PTEN c.105G>T, p.Met35Ile

Reasoning

The PTEN VCEP applies PM2 at Supporting strength when a variant is absent in population databases 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 both recorded non-cancer subsets, and gnomAD v4.1 has total AF 0.0 with 0/1,612,312 alleles and 0 homozygotes, satisfying PM2 Supporting.

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: 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%).
• gnomAD v2.1: variant absent.
• GNOMAD_V2_1_NON_CANCER: variant absent; the source reported no allele count or frequency because the variant was not found.
• GNOMAD_V3_1_NON_CANCER: variant absent; the source reported no allele count or frequency because the variant was not found.
• gnomAD v4.1: total AC=0, AN=1,612,312, AF=0.0, homozygotes=0; all listed subpopulations also had AF=0.0.

Sources

Reasoning

The PTEN VCEP PP3 rule applies to missense variants using REVEL, with a pathogenic computational threshold of >0.7. NM_000314.8:c.105G>T is a missense variant producing p.(Met35Ile), and the available REVEL score is 0.947, which exceeds the VCEP threshold. The SpliceAI result is not used for PP3 because the VCEP's missense path names REVEL rather than a splice predictor; this prediction is not also counted for BP4 or BP7.

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

• PTEN VCEP PP3 Supporting rule: for missense variants, REVEL score > 0.7.
• Local REVEL predictor lookup for NM_000314.8:c.105G>T (p.(Met35Ile)): REVEL score = 0.947.

Sources

Reasoning

The variant is NP_000305.3:p.(Met35Ile). The reviewed evidence contains no previously established pathogenic variant producing the same amino-acid change, p.Met35Ile, by a different nucleotide substitution. PMID:25875300 and PMID:21828076 report other substitutions at residue 35, including M35R and M35A, but those are different amino-acid changes and therefore do not satisfy PS1.

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

• PMID:21828076, Results/Figure 6A: reports M35R among PHTS-associated mutations but does not report p.Met35Ile.
• PMID:25875300, Results, Functional analysis of tumor-associated N-terminal PTEN mutations, Fig. 2/Table 2: reports M35R and separately M35A, not p.Met35Ile.

Sources

Reasoning

The governing PTEN VCEP rule permits PS3_Moderate for the Mighell et al. 2018 massively parallel phosphatase assay when the Table S2 variant is high confidence and has cumulative phosphatase fitness score <= -1.11. The M35I / Met35Ile row in mmc2.xlsx Table S2 is High_conf=True (Pass SE Filter) with Cum_score=-0.876619465, which does not meet the <= -1.11 PS3_Moderate threshold. The available table row does not establish the separate PS3_Supporting requirement of phosphatase activity <50% of wild type or an abnormal cellular/transgenic assay, so PS3 is not met at a specified strength.

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 mmc2.xlsx, Table S2, row M35I / Met35Ile: Cum_score=-0.876619465; High_conf=True; High_conf_nature=Pass SE Filter; Imputed_score=NA.
• The mmc2.xlsx text identifies columns 9-12 as Cum_score, Cum_SE, High_conf, and High_conf_nature; the M35I row is a missense variant with direct measured score and high-confidence status.
• The variant normalizes as NM_000314.8:c.105G>T, NP_000305.3:p.(Met35Ile), also represented as M35I; c.105G>T and p.Met35Ile were not literal row keys in mmc2.xlsx, but the M35I/Met35Ile row is the matching entry.

Sources

Reasoning

The variant is p.Met35Ile at residue 35. The PTEN VCEP's complete approved catalytic-motif list is residues 90-94, 123-130, and 166-168 (NP_000305.3); residue 35 is outside all three regions. Although the case evidence reports a statistically significant cancer hotspot at PTEN M35, the PTEN rule defines the functional-domain route only through the listed catalytic motifs and does not name a hotspot source or hotspot route, so that finding cannot establish PM1 under this VCEP specification.

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: approved catalytic motifs are residues 90-94, 123-130, and 166-168 (NP_000305.3); p.Met35Ile is at residue 35.
• The case hotspot result identifies PTEN M35 as a statistically significant hotspot, but the VCEP PM1 specification does not name cancerhotspots.org, COSMIC, or a general hotspot route.

Sources

Reasoning

The PTEN VCEP BA1 threshold is a gnomAD filtering allele frequency greater than 0.00056 (0.056%). The variant is absent from gnomAD v2.1 and has gnomAD v4.1 total allele frequency 0.00000 (0/1,612,312 alleles), with 0 homozygotes; therefore the BA1 threshold is not reached.

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: variant absent.
• gnomAD v4.1: total AC=0, AN=1,612,312, AF=0.0, homozygotes=0; highest observed subpopulation AF was also 0.0 in African/African American individuals (AC=0, AN=74,964).

Sources

Reasoning

The PTEN VCEP defines 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 has gnomAD v4.1 total allele frequency 0.00000 (0/1,612,312 alleles), so it falls below both BS1 ranges.

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: variant absent.
• gnomAD v4.1: total AC=0, AN=1,612,312, AF=0.0, homozygotes=0.

Sources

Reasoning

The PTEN VCEP BS2 rule requires observation in the homozygous state in a healthy or PHTS-unaffected individual, with one observation if homozygous status is confirmed or two if it is not confirmed; it specifies Supporting strength for two homozygous observations without clinical data or when BS1 is also applied. gnomAD v4.1 reports 0 homozygotes and AF 0.0, while gnomAD v2.1 is absent, so no BS2 observation is available.

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 Strong 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.
• ClinGen PTEN VCEP Version 3.2, BS2 Supporting rule: Two homozygous observations with no clinical data provided, or meets criteria for BS2 but BS1 is also applied.
• gnomAD v4.1: homozygotes=0, total AC=0, total AN=1,612,312, total AF=0.0.
• gnomAD v2.1: variant absent.

Sources

Reasoning

The governing PTEN VCEP rule permits BS3_Supporting for the Mighell et al. 2018 Table S2 phosphatase assay when the high-confidence variant has phosphatase activity score >0. The matching M35I / Met35Ile row is high confidence but has Cum_score=-0.876619465, not >0; therefore the specified BS3_Supporting rule is not met. No evidence in the consulted VCEP functional file establishes the separate BS3_Strong requirement of well-established functional studies showing no 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 mmc2.xlsx, Table S2, row M35I / Met35Ile: Cum_score=-0.876619465; High_conf=True; High_conf_nature=Pass SE Filter.
• The M35I row is the matching entry for NM_000314.8:c.105G>T / NP_000305.3:p.(Met35Ile); the file contains no positive phosphatase score for this variant.

Sources

Reasoning

The PTEN VCEP BP4 rule applies to missense variants using REVEL, with a benign computational threshold of <0.5. NM_000314.8:c.105G>T is missense and has a REVEL score of 0.947, which does not meet the VCEP benign threshold; therefore BP4 is not met. The SpliceAI result is not used for BP4 because the VCEP's missense path names REVEL rather than a splice predictor.

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

• PTEN VCEP BP4 Supporting rule: for missense variants, REVEL score < 0.5.
• Local REVEL predictor lookup for NM_000314.8:c.105G>T (p.(Met35Ile)): REVEL score = 0.947, above the benign threshold.

Sources

Reasoning

The available case evidence does not document a de novo observation for NM_000314.8:c.105G>T (NP_000305.3:p.(Met35Ile)), confirmation of both maternity and paternity, or absence of family history. The VCEP PS2 rule therefore cannot be evaluated.

Still missing

• Documented proband disease phenotype
• Parental testing showing the variant absent in both parents
• Confirmation of maternity and paternity
• Documented absence of family history

Reasoning

The PTEN VCEP permits PS4 from a sufficiently powered case-control study with odds ratio >2 for a PHTS component phenotype, p<0.05, and 95% confidence-interval lower limit >1.5, or from aggregated phenotype-specificity scores. The available evidence reports no affected-case carrier count, unaffected/control carrier count, odds ratio, confidence interval, p-value, Cleveland Clinic score, or pediatric phenotype score for NM_000314.8:c.105G>T (p.Met35Ile). gnomAD v4.1 reports 0/1,612,312 alleles, but a population-control frequency alone is not a case-control comparison without affected-case data. The reviewed literature packet contained no AI-classified PS4 papers; extracted papers either did not mention this variant or reported different M35 substitutions, so no qualifying case-control or phenotype-specificity evidence can 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. ... **PTEN EP Specifications:** This criterion may not be applied if BS1 applies.  Phenotype specificity scores are added across independent probands and calculated as follows: ... 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 PS4 rule states: case-control evidence requires a sufficiently powered study with odds ratio >2 for a PHTS component phenotype, p<0.05, and a 95% confidence-interval lower limit >1.5; its alternative path uses summed adult Cleveland Clinic or pediatric phenotype-specificity scores.
• gnomAD v4.1: NM_000314.8:c.105G>T has total AC=0 and AN=1,612,312 (AF=0.0), including 0/74,964 African/African American alleles; this supplies control absence but no affected-case frequency.
• No case-control counts, OR, 95% CI, p-value, Cleveland Clinic score, or pediatric phenotype score were present in the case evidence.
• PMID:21828076 reports different PTEN substitutions including M35R, not p.Met35Ile; its functional findings do not establish a PS4 case-control or phenotype-specificity result for this variant.
• PMID:25875300 reports M35R and M35A, not p.Met35Ile, and provides no qualifying PS4 evidence for this variant.
• PMID:23788249, PMID:25356965, PMID:25645574, and PMID:27854360 do not provide variant-specific PTEN p.Met35Ile case-control or phenotype-specificity evidence.

Still missing

• Affected-case carrier count or frequency for NM_000314.8:c.105G>T.
• Matched control carrier count or frequency, with a computed OR and 95% CI and p-value if using case-control evidence.
• Cleveland Clinic score or pediatric phenotype score for independent probands, if using the phenotype-specificity path.

Sources

Reasoning

A different missense change at the same residue, M35R, is listed as pathogenic in the PTEN saturation-mutagenesis reference file's Table S3 summary, and PMID:25875300 reports damaging functional effects for M35R and M35A. However, the available case evidence does not provide the authoritative BLOSUM62 values needed to verify that p.Met35Ile has a score equal to or less than the qualifying known variant, so the VCEP PM5 rule cannot be completed.

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 S3 in mmc2, PTEN c.104T>G (p.Met35Arg) row: Pathogenic (last reviewed Sep 1, 2003).
• PMID:25875300, Results, Fig. 2/Table 2: M35R totally abrogated PTEN activity in the yeast model; the paper also reports M35A functional impairment.
• The VCEP PM5 instruction requires the interrogated variant's BLOSUM62 score to be equal to or less than the known variant; the needed matrix values are not present in the available case evidence.

Still missing

• Authoritative BLOSUM62 values for p.Met35Ile and the qualifying same-residue comparator are not available in the case evidence.

Sources

Reasoning

The available case evidence does not document an assumed de novo observation for NM_000314.8:c.105G>T (NP_000305.3:p.(Met35Ile)), a disease phenotype, or no family history, and it does not provide the number of proven or assumed de novo observations needed for the VCEP PM6 strengths. The criterion cannot be evaluated.

Still missing

• Documented proband disease phenotype
• Evidence supporting presumed de novo status
• Parental testing or explicit statement that maternity and paternity are unconfirmed
• Documented absence of family history
• Number of proven or assumed de novo observations

Reasoning

The available case evidence contains no affected family-member genotypes, phenotype-linked segregation observations, or meiosis count for NM_000314.8:c.105G>T (NP_000305.3:p.(Met35Ile)). The VCEP PP1 thresholds of 3 or 4, 5 or 6, or at least 7 observed meioses therefore cannot be evaluated.

Still missing

• Affected family-member phenotypes
• Variant genotypes in affected relatives
• Informative meiosis count
• Family-level segregation data

Reasoning

The PTEN VCEP marks PP2 applicable at supporting strength, but the available case evidence does not establish both required elements of the rule: a low rate of benign missense variation and missense variants as a common disease mechanism. Variant-specific functional, ClinVar, hotspot, and population observations do not supply the missing gene-level benign-missense-rate assessment.

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

• ClinGen PTEN VCEP specification v3.2 lists PP2 as applicable at Supporting strength but provides no case-specific measurement or threshold for the required gene-level conditions.

Still missing

• A VCEP-supported gene-level assessment of benign missense variation rate and missense disease mechanism is unavailable.

Sources

Reasoning

The available case evidence does not document lack of segregation of NM_000314.8:c.105G>T (NP_000305.3:p.(Met35Ile)) in affected members of one or more families. The VCEP BS4 rule cannot be evaluated.

Still missing

• Affected family-member phenotypes
• Variant status in affected relatives
• Documented non-segregation in at least one family

Reasoning

The PTEN VCEP BP2 rule requires observation of this 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 an affected-proband observation with a second PTEN variant, its pathogenic/likely pathogenic status, or phase, 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

• The case evidence documents NM_000314.8:c.105G>T (p.Met35Ile), but provides no qualifying trans observation, no three qualifying cis or phase-unknown observations with different pathogenic/likely pathogenic PTEN variants, and no phase or inheritance data for this criterion.

Still missing

• Documented affected-proband or family observations involving a second PTEN variant
• Pathogenic or likely pathogenic classification of the second PTEN variant
• Phase determination showing trans, cis, or qualifying phase-unknown observations

Reasoning

BP5 is applicable under the PTEN VCEP 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 evidence contains no two qualifying cases, alternate-gene diagnoses, penetrance documentation, or non-overlapping personal/family histories; therefore BP5 cannot be evaluated.

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 personal/family histories demonstrating no overlap between the alternate disorder and PTEN.
• No qualifying alternate molecular diagnosis or the required case-level phenotype and family-history documentation is available.

Still missing

• At least two independent cases carrying the variant with a documented alternate molecular basis for disease.
• Evidence that each alternate gene/disorder is highly penetrant.
• Personal and family histories showing no overlap between the alternate disorder and PTEN for each case.

Sources