PTEN c.771C>A, p.(Phe257Leu)

Reasoning

The PTEN VCEP governs PS3_Moderate when phosphatase activity is ≤ -1.11 per Mighell et al. 2018. In mmc2.xlsx Table S2, the exact one-letter and three-letter matches are F257L/Phe257Leu, a missense variant with Cum_score -2.055963973 and High_conf True (Pass SE Filter). The pre-assigned functional result therefore meets the VCEP PS3 Moderate threshold; no imputed score is used because a direct high-confidence cumulative score is available.

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

• mmc2.xlsx, Table S2, row F257L/Phe257Leu: missense; Cum_score=-2.055963973; High_conf=True; Pass SE Filter; Imputed_score=NA.
• The PTEN VCEP file index declares mmc2.xlsx as governing PS3 and specifies PS3_Moderate when Cum_score <= -1.11 for this saturation-mutagenesis phosphatase assay.
• The assay is a massively parallel PTEN phosphatase-activity functional screen with cumulative fitness scores and high-confidence filtering as described in the governing file index.

Sources

Reasoning

PM2 is met at supporting strength under the ClinGen PTEN Expert Panel specification v3.2. The rule requires absence or a database allele frequency below 0.00001 (0.001%) in gnomAD or another large sequenced population, with a lower subpopulation threshold when multiple alleles are present; this variant is absent from gnomAD v2.1 and gnomAD v4.1, and therefore has no observed allele frequency or subpopulation allele count in the available records.

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 Expert Panel specification v3.2 assigns PM2 at supporting strength when databases are 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 reports NM_000314.8:c.771C>A as absent, with no allele count, allele frequency, or subpopulation observation.
• GNOMAD_V4_1 reports NM_000314.8:c.771C>A as absent, with no allele count, allele frequency, or subpopulation observation.

Sources

Reasoning

The governing PTEN PM1 rule defines the criterion as location in a mutational hotspot and/or critical, well-established functional domain, specifically including catalytic-motif residues 90-94, 123-130, and 166-168 of NP_000305.3. The variant is p.Phe257Leu, outside all three VCEP-defined catalytic intervals. vcep_materials.json contains an empty domain_tables object for PTEN, so there are no additional authoritative approved domain-table entries to check. The case evidence also states that the variant does not lie in a statistically significant hotspot. PM1 is therefore not met.

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: critical catalytic-motif residues are 90-94, 123-130, and 166-168 on NP_000305.3.
• Variant normalization: NP_000305.3:p.(Phe257Leu), residue 257; this is outside every VCEP-listed catalytic interval.
• vcep_materials.json domain_tables for PTEN: empty; no additional authoritative approved functional-domain entries are present.
• Cancer Hotspots case evidence states that the variant does not lie in a statistically significant hotspot.

Sources

Reasoning

The variant is a missense substitution, NM_000314.8:c.771C>A, NP_000305.3:p.(Phe257Leu). Under the PTEN VCEP PP3 rule, the missense path is evaluated with REVEL and requires a score > 0.7; it does not require a splice prediction in addition to the missense predictor. The available REVEL score is 0.631, below the VCEP cutoff, so PP3 is not met. The VCEP's SpliceAl/VarSeak concordance path applies to splicing variants and is not used to supplement or double-count the missense REVEL path for this variant.

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 missense pathway names REVEL and requires a REVEL score > 0.7; the splicing pathway separately names concordant SpliceAl and VarSeak predictions.
• Local REVEL predictor lookup for NM_000314.8:c.771C>A / NP_000305.3:p.(Phe257Leu): REVEL score = 0.631, which is below the VCEP PP3 threshold of > 0.7.
• Variant Validator consequence: NM_000314.8:c.771C>A produces the missense change NP_000305.3:p.(Phe257Leu); the available SpliceAI result was not used for PP3 because the governing missense path names REVEL and the splicing path requires SpliceAl plus VarSeak concordance.

Sources

Reasoning

BA1 is not met under the ClinGen PTEN Expert Panel specification v3.2. The governing rule requires a gnomAD filtering allele frequency greater than 0.00056 (0.056%); the variant is absent from both gnomAD v2.1 and gnomAD v4.1, so no frequency approaches the threshold.

VCEP rule

gnomAD Filtering allele frequency >0.00056 (0.056%)

Evidence considered

• ClinGen PTEN Expert Panel specification v3.2 defines BA1 as gnomAD Filtering allele frequency >0.00056 (0.056%).
• GNOMAD_V2_1 reports NM_000314.8:c.771C>A as absent; no allele count, allele frequency, homozygote count, or population-specific frequency is reported.
• GNOMAD_V4_1 reports NM_000314.8:c.771C>A as absent; no allele count, allele frequency, homozygote count, or population-specific frequency is reported.

Sources

Reasoning

BS1 is not met under the ClinGen PTEN Expert Panel specification v3.2. The governing BS1 ranges begin at 0.0000043 for supporting strength and 0.000043 for strong strength; the variant is absent from both gnomAD v2.1 and gnomAD v4.1, with no observed allele frequency in either dataset.

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 Expert Panel specification v3.2 defines BS1 Strong as gnomAD Filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%).
• ClinGen PTEN Expert Panel specification v3.2 defines BS1 Supporting as allele frequency from 0.0000043 (0.00043%) up to 0.000043 (0.0043%).
• GNOMAD_V2_1 reports NM_000314.8:c.771C>A as absent, with no allele frequency or population-specific frequency reported.
• GNOMAD_V4_1 reports NM_000314.8:c.771C>A as absent, with no allele frequency or population-specific frequency reported.

Sources

Reasoning

The PTEN VCEP governs BS3_Supporting when phosphatase activity is >0 per Mighell et al. 2018. The exact Table S2 row for F257L/Phe257Leu reports Cum_score -2.055963973 with High_conf True, which is not greater than zero and instead supports the damaging PS3 path. Therefore BS3 is not met.

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

• mmc2.xlsx, Table S2, row F257L/Phe257Leu: missense; Cum_score=-2.055963973; High_conf=True; Pass SE Filter.
• The PTEN VCEP file index declares mmc2.xlsx as governing BS3, and the CSPEC BS3 Supporting rule specifies phosphatase activity >0 per Mighell et al. 2018.

Sources

Reasoning

The variant is a missense substitution, NM_000314.8:c.771C>A, NP_000305.3:p.(Phe257Leu). Under the PTEN VCEP BP4 rule, the missense path is evaluated with REVEL and requires a score < 0.5; it does not require a splice prediction in addition to the missense predictor. The available REVEL score is 0.631, above the VCEP cutoff, so BP4 is not met. The VCEP's SpliceAl/VarSeak concordance path applies to splicing variants and is not used to supplement or double-count the missense REVEL path for this variant.

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 missense pathway names REVEL and requires a REVEL score < 0.5; the splicing pathway separately names concordant SpliceAl and VarSeak predictions.
• Local REVEL predictor lookup for NM_000314.8:c.771C>A / NP_000305.3:p.(Phe257Leu): REVEL score = 0.631, which is above the VCEP BP4 threshold of < 0.5.
• Variant Validator consequence: NM_000314.8:c.771C>A produces the missense change NP_000305.3:p.(Phe257Leu); the available SpliceAI result was not used for BP4 because the governing missense path names REVEL and the splicing path requires SpliceAl plus VarSeak concordance.

Sources

Reasoning

The variant is NP_000305.3:p.(Phe257Leu). The governing PTEN rule requires either the same amino acid change as a previously established pathogenic variant or a different variant at the same nucleotide position as a pathogenic splicing variant with at least equivalent predicted impact. The available evidence identifies no verified previously established pathogenic p.Phe257Leu comparator and no qualifying pathogenic splicing comparator at c.771; ClinVar records this variant as uncertain significance, and the collected comparator search reports zero same-residue candidates. Therefore PS1 cannot be evaluated as met from the available VCEP-governed evidence.

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

• ClinGen PTEN VCEP specification v3.2, PS1: requires the same amino acid change as a previously established pathogenic variant, or a different variant at the same nucleotide position as a pathogenic splicing variant with equivalent or greater predicted impact.
• Variant normalization: NM_000314.8:c.771C>A produces NP_000305.3:p.(Phe257Leu).
• PM5 comparator candidate collection: total candidates seen 1, same-residue candidates 0, expert-panel pathogenic/likely pathogenic candidates 0, and non-expert majority pathogenic/likely pathogenic candidates 0.
• ClinVar variation 492340 classifies this variant as uncertain significance with a single submitter; it is not an established pathogenic comparator.

Still missing

• A verified previously established pathogenic PTEN p.Phe257Leu variant or qualifying pathogenic splicing comparator is unavailable.

Sources

Reasoning

The available case evidence contains no proband-level de novo observation for NM_000314.8:c.771C>A (NP_000305.3:p.(F257L)), no documented confirmation of both maternity and paternity, and no documented absence of family history. The available literature extracts also do not report this variant or a relevant de novo observation. Therefore the PTEN VCEP PS2 requirement cannot be evaluated.

Evidence considered

• No proband, parental-testing, or family-history evidence for this variant is present in the case evidence bundle.
• PMID:25394175 does not mention c.771C>A or p.(F257L) and provides no variant-specific de novo evidence.
• PMID:25741868 does not mention PTEN p.(F257L) and provides no variant-specific de novo evidence.

Still missing

• Proband-level clinical report for NM_000314.8:c.771C>A
• Parental testing confirming maternity and paternity
• Documented family-history assessment

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 summed across independent probands. The available evidence contains no case-control odds ratio, p-value, confidence interval, Cleveland Clinic scores, pediatric phenotype scores, or documented independent proband set for NM_000314.8:c.771C>A. The variant is absent from gnomAD v2.1 and v4.1, but absence in population databases does not satisfy the VCEP's PS4 rule by itself, and the VCEP also states that PS4 may not be applied in combination with PP4.

VCEP rule

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 (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

• The VCEP PS4 instructions require a sufficiently powered case-control study with odds ratio >2, p<0.05, and 95% confidence-interval lower limit >1.5, or phenotype-specificity scoring across independent probands.
• No case-control statistics or phenotype-specificity scores for this variant are present in the available case evidence.
• The variant is absent from gnomAD v2.1 and v4.1, but the VCEP PS4 rule does not authorize applying PS4 from population absence alone.

Still missing

• Case-control odds ratio, p-value, and 95% confidence interval are unavailable.
• Cleveland Clinic scores or pediatric phenotype scores for independent probands are unavailable.

Sources

Reasoning

The variant is a missense change at residue 257. The governing PTEN rule requires a different missense change at the same amino acid residue that has been determined pathogenic or likely pathogenic, and requires the interrogated variant's BLOSUM62 score to be equal to or less than that known variant. The collected evidence reports zero same-residue candidates and provides no qualifying pathogenic or likely pathogenic comparator or BLOSUM62 comparison. PM5 cannot therefore 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

• ClinGen PTEN VCEP specification v3.2, PM5: requires a different missense change at the same amino acid residue determined pathogenic or likely pathogenic, with the interrogated variant's BLOSUM62 score equal to or less than the known variant.
• Variant normalization: NP_000305.3:p.(Phe257Leu), residue 257.
• PM5 comparator candidate collection: same-residue candidates 0; expert-panel pathogenic/likely pathogenic candidates 0; non-expert majority pathogenic/likely pathogenic candidates 0.

Still missing

• A verified pathogenic or likely pathogenic alternate missense variant at residue 257 and its BLOSUM62 score are unavailable.

Sources

Reasoning

The available case evidence contains no presumed or assumed de novo observation for NM_000314.8:c.771C>A (NP_000305.3:p.(F257L)), no proband-level disease documentation, no family-history assessment, and no count of independent de novo observations. The available literature extracts do not report this variant. Therefore the PTEN VCEP PM6 requirement cannot be evaluated at any strength.

Evidence considered

• No presumed or assumed de novo observations for this variant are present in the case evidence bundle.
• PMID:25394175 does not mention c.771C>A or p.(F257L) and provides no variant-specific de novo evidence.
• PMID:25741868 does not mention PTEN p.(F257L) and provides no variant-specific de novo evidence.

Still missing

• Proband-level report documenting disease and presumed de novo occurrence
• Family-history assessment
• Number of independent presumed de novo observations
• Maternity and paternity status for each observation

Sources

Reasoning

The available case evidence contains no affected relatives, family structure, variant-positive relatives, variant-negative relatives, or meiosis count for NM_000314.8:c.771C>A (NP_000305.3:p.(F257L)). Accordingly, co-segregation cannot be evaluated against the PTEN VCEP thresholds of 3 or 4, 5 or 6, or at least 7 meioses.

Evidence considered

• No family segregation results or informative meioses for this variant are present in the case evidence bundle.

Still missing

• Pedigree and affected-family-member information
• Variant testing results for relatives
• Count of informative meioses across families

Sources

Reasoning

The variant is missense, satisfying the variant-type portion of the governing rule. However, the rule also requires that the gene have a low rate of benign missense variation and that missense variants be a common disease mechanism. The available VCEP-governed evidence does not establish both gene-level conditions for PTEN, and no generic threshold or external calibration may be substituted. PP2 is therefore not assessed.

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, PP2: requires a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease.
• Variant consequence: missense, NP_000305.3:p.(Phe257Leu).
• The supplied VCEP materials do not establish both gene-level PP2 conditions; generic population or predictor thresholds were not substituted.

Still missing

• VCEP-governed evidence that PTEN has a low rate of benign missense variation and that missense variants are a common disease mechanism is unavailable.

Sources

Reasoning

BS2 cannot be assessed. The PTEN VCEP requires the variant to be observed in the homozygous state in a healthy or PHTS-unaffected individual, with one observation when homozygosity is confirmed and two when it is not confirmed; the available gnomAD records report absence and provide no individual-level homozygous, health, PHTS-status, or parental-confirmation evidence.

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 Expert Panel specification v3.2 requires an observation in the homozygous state in a healthy or PHTS-unaffected individual; one observation is required when homozygous status is confirmed and two when it is not confirmed.
• The same PTEN rule states that BS2 is applied at supporting evidence level if BS1 is also applied; BS1 is not met here.
• GNOMAD_V2_1 and GNOMAD_V4_1 both report the variant as absent and provide no individual-level phenotype, homozygous genotype, or parental-confirmation data.

Still missing

• Individual-level evidence of the variant in a homozygous healthy or PHTS-unaffected person, including confirmation status, is unavailable.

Sources

Reasoning

The available case evidence contains no affected family members who lack the variant, no family-level non-segregation analysis, and no information for one or more families. Therefore lack of segregation cannot be evaluated at supporting strength for one family or strong strength for two or more families. The PTEN VCEP caveat concerning phenocopies and more than one pathogenic variant also cannot be assessed from the available evidence.

Evidence considered

• No affected-relative testing or non-segregation evidence for this variant is present in the case evidence bundle.

Still missing

• Affected relatives tested for the variant
• Family-level genotype-phenotype results
• Assessment of phenocopies and additional pathogenic variants

Sources

Reasoning

The PTEN VCEP permits BP2 at supporting strength when the variant is 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 or likely pathogenic PTEN variants. The available case evidence documents no qualifying trans observation and no set of three qualifying cis or phase-unknown observations for NM_000314.8:c.771C>A (NP_000305.3:p.(Phe257Leu)); therefore BP2 cannot be evaluated as met or 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 CSPEC v3.2, criteria entry BP2: supporting-level rule requires 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.
• Case evidence for NM_000314.8:c.771C>A (NP_000305.3:p.(Phe257Leu)) contains no documented qualifying trans, cis, or phase-unknown observation with another pathogenic or likely pathogenic PTEN variant.

Still missing

• Documented phase and affected-proband observations pairing NM_000314.8:c.771C>A with pathogenic or likely pathogenic PTEN variants are unavailable.
• No qualifying trans observation or at least three qualifying cis and/or phase-unknown observations were identified.

Sources

Reasoning

The PTEN VCEP BP5 rule requires the variant to be found in a case with an alternate molecular basis for disease, at least two such cases, a highly penetrant alternate gene/disorder, and personal or family history demonstrating no overlap with PTEN. The available evidence documents no alternate molecular diagnosis, qualifying cases, penetrance assessment, or non-overlapping personal/family history.

VCEP rule

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 available case evidence does not document an alternate molecular basis for disease or the required clinical-history exclusions.

Still missing

• At least two qualifying cases with an alternate molecular basis are unavailable.
• Evidence that the alternate gene/disorder is highly penetrant and that personal/family histories do not overlap PTEN is unavailable.

Sources