PTEN c.18A>C, p.Lys6Asn

Reasoning

The PTEN VCEP permits PM2 at Supporting strength when a variant is absent or present below 0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population; if multiple alleles occur in a subpopulation, that subpopulation must remain below 0.00002 (0.002%). The variant is absent from gnomAD v2.1 and gnomAD v4.1, satisfying the VCEP PM2 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: absent in population; databases present at <0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population, with <0.00002 (0.002%) required in a subpopulation if multiple alleles are present.
• gnomAD v2.1: NM_000314.8:c.18A>C / GRCh37 10:89624244 A>C absent.
• gnomAD v4.1: GRCh38 10:87864487 A>C absent.

Sources

Reasoning

The variant is a missense change, NM_000314.8:c.18A>C (p.Lys6Asn). The PTEN VCEP's PP2 rule applies to a missense variant in a gene with a low rate of benign missense variation where missense variants are a common disease mechanism; PTEN is governed by this VCEP PP2 specification.

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 case normalization identifies NM_000314.8:c.18A>C as NP_000305.3:p.(Lys6Asn), a missense variant.
• PTEN VCEP CSPEC PP2 specifies supporting evidence for a missense variant in a gene with a low rate of benign missense variation where missense variants are a common mechanism of disease; PP2 is an applicable PTEN criterion in this VCEP specification.

Sources

Reasoning

The PTEN VCEP permits BP4 for missense variants when the REVEL score is less than 0.5. NM_000314.8:c.18A>C is a missense variant producing p.(Lys6Asn), and its REVEL score is 0.489, which is below the VCEP cutoff. The SpliceAI result is not used for BP4 in this missense path because the governing rule names REVEL for missense variants.

VCEP rule

**PTEN EP Specification:** To be applied to synonymous or intronic variants where SpliceAl and VarSeak _in silico_ models predict no splicing impact (SpliceAl: scores 0-0.2 are considered evidence of benign. VarSeak: Class 1 and 2 are considered evidence of benign). Not to be applied for variants which may impact the intron 1 splice donor or acceptor sites, and to be used cautiously for variants which may impact the intron 6 splice acceptor. May also be applied to missense variants with REVEL score < 0.5.

Evidence considered

• PTEN VCEP BP4 specification: may be applied to missense variants with REVEL score < 0.5.
• Local REVEL predictor lookup for NM_000314.8:c.18A>C (p.(Lys6Asn)): REVEL = 0.489; this is below the VCEP BP4 cutoff and supports BP4 at supporting strength.
• Variant consequence: missense, NM_000314.8:c.18A>C, NP_000305.3:p.(Lys6Asn).

Sources

Reasoning

The governing PTEN VCEP assay is Mighell et al. 2018 Table S2. The exact variant is present as K6N / Lys6Asn with High_conf=True and Cum_score=-1.045273268. Although the result is high confidence, it does not meet the VCEP PS3_Moderate cutoff of phosphatase activity <= -1.11. The additional K6N gene-expression study in PMID:27147599 reported correlation with the wild-type PTEN expression signature and stated that K6N did not completely inactivate PTEN function, but it did not establish a VCEP-defined damaging functional result or provide a quantitative phosphatase result meeting PS3_Supporting.

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 K6N / Lys6Asn: Cum_score=-1.045273268, High_conf=True, Imputed_Score=NA; the row is high confidence but does not satisfy the <= -1.11 PS3_Moderate threshold.
• PMID:27147599, Results, Gene expression correlation analysis differentiates allele function, Fig. 3E paragraph: K6N correlated with the signature induced by overexpressing wild-type PTEN, suggesting that it did not completely inactivate PTEN function; this is not a VCEP-defined damaging functional result or a quantitative phosphatase result.
• The literal notations c.18A>C and p.Lys6Asn were not found in the converted mmc2 text; the governing Table S2 entry was found under the alternate keys K6N and Lys6Asn.

Sources

Reasoning

The PTEN VCEP defines PM1 for 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.Lys6Asn at residue 6, outside all three listed catalytic-motif intervals, and the case hotspot assessment reports that it does not lie in a statistically significant hotspot. vcep_materials.json has no domain_tables key for PTEN, so no additional authoritative domain-table entries are available 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

• PTEN VCEP PM1 specifies catalytic-motif residues 90-94, 123-130, and 166-168 in NP_000305.3; the variant is p.Lys6Asn at residue 6.
• The case hotspot result states that this variant does not lie in a statistically significant hotspot.
• vcep_materials.json contains no domain_tables key for PTEN; therefore the VCEP CSPEC catalytic-motif list is the available domain/location rule.

Sources

Reasoning

The PTEN VCEP permits PP3 for missense variants when the REVEL score is greater than 0.7. NM_000314.8:c.18A>C is a missense variant producing p.(Lys6Asn), and its REVEL score is 0.489, which does not exceed the VCEP cutoff. The SpliceAI result is not used for PP3 in this missense path because the governing rule names REVEL for missense variants.

VCEP rule

**PTEN EP Specification:** To be applied to synonymous or intronic variants where SpliceAl and VarSeak _in silico_ models predict a splicing impact (SpliceAl: scores 0.5-1 are consider evidence of pathogenic. VarSeak: Class 4 and 5 are consider evidence of pathogenic). May also be applied to missense variants with REVEL score > 0.7.

Evidence considered

• PTEN VCEP PP3 specification: may be applied to missense variants with REVEL score > 0.7.
• Local REVEL predictor lookup for NM_000314.8:c.18A>C (p.(Lys6Asn)): REVEL = 0.489; this is below the VCEP PP3 cutoff and therefore does not support PP3.
• Variant consequence: missense, NM_000314.8:c.18A>C, NP_000305.3:p.(Lys6Asn).

Sources

Reasoning

The PTEN VCEP BA1 threshold is a gnomAD filtering allele frequency greater than 0.00056 (0.056%). The variant is absent from both default all-comers population datasets available for this case: gnomAD v2.1 and gnomAD v4.1. Therefore, the BA1 threshold is not met.

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: NM_000314.8:c.18A>C / GRCh37 10:89624244 A>C absent; no allele frequency was reported.
• gnomAD v4.1: GRCh38 10:87864487 A>C absent; no allele frequency was reported.

Sources

Reasoning

The PTEN VCEP defines BS1 Strong as a gnomAD filtering allele frequency from 0.000043 (0.0043%) up to 0.00056 (0.056%), and BS1 Supporting as 0.0000043 (0.00043%) up to 0.000043 (0.0043%). The variant is absent from gnomAD v2.1 and gnomAD v4.1, so no frequency falls within either BS1 interval.

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 and gnomAD v4.1 both report the variant as absent, with no allele frequency available for comparison.

Sources

Reasoning

The PTEN VCEP requires observation of the variant in the homozygous state in a healthy or PHTS-unaffected individual, with one observation if homozygosity is confirmed or two if it is unconfirmed; two homozygous observations without clinical data support BS2 Supporting. The available gnomAD records report the variant as absent in both v2.1 and v4.1, with no homozygous observation, so BS2 is not met.

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. ... Two homozygous observations with no clinical data provided, or meets criteria for BS2 but BS1 is also applied.

Evidence considered

• ClinGen PTEN VCEP Version 3.2 BS2 Strong rule requires the variant to be observed homozygously in a healthy or PHTS-unaffected individual; one observation is sufficient if homozygosity is confirmed and two if it is not confirmed.
• ClinGen PTEN VCEP Version 3.2 BS2 Supporting rule requires two homozygous observations with no clinical data, or BS2 evidence when BS1 is also applied.
• gnomAD v2.1 and gnomAD v4.1 both report the variant as absent; therefore no homozygous population observation is available.

Sources

Reasoning

The governing PTEN VCEP benign functional path requires high-confidence Mighell et al. 2018 Table S2 evidence with phosphatase activity >0 for BS3_Supporting. The exact K6N / Lys6Asn row is high confidence but has Cum_score=-1.045273268, which is not >0. The additional K6N gene-expression study reported a wild-type-correlated expression signature and incomplete functional inactivation, but it did not demonstrate the VCEP-required absence of damaging effect together with a qualifying second assay for a missense variant.

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 K6N / Lys6Asn: Cum_score=-1.045273268, High_conf=True, Imputed_Score=NA; the score is not >0, so the VCEP BS3_Supporting threshold is not met.
• PMID:27147599, Results, Gene expression correlation analysis differentiates allele function, Fig. 3E paragraph: K6N correlated with the wild-type PTEN expression signature and was interpreted as not completely inactivating PTEN function, but this alone does not demonstrate no damaging effect under the VCEP missense BS3 specification.

Sources

Reasoning

The PTEN VCEP requires the same amino acid change as a previously established pathogenic variant, regardless of nucleotide change. The case evidence identifies p.Lys6Asn as a ClinVar uncertain-significance variant and provides no verified previously established pathogenic PTEN variant producing the same amino-acid change. The available PM5 comparator collection found no same-residue candidates but also reports a soft retrieval failure, so absence of a qualifying comparator cannot be established from the available 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

• PTEN VCEP CSPEC PS1 rule requires the same amino acid change as a previously established pathogenic variant regardless of nucleotide change.
• ClinVar exact match NM_000314.8(PTEN):c.18A>C (p.Lys6Asn), Variation ID 2561217, is classified as Uncertain significance with single-submitter review status.
• pm5_candidates.json reports total_candidates_seen=6, same_residue_candidates=0, and an HTTP 429 soft retrieval failure; it does not establish that no qualifying pathogenic comparator exists.

Still missing

• A verified database or literature record of a previously established pathogenic PTEN variant producing p.Lys6Asn is needed to assess PS1.

Sources

Reasoning

The ClinGen PTEN VCEP PS2 rule requires a de novo observation in a patient with disease and no family history, with both maternity and paternity confirmed for Strong PS2; the available case evidence contains no proband-level de novo observation, parental testing, maternity/paternity confirmation, or family-history information. Therefore PS2 cannot be assessed.

VCEP rule

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

Evidence considered

• No proband, parental-testing, maternity/paternity, de novo, or family-history data are present in the reviewed case evidence.
• The available literature extracts do not report a de novo observation or parental testing for PTEN c.18A>C (p.Lys6Asn).

Still missing

• Proband-level clinical diagnosis and phenotype documentation
• Variant testing results for both biological parents
• Confirmation of maternity and paternity
• Family history and evidence that no family history is present

Sources

Reasoning

The ClinGen PTEN VCEP permits PS4 for a sufficiently powered case-control study with odds ratio >2 for a PHTS component phenotype, p<0.05, and a 95% confidence-interval lower limit >1.5, or by summing phenotype-specificity points across independent probands. The available case evidence contains no case-control odds ratio, p-value, confidence interval, Cleveland Clinic scores, pediatric phenotype scores, or independent-proband specificity total. Therefore PS4 cannot be evaluated under the VCEP rule. The variant is absent from gnomAD v2.1 and v4.1, but that does not establish PS4 and no PS4 conclusion is made from it.

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 case materials identify NM_000314.8:c.18A>C (p.Lys6Asn) and provide no case-control study statistics or patient-level phenotype scoring data.
• The VCEP source specifies case-control OR >2, p<0.05, and 95% confidence-interval lower limit >1.5 for the sufficiently powered case-control path, and specificity-score thresholds for the proband path.

Still missing

• Case-control odds ratio, p-value, and 95% confidence interval for a PHTS component phenotype are unavailable.
• Independent-proband Cleveland Clinic scores or pediatric phenotype scores needed to calculate the specificity total are unavailable.
• The VCEP's PS4 rule states that this criterion may not be applied if BS1 applies; BS1 status was not evaluated in this group.

Sources

Reasoning

The PTEN VCEP requires a different missense change at the same amino-acid residue that has previously been determined pathogenic or likely pathogenic, with a BLOSUM62 score equal to or less than that of the known variant. The available PM5 candidate collection reports zero same-residue candidates and a soft HTTP 429 retrieval failure, so no qualifying comparator and no required BLOSUM62 comparison can be verified.

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

• PTEN VCEP PM5 requires a different missense change at the same amino-acid residue previously determined pathogenic or likely pathogenic and a BLOSUM62 score equal to or less than the known variant.
• pm5_candidates.json reports same_residue_candidates=0 and an HTTP 429 soft retrieval failure; it does not provide a qualifying comparator or BLOSUM62 values.

Still missing

• A verified pathogenic or likely pathogenic alternate missense variant at PTEN residue Lys6 and its BLOSUM62 comparison with Lys6Asn are needed.

Sources

Reasoning

The ClinGen PTEN VCEP PM6 rules require specified counts of proven, presumed, or assumed de novo observations in patients with disease and no family history; the available case evidence contains no de novo observations, proband-level disease information, parental testing, or family-history information. Therefore PM6 cannot be assessed.

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

Evidence considered

• No proven, presumed, or assumed de novo observations for PTEN c.18A>C (p.Lys6Asn) are documented in the reviewed case evidence.
• The available literature extracts do not report de novo observations, parental testing, or family-history data for this variant.

Still missing

• Documented de novo observations and their number
• Proband-level clinical diagnosis and phenotype documentation
• Parental testing and whether maternity/paternity were confirmed
• Family history for each reported proband

Sources

Reasoning

The ClinGen PTEN VCEP PP1 rules require co-segregation with disease in multiple affected family members and specify thresholds of 3–4, 5–6, or at least 7 meioses; the available case evidence contains no affected relatives, segregation observations, or meiosis count. Therefore PP1 cannot be assessed.

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 family pedigree, affected-relative testing, segregation results, or informative meioses are present in the reviewed case evidence.
• The available literature extracts do not report segregation or meiosis data for PTEN c.18A>C (p.Lys6Asn).

Still missing

• Affected family members tested for the variant
• Unaffected relatives tested for the variant where informative
• Pedigree and phenotype consistency for each relative
• Count of informative meioses and family count

Sources

Reasoning

The ClinGen PTEN VCEP BS4 rules require lack of segregation in affected members of one family for Supporting or two or more families for Strong; the available case evidence contains no affected-family testing or non-segregation observations. Therefore BS4 cannot be assessed.

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-family testing, non-segregation observation, or family count is present in the reviewed case evidence.
• The available literature extracts do not report non-segregation data for PTEN c.18A>C (p.Lys6Asn).

Still missing

• Affected family members tested for the variant
• Clinical phenotype of each tested affected relative
• Family-specific non-segregation results
• Number of families with affected non-carriers

Sources

Reasoning

The PTEN VCEP BP2 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 identifies NM_000314.8:c.18A>C (p.Lys6Asn) but provides no second PTEN variant, cis/trans relationship, phase information, or qualifying series of three observations. BP2 therefore cannot be assessed; absence of such documentation is not evidence against the criterion.

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, Version 3.2, BP2 rule: the required evidence is a trans observation with a pathogenic or likely pathogenic PTEN variant, or at least three cis and/or phase-unknown observations with different pathogenic or likely pathogenic PTEN variants.
• ClinVar exact-match record NM_000314.8(PTEN):c.18A>C (p.Lys6Asn), Variation ID 2561217, is classified as uncertain significance and contains no documented cis/trans or phase relationship to another PTEN variant.

Still missing

• Documented observation of NM_000314.8:c.18A>C in trans with a pathogenic or likely pathogenic PTEN variant, or three qualifying cis/phase-unknown observations with different pathogenic or likely pathogenic PTEN variants.

Sources

Reasoning

The ClinGen PTEN VCEP requires at least two cases in which the variant is found with an alternate molecular basis for disease, with the other gene/disorder highly penetrant and the patient's personal or family history showing no overlap between that disorder and PTEN. The available case materials do not document two such cases, the alternate gene or disorder, its penetrance, or non-overlapping personal/family histories. BP5 therefore cannot be assessed under the VCEP rule.

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_. ... 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 case materials do not document an alternate molecular diagnosis, highly penetrant alternate disorder, or qualifying personal/family histories for two cases.
• The PTEN VCEP instructions add that at least two such cases are required before BP5 can apply.

Still missing

• At least two cases with the variant and an alternate molecular basis are required.
• The alternate gene/disorder and evidence that it is highly penetrant are unavailable.
• Personal and family histories demonstrating no overlap with PTEN are unavailable for the qualifying cases.

Sources