NOTCH2 c.5625del, p.(Thr1876LeufsTer54)

Reasoning

NM_024408.3:c.5625del is a single-nucleotide frameshift deletion in exon 31 of NOTCH2, predicted to produce NP_077719.2:p.(Thr1876LeufsTer54). No official ClinGen NOTCH2 VCEP/CSPEC PVS1 specification is available for this case (framework_mode: generic_acmg; cspec.found: false), so the generic ClinGen SVI PVS1 decision-tree framework (PMC6185798) was applied, consistent with the case's own pvs1_variant_assessment scaffold, which flags apply_generic_pvs1_framework=true and a suggested_default_strength of PVS1 for this frameshift. Gene-level gating: pvs1_gene_context establishes that NOTCH2 has a germline loss-of-function disease mechanism (haploinsufficiency causing Alagille syndrome via heterozygous truncating/frameshift variants), corroborated by PMID:16773578, which reports NOTCH2 truncating mutations segregating with Alagille syndrome in two families, directly supporting LOF as a bona fide germline pathogenic mechanism for this gene. Transcript relevance: NM_024408.3 is the RefSeq transcript used for variant description and matches VariantValidator/Mutalyzer annotation with no evidence of an alternative, more clinically relevant transcript being missed. NMD prediction: the new stop codon falls in exon 31 of a transcript with the terminal coding exon at exon 34 (CDS extends into c.6028-*3753); the variant is therefore several exon-exon junctions upstream of the last junction and well beyond the ~50-55 nt NMD escape boundary, so the transcript is predicted to undergo nonsense-mediated decay, consistent with a null allele. Exon importance: exon 31 lies within the constitutively-included coding region of NOTCH2 (not among the terminal 1-2 exons and not annotated as an alternatively-spliced/dispensable exon in the materials reviewed), so there is no basis to downgrade for exon skippability. Protein length change/domain impact: even absent NMD, the frameshift would truncate/scramble the final ~596 of 2472 residues (position_last_predicted 1929 vs position_last_original 2472), removing substantial C-terminal coding sequence; there is no evidence this region is dispensable. Repeat-region context: this is a single-nucleotide deletion in unique coding sequence, not in a low-complexity/repetitive region requiring special repeat-region caution. Taken together (frameshift + NMD-predicted + established LOF disease mechanism + no exon-skipping/rescue evidence), PVS1 applies at full (very strong) strength per the generic PVS1 framework's default disposition for this configuration.

Rule

PVS1_generic_frameshift_NMD_predicted

Evidence considered

• pvs1_variant_assessment.json classifies c.5625del as consequence_class=frameshift, variant_bucket=frameshift, and recommends applying the generic PVS1 framework at suggested_default_strength=PVS1, citing PMC6185798 (ClinGen SVI PVS1 recommendations) as the governing framework since no NOTCH2 CSPEC/VCEP PVS1 rule exists for this case.
• pvs1_gene_context.json confirms NOTCH2 germline loss-of-function is an established disease mechanism (pvs1_gene_gate=eligible, lof_mechanism_supported=true), based on targeted literature review of NOTCH2-related Alagille syndrome and related disorders.
• VariantValidator/Mutalyzer exon mapping (prefetch.json) places c.5625 within exon 31 of NOTCH2 (c.5480-5781), with the terminal coding exon being exon 34 (c.6028-*3753); the premature termination codon falls multiple exon-exon junctions upstream of the last junction, consistent with NMD-predicted decay per the >50-55nt rule in the generic PVS1 decision tree.
• Predicted protein consequence NP_077719.2:p.(Thr1876LeufsTer54) with a reference protein length of 2472 aa (position_last_original=2472) and truncated/frameshifted region beginning at residue 1875 (position_first=1875), reflecting loss of a substantial C-terminal portion of the protein.
• PMID:16773578 abstract: NOTCH2 mutation screening in Alagille syndrome (AGS) probands identified NOTCH2 mutations segregating in two families, establishing NOTCH2 loss-of-function/truncating variants as a germline disease mechanism for Alagille syndrome, supporting eligibility for the PVS1 framework in this gene.
• No canonical splice-site involvement was noted (canonical_splice_consensus=false in pvs1_variant_assessment.json), so this is evaluated purely as a coding frameshift deletion, not a splice-altering event; SpliceAI (max delta 0.17) also shows no strong splice signal, consistent with a straightforward frameshift/NMD mechanism rather than an aberrant splicing mechanism.
• No evidence in the case bundle (gnomAD v2.1/v4.1 absent for this variant; no ClinVar entry) suggesting the affected exon is enriched for benign/population loss-of-function variation that would argue against exon importance.

Still missing

• No official NOTCH2 ClinGen VCEP-specific PVS1 specification/CSPEC was available in this case; generic PVS1 framework (PMC6185798) was applied by necessity.
• No gene-specific exon-level population constraint (e.g., LOEUF/pext) or minigene/RNA data was available in the case bundle to directly confirm exon 31 biological importance or rule out exon skipping; this rests on the absence of any exon-skipping/dispensability evidence rather than a positive constraint metric.
• No direct experimental (RT-PCR/RNA-seq) NMD confirmation was available for this specific variant; NMD status is inferred from exon position relative to the terminal exon-exon junction per standard rules.

Sources

Reasoning

The normalized deletion is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, meeting generic ACMG/AMP PM2 absent-from-controls evidence at moderate strength.

Rule

Generic ACMG/AMP PM2: absent from controls (or at extremely low frequency if recessive) supports moderate pathogenic evidence.

Evidence considered

• NM_024408.3:c.5625del was queried as GRCh37 1-120462090-TC-T and GRCh38 1-119919467-TC-T and was reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0.
• No NOTCH2-specific VCEP/ClinGen specification was retrieved; generic ACMG/AMP 2015 PM2 applies.

Sources

Reasoning

No ClinGen NOTCH2/Alagille-Hajdu-Cheney VCEP CSPEC or gene-specific PP3/BP4 lookup spreadsheet was found for this case (cspec.found=false; vcep_materials.note='no cspec url available'; final_classification_framework.source='generic_acmg_fallback'), so generic ACMG in-silico evidence rules apply. This variant is a single-nucleotide coding deletion (NM_024408.3:c.5625del) producing a frameshift (p.(Thr1876LeufsTer54)); it is not a missense or synonymous substitution, so the REVEL/BayesDel/aGVGD missense in-silico path and the BP7 synonymous path are not applicable to it (prefetch.json confirms revel and bayesdel were both skipped as 'not snv or no coords'). The only relevant computational-evidence path is the SpliceAI splice-impact score. SpliceAI predicts a maximum delta score of 0.173 (DS_DG=0.173, all other delta scores <=0.016), which falls below the commonly used ~0.2 high-confidence splice-altering threshold derived from the original SpliceAI validation (Jaganathan et al., Cell 2019, PMID 30661751), so it does not reach the level needed to support a splice-altering prediction for PP3.

Evidence considered

• SpliceAI Lookup evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.17).' with underlying scores DS_AG=0.016, DS_AL=0.005, DS_DG=0.173, DS_DL=0.011.
• Using the original SpliceAI high-recall threshold of ~0.2 (Jaganathan et al., Cell 2019, PMID 30661751), a max delta of 0.173 does not clear the bar needed to support a pathogenic splice-altering effect.

Still missing

• No gene-specific (NOTCH2/Alagille/Hajdu-Cheney VCEP) PP3/BP4 SpliceAI lookup table is available in this case bundle to confirm gene-specific thresholds.
• SpliceAI max delta of 0.173 sits in an indeterminate zone between commonly used ~0.1 (no-impact) and ~0.2 (splice-altering) thresholds, so the prediction is not strongly informative either direction.
• No RNA/minigene splicing functional data available in this bundle to corroborate or refute the SpliceAI prediction.

Sources

Reasoning

The normalized deletion is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; it therefore does not meet the generic ACMG/AMP BA1 high-frequency benign criterion.

Rule

Generic ACMG/AMP BA1: stand-alone benign evidence requires an allele frequency greater than 5% in an appropriate population dataset.

Evidence considered

• NM_024408.3:c.5625del was queried as GRCh37 1-120462090-TC-T and GRCh38 1-119919467-TC-T and was reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0.
• No NOTCH2-specific VCEP/ClinGen specification was retrieved; generic ACMG/AMP 2015 frequency logic applies.

Sources

Reasoning

The variant is absent from all three queried population datasets, so its frequency cannot be greater than expected for a NOTCH2-related disorder and BS1 is not met.

Rule

Generic ACMG/AMP BS1: apply only when allele frequency is greater than expected for the disorder.

Evidence considered

• NM_024408.3:c.5625del was absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; no population frequency above an expected-disease threshold was observed.
• No NOTCH2-specific VCEP/ClinGen specification was retrieved; generic ACMG/AMP 2015 frequency logic applies.

Still missing

• No NOTCH2 disease-specific BS1 maximum credible allele-frequency threshold was available because no applicable VCEP/gene-specific specification was retrieved.

Sources

Reasoning

Same generic-ACMG-fallback context as PP3 (no CSPEC/VCEP PP3-BP4 lookup spreadsheet found for NOTCH2 in this case). The variant is a frameshift deletion, not a missense or synonymous change, so the missense in-silico (REVEL/BayesDel/aGVGD) and BP7 synonymous paths do not apply; REVEL and BayesDel were both explicitly skipped in prefetch ('not snv or no coords'). For the splice-impact path, SpliceAI's max delta score of 0.173 is above the commonly used ~0.1 threshold typically used to support 'no splice impact' (BP4), so the score is not low enough to confidently support a benign computational prediction; it falls in an indeterminate zone rather than clearly below a benign-supporting cutoff.

Evidence considered

• SpliceAI Lookup evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.17).' with underlying scores DS_AG=0.016, DS_AL=0.005, DS_DG=0.173, DS_DL=0.011.
• A max delta of 0.173 is above the ~0.1 threshold commonly used to support a confident 'no impact' call under the original SpliceAI calibration (Jaganathan et al., Cell 2019, PMID 30661751), so it is treated as indeterminate rather than clearly benign.

Still missing

• SpliceAI max delta 0.173 is in an indeterminate range (not clearly <0.1) so cannot be confidently used to support BP4.
• No gene-specific VCEP PP3/BP4 lookup spreadsheet available to adjudicate this borderline score under a validated gene-specific rule.
• BayesDel score not available/not calibrated for this variant type and pipeline; treated as not_available per standing calibration policy regardless of any score that might otherwise be recalled.

Sources

Reasoning

No proband phenotype, parental genotypes, parentage confirmation, or germline de novo testing result is present. PS2 cannot be assessed.

Rule

Generic ACMG/AMP 2015 PS2: de novo occurrence requires the variant in an affected individual with confirmed parental absence; strength requires case-specific de novo and phenotype information.

Evidence considered

• No NOTCH2 VCEP/ClinGen gene-specific specification was retrieved; the case framework designates generic ACMG/AMP 2015 fallback.
• The consolidated case-evidence bundle contains no proband phenotype, parental testing, or parentage data for this variant.

Still missing

• Obtain a laboratory-confirmed result for the proband and both biological parents for NM_024408.3:c.5625del.
• Confirm biological parentage and determine whether the variant is absent in parental germline samples; assess parental mosaicism if clinically indicated.
• Document whether the proband phenotype is consistent with a NOTCH2-associated disorder.

Sources

Reasoning

No published functional/biochemical assay data (e.g., reporter/luciferase Notch signaling assays, minigene splicing assays, complementation assays) for this specific variant (NM_024408.3:c.5625del, p.(Thr1876LeufsTer54)) were located anywhere in the case bundle. The three high-priority 'functional' theme literature hits (PMID:15994325, PMID:16773578, PMID:22006338) are gene-level NOTCH2/Notch pathway functional/disease papers; full text was available for PMID:15994325 and PMID:22006338 and searched specifically for this variant/protein change, with no mention found. No CSPEC/VCEP-specific functional evidence framework is present for NOTCH2 (framework_mode: generic_acmg, cspec.found: false), so no gene-specific PS3 assay-validation rules apply either. This variant is a frameshift/truncating change already addressed under PVS1; no independent validated functional assay evidence exists to invoke PS3.

Evidence considered

• Full text of PMID:15994325 (Drosophila Notch EGF-domain O-fucosylation/ligand-binding deletion mapping assay) was searched for this variant's HGVS/protein notation and residue number; no mention of c.5625del or p.Thr1876Lfs*54 found, so this source cannot be cited for PS3/BS3 per the strict grounding rule.
• Full text of PMID:22006338 (NOTCH1/NOTCH2 loss-of-function mutation survey in cutaneous/lung SCC) was searched for this variant; the paper catalogs somatic NOTCH2 LOF mutations broadly but does not mention this specific germline variant or a matched functional assay result for it.
• PMID:16773578 (NOTCH2 mutations causing Alagille syndrome) is a case-report/segregation paper describing NOTCH2 mutation discovery in AGS families, not a functional assay of variant effect; full text was not available in this bundle to confirm any variant-specific assay content.

Still missing

• No variant-specific functional assay (in vitro or in vivo) identified for NM_024408.3:c.5625del / p.(Thr1876LeufsTer54).
• No CSPEC/VCEP NOTCH2-specific functional assay framework available to calibrate assay validity or strength.

Sources

Reasoning

No case-control, cohort-enrichment, or statistically interpretable affected-versus-control data for NM_024408.3:c.5625del (p.Thr1876LeufsTer54) were provided. PS4 cannot be assessed from variant absence in population databases or from gene-level disease literature.

Rule

Generic ACMG/AMP 2015 PS4: significantly increased prevalence of the exact variant in affected individuals compared with controls.

Evidence considered

• The consolidated literature packet contains no variant-specific case-observation or case-control study for NM_024408.3:c.5625del.
• No NOTCH2-specific VCEP/ClinGen specification was retrieved; generic ACMG/AMP assessment is the governing fallback.

Still missing

• Variant-specific affected-case counts, ancestry-matched control counts, phenotype definitions, and an enrichment analysis are required.

Reasoning

No reported de novo occurrence or parental testing is present. PM6 cannot be assessed.

Rule

Generic ACMG/AMP 2015 PM6: assumed de novo occurrence may be considered only when parental absence is not confirmed and relevant case information supports the observation.

Evidence considered

• No NOTCH2 VCEP/ClinGen gene-specific specification was retrieved; the case framework designates generic ACMG/AMP 2015 fallback.
• The consolidated case-evidence bundle contains no parental genotype data or reported de novo observation.

Still missing

• Obtain parental testing for NM_024408.3:c.5625del.
• If parents are unavailable, obtain documentation supporting an assumed de novo occurrence and the proband phenotype.

Sources

Reasoning

No family pedigree, affected relative genotypes, phenotype data, or informative meioses are present. PP1 cannot be assessed.

Rule

Generic ACMG/AMP 2015 PP1: co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease supports pathogenicity; assess the strength from documented informative segregation data.

Evidence considered

• No NOTCH2 VCEP/ClinGen gene-specific specification was retrieved; the case framework designates generic ACMG/AMP 2015 fallback.
• The consolidated case-evidence bundle contains no pedigree, relative testing, phenotype co-segregation, or meiosis information.

Still missing

• Obtain a pedigree with affected and unaffected relatives, their phenotypes, and variant testing results.
• Determine the number of informative meioses in which the variant segregates with a phenotype consistent with a NOTCH2-associated disorder.

Sources

Reasoning

No proband phenotype, diagnostic findings, or exclusion of alternative etiologies was supplied. Therefore, phenotype specificity for a NOTCH2-associated disorder cannot be evaluated for this variant.

Rule

Generic ACMG/AMP 2015 PP4: the patient's phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• The consolidated case evidence contains variant normalization and database results but no patient phenotype or diagnostic workup.
• No NOTCH2-specific VCEP/ClinGen specification was retrieved; generic ACMG/AMP assessment is the governing fallback.

Still missing

• Structured proband phenotype data, including key diagnostic findings and differential-diagnosis evaluation, are required to assess PP4.

Reasoning

The exact variant has no ClinVar record; consequently, there is no exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion to support PP5.

Rule

Restricted rule: apply only to an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic assertion.

Evidence considered

• ClinVar search for NM_024408.3:c.5625del returned no result and no variation ID.

Still missing

• An exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification would be required.

Sources

Reasoning

No data identify this variant in healthy adult individuals, nor are homozygote/carrier observations in unaffected individuals available; BS2 cannot be assessed.

Rule

Generic ACMG/AMP BS2: requires observation in a healthy adult individual for a disorder expected to be fully penetrant at an early age.

Evidence considered

• The population resources report the variant as absent and provide no healthy-adult carrier or homozygote observations for this variant.
• No NOTCH2-specific VCEP/ClinGen specification was retrieved; generic ACMG/AMP 2015 frequency logic applies.

Still missing

• Variant-specific observations in well-phenotyped unaffected adults, including homozygote status and age, are needed.
• Disease-specific penetrance and age-of-onset information sufficient to determine whether healthy-carrier evidence would be informative was not retrieved.

Reasoning

No functional assay data of any kind (variant-specific or otherwise) demonstrating normal/wild-type function for this variant were identified in the case bundle. Absent any assay results, BS3 cannot be met or ruled not_met with evidentiary support; there is simply no functional evidence to evaluate.

Still missing

• No functional assay data (validated or otherwise) available for this variant to assess normal-function evidence for BS3.

Reasoning

No tested affected non-carriers, unaffected carriers, pedigree data, or informative non-segregation observations are present. BS4 cannot be assessed.

Rule

Generic ACMG/AMP 2015 BS4: lack of segregation in affected family members may support a benign interpretation only when the family data are informative and the phenotype-genotype relationship has been evaluated.

Evidence considered

• No NOTCH2 VCEP/ClinGen gene-specific specification was retrieved; the case framework designates generic ACMG/AMP 2015 fallback.
• The consolidated case-evidence bundle contains no pedigree, relative testing, or phenotype data demonstrating lack of segregation.

Still missing

• Obtain a pedigree and genotype-phenotype data identifying informative relatives in whom the variant does not segregate with a phenotype consistent with a NOTCH2-associated disorder.
• Account for variable expressivity, age-dependent manifestations, reduced penetrance, and possible phenocopies before using non-segregation evidence.

Sources

Reasoning

BP2 cannot be assessed: the bundle contains no observation of NM_024408.3:c.5625del in cis or trans with a second pathogenic variant, no phase determination, and no unaffected-carrier evidence relevant to this variant.

Rule

Generic ACMG/AMP BP2: observed in trans with a pathogenic variant for a fully penetrant dominant disorder, or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• No NOTCH2 VCEP/CSPEC or local gene-specific framework was retrieved; the case specifies generic ACMG/AMP fallback.
• No phase, parental segregation, second-allele, or unaffected-carrier evidence is present in the consolidated case-evidence bundle.

Still missing

• Obtain genotype and phase information for any additional pathogenic/likely pathogenic variant in the proband.
• If a trans observation is considered, document the applicable disorder's penetrance and inheritance context; if a cis observation is considered, document that the co-occurring variant is pathogenic/likely pathogenic.

Reasoning

No proband-level molecular diagnosis or phenotype attribution to an alternative cause was provided. BP5 cannot be assessed.

Rule

Generic ACMG/AMP 2015 BP5: variant found in a case with an alternate molecular basis for disease.

Evidence considered

• The consolidated case evidence contains no patient phenotype, testing report, or alternative molecular diagnosis.

Still missing

• A confirmed alternative molecular diagnosis that fully explains the phenotype, together with the relevant proband phenotype, is required.

Reasoning

The exact variant has no ClinVar record; consequently, there is no exact-variant ClinVar expert-panel Benign or Likely benign assertion to support BP6.

Rule

Restricted rule: apply only to an exact-variant ClinVar expert-panel Benign or Likely benign assertion.

Evidence considered

• ClinVar search for NM_024408.3:c.5625del returned no result and no variation ID.

Still missing

• An exact-variant ClinVar expert-panel Benign or Likely benign classification would be required.

Sources