PTEN c.964A>G, p.Lys322Glu

Reasoning

The PTEN VCEP permits PM2 at Supporting strength when the variant is 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%). NM_000314.8:c.964A>G is absent from gnomAD v2.1 and gnomAD v4.1, giving an observed allele frequency of 0 in both datasets and satisfying the <0.00001 threshold. The VCEP does not specify a non-cancer or exome-only source for PM2, so the all-comers gnomAD entries are the governing default; the separately reported non-cancer subsets are also absent.

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 v3.2 PM2 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, that subpopulation allele frequency must be <0.00002 (0.002%).
• gnomAD v2.1 record for 10-89720813-A-G: absent, equivalent to observed allele frequency 0 in the queried dataset.
• gnomAD v4.1 record for 10-87961056-A-G: absent, equivalent to observed allele frequency 0 in the queried dataset.
• The case evidence also reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; the VCEP does not require either subset for PM2.

Sources

Reasoning

The variant is a missense substitution, so the PTEN VCEP missense branch applies. The VCEP PP3 rule requires a REVEL score > 0.7 for missense variants. The recorded REVEL score is 0.877, which exceeds that cutoff; the splice-prediction branch is not used for this missense assessment.

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.964A>G (NP_000305.3:p.(Lys322Glu)).
• Local REVEL predictor lookup: score 0.877 for NC_000010.11:g.87961056A>G.
• ClinGen PTEN VCEP Version 3.2 PP3 rule: for missense variants, REVEL score > 0.7; 0.877 satisfies this threshold.

Sources

Reasoning

The governing PTEN VCEP rule permits PS3_Moderate from the Mighell et al. 2018 massively parallel phosphatase assay only when Table S2 lists the variant as high confidence and its Cum_score is <= -1.11. The matching Table S2 row is K322E / Lys322Glu, with High_conf=True, High_conf_nature=Pass SE Filter, and Cum_score=-0.166801504. Because the high-confidence score is above -1.11, the VCEP PS3_Moderate threshold is not met; no separate VCEP-qualified assay result supporting PS3 at another strength was identified.

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

• Mighell et al. 2018 supplementary data mmc2, Table S2, row K322E / Lys322Glu: High_conf=True; High_conf_nature=Pass SE Filter; Cum_score=-0.166801504; Cum_SE=0.134526022.
• The equivalent variant row was found under the one-letter and three-letter notations K322E and Lys322Glu; literal searches for c.964A>G, p.Lys322Glu, and p.K322E returned no separate literal row.

Sources

Reasoning

The VCEP defines PM1 for residues in the WPD loop (90-94), P-loop/phosphatase core (123-130), or TI-loop (166-168) of NP_000305.3, and permits a mutational-hotspot path. The queried residue is Lys322, outside all three specified catalytic motifs; the collected hotspot result reports no statistically significant hotspot at PTEN K322. vcep_materials.json has an empty domain_tables object, so there is no additional authoritative PTEN domain-table entry to check.

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

• CSPEC PM1 instructions define the PTEN catalytic motifs as WPD loop residues 90-94, P-loop/phosphatase core residues 123-130, and TI-loop residues 166-168; the variant is p.Lys322Glu.
• vcep_materials.json domain_tables is empty for PTEN, so no additional authoritative domain-table entry exists for this gene.
• The collected PTEN K322 hotspot query returned no statistically significant hotspot row.

Sources

Reasoning

The PTEN VCEP requires BA1 when the gnomAD filtering allele frequency is >0.00056 (0.056%). NM_000314.8:c.964A>G is absent from gnomAD v2.1 and gnomAD v4.1, so no filtering allele frequency exceeding the BA1 threshold is present. The VCEP does not specify a non-cancer or exome-only source for BA1, so the all-comers gnomAD entries are the governing default; the separately reported non-cancer subsets are also absent.

VCEP rule

gnomAD Filtering allele frequency >0.00056 (0.056%)

Evidence considered

• ClinGen PTEN VCEP v3.2 BA1 rule: gnomAD Filtering allele frequency >0.00056 (0.056%).
• gnomAD v2.1 record for 10-89720813-A-G: absent; no filtering allele frequency reported.
• gnomAD v4.1 record for 10-87961056-A-G: absent; no filtering allele frequency reported.
• The case evidence also reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; the VCEP does not require either subset for BA1.

Sources

Reasoning

The PTEN VCEP requires BS1 Strong for a gnomAD filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%), or BS1 Supporting from 0.0000043 (0.00043%) up to 0.000043 (0.0043%). NM_000314.8:c.964A>G is absent from gnomAD v2.1 and gnomAD v4.1, so it falls in neither frequency interval. The VCEP does not specify a non-cancer or exome-only source for BS1, so the all-comers gnomAD entries are the governing default; the separately reported non-cancer subsets are also absent.

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 v3.2 BS1 Strong rule: gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%).
• ClinGen PTEN VCEP v3.2 BS1 Supporting rule: Allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%).
• gnomAD v2.1 record for 10-89720813-A-G: absent; no filtering allele frequency reported.
• gnomAD v4.1 record for 10-87961056-A-G: absent; no filtering allele frequency reported.
• The case evidence also reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; the VCEP does not require either subset for BS1.

Sources

Reasoning

The governing PTEN VCEP rule permits BS3_Supporting from the Mighell et al. 2018 massively parallel phosphatase assay only when Table S2 lists the variant as high confidence and its Cum_score is >0. The matching Table S2 row is K322E / Lys322Glu, with High_conf=True, High_conf_nature=Pass SE Filter, and Cum_score=-0.166801504. The score is not greater than zero, so the VCEP BS3_Supporting threshold is not met; the available functional result does not qualify as benign functional evidence under this rule.

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 data mmc2, Table S2, row K322E / Lys322Glu: High_conf=True; High_conf_nature=Pass SE Filter; Cum_score=-0.166801504; Cum_SE=0.134526022.
• The equivalent variant row was found under the one-letter and three-letter notations K322E and Lys322Glu; literal searches for c.964A>G, p.Lys322Glu, and p.K322E returned no separate literal row.

Sources

Reasoning

The variant is a missense substitution, so the PTEN VCEP BP4 missense branch applies. That branch requires a REVEL score < 0.5. The recorded REVEL score is 0.877, which does not meet the benign computational threshold. The SpliceAI result is not used to satisfy BP4 because the governing rule names REVEL for the missense situation.

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.964A>G (NP_000305.3:p.(Lys322Glu)).
• Local REVEL predictor lookup: score 0.877 for NC_000010.11:g.87961056A>G.
• ClinGen PTEN VCEP Version 3.2 BP4 rule: for missense variants, REVEL score < 0.5; 0.877 does not satisfy this threshold.
• SpliceAI max delta score 0.009 is not counted for BP4 because the VCEP rule names REVEL for this missense branch.

Sources

Reasoning

The variant is NM_000314.8:c.964A>G, p.(Lys322Glu). The VCEP requires the same amino acid change as a previously established pathogenic variant, regardless of nucleotide change, or a qualifying splice-site comparison. The available ClinVar record classifies p.Lys322Glu as uncertain significance, and the reviewed paper extracts do not report this variant or a pathogenic p.Lys322Glu comparator. Therefore, the required pathogenic same-amino-acid evidence is not available; this criterion is not assessed rather than inferred from absence of a report.

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

• Variant normalization: NM_000314.8:c.964A>G produces NP_000305.3:p.(Lys322Glu).
• ClinVar variation 2504932 reports p.Lys322Glu as uncertain significance with two submitters and no expert-panel submission.
• PMID:26467025 does not mention PTEN or this variant; PMID:32350270 does not explicitly report c.964A>G or p.(K322E).

Still missing

• A verified previously established pathogenic PTEN p.Lys322Glu variant or qualifying pathogenic splice-site comparator is needed.

Sources

Reasoning

The PTEN VCEP requires either a proven de novo observation with both maternity and paternity confirmed, or the specified multiple-observation pathway. The available case evidence does not document a proband, disease phenotype, family history, parental testing, or maternity/paternity confirmation for NM_000314.8:c.964A>G (p.Lys322Glu). The two reviewed publications do not report this exact variant or provide variant-specific de novo evidence. Therefore PS2 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 documents the variant as NM_000314.8:c.964A>G (NP_000305.3:p.(Lys322Glu)) but provides no proband-level de novo observation or parental testing result.
• PMID:26467025 does not mention PTEN c.964A>G or p.(K322E) and provides no variant-specific clinical or segregation evidence.
• PMID:32350270 does not explicitly mention c.964A>G or p.(K322E) and provides no variant-specific clinical or de novo evidence.

Still missing

• Proband disease phenotype and clinical diagnosis
• Family history information
• Parental testing results confirming or excluding the variant in both parents
• Maternity and paternity confirmation
• Number of independent de novo observations

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 accumulated phenotype-specific proband scores. No case-control statistics, Cleveland Clinic scores, pediatric phenotype scores, or documented phenotype/family-history observations for this variant are available. The two cited papers do not mention c.964A>G or p.(K322E), and ClinVar reports only an uncertain-significance submission with no phenotype provided. The VCEP also states that PS4 may not be applied in combination with PP4; PP4 is not applicable here.

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. * Use 2: Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. **PTEN EP Specifications:** 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 (see text). PS4: Probands with specificity score of 4-15.5 (see text). PS4\_Moderate: Probands with specificity score of 2-3.5 (see text). PS4\_Supporting: Proband(s) with specificity score of 1-1.5 (see text).

Evidence considered

• ClinVar exact-match record NM_000314.8:c.964A>G (p.Lys322Glu): uncertain significance; conditions listed as not provided.
• PMID:26467025: the extracted full text does not mention PTEN, c.964A>G, or p.(K322E), so it provides no variant-specific phenotype or case-control evidence.
• PMID:32350270: the extracted full text does not individually mention c.964A>G, p.(K322E), or K322E, so it provides no variant-specific phenotype or case-control evidence.

Still missing

• A sufficiently powered case-control analysis with odds ratio, p-value, and 95% confidence interval is unavailable.
• Independent proband Cleveland Clinic or pediatric phenotype scores are unavailable.
• Variant-specific phenotype and family-history information is unavailable.

Sources

Reasoning

The VCEP requires a different missense change at the same amino acid residue that was determined pathogenic or likely pathogenic, with the assessed variant's BLOSUM62 score equal to or less than the known variant; the VCEP also cautions against using PM5 when it would produce a higher pathogenic classification. The collected comparator set contains no same-residue qualifying pathogenic or likely pathogenic comparator for Lys322, so the criterion cannot be evaluated from the available evidence.

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

• PM5 candidate collection recorded five candidates but zero same-residue candidates and zero expert-panel or non-expert-majority pathogenic/likely pathogenic candidates.
• The queried variant is p.(Lys322Glu), so a qualifying comparator must be a different missense substitution at residue 322 and meet the VCEP pathogenicity and BLOSUM62 requirements.

Still missing

• A verified pathogenic or likely pathogenic alternate missense variant at PTEN Lys322, with its BLOSUM62 score, is needed.

Sources

Reasoning

The PTEN VCEP permits PM6 for an assumed de novo observation without confirmation of paternity and maternity in a proband with the disease and no family history, with stronger levels requiring the stated numbers of proven or assumed observations. The available case evidence contains no documented proband disease status, family history, parental testing, or assumed/proven de novo observation for NM_000314.8:c.964A>G (p.Lys322Glu). The reviewed publications do not report this exact variant or variant-specific de novo evidence. Therefore PM6 cannot be evaluated.

VCEP rule

Assumed _de novo,_ but without confirmation of paternity and maternity, in proband with the disease and no family history.

Evidence considered

• The case evidence does not document an assumed or proven de novo occurrence for NM_000314.8:c.964A>G (NP_000305.3:p.(Lys322Glu)).
• PMID:26467025 does not mention PTEN c.964A>G or p.(K322E) and provides no variant-specific de novo evidence.
• PMID:32350270 does not explicitly mention c.964A>G or p.(K322E) and provides no variant-specific de novo evidence.

Still missing

• Proband disease phenotype and clinical diagnosis
• Family history information
• Parental testing results or basis for assuming de novo status
• Number of proven and assumed de novo observations

Sources

Reasoning

The PTEN VCEP requires co-segregation with disease in multiple affected family members and assigns supporting strength only when 3 or 4 meioses are observed, with higher strengths requiring 5–6 or at least 7 meioses across at least two families. No affected relatives, genotype-phenotype relationships, family structure, or meioses are documented for NM_000314.8:c.964A>G (p.Lys322Glu). The reviewed publications do not report this exact variant or variant-specific segregation evidence. Therefore PP1 cannot be evaluated.

VCEP rule

Co-segregation with disease in multiple affected family members, with 3 or 4 meioses observed.

Evidence considered

• The case evidence provides no family segregation data, affected relatives, or meiosis count for NM_000314.8:c.964A>G (NP_000305.3:p.(Lys322Glu)).
• PMID:26467025 does not mention PTEN c.964A>G or p.(K322E) and provides no variant-specific segregation evidence.
• PMID:32350270 does not explicitly mention c.964A>G or p.(K322E) and provides no variant-specific segregation evidence.

Still missing

• Affected family members carrying the variant
• Unaffected or non-carrier relatives tested
• Family structure and informative meioses
• Variant-specific segregation observations across families

Sources

Reasoning

The VCEP retains PP2 at supporting strength for a missense variant in a gene with a low rate of benign missense variation where missense variants are a common disease mechanism. The case evidence establishes that this is a PTEN missense variant, but it does not provide VCEP-specific evidence establishing both the required low benign-missense rate and common missense disease mechanism under this criterion. Generic gene knowledge and unrelated predictor or frequency thresholds cannot substitute for the missing VCEP-supported evidence.

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 normalized consequence is missense, p.(Lys322Glu).
• The PTEN CSPEC lists PP2 as applicable at supporting strength but provides no additional PTEN-specific instructions or quantitative evidence in the PP2 entry.

Still missing

• VCEP-supported evidence must establish both a low rate of benign PTEN missense variation and missense variants as a common disease mechanism.

Sources

Reasoning

The PTEN VCEP requires observation of the variant in the homozygous state in a healthy or PHTS-unaffected individual. The available gnomAD records report only that the variant is absent and provide no homozygote observations, parental confirmation, or clinical status; therefore BS2 cannot be assessed.

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 v3.2 BS2 rule: observed in the homozygous state in a healthy or PHTS-unaffected individual; one observation if homozygous status is confirmed and two if not confirmed.
• gnomAD v2.1 and v4.1 both report the variant as absent and contain no homozygote or clinical-status data for this variant.

Still missing

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

Sources

Reasoning

The PTEN VCEP requires lack of segregation in affected members of one family for supporting BS4 or two or more families for strong BS4. The available case evidence contains no affected relatives, family testing, non-segregation result, or number of families for NM_000314.8:c.964A>G (p.Lys322Glu). The reviewed publications do not report this exact variant or variant-specific non-segregation evidence. Therefore BS4 cannot be evaluated.

VCEP rule

Lack of segregation in affected members of one family.

Evidence considered

• The case evidence provides no family testing or non-segregation observation for NM_000314.8:c.964A>G (NP_000305.3:p.(Lys322Glu)).
• PMID:26467025 does not mention PTEN c.964A>G or p.(K322E) and provides no variant-specific non-segregation evidence.
• PMID:32350270 does not explicitly mention c.964A>G or p.(K322E) and provides no variant-specific non-segregation evidence.

Still missing

• Affected relatives tested for the variant
• Non-segregation result in one or more families
• Number of informative families

Sources

Reasoning

BP2 is applicable at Supporting strength under the PTEN Expert Panel specification, but the available evidence does not document the phase of NM_000314.8:c.964A>G relative to a pathogenic or likely pathogenic PTEN variant, nor at least three observations in cis and/or phase unknown with different pathogenic or likely pathogenic PTEN variants. ClinVar lists the variant as uncertain significance and the reviewed papers do not report this variant or provide phase/co-occurrence observations. The BP2 rule therefore cannot be evaluated.

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 BP2 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.
• ClinVar variation 2504932 records NM_000314.8:c.964A>G (p.Lys322Glu) as uncertain significance in two clinical-laboratory submissions; no phase, co-occurring PTEN variant, or pathogenic/likely pathogenic classification for an accompanying PTEN allele is provided.
• Karbassi et al. 2016 (PMID:26467025) does not mention PTEN, c.964A>G, or p.(K322E), and provides no variant-specific co-occurrence or phase observation.
• Post et al. 2020 (PMID:32350270) does not explicitly mention PTEN c.964A>G or p.(K322E), and provides no variant-specific clinical, phase, or co-occurrence observation.

Still missing

• Documented phase relative to a pathogenic or likely pathogenic PTEN variant is unavailable.
• At least three cis and/or phase-unknown observations with different pathogenic or likely pathogenic PTEN variants are unavailable.

Sources

Reasoning

BP5 is applicable only when the variant is found in at least two cases with an alternate molecular basis, the other gene/disorder is highly penetrant, and each patient’s personal or family history demonstrates no overlap between that disorder and PTEN. The available case record contains no qualifying alternate-gene cases, penetrance assessment, or non-overlapping clinical histories.

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, a highly penetrant alternate gene/disorder, and patient histories showing no overlap between the alternate disorder and PTEN; none are documented in the available case evidence.

Still missing

• At least two 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 with PTEN are unavailable.

Sources