PTEN c.-326_-324delinsCCC, p.?

Reasoning

The PTEN VCEP permits PM2 at Supporting strength when the variant is absent or present at <0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population; if multiple alleles occur in a subpopulation, that subpopulation frequency must be <0.00002 (0.002%). The variant was absent from gnomAD v2.1 and v4.1, satisfying the applicable absence/frequency requirement.

VCEP 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%).

Evidence considered

• ClinGen PTEN VCEP Version 3.2, PM2 Supporting rule: 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%).
• gnomAD v2.1 result for NC_000010.10:g.89623901_89623903delinsCCC: absent.
• gnomAD v4.1 result for NC_000010.11:g.87864144_87864146delinsCCC: absent.

Sources

Reasoning

The PTEN VCEP specifies BA1 only when the gnomAD filtering allele frequency is >0.00056 (0.056%). The variant was absent from gnomAD v2.1 and gnomAD v4.1, so no frequency exceeding the BA1 threshold is observed.

VCEP rule

gnomAD Filtering allele frequency >0.00056 (0.056%)

Evidence considered

• ClinGen PTEN VCEP Version 3.2, BA1 rule: gnomAD Filtering allele frequency >0.00056 (0.056%).
• gnomAD v2.1 result for NC_000010.10:g.89623901_89623903delinsCCC: absent; no allele frequency reported.
• gnomAD v4.1 result for NC_000010.11:g.87864144_87864146delinsCCC: absent; no allele frequency reported.

Sources

Reasoning

The PTEN VCEP specifies BS1 Strong for a gnomAD filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%), and BS1 Supporting from 0.0000043 (0.00043%) up to 0.000043 (0.0043%). The variant was absent from gnomAD v2.1 and v4.1, so neither frequency interval is met.

VCEP rule

gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%) ... Allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%).

Evidence considered

• ClinGen PTEN VCEP Version 3.2, BS1 Strong rule: gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%).
• ClinGen PTEN VCEP Version 3.2, BS1 Supporting rule: Allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%).
• gnomAD v2.1 result for NC_000010.10:g.89623901_89623903delinsCCC: absent; no allele frequency reported.
• gnomAD v4.1 result for NC_000010.11:g.87864144_87864146delinsCCC: absent; no allele frequency reported.

Sources

Reasoning

The available case evidence contains no proband-level de novo observation, parental testing, maternity or paternity confirmation, disease phenotype, or family-history information. Therefore, the PTEN VCEP PS2 rule cannot be evaluated.

VCEP rule

De novo (both maternity and paternity confirmed) observation in a patient with the disease and no family history.

Evidence considered

• The case evidence bundle reports no pedigree, proband de novo, parental testing, maternity, paternity, phenotype, or family-history data for this variant.
• The governing PTEN VCEP rule requires a de novo observation in a patient with the disease and no family history, with both maternity and paternity confirmed for PS2 Strong.

Still missing

• Proband disease phenotype and confirmation of PTEN-related disease
• Variant testing results for both biological parents
• Maternity and paternity confirmation
• Family history and absence of disease in relatives

Sources

Reasoning

The PTEN VCEP permits PS3 for a damaging splicing assay, for phosphatase activity <= -1.11 per Mighell et al. 2018, or for a damaging functional study below that moderate threshold. The case variant is NM_000314.8:c.-326_-324delinsCCC, normalized as NP_000305.3:p.? with unknown consequence class; the governing mmc2.xlsx file is indexed for missense variants, and searches of its full text for c.-326_-324delinsCCC and p.? found no matching entry. No case-specific RNA, mini-gene, phosphatase, or other functional result establishes a PS3 finding, so PS3 cannot be assessed.

VCEP rule

Well-established _in vitro_ or _in vivo_ functional studies supportive of a damaging effect on the gene or gene product. * Phosphatase activity ≤ -1.11 per Mighell et al. 2018, PMID: 29706350.

Evidence considered

• ClinGen PTEN VCEP Version 3.2 PS3 rule: damaging RNA, mini-gene, or other splicing assay may support PS3 Strong; phosphatase activity <= -1.11 per Mighell et al. 2018 may support PS3 Moderate; a damaging functional study below that moderate threshold may support PS3 Supporting.
• Case normalization: NM_000314.8:c.-326_-324delinsCCC; NP_000305.3:p.?; consequence class unknown.
• Mighell et al. 2018 supplementary file mmc2.xlsx, README and indexed Tables S2-S5: the file is specified for PTEN missense functional data; full-text searches for c.-326_-324delinsCCC and p.? returned no matching row.

Still missing

• No matching c.-326_-324delinsCCC entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PTEN/fulltext/mmc2.xlsx.txt.
• No matching p.? entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PTEN/fulltext/mmc2.xlsx.txt.
• A variant-specific RNA, mini-gene, phosphatase, or other validated functional assay result is unavailable.

Sources

Reasoning

The PTEN VCEP permits PS4 from a sufficiently powered case-control study or phenotype-specificity scores across independent probands. The available evidence contains no case-control odds ratio, p-value, confidence interval, Cleveland Clinic scores, pediatric phenotype scores, proband count, or qualifying phenotype/family-history data for this variant. Therefore PS4 cannot be evaluated. The VCEP also states that PS4 may not be applied with PP4 and may not be applied if BS1 applies; neither interaction changes the present evidence gap.

VCEP rule

This criterion may not be applied if BS1 applies. Phenotype specificity scores are added across independent probands and calculated as follows: Adults: 1 point per proband with Cleveland Clinic (CC) score ≥ 30 (Tan 2011); 0.5 points per proband with CC score of 25-29. Children: 1 point per proband with pediatric phenotype score ≥ 5 (please see supplementary information in manuscript for scoring rubric). 0.5 points per proband with pediatric phenotype score of 4, but autism/developmental delay/intellectual disability may not contribute to the score. PS4_Very Strong: Probands with specificity score ≥16. PS4: Probands with specificity score of 4-15.5. PS4_Moderate: Probands with specificity score of 2-3.5. PS4_Supporting: Proband(s) with specificity score of 1-1.5.

Evidence considered

• No case-control study result, odds ratio, p-value, confidence interval, proband specificity score, Cleveland Clinic score, or pediatric phenotype score was present in the available case evidence.
• Variant facts identify NM_000314.8:c.-326_-324delinsCCC as an upstream noncoding variant with predicted protein consequence p.(=), but provide no patient phenotype or family-history observations usable for PS4.

Still missing

• Case-control odds ratio, p-value, and 95% confidence interval for a PHTS component phenotype are unavailable.
• Independent proband phenotype data and Cleveland Clinic or pediatric phenotype scores are unavailable.
• BS1 status is not relevantly documented for this criterion, and the VCEP's PS4-BS1 restriction cannot be independently assessed from phenotype evidence alone.

Sources

Reasoning

The available case evidence contains no presumed or assumed de novo observations, proband disease phenotype, family history, or parental testing information. Consequently, none of the PTEN VCEP PM6 de novo observation thresholds can be evaluated.

VCEP rule

Two proven OR four assumed OR one proven + two assumed de novo observations in a patient with the disease and no family history. ... 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) ... Assumed _de novo,_ but without confirmation of paternity and maternity, in proband with the disease and no family history.

Evidence considered

• The case evidence bundle reports no de novo observations, parental testing, proband phenotype, or family-history data for this variant.
• The PTEN VCEP PM6 rule specifies separate thresholds for proven and assumed de novo observations in patients with disease and no family history.

Still missing

• Number of de novo observations
• Whether each de novo observation is proven or assumed
• Proband disease phenotype
• Family history
• Maternity and paternity confirmation

Sources

Reasoning

The available case evidence contains no affected relatives, segregation observations, family structure, or meiosis count. Therefore, the PTEN VCEP PP1 co-segregation thresholds cannot be evaluated.

VCEP rule

Co-segregation with disease in multiple affected family members, with ≥7 meioses observed across at least two families. ... Co-segregation with disease in multiple affected family members, with 5 or 6 meioses observed. ... Co-segregation with disease in multiple affected family members, with 3 or 4 meioses observed.

Evidence considered

• The case evidence bundle reports no affected family members, segregation results, family structure, or meiosis count for this variant.
• The PTEN VCEP PP1 rule assigns strength according to the number of observed co-segregating meioses across affected family members and families.

Still missing

• Affected family members with the variant
• Unaffected or non-carrier relatives where informative
• Family structure and phase information
• Observed meiosis count across families

Sources

Reasoning

The variant is a noncoding 5'-UTR change with no missense consequence, so the PTEN VCEP's splicing-variant computational path is the relevant path. That path requires concordance of SpliceAl and VarSeak to support a deleterious effect. SpliceAI is available and reports a maximum delta score of 0.015, but no VarSeak result is present; therefore concordance cannot be evaluated and PP3 cannot be assigned.

VCEP rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product. * Splicing variants: Concordance of SpliceAl and VarSeak * Missense variants: REVEL score > 0.7

Evidence considered

• ClinGen PTEN VCEP Version 3.2 PP3 rule: the splicing-variant path requires concordance of SpliceAl and VarSeak.
• SpliceAI Lookup for NM_000314.8:c.-326_-324delinsCCC reports DS_AG 0.014, DS_AL 0.015, DS_DG 0.001, and DS_DL 0.004; maximum delta score is 0.015.
• No VarSeak result is present for this variant, so the required predictor concordance cannot be determined.

Still missing

• VarSeak prediction for NM_000314.8:c.-326_-324delinsCCC is unavailable, so the VCEP-required SpliceAl/VarSeak concordance cannot be assessed.

Sources

Reasoning

The PTEN VCEP requires observation of the variant in the homozygous state in a healthy or PHTS-unaffected individual; one observation is sufficient if homozygous status is confirmed and two are required if it is not confirmed. The available gnomAD results report absence and provide no homozygote observations or clinical status, so BS2 cannot be evaluated.

VCEP rule

Observed in the homozygous state in a healthy or PHTS-unaffected individual. One observation if homozygous status confirmed, two if not confirmed. To be applied at supporting evidence level if BS1 is also applied.

Evidence considered

• ClinGen PTEN VCEP Version 3.2, BS2 rule: observed in the homozygous state in a healthy or PHTS-unaffected individual; one observation if homozygous status confirmed, two if not confirmed.
• gnomAD v2.1 and gnomAD v4.1 report the variant as absent and contain no homozygote or clinical-status observation for this variant.

Still missing

• No documented homozygous observations with confirmation status and healthy or PHTS-unaffected clinical status were available.

Sources

Reasoning

The PTEN VCEP permits BS3 for a well-established functional study showing no damaging effect, including phosphatase activity >0 per Mighell et al. 2018 for BS3 Supporting. The case variant is normalized as NP_000305.3:p.? with unknown consequence class; searches of the governing mmc2.xlsx full text for c.-326_-324delinsCCC and p.? found no matching entry, and no variant-specific benign functional assay result is available. Therefore BS3 cannot be assessed.

VCEP rule

Well-established _in vitro_ or _in vivo_ functional studies shows no damaging effect on protein function. To be applied to intronic or synonymous variants, RNA, mini-gene or other splicing assay demonstrating no splicing impact. ... _In vitro_ or _in vivo_ functional study or studies showing no damaging effect on protein function. * Phosphatase activity >0 per Mighell et al. 2018, PMID: 29706350.

Evidence considered

• ClinGen PTEN VCEP Version 3.2 BS3 rule: a well-established functional study showing no damaging effect may support BS3 Strong; an in vitro or in vivo study showing no damaging effect, including phosphatase activity >0 per Mighell et al. 2018, may support BS3 Supporting.
• Case normalization: NM_000314.8:c.-326_-324delinsCCC; NP_000305.3:p.?; consequence class unknown.
• Mighell et al. 2018 supplementary file mmc2.xlsx, README and indexed Tables S2-S5: the file is specified for PTEN missense functional data; full-text searches for c.-326_-324delinsCCC and p.? returned no matching row.

Still missing

• No matching c.-326_-324delinsCCC entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PTEN/fulltext/mmc2.xlsx.txt.
• No matching p.? entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PTEN/fulltext/mmc2.xlsx.txt.
• A variant-specific functional study demonstrating no damaging effect or phosphatase activity >0 is unavailable.

Sources

Reasoning

The available case evidence contains no affected-family-member genotypes or segregation results, so there is no evidence of lack of segregation in one or more families. The PTEN VCEP BS4 rule therefore cannot be evaluated.

VCEP rule

Lack of segregation in affected members of two or more families. ... Lack of segregation in affected members of one family.

Evidence considered

• The case evidence bundle reports no affected-family-member genotypes, phenotypes, or segregation results for this variant.
• The PTEN VCEP BS4 rule requires lack of segregation in affected members of one or more families, with strength determined by the number of families.

Still missing

• Genotypes and phenotypes of affected family members
• Segregation results within each family
• Number of families with informative non-segregation

Sources

Reasoning

The PTEN VCEP Version 3.2 BP2 rule requires either observation 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. The available case evidence contains no affected-proband observations, phase information, or documented cis/trans pairing with another pathogenic or likely pathogenic PTEN variant, so BP2 cannot be assessed.

VCEP rule

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 to the ACMG/AMP Variant Interpretation Guidelines for PTEN Version 3.2, criteria entry BP2: "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."
• The case evidence records no affected-proband, inheritance, phase, cis, trans, or qualifying second-variant observations for NM_000314.8:c.-326_-324delinsCCC.

Still missing

• Affected-proband observations with a second PTEN pathogenic or likely pathogenic variant are not documented.
• Phase is not established as trans, cis, or phase unknown for any qualifying PTEN variant pair.
• No qualifying count of at least three cis and/or phase-unknown observations with different pathogenic/likely pathogenic PTEN variants is documented.

Sources

Reasoning

The variant is a noncoding 5'-UTR change with no missense consequence, so the PTEN VCEP's splicing-variant computational path is the relevant path. That path requires concordance of SpliceAl and VarSeak to support no impact on the gene or gene product. SpliceAI is available and reports a maximum delta score of 0.015, but no VarSeak result is present; therefore concordance cannot be evaluated and BP4 cannot be assigned.

VCEP rule

Multiple lines of computational evidence suggest no impact on gene or gene product.  * Splicing variants: Concordance of SpliceAl and VarSeak * Missense variants: REVEL scores < 0.5

Evidence considered

• ClinGen PTEN VCEP Version 3.2 BP4 rule: the splicing-variant path requires concordance of SpliceAl and VarSeak.
• SpliceAI Lookup for NM_000314.8:c.-326_-324delinsCCC reports DS_AG 0.014, DS_AL 0.015, DS_DG 0.001, and DS_DL 0.004; maximum delta score is 0.015.
• No VarSeak result is present for this variant, so the required predictor concordance cannot be determined.

Still missing

• VarSeak prediction for NM_000314.8:c.-326_-324delinsCCC is unavailable, so the VCEP-required SpliceAl/VarSeak concordance cannot be assessed.

Sources

Reasoning

The PTEN VCEP requires at least two cases with an alternate molecular basis, a highly penetrant other gene/disorder, and personal or family history showing no overlap between that disorder and PTEN. No qualifying alternate molecular diagnosis, case count, penetrance evidence, or non-overlapping personal/family history is available for this variant. Therefore BP5 cannot be evaluated.

VCEP 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.

Evidence considered

• The available case evidence contains no qualifying alternate molecular diagnosis, case series, penetrance assessment, or personal/family history for BP5.

Still missing

• At least two qualifying cases with an alternate molecular basis are unavailable.
• Evidence that the alternate gene/disorder is highly penetrant is unavailable.
• Personal and family histories demonstrating no overlap between the alternate disorder and PTEN are unavailable.

Sources