PTEN c.377C>A, p.Ala126Asp

Reasoning

PS3 is met at supporting strength. The VCEP permits PS3_Supporting for phosphatase activity below 50% of wild type or an abnormal in vitro cellular assay that does not meet PS3_Moderate. Mighell et al. Table S2 contains a high-confidence A126D entry (High_conf=True) with Cum_score -1.027466629, which does not meet the VCEP PS3_Moderate cutoff of <= -1.11. Independently, Rodriguez-Escudero et al. directly tested A126D in a yeast heterologous reconstitution assay and reported that the mutation fully abrogated PTEN PIP3 phosphatase activity. The VCEP specifically identifies this study among phosphatase assays eligible for PS3_Supporting review.

VCEP rule

Phosphatase activity <50% of wild-type or abnormal _in vitro_ cellular assay or transgenic model with phenotype different from wild type that does not meet PS3\_moderate.

Evidence considered

• Mighell et al. 2018 supplementary file mmc2.xlsx, Table S2, row A126D/Ala126Asp: Type=missense; Cum_score=-1.027466629; Cum_SE=0.405124511; High_conf=True; High_conf_nature=Pass SE Filter.
• Rodriguez-Escudero et al. 2011, PMID:21828076, Results section, Functional analysis of mutations at the PTEN P-loop mimicking PTEN-like proteins: the A126D P-loop mutation fully abrogated PTEN PIP3 phosphatase activity in yeast.
• The variant is a missense substitution at PTEN residue A126, and the yeast assay measured in vivo PIP3 phosphatase activity in a Saccharomyces cerevisiae heterologous reconstitution system.
• The Mighell Table S2 score is -1.027466629, so it does not satisfy the VCEP PS3_Moderate threshold of <= -1.11; the direct damaging assay supports PS3_Supporting instead.

Sources

Reasoning

The PTEN VCEP defines PM1 as a variant located in a mutational hot spot and/or critical, well-established functional domain and explicitly includes residues 123-130 of NP_000305.3. The variant is p.Ala126Asp, so residue 126 lies within the authoritative VCEP catalytic-motif interval 123-130. The paper extract independently describes A126D as a P-loop mutation and reports complete loss of PIP3 phosphatase activity, consistent with this functional region.

VCEP 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)

Evidence considered

• ClinGen PTEN VCEP specification v3.2, PM1 rule: critical catalytic-motif residues are 90-94, 123-130, and 166-168 of NP_000305.3; p.Ala126Asp is at residue 126.
• Rodríguez-Escudero et al. 2011 (PMID:21828076), Results, 'Functional analysis of mutations at the PTEN P-loop mimicking PTEN-like proteins,' paragraph 1: the paper reports A126D among mutations that fully abrogated PTEN PIP3 phosphatase activity in yeast.

Sources

Reasoning

The governing PTEN VCEP permits PM2 Supporting when a variant is present at less than 0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population; if multiple alleles are present in a subpopulation, that subpopulation frequency must be less than 0.00002 (0.002%). The variant is absent from gnomAD v2.1 and v4.1, satisfying the stated frequency condition.

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 specification v3.2, 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: NM_000314.8:c.377C>A / GRCh37 10-89692893-C>A absent; observed allele frequency is therefore below the VCEP threshold.
• gnomAD v4.1: GRCh38 10-87933136-C>A absent; observed allele frequency is therefore below the VCEP threshold.

Sources

Reasoning

The variant is a missense substitution, so the PTEN VCEP PP3 missense path applies: REVEL score > 0.7. The recorded REVEL score is 0.96, which exceeds the VCEP threshold. The splice-prediction path is not used for this missense assessment, and the REVEL result is not counted again as independent evidence for BP4.

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

• Variant consequence: missense, NM_000314.8:c.377C>A (NP_000305.3:p.(Ala126Asp)).
• PTEN VCEP PP3 missense path: REVEL score > 0.7.
• Local REVEL predictor lookup for NM_000314.8:c.377C>A: score 0.96, exceeding the >0.7 threshold.

Sources

Reasoning

The governing PTEN VCEP defines BA1 as a stand-alone benign criterion when the gnomAD filtering allele frequency is greater than 0.00056 (0.056%). NM_000314.8:c.377C>A (p.Ala126Asp) is absent from gnomAD v2.1 and gnomAD v4.1, so no frequency above the BA1 threshold is present.

VCEP rule

gnomAD Filtering allele frequency >0.00056 (0.056%)

Evidence considered

• ClinGen PTEN VCEP specification v3.2, BA1 rule: gnomAD Filtering allele frequency >0.00056 (0.056%).
• gnomAD v2.1: NM_000314.8:c.377C>A / GRCh37 10-89692893-C>A absent; no allele frequency reported.
• gnomAD v4.1: GRCh38 10-87933136-C>A absent; no allele frequency reported.

Sources

Reasoning

The governing PTEN VCEP assigns 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 is 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 specification v3.2, BS1 Strong rule: gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%).
• ClinGen PTEN VCEP specification v3.2, BS1 Supporting rule: Allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%).
• gnomAD v2.1: NM_000314.8:c.377C>A / GRCh37 10-89692893-C>A absent; no allele frequency reported.
• gnomAD v4.1: GRCh38 10-87933136-C>A absent; no allele frequency reported.

Sources

Reasoning

BS3 is not met. The governing VCEP supporting rule requires phosphatase activity >0 in the Mighell et al. 2018 assay, but the high-confidence A126D row in Table S2 has Cum_score -1.027466629, not a value >0. The independent A126D yeast assay demonstrated complete loss rather than absence of a damaging effect.

VCEP rule

_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

• Mighell et al. 2018 supplementary file mmc2.xlsx, Table S2, row A126D/Ala126Asp: Cum_score=-1.027466629 and High_conf=True; this is not >0.
• Rodriguez-Escudero et al. 2011, PMID:21828076, Results section, Functional analysis of mutations at the PTEN P-loop mimicking PTEN-like proteins: A126D fully abrogated PTEN PIP3 phosphatase activity in yeast, which is inconsistent with a no-damaging-effect result.

Sources

Reasoning

The variant is a missense substitution, so the PTEN VCEP BP4 missense path applies: REVEL scores < 0.5. The recorded REVEL score is 0.96, which does not meet the benign threshold. The VCEP's splicing path is not applicable to this missense assessment, and the same REVEL prediction is not double-counted with PP3.

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

• Variant consequence: missense, NM_000314.8:c.377C>A (NP_000305.3:p.(Ala126Asp)).
• PTEN VCEP BP4 missense path: REVEL scores < 0.5.
• Local REVEL predictor lookup for NM_000314.8:c.377C>A: score 0.96, not below the <0.5 threshold.

Sources

Reasoning

The variant is NP_000305.3:p.(Ala126Asp), but the available evidence does not establish a previously established pathogenic variant with the same amino-acid change. The ClinVar record is a single-submitter likely pathogenic classification with a conflicting uncertain-significance submission, and the reviewed literature does not establish a prior pathogenic p.Ala126Asp variant. Therefore the VCEP PS1 condition cannot be evaluated as met.

VCEP 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.

Evidence considered

• The case variant is NM_000314.8:c.377C>A, NP_000305.3:p.(Ala126Asp).
• ClinVar variation 2674280 contains one single-submitter likely pathogenic submission and one uncertain-significance submission; this is not an expert-panel-established pathogenic comparator.
• Rodríguez-Escudero et al. 2011 (PMID:21828076) experimentally studied A126D but did not establish it as a previously pathogenic clinical variant.
• Post et al. 2020 (PMID:32350270) names A126D in a stability experiment but does not establish a prior pathogenic classification.

Still missing

• A verified previously established pathogenic p.Ala126Asp comparator is not available.

Sources

Reasoning

No documented germline de novo observation is available for NM_000314.8:c.377C>A (NP_000305.3:p.(Ala126Asp)). The available variant-specific publication reports the variant in breast carcinoma and adjacent atypical hyperplasia tissue as a somatic observation, without parental testing, maternity or paternity confirmation, family-history information, or a qualifying de novo observation in a patient with PTEN hamartoma tumor syndrome. Therefore, the VCEP PS2 thresholds cannot 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. ... De novo (both maternity and paternity confirmed) observation in a patient with the disease and no family history.

Evidence considered

• No case evidence documents a germline proband with c.377C>A, no family history, or confirmed maternity and paternity.
• PMID:20300775 reports c.377C>A in patient 053383 with invasive breast carcinoma and adjacent atypical ductal hyperplasia tissue; the report describes tumor-tissue sequencing and does not provide germline parental testing or a de novo observation.

Still missing

• A documented PTEN hamartoma tumor syndrome proband with the variant, disease status, family history, and parental testing showing confirmed or presumed de novo occurrence.

Sources

Reasoning

The PTEN VCEP permits PS4 from a sufficiently powered case-control study with odds ratio >2, p<0.05, and a 95% confidence-interval lower limit >1.5, or from phenotype-specificity scores accumulated across probands. The available evidence contains no qualifying case-control statistics and no Cleveland Clinic or pediatric phenotype scores for this variant, so PS4 cannot be evaluated.

VCEP rule

**PTEN EP Commentary:** This criterion is unlikely to be used in this manner for a condition as rare as PHTS. However, if sufficiently powered, a case-control study finding an odds ratio >2 for a PHTS component phenotype with p\<0.05 and 95% confidence interval with lower limit >1.5, this criteria may be applied. However, this criterion may _not_ be applied in combination with PP4. ... 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 ... 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

• The PTEN VCEP commentary permits a sufficiently powered case-control study with odds ratio >2, p<0.05, and 95% confidence-interval lower limit >1.5.
• The PTEN VCEP specifies adult Cleveland Clinic score thresholds of >=30 or 25-29 and pediatric phenotype score thresholds of >=5 or 4, but no such scores are reported for this case.
• PMID:25190698 and PMID:25645574 were reviewed as candidate case-observation sources; their extracted text does not mention PTEN c.377C>A or p.A126D.

Still missing

• A sufficiently powered case-control study with odds ratio, p-value, and 95% confidence interval is not available.
• No adult Cleveland Clinic scores, pediatric phenotype scores, or accumulated proband specificity score are available.

Sources

Reasoning

A different missense change at the same residue is documented: PTEN c.377C>T (p.Ala126Val) is listed as likely pathogenic in Mighell et al. 2018 Supplementary Table S3. However, the VCEP PM5 rule additionally requires the interrogated variant's BLOSUM62 score to be equal to or less than the known comparator's score, and the case materials do not provide the applicable BLOSUM62 values. The criterion therefore cannot be completed without importing an unverified score.

VCEP rule

Missense change at an amino acid residue where a different missense change determined to be pathogenic or likely pathogenic has been seen before. In addition, variant being interrogated must have BLOSUM62 score equal to or less than the known variant.

Evidence considered

• Mighell et al. 2018 Supplementary Table 3, row NM_000314.6(PTEN):c.377C>T (p.Ala126Val): clinical significance Likely pathogenic, criteria provided, single submitter, VariationID 428234.
• The case variant and comparator are both missense changes at residue 126, but the required BLOSUM62 score comparison cannot be verified from the supplied VCEP materials.

Still missing

• BLOSUM62 scores for Ala126Asp and the Ala126Val comparator are not available in the case materials.

Sources

Reasoning

No presumed de novo germline observation is available for NM_000314.8:c.377C>A (NP_000305.3:p.(Ala126Asp)). The available variant-specific publication reports a somatic tumor observation and does not establish a patient with PTEN hamartoma tumor syndrome, no family history, or unconfirmed maternity and paternity. Thus, the VCEP PM6 thresholds cannot 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

• No case evidence documents presumed de novo occurrence, an affected PTEN hamartoma tumor syndrome proband, or absence of family history.
• PMID:20300775 reports c.377C>A in breast carcinoma and adjacent atypical ductal hyperplasia tissue, not as a presumed de novo germline observation, and provides no maternity or paternity information.

Still missing

• A documented affected proband with the variant, no family history, and evidence supporting presumed de novo status despite unconfirmed maternity and paternity.

Sources

Reasoning

No informative familial co-segregation data are available for NM_000314.8:c.377C>A (NP_000305.3:p.(Ala126Asp)). The case evidence contains no affected relatives carrying the variant, unaffected relatives lacking it, pedigree, or count of informative meioses; therefore, none of the VCEP PP1 thresholds can be assigned.

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

• No familial segregation results, affected-relative testing, pedigree, or informative meiosis count is documented for this variant.

Still missing

• Pedigree-based testing results for affected relatives and the number of informative meioses across families.

Sources

Reasoning

The VCEP PP2 rule requires both a low rate of benign missense variation and missense variants being a common mechanism of disease. The available case materials do not provide VCEP-specific evidence establishing both conditions, and no generic population or mechanism threshold may be substituted.

VCEP rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• The VCEP PP2 rule requires two gene-level conditions, but the supplied case evidence does not provide a VCEP-assigned determination of either condition.

Still missing

• VCEP-specific evidence demonstrating both PP2 conditions is unavailable.

Sources

Reasoning

The governing PTEN VCEP requires observation of the variant in the homozygous state in a healthy or PHTS-unaffected individual, with one confirmed observation or two if parental confirmation is unavailable. The available evidence reports population absence, not any homozygous individual, 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.

Still missing

• A confirmed homozygous observation in a healthy or PHTS-unaffected individual, or two independent unconfirmed homozygous observations, is unavailable.

Reasoning

No familial non-segregation data are available for NM_000314.8:c.377C>A (NP_000305.3:p.(Ala126Asp)). The case evidence does not identify affected family members who lack the variant, nor does it provide one-family or two-family counts; consequently, the VCEP BS4 thresholds 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

• No affected-relative non-segregation results or family counts are documented for this variant.

Still missing

• Family-based testing showing lack of the variant in affected members, with the number of informative families documented.

Sources

Reasoning

The PTEN VCEP rule requires either observation of the tested variant in trans with a pathogenic or likely pathogenic PTEN variant, or at least three observations in cis and/or phase unknown with different pathogenic or likely pathogenic PTEN variants. The available case evidence does not document a phase-resolved pathogenic/likely pathogenic PTEN variant in trans, nor three qualifying cis or phase-unknown observations with different pathogenic/likely pathogenic PTEN variants. The available literature findings concern functional or somatic observations and do not establish the required allelic phase relationships. Because the VCEP rule cannot be evaluated from the available evidence, BP2 is not assessed rather than not met.

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 Version 3.2 CSPEC BP2 entry: Supporting when observed in trans with a pathogenic or likely pathogenic PTEN variant, or when there are at least three observations in cis and/or phase unknown with different pathogenic/likely pathogenic PTEN variants.
• Case evidence documents PTEN c.377C>A (p.Ala126Asp) and unrelated functional or somatic reports, but provides no qualifying phase-resolved trans observation or three qualifying cis/phase-unknown observations with different pathogenic/likely pathogenic PTEN variants.

Still missing

• Phase-resolved evidence showing NM_000314.8:c.377C>A in trans with a pathogenic or likely pathogenic PTEN variant.
• Alternatively, at least three observations in cis and/or phase unknown with different pathogenic or likely pathogenic PTEN variants.

Sources

Reasoning

The PTEN VCEP requires at least two cases with an alternate molecular basis, a highly penetrant other gene/disorder, and a personal or family history demonstrating no overlap between that disorder and PTEN. The available evidence does not establish these requirements.

VCEP rule

**PTEN EP Specifications:** At least two such cases are required for criteria to apply. In addition, the other gene/disorder must be considered highly penetrant AND the patient’s personal/family history must demonstrate no overlap between the other gene and PTEN.

Evidence considered

• The PTEN VCEP requires at least two cases and additionally requires a highly penetrant alternate gene/disorder plus no personal or family-history overlap with PTEN; no such evidence is available.

Still missing

• At least two qualifying cases with an alternate molecular basis are not documented.
• A highly penetrant alternate gene/disorder and non-overlapping personal or family histories are not documented.

Sources