NM_017617.5:c.3225G>A

NOTCH1 · NP_060087.3:p.(Trp1075Ter) · NM_017617.5

GRCh37: chr9:139402784 C>T · GRCh38: chr9:136508332 C>T

Gene: NOTCH1 Transcript: NM_017617.5

Final call

VUS

PVS1 very strong PM2 supporting

Variant details

Gene

NOTCH1

Transcript

NM_017617.5

Protein

NP_060087.3:p.(Trp1075Ter)

gnomAD AF

ClinVar

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_017617.5:c.3225G>A (p.Trp1075Ter) is a nonsense variant in NOTCH1 predicted to undergo nonsense-mediated decay, meeting PVS1 at very strong strength.

The variant is absent from all gnomAD population databases (v2.1, v4.1, gnomAD-Canada), meeting PM2 at supporting strength.

No variant-specific functional studies, de novo observations, case-control data, or cosegregation evidence are available. ClinVar contains no entry for this variant.

Five publications identified via OncoKB discuss NOTCH1 loss-of-function in squamous cell carcinoma at the gene level, but none mention NM_017617.5:c.3225G>A specifically. COSMIC reports two somatic occurrences (COSV53100468) without functional characterization.

The met criteria are PVS1 (very_strong) and PM2 (supporting). Under generic ACMG/AMP 2015 combination rules, one very-strong criterion plus one supporting criterion does not meet the threshold for Pathogenic (requires 1 Strong, 2 Moderate, or 1 Moderate + 1 Supporting in addition to PVS1) or Likely Pathogenic (requires at least PVS1 + 1 Moderate). The variant is classified as 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	Met	Nonsense variant NM_017617.5:c.3225G>A (p.Trp1075Ter) in exon 20/34 of NOTCH1 creates a premature termination codon predicted to trigger nonsense-mediated decay (NMD). Under PMC6185798, nonsense variants in genes where loss of function is an established disease mechanism are assigned PVS1 at full strength. NOTCH1 loss of function is supported for germline disease (Aortic Valve Disease 1, Adams-Oliver syndrome, congenital heart defects). The variant is absent from gnomAD and the stop codon is >50bp upstream of the last exon-exon junction, favoring NMD. The truncation removes all intracellular signaling domains including ANK repeats, TAD, and PEST.	pvs1_generic_framework pvs1_gene_context pvs1_variant_assessment gnomad_v2 gnomad_v4
PS1	Not met	No established pathogenic variant at this nucleotide position with the same predicted protein change (p.Trp1075Ter). ClinVar is absent for this variant, and no literature reports a pathogenic change at this codon. A different nucleotide substitution producing the same premature stop (e.g., c.3224G>A) would qualify, but none has been reported.	clinvar
PS2	Not assessed	No de novo testing data (maternity and paternity confirmation) provided for this case.
PS3	Not met	No variant-specific functional assay data exist for NM_017617.5:c.3225G>A (p.Trp1075Ter). Five publications identified via OncoKB discuss NOTCH1 loss-of-function in squamous cell carcinoma at the gene level, but none directly tested this nonsense variant. No systematic range characterization (tiling screen, saturation mutagenesis) includes position 1075. COSMIC reports 2 somatic occurrences but without functional characterization. The OncoKB 'Likely Oncogenic' classification reflects somatic tumor-suppressive context, not germline functional evidence.	oncokb
PS4	Not assessed	No case-control comparison data or disease-specific prevalence statistics provided for this variant in the phenotype associated with NOTCH1 germline disease.
PS5	Not met	No reputable germline source reports this variant as pathogenic. The variant is absent from ClinVar. OncoKB classifies it as 'Likely Oncogenic' in a somatic context, which does not satisfy PS5 requirements for a germline classification. COSMIC presence (2 somatic occurrences) does not constitute a reputable germline pathogenicity assertion.	clinvar oncokb
PM1	Not met	The nonsense variant at position 1075 lies within the EGF repeat region of NOTCH1, which is functionally critical for ligand binding (Delta/Serrate). However, this is a nonsense variant predicted to undergo NMD, resulting in no protein expression. The PVS1 criterion already fully captures the deleterious effect of complete protein loss. Applying PM1 for a domain that is never expressed would constitute double-counting of evidence that is not independent. The residue is not a statistically significant hotspot at cancerhotspots.org. PM1 for truncating variants is more appropriately applied when the variant escapes NMD and produces a truncated protein that specifically removes a functionally characterized domain (e.g., PEST domain truncations in T-ALL).	pm5_candidates PMID:22006338 PMID:1657403
PM2	Met	The variant is absent from all gnomAD population databases (v2.1 exomes, v4.1 exomes, and gnomAD-Canada v1.0 genomes), meeting the PM2 threshold of allele frequency <0.1% in population databases under generic ACMG rules.	gnomad_v2 gnomad_v4 gnomad_canada
PM5	N/A	PM5 applies to missense variants at an amino acid residue where a different pathogenic missense change has been observed. This variant is a nonsense substitution (p.Trp1075Ter), not a missense change. No same-residue missense comparator candidates were identified.	pm5_candidates
PM6	Not assessed	No de novo observation data (with or without confirmation of paternity/maternity) provided for this variant.
PP1	Not assessed	No family cosegregation data provided for this variant.
PP2	N/A	PP2 applies to missense variants in genes with a low rate of benign missense variation. This is a nonsense variant, not a missense change.
PP3	Not met	SpliceAI predicts no splice impact (max delta score = 0.00). BayesDel score of 0.66 is designed for missense variant deleteriousness prediction and is not informative for a nonsense change. REVEL and HCI prior scores are unavailable. The deleterious effect of this nonsense variant is already captured by PVS1 (which incorporates the anticipated null effect) and should not be independently credited through PP3 to avoid double-counting.	spliceai bayesdel
PP4	Not assessed	No patient phenotype data provided to evaluate whether the clinical presentation is highly specific for NOTCH1-related disease (AOVD1, Adams-Oliver syndrome, congenital heart defects).
PP5	Not met	The variant is absent from ClinVar. No expert panel or reputable germline source has classified this variant. PP5 requires a reputable source asserting pathogenicity, which is not available.	clinvar
BA1	Not met	The variant is absent from gnomAD v2.1 and v4.1. An allele frequency >1% (BA1 threshold) is not observed.	gnomad_v2 gnomad_v4
BS1	Not met	The variant is absent from gnomAD. An allele frequency >0.3% (BS1 threshold for non-VCEP) is not observed.	gnomad_v2 gnomad_v4
BS2	Not met	The variant is absent from gnomAD. No observation in healthy adults at significant frequency is available. This variant has not been observed in any population database.	gnomad_v2 gnomad_v4 gnomad_canada
BS3	Not met	No well-established functional studies demonstrate a neutral effect for this variant. The available literature on NOTCH1 loss-of-function mutations (PMID:22006338, PMID:21798897) indicates that truncating NOTCH1 variants abrogate receptor signaling in squamous cell carcinomas, consistent with a tumor-suppressive loss-of-function mechanism. This evidence is directionally consistent with pathogenicity, not benign effect.	PMID:22006338 PMID:21798897
BS4	Not assessed	No family segregation data available to evaluate lack of cosegregation with disease.
BP1	N/A	BP1 applies to missense variants in genes where primarily truncating variants are the established disease mechanism. This variant is itself a truncating (nonsense) variant.
BP2	Not assessed	No data on observation in trans with a known pathogenic variant in NOTCH1 (autosomal dominant AOVD1/Adams-Oliver syndrome).
BP4	Not met	SpliceAI predicts no splicing impact (max delta = 0.00), indicating no cryptic splice site creation or disruption. However, this variant's primary mechanism is protein truncation via a premature stop codon/NMD, not splice alteration. The SpliceAI benign prediction does not negate the established deleterious mechanism of nonsense-mediated decay, which is already addressed by PVS1.	spliceai
BP5	Not assessed	No data on an alternate molecular basis for disease in an individual carrying this variant.
BP6	Not met	The variant is absent from ClinVar. No reputable source reports this variant as benign.	clinvar
BP7	N/A	BP7 applies to synonymous variants with no predicted splice impact. This is a nonsense substitution (c.3225G>A, p.Trp1075Ter), not a synonymous change.
BP3	N/A	BP3 applies to in-frame indels in repetitive regions without known function. This is a single-nucleotide substitution, not an in-frame indel.
PM3	N/A	PM3 applies to recessive disorders with a pathogenic variant in trans. NOTCH1-related disorders (AOVD1, Adams-Oliver syndrome) are autosomal dominant. No recessive disease association is established.
PM4	N/A	PM4 applies to in-frame deletions/insertions or stop-loss variants altering protein length. This is a single-nucleotide nonsense substitution resulting in protein truncation via premature stop codon, classified under PVS1.

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