PTEN c.674_675dup, p.(Ser226IlefsTer31)

Reasoning

NM_000314.6:c.674_675dupAT (normalized c.674_675dup) is a 2bp duplication in exon 7 of 9 causing a frameshift, NP_000305.3:p.(Ser226IlefsTer31). The premature termination codon falls in exon 7, well upstream of the last exon-exon junction (exon 9), so the transcript is predicted to be a substrate for nonsense-mediated decay (NMD) under standard NMD rules. Applying the PTEN VCEP PVS1 decision tree (PVS1_DecisionTree_PTEN.pdf, using biologically-relevant transcript NM_000314.8): for variants predicted to undergo NMD, the tree asks whether the stop codon/disruption occurs at or 5' to p.D375 (c.1121). The new stop here occurs at predicted residue 256 (well 5' of D375/c.1121), so the decision tree assigns PVS1 at Very Strong strength.

Rule

PTEN VCEP PVS1 (cspec v3.2): 'Use PTEN PVS1 decision tree' -> NMD-predicted frameshift with stop at or 5' to p.D375 (c.1121) = PVS1 Very Strong.

Evidence considered

• PTEN VCEP PVS1 decision tree (PVS1_DecisionTree_PTEN.pdf) markdown extract: 'Predicted to undergo NMD: Stop codon or disruption at or 5' to Exon is present in biologically-relevant transcript NM_000314.8 PVS1 p.D375 (c.1121)... Not predicted to undergo NMD: Stop codon 3' to p.D375 (c.1121) PVS1_Moderate'.
• Mutalyzer/VariantValidator normalization: NM_000314.6:c.674_675dup gives protein consequence NP_000305.3:p.(Ser226IlefsTer31); variant_exonic_positions places the variant in exon 7 of the 9-exon transcript (NC_000010.10/NC_000010.11 start_exon=end_exon=7).
• Predicted protein sequence from prefetch shows the frameshifted ORF terminates at predicted residue position 256 (position_last_predicted=256), which is 5' to the p.D375/c.1121 threshold defined by the PTEN VCEP.
• Because the new stop is in exon 7 of 9 and far upstream of the last exon-exon junction, the transcript meets standard NMD-competent criteria (PTC >50-55 nt upstream of the final exon-exon junction), matching the VCEP tree's 'Predicted to undergo NMD' branch.

Sources

Reasoning

PM2_Supporting is met. This variant is absent from the queried gnomAD v2.1 and v4.1 datasets and is also absent from gnomAD-Canada v1.0, satisfying the PTEN Expert Panel population-absence requirement.

Rule

PTEN EP v3.2 PM2_Supporting: absent from population databases or gnomAD AF <0.00001; if multiple alleles are present in a subpopulation, subpopulation AF <0.00002.

Evidence considered

• ClinGen PTEN Expert Panel Specifications version 3.2 apply PM2 at Supporting strength for absence from population databases, or allele frequency <0.00001 (0.001%) in gnomAD or another large sequenced population; where multiple alleles occur in a subpopulation, subpopulation allele frequency must be <0.00002 (0.002%).
• The normalized variant 10-89717644-G-GAT is absent from gnomAD v2.1; 10-87957887-G-GAT is absent from gnomAD v4.1 and gnomAD-Canada v1.0.

Sources

Reasoning

BA1 is not met. The variant is absent from the queried gnomAD v2.1 and v4.1 datasets, therefore it does not exceed the PTEN Expert Panel BA1 filtering allele-frequency threshold.

Rule

PTEN EP v3.2 BA1: gnomAD filtering allele frequency >0.00056 (0.056%).

Evidence considered

• ClinGen PTEN Expert Panel Specifications version 3.2 define BA1 as gnomAD filtering allele frequency >0.00056 (0.056%).
• The normalized variant 10-89717644-G-GAT is absent from gnomAD v2.1, and 10-87957887-G-GAT is absent from gnomAD v4.1.

Sources

Reasoning

BS1 is not met. The variant is absent from the queried gnomAD v2.1 and v4.1 datasets, so no filtering allele frequency reaches either PTEN Expert Panel BS1 range.

Rule

PTEN EP v3.2 BS1: Strong at filtering AF 0.000043 to 0.00056; Supporting at AF 0.0000043 to 0.000043.

Evidence considered

• ClinGen PTEN Expert Panel Specifications version 3.2 define BS1_Strong as gnomAD filtering allele frequency 0.000043 to 0.00056 and BS1_Supporting as 0.0000043 to 0.000043.
• The normalized variant is absent from gnomAD v2.1 and gnomAD v4.1.

Sources

Reasoning

No case-specific observation documents the variant in an affected proband with both parents tested negative, parentage confirmed, and no relevant family history; PS2 cannot be assigned.

Rule

PTEN EP PS2: Strong requires confirmed maternity and paternity for a de novo observation in an affected patient without family history; Very Strong requires two PS2 observations or two PM6 plus one PS2 observation.

Evidence considered

• The PTEN Expert Panel requires a de novo observation in a patient with disease, no family history, and confirmation of both maternity and paternity for PS2 at Strong; PS2_Very Strong requires two PS2 observations or two PM6 plus one PS2 observation.
• The consolidated case-evidence bundle supplies no case-level proband, parental-testing, parentage, family-history, or de novo observation data for this variant.

Still missing

• Obtain the proband phenotype and PTEN testing result.
• Obtain parental PTEN results and maternity/paternity confirmation.
• Document family history and additional independent proven de novo observations, if present.

Sources

Reasoning

NM_000314.6:c.674_675dupAT (p.Ser226IlefsTer31) is a frameshift duplication, not a missense or splicing variant. The PTEN VCEP (cspec v3.2) PS3 rule ties Moderate/Supporting strength to phosphatase activity scores from Mighell et al. 2018 (PMID: 29706350), whose saturation mutagenesis dataset (mmc2.xlsx Table S2) covers only missense substitutions, and ties Strong strength to RNA/mini-gene splicing assay evidence, which does not apply here since this variant is not a splice-region change (SpliceAI predicts no significant splice impact, max delta 0.10). No in vitro or in vivo functional study of this specific frameshift variant or its truncated protein product was identified anywhere in the case bundle, source registry, or reviewed literature.

Rule

PTEN VCEP (ClinGen cspec v3.2) PS3: Strong for RNA/mini-gene splicing assay showing splicing impact; Moderate for phosphatase activity <=-1.11 per Mighell et al. 2018 (PMID: 29706350); Supporting for phosphatase activity <50% of wild-type or other abnormal in vitro/transgenic model. Mighell dataset (mmc2.xlsx) is restricted to missense variants and is not applicable to this frameshift duplication.

Still missing

• No functional (phosphatase activity, splicing, or other in vitro/in vivo) assay data available for this specific frameshift duplication variant; the VCEP Mighell et al. 2018 dataset used for PTEN PS3/BS3 applies only to missense variants.

Reasoning

The PTEN Expert Panel requires either a sufficiently powered case-control result for a PHTS component phenotype or qualifying, independent phenotype-specific probands. No case-control enrichment statistics or qualifying proband phenotype-specificity score is available for this exact variant.

Rule

PTEN EP PS4: phenotype-specificity scores across independent probands determine strength; alternatively, a sufficiently powered PHTS component-phenotype case-control study may support strong evidence. PS4 cannot be combined with PP4.

Evidence considered

• PTEN EP PS4 permits phenotype-specificity scoring across independent probands: adult CC score >=30 contributes 1 point and 25-29 contributes 0.5 points; pediatric score >=5 contributes 1 point and score 4 contributes 0.5 points under the stated restrictions. Strength requires a total score of 1-1.5 (supporting), 2-3.5 (moderate), 4-15.5 (strong), or >=16 (very strong).
• The PTEN EP also permits strong PS4 from a sufficiently powered case-control study with odds ratio >2, p<0.05, and 95% CI lower limit >1.5 for a PHTS component phenotype.
• PMID:21194675 was searched for NM_000314.6:c.674_675dup, c.674_675dup, p.Ser226IlefsTer31, and p.S226Ifs*31; the publication reports aggregate PTEN mutation cohorts and does not report this exact variant.

Still missing

• Independent proband-level phenotypes and adult Cleveland Clinic or pediatric phenotype scores for this exact variant are not available.
• No sufficiently powered case-control study reporting an odds ratio, p value, and confidence interval for this exact variant is available.

Sources

Reasoning

No case-specific presumed de novo observation is documented; therefore PM6 cannot be assigned.

Rule

PTEN EP PM6: Moderate requires an assumed de novo occurrence in an affected proband without family history and without confirmed maternity/paternity; Strong and Very Strong require the specified additional observations or phenotype specificity.

Evidence considered

• The PTEN Expert Panel permits PM6 for assumed de novo occurrence without confirmation of maternity and paternity in an affected proband without family history; higher strengths require the specified number of independent presumed/proven de novo observations or a highly specific phenotype.
• The consolidated case-evidence bundle supplies no case-level proband, parental-testing, parentage, family-history, or presumed de novo observation data for this variant.

Still missing

• Obtain the proband phenotype and PTEN testing result.
• Obtain parental PTEN testing showing the variant absent in both parents, with parentage status recorded.
• Document family history and any additional independent presumed de novo observations.

Sources

Reasoning

No informative affected relatives, genotypes, phenotypes, pedigrees, or meiosis count are documented; PP1 cannot be assigned.

Rule

PTEN EP PP1: Supporting for 3-4 meioses, Moderate for 5-6 meioses, and Strong for at least 7 meioses across at least two families.

Evidence considered

• The PTEN Expert Panel requires co-segregation in affected relatives across 3-4 meioses for PP1 Supporting, 5-6 meioses for PP1 Moderate, and at least 7 meioses across at least two families for PP1 Strong.
• The consolidated case-evidence bundle contains no family segregation data or meiosis count for this variant.

Still missing

• Obtain pedigrees with affected relative phenotypes and PTEN genotypes.
• Establish the number of informative meioses and number of independent families.
• Evaluate potential phenocopies and age-dependent penetrance before segregation counting.

Sources

Reasoning

BS2 cannot be assessed because no observation of this variant in a homozygous healthy or PHTS-unaffected individual is provided. The variant's absence from the population datasets supplies no homozygote observation.

Rule

PTEN EP v3.2 BS2: homozygous observation(s) in healthy or PHTS-unaffected individual(s), with specified confirmation requirements.

Evidence considered

• ClinGen PTEN Expert Panel Specifications version 3.2 permit BS2_Strong for a homozygous healthy or PHTS-unaffected individual (one observation if homozygosity is confirmed, two if not confirmed); if BS1 is also applied, BS2 is Supporting.
• The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; no homozygote record or phenotype information is available.

Still missing

• Confirmed homozygous observation(s) with health or PHTS-unaffected status are required for BS2 assessment.

Sources

Reasoning

Same rationale as PS3: NM_000314.6:c.674_675dupAT is a frameshift duplication outside the scope of the PTEN VCEP functional-evidence data source. The VCEP BS3 rule requires either a splicing assay showing no impact (applicable to intronic/synonymous variants) or phosphatase activity >0 per Mighell et al. 2018 (PMID: 29706350), whose missense-only saturation mutagenesis dataset does not cover this variant. No functional study of this variant or its truncated protein product showing normal/wild-type-like function was found in the case bundle or reviewed literature.

Rule

PTEN VCEP (ClinGen cspec v3.2) BS3: Strong for well-established in vitro/in vivo functional study (or splicing assay for intronic/synonymous variants) showing no damaging effect; Supporting for phosphatase activity >0 per Mighell et al. 2018 (PMID: 29706350). Mighell dataset (mmc2.xlsx) is restricted to missense variants and is not applicable to this frameshift duplication.

Still missing

• No functional assay data available for this specific frameshift duplication variant; the applicable VCEP functional dataset (Mighell et al. 2018) is restricted to missense variants.

Reasoning

No affected relative who lacks the variant is documented, so non-segregation evidence is unavailable and BS4 cannot be assigned.

Rule

PTEN EP BS4: Supporting requires lack of segregation in affected members of one family; Strong requires lack of segregation in affected members of two or more families.

Evidence considered

• The PTEN Expert Panel specifies BS4 Supporting for lack of segregation in affected members of one family and BS4 Strong for lack of segregation in affected members of two or more families.
• The consolidated case-evidence bundle contains no affected-relative genotype/phenotype data demonstrating non-segregation.

Still missing

• Obtain pedigrees with affected relative phenotypes and PTEN genotypes.
• Document informative non-segregation in one or more families and assess phenocopies or other molecular diagnoses.

Sources

Reasoning

No case-level observation establishes this variant in trans with a pathogenic/likely pathogenic PTEN variant, nor are three qualifying cis or phase-unknown observations with different pathogenic/likely pathogenic PTEN variants documented.

Rule

PTEN Expert Panel BP2 Supporting: observed in trans with a pathogenic/likely pathogenic PTEN variant, or at least three cis/phase-unknown observations with different pathogenic/likely pathogenic PTEN variants.

Evidence considered

• The PTEN Expert Panel permits BP2 for an observation in trans with a pathogenic or likely pathogenic PTEN variant, or for at least three observations in cis and/or phase unknown with different pathogenic or likely pathogenic PTEN variants.
• The consolidated case-evidence bundle contains no qualifying co-occurring PTEN variant, phase determination, or affected-proband observation for this criterion.

Still missing

• Phase-resolved case observations with a pathogenic/likely pathogenic PTEN variant, or at least three distinct qualifying cis/phase-unknown observations, are needed.

Sources

Reasoning

No reports identify this variant in at least two cases with a highly penetrant alternate molecular diagnosis and a personal/family history lacking overlap with PTEN-associated disease.

Rule

PTEN EP BP5 supporting: at least two cases with a highly penetrant alternate molecular diagnosis and documented absence of phenotype overlap with PTEN.

Evidence considered

• PTEN EP requires at least two such cases; the alternate gene/disorder must be highly penetrant, and the patient's personal/family history must show no overlap between that disorder and PTEN. No such cases are present in the supplied evidence.

Still missing

• At least two independent cases with the exact variant, a highly penetrant alternate molecular diagnosis, and documented non-overlapping PTEN and alternate-disorder phenotypes are required.

Sources