observation of this variant without confirmation of maternity and paternity was identified.
PP1	Not assessed	No family cosegregation data were identified for this variant.
PP2	Not met	NOTCH3 is not a gene with a low rate of benign missense variation. While missense variants are a common mechanism for CADASIL (particularly cysteine-altering substitutions in EGF repeats), the gene tolerates substantial missense variation in the general population and does not meet the PP2 requirement of having a low rate of benign missense variation.	gnomad_v2
PP3	Not met	Multiple lines of computational evidence do not support a deleterious effect. REVEL score is 0.08 (below the 0.5 clinical threshold favoring pathogenicity). BayesDel score is -0.37 (below the 0.27 pathogenic threshold, consistent with a benign prediction). SpliceAI predicts no splicing impact (max delta = 0.00).	revel bayesdel spliceai
PP4	Not assessed	No patient phenotype or family history data are available for this case.
PP5	Not met	No reputable source (ClinVar, published literature) reports this variant as pathogenic with accessible evidence. The variant is absent from ClinVar, and OncoKB classifies it as 'Unknown Oncogenic Effect.'	clinvar oncokb
BA1	Not met	The variant has an allele frequency of 6.24e-7 in gnomAD v4.1 (1/1,602,340 alleles), far below the BA1 threshold of >1% (or >5% in some frameworks).	gnomad_v4
BS1	Not met	The variant allele frequency (6.24e-7 in gnomAD v4.1; absent in v2.1) is far below the 0.3% threshold for BS1. CADASIL is a rare dominant disorder, and an allele frequency of this magnitude does not exceed what is expected for disease prevalence.	gnomad_v2 gnomad_v4
BS2	Not assessed	A single allele is observed in gnomAD v4.1, but CADASIL has adult-onset with variable expressivity and the phenotype of the gnomAD individual is unknown. The ultra-low frequency (1/1,602,340) does not provide sufficient confidence that the individual is a healthy adult unaffected by a NOTCH3-related disorder.	gnomad_v4
BS3	Not assessed	No well-established functional studies demonstrating no damaging effect for the exact variant NM_000435.2:c.4688C>T were identified.
BS4	Not assessed	No family segregation data (either positive or negative) were identified for this variant.
BP1	Not met	CADASIL is primarily caused by missense variants in NOTCH3, not exclusively by truncating variants. BP1 applies only when disease is caused exclusively by truncating variants in the gene.	pvs1_gene_context
BP2	Not assessed	No data on co-occurrence of this variant in trans with a known pathogenic NOTCH3 variant were identified.
BP4	Met	Multiple lines of computational evidence support a benign impact. REVEL score is 0.08 (below 0.5 threshold), BayesDel score is -0.37 (below pathogenic threshold), and SpliceAI predicts no splicing impact (max delta = 0.00). All three in silico predictors are consistent with a benign or neutral effect.	revel bayesdel spliceai
BP5	Not assessed	No data are available on whether this variant has been observed in a case where an alternate molecular basis for disease has been identified.
BP6	Not met	No reputable source reports this variant as benign. The variant is absent from ClinVar.	clinvar
BP7	N/A	NM_000435.2:c.4688C>T is a missense variant (p.Pro1563Leu), not a synonymous/silent variant. BP7 applies only to silent variants with no predicted splice impact.	spliceai

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