PIK3CA c.1346C>T, p.(Pro449Leu)

Reasoning

PIK3CA p.Pro449Leu affects residue 449, which lies within the PIK3CA kinase-domain interval AA 322-483 approved in Table 4 of the ClinGen Brain Malformations VCEP specification. The VCEP specifies PM1 at Supporting strength for residues in these critical functional domains and does not require separate hotspot recurrence when the approved-domain criterion is met.

Rule

Residues affecting critical functional domains provided in Table 4 for each gene. Supporting.

Evidence considered

• The governing ClinGen Brain Malformations VCEP specification lists PIK3CA kinase-domain AA 322-483 as an approved critical functional domain for PM1_Supporting; residue 449 is within this interval.

Sources

Reasoning

Under the PIK3CA Brain Malformations VCEP, PM2 is Supporting when a variant is absent/rare from controls in an ethnically matched cohort population sample, with a maximum of one person. The exact variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, supporting PM2 at Supporting strength.

Rule

Absent/rare from controls in an ethnically-matched cohort population sample (≥1).

Evidence considered

• The governing Brain Malformations VCEP specifies PM2 at Supporting strength for a variant absent/rare from controls in an ethnically matched cohort population sample, with a maximum of one person.
• The exact variant NM_006218.4:c.1346C>T (p.Pro449Leu) is reported absent from gnomAD v2.1.
• The exact variant is reported absent from gnomAD v4.1.
• The exact variant is reported absent from gnomAD-Canada v1.0; these concordant population observations support absence from available control cohorts without invoking any disease classification from another source.

Sources

Reasoning

The Brain Malformations VCEP PS4 framework requires PM2 and phenotype-point accumulation. Although the variant is absent from the assessed population datasets, the bundle supplies no reportable affected-individual phenotype data that can be assigned Table 2A points. Two somatic-cancer observations are reported in the bundle; per the VCEP material, tumor evidence contributes only 0.25 point per occurrence category and does not reach the 0.5-point PS4_Supporting threshold.

Rule

ClinGen Brain Malformations VCEP PS4: assign Table 2A phenotype points only when PM2 is met; apply Supporting at 0.5-1.25 points, Moderate at 1.5-3.25, Strong at 3.5-15.75, and Very Strong at >=16.

Evidence considered

• The Brain Malformations VCEP specification makes PS4 phenotype-point based, requires PM2, and defines PS4_Supporting as 0.5-1.25 points, PS4_Moderate as 1.5-3.25 points, PS4_Strong as 3.5-15.75 points, and PS4_VeryStrong as at least 16 points.
• The bundle reports the exact variant absent from gnomAD v2.1 and v4.1, satisfying the population-data prerequisite for considering PS4 but not establishing PS4 on its own.
• The bundle reports two somatic-cancer observations and states that the VCEP Table 2A limits tumor evidence to 0.25 point per occurrence category; this is below the supporting threshold without qualifying clinical phenotype evidence.

Still missing

• Exact-variant affected-individual reports with sufficient phenotype, tissue/mosaicism, and independence data to assign Table 2A points were not available in the bundle.
• The listed PS4 candidate papers have no full-text extraction or text file in this case; only publication images are present, so they cannot be audited for exact-variant case counts or phenotype points.

Sources

Reasoning

The PIK3CA Brain Malformations VCEP specifies BA1 at allele frequency >0.0926%. The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, so the BA1 threshold is not reached.

Rule

Allele frequency (>0.0926%).

Evidence considered

• The governing Brain Malformations VCEP specification for PIK3CA sets BA1 at allele frequency >0.0926%.
• The case bundle reports the exact variant NM_006218.4:c.1346C>T (p.Pro449Leu) as absent from gnomAD v2.1.
• The case bundle reports the exact variant as absent from gnomAD v4.1.
• The case bundle reports the exact variant as absent from gnomAD-Canada v1.0; therefore no population allele frequency approaches the BA1 threshold.

Sources

Reasoning

The PIK3CA Brain Malformations VCEP specifies BS1 at allele frequency >0.0185%. The variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, so the BS1 threshold is not reached.

Rule

Allele frequency (>0.0185%).

Evidence considered

• The governing Brain Malformations VCEP specification for PIK3CA sets BS1 at allele frequency >0.0185%.
• The case bundle reports the exact variant NM_006218.4:c.1346C>T (p.Pro449Leu) as absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; there is no observed allele frequency exceeding the BS1 threshold.

Sources

Reasoning

The VCEP requires at least 3 homozygotes in gnomAD/ExAC or at least 3 heterozygous occurrences in well-phenotyped family members. The variant is absent from all three population datasets in the case bundle, providing no homozygote observations, and no qualifying well-phenotyped family-member observations are supplied.

Rule

Award BS2 if ≥3 homozygotes present in gnomAD or ≥3 heterozygous in well phenotyped family members.

Evidence considered

• The governing Brain Malformations VCEP rule awards BS2 only for at least 3 homozygotes in gnomAD or ExAC, or at least 3 heterozygous observations in well-phenotyped family members.
• The exact variant is reported absent from gnomAD v2.1 and gnomAD v4.1, so the bundle contains no homozygote observations.
• The exact variant is also reported absent from gnomAD-Canada v1.0, and the bundle contains no qualifying well-phenotyped family-member observations.

Still missing

• No independent well-phenotyped family-member dataset was provided; such data would not establish BS2 unless at least 3 qualifying heterozygous observations were available.

Sources

Reasoning

The variant is PIK3CA p.Pro449Leu. The available same-residue paper reports p.Pro449Thr, not the same amino-acid change, and the bundle provides no independent source establishing p.Pro449Leu as a previously established pathogenic variant under PS1. The exact variant's ClinVar classifications cannot be used as independent evidence for this criterion.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. No change.

Evidence considered

• PMID:20530683 was checked for the exact variant and same-amino-acid change; it reports p.Pro449Thr rather than p.Pro449Leu.

Still missing

• An independent, clinically established pathogenic PIK3CA variant producing p.Pro449Leu through a different nucleotide change was not identified.

Sources

Reasoning

The applicable Brain Malformations VCEP PS2 rule requires evidence that the variant is present in the proband and absent from parental samples with confirmed maternity and paternity; PS2_Strong additionally requires tissue-specific allele-fraction evidence, while PS2_Moderate requires the parental or tissue condition specified by the VCEP. The bundle contains no proband assay details, parental genotypes, confirmation of maternity/paternity, allele fractions, or affected-tissue versus other-tissue comparison sufficient to adjudicate PS2.

Rule

Award PS2_Strong if Criteria 1 and 2 are fulfilled: the variant is present at a detectable allele fraction and absent from parental samples with confirmed maternity and paternity, and is present at a detectable allele fraction in affected tissue but absent from or detected at a lower allelic fraction in another tissue. Award PS2_Moderate if Criteria 1 is fulfilled, or if parents are unavailable but Criteria 2 is fulfilled.

Evidence considered

• The Brain Malformations VCEP specifies PS2_Strong only when both parental absence with confirmed maternity/paternity and the required affected-tissue versus other-tissue allele-fraction pattern are demonstrated; PS2_Moderate requires the specified de novo parental or tissue evidence.
• The case bundle does not provide the family-testing, proband allele-fraction, or tissue-distribution data needed for either PS2 strength.

Still missing

• Proband variant-detection method, quality metrics, and allele fraction
• Maternal and paternal test results with confirmed maternity and paternity
• For PS2_Strong, affected-tissue and comparison-tissue allele fractions and assay validation details

Sources

Reasoning

The applicable ClinGen Brain Malformations VCEP specification permits PS3 for validated functional assays meeting SVI/BMVCEP quality requirements. The only extracted functional paper was directly reviewed, but it tested PIK3CA p.P449T rather than the case variant p.Pro449Leu; its gain-of-function result therefore cannot be transferred to this variant. No target-specific validated assay result, assay controls, replicate information, or variant-specific biological readout for p.Pro449Leu is available in the case bundle.

Rule

Follow recommendations set forth by the SVI in conjunction with specifications added by the BMVCEP for quality metrics and minimum validation controls required. (Supplemental Document 1).

Evidence considered

• The Brain Malformations VCEP specification states that PS3 follows SVI recommendations with BMVCEP quality metrics and minimum validation controls; it identifies phosphorylation, DEPTOR binding, cell survivability, and cell proliferation as broadly accepted assay classes.
• The extracted PMID:20530683 finding explicitly states that the paper does not report c.1346C>T or p.(P449L); it reports the different same-residue substitution P449T.
• PMID:20530683 reports that P449T showed gain of function (>2-fold) versus wild-type PI3Kalpha in a relative kinase-activity assay, but this result is not variant-identical evidence for p.Pro449Leu and is not used to award PS3.
• The case bundle contains no variant-specific functional assay controls, replicate data, or validated assay result for p.Pro449Leu.

Still missing

• A functional assay directly testing PIK3CA p.Pro449Leu is needed.
• The assay report must document the relevant positive and negative controls, including wild-type and an appropriate benign/likely benign control independent of BS3.
• For strength above Supporting, the VCEP requires documented technical replication and sufficient validated variant controls; these data are unavailable for p.Pro449Leu.

Sources

Reasoning

The collected same-residue comparator is PIK3CA p.Pro449Thr. The paper documents gain of function for p.Pro449Thr, but does not establish that comparator as a pathogenic clinical variant, and the PM5 candidate summary found no useful qualifying same-residue candidates. Therefore PM5 cannot be assigned from the available evidence.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. No change. Moderate.

Evidence considered

• PMID:20530683 reports p.Pro449Thr and a greater-than-twofold gain of relative PI3K-alpha kinase activity compared with wild type, but does not provide an independent clinical pathogenic classification for p.Pro449Thr.
• The PM5 candidate summary reports four candidates but zero same-residue candidates and no useful qualifying candidates.

Still missing

• A different missense change at Pro449 that is independently established as pathogenic or likely pathogenic under the governing PM5 rule was not identified.

Sources

Reasoning

The PIK3CA VCEP rule requires a missense-constraint z-score greater than 3.09. No ExAC/gnomAD missense-constraint z-score for PIK3CA is present in the case-evidence bundle, so PP2 cannot be evaluated.

Rule

Missense constraint computed in ExAC/gnomAD was utilized. Award PP2 if the z-score > 3.09. Supporting.

Evidence considered

• The governing VCEP specifies PP2_Supporting for PIK3CA when the ExAC/gnomAD missense-constraint z-score is greater than 3.09; the bundle does not provide that score.

Still missing

• A verified PIK3CA ExAC/gnomAD missense-constraint z-score is required to compare with the VCEP threshold of >3.09.

Sources

Reasoning

The applicable ClinGen Brain Malformations VCEP specification requires well-established functional evidence showing no damaging effect and directs curators to apply SVI/BMVCEP quality metrics and minimum validation controls. No assay directly testing p.Pro449Leu and demonstrating a benign or otherwise non-damaging effect is available. The extracted functional paper concerns P449T, a different substitution, and reports gain of function rather than a benign result; it cannot support BS3 for p.Pro449Leu.

Rule

Follow recommendations set forth by the SVI in conjunction with specifications added by the Brain Malformation Group for quality metrics and minimum validation controls required.

Evidence considered

• The Brain Malformations VCEP specification defines BS3 as well-established in vitro or in vivo functional evidence showing no damaging effect and requires SVI/BMVCEP quality metrics and minimum validation controls.
• The extracted PMID:20530683 finding states that the tested variant was P449T, not the case variant p.Pro449Leu.
• PMID:20530683 reports gain of function for P449T (>2-fold versus wild type), not a non-damaging result for p.Pro449Leu; no BS3 inference is made from it.

Still missing

• A validated assay directly testing PIK3CA p.Pro449Leu and showing no damaging effect is needed.
• The assay report must document wild-type and appropriate negative/benign controls, assay quality metrics, and replication sufficient for the claimed BS3 strength.

Sources

Reasoning

The VCEP permits BP2 at Supporting strength when the variant is observed in cis or trans with a known pathogenic variant in the same gene. The available case evidence identifies NM_006218.4:c.1346C>T (p.Pro449Leu) as de novo in one ClinVar submission and somatic in another, but provides no second pathogenic PIK3CA variant, affected-proband co-occurrence, or phase information. BP2 therefore cannot be applied from the available evidence.

Rule

Observed in cis or trans with a known pathogenic variant in the same gene.

Evidence considered

• The governing VCEP rule defines BP2 as observed in cis or trans with a known pathogenic variant in the same gene.
• ClinVar records the exact variant, with one submission describing de novo origin and another describing somatic origin, but the available record does not document a second pathogenic PIK3CA variant or phase.

Still missing

• No documented pathogenic PIK3CA variant in the same affected proband or sample for cis/trans comparison.
• No parental or molecular phasing data establishing cis or trans configuration.
• No adequately detailed affected-proband segregation or allelic observation report for this variant.

Sources

Reasoning

BP5 remains applicable without VCEP modification, but the bundle contains no exact-variant case demonstrating an alternate molecular diagnosis that explains the presenting disease.

Rule

ClinGen Brain Malformations VCEP BP5: applicable as originally described; require an alternate molecular basis for disease in a case carrying the variant.

Evidence considered

• The VCEP retains BP5 unchanged: a variant found in a case with an alternate molecular basis for disease. No such case-level evidence appears in the supplied bundle.

Still missing

• Case-level molecular diagnostic results establishing an alternate cause of disease in an individual carrying this exact variant were not provided.

Sources