PIK3CA c.1624G>A, p.Glu542Lys

Reasoning

PS3_Supporting is met. The governing Brain Malformations VCEP rule requires functional assay evidence meeting the SVI/BMVCEP quality and validation recommendations. PMID:20593314 explicitly studied PIK3CA p.E542K and reported increased PI3Kα enzymatic activity, with activation similar in magnitude to physiological PI3Kα effectors. This is biologically relevant functional evidence supporting a damaging gain-of-function effect. The available extraction does not document the assay's positive and negative validation controls, technical replicate count, or the publication's inclusion in the VCEP functional-assay spreadsheet, so the evidence cannot be calibrated to Moderate or Strong; the VCEP's Strong-level minimum-control and multi-variant requirements are not established here.

VCEP rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product ... Brain Malformation Recommendation: Follow recommendations set forth by the SVI in conjunction with specifications added by the BMVCEP for quality metrics and minimum validation controls required (PMID: 31892348)

Evidence considered

• PMID:20593314, Introduction, paragraph 4; Cancer-specific mutations discussion: the paper explicitly describes PIK3CA p.E542K and reports that E542K increased PI3Kα enzymatic activity, with an increase similar in magnitude to activation by physiological effectors.
• PMID:20593314 direct quote: "Subsequently, it was determined that two additional high frequency mutations, E542K and E545K, also increased enzymatic activity [11]. The increase in activity produced by the mutations was similar in magnitude to the increase that is observed when PI3Kα is activated by its physiological effectors, including phosphorylated tyrosine kinase receptors and their substrates [12]."
• ClinGen Brain Malformations VCEP specification v1.1, PS3_Supporting: the criterion follows SVI recommendations with BMVCEP quality metrics and minimum validation controls; the variant-specific assay result supports the Supporting level, while stronger-level controls and calibration are not documented.

Still missing

• The available PMID:20593314 extraction does not document positive and negative validation controls or technical replicate counts required for strength above Supporting.
• The available case materials do not establish that PMID:20593314 is included in the VCEP functional-assay spreadsheet referenced by the PS3 rule.
• No entry for c.1624G>A, p.Glu542Lys, or p.E542K was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched under all three notations.

Sources

Reasoning

The governing VCEP requires PM2 before phenotype points can be used and assigns PS4 Supporting at 0.5-1.25 points. The variant is absent from gnomAD v2.1 and v4.1, satisfying the available PM2 prerequisite. In PMID:25599672, PIK3CA p.E542K was reported in HME-3, a patient with hemimegalencephaly, supporting one Table 2A phenotype point for cortical malformation; the case also provides direct variant-specific disease phenotype evidence. COSMIC reports the variant in somatic tumors (n=2043), supporting one Table 2A tumor-occurrence point of 0.25, but the record does not establish 2043 independent individuals or provide a case-control denominator. Total conservatively counted point score is 1.25 (1 + 0.25), which satisfies the VCEP PS4 Supporting range. No odds ratio or 95% confidence interval could be calculated because the available literature does not provide matched affected-control counts; this is not required by the VCEP point-based path used here.

VCEP rule

Points are assigned for phenotype according to (Table 2A). Phenotype criteria can only be used if the variant meets criteria for (PM2). Strength of evidence is determined by points according to (Table 2B). ... PS4_Supporting = .5-1.25 points

Evidence considered

• ClinGen Brain Malformations VCEP Table 2A assigns 1 point for neuroimaging demonstrating at least one large cerebral hemisphere with cortical malformation(s), and 0.25 points for presence of the variant in a tumor sample.
• ClinGen Brain Malformations VCEP Table 2B defines PS4 Supporting as 0.5-1.25 points; the PS4 rule requires PM2 before phenotype points are used.
• PMID:25599672, Results paragraph 4 and Table 1/Supplementary Table 3, reports mosaic PIK3CA E542K (c.1624G>A) in patient HME-3 with hemimegalencephaly; the study analyzed 53 patients with focal cortical dysplasia or hemimegalencephaly.
• COSMIC record COSV55873227 reports this variant in somatic cancers with a displayed count of 2043; because independent individuals and control counts are not provided, only the VCEP tumor-occurrence category of 0.25 was counted.
• PM2 prerequisite: gnomAD v2.1 and gnomAD v4.1 report the variant as absent.

Still missing

• No case-control counts or matched-ancestry control frequency were reported for PMID:25599672, so an odds ratio and 95% confidence interval could not be calculated.
• The governing VCEP full-text file contains no variant-specific row for c.1624G>A, p.Glu542Lys, or p.E542K; its Table 2A/2B rules were applied instead: /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

Expert panel ClinGen Brain Malformations Variant Curation Expert Panel classified as Pathogenic.

VCEP rule

This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.

Evidence considered

• The VCEP states: ‘This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.’
• ClinVar expert panel classification

Sources

Reasoning

PS1 is not met. The governing rule requires the same amino-acid change as a previously established pathogenic variant regardless of nucleotide change. The available pathogenic record is the same variant, NM_006218.4:c.1624G>A (p.Glu542Lys/p.E542K), and no qualifying alternate-nucleotide Glu542Lys record was identified in the searched VCEP full-text file or case evidence. The VCEP full-text search returned no match for c.1624G>A, p.Glu542Lys, or p.E542K in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

VCEP rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change ... Brain Malformation Recommendation: Previously established variant must meet criteria as pathogenic per BMVCEP criteria independent of this point.

Evidence considered

• ClinGen Brain Malformations VCEP PS1 rule: same amino-acid change must be established as pathogenic independently of PS1; the available ClinVar expert-panel record is NM_006218.4:c.1624G>A (p.Glu542Lys), the variant under review, not a different nucleotide change producing the same amino-acid change.
• The governing VCEP full-text file was searched for c.1624G>A, p.Glu542Lys, and p.E542K; no entry for this variant notation was found in that file.

Still missing

• No alternate-nucleotide p.Glu542Lys/p.E542K record was identified in the available evidence.
• No entry for c.1624G>A, p.Glu542Lys, or p.E542K was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

PM1 is not met. The VCEP limits PM1_Supporting to an exon within an approved Table 4 functional domain and states that recurrent gain-of-function residues are accounted for by PS4 instead. PIK3CA residue 542 is outside the authoritative domain-table intervals 31-108, 173-292, 322-483, and 797-1068; the literature describes Glu542 as a helical-domain hotspot, but that does not create a separate PM1 route under this VCEP.

VCEP rule

A supporting point may be awarded if the variant of interest is located in an exon within one of the below approved functional domains Table 4. Please note that specific residues subject to recurrent gain-of-function mutations are not covered by this criterion; these are instead accounted for by PS4.

Evidence considered

• vcep_materials.json domain_tables lists PIK3CA approved intervals 31-108, 173-292, 322-483, and 797-1068; residue 542 lies in none of them.
• Table 4 PM1_Supporting rule: the point may be awarded for a variant in an exon within an approved functional domain, while recurrent gain-of-function residues are accounted for by PS4.
• PMID:20593314 describes Glu542 as a recurrent PIK3CA helical-domain mutation, but the VCEP-approved PM1 domain list does not include residue 542.

Sources

Reasoning

The governing VCEP rule assigns BA1 when allele frequency is >0.0926%. The variant was absent from gnomAD v2.1, gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer, so no allele frequency exceeds the BA1 threshold. No variant-specific row was found in the governing VCEP full-text file.

VCEP rule

Allele frequency (>0.0926%). An allele frequency (>0.0926%) was approved. ... Note: this was adjusted from ACMG Guidelines due to maintaining the 5x threshold for benign (consistent with previously established guidelines)

Evidence considered

• ClinGen Brain Malformations VCEP specification, benign stand-alone criterion: allele frequency >0.0926% is required for BA1.
• GNOMAD_V2_1: query 3-178936082-G-A was absent; no allele count, allele number, allele frequency, or homozygote count was reported.
• GNOMAD_V4_1: query 3-179218294-G-A was absent; no allele count, allele number, allele frequency, or homozygote count was reported.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER were also recorded as absent for the variant.

Still missing

• No c.1624G>A, p.Glu542Lys, or p.E542K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

The governing VCEP rule assigns BS1 when allele frequency is >0.0185%. The variant was absent from gnomAD v2.1, gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer, so no allele frequency exceeds the BS1 threshold. No variant-specific row was found in the governing VCEP full-text file.

VCEP rule

Allele frequency (>0.0185%). An allele frequency (>0.0185%) was approved. (Supplemental Table 3).

Evidence considered

• ClinGen Brain Malformations VCEP specification, strong benign criterion: allele frequency >0.0185% is required for BS1.
• GNOMAD_V2_1: query 3-178936082-G-A was absent; no allele count, allele number, allele frequency, or homozygote count was reported.
• GNOMAD_V4_1: query 3-179218294-G-A was absent; no allele count, allele number, allele frequency, or homozygote count was reported.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER were also recorded as absent for the variant.

Still missing

• No c.1624G>A, p.Glu542Lys, or p.E542K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

The governing VCEP rule requires at least 3 homozygotes in gnomAD or at least 3 heterozygous observations in well-phenotyped family members. The available population records report the variant as absent and contain no homozygote count; no qualifying family observations are available. No variant-specific row was found in the governing VCEP full-text file.

VCEP rule

Award BS2 if ≥3 homozygotes present in gnomAD or ≥3 heterozygous in well phenotyped family members. ... Homozygous occurrences in gnomAD or ExAC can also be counted for this point (≥3).

Evidence considered

• ClinGen Brain Malformations VCEP specification: BS2 requires ≥3 homozygotes in gnomAD or ≥3 heterozygous observations in well-phenotyped family members; homozygous occurrences in gnomAD or ExAC may be counted.
• GNOMAD_V2_1 and GNOMAD_V4_1 reported the variant as absent and supplied no homozygote observations.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER also reported the variant as absent; no qualifying family-member observations were present in the reviewed case evidence.

Still missing

• No c.1624G>A, p.Glu542Lys, or p.E542K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

The VCEP requires PS2_Strong only when both Criteria 1 and Criteria 2 are fulfilled: the variant must be detectable and absent from parental samples with confirmed maternity and paternity, and detectable in affected tissue but absent from or lower in another tissue. PMID:25599672 reports mosaic p.E542K in patient HME-3, with 28% alternate allele frequency by next-generation sequencing and 8.5–9% by ddPCR, but does not establish confirmed parental absence or the required affected-versus-other-tissue comparison. Therefore the available evidence is insufficient to assess PS2; it does not establish de novo origin.

VCEP rule

Award the PS2_Strong point if Criteria 1 AND Criteria 2 are fulfilled. ... Criteria 1. The variant is present at a detectable allele fraction but is absent from parental samples with confirmed maternity and paternity. ... Criteria 2. The variant is present at a detectable allele fraction in an affected tissue sample but is absent from or detected at a lower allelic fraction in another tissue (e.g. if present in 5% of brain tissue but absent from the blood or skin this point can be awarded).

Evidence considered

• ClinGen Brain Malformations VCEP specification, PS2 rule: PS2_Strong requires both parental absence with confirmed maternity and paternity and the affected-tissue versus another-tissue allele-fraction criterion.
• Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia, Results paragraph 4 and Table 1, patient HME-3: mosaic PIK3CA E542K (c.1624G>A), 28% alternate allele frequency by next-generation sequencing and 8.5–9% by ddPCR; parental testing and the required cross-tissue comparison are not reported.

Still missing

• Confirmed maternity and paternity with parental samples tested for absence of NM_006218.4:c.1624G>A are not reported.
• A comparison showing the variant detectable in affected tissue and absent or at a lower allelic fraction in another tissue is not reported.
• The governing VCEP file was searched for c.1624G>A, p.Glu542Lys, and p.E542K; no variant-specific entry was found.

Sources

Reasoning

The governing VCEP rule permits PM2_Supporting for a variant absent or rare in an ethnically matched cohort population sample, with one person maximum. The variant was absent from gnomAD v2.1, v4.1, v2.1 non-cancer, and v3.1 non-cancer, but the available records do not establish that any queried cohort is ethnically matched to the affected individual or family. Therefore the VCEP's required ethnic-match condition cannot be evaluated.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP specification: PM2_Supporting is absent/rare from controls in an ethnically-matched cohort population sample (≤1), with the Brain Malformation recommendation allowing one person maximum to account for sequencing artifacts.
• GNOMAD_V2_1 and GNOMAD_V4_1 reported the variant as absent.
• GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER also reported the variant as absent, but the records do not provide the ancestry-match information required by the VCEP rule.

Still missing

• No documented ancestry for the affected individual or family and no evidence identifying a queried gnomAD cohort as ethnically matched.
• No c.1624G>A, p.Glu542Lys, or p.E542K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

PM5 could not be fully assessed. The governing rule requires a different missense change at residue 542 that has independently met the VCEP pathogenic criteria. The available PM5 candidate artifact reports zero same-residue candidates, but its retrieval ended with HTTP 429 and therefore does not establish that no qualifying comparator exists.

VCEP rule

Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before ... Brain Malformation Recommendation: Previously established variant must meet criteria as pathogenic per BMVCEP criteria independent of this point.

Evidence considered

• The VCEP PM5 rule requires a different missense change at the same amino-acid residue and requires that previously established variant to meet BMVCEP pathogenic criteria independently.
• pm5_candidates.json reports same_residue_candidates: 0 and no candidates, but also records HTTP Error 429: Too Many Requests; this is an incomplete search rather than evidence that no comparator exists.

Still missing

• A complete independently verified search for pathogenic alternate missense changes at PIK3CA residue 542 is unavailable because PM5 candidate retrieval ended with HTTP 429.

Sources

Reasoning

PP2 could not be assessed because the VCEP requires the PIK3CA missense-constraint z-score to exceed 3.09, and no ExAC/gnomAD missense-constraint z-score is present in the available case evidence.

VCEP rule

Missense constraint computed in ExAC/gnomAD was utilized. Award PP2 if the z-score > 3.09. (applicable to MTOR, PIK3CA and AKT3 but not PIK3R2).

Evidence considered

• The VCEP PP2 rule requires a missense-constraint z-score greater than 3.09 for PIK3CA; the case evidence contains no such z-score.

Still missing

• No ExAC/gnomAD missense-constraint z-score for PIK3CA was available in the case evidence.

Sources

Reasoning

BS3 is not assessed. The governing Brain Malformations VCEP rule requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function, evaluated with the specified quality metrics and minimum validation controls. The available variant-specific functional evidence for p.E542K instead reports increased PI3Kα enzymatic activity, and no benign/neutral assay result for this variant was identified in the reviewed evidence.

VCEP 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 (PMID: 31892348) (Supplementary Document 1)

Evidence considered

• PMID:20593314, Introduction, paragraph 4; Cancer-specific mutations discussion: E542K increased PI3Kα enzymatic activity, which is opposite to the no-damaging-effect direction required for BS3.
• ClinGen Brain Malformations VCEP specification v1.1, BS3: the criterion concerns functional studies showing no damaging effect and requires the specified quality metrics and minimum validation controls.

Still missing

• No validated benign or neutral functional assay result for p.E542K was available in the reviewed evidence.
• The available case materials do not document a BS3-eligible assay with the VCEP-required quality metrics and minimum validation controls.

Sources

Reasoning

The VCEP marks BP2 as applicable as originally described, so it requires the variant to be observed in trans with a pathogenic variant for a fully penetrant dominant disorder or in cis with a pathogenic variant in any inheritance pattern. The available variant-specific extraction reports mosaic PIK3CA p.E542K in patient HME-3, but provides no cis/trans phase and no pathogenic same-gene partner; BP2 therefore cannot be assessed.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• ClinGen Brain Malformations VCEP specification v1.1, BP2 original ACMG summary: "Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern."
• PMID:25599672, Results paragraph 4, Table 1 HME-3, and Supplementary Table 3: the paper reports mosaic PIK3CA E542K (c.1624G>A) in an affected patient, but the paper-extract record provides no cis/trans phase or pathogenic same-gene partner.
• The case evidence contains no documented cis/trans phase, parental phase result, or same-gene pathogenic allele for NM_006218.4:c.1624G>A.

Still missing

• No case evidence documents cis or trans phase between NM_006218.4:c.1624G>A (p.Glu542Lys/p.E542K) and a known pathogenic PIK3CA variant.
• No parental, offspring, haplotype, or other phase result identifies a pathogenic PIK3CA allele in cis or trans.
• No c.1624G>A, p.Glu542Lys, or p.E542K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

BP5 remains applicable as originally described, with the VCEP specifying an alternate molecular basis in a different gene. The reviewed evidence documents PIK3CA p.E542K in a patient with hemimegalencephaly but does not document a confirmed alternate disease-causing variant in a different gene for that same case; therefore BP5 cannot be assessed.

VCEP rule

Observed in a case with an alternate molecular basis for disease in a different gene

Evidence considered

• The VCEP BP5 rule is ‘Observed in a case with an alternate molecular basis for disease in a different gene.’
• PMID:25599672 reports PIK3CA E542K in HME-3 but does not document a confirmed alternate disease-causing variant in a different gene for HME-3.

Still missing

• No confirmed alternate molecular basis in a different gene was documented for the same variant-positive case in the reviewed evidence.

Sources