SETD2 c.4260del, p.(Glu1420AspfsTer12)

Reasoning

NM_014159.6:c.4260del is a single-nucleotide deletion producing NP_054878.5:p.(Glu1420AspfsTer12), a frameshift with a premature termination codon (PTC) 12 residues downstream of the frame shift. No official CSPEC/VCEP framework is available for SETD2 (framework_mode=generic_acmg; cspec.found=false), so the generic ClinGen SVI PVS1 decision tree (PMC6185798) is applied, as directed by the case's own pvs1_variant_assessment scaffold. Gene-disease mechanism gate: germline loss-of-function is an established SETD2 disease mechanism (overgrowth/Sotos-like syndrome and related SETD2 chromatin disorders caused by intragenic LoF variants per pvs1_gene_context, lof_mechanism_supported=true, pvs1_gene_gate=eligible), so PVS1 is eligible to apply. Transcript relevance: NM_014159.6 is the RefSeq-Select transcript for SETD2 (refseq_select=true, MANE-adjacent select transcript), confirming the frameshift is called on a clinically relevant transcript. Exon/NMD analysis: using the mutalyzer exon map (c.88-4454 spans the exon containing c.4260, an internal exon far from the terminal exon) and the CDS terminal exon boundary (final coding exon c.7534-*685, last exon-exon junction at c.7533), the PTC introduced by the frameshift (approximately c.4260-4296, well under half-way through the transcript) falls thousands of nucleotides upstream of the final exon-exon junction, far in excess of the 50-nt NMD rule threshold. NMD is therefore predicted, consistent with the standard SVI frameshift pathway to PVS1 Very Strong when NMD is predicted and the exon is not known to be dispensable. Exon importance / protein truncation: even in an NMD-escape scenario, the truncation at residue 1420 (of a ~2564 aa protein, predicted truncated product ~1431 aa) occurs immediately upstream of and would eliminate the catalytic SET methyltransferase domain and the C-terminal SRI/WW domains required for H3K36 trimethylation and RNA Pol II CTD interaction, so the truncated region is not a dispensable distal segment. No repeat-region or low-complexity-region caveat applies: the deleted nucleotide and resulting frameshift are not in a homopolymer/tandem-repeat context flagged anywhere in the bundle, and this exon is not noted as enriched for population-observed LoF variation; the variant itself is absent from gnomAD v2.1, v4.1 and gnomAD-Canada, providing no contradicting population evidence. All PVS1 decision-tree gates (gene-disease LoF mechanism, correct/relevant transcript, non-terminal-exon PTC with NMD predicted, non-dispensable exon, no repeat-region exemption) are satisfied, supporting PVS1 at full (Very Strong) strength.

Rule

PVS1_frameshift_NMD_predicted_generic_SVI

Evidence considered

• case_summary.json normalization: NM_014159.6:c.4260del produces NP_054878.5:p.(Glu1420AspfsTer12), a frameshift consequence (consequence_class=frameshift, variant_bucket=frameshift).
• pvs1_variant_assessment.json: framework_source is ClinGen SVI PVS1 recommendations (PMC6185798); apply_generic_pvs1_framework=true; suggested_default_strength=PVS1; rationale confirms frameshift variants are evaluated with the generic PVS1 framework once germline LoF is established for the gene.
• pvs1_gene_context.json: germline_disease_context_found=true; lof_mechanism_supported=true; pvs1_gene_gate=eligible; mechanism_rationale states targeted germline literature review identified disease-focused publications supporting SETD2 loss of function as a germline disease mechanism (SETD2-related overgrowth condition/Sotos-like syndrome, citing PMID 24852293 and PMID 31643139 among supporting_sources), so generic PVS1 framework assessment is eligible.
• prefetch.json variant_validator annotations: NM_014159.6 has refseq_select=true (RefSeq Select transcript), confirming clinical/transcript relevance for PVS1 application.
• prefetch.json mutalyzer selector_short exon map (c.-coordinates): the variant position c.4260 falls within the exon spanning c.88-4454, an internal exon; the terminal coding exon spans c.7534-*685 with the last exon-exon junction at c.7533, meaning the PTC produced at approximately c.4260-4296 is thousands of nucleotides upstream of that junction, far exceeding the 50-nt threshold used to predict NMD under the ClinGen SVI PVS1 framework.
• prefetch.json protein prediction: predicted truncated protein length ~1431 aa versus a ~2564 aa original (position_last_original=2565), truncating at residue 1420 -- immediately N-terminal to the SETD2 catalytic SET methyltransferase domain and upstream of the C-terminal SRI/WW domains, so even under an NMD-escape scenario the truncated/degraded product would lack functionally essential domains.
• case_summary.json gnomAD panel: variant is absent from gnomAD v2.1 exomes, gnomAD v4.1 exomes, and gnomAD-Canada v1.0, providing no population evidence that this exon/region tolerates LoF variation.
• No CSPEC/VCEP-specific PVS1 rules were found for SETD2 (case_summary.json vcep_materials.final_classification_framework.found=false, source='generic_acmg_fallback'), so the generic ACMG/SVI PVS1 framework governs per the required precedence (VCEP first, then gene-specific, then generic ACMG).

Still missing

• No official CSPEC/VCEP-specific PVS1 gene framework exists for SETD2; assessment relies on the generic SVI PVS1 decision tree (PMC6185798) applied via the case's own gene-context/variant-assessment scaffolding rather than an expert-panel-curated exon-criticality map.
• Exact exon number was not explicitly labeled in the source data (exon field null); exon position was derived from mutalyzer c.-coordinate exon boundaries rather than a pre-computed exon index.
• No pLI/LOEUF or curated exon-dispensability data were available in the bundle to formally confirm this exon is not subject to naturally occurring biallelic LoF tolerance.

Sources

Reasoning

No SETD2-specific VCEP/CSPEC population rule was retrieved, so generic ACMG/AMP evidence logic applies. The exact normalized deletion is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. This satisfies PM2 at supporting strength as absence from population controls; no elevated-frequency or ancestry-specific frequency artifact was reported in the available population evidence.

Rule

generic_acmg_2015: PM2 applies to variants absent from population databases or present at extremely low frequency, at supporting strength.

Evidence considered

• The exact normalized deletion was queried in gnomAD v2.1 (GRCh37), gnomAD v4.1 (GRCh38), and gnomAD-Canada v1.0 (GRCh38); each source reported the variant as absent.

Sources

Reasoning

SpliceAI, applied under the generic ACMG framework (no gene-specific SETD2 VCEP PP3/BP4 lookup material found), predicts no splice impact for NM_014159.6:c.4260del: max delta score = 0.014 (DS_AG=0.0, DS_AL=0.0, DS_DG=0.014, DS_DL=0.001), well below the ~0.2 delta-score threshold widely used (per the original SpliceAI publication, Jaganathan et al. 2019, Cell, PMID:30661751, describing high-precision score bands of 0.2/0.5/0.8) to flag a plausible splice-altering effect, and also below the conservative ~0.1 no-effect band. This supports BP4 at supporting strength for the splicing computational path. The missense in-silico path (REVEL/BayesDel) does not apply because this variant is a frameshift deletion, not a missense substitution; REVEL and BayesDel scores are both absent from the case bundle regardless.

Rule

generic_acmg_bp4

Evidence considered

• case_summary.compact_evidence.spliceai.evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01).' with scores DS_AG=0.0, DS_AL=0.0, DS_DG=0.014, DS_DL=0.001, DP values all indicating distant/low-confidence positions, max_delta=0.014.
• SpliceAI delta score of 0.014 falls well below the ~0.2 threshold band associated with likely splice-altering variants described in the original SpliceAI publication (Jaganathan et al. 2019, Cell, PMID:30661751), supporting a benign/no-impact splicing prediction at supporting strength.

Still missing

• No gene-specific SETD2 VCEP PP3/BP4 lookup table was located to confirm or override this generic-framework call.
• PMID:30661751 (Jaganathan et al. 2019) is cited from established knowledge of the SpliceAI publication's score bands, not retrieved/verified as full text in this case's source registry.

Sources

Reasoning

No CSPEC/VCEP-specific PP3/BP4 lookup material is available for SETD2 (framework_mode=generic_acmg, cspec.found=false, vcep_materials empty), so generic ACMG in-silico evidence applies. This variant, NM_014159.6:c.4260del (p.Glu1420AspfsTer12), is a frameshift deletion, not a missense substitution, so the missense computational path (REVEL/BayesDel/aGVGD) is not applicable: REVEL score is null/not found and BayesDel score is null/not found in the case bundle, and per pipeline policy BayesDel is treated as insufficiently calibrated regardless of any recalled cutoff. For the splice-impact path, SpliceAI predicts no significant splice impact (max delta score 0.014; DS_AG=0.0, DS_AL=0.0, DS_DG=0.014, DS_DL=0.001), far below any threshold used to support a deleterious splicing prediction. There is therefore no computational evidence (splice or missense) supporting a deleterious effect for this variant.

Rule

generic_acmg_pp3

Evidence considered

• case_summary.compact_evidence.spliceai.evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.01).' with scores DS_AG=0.0, DS_AL=0.0, DS_DG=0.014, DS_DL=0.001, max_delta=0.014.
• Variant is a frameshift deletion (NP_054878.5:p.(Glu1420AspfsTer12)), not a missense change, so the missense in-silico path (REVEL/BayesDel/aGVGD) is not applicable; case_summary.compact_evidence.revel.score and .bayesdel.score are both null.

Still missing

• No gene-specific VCEP PP3/BP4 lookup table was available for SETD2 to check for a pre-assigned code.
• REVEL and BayesDel scores are not populated in this case's evidence bundle (variant is a frameshift, so the missense predictor path is not expected to apply).

Sources

Reasoning

The exact variant is absent from ClinVar; consequently, there is no exact-variant pathogenic or likely pathogenic expert-panel assertion to support PP5.

Rule

PP5 is eligible only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Evidence considered

• ClinVar exact-variant retrieval reported no result for NM_014159.6:c.4260del. No expert-panel classification is present.
• Ordinary laboratory submissions, aggregate labels, and non-expert assertions were not used.

Sources

Reasoning

No SETD2-specific VCEP/CSPEC population rule was retrieved, so generic ACMG/AMP evidence logic applies. NM_014159.6:c.4260del (GRCh37 3-47161865-GC-G; GRCh38 3-47120375-GC-G) was absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0. It therefore has no population allele frequency that could satisfy BA1.

Rule

generic_acmg_2015: BA1 requires an allele frequency too high for the disorder.

Evidence considered

• The exact normalized deletion was queried in gnomAD v2.1 (GRCh37), gnomAD v4.1 (GRCh38), and gnomAD-Canada v1.0 (GRCh38); each source reported the variant as absent.

Sources

Reasoning

No SETD2-specific VCEP/CSPEC population rule was retrieved, so generic ACMG/AMP evidence logic applies. The variant was absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; there is no observed allele frequency that exceeds a disease-compatible BS1 threshold.

Rule

generic_acmg_2015: BS1 requires an allele frequency greater than expected for the disorder.

Evidence considered

• The exact normalized deletion was queried in gnomAD v2.1 (GRCh37), gnomAD v4.1 (GRCh38), and gnomAD-Canada v1.0 (GRCh38); each source reported the variant as absent.

Sources

Reasoning

The exact variant is absent from ClinVar; consequently, there is no exact-variant benign or likely benign expert-panel assertion to support BP6.

Rule

BP6 is eligible only for an exact-variant ClinVar expert-panel Benign or Likely benign classification.

Evidence considered

• ClinVar exact-variant retrieval reported no result for NM_014159.6:c.4260del. No expert-panel classification is present.
• Ordinary laboratory submissions, aggregate labels, and non-expert assertions were not used.

Sources

Reasoning

No case-level parental testing or confirmed de novo occurrence is provided for NM_014159.6:c.4260del.

Rule

Generic ACMG/AMP 2015 PS2: de novo occurrence in a patient with the disease and no family history, with maternity and paternity confirmed.

Evidence considered

• The consolidated case-evidence bundle identifies no SETD2 CSPEC/VCEP or local gene-specific framework; generic ACMG/AMP assessment is therefore the applicable framework.
• The bundle contains variant normalization and population/functional-source results but no proband phenotype, parental genotype, parentage confirmation, or de novo testing result for this variant.

Still missing

• Parental genotyping with confirmed biological parentage is required to assess a confirmed de novo occurrence.
• Proband phenotype information is required to evaluate phenotype consistency for de novo evidence.

Reasoning

No ClinGen VCEP or gene-specific functional-evidence framework is available for SETD2 in this case (cspec.found = false; framework_mode = generic_acmg), so generic ACMG PS3 criteria apply. PS3 requires a validated functional assay (with appropriate positive/negative controls) that was performed specifically on this variant, NM_014159.6:c.4260del (p.(Glu1420AspfsTer12)), and shown to abolish gene/protein function in a manner consistent with the established disease mechanism. The three literature-pass PMIDs retrieved for this case (23417712, 24509477, 25728682) are general SETD2 somatic-mutation studies in pediatric high-grade glioma, acute leukemia, and clear-cell renal carcinoma cohorts; none of them mention this specific variant (searched for c.4260del/4260del, E1420Dfs*12/1420Dfs, and Glu1420 across all three full-text files with no matches). No variant-specific functional/complementation/rescue assay data were found in the case bundle. Because there is no assay evidence tied to this exact variant, PS3 cannot be assessed.

Still missing

• No variant-specific functional assay (e.g., methyltransferase activity assay, H3K36me3 rescue/complementation assay, or minigene splicing assay) for NM_014159.6:c.4260del was identified in the case bundle or the three retrieved publications.
• The retrieved PMIDs discuss SETD2 loss-of-function broadly in somatic cancer cohorts but do not report data on this specific frameshift variant.
• No ClinGen SETD2 VCEP specification with a PS3 functional-evidence rule (assay list, strength calibration) is available for this case.

Sources

Reasoning

No case-control or statistically interpretable enrichment data for the exact variant were identified.

Rule

Generic ACMG/AMP PS4 requires statistically increased prevalence of the variant in affected individuals compared with controls.

Evidence considered

• The three retrieved publications were searched for exact variant identifiers (c.4260del, p.Glu1420AspfsTer12/p.E1420Dfs*12, and genomic deletion aliases); none mentioned the exact variant. They therefore provide no exact-variant case count or control comparison for PS4.
• No SETD2 VCEP/ClinGen gene-specific specification was retrieved; generic ACMG/AMP assessment applies.

Still missing

• A phenotype-defined case series or case-control dataset reporting NM_014159.6:c.4260del, with affected and control counts sufficient to assess enrichment.

Sources

Reasoning

PM3 cannot be assessed. The case bundle contains no affected-proband observation of NM_014159.6:c.4260del with a second pathogenic or likely pathogenic SETD2 variant, no phase result demonstrating trans configuration, and no evidence establishing a relevant autosomal-recessive SETD2 disorder for this case.

Rule

Generic ACMG/AMP PM3: allelic pathogenic evidence requires a pathogenic or likely pathogenic variant observed in trans with the variant under assessment in an affected individual with a recessive disorder.

Evidence considered

• No SETD2 VCEP/CSPEC or local gene-specific framework was retrieved; the bundle designates generic ACMG/AMP fallback.
• The consolidated bundle provides only the index frameshift, NM_014159.6:c.4260del (p.Glu1420AspfsTer12), and contains no proband-level co-occurring SETD2 genotype, phase, or parental-segregation data.
• The germline SETD2 context in the bundle does not specify an inheritance mode.

Still missing

• Affected-proband genotype data identifying any second SETD2 variant.
• Parental testing or other phase evidence establishing whether any second variant is in trans.
• Disease-specific evidence that the pertinent SETD2 condition has autosomal-recessive inheritance and that the second allele is pathogenic or likely pathogenic.

Reasoning

No reported de novo occurrence without confirmed parentage is provided for NM_014159.6:c.4260del.

Rule

Generic ACMG/AMP 2015 PM6: assumed de novo occurrence, without confirmation of paternity and maternity.

Evidence considered

• The consolidated case-evidence bundle identifies no SETD2 CSPEC/VCEP or local gene-specific framework; generic ACMG/AMP assessment is therefore the applicable framework.
• No parental test result, parental relationship information, or reported de novo assertion for this variant is present in the bundle.

Still missing

• Parental genotyping demonstrating absence of the variant is required to assess presumed de novo evidence.
• Proband phenotype information is required to evaluate phenotype consistency.

Reasoning

No family pedigree, affected-relative genotypes, phenotypes, or informative meioses are provided for segregation assessment.

Rule

Generic ACMG/AMP 2015 PP1: cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• The consolidated case-evidence bundle identifies no SETD2 CSPEC/VCEP or local gene-specific framework; generic ACMG/AMP assessment is therefore the applicable framework.
• No family segregation data, relative testing results, pedigree, or meiosis count is present in the bundle.

Still missing

• Pedigree and genotype/phenotype data for informative relatives are required to evaluate cosegregation.
• The number of informative meioses and the clinical status of tested relatives must be documented.

Reasoning

The evidence bundle contains no proband phenotype, diagnosis, or phenotype-exclusion information for this exact variant; phenotype specificity cannot be assessed.

Rule

Generic ACMG/AMP PP4 requires a patient phenotype or family history highly specific for a disease with a single genetic etiology.

Evidence considered

• The bundle identifies a SETD2-related overgrowth condition at the gene level, but supplies no phenotype for the individual carrying NM_014159.6:c.4260del. Gene-disease validity alone is not PP4 evidence.
• No SETD2 VCEP/ClinGen gene-specific specification was retrieved; generic ACMG/AMP assessment applies.

Still missing

• Phenotype and clinical diagnostic information for the tested individual, including whether the presentation is highly specific for SETD2-related disorder and whether relevant alternative causes have been excluded.

Sources

Reasoning

No SETD2-specific VCEP/CSPEC BS2 rule was retrieved. The population sources report the variant as absent and therefore provide neither adult genotype observations nor homozygote observations. BS2 cannot be assessed from absence data.

Rule

generic_acmg_2015: BS2 requires observation in healthy adult individual(s) for a fully penetrant early-onset disorder.

Evidence considered

• The exact normalized deletion was absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; consequently, these sources contain no carriers or homozygotes from which BS2 evidence can be derived.

Still missing

• Variant-level observations in well-phenotyped unaffected adults, including genotype and age/phenotype context, are required to assess BS2 under generic ACMG/AMP guidance.

Sources

Reasoning

No ClinGen VCEP or gene-specific functional-evidence framework is available for SETD2 in this case (cspec.found = false; framework_mode = generic_acmg), so generic ACMG BS3 criteria apply. BS3 requires a validated functional assay showing no damaging effect on protein function for this specific variant. No such assay data for NM_014159.6:c.4260del were found; the three retrieved PMIDs (23417712, 24509477, 25728682) are general SETD2 somatic-cancer mutation studies that do not mention this variant (confirmed by text search for c.4260del, E1420Dfs*12, and Glu1420 with no matches). Additionally, this variant is a frameshift predicted to cause p.(Glu1420AspfsTer12), consistent with loss of function, which is not the expected molecular consequence supporting a benign functional-evidence conclusion. There is no basis to assess BS3.

Still missing

• No variant-specific functional assay data (of any kind) exist in the case bundle for this variant.
• No ClinGen SETD2 VCEP specification with a BS3 rule/assay list is available.

Sources

Reasoning

No informative non-segregation observation is provided; unaffected-carrier and affected-noncarrier data are absent.

Rule

Generic ACMG/AMP 2015 BS4: lack of segregation in affected members of a family.

Evidence considered

• The consolidated case-evidence bundle identifies no SETD2 CSPEC/VCEP or local gene-specific framework; generic ACMG/AMP assessment is therefore the applicable framework.
• No pedigree, affected-noncarrier observation, or unaffected-carrier observation for this variant is present in the bundle.

Still missing

• Genotype and disease-status data from informative family members are required to establish lack of segregation.
• Age, penetrance, and phenotype ascertainment information are needed to interpret unaffected carriers.

Reasoning

BP2 cannot be assessed. The case bundle contains no observation of NM_014159.6:c.4260del in cis or in trans with a pathogenic or likely pathogenic SETD2 variant, and no phenotype or inheritance information that would establish that a documented co-occurrence is non-contributory.

Rule

Generic ACMG/AMP BP2: benign allelic evidence requires a documented observation of the variant with a pathogenic or likely pathogenic variant in a phase and disease context where that co-occurrence supports non-pathogenicity.

Evidence considered

• No SETD2 VCEP/CSPEC or local gene-specific framework was retrieved; the bundle designates generic ACMG/AMP fallback.
• The consolidated bundle provides no co-occurring SETD2 variant, phase result, affected-proband observation, or parental-segregation information for the index variant.
• The germline SETD2 context in the bundle does not specify an inheritance mode.

Still missing

• Affected or unaffected individual-level data documenting a co-occurring pathogenic or likely pathogenic SETD2 variant.
• Phase evidence establishing cis or trans configuration.
• Phenotype and inheritance information needed to determine whether a confirmed co-occurrence is compatible with non-pathogenicity of the index variant.

Reasoning

No individual-level phenotype, molecular findings, or alternative causal diagnosis is provided for this exact variant.

Rule

Generic ACMG/AMP BP5 requires that a variant be found in an individual with an alternative molecular basis for disease.

Evidence considered

• No case-level alternate molecular diagnosis was available in the consolidated evidence bundle.
• No SETD2 VCEP/ClinGen gene-specific specification was retrieved; generic ACMG/AMP assessment applies.

Still missing

• Case-level evidence documenting that the individual has an alternative molecular diagnosis that fully explains the phenotype.