TP53 c.266_267del, p.(Pro89LeufsTer59)

Reasoning

PS1 in the TP53 VCEP (v2.4) applies only to compare the amino acid change of the variant under assessment against a previously established pathogenic/likely pathogenic variant causing the SAME amino acid change via a different nucleotide substitution (classic missense-vs-missense comparison). NM_000546.6:c.266_267del is a 2-bp deletion producing a frameshift, NP_000537.3:p.(Pro89LeufsTer59) -- not a missense substitution with a single defined resultant amino acid to compare against prior pathogenic missense calls. The VCEP PS1 rule text and originalACMGSummary both frame the code strictly around 'same amino acid change' from an alternate codon (e.g. Val->Leu via G>C or G>T), which structurally does not apply to a frameshift variant.

Rule

TP53 VCEP v2.4 PS1: same amino acid change as an established pathogenic/likely pathogenic missense variant.

Evidence considered

• TP53 VCEP v2.4 PS1 rule: applicability Applicable but instructionsToUse and originalACMGSummary restrict use to same-amino-acid-change comparisons between missense variants; caveat also flags need to rule out splicing impact, which is not the mechanism here.
• Variant normalization: NM_000546.6:c.266_267del -> NP_000537.3:p.(Pro89LeufsTer59), a frameshift, not a missense substitution.

Still missing

• No PS1 comparator search performed since the variant type (frameshift) is categorically outside PS1's missense-comparison scope.

Sources

Reasoning

NM_000546.6:c.266_267del (NP_000537.3:p.(Pro89LeufsTer59)) is a frameshift/null variant. The TP53 VCEP functional rule-code flowchart and Functional-worksheet.xlsx (Supplementary Table S3) restrict PS3/BS3 application to missense variants and small in-frame deletions assessed by the eligible protein-function assays (Kato PMID:12826609, Funk PMID:39774325, Giacomelli PMID:30224644, Kotler PMID:29979965, Kawaguchi PMID:16007150); a frameshift is neither a missense substitution nor an in-frame deletion, so it falls outside this functional framework's scope by variant type alone. In addition, none of these eligible assays, nor the worksheet, contain an entry for this specific frameshift variant or its protein change - no functional assay data exists for it in the case bundle. The VCEP functional instructions also state that PS3 should not be applied at any strength when PVS1 is applied at full strength for the variant, which is the applicable pathway for this class of null variant. No functional assay evidence is available or applicable to invoke PS3.

Rule

TP53 VCEP PS3 (cspec v2.4): Non-functional on Kato et al. data AND LOF by majority of other eligible assays = Strong; Partially functional on Kato AND LOF by majority of others = Moderate; Non-functional on Kato AND abnormal on Kawaguchi = Supporting (also PS3_Supporting for small deletions with LOF on majority of available assays). Scope limited to missense variants and small in-frame deletions per Flowchart-for-application-of-functional-rule-codes.pdf; frameshift variants are out of scope.

Evidence considered

• TP53 VCEP Functional-worksheet.xlsx (Supplementary Table S3) and Flowchart-for-application-of-functional-rule-codes.pdf restrict PS3/BS3 to missense variants and small in-frame deletions assessed via Kato, Funk, Giacomelli, Kotler, and Kawaguchi assays; this variant is a frameshift deletion (p.Pro89LeufsTer59), not a missense substitution or in-frame (non-frameshifting) small deletion, so it is out of scope for this evidence code's assay-based framework.
• TP53 VCEP cSpec v2.4 PS3 instructionsToUse states functional missense codes (including PS3) should not be applied if PVS1 is applied for the variant at full strength; this frameshift variant's evaluation pathway is PVS1, not PS3, per the VCEP's own functional-rule caveats.
• No entry for P89 frameshift/deletion is present in the retrieved Functional-worksheet.xlsx excerpt (only a P89A missense substitution entry was found, which is a different variant with a different consequence).

Still missing

• No functional assay data (Kato, Funk, Giacomelli, Kotler, or Kawaguchi) exists for this specific frameshift variant; the VCEP functional worksheet only covers missense substitutions and single amino-acid in-frame deletions.
• This variant class (frameshift/null, NMD-predicted) is evaluated through the PVS1 pathway, not the PS3/BS3 functional-assay pathway, per VCEP specification caveats.

Sources

Reasoning

TP53 VCEP PM1 applies to missense variants located within codons 175, 245, 248, 249, 273, or 282, or to missense variants seen in cancerhotspots.org with >=2 somatic occurrences for the same amino acid change. The variant under review is a frameshift (p.Pro89LeufsTer59) at codon 89, which is (a) not one of the six listed hotspot codons and (b) not a missense variant, so the cancerhotspots.org same-amino-acid-change somatic-occurrence pathway is also structurally inapplicable (there is no single defined missense amino acid change to look up).

Rule

TP53 VCEP v2.4 PM1: missense hotspot codons 175/245/248/249/273/282, or cancerhotspots.org same-amino-acid-change somatic occurrence thresholds.

Evidence considered

• TP53 VCEP v2.4 PM1: applies to missense variants in codons 175/245/248/249/273/282, or missense variants with >=10 (Moderate) or 2-9 (Supporting) somatic occurrences of the same amino acid change in cancerhotspots.org.
• Variant is a frameshift deletion at codon 89 (p.Pro89LeufsTer59), outside the listed hotspot codon list and not a missense substitution eligible for the cancerhotspots.org pathway.

Still missing

• No cancerhotspots.org lookup performed given the variant class is not missense; not required since PM1 explicitly targets missense hotspot codons/amino acid changes.

Sources

Reasoning

The ClinGen TP53 Expert Panel Specifications, version 2.4, explicitly state that PM3 does not apply to TP53/Li-Fraumeni syndrome. Therefore, phase or trans observations cannot be used to apply PM3 in this gene-disease context.

VCEP rule

This rule does not apply to TP53/Li-Fraumeni syndrome.

Evidence considered

• The TP53 VCEP framework lists PM3 as not applicable and states: 'This rule does not apply to TP53/Li-Fraumeni syndrome.' The framework identifies Li-Fraumeni syndrome as autosomal dominant.
• The case variant is a TP53 frameshift, NM_000546.6:c.266_267del, but variant consequence and available phase observations do not alter the explicit VCEP exclusion of PM3.

Sources

Reasoning

The TP53 VCEP specification (cspec v2.4) explicitly marks PM4 (protein length change due to in-frame deletion/insertion or stop-loss) as 'Not Applicable' for this framework. This variant, c.266_267del, is a frameshift deletion (not in-frame) that produces a premature termination codon via reading-frame disruption, not a net protein length change through in-frame indel or stop-loss mechanism. Under the VCEP framework such variants are captured entirely by the PVS1 pathway; PM4 has no defined role and is excluded from application.

VCEP rule

TP53 VCEP (cspec v2.4): PM4 = Not Applicable.

Evidence considered

• case_summary.json cspec criteria list: entry {"label": "PM4", "applicability": "Not Applicable"} under TP53 VCEP specifications v2.4.
• pvs1_variant_assessment.json: consequence_class=frameshift (not an in-frame deletion/insertion or stop-loss variant, so PM4's protein-length-change rationale does not apply).

Sources

Reasoning

TP53 VCEP PM5 applies to a novel missense change at a residue where a different missense change at the same residue has already been established pathogenic/likely pathogenic, using Grantham distance comparison. The variant under review, p.(Pro89LeufsTer59), is a frameshift, not a missense substitution -- there is no single resultant amino acid at codon 89 to compare via Grantham score against other pathogenic missense variants at that residue. This is corroborated by the case's own pm5_candidates artifact, which flags the variant as ineligible for the classic same-residue missense PM5 search precisely because the consequence class is frameshift, not missense.

Rule

TP53 VCEP v2.4 PM5: missense variant at a residue with 1 (Moderate/Supporting per LP evidence tier) or >=2 (Strong) prior pathogenic missense variants, compared via Grantham distance.

Evidence considered

• TP53 VCEP v2.4 PM5: applies to a missense change at a residue with an established pathogenic/likely pathogenic missense change at that same residue, compared via Grantham distance; caveat to rule out splicing impact.
• pm5_candidates.json pm5_candidates_summary: pm5_mode='classic_same_residue_missense', eligible_for_classic_pm5_search=false, recommendation.level='not_applicable', recommendation.summary='Framework is classic same-residue missense PM5, but the variant consequence class (frameshift) is not missense.'

Still missing

• pm5_candidates.json reports zero same-residue candidates were even searched, consistent with categorical inapplicability rather than an evidentiary gap.

Sources

Reasoning

PM6 is not applicable under the TP53 VCEP Version 2.4 framework. The VCEP explicitly discontinued PM6 and uses PS2 exclusively for de novo evidence, including cases without confirmed parental origin only insofar as they are handled by the PS2 de novo point framework.

VCEP rule

TP53 VCEP PM6: not applicable; see the combined PS2 de novo rule.

Evidence considered

• The TP53 VCEP Version 2.4 specification states that PM6 is not applicable and that the VCEP has dropped PM6 to use PS2 exclusively for de novo evidence.

Sources

Reasoning

The TP53 VCEP v2.4 specification explicitly marks PP2 as Not Applicable for this gene across all strength levels (applicability='Not applicable', instructionsToUse='Not applicable'), superseding the generic ACMG PP2 criterion regardless of the variant's consequence type.

VCEP rule

TP53 VCEP v2.4 PP2: Not Applicable for this gene.

Evidence considered

• TP53 VCEP v2.4: PP2 label entry lists applicability='Not applicable' at every strength level (Stand Alone/Very Strong/Strong/Moderate/Supporting), with instructionsToUse literally stating 'Not applicable'.

Sources

Reasoning

NM_000546.6:c.266_267del is a 2-nucleotide out-of-frame deletion producing a frameshift, NP_000537.3:p.(Pro89LeufsTer59) - not a missense substitution, synonymous variant, single amino-acid in-frame deletion, or intronic/splice-region variant. The TP53 VCEP PP3/BP4/BP7 flowchart and its supporting spreadsheets (PP3-BP4-codes.xlsx for missense/synonymous/intronic; TP53-single-amino-acid-deletions-BayesDel-scores spreadsheet for single-aa in-frame deletions) are scoped only to those variant classes; this frameshift deletion falls outside that scope entirely and is instead adjudicated under the TP53 VCEP PVS1 null-variant flowchart (a different criterion group). SpliceAI was queried and returned a max delta score of 0.116 (DS_AG=0.016, DS_AL=0.015, DS_DG=0.001, DS_DL=0.116), below the VCEP PP3 SpliceAI threshold of >=0.2, but this is immaterial because the variant type itself is not eligible for PP3 under the VCEP's in silico flowchart.

Rule

TP53 VCEP v2.4 PP3/BP4/BP7 flowchart - variant type (out-of-frame frameshift deletion) not covered by PP3/BP4/BP7 scope; adjudicated instead under PVS1.

Evidence considered

• Case summary normalization shows NM_000546.6:c.266_267del predicts NP_000537.3:p.(Pro89LeufsTer59), a 2-nucleotide deletion causing a frameshift and premature termination, not a missense, synonymous, in-frame deletion, or splice-region variant.
• TP53 VCEP Flowchart-for-application-of-PP3,-BP4,-and-BP7.pdf scopes PP3/BP4/BP7 application to missense variants, synonymous/silent variants, single amino acid in-frame deletions, and intronic/exonic splice-region variants only; a 2-bp out-of-frame deletion is not among the covered variant types.
• SpliceAI Lookup query for NM_000546.6:c.266_267del returned max delta score 0.116 (DS_DL=0.116, DS_AG=0.016, DS_AL=0.015, DS_DG=0.001), which is below the VCEP PP3 SpliceAI threshold of >=0.2, though not determinative here because the variant type itself is out of PP3/BP4/BP7 scope.

Still missing

• No missense/BayesDel/aGVGD in silico path applies (variant is not missense).
• No single-amino-acid in-frame deletion BayesDel lookup applies (deletion is out-of-frame, not a single-aa in-frame deletion).

Sources

Reasoning

PP5 is not applicable under the TP53 VCEP specification. Independently, the exact-variant ClinVar record has no expert-panel submission; its sole Pathogenic assertion is from a single clinical laboratory and cannot trigger PP5.

VCEP rule

TP53 VCEP: PP5 is Not Applicable. An exact-variant ClinVar Pathogenic/Likely pathogenic expert-panel assertion would otherwise be required for PP5 consideration.

Evidence considered

• TP53 VCEP version 2.4 designates PP5 as Not Applicable.
• ClinVar variation 3328161 is an exact match but has one Pathogenic submission from Ambry Genetics with review status 'criteria provided, single submitter'; the submission audit identifies zero expert-panel submissions.

Sources

Reasoning

Same scope limitation as PS3: the TP53 VCEP functional rule-code framework (Flowchart-for-application-of-functional-rule-codes.pdf and Functional-worksheet.xlsx) applies BS3 only to missense variants and small in-frame deletions using the eligible assay panel (Kato, Funk, Giacomelli, Kotler, Kawaguchi). NM_000546.6:c.266_267del is a frameshift deletion (p.Pro89LeufsTer59), not a missense substitution or in-frame deletion, and no functional assay data exists for this specific variant in any eligible or non-systematic assay reviewed. BS3 cannot be assessed or applied.

Rule

TP53 VCEP BS3 (cspec v2.4): Functional on Kato et al. data AND no LOF by majority of eligible assays = Strong; Partially functional on Kato AND no evidence of LOF by all available assays = Supporting (also BS3_Supporting for small deletions with available Kotler data showing no LOF by majority of assays). Scope limited to missense variants and small in-frame deletions per Flowchart-for-application-of-functional-rule-codes.pdf; frameshift variants are out of scope.

Evidence considered

• TP53 VCEP Functional-worksheet.xlsx (Supplementary Table S3) and Flowchart-for-application-of-functional-rule-codes.pdf restrict PS3/BS3 to missense variants and small in-frame deletions; this frameshift variant (p.Pro89LeufsTer59) is out of scope for the assay-based BS3 pathway.
• No entry for a P89 frameshift/deletion consequence is present in the retrieved Functional-worksheet.xlsx excerpt; only an unrelated P89A missense substitution entry (BS3, Functional on Kato, noLOF on Giacomelli) was found, which does not apply to this frameshift variant.

Still missing

• No functional assay data (Kato, Funk, Giacomelli, Kotler, or Kawaguchi) exists for this specific frameshift variant.
• Frameshift/null variant class is outside the VCEP functional (PS3/BS3) framework's scope, which is restricted to missense and small in-frame deletion variant types.

Sources

Reasoning

The TP53 VCEP v2.4 specification explicitly marks BP1 as Not Applicable for this gene (case_summary.json cspec criteria list: {'label':'BP1','applicability':'Not Applicable'}), superseding the generic ACMG BP1 criterion regardless of the variant's consequence type.

VCEP rule

TP53 VCEP v2.4 BP1: Not Applicable for this gene.

Evidence considered

• TP53 VCEP v2.4 cspec criteria list marks BP1 with applicability='Not Applicable', with no strength rules or instructionsToUse provided.

Sources

Reasoning

The ClinGen TP53 Expert Panel Specifications, version 2.4, explicitly list BP2 as not applicable for TP53/Li-Fraumeni syndrome. Consequently, no BP2 evidence is applied, even though the framework describes trans-phase and multiple-observation scenarios in its instructions.

VCEP rule

Evidence code can be applied in either scenario: variant is observed in trans with a pathogenic or likely pathogenic TP53 variant (phase confirmed) OR there are 3 or more observations with a pathogenic or likely pathogenic variant when phase is unknown; in the latter scenario, the variant must be seen with at least two different pathogenic or likely pathogenic TP53 variants. The TP53 VCEP applicability is not applicable.

Evidence considered

• The TP53 VCEP framework lists BP2 as not applicable. Its instructions describe BP2 scenarios as a phase-confirmed trans observation with a pathogenic or likely pathogenic TP53 variant, or at least three observations involving at least two different pathogenic or likely pathogenic TP53 variants when phase is unknown, but those scenarios are not usable under the explicit TP53 VCEP applicability designation.
• The framework identifies the TP53/Li-Fraumeni syndrome context as autosomal dominant.

Sources

Reasoning

The TP53 VCEP specification (cspec v2.4) explicitly marks BP3 (in-frame indel in a repetitive region without known function) as 'Not Applicable' for this framework. Additionally, c.266_267del is a frameshift variant, not an in-frame deletion, and therefore falls outside BP3's scope by variant type as well as by explicit VCEP exclusion.

VCEP rule

TP53 VCEP (cspec v2.4): BP3 = Not Applicable.

Evidence considered

• case_summary.json cspec criteria list: entry {"label": "BP3", "applicability": "Not Applicable"} under TP53 VCEP specifications v2.4.
• pvs1_variant_assessment.json: consequence_class=frameshift, not an in-frame repeat-region deletion, so BP3's repeat-region rationale is not applicable regardless of the explicit VCEP exclusion.

Sources

Reasoning

Same variant-type mismatch as PP3: NM_000546.6:c.266_267del is an out-of-frame frameshift deletion (p.Pro89LeufsTer59), not a missense, synonymous, intronic, or single amino-acid in-frame deletion variant. The TP53 VCEP BP4 rule (BayesDel/aGVGD for missense and single-aa in-frame deletions; SpliceAI <=0.1 for silent/intronic variants) does not apply to this variant class. SpliceAI max delta score is 0.116, which does not meet the BP4 SpliceAI <=0.1 threshold that applies to silent/intronic variants even if this variant were in that category, so BP4 could not be applied on splicing grounds regardless.

Rule

TP53 VCEP v2.4 PP3/BP4/BP7 flowchart - variant type (out-of-frame frameshift deletion) not covered by PP3/BP4/BP7 scope; adjudicated instead under PVS1.

Evidence considered

• Case summary normalization confirms this is a 2-nucleotide out-of-frame deletion causing a frameshift (p.Pro89LeufsTer59), not a variant class covered by the TP53 VCEP BP4 rule (missense BayesDel/aGVGD; single-aa in-frame deletion BayesDel; or silent/intronic SpliceAI<=0.1).
• TP53 VCEP Flowchart-for-application-of-PP3,-BP4,-and-BP7.pdf restricts BP4 to missense variants, single amino acid in-frame deletions, and silent/intronic variants outside the core splice motif; this frameshift deletion is none of those.
• SpliceAI Lookup max delta score for this variant was 0.116, above the BP4 silent/intronic threshold of <=0.1, so even under a hypothetical silent/intronic reading BP4 would not be met on splicing grounds.

Still missing

• No missense/BayesDel/aGVGD in silico path applies (variant is not missense).
• No single-amino-acid in-frame deletion BayesDel lookup applies (deletion is out-of-frame).
• Variant is not silent/intronic, so the SpliceAI<=0.1 BP4 path also does not apply as a primary basis, though the observed score (0.116) would not meet it anyway.

Sources

Reasoning

BP5 is not applicable under the TP53 VCEP specification.

VCEP rule

TP53 VCEP: BP5 is Not Applicable.

Evidence considered

• TP53 VCEP version 2.4 designates BP5 as Not Applicable.

Sources

Reasoning

BP6 is not applicable under the TP53 VCEP specification. The exact-variant ClinVar record has no expert-panel submission and no Benign/Likely benign assertion.

VCEP rule

TP53 VCEP: BP6 is Not Applicable. An exact-variant ClinVar Benign/Likely benign expert-panel assertion would otherwise be required for BP6 consideration.

Evidence considered

• TP53 VCEP version 2.4 designates BP6 as Not Applicable.
• ClinVar variation 3328161 has one Pathogenic single-submitter assertion from Ambry Genetics and zero expert-panel submissions; it cannot trigger BP6.

Sources

Reasoning

BP7 under the TP53 VCEP specification applies only to synonymous (silent) or intronic variants. NM_000546.6:c.266_267del is a coding frameshift deletion (p.Pro89LeufsTer59) affecting the reading frame, not a synonymous or intronic substitution, so BP7 is not applicable to this variant class regardless of the SpliceAI result.

Rule

TP53 VCEP v2.4 BP7 - restricted to synonymous/intronic variants; this variant class (frameshift deletion) is out of scope.

Evidence considered

• TP53 VCEP specification (cspec v2.4) restricts BP7 to synonymous (silent) or intronic variants with no predicted splicing impact; this variant is a coding frameshift deletion, not synonymous or intronic.

Sources