PIK3CA c.1633G>A, p.Glu545Lys

Reasoning

PS3_Supporting is met. PMID:15930273 directly tested E545K in engineered p110alpha and reported greater lipid kinase activity than wild type, increased Akt and p70S6K phosphorylation, increased transformed foci, and anchorage-independent colony formation. PMID:26637981 directly tested p.Glu545Lys in human umbilical vein endothelial cells and reported AKT activation, abnormal endothelial morphology, altered extracellular-matrix fibronectin, and reversal of abnormal phenotypes by the PI3Kalpha inhibitor BYL719. These are functional assays showing a damaging or activating effect and therefore support PS3 at the VCEP-defined supporting level. The available extracts do not document the positive and negative validation controls or replicate requirements needed for evidence above supporting, so moderate or strong PS3 is not assigned.

VCEP rule

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). ... Any publication within the spreadsheet can be used as evidence for a supporting level of evidence (PS3). ... Any paper must have validation controls (positive and negative) in order to be used as evidence for a level above supporting.

Evidence considered

• PMID:15930273, Functional assays using engineered Myc-tagged p110alpha E545K in HEK293T cells and NIH 3T3 cells: E545K had greater lipid kinase activity than wild type, increased Akt and p70S6K phosphorylation, produced 2.0- to 3.4-fold more transformed foci than wild type, and enabled anchorage-independent colony formation; direct quote: "The hotspot mutations examined in this study, E542K, E545K, and H1047R, all had high enzymatic and transforming activities."
• PMID:26637981, Targeted deep sequencing of venous-malformation tissue plus retroviral expression in human umbilical vein endothelial cells: p.Glu545Lys activated AKT, disrupted endothelial monolayer morphology, altered fibronectin and ANGPT2/PDGFB expression, and the abnormal phenotypes were reversed by BYL719; direct quote: "The p110 a-helical-domain substitutions p.Glu542Lys (c.1624G>A, NM_006218.2) and p.Glu545Lys (c.1633G>A) were identified in eight and four individuals, respectively (together, 44.4%)."
• ClinGen Brain Malformations VCEP specification v1.1, Supplemental Document 1: any publication in the specified spreadsheet may support PS3, whereas evidence above supporting requires positive and negative validation controls and additional assay-quality requirements.

Still missing

• The available evidence does not document the VCEP-required positive and negative validation controls for either functional publication.
• The available evidence does not document the VCEP-required technical replicate details needed to calibrate PS3 above supporting.

Sources

Reasoning

The VCEP requires the PS4 phenotype-point framework to be used only when the variant meets PM2. gnomAD v2.1 reports 1/247,990 alleles and gnomAD v4.1 reports absence, satisfying the available rare-control prerequisite. Applying each reported occurrence to the single highest applicable Table 2A category, PMID:22729223 reports four variant-positive hemimegalencephaly cases, each with neuroimaging demonstrating a large cerebral hemisphere with cortical malformation(s), for 4.0 points; PMID:26637981 reports four variant-positive vascular-malformation individuals, for 3.0 points; and PMID:15608678 reports six E545K-positive primary tumors, for 1.5 points. Total = 8.5 points. The governing Table 2B interval is 3.5-15.75 points for PS4_Strong, so PS4_Strong is met. The named VCEP full-text file contains the governing tables and rules but no exact c.1633G>A, p.Glu545Lys, or p.E545K row; the case counts were therefore taken from the cited papers. No matched unaffected case-control counts or odds ratio were available, but the governing VCEP provides this point-based route for literature cases.

VCEP rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls ... Brain Malformation Recommendation: For PS4, for cases reported in the literature, we recommend assigning each one to the SINGLE category below that is associated with the highest point value (Table 2A). The total score obtained for all reported cases with a particular variant will determine the strength of PS4 assigned according to the scale (Table 2B).* ... PS4_Strong = 3.5-15.75 points

Evidence considered

• VCEP Table 2A assigns 1 point to neuroimaging demonstrating at least one large cerebral hemisphere with cortical malformation(s); PMID:22729223 reports four variant-positive hemimegalencephaly cases, counted once each = 4.0 points.
• VCEP Table 2A assigns 0.75 points to segmental overgrowth or vascular malformation of a limb or body region; PMID:26637981 reports four variant-positive venous-malformation individuals = 3.0 points.
• VCEP Table 2A assigns 0.25 points to presence of the variant in a tumor sample; PMID:15608678 reports six E545K-positive tumors = 1.5 points.
• Total PS4 score = 4.0 + 3.0 + 1.5 = 8.5 points; VCEP Table 2B assigns 3.5-15.75 points to PS4_Strong.
• gnomAD v2.1: 1/247,990 alleles; gnomAD v4.1: absent. These data satisfy the VCEP PS4 prerequisite that the variant be absent/rare from controls according to PM2.
• The VCEP full-text file was searched for c.1633G>A, p.Glu545Lys, and p.E545K; no exact-variant table row was found.
• PMID:22729223 paper_extracts: a recurrent PIK3CA c.1633G>A mutation was found in four separate cases with hemimegalencephaly.
• PMID:26637981 paper_extracts: p.Glu545Lys was identified in four individuals with unifocal venous malformations.
• PMID:15608678 paper_extracts: E545K was detected in three gastric cancers, two breast cancers, and one lung cancer.

Still missing

• No exact c.1633G>A, p.Glu545Lys, or p.E545K entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the file was searched for all three notations.
• No matched unaffected case-control counts or odds ratio were available in the reviewed evidence.

Sources

Reasoning

The governing VCEP assigns PM2 at Supporting strength for a variant absent or rare in controls, with a one-person maximum to account for possible population-database sequencing or calling artifacts. gnomAD v2.1 exomes contains 1 alternate allele in 247,990 alleles (AF 4.03242e-06; 0 homozygotes), while the highest observed European non-Finnish subgroup frequency is 1/112,622 (8.87926e-06); gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer report absence. This is within the VCEP one-person maximum, so PM2_Supporting is met.

VCEP rule

Brain Malformation Recommendation:(One person maximum) This criterion accounts for sequencing artifacts that may have been included in population databases. This number accounts for false calls due to sequencing/ calling errors since the data sets from ExAC/gnomAD are from various sources and GATK calling is also known to call false positives. (PMID: 22827831)

Evidence considered

• ClinGen Brain Malformations VCEP specification, PM2_Supporting: absent from controls or at extremely low frequency in ExAC/gnomAD, with a one-person maximum to account for sequencing and calling artifacts.
• gnomAD v2.1 exomes for NM_006218.4:c.1633G>A (p.Glu545Lys): 1/247990 alleles, AF 4.03242e-06, 0 homozygotes; European non-Finnish subgroup 1/112622 alleles, AF 8.87926e-06, 0 homozygotes.
• gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer report the variant absent.

Still missing

• No variant-specific entry for c.1633G>A, p.Glu545Lys, or p.E545K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the criterion rule itself was found and applied.

Sources

Reasoning

The VCEP marks BP5 as applicable as originally described. PMID:15608678 reports six E545K-positive tumors, including one advanced gastric carcinoma and one invasive ductal breast carcinoma in which E545K co-occurred with a second PIK3CA mutation. Those cases provide an alternate molecular basis for disease, so BP5_Supporting is met.

Rule

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

Evidence considered

• PMID:15608678 paper_extracts: E545K was detected in six tumors; one advanced gastric carcinoma and one invasive ductal breast carcinoma carried E545K together with a second PIK3CA mutation.
• The VCEP BP5 entry is applicable as originally described and states No change.

Sources

Reasoning

Residue 545 is outside every authoritative PIK3CA domain listed in the VCEP Table 4 extraction: Ras-binding domain 173-292, kinase domains 322-483 and 797-1068, and adaptor-binding domain 31-108. The VCEP permits PM1_Supporting when the variant is in an exon within an approved functional domain and explicitly states that recurrent gain-of-function residues are accounted for by PS4 instead. Although PMID:15930273 and PMID:26637981 report E545K as a recurrent hotspot, that hotspot recurrence does not provide a separate PM1 route under this VCEP rule.

VCEP rule

PM1_Supporting Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation ... 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

• VCEP Table 4 approved PIK3CA domains: Kinase Ras-binding domain AA 173-292; kinase domains AA 322-483 and AA 797-1068; adaptor binding domain (PI3K ABD) AA 31-108. Residue 545 is in none of these intervals.
• The VCEP PM1_Supporting text states that a supporting point may be awarded for a variant in an exon within an approved functional domain and that recurrent gain-of-function residues are instead accounted for by PS4.
• PMID:15930273 reports E545K as a PIK3CA helical-domain hotspot; PMID:26637981 reports p.Glu545Lys among recurrent PIK3CA mutations. These observations do not override the VCEP's domain-limited PM1 rule.

Still missing

• No exact c.1633G>A, p.Glu545Lys, or p.E545K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the Table 4 domain rules were available and applied.

Sources

Reasoning

The governing VCEP threshold for BA1 is an allele frequency greater than 0.0926%. gnomAD v2.1 exomes reports 1 of 247,990 alleles, AF 4.03242e-06 (0.000403242%), with 0 homozygotes; the highest observed European non-Finnish subgroup frequency is 8.87926e-06 (0.000887926%), also below 0.0926%. gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer report the variant as absent. Therefore BA1 is not met.

VCEP rule

Brain Malformation Recommendation: 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: allele frequency >0.0926% is the approved stand-alone threshold.
• gnomAD v2.1 exomes for NM_006218.4:c.1633G>A (p.Glu545Lys): AF 4.03242e-06, 1/247990 alleles, 0 homozygotes; European non-Finnish AF 8.87926e-06, 1/112622 alleles, 0 homozygotes.
• gnomAD v4.1 reports the variant absent; the case population evidence also reports absence in gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer.

Still missing

• No variant-specific entry for c.1633G>A, p.Glu545Lys, or p.E545K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the criterion rule itself was found and applied.

Sources

Reasoning

The governing VCEP threshold for BS1 is an allele frequency greater than 0.0185%. gnomAD v2.1 exomes reports AF 4.03242e-06 (0.000403242%), and the highest observed European non-Finnish subgroup frequency is 8.87926e-06 (0.000887926%); both are below 0.0185%. gnomAD v4.1 and the non-cancer subsets report the variant as absent. Therefore BS1 is not met.

VCEP rule

Brain Malformation Recommendation: An allele frequency (>0.0185%) was approved. (Supplemental Table 3)

Evidence considered

• ClinGen Brain Malformations VCEP specification, BS1: allele frequency >0.0185% is the approved strong threshold.
• gnomAD v2.1 exomes for NM_006218.4:c.1633G>A (p.Glu545Lys): AF 4.03242e-06 (0.000403242%), 1/247990 alleles; European non-Finnish AF 8.87926e-06 (0.000887926%), 1/112622 alleles.
• gnomAD v4.1 reports the variant absent; the case population evidence also reports absence in gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer.

Still missing

• No variant-specific entry for c.1633G>A, p.Glu545Lys, or p.E545K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the criterion rule itself was found and applied.

Sources

Reasoning

The governing VCEP permits BS2 with at least 3 homozygotes in gnomAD or ExAC, or at least 3 heterozygous instances in well-phenotyped family members; the variant should be germline or somatic in a relevant tissue. gnomAD v2.1 records 0 homozygotes, and no qualifying well-phenotyped family-member observations are available. Therefore BS2 is not met.

VCEP rule

Brain Malformation Recommendation: 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: at least 3 homozygous occurrences in gnomAD or ExAC, or at least 3 heterozygous instances in well-phenotyped family members, may support BS2.
• gnomAD v2.1 exomes for NM_006218.4:c.1633G>A (p.Glu545Lys): 0 homozygotes; the record contains 1 heterozygous allele, not 3 homozygotes.
• No qualifying well-phenotyped family-member observations were present in the available case evidence.

Still missing

• No variant-specific entry for c.1633G>A, p.Glu545Lys, or p.E545K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the criterion rule itself was found and applied.
• No qualifying well-phenotyped family-member observations were available in the reviewed case evidence.

Sources

Reasoning

The VCEP defines PS1 as the same amino acid change as a previously established pathogenic variant regardless of nucleotide change, and requires that the previously established variant meet BMVCEP pathogenic criteria independently. The available evidence identifies this exact p.Glu545Lys variant in functional and clinical literature, but does not provide a distinct nucleotide change producing p.Glu545Lys with an independent BMVCEP pathogenic classification. The governing VCEP text was searched for c.1633G>A, p.Glu545Lys, and p.E545K; no exact variant entry was found 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 as a previously established pathogenic variant regardless of nucleotide change; the previously established variant must meet BMVCEP pathogenic criteria independently.
• The available literature records p.Glu545Lys/E545K itself, but does not document an independent alternate-nucleotide p.Glu545Lys comparator with a BMVCEP pathogenic classification.

Still missing

• No independently BMVCEP-pathogenic alternate nucleotide change producing p.Glu545Lys was available.
• No exact c.1633G>A, p.Glu545Lys, or p.E545K 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 permits PS2_Moderate when Criteria 1 is fulfilled, or when parents are not available but Criteria 2 is fulfilled. PMID:22729223 reports PIK3CA c.1633G>A (p.Glu545Lys) in four individuals with hemimegalencephaly, with the variant detected in affected brain tissue and absent from matched blood, supporting Criteria 2. However, the available extraction does not document whether parents were unavailable, nor does it document confirmed maternity and paternity or absence of the variant from parental samples; therefore the VCEP PS2_Moderate rule cannot be applied. The paper does not establish the VCEP PS2_Strong requirement that both criteria be fulfilled with confirmed parental testing. The governing VCEP file was searched for c.1633G>A, p.Glu545Lys, and p.E545K, with no matching variant entry found.

VCEP rule

Award the PS2_Moderate point if Criteria 1 is fulfilled, OR if parents are not available but Criteria 2 is fulfilled.

Evidence considered

• PMID:22729223 paper_extracts entry: the variant was reported as a recurrent de novo somatic mutation in four individuals with hemimegalencephaly; it was absent from matched blood and detected in affected brain tissue, with approximately 21–40% mutation burden across sampled regions in HME-1573 and approximately 30% in three additional cases.
• PMID:22729223, Abstract; Results, mutation identification and validation; Table 1; Figure 2; Discussion: "A recurrent PIK3CA c.1633G>A mutation was found in four separate cases."

Still missing

• Parental availability, confirmed maternity and paternity, and parental variant testing are not documented.
• No matching c.1633G>A, p.Glu545Lys, or p.E545K entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

The VCEP leaves PM5 unchanged but requires a missense change at the same amino acid residue where a different missense change previously determined to be pathogenic has been seen, with the comparator meeting BMVCEP pathogenic criteria independently. The collected PM5 candidate record contains no same-residue candidates, but its retrieval ended with an HTTP 429 error; therefore the available evidence cannot 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 that was previously determined pathogenic; the governing text further requires the previously established variant to meet BMVCEP pathogenic criteria independently.
• pm5_candidates.json reported same_residue_candidates = 0 and no useful candidates found, with an HTTP 429 retrieval error; this is insufficient to conclude that no comparator exists.

Still missing

• A complete, independently BMVCEP-pathogenic alternate missense search at PIK3CA residue 545 is unavailable.
• The PM5 candidate retrieval reported HTTP Error 429: Too Many Requests and returned zero same-residue candidates.
• No exact c.1633G>A, p.Glu545Lys, or p.E545K 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 allows PP2 for PIK3CA only when the missense constraint z-score computed in ExAC/gnomAD is greater than 3.09. No applicable ExAC/gnomAD missense-constraint z-score is present in the available case evidence, so PP2 cannot be evaluated without importing an unapproved value or threshold.

VCEP rule

Brain Malformation Recommendation: 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 states: missense constraint computed in ExAC/gnomAD was utilized, and PP2 is awarded if the z-score is >3.09; this applies to PIK3CA.
• The available case evidence contains population allele counts for this variant but no PIK3CA missense-constraint z-score.

Still missing

• The ExAC/gnomAD missense-constraint z-score for PIK3CA is not available in the case evidence.
• No exact c.1633G>A, p.Glu545Lys, or p.E545K entry was found in /Users/gs_agent/LYFESCI_HERMES_v11/vcep_db/PIK3CA/fulltext/clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

BS3 is not assessed because the reviewed variant-specific functional evidence reports increased enzymatic, signaling, transforming, or endothelial activity rather than a well-established absence of damaging effect. No reviewed source provides a benign functional result for p.Glu545Lys, and the available evidence does not establish the VCEP-required BS3 assay-quality basis for a benign conclusion.

VCEP rule

Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. ... Brain Malformation Recommendation: 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:15930273 reports high enzymatic and transforming activity for E545K rather than a benign or neutral functional result.
• PMID:26637981 reports AKT activation and abnormal endothelial phenotypes for p.Glu545Lys rather than absence of damaging effect.
• ClinGen Brain Malformations VCEP specification v1.1, BS3 rule: functional studies must show no damaging effect, with SVI/Brain Malformation Group quality metrics and minimum validation controls.

Still missing

• No variant-specific assay showing no damaging effect on protein function or splicing was identified in the reviewed evidence.
• No VCEP-compliant benign functional assay with documented quality metrics and validation controls was available for BS3 assessment.

Sources

Reasoning

The VCEP permits BP2 when this variant is observed in cis or trans with a known pathogenic variant in the same gene. The available variant-specific evidence does not establish such a configuration. PMID:15608678 reports that E545K co-occurred with a second PIK3CA mutation in one gastric carcinoma and one breast carcinoma, but does not establish phase or that the second variant was known pathogenic. PMID:22729223 reports the target variant in four affected individuals but provides no cis/trans co-occurrence with another pathogenic PIK3CA variant. Therefore BP2 cannot be evaluated from the available evidence.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP specification v1.1, BP2 entry: the criterion requires observation in cis or trans with a known pathogenic variant in the same gene.
• Campbell et al. 2004 (PMID:15608678), Results/Table 1: E545K was detected in six tumors; one advanced gastric carcinoma and one invasive ductal breast carcinoma also carried a second PIK3CA mutation, without phase information or classification of that partner as known pathogenic.
• Lee et al. 2012 (PMID:22729223), Abstract/Results/Table 1: PIK3CA c.1633G>A was found in four separate hemimegalencephaly cases, with the variant absent from matched blood and detected in affected brain tissue; no cis/trans pathogenic partner was reported.
• The governing VCEP full-text file was searched under all three variant notations—c.1633G>A, p.Glu545Lys, and p.E545K—and contained no variant-specific table entry.

Still missing

• No evidence establishes whether the second PIK3CA mutation reported with E545K in PMID:15608678 was in cis or trans.
• No evidence establishes that the second PIK3CA mutation reported with E545K in PMID:15608678 was a known pathogenic variant.
• The governing VCEP file clingen_brainmalform_acmg_specifications_v1_1.pdf was searched for c.1633G>A, p.Glu545Lys, and p.E545K; no variant-specific entry was found.

Sources