PIK3CA c.1634A>G, p.Glu545Gly

Reasoning

PS2_Moderate is met under the VCEP rule because the variant was present at a detectable allele fraction in affected epidermal-nevus tissue and absent from unaffected tissue and leukocyte DNA, satisfying Criteria 2 when parents are not available. The study reports heterozygous E545G in nine of 33 epidermal nevi and wild-type sequence in unaffected tissue from four patients and leukocyte DNA from one additional case; independent PCR and bidirectional sequencing confirmed the findings. The VCEP notes that the rule is intended for high-confidence somatic mutations identified by the reporting CLIA laboratory, so human review is warranted because this evidence comes from a published research study rather than a documented CLIA report. The governing VCEP file contained no exact c.1634A>G, p.Glu545Gly, or p.E545G entry.

VCEP rule

Award the PS2_Moderate point if Criteria 1 is fulfilled, OR if parents are not available but Criteria 2 is fulfilled. ... 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). ... For the sake of implementation, these criteria are intended to apply to high-confidence somatic mutations identified by the reporting CLIA laboratory.

Evidence considered

• PMID:17673550, Results subsection ‘Activating PIK3CA Mutations Are Common in EN,’ Table 1: E545G was detected in nine of 33 epidermal nevi (27%).
• PMID:17673550: every affected lesion was heterozygous, while unaffected tissue from four patients and leukocyte DNA from one additional case showed only wild-type sequence; mutations were confirmed by independent PCR and bidirectional sequencing.
• The VCEP PS2 rule permits the Moderate point when parents are not available but the affected-tissue distribution criterion is fulfilled; the VCEP implementation note limits the intended use to high-confidence somatic mutations identified by the reporting CLIA laboratory.

Still missing

• No exact c.1634A>G, p.Glu545Gly, or p.E545G entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.
• The available publication does not document testing by a reporting CLIA laboratory or confirmed maternity and paternity.

Sources

Reasoning

The VCEP requires PM2 before PS4; the variant is absent from gnomAD v2.1 (0/247982 alleles; zero homozygotes) and absent from gnomAD v4.1. Table 2A assigns 0.25 points for presence in a tumor sample, permits COSMIC, and limits counting to 9 tumor samples for one variant. COSMIC reports this exact variant as COSV55873220 with n=213 somatic occurrences; applying the VCEP cap gives 9 x 0.25 = 2.25 points. The required inequality is 1.5 <= 2.25 <= 3.25? yes, so PS4_Moderate is met. PMID:15520168 reports one E545G primary cancer and PMID:17673550 reports E545G in 9 of 33 epidermal nevi, but neither provides a matched-ancestry population case-control comparison or odds ratio; the PS4 decision therefore uses the VCEP's explicitly permitted tumor-occurrence category, not a computed case-control estimate.

VCEP rule

Points are assigned for phenotype according to (Table 2A). Phenotype criteria can only be used if the variant is absent from controls (PM2). Strength of evidence is determined by points according to (Table 2B). PS4_Moderate = 1.5-3.25 points. ... Presence of this variant in a tumor sample (databases such as COSMIC can be used) 0.25 ... A maximum of 9 tumor samples can be counted for an individual variant keeping with the idea that tumor data cannot be utilized for any strength above supporting.

Evidence considered

• ClinGen Brain Malformations VCEP specification v1.1, Table 2A: Presence of this variant in a tumor sample (databases such as COSMIC can be used) = 0.25 points; a maximum of 9 tumor samples can be counted for an individual variant.
• COSMIC evidence for PIK3CA c.1634A>G / p.E545G reports COSV55873220 with somatic count n=213; applying the VCEP cap gives 9 counted occurrences x 0.25 = 2.25 points.
• ClinGen Brain Malformations VCEP specification v1.1, Table 2B: PS4_Moderate = 1.5-3.25 points; explicit comparison: 1.5 <= 2.25 <= 3.25? yes.
• PMID:15520168, Table 1, row A1634G E545G: one primary cancer; no usable population-control frequency is provided for this exact variant.
• PMID:17673550, Results/Table 1: E545G detected in 9 of 33 epidermal nevi (27%); unaffected tissue and leukocyte comparisons were wild type, but this is not a matched population case-control comparison.
• gnomAD v2.1 reports 0/247982 alleles and zero homozygotes; gnomAD v4.1 reports the variant absent, satisfying the VCEP PS4 prerequisite that the variant be absent/rare from controls according to PM2.

Still missing

• No formal matched-ancestry case-control odds ratio or confidence interval was available in PMID:15520168 or PMID:17673550.
• The literature sources do not establish a variant-specific brain-malformation phenotype; PS4 rests on the VCEP tumor-occurrence category.
• The governing VCEP file had no exact variant row under c.1634A>G, p.Glu545Gly, or p.E545G; the answer comes from its criterion tables and rules.

Sources

Reasoning

The Brain Malformations VCEP applies PM2 at Supporting strength for a variant absent or rare in an ethnically matched control population, with a maximum of one person. NM_006218.4:c.1634A>G (p.Glu545Gly/p.E545G) is absent from gnomAD v4.1 and has AF=0 with 0 observed alleles and 0 homozygotes in gnomAD v2.1 all-comers exomes and the non-cancer exome subset; the highest observed subpopulation frequency is also 0. This satisfies the VCEP population requirement.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP specification, PM2 rule: absent/rare from controls in an ethnically-matched cohort population sample (≤1), with the Brain Malformation recommendation limiting the criterion to one person maximum.
• gnomAD v2.1 all-comers exomes: AC=0, AN=247982, AF=0.0, homozygotes=0; highest observed subpopulation was African/African American with AF=0.0.
• gnomAD v2.1 non-cancer exomes: AC=0, AN=233496, AF=0.0, homozygotes=0.
• gnomAD v4.1: variant absent.

Still missing

• No entry for c.1634A>G, p.Glu545Gly, or p.E545G was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched; the VCEP rule was nevertheless available from its criteria text.

Sources

Reasoning

The VCEP permits PM1_Supporting when the variant is located in an exon within an approved Table 4 functional domain and states that recurrent gain-of-function residues are handled by PS4 instead. PIK3CA p.Glu545Gly is at residue 545, outside every authoritative domain listed for PIK3CA: Ras-binding domain 173-292, kinase domains 322-483 and 797-1068, and adaptor-binding domain 31-108. The E545 hotspot therefore does not qualify for PM1 under this VCEP specification.

VCEP rule

Brain Malformation Recommendation: 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

• ClinGen Brain Malformations VCEP Table 4/domain table lists PIK3CA Ras-binding domain 173-292, kinase domains 322-483 and 797-1068, and adaptor-binding domain 31-108; residue 545 is in none of them.
• The VCEP PM1 specification states that recurrent gain-of-function residues are not covered by PM1 and are instead accounted for by PS4.
• Huang et al. 2010 discusses recurrent Glu545 cancer mutations and structural/biochemical effects, but does not establish an additional VCEP-approved PM1 route for residue 545.

Still missing

• No c.1634A>G, p.Glu545Gly, or p.E545G entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt after searching all three notations.

Sources

Reasoning

The Brain Malformations VCEP assigns BA1 when allele frequency is >0.0926%. NM_006218.4:c.1634A>G (p.Glu545Gly/p.E545G) has gnomAD v2.1 all-comers exome AC=0/247,982, AF=0, and gnomAD v2.1 non-cancer exome AC=0/233,496, AF=0; it is also absent from gnomAD v4.1. The observed frequency is therefore below the VCEP BA1 threshold.

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, BA1 rule: allele frequency >0.0926% is the approved stand-alone threshold.
• gnomAD v2.1 all-comers exomes: variant 3-178936092-A-G, AC=0, AN=247982, AF=0.0, homozygotes=0.
• gnomAD v2.1 non-cancer exomes: AC=0, AN=233496, AF=0.0, homozygotes=0.
• gnomAD v4.1: variant absent.

Still missing

• No entry for c.1634A>G, p.Glu545Gly, or p.E545G was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched; the VCEP rule was nevertheless available from its criteria text.

Sources

Reasoning

The Brain Malformations VCEP assigns BS1 when allele frequency is >0.0185%. NM_006218.4:c.1634A>G (p.Glu545Gly/p.E545G) has AF=0 in gnomAD v2.1 all-comers exomes (0/247,982 alleles), AF=0 in the non-cancer exome subset (0/233,496), and is absent from gnomAD v4.1. This is below the VCEP BS1 threshold.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP specification, BS1 rule: allele frequency >0.0185% is the approved strong threshold.
• gnomAD v2.1 all-comers exomes: variant 3-178936092-A-G, AC=0, AN=247982, AF=0.0, homozygotes=0.
• gnomAD v2.1 non-cancer exomes: AC=0, AN=233496, AF=0.0, homozygotes=0.
• gnomAD v4.1: variant absent.

Still missing

• No entry for c.1634A>G, p.Glu545Gly, or p.E545G was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched; the VCEP rule was nevertheless available from its criteria text.

Sources

Reasoning

The Brain Malformations VCEP requires at least 3 homozygotes in gnomAD or at least 3 heterozygous observations in well-phenotyped family members. gnomAD v2.1 all-comers exomes and its non-cancer subset each show 0 homozygotes, and the variant is absent from gnomAD v4.1; no qualifying family observations are available. Therefore the BS2 requirement is not met.

VCEP rule

Award BS2 if ≥3 homozygotes present in gnomAD or ≥3 heterozygous in well phenotyped family members. Clinical laboratories are encouraged to accumulate more than 2 (≥3) instances of well phenotyped family members before applying this strong criterion. To be considered for this point, the variant should be either germline (most common), or somatic in a relevant tissue. Homozygous occurrences in gnomAD or ExAC can also be counted for this point (≥3).

Evidence considered

• ClinGen Brain Malformations VCEP specification, BS2 rule: at least 3 homozygotes in gnomAD or at least 3 heterozygous well-phenotyped family members are required.
• gnomAD v2.1 all-comers exomes: homozygotes=0 for 3-178936092-A-G.
• gnomAD v2.1 non-cancer exomes: homozygotes=0.
• gnomAD v4.1: variant absent.

Still missing

• No entry for c.1634A>G, p.Glu545Gly, or p.E545G was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched; the VCEP rule was nevertheless available from its criteria text.
• No well-phenotyped family-member observations were available for the alternative BS2 path.

Sources

Reasoning

The VCEP requires the same amino acid change as a previously established pathogenic variant and independently pathogenic status under BMVCEP criteria. The reviewed literature reports the exact E545G variant in somatic cancer and epidermal nevi, but no independently established BMVCEP-pathogenic comparator with a different nucleotide change was available. Therefore PS1 cannot be assessed from the available evidence.

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 as a previously established pathogenic variant, with the VCEP requirement that the comparator independently meet BMVCEP pathogenic criteria.
• Campbell et al. 2004, Table 1: A1634G E545G was reported once in primary cancer; this is the exact variant, not a different-nucleotide same-amino-acid comparator.
• Hafner et al. 2007, Results/Table 1: E545G was detected in nine epidermal nevi (27%); this is the exact variant, not an independently established different-nucleotide comparator.

Still missing

• No c.1634A>G, p.Glu545Gly, or p.E545G entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt after searching all three notations.
• An independently BMVCEP-established pathogenic PIK3CA variant producing the same amino acid change through a different nucleotide change was not available.

Sources

Reasoning

The governing ClinGen Brain Malformations VCEP specification requires PS3 functional evidence to follow the SVI/BMVCEP quality and validation-control recommendations. The case literature packet identifies PMID:17376864 as a possible variant-specific functional study and contains an unverified classifier lead describing E545G gain-of-function findings, but that lead is not independently usable evidence. paper_extracts.json has no entry for PMID:17376864, and the publications directory contains only a PNG artifact rather than readable full text or an auditable extraction. Therefore the assay design, biological controls, technical replication, and exact variant-specific result cannot be verified, so PS3 is not assessed.

Evidence considered

• ClinGen Brain Malformations VCEP specification, Document 1 (PS3): the four assay classes are phosphorylation, DEPTOR binding, cell survivability, and cell proliferation; any publication in the spreadsheet may support PS3, while evidence above supporting requires positive and negative validation controls.
• The case literature packet lists PMID:17376864 as a functional-study candidate and reports an unverified classifier lead for E545G; this lead was not treated as evidence because the underlying paper could not be audited from paper_extracts.json or readable full text.

Still missing

• The VCEP file clingen_brainmalform_acmg_specifications_v1_1.pdf was searched for c.1634A>G, p.Glu545Gly, and p.E545G; no variant-specific entry was found.
• PMID:17376864 has no paper_extracts.json entry, and its case publication artifact is only publications/17376864.png; the variant-specific assay details, controls, and replication cannot be audited.
• No independently verifiable functional assay report with the required biological relevance and assay-quality details was available for this criterion.

Sources

Reasoning

The VCEP leaves PM5 unchanged but requires a different missense change at the same amino acid residue that was independently determined pathogenic under BMVCEP criteria. The available PM5 candidate collection contains zero same-residue comparator candidates, and the reviewed exact-variant papers do not establish a different missense substitution at Glu545 as BMVCEP-pathogenic. PM5 is therefore not assessed.

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 its recommendation requires that previously established variant to meet BMVCEP pathogenic criteria independently.
• The PM5 candidate collection reported zero same-residue candidates for PIK3CA residue 545.
• The reviewed literature discusses other Glu545 substitutions, especially E545K, but does not provide an independently BMVCEP-established pathogenic comparator for this criterion.

Still missing

• No c.1634A>G, p.Glu545Gly, or p.E545G entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt after searching all three notations.
• No usable same-residue comparator was available in the PM5 candidate collection; it reported zero same-residue candidates.

Sources

Reasoning

The VCEP specifies PP2 for PIK3CA when the ExAC/gnomAD missense-constraint z-score is greater than 3.09. The case evidence provides no ExAC/gnomAD missense-constraint z-score, so the criterion cannot be evaluated without substituting an unapproved value or threshold.

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 specification applies to PIK3CA and requires a missense-constraint z-score greater than 3.09; no such z-score is available in the case evidence.

Still missing

• No c.1634A>G, p.Glu545Gly, or p.E545G entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt after searching all three notations.
• The required PIK3CA ExAC/gnomAD missense-constraint z-score is not present in the available case evidence.

Sources

Reasoning

The governing ClinGen Brain Malformations VCEP specification defines BS3 through well-established functional studies showing no damaging effect and requires the SVI/BMVCEP quality metrics and minimum validation controls. The available case evidence contains no independently verifiable benign or neutral functional assay for NM_006218.4:c.1634A>G (p.Glu545Gly), and the available functional-study lead is described as gain-of-function rather than a no-damaging-effect result. BS3 therefore cannot be evaluated.

Evidence considered

• ClinGen Brain Malformations VCEP specification, BS3 rule: follow SVI recommendations together with Brain Malformation Group specifications for quality metrics and minimum validation controls.
• The available functional-study lead describes possible gain-of-function evidence, not a validated no-damaging-effect result, and therefore does not support BS3.

Still missing

• The VCEP file clingen_brainmalform_acmg_specifications_v1_1.pdf was searched for c.1634A>G, p.Glu545Gly, and p.E545G; no variant-specific entry was found.
• No variant-specific assay with a validated no-damaging-effect result, appropriate controls, and auditable methodology was available in the case evidence.
• PMID:17376864 has no paper_extracts.json entry, and its case publication artifact is only publications/17376864.png; its functional result cannot be independently audited.

Sources

Reasoning

The VCEP permits BP2 when the variant is observed in cis or trans with a known pathogenic variant in the same gene. The available case evidence and exact-variant paper extracts report E545G in somatic tumors or epidermal nevi, but provide no documented cis/trans phase with a known pathogenic PIK3CA variant; therefore BP2 cannot be assessed.

VCEP rule

Brain Malformation Recommendation: Observed in cis or trans with a known pathogenic variant in the same gene

Evidence considered

• ClinGen Brain Malformations VCEP specification, BP2 section: 'Observed in cis or trans with a known pathogenic variant in the same gene.'
• PMID:15520168, Table 1, row 'A1634G E545G 1 1': E545G was identified in one primary cancer, but the extract reports no cis/trans phase or second pathogenic PIK3CA variant.
• PMID:17673550, Results subsection 'Activating PIK3CA Mutations Are Common in EN', Table 1: 'The E545G mutation was detected in nine cases (27%); no other alterations were found.' The reported lesions were heterozygous and the extract provides no cis/trans observation with a known pathogenic PIK3CA variant.

Still missing

• No phase-resolved observation of NM_006218.4:c.1634A>G/p.E545G with a known pathogenic PIK3CA variant was found in the case evidence bundle.
• No entry for c.1634A>G, p.Glu545Gly, or p.E545G was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; that file supplies the rule but no case-specific phase data.

Sources

Reasoning

The VCEP applies BP5 as originally described. The governing ACMG/AMP criterion requires the variant to be found in a case with an alternate molecular basis for disease. PMID:15520168 and PMID:17673550 document the exact E545G variant but do not establish whether an alternate pathogenic variant was present; no case-level molecular workup demonstrating an alternate basis was available. The evidence is therefore insufficient to call BP5 met or not met.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• ClinGen Brain Malformations VCEP specification v1.1, BP5 clarification: observed in a case with an alternate molecular basis for disease in a different gene.
• PMID:15520168 and PMID:17673550 report the exact E545G variant, but their extracted findings do not document an alternate molecular diagnosis in another gene.

Still missing

• No case-level molecular workup was available showing whether an alternate pathogenic variant in a different gene was present.

Sources