NOTCH1 c.7541_7542del, p.Pro2514ArgfsTer4

Reasoning

PM2 is met at supporting strength under the supplied generic SVI threshold. The variant is rare in population controls: gnomAD v2.1 shows AF = 2.07477e-05 with grpmax FAF = 1.158e-05, and gnomAD v4.1 shows AF = 1.11795e-05 with grpmax FAF = 7.64e-06; both are <= 0.0001. No homozygotes are observed in either dataset. Although this is a two-base deletion and the ACMG/AMP rule cautions that indels may be poorly called by next-generation sequencing, the variant is observed with consistent low frequency in large datasets, so the population data are not an absence claim and support the supplied extremely-low-frequency PM2 threshold.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: 5/240,990 alleles, AF 2.07477e-05, grpmax FAF 1.158e-05, homozygotes 0.
• gnomAD v4.1: 18/1,610,094 alleles, AF 1.11795e-05, grpmax FAF 7.64e-06, homozygotes 0.
• Generic calibration supplied for this case: PM2 supporting threshold <= 0.0001, ClinGen SVI recommendation cited in the case as PMID:25741868. The variant is rare rather than absent, so the extremely-low-frequency path is used.
• The ACMG/AMP indel caveat was considered; concordant observations in large gnomAD datasets reduce concern that this specific call is solely a sequencing artifact.

Sources

Reasoning

NM_017617.5:c.7541_7542del is a frameshift in exon 34 that predicts NP_060087.3:p.(Pro2514ArgfsTer4), but exon 34 is the terminal coding exon and the premature stop is near the protein terminus (predicted residue 2517 versus 2556 original residues), making substantial nonsense-mediated decay unlikely. The resulting truncation removes the C-terminal PEST domain rather than producing a demonstrated null transcript or protein. The exact c.7541_7542delCT/P2514fs*4 variant is reported in NOTCH1 exon 34 and is described as increasing NOTCH1 intracellular-domain stability and prolonging activation through loss of the FBXW7 and WSSSSP degron regions, an activating mechanism inconsistent with PVS1 null-variant evidence. Therefore the generic PVS1 definition is not satisfied; no PVS1 strength is assigned. The available germline LOF context is not sufficient to override the variant-specific terminal-exon and activating-mechanism evidence.

Rule

PVS1: Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.

Evidence considered

• NM_017617.5:c.7541_7542del is normalized as NP_060087.3:p.(Pro2514ArgfsTer4), a frameshift in exon 34; the predicted protein ends at residue 2517 compared with 2556 residues in the reference protein.
• The exact c.7541_7542delCT/P2514fs*4 variant is described in NOTCH1 exon 34 as truncating the C-terminal PEST domain and removing the FBXW7 and WSSSSP degron regions, with increased NOTCH1 intracellular-domain stability and prolonged activation.
• The generic ACMG/AMP PVS1 rule requires a null variant in a gene where loss of function is a known disease mechanism; this terminal-exon variant has variant-specific evidence for an activating, non-null mechanism.

Still missing

• No direct RNA or protein study for this exact germline specimen was available to experimentally confirm transcript stability or the precise cellular consequence.

Sources

Reasoning

The available literature describes the variant in chronic lymphocytic leukemia and reports NOTCH1 mutation enrichment in CLL subgroups, but it does not establish a patient phenotype or family history that is highly specific for a disease with a single genetic etiology. CLL is genetically heterogeneous, and no case-specific clinical phenotype or uniquely diagnostic syndrome presentation is supplied.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• PMID:30087429: the exact variant was found in two newly diagnosed CLL patients in a 12-patient sequencing cohort, without a syndrome-specific phenotype or family-history description.
• PMID:29998084: NOTCH1 P2514fs*4 is discussed as a recurrent CLL mutation associated with NOTCH1 activation; the review does not define CLL as a phenotype highly specific to a single genetic etiology.
• PMID:22237449: the reviewed NOTCH1 cardiovascular discussion concerns other variants and does not provide an exact-variant, highly specific phenotype for this case.

Sources

Reasoning

Exact-variant ClinVar records show three clinical-laboratory submissions, all classified as Uncertain significance, with zero expert-panel submissions. The literature does not provide a reputable-source pathogenic classification for this exact variant that is unavailable for independent evaluation. OncoKB's likely-oncogenic somatic context and publications discussing other NOTCH1 variants do not satisfy the required exact-variant ClinVar expert-panel rule.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 581973 and submission audit: exact variant NM_017617.5:c.7541_7542del has 3 clinical-laboratory submissions, all Uncertain significance, and expert_panel_submissions = 0.
• PMID:30087429 and PMID:29998084 discuss the exact variant in CLL, but neither is an exact-variant ClinVar expert-panel classification reporting Pathogenic or Likely pathogenic.

Sources

Reasoning

BA1 is not met. The governing generic rule requires an allele frequency above 5%. Using the required filtering metric rather than a population point estimate, gnomAD v2.1 reports grpmax FAF = 1.158e-05 and gnomAD v4.1 reports grpmax FAF = 7.64e-06; both are far below 0.05. The highest observed subpopulation point estimates are also only 4.83232e-05 in Finnish individuals (1/20,694 alleles) and 3.41018e-05 in Ashkenazi Jewish individuals (1/29,324 alleles), and neither supports BA1. The variant is an indel, but the large allele counts and concordant observations across gnomAD datasets provide usable population evidence; no non-cancer or exome-only governing source was specified for this generic interpretation.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: 5/240,990 alleles, total AF 2.07477e-05, homozygotes 0, grpmax FAF 1.158e-05; highest point estimate Finnish AF 4.83232e-05 from 1/20,694 alleles.
• gnomAD v4.1: 18/1,610,094 alleles, total AF 1.11795e-05, homozygotes 0, grpmax FAF 7.64e-06; highest point estimate Ashkenazi Jewish AF 3.41018e-05 from 1/29,324 alleles.
• Generic calibration supplied for this case: BA1 stand-alone threshold >= 0.05, ACMG/AMP 2015 (PMID:25741868). The required filtering allele frequency is used; popmax point estimates are not used for the decision.

Sources

Reasoning

BS1 is not met. The generic default threshold supplied for this case is >= 0.01, and the required filtering metric is the grpmax FAF. The observed values are 1.158e-05 in gnomAD v2.1 and 7.64e-06 in gnomAD v4.1, both substantially below 0.01. The maximum observed subpopulation point estimates are also very low and are not used in place of FAF. Therefore, the population data do not show an allele frequency greater than expected for the disorder, and a maximum-credible-frequency calculation is not needed to reject this criterion.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: grpmax FAF 1.158e-05 from exome data; total AF 2.07477e-05 across 240,990 alleles.
• gnomAD v4.1: grpmax FAF 7.64e-06 using the combined dataset; total AF 1.11795e-05 across 1,610,094 alleles.
• Generic calibration supplied for this case: BS1 default threshold >= 0.01; the criterion is allele frequency greater than expected for the disorder. No elevated ancestry-specific FAF is present, so prevalence, penetrance, and allelic-heterogeneity adjustments do not change the conclusion.

Sources

Reasoning

Exact-variant ClinVar records show zero expert-panel submissions and no benign or likely-benign expert-panel classification. The three clinical-laboratory submissions classify the exact variant as Uncertain significance, so ordinary laboratory submissions and aggregate labels cannot trigger BP6.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 581973 and submission audit: exact variant NM_017617.5:c.7541_7542del has expert_panel_submissions = 0; all 3 clinical-laboratory submissions classify it as Uncertain significance.

Sources

Reasoning

No case-level evidence is provided showing that the variant is de novo with both maternity and paternity confirmed, and no documented absence of family history is available. The exact-variant literature reviewed reports somatic CLL observations rather than germline parent-child testing. Therefore PS2 cannot be assessed.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• PMID:30087429 reports the exact p.P2514fs*4 deletion in two newly diagnosed CLL patients, but provides no germline parent testing, maternity or paternity confirmation, or family-history assessment.
• PMID:29998084 describes the exact c.7541_7542delCT/p.P2514fs*4 alteration in compiled CLL studies and does not provide a germline de novo pedigree or parental testing for this variant.

Still missing

• Proband-level germline phenotype and disease diagnosis
• Variant testing in both biological parents with maternity and paternity confirmation
• Documented family history or absence of family history

Sources

Reasoning

No qualifying variant-specific functional assay was identified for NOTCH1 NM_017617.5:c.7541_7542del (p.P2514Rfs*4). PMID:30087429 reports detection of the exact deletion by MinION sequencing and Sanger confirmation, but this is an identification assay rather than a functional study. PMID:29998084 provides mechanistic review context that the exact PEST-domain frameshift is associated with loss of degron sequences, increased NOTCH1-ICD stability, and prolonged activation, while PMID:25564152 describes functional effects of other NOTCH PEST-domain mutations; neither establishes a validated, reproducible assay result for this exact variant. Therefore the evidence is insufficient to apply PS3 at any strength.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• PMID:30087429, Results and Table 3: the exact NOTCH1 p.P2514fs*4 deletion was detected in 2 of 12 newly diagnosed CLL patients by targeted MinION sequencing and confirmed by Sanger sequencing; this demonstrates variant detection, not altered protein function.
• PMID:29998084, Section 'Types of NOTCH1 Mutations in CLL' and Table 1: the exact c.7541_7542delCT/p.P2514fs*4 variant is described as truncating the C-terminal PEST domain and removing FBXW7/WSSSSP degron regions, with increased NOTCH1-ICD stability and prolonged activation; this is review-level mechanistic context rather than a variant-specific validated functional assay.
• PMID:25564152: functional studies of other NOTCH PEST-domain mutations reported increased NICD1 stability, pathway activation, and xenograft/GSI-response effects, but the exact c.7541_7542del variant was not mentioned.
• PMID:19604236: a different NOTCH1 PEST-domain deletion was identified by exon sequencing in 2 of 43 B-CLL patients; no functional assay of the case variant was provided.

Still missing

• A validated, biologically relevant in vitro or in vivo assay testing NOTCH1 p.P2514Rfs*4 is needed, with appropriate wild-type and assay-performance controls and reproducible results.

Sources

Reasoning

The exact variant was reported in 2 of 12 newly diagnosed CLL patients in PMID:30087429, but that study provides no unaffected or population control cohort and no matched-ancestry control frequency. PMID:29998084 reports 646 compiled patients across studies but is a review, not an independent case-control comparison. The available gnomAD v4.1 frequency is 18/1,610,094 alleles, but case ancestry and comparable carrier-level control data are not established; therefore a valid case-control OR and 95% CI cannot be calculated under the governing rule. The literature_pass.json packet has no ai_classified_papers to verify as PS4.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• PMID:30087429, Table 3: the exact NOTCH1 p.P2514fs*4 variant was detected in 2 of 12 newly diagnosed CLL patients; both had an allelic ratio of 20.69%.
• PMID:29998084, Table 1 and the section 'Types of NOTCH1 Mutations in CLL': a review compiles P2514fs*4 in 646 patients (78.78%) across published NOTCH1 studies, but does not provide a matched unaffected-control comparison for this exact variant.
• gnomAD v4.1: 18/1,610,094 alleles, AF 1.11795e-05; this population frequency cannot be used here as a matched-ancestry carrier-control comparison because the case ancestry and comparable control denominators are unavailable.

Still missing

• A case-control study or matched-ancestry control cohort with comparable carrier counts is required to calculate the OR and 95% CI.

Sources

Reasoning

PM3 cannot be assessed because the available evidence does not establish that the relevant NOTCH1 disorder is recessive, does not identify a pathogenic variant on the other allele, and provides no parental or offspring testing to establish trans phase. The exact variant is reported in somatic CLL cohorts, but those observations do not establish germline allelic configuration or recessive disease evidence.

Rule

For recessive disorders, detected in trans with a pathogenic variant ... Note: This requires testing of parents (or offspring) to determine phase.

Evidence considered

• PMID:30087429 reports the exact p.P2514fs*4 variant in two newly diagnosed CLL patients, with allelic ratio 20.69% in each case, but does not provide germline inheritance, a second pathogenic allele, or parental/offspring phase testing.
• PMID:29998084 compiles the exact c.7541_7542delCT/p.P2514fs*4 NOTCH1 mutation in somatic CLL studies and describes its PEST-domain effect, but provides no germline trans-allele or phase evidence for this case.

Still missing

• Confirm the relevant germline NOTCH1 disorder and its inheritance mode.
• Identify a pathogenic or likely pathogenic variant on the other allele, if present.
• Obtain parental or offspring testing, or equivalent validated phasing, to establish trans phase.

Sources

Reasoning

No case-level evidence is provided that the variant was assumed de novo without parental confirmation. The exact-variant literature reviewed concerns somatic CLL cohorts and does not document an unconfirmed germline de novo observation. Therefore PM6 cannot be assessed.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• PMID:30087429 reports the exact variant in CLL patients using targeted sequencing, but does not characterize a germline de novo event or parental testing.
• PMID:29998084 reviews the exact variant in CLL and provides no assumed-germline-de-novo pedigree evidence.

Still missing

• A germline proband case with a phenotype consistent with a NOTCH1-related disorder
• Evidence that the variant is absent from tested parents or an explicit unconfirmed de novo statement

Sources

Reasoning

No affected family members, informative meioses, genotype-phenotype cosegregation, or pedigree data are provided for this variant. The exact-variant papers reviewed describe somatic CLL cohorts rather than familial germline segregation. Therefore PP1 cannot be assessed.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• PMID:30087429 reports two exact-variant CLL cases but contains no familial segregation analysis or affected-relative testing.
• PMID:29998084 compiles exact-variant CLL observations and does not report germline pedigrees, informative meioses, or cosegregation.

Still missing

• Pedigree with clinically characterized affected and unaffected relatives
• Variant genotypes for informative relatives
• Disease status and phenotype assessment for each tested relative

Sources

Reasoning

BS2 is not assessed. gnomAD reports zero homozygotes in both v2.1 and v4.1, but those aggregate population counts do not document a healthy adult individual carrying the variant in the inheritance state required by the criterion. The available case evidence does not establish the relevant disorder's expected early-age full penetrance or provide a phenotype-reviewed healthy adult carrier observation. Consequently, absence of homozygotes cannot be converted into BS2, and no direct healthy-adult observation is available to apply the rule.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v2.1 reports homozygotes = 0 and gnomAD v4.1 reports homozygotes = 0, but these are aggregate genotype counts rather than phenotype-reviewed healthy adult observations.
• The available case materials do not provide a healthy adult carrier report or establish full early-age penetrance for the governing NOTCH1 disorder.

Still missing

• A documented healthy adult carrier observation in the relevant inheritance state is unavailable.
• The disorder-specific expectation of full penetrance at an early age is not established in the available evidence.

Sources

Reasoning

No qualifying variant-specific functional study demonstrating absence of a damaging effect on NOTCH1 protein function or splicing was identified. PMID:30087429 confirms the exact deletion by sequencing but does not test function or splicing. PMID:29998084 discusses the exact frameshift as a PEST-domain truncation with increased NOTCH1-ICD stability and prolonged activation, which is not benign functional evidence, and PMID:25564152 concerns other PEST-domain mutations. Consequently, the available evidence cannot establish BS3 at any strength.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• PMID:30087429, Results and Table 3: the exact p.P2514fs*4 deletion was detected and Sanger-confirmed, but the study provides no protein-function or splicing result that could support BS3.
• PMID:29998084, Section 'Types of NOTCH1 Mutations in CLL' and Table 1: the exact c.7541_7542delCT/p.P2514fs*4 variant is discussed as removing degron regions and prolonging NOTCH1 activation, not as demonstrating preserved function.
• PMID:25564152: functional results concern other NOTCH PEST-domain mutations and therefore cannot establish a benign effect for this exact variant.
• PMID:19604236: the reported PEST-domain deletion differs from the case variant and was not subjected to a qualifying benign functional assay.

Still missing

• A validated, biologically relevant assay showing no damaging effect of NOTCH1 p.P2514Rfs*4 on protein function or splicing is needed, with suitable controls and reproducibility.

Sources

Reasoning

No affected family members who lack the variant, non-segregating pedigree, or other informative family-level genotype-phenotype discordance is documented. The reviewed exact-variant literature contains no familial germline segregation dataset. Therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• PMID:30087429 provides exact-variant somatic CLL cases but no affected-relative testing or familial non-segregation analysis.
• PMID:29998084 provides a review of exact-variant CLL reports without a germline pedigree or affected-relative genotype comparison.

Still missing

• Affected relatives tested for the variant
• Reliable clinical phenotyping of those relatives
• Pedigree and segregation results sufficient to distinguish non-segregation from phenocopy or locus heterogeneity

Sources

Reasoning

BP2 cannot be assessed because no pathogenic variant in cis or trans with NM_017617.5:c.7541_7542del is documented, and the available reports concern somatic CLL observations rather than a germline affected proband with validated phase. There is therefore no evidence for either the fully penetrant dominant trans configuration or the cis configuration described by the rule.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• PMID:30087429 reports the exact variant in two CLL patients but does not report a phased pathogenic germline partner in cis or trans.
• PMID:29998084 reviews the exact somatic NOTCH1 PEST-domain deletion in CLL and does not provide the phase or affected-proband allelic evidence required for BP2.

Still missing

• Document a pathogenic or likely pathogenic second variant, if present.
• Obtain validated phasing showing cis or trans configuration.
• For a trans observation, establish that the relevant dominant disorder is fully penetrant.

Sources

Reasoning

The reviewed records do not document this exact variant in a case with a confirmed alternate molecular basis for the disease. The CLL reports describe NOTCH1 mutation findings but do not establish an alternate molecular diagnosis for an individual carrying this variant.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• PMID:30087429 reports the exact variant in two CLL patients but does not report an alternate molecular basis for either patient's disease.
• PMID:29998084 reviews NOTCH1 mutations in CLL and does not document an alternate molecular diagnosis linked to this exact variant.

Still missing

• A case report or clinical record documenting the exact variant together with a confirmed alternate molecular diagnosis is required.

Sources