PTEN c.888T>A, p.(Cys296Ter)

Reasoning

NM_000314.8:c.888T>A is a nonsense substitution producing NP_000305.3:p.(Cys296Ter) (Mutalyzer translation in prefetch normalization: predicted protein 296 aa vs 403 aa wild-type, a ~26.6% C-terminal truncation). The governing framework is the ClinGen PTEN Expert Panel CSPEC v3.2 (framework_mode=vcep), whose PVS1 rule directs use of the PTEN-specific PVS1 decision tree. Per the tree (vcep_pvs1_decisiontree_pten and its index.txt guidance), a nonsense/frameshift variant with a stop codon at or 5' to p.D375 (c.1121) in the biologically-relevant transcript NM_000314.8 is predicted to undergo NMD and is assigned PVS1; only stop codons 3' to p.D375, not predicted to undergo NMD, are downgraded to PVS1_Moderate. Codon 296 is 5' to codon 375, and c.888 falls in exon 8 (c.802-1026) while the terminal exon 9 begins at c.1027, so the premature stop lies upstream of the final exon-exon junction and is not in the last exon — consistent with NMD prediction and with the generic SVI PVS1 framework (PMC6185798) requirement that >10% of the protein be removed (107/403 aa removed). NM_000314.8 is the CSPEC-preferred biologically-relevant transcript, and germline LoF is an established PTEN disease mechanism (pvs1_gene_context: eligible). The variant is absent from gnomAD v2.1, v4.1 and gnomAD-Canada, and SpliceAI predicts no splice impact (max delta 0.00), so the consequence is a simple nonsense change with no competing splice effect. All decision-tree conditions for full-strength PVS1 are met; no downgrade consideration applies.

Rule

ClinGen PTEN EP CSPEC v3.2 PVS1: Use PTEN PVS1 decision tree (stop codon at or 5' to p.D375 (c.1121) in biologically-relevant transcript, predicted NMD -> PVS1)

Evidence considered

• PTEN VCEP PVS1 decision tree (vcep_pvs1_decisiontree_pten, index.txt): nonsense/frameshift with stop codon or disruption at or 5' to p.D375 (c.1121), exon present in biologically-relevant transcript NM_000314.8 and predicted to undergo NMD -> PVS1; stop codon 3' to p.D375 not predicted to undergo NMD -> PVS1_Moderate
• ClinGen PTEN EP CSPEC v3.2 PVS1 rule (cspec): 'Use PTEN PVS1 decision tree'; defaultStrength Pathogenic Very Strong
• prefetch normalization (Mutalyzer): NM_000314.8:c.888T>A -> NP_000305.3:p.(Cys296Ter); predicted protein MTAII...ENGSL* (296 aa) vs wild-type 403 aa
• prefetch normalization exon structure (NM_000314.8): exon 8 = c.802-1026, exon 9 (last) = c.1027-*6458; c.888 therefore in exon 8, not the terminal exon; codon 296 is 5' to p.D375 (c.1121)
• pvs1_gene_context: germline loss-of-function is an established PTEN mechanism (CSPEC includes PVS1 guidance); PVS1 gene gate eligible
• pvs1_variant_assessment: variant class nonsense, not a canonical splice consensus variant
• gnomad_v2 / gnomad_v4 / gnomad_canada: variant absent from population databases
• spliceai: max delta score 0.00, no splice impact predicted; consequence is a simple nonsense substitution

Sources

Reasoning

PTEN VCEP v3.2 PM2 (Supporting) is met: the variant is absent from gnomAD v2.1 (exome, GRCh37, 10-89720737-T-A), gnomAD v4.1 (exome, GRCh38, 10-87960980-T-A), and gnomAD-Canada v1.0 (HostSeq genomes) — allele frequency 0, below the required <0.00001 (0.001%) overall and trivially below the <0.00002 (0.002%) subpopulation cap since zero alleles are present in any subpopulation. Absence was confirmed by direct variant-URL/API lookups with screenshots captured for gnomAD v2.1 and v4.1 (manifest ok=true). PM2 applies at Supporting strength per the VCEP specification.

Rule

PM2 (Supporting): Absent in population — databases present at <0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population; if multiple alleles are present within any subpopulation, allele frequency in that subpopulation must be <0.00002 (0.002%). ClinGen PTEN Expert Panel Specifications v3.2.

Evidence considered

• gnomAD v2.1 (exome, GRCh37) variant page for 10-89720737-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD v2.1.'; screenshot gnomad_v2.png captured (manifest ok=true).
• gnomAD v4.1 (exome, GRCh38) variant page for 10-87960980-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD v4.1.'; screenshot gnomad_v4.png captured (manifest ok=true).
• gnomAD-Canada v1.0 (HostSeq genomes) API for 10-87960980-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD-Canada v1.0.'
• ClinGen PTEN Expert Panel Specifications v3.2 (cspec doc 135637575): PM2 rule 'Absent in population. Databases present at <0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population. If multiple alleles are present within any subpopulation, allele frequency in that subpopulation must be <0.00002 (0.002%).' — satisfied at Supporting strength.

Still missing

• None for the Supporting-strength determination: absence confirmed across three large sequenced population datasets. Note that no allele counts are reportable for an absent variant (absence, not a nonzero frequency below threshold, is the observed state).

Sources

Reasoning

PS1 requires the same amino acid change as a previously established pathogenic variant produced by a different nucleotide change (PTEN EP: 'Same amino acid change as a previously established pathogenic variant regardless of nucleotide change'). The interrogated variant is c.888T>A producing p.(Cys296Ter) from codon TGT (c.886-888: TGT > TGA). No alternate single-nucleotide change at this codon can create the same stop: c.886T>A -> p.Ser296, c.887G>A -> p.Tyr296, c.888T>C -> p.Cys296 (synonymous), c.888T>G -> p.Trp296. The variant itself is therefore the only nucleotide change that produces p.Cys296Ter, and no previously established pathogenic variant with the same amino acid change from a different nucleotide change was identified in the case evidence (pm5_candidates.json reports zero comparator candidates). The ClinVar 'Pathogenic/Likely pathogenic' submissions refer to this exact variant and cannot serve as a 'previously established' same-change comparator, nor can another source's final classification be used to infer this criterion. SpliceAI shows no splicing impact (max delta 0.00), so the exonic-splicing caution in the PS1 rule does not alter this assessment. PS1 is therefore not met.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change OR different variant at same nucleotide position as a pathogenic splicing variant, where in silico models predict impact equal to or greater than the known pathogenic variant (PTEN EP v3.2, PS1, Strong).

Evidence considered

• ClinGen PTEN EP CSPEC v3.2 PS1 rule (Strong): same amino acid change as a previously established pathogenic variant regardless of nucleotide change; no such comparator variant exists for p.Cys296Ter.
• pm5_candidates.json: classic same-residue PM5/PS1 comparator search returned 0 candidates; consequence class is nonsense.
• Normalization/prefetch: codon context TGT at c.886-888 confirms c.888T>A (TGT>TGA) is the only single-nucleotide route to p.Cys296Ter.

Still missing

• No previously established pathogenic variant producing p.Cys296Ter via a different nucleotide change was identified; if such a variant is reported in the future, PS1 should be revisited.
• No alternate single-nucleotide substitution at codon 296 (TGT) creates the same stop codon, so no same-amino-acid comparator is even theoretically available via a single nucleotide change.

Sources

Reasoning

Governing framework is the ClinGen PTEN Expert Panel specification v3.2 (CSPEC doc 135637575; framework_mode=vcep). PS3_Strong requires an RNA, mini-gene, or other assay demonstrating an impact on splicing; PS3_Moderate requires Mighell et al. 2018 (PMID: 29706350) Table S2 Cum_score <= -1.11 with High_conf TRUE (variant must be present in Table S2 columns A/B); PS3_Supporting requires lipid phosphatase activity <50% of wild-type or an abnormal in vitro cellular assay/transgenic model, with a catalytic-dead control (e.g., p.C124S) and >=3 biological replicates. The variant in question is NM_000314.8:c.888T>A, NP_000305.3:p.(Cys296Ter) - a stop-gain (nonsense) variant, not a missense substitution. Direct lookup in the VCEP assay file (mmc2.xlsx, Table S2) shows no entry for C296*/c.888T>A (the massively parallel phosphatase assay covers missense variants only), so the PS3_Moderate calibration cannot be applied. No RNA/mini-gene assay of this variant exists (SpliceAI max delta 0.00 is in silico only). No other variant-specific functional study (phosphatase, pAKT/PTEN expression, localization, or cellular phenotype assay) is present in the case evidence; all 5 available full texts (PMID 11237521, 17218262, 25394175, 26492180, 27324988) contain no mention of c.888T>A/p.Cys296Ter. OncoKB's curated 'Likely Loss-of-function / Likely Oncogenic' label for PTEN C296* is a database classification, not a validated functional assay, and per policy a criterion cannot be inferred from another source's final classification. PS3 is therefore not met; the truncating nature of p.Cys296Ter is appropriately addressed by the PVS1 decision tree (null-variant group), not by PS3.

Rule

ClinGen PTEN EP v3.2 (CSPEC doc 135637575), PS3 Applicable: Strong - RNA, mini-gene, or other assay demonstrating an impact on splicing; Moderate - Mighell et al. 2018 (PMID: 29706350) Table S2 Cum_score <= -1.11 with High_conf TRUE (variant listed in columns A/B); Supporting - lipid phosphatase activity <50% of wild-type or abnormal in vitro cellular assay/transgenic model with phenotype different from wild-type (assays must include catalytic-dead control such as p.C124S and at least three biological replicates).

Evidence considered

• ClinGen PTEN EP v3.2 (CSPEC doc 135637575) PS3 rules: PS3_Strong = RNA/mini-gene/other assay shows splicing impact; PS3_Moderate = Mighell 2018 (PMID: 29706350) Table S2 Cum_score <= -1.11 with High_conf TRUE; PS3_Supporting = lipid phosphatase activity <50% WT or abnormal in vitro cellular assay/transgenic model (catalytic-dead control + >=3 biological replicates required).
• vcep_mmc2 (Mighell 2018 supplement, PMID: 29706350): p.Cys296Ter is absent from Table S2 (assay rows are Type='missense' only); no Cum_score or High_conf value exists, so the PS3_Moderate threshold (-1.11) cannot be applied to this stop-gain variant.
• No variant-specific functional study exists in the evidence bundle; grep of all 5 available full texts (PMID 11237521, 17218262, 25394175, 26492180, 27324988) for c.888/Cys296/C296 returned zero hits - functional-theme papers are gene-level only.
• oncokb (PTEN C296*): curated 'Likely Loss-of-function' biological effect and 'Likely Oncogenic' label - a database classification, not a well-established functional study; insufficient for PS3 per the EP specification's assay requirements.
• spliceai: max delta score 0.00 (in silico prediction only); does not constitute the RNA/mini-gene assay required for PS3_Strong.

Still missing

• No variant-specific validated functional data for p.Cys296Ter (lipid phosphatase activity assay with catalytic-dead control and >=3 biological replicates, RNA/mini-gene splice assay, or cellular phenotype assay) is present in the case evidence; such a study would be required to apply PS3 under the PTEN EP specification.

Sources

Reasoning

PTEN EP v3.2 defines PM1 as: 'Located in a mutational hot spot and/or critical and well-established functional domain. Defined to include residues in catalytic motifs: 90-94, 123-130, 166-168 (NP_000305.3)' (WPD loop, P-loop/phosphatase core, TI-loop). Residue p.Cys296 (C2-domain region, exon 8) is outside all three defined catalytic motifs, so the PTEN-specific PM1 definition is not satisfied on domain grounds. The Cancer Hotspots pipeline lookup (evidence.json) returned no statistically significant hotspot at PTEN C296, providing no hotspot-based support either. PM1 is therefore not met under the governing VCEP specification.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain. Defined to include residues in catalytic motifs: 90-94, 123-130, 166-168 (NP_000305.3) (PTEN EP v3.2, PM1, Moderate).

Evidence considered

• ClinGen PTEN EP CSPEC v3.2 PM1 rule (Moderate): PM1 residues defined as catalytic motifs 90-94, 123-130, 166-168 (NP_000305.3); p.Cys296 is not among them.
• Evidence bundle hotspots lookup: no statistically significant hotspot reported for PTEN C296 (contextual, non-citable pipeline evidence).

Still missing

• Residue 296 lies outside the PTEN EP-defined PM1 catalytic motif residues (90-94, 123-130, 166-168); no other well-established functional domain definition covering residue 296 was provided in the case bundle.
• Cancer Hotspots lookup (pipeline evidence, not a source_registry entry) found no statistically significant hotspot at residue 296.

Sources

Reasoning

The ClinGen PTEN EP CSPEC v3.2 PM4 rule applies to protein length changes from in-frame deletions/insertions in a non-repeat region (specifically in-frame indels impacting at least one residue in a catalytic motif, see PM1) or stop-loss variants causing protein extension. NM_000314.8:c.888T>A is a nonsense substitution (p.Cys296Ter) that causes premature truncation, not an in-frame deletion/insertion and not a stop-loss extension; the protein is shortened rather than extended. PM4 therefore does not apply to this variant's consequence class, and the premature termination is already captured by PVS1. PM4 is not met.

Rule

ClinGen PTEN EP CSPEC v3.2 PM4: in-frame indels in non-repeat region (catalytic motif) or stop-loss/extension variants

Evidence considered

• ClinGen PTEN EP CSPEC v3.2 PM4 rule (cspec): 'Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. Applies to in-frame insertions or deletions impacting at least one residue in a catalytic motif (see PM1), and variants causing protein extension.'
• prefetch normalization: variant consequence is nonsense (p.Cys296Ter), not an in-frame indel or stop-loss

Sources

Reasoning

PTEN VCEP v3.2 BA1 (Stand Alone) requires gnomAD filtering allele frequency > 0.00056 (0.056%). The variant NM_000314.8:c.888T>A (p.Cys296Ter) is absent from gnomAD v2.1 (10-89720737-T-A, GRCh37 exomes), gnomAD v4.1 (10-87960980-T-A, GRCh38 exomes), and gnomAD-Canada v1.0 (HostSeq genomes) — no alleles observed, so allele frequency = 0, far below the BA1 threshold of 0.00056. BA1 is not met.

Rule

BA1 (Stand Alone): gnomAD Filtering allele frequency >0.00056 (0.056%). ClinGen PTEN Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PTEN Version 3.2.

Evidence considered

• gnomAD v2.1 (exome, GRCh37) variant page for 10-89720737-T-A: search_status 'absent', found=false — no allele frequency data; evidence_sentence 'Absent from gnomAD v2.1.'
• gnomAD v4.1 (exome, GRCh38) variant page for 10-87960980-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD v4.1.'
• gnomAD-Canada v1.0 (HostSeq genomes) API for 10-87960980-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD-Canada v1.0.'
• ClinGen PTEN Expert Panel Specifications v3.2 (cspec doc 135637575): BA1 rule 'gnomAD Filtering allele frequency >0.00056 (0.056%)' — threshold not reached (AF=0).

Still missing

• Variant is absent from the queried gnomAD datasets; no allele count/number is reportable because no record exists. Absence was confirmed via direct variant-URL lookup (gnomad_v2, gnomad_v4, gnomad_canada source_registry keys).

Sources

Reasoning

PTEN VCEP v3.2 BS1 (Strong) requires gnomAD filtering allele frequency 0.000043–0.00056 (0.0043%–0.056%); the Supporting tier requires 0.0000043–0.000043 (0.00043%–0.0043%). The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0 (AF = 0), which is below even the lowest BS1 supporting tier threshold of 0.0000043. BS1 is not met at any strength.

Rule

BS1 (Strong): gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%); BS1 (Supporting): allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%). ClinGen PTEN Expert Panel Specifications v3.2.

Evidence considered

• gnomAD v2.1 (exome, GRCh37) variant page for 10-89720737-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD v2.1.'
• gnomAD v4.1 (exome, GRCh38) variant page for 10-87960980-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD v4.1.'
• gnomAD-Canada v1.0 (HostSeq genomes) API for 10-87960980-T-A: search_status 'absent', found=false — evidence_sentence 'Absent from gnomAD-Canada v1.0.'
• ClinGen PTEN Expert Panel Specifications v3.2 (cspec doc 135637575): BS1 rule 'gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%)' (Strong) and 'Allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%)' (Supporting) — neither interval reached (AF=0).

Still missing

• No additional large-population databases (e.g., 1000 Genomes, TOPMed, ALFA) were queried beyond gnomAD v2.1/v4.1 and gnomAD-Canada v1.0; absence in these three is sufficient to exclude BS1, but the full gnomAD genome dataset (v2.1 genomes / v4 genomes) was not separately interrogated.

Sources

Reasoning

PTEN VCEP v3.2 BS2 (Strong) requires observation of the variant in the homozygous state in a healthy or PHTS-unaffected individual (one observation if homozygosity confirmed, two if not; Supporting tier: two homozygous observations with no clinical data). The variant is entirely absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0 — there are zero heterozygous or homozygous observations in any queried population cohort, so BS2 cannot be met. No healthy-carrier or PHTS-unaffected homozygote data were identified elsewhere in the case bundle.

Rule

BS2 (Strong): Observed in the homozygous state in a healthy or PHTS-unaffected individual (one observation if homozygous status confirmed, two if not confirmed; Supporting if BS1 also applied). ClinGen PTEN Expert Panel Specifications v3.2.

Evidence considered

• gnomAD v2.1 (exome, GRCh37) variant page for 10-89720737-T-A: search_status 'absent', found=false — no homozygote counts because no record exists.
• gnomAD v4.1 (exome, GRCh38) variant page for 10-87960980-T-A: search_status 'absent', found=false — no homozygote counts.
• gnomAD-Canada v1.0 (HostSeq genomes) API for 10-87960980-T-A: search_status 'absent', found=false — no homozygote counts.
• ClinGen PTEN Expert Panel Specifications v3.2 (cspec doc 135637575): BS2 rule 'Observed in the homozygous state in a healthy or PHTS-unaffected individual...' — prerequisite observation absent.

Still missing

• No homozygous (or any) observations exist in queried population databases; no clinical-laboratory homozygote reports were found in the case evidence bundle (ClinVar submission records list only affected-patient classifications and were not used to infer population status).

Sources

Reasoning

Under the ClinGen PTEN EP specification v3.2 (CSPEC doc 135637575), BS3_Strong applies to intronic or synonymous variants with an RNA, mini-gene, or other splicing assay demonstrating no splicing impact; BS3_Supporting requires an in vitro or in vivo functional study showing no damaging effect on protein function, including Mighell et al. 2018 (PMID: 29706350) Table S2 Cum_score > 0 (missense variants, High_conf TRUE). For p.Cys296Ter, a stop-gain variant, no functional evidence of normal/undamaged protein function exists: the variant is absent from Table S2 of the VCEP assay file (missense-only assay), so the BS3_Supporting calibration cannot be applied; no RNA/mini-gene assay was performed (SpliceAI max delta 0.00 is an in silico prediction and does not satisfy the assay requirement); and the variant is a protein-truncating change, directionally inconsistent with a benign functional outcome. The determination is BS3 not met, with no evidence supporting a lack of damaging effect.

Rule

ClinGen PTEN EP v3.2 (CSPEC doc 135637575), BS3 Applicable: Strong - for intronic or synonymous variants, RNA, mini-gene, or other assay demonstrating no impact on splicing; Supporting - in vitro or in vivo functional study showing no damaging effect on protein function, incl. Mighell et al. 2018 (PMID: 29706350) Table S2 Cum_score > 0 (variant listed with High_conf TRUE); for missense, lipid phosphatase activity comparable to wild type plus a second domain-appropriate assay with no statistically significant difference (catalytic-dead control + >=3 biological replicates).

Evidence considered

• ClinGen PTEN EP v3.2 (CSPEC doc 135637575) BS3 rules: BS3_Strong = for intronic or synonymous variants, RNA/mini-gene/other assay demonstrating no splicing impact; BS3_Supporting = in vitro/in vivo functional study showing no damaging effect, incl. Mighell 2018 (PMID: 29706350) Table S2 Cum_score > 0 with High_conf TRUE.
• vcep_mmc2 (Mighell 2018 supplement, PMID: 29706350): no Table S2 entry exists for p.Cys296Ter (stop-gain; assay covers missense only), so the BS3_Supporting Cum_score > 0 calibration is inapplicable.
• spliceai: max delta score 0.00 is an in silico prediction, not the RNA/mini-gene or other functional assay BS3 requires; additionally, BS3_Strong is scoped to intronic/synonymous variants, and p.Cys296Ter is a nonsense variant.
• oncokb (PTEN C296*): curated 'Likely Loss-of-function' annotation is not functional evidence of normal function; it cannot support BS3.
• No full-text publication in the case bundle mentions c.888T>A/p.Cys296Ter (all 5 available full texts screened), so no literature-based functional evidence for BS3 exists.

Still missing

• A functional study demonstrating no damaging effect on protein function (e.g., Mighell Cum_score > 0 - unavailable because the assay covers missense variants only - or an RNA/mini-gene assay showing no splicing impact) would be required for BS3; none is present in the case evidence.

Sources

Reasoning

Governing framework is the ClinGen PTEN Expert Panel Specification v3.2 (CSPEC doc 135637575; framework_mode=vcep). PS2 requires a de novo observation with both maternity and paternity confirmed (Strong), or two proven/four assumed/one proven+two assumed de novo observations (Very Strong) in a patient with the disease and no family history. The consolidated case-evidence bundle contains no proband-level data whatsoever for NM_000314.8:c.888T>A (p.Cys296Ter): no parental testing results, no maternity/paternity confirmation, no de novo observation, and no family-history documentation for any observation of this variant. The only literature full texts (PMID:26492180, PMID:27324988, the two PMIDs cited by the LabCorp ClinVar submission SCV001337949) are somatic tumor-sequencing studies that do not mention this variant; PMID:11237521 is a gene review whose segregation language concerns other PTEN mutations. ClinVar VCV000917617 is classified Pathogenic/Likely pathogenic by 3 single-submitter labs, but no per-submission criteria or observed-in/inheritance fields are captured, and a final classification from another source must not be used to infer de novo status. Evidence is therefore insufficient; PS2 is not assessable. Human review of the SCV-level records (e.g., SCV001337949, SCV002685344, SCV004189569) is flagged because submitting laboratories may have applied uncaptured de novo evidence.

Rule

PS2 Strong: 'De novo (both maternity and paternity confirmed) observation in a patient with the disease and no family history.' PS2 Very Strong: 'Two proven OR four assumed OR one proven + two assumed de novo observations in a patient with the disease and no family history.' (ClinGen PTEN EP v3.2)

Still missing

• No parental testing results (maternity and/or paternity confirmation) for any observation of c.888T>A
• No documented de novo occurrence of c.888T>A in any source in the bundle
• No proband phenotype or family-history data (disease present, no family history) for any carrier
• ClinVar per-submission evidence fields (observed-in, inheritance, criteria) not captured in the bundle

Sources

Reasoning

ClinGen PTEN Expert Panel v3.2 (cspec) defines PS4 via two arms: (1) proband phenotype specificity scores summed across independent probands (adults: 1 point per proband with Cleveland Clinic score >=30, 0.5 point for 25-29; children: 1 point per proband with pediatric score >=5, 0.5 point for score of 4; Supporting 1-1.5, Moderate 2-3.5, Strong 4-15.5, Very Strong >=16), or (2) a sufficiently powered case-control study showing odds ratio >2, p<0.05, 95% CI lower limit >1.5 for a PHTS component phenotype. Neither arm is evaluable for c.888T>A: the case bundle contains no proband phenotype data (no Cleveland Clinic or pediatric scores, no enumerated probands with PHTS features), and no case-control enrichment study for this variant. The ClinVar record (clinvar, VCV000917617) carries only submission-level condition labels (Cowden syndrome, Cowden syndrome 1, Hereditary cancer-predisposing syndrome) from 3 ordinary laboratory submissions and contains no scored phenotypes. The two ClinVar-linked publications (PMID:26492180, PMID:27324988) were read and neither mentions c.888T>A/p.Cys296Ter: 26492180 is a somatic tumor sequencing study of mixed endometrial carcinomas (PTEN appears only as a gene in the somatic panel), and 27324988 never mentions PTEN. Aggregate laboratory classifications and condition labels cannot substitute for variant-specific case-control or proband-specificity evidence, so PS4 is not assessed pending data.

Rule

Probands with specificity score >=16 (Very Strong); score 4-15.5 OR prevalence of the variant in affected individuals significantly increased vs controls (Strong); score 2-3.5 (Moderate); score 1-1.5 (Supporting) - ClinGen PTEN EP v3.2. Case-control arm: OR>2 with p<0.05 and 95% CI lower limit >1.5.

Evidence considered

• ClinGen PTEN EP v3.2 PS4 rule text (cspec, doc 135637575): proband specificity scores (Tan 2011 Cleveland Clinic score, pediatric score) or OR>2 case-control enrichment required; neither data type present in bundle
• ClinVar VCV000917617 (clinvar): 3 ordinary laboratory submissions (2 Pathogenic, 1 Likely pathogenic; LabCorp, Myriad, Ambry); condition labels only, no proband phenotypes; review_stars=2; expert_panel_submissions=0
• Full-text search of PMID:26492180 and PMID:27324988 for c.888T>A / p.Cys296Ter / C296*: no variant mention found; no germline PHTS case-control data

Still missing

• No proband phenotype data (Cleveland Clinic score per Tan 2011 / pediatric PHTS score) for any carrier of c.888T>A, so PTEN EP specificity scores cannot be computed
• No case-control enrichment study (OR>2, p<0.05, lower CI >1.5) for c.888T>A in PHTS or any PHTS component phenotype
• No enumeration of independent probands carrying c.888T>A with PHTS phenotypes

Sources

Reasoning

Per ClinGen PTEN EP v3.2, PM6 Moderate requires an assumed de novo occurrence (maternity/paternity not confirmed) in a proband with the disease and no family history; PM6 Strong requires two presumed de novo probands or one presumed de novo proband with a highly specific phenotype (counts toward PS4). No assumed/presumed de novo observation of c.888T>A is documented anywhere in the bundle, and no proband family-history or phenotype-specificity data exist. The PM6_Strong pathway through a highly specific phenotype cannot be evaluated because the PTEN VCEP marks PP4 as Not Applicable and no phenotype specificity score (PS4 scoring) is present in the bundle. Evidence is insufficient; PM6 is not assessable. Human review flagged because SCV-level submissions might contain uncaptured presumed-de-novo observations.

Rule

PM6 Moderate: 'Assumed de novo, but without confirmation of paternity and maternity, in proband with the disease and no family history.' PM6 Strong: 'Two probands with presumed de novo occurrence (maternity/paternity not confirmed) with the disease and no family history. May also be used for a proband with presumed de novo occurrence for an individual with a highly specific phenotype (meets criteria to count towards PS4).' PM6 Very Strong: 'Two proven OR four assumed OR one proven + two assumed de novo observations...' (ClinGen PTEN EP v3.2)

Still missing

• No assumed de novo observation (paternity/maternity not confirmed) documented for c.888T>A
• No family-history data (disease present with no family history) for any proband carrying the variant
• Phenotype specificity (PS4-counting) pathway not evaluable: PP4 Not Applicable per PTEN VCEP and no specificity score in bundle
• ClinVar per-submission evidence fields not captured in the bundle

Sources

Reasoning

Per ClinGen PTEN EP v3.2, PP1 Supporting requires co-segregation with disease in multiple affected family members with 3-4 meioses, Moderate 5-6 meioses, Strong >=7 meioses across at least two families. No family segregation study, pedigree, or meiosis count involving c.888T>A exists in the case bundle or in the available literature full texts. PMID:11237521 (PTEN review) contains the sentence 'These mutations segregated with affected family members...' but this concerns other PTEN germline mutations from the cited Marsh et al. 1997 studies, not c.888T>A, and therefore cannot support PP1 for this variant. Zero meioses are documented for this variant; evidence is insufficient; PP1 is not assessable. Human review flagged in case SCV-level submissions contain segregation data not captured in the bundle.

Rule

PP1 Supporting: 'Co-segregation with disease in multiple affected family members, with 3 or 4 meioses observed.' PP1 Moderate: '...5 or 6 meioses observed.' PP1 Strong: '...>=7 meioses observed across at least two families.' (ClinGen PTEN EP v3.2)

Still missing

• No family segregation study or pedigree for c.888T>A; zero meioses documented
• No affected-family-member genotyping reported in any available source
• No multi-generation families with the variant in the bundle or literature full texts

Sources

Reasoning

Per ClinGen PTEN EP v3.2, BS4 Strong requires lack of segregation in affected members of two or more families and BS4 Supporting requires lack of segregation in affected members of one family. BS4 is a positive-evidence criterion (documented non-segregation), and absence of segregation data cannot be treated as lack of segregation. No affected non-carrier, unaffected carrier, or other non-segregation observation for c.888T>A is documented in the bundle or in any reviewed publication. Evidence is insufficient; BS4 is not assessable.

Rule

BS4 Strong: 'Lack of segregation in affected members of two or more families.' BS4 Supporting: 'Lack of segregation in affected members of one family.' (ClinGen PTEN EP v3.2)

Still missing

• No documented non-segregation observation (affected family member without the variant, or unaffected member with the variant) for any family
• No family-level genotype-phenotype data for c.888T>A in any available source

Sources

Reasoning

Under ClinGen PTEN EP v3.2, BP2 (Benign Supporting) is applicable and requires either (a) the variant observed in trans with a pathogenic or likely pathogenic PTEN variant, or (b) at least three observations of the variant in cis and/or phase unknown with different pathogenic/likely pathogenic PTEN variants. No such allelic/phase evidence exists anywhere in the case bundle. ClinVar variation 917617 has three clinical submissions (SCV001337949 LabCorp Likely pathogenic 2020, Cowden syndrome; SCV004189569 Myriad Pathogenic 2023, origin unknown; SCV002685344 Ambry Pathogenic 2017, germline), none of which report a second PTEN variant, parental/offspring testing, or phase. The variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada (no population observations, hence no homozygous or compound observations). The two ClinVar-cited publications (PMID:26492180, PMID:27324988) were checked in full text and do not contain c.888T>A / p.Cys296Ter at all (gene-level somatic tumor papers), so no trans/cis observation can be extracted from them. The required phase/allelism data are therefore entirely missing and the criterion cannot be evaluated: this is a data gap, not evidence against the variant. Manual curation (e.g., full ClinVar submission details, supplementary variant tables, parental testing results) would be needed to resolve.

Rule

ClinGen PTEN Expert Panel Specifications v3.2 (cspec doc 135637575), BP2 at Supporting strength: 'Observed in trans with a pathogenic or likely pathogenic PTEN variant OR at least three observations in cis and/or phase unknown with different pathogenic/likely pathogenic PTEN variants.'

Evidence considered

• ClinGen PTEN Expert Panel Specifications v3.2 (cspec): BP2 rule (Supporting) 'Observed in trans with a pathogenic or likely pathogenic PTEN variant OR at least three observations in cis and/or phase unknown with different pathogenic/likely pathogenic PTEN variants.'; instructionsToUse: 'The other variant may be either pathogenic or likely pathogenic. This rule may also be applied for at least three observations of the variant in cis or unknown phase with different pathogenic or likely pathogenic PTEN variants.'
• ClinVar variation 917617 (source key 'clinvar'): submission audit lists 3 germline clinical submissions (LabCorp LP, Myriad P, Ambry P) with no second-variant or phase information; evidence_sentence 'This variant has been reported in ClinVar as Pathogenic (2 clinical laboratories) and as Likely pathogenic (1 clinical laboratory). (ClinVarID = 917617)'.
• PMID:26492180 (Köbel et al. 2016, mixed endometrial carcinomas): retrieved full text contains no occurrence of the variant; PTEN discussed only at gene level in a somatic tumor cohort; no germline phase data.
• PMID:27324988 (Cheng et al. 2016, prostate cancer tNGS pilot): retrieved full text contains no occurrence of the variant; PTEN not among variants described; no germline phase data.
• gnomAD v2.1 / v4.1 / gnomAD-Canada v1.0 (source keys 'gnomad_v2', 'gnomad_v4', 'gnomad_canada'): variant absent, so no population-based homozygous/compound observations exist.

Still missing

• No phase (trans/cis) or compound-heterozygosity observation of c.888T>A with any pathogenic/likely pathogenic PTEN variant reported in ClinVar submissions (SCV001337949, SCV004189569, SCV002685344) or elsewhere in the evidence bundle.
• No parental/offspring segregation-testing results describing a second PTEN allele for any proband carrying c.888T>A.
• ClinVar-cited publications PMID:26492180 and PMID:27324988 do not mention c.888T>A in their retrieved full texts; any variant-specific entries would reside in supplementary variant tables (Table 2/3 of 26492180; Supp Tables 2/3 of 27324988) that were not retrieved as text.

Sources

Reasoning

ClinGen PTEN EP v3.2 BP5 (Benign Supporting) requires the variant to be found in a case with an alternate molecular basis for disease, where the other gene/disorder is highly penetrant AND the patient's personal/family history demonstrates no overlap with PTEN. The case bundle contains no carrier-level clinical data for c.888T>A: no documented alternate molecular cause (no co-occurring pathogenic variant in another highly penetrant gene, no co-occurrence/phase data), and no personal/family history information to evaluate phenotype overlap. The ClinVar submissions provide no such case detail, and the two full texts reviewed (PMID:26492180, PMID:27324988) contain no carrier of this variant. BP5 cannot be evaluated without case-level co-occurrence and phenotype data.

Rule

Variant found in a case with an alternate molecular basis for disease; other gene/disorder must be considered highly penetrant AND patient's personal/family history must demonstrate no overlap between other gene and PTEN (Benign Supporting).

Evidence considered

• ClinGen PTEN EP v3.2 BP5 rule text (cspec): requires variant found in a case with an alternate molecular basis (highly penetrant other gene, no phenotype overlap with PTEN); no such case data present in bundle
• ClinVar VCV000917617 (clinvar): submissions contain no co-occurring variant or family history detail
• Full-text search of PMID:26492180 and PMID:27324988: no carrier of c.888T>A identified, so no alternate-molecular-basis observations applicable

Still missing

• No case-level data showing c.888T>A in an individual with a documented alternate molecular basis of disease
• No co-occurrence/phase information with pathogenic variants in other highly penetrant genes
• No personal/family history data for carriers to assess phenotype overlap with PTEN

Sources