PIK3CA c.3140A>T, p.His1047Leu

Reasoning

PS3 Supporting is met. The VCEP permits supporting-level PS3 when a publication within its functional-assay evidence set provides functional evidence, while evidence above Supporting requires positive and negative validation controls and 8-34 tested variants for Moderate or 35+ for Strong. PMID:22120714 directly studied the exact PIK3CA p.H1047L variant (c.3140A>T) in recombinant p110α/p85α biochemical assays and found hydrophobic and electrostatic lipid binding several-fold higher than activated wild-type enzyme after phosphopeptide activation. This is a validated, biologically relevant functional assay supporting an activating effect. The available evidence does not establish the VCEP requirements for Moderate or Strong strength, so PS3 is limited to Supporting. No exact entry for c.3140A>T, p.His1047Leu, or p.H1047L was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

VCEP rule

Any publication within the spreadsheet can be used as evidence for a supporting level of evidence (PS3). ... 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

• ClinGen Brain Malformations VCEP specification v1.1, Document 1 (PS3), Functional Assay Validation: any publication within the spreadsheet can support PS3 at Supporting level; evidence above Supporting requires validation controls.
• PMID:22120714, Results, Effects of cancer-linked mutations in p110α/p85α on lipid binding, paragraph 3: recombinant p110α/p85α complexes carrying H1047L showed similarly high hydrophobic binding to neutral lipids and electrostatic binding to PS/PtdIns(4,5)P2-containing lipids, with binding levels a few-fold higher than activated WT enzyme.
• PMID:17376864, Results and Table 1: H1047L induced oncogenic transformation in primary chicken embryo fibroblasts and was included among rare gain-of-function mutants with elevated in-vitro lipid kinase activity.
• PMID:26627007, Results: the exact c.A3140T (p.H1047L) variant was included in a Ba/F3 growth-factor withdrawal functional assay and was among variants with greater than 40-fold increased growth-promoting activity relative to negative controls.
• The governing VCEP file was searched for c.3140A>T, p.His1047Leu, and p.H1047L; no exact variant-specific table entry was found.

Still missing

• No exact entry for c.3140A>T, p.His1047Leu, or p.H1047L was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.
• The available functional evidence does not document the VCEP-required validation controls and variant counts needed for PS3 Moderate or Strong.

Sources

Reasoning

The Brain Malformations VCEP requires PM2 for PS4 and assigns phenotype points using Table 2A, with PS4_Supporting at 0.5-1.25 points. The variant meets the VCEP PM2 prerequisite because gnomAD v2.1 reports one allele and no homozygotes, while gnomAD v4.1 reports it absent. PMID:15520168 explicitly reports p.H1047L in 4 primary breast-cancer samples; under Table 2A, each tumor sample contributes 0.25 points, for 1.00 point total. The paper reports affected-cohort mutation counts but does not provide unaffected-control carrier counts or an odds ratio, so no case-control OR/95% CI can be computed. The 1.00-point total is within the VCEP PS4_Supporting interval.

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_Supporting = 0.5 – 1.25 points

Evidence considered

• ClinGen Brain Malformations VCEP Table 2A: presence of the variant in a tumor sample receives 0.25 points; tumor occurrences may be counted separately, with a maximum of 9 tumor samples per individual.
• ClinGen Brain Malformations VCEP Table 2B and PS4 rule: 0.5-1.25 points corresponds to PS4_Supporting, and phenotype points may be used only when PM2 is met.
• gnomAD v2.1 reports 1/248274 alleles and 0 homozygotes; gnomAD v4.1 reports the variant absent, satisfying the VCEP PM2 prerequisite of no more than one person.
• PMID:15520168, Results and Discussion/Table 1: the exact p.H1047L variant was found in 4 primary breast-cancer samples; these are counted as four tumor samples at 0.25 points each, totaling 1.00 point. The paper does not provide unaffected-control carrier counts or an OR/95% CI.

Still missing

• No entry for c.3140A>T, p.His1047Leu, or p.H1047L was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; this is not an evidence-absence claim and does not alter the rule-based assessment.
• PMID:15520168 does not provide unaffected-control carrier counts or an odds ratio, so a case-control OR and 95% CI cannot be calculated from that paper.

Sources

Reasoning

The governing VCEP permits PM2 Supporting when the variant is absent or rare in an ethnically matched control population sample with no more than 1 allele. gnomAD v2.1 exomes show 1 allele among 248274 total alleles (AF=4.027807986337675e-06), with the highest observed population count also 1 allele in the European (non-Finnish) population; gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer searches report absence, and gnomAD v4.1 reports absence. The observed control count is within the VCEP maximum of 1 allele. The governing VCEP full-text file was searched under c.3140A>T, p.His1047Leu, and p.H1047L, with no exact variant entry found.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP PM2 Supporting rule: absent/rare from controls in an ethnically-matched cohort population sample (≤1).
• gnomAD v2.1 exomes: AC=1, AN=248274, AF=4.027807986337675e-06; European (non-Finnish) subgroup AC=1, AN=112236, AF=8.909797213015431e-06; homozygotes=0.
• gnomAD v2.1 non-cancer: variant absent.
• gnomAD v3.1 non-cancer: variant absent.
• gnomAD v4.1: variant absent.

Still missing

• No c.3140A>T, p.His1047Leu, or p.H1047L entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the governing PM2 rule was identified in the VCEP criteria text.
• The non-cancer subset records provide absence calls without allele-number denominators or exome-level numerical counts.

Sources

Reasoning

Residue His1047 is inside the authoritative PIK3CA kinase-domain range AA 797-1068 in vcep_materials.json and Table 4. However, the governing VCEP explicitly excludes specific residues subject to recurrent gain-of-function mutations from PM1, directing those residues to PS4 instead. H1047L is documented as a recurrent gain-of-function mutation in the consulted variant-specific literature and somatic records, so the domain location does not yield PM1 for this residue.

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

• vcep_materials.json domain_tables lists PIK3CA kinase domains AA 322-483 and AA 797-1068; residue 1047 is within AA 797-1068.
• ClinGen Brain Malformations VCEP Table 4 lists PIK3CA kinase domains AA 322-483 and AA 797-1068.
• The VCEP PM1_Supporting rule permits a supporting point for a variant in an exon within an approved functional domain, but explicitly states that specific residues subject to recurrent gain-of-function mutations are not covered by PM1 and are accounted for by PS4.
• PMID:15520168 reports H1047L among recurrent exon 20 cancer mutations; PMID:22120714 identifies H1047L as a cancer-linked kinase-domain mutation; PMID:17376864 reports H1047L as a gain-of-function mutant; COSMIC records 610 somatic occurrences.

Sources

Reasoning

The governing VCEP threshold for BA1 is an allele frequency greater than 0.0926%. The highest documented gnomAD v2.1 frequency is 8.909797213015431e-06 in the European (non-Finnish) population (0.00089098%), with 1/112236 alleles and 0 homozygotes; the total exome frequency is 4.027807986337675e-06 (0.00040278%), and the variant is absent from gnomAD v4.1. These values are far below the VCEP BA1 threshold. The governing VCEP full-text file was searched under c.3140A>T, p.His1047Leu, and p.H1047L, with no exact variant entry found.

VCEP rule

Allele frequency (>0.0926%). An allele frequency (>0.0926%) was approved.

Evidence considered

• ClinGen Brain Malformations VCEP BA1 rule: allele frequency (>0.0926%).
• gnomAD v2.1 exomes: total AC=1, AN=248274, AF=4.027807986337675e-06 (0.00040278%), homozygotes=0.
• gnomAD v2.1 highest observed population frequency: European (non-Finnish), AC=1, AN=112236, AF=8.909797213015431e-06 (0.00089098%), homozygotes=0.
• gnomAD v4.1: variant absent.

Still missing

• No c.3140A>T, p.His1047Leu, or p.H1047L entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the governing threshold was identified in the VCEP criteria text.
• gnomAD-Canada was unavailable because the query timed out; this does not affect the observed gnomAD frequencies relative to the BA1 threshold.

Sources

Reasoning

The governing VCEP threshold for BS1 is an allele frequency greater than 0.0185%. The highest documented gnomAD v2.1 frequency is 8.909797213015431e-06 in the European (non-Finnish) population (0.00089098%), with a total exome frequency of 4.027807986337675e-06 (0.00040278%); the variant is absent from gnomAD v4.1. The observed frequencies are below the VCEP BS1 threshold. The governing VCEP full-text file was searched under c.3140A>T, p.His1047Leu, and p.H1047L, with no exact variant entry found.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP BS1 rule: allele frequency (>0.0185%).
• gnomAD v2.1 exomes: total AF=4.027807986337675e-06 (0.00040278%), AC=1/248274 alleles, homozygotes=0.
• gnomAD v2.1 highest observed population frequency: European (non-Finnish), AF=8.909797213015431e-06 (0.00089098%), AC=1/112236 alleles, homozygotes=0.
• gnomAD v4.1: variant absent.

Still missing

• No c.3140A>T, p.His1047Leu, or p.H1047L entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the governing threshold was identified in the VCEP criteria text.
• gnomAD-Canada was unavailable because the query timed out; this does not affect the observed gnomAD frequencies relative to the BS1 threshold.

Sources

Reasoning

The governing VCEP requires at least 3 homozygotes in gnomAD or at least 3 heterozygous observations in well-phenotyped family members. gnomAD v2.1 reports 0 homozygotes and gnomAD v4.1 reports the variant as absent; no qualifying family observations are documented. Therefore the BS2 threshold is not met. The governing VCEP full-text file was searched under c.3140A>T, p.His1047Leu, and p.H1047L, with no exact variant entry found.

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 BS2 rule: award BS2 if ≥3 homozygotes are present in gnomAD or ≥3 heterozygous observations occur in well-phenotyped family members.
• gnomAD v2.1 exomes: homozygotes=0; total AC=1 and AN=248274.
• gnomAD v4.1: variant absent.
• No qualifying well-phenotyped family observations are documented.

Still missing

• No c.3140A>T, p.His1047Leu, or p.H1047L entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; the governing BS2 rule was identified in the VCEP criteria text.
• No well-phenotyped family segregation observations are available in the case evidence.

Sources

Reasoning

BS3 is not met. The VCEP requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function, with the specified quality metrics and minimum validation controls. Instead, the exact H1047L variant showed increased lipid-membrane binding relative to activated wild-type enzyme, oncogenic transformation with elevated lipid kinase activity, and increased growth-promoting activity in independent functional assays. These results are directionally incompatible with a no-damaging-effect conclusion. No exact entry for c.3140A>T, p.His1047Leu, or p.H1047L was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

VCEP rule

Well-established in vitro or in vivo functional studies shows no damaging effect on protein function. ... 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

• ClinGen Brain Malformations VCEP specification v1.1, BS3 rule: functional studies must show no damaging effect on protein function and follow the specified quality metrics and minimum validation controls.
• PMID:22120714, Results, Effects of cancer-linked mutations in p110α/p85α on lipid binding: H1047L showed lipid binding a few-fold higher than activated WT enzyme.
• PMID:17376864, Results and Discussion: H1047L induced oncogenic transformation and was among rare gain-of-function mutants with elevated in-vitro lipid kinase activity.
• PMID:26627007, Results: H1047L showed greater than 40-fold increased growth-promoting activity relative to negative controls in the Ba/F3 assay.
• The governing VCEP file was searched for c.3140A>T, p.His1047Leu, and p.H1047L; no exact variant-specific table entry was found.

Still missing

• No exact entry for c.3140A>T, p.His1047Leu, or p.H1047L was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

Sources

Reasoning

The queried variant is p.His1047Leu/p.H1047L. The VCEP requires the previously established pathogenic variant to meet BMVCEP pathogenic criteria independently of PS1. The consulted evidence reports H1047L in cancer cohorts and functional assays, but no independent BMVCEP pathogenic assessment or pre-assigned BMVCEP code is available for the required PS1 determination.

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 specification: PS1 requires the same amino acid change as a previously established pathogenic variant, and its Brain Malformation Recommendation requires that the previously established variant meet BMVCEP pathogenic criteria independently of PS1.
• PMID:15520168 reports c.3140A>T/p.H1047L in 4 of 9 exon 20 mutations detected in breast cancers, but does not establish a BMVCEP pathogenic assessment.
• PMID:22120714 and PMID:26627007 provide variant-specific functional evidence for H1047L, but are not independent BMVCEP pathogenic classifications.
• ClinVar reports pathogenic submissions for the variant, but the case evidence records no expert-panel submission.

Still missing

• No independent BMVCEP pathogenic criteria assessment or pre-assigned pathogenic code for a previously established p.H1047L variant was found in the consulted case evidence.

Sources

Reasoning

The VCEP PS2 rule requires detectable variant allele fraction in an affected proband and absence from parental samples with confirmed maternity and paternity for Criteria 1; PS2_Strong additionally requires detectable variant in affected tissue and absence from or lower fraction in another tissue for Criteria 2. The reviewed exact-variant reports do not document confirmed maternity and paternity, parental testing results, affected-tissue versus other-tissue allele fractions, or no family history. Therefore PS2 cannot be assigned at Strong or Moderate strength from the available evidence. The governing VCEP file was searched for c.3140A>T, p.His1047Leu, and p.H1047L, with no variant-specific table entry found.

VCEP rule

Award the PS2_Strong point if Criteria 1 AND Criteria 2 are fulfilled. ... Criteria 1: if the variant is present at a detectable allelic fraction in a proband with the disease but is absent from parental samples with confirmed maternity and paternity. Criteria 2: can be awarded if 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 ... Award the PS2_Moderate point if Criteria 1 is fulfilled, OR if parents are not available but Criteria 2 is fulfilled.

Evidence considered

• ClinGen Brain Malformations VCEP specification, PS2: Criteria 1 requires a detectable variant allele fraction in a proband with disease, absence from parental samples, and confirmed maternity and paternity; Criteria 2 concerns detectable variant in affected tissue with absence or lower allele fraction in another tissue.
• Campbell et al. 2004, PMID:15520168, Table 1: exact H1047L was reported in breast-cancer cases, but no parental testing, de novo determination, or tissue allele-fraction comparison was reported in the extracted finding.
• Dogruluk et al. 2015, PMID:26627007: exact c.A3140T (p.H1047L) was functionally tested and observed once in ExAC, but no parental testing, de novo determination, or segregation data were reported in the extracted finding.
• Mandelker et al. 2012, PMID:22120714: exact H1047L was evaluated in biochemical assays, but no patient, parental, de novo, or segregation data were reported in the extracted finding.
• Gymnopoulos et al. 2007, PMID:17376864: exact H1047L was studied in cellular transformation and signaling assays, but no parental, de novo, or segregation data were reported in the extracted finding.

Still missing

• Confirmed maternity and paternity with parental samples are not documented.
• Affected-tissue and comparator-tissue allele fractions are not documented.
• No c.3140A>T, p.His1047Leu, or p.H1047L entry was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched.

Sources

Reasoning

The VCEP requires a different missense change at the same amino acid residue that has independently met BMVCEP pathogenic criteria. H1047R is discussed in the consulted literature, but no independent BMVCEP pathogenic assessment for an alternate His1047 missense variant is available in the case evidence. Therefore PM5 cannot be evaluated under the VCEP rule.

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 missense change at the same amino acid residue where a different missense change determined to be pathogenic has been seen, and requires the previously established variant to meet BMVCEP pathogenic criteria independently of PM5.
• PMID:20593314 discusses the alternate H1047R substitution, but does not provide an independent BMVCEP pathogenic assessment.
• PMID:22120714 reports H1047R alongside H1047L in biochemical studies, but does not provide an independent BMVCEP pathogenic assessment for H1047R.

Still missing

• Independent BMVCEP pathogenic criteria assessment for an alternate missense variant at His1047 is unavailable.

Sources

Reasoning

PP2 is applicable to PIK3CA only when the ExAC/gnomAD missense-constraint z-score is greater than 3.09. The consulted case evidence contains no PIK3CA missense-constraint z-score, so the VCEP threshold cannot be evaluated.

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 specifies that missense constraint is computed in ExAC/gnomAD and PP2 is awarded for PIK3CA if the z-score is greater than 3.09.

Still missing

• PIK3CA ExAC/gnomAD missense-constraint z-score is not available in the case evidence.

Sources

Reasoning

The governing VCEP BP2 recommendation is met only when the queried variant is observed in cis or trans with a known pathogenic variant in the same gene. The available exact-variant reports document c.3140A>T/p.His1047Leu or p.H1047L in cancer cohorts and functional studies, but none documents phase with another known pathogenic PIK3CA variant. BP2 is therefore not assessed rather than not met.

VCEP rule

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

Evidence considered

• ClinGen Brain Malformations VCEP BP2 recommendation: 'Observed in cis or trans with a known pathogenic variant in the same gene.'
• PMID:15520168 reports p.H1047L in four primary breast cancers but provides no cis/trans phase with another pathogenic PIK3CA variant.
• PMID:26627007 reports c.A3140T/p.H1047L in functional assays and ExAC but provides no cis/trans phase with another pathogenic PIK3CA variant.
• PMID:22120714 and PMID:17376864 report biochemical or transformation findings for H1047L but provide no cis/trans phase with another pathogenic PIK3CA variant.

Still missing

• No cis/trans phase result linking NM_006218.4:c.3140A>T (p.His1047Leu/p.H1047L) to another known pathogenic PIK3CA variant is available in the case evidence.
• No c.3140A>T, p.His1047Leu, or p.H1047L entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; this is a search result for the governing file, not a claim that the evidence is absent.

Sources

Reasoning

The VCEP applies BP5 as originally described and further specifies that the alternate molecular basis must be in a different gene. The reviewed exact-variant evidence documents breast cancer and PIK3CA-related somatic findings, but does not document a case in which disease was explained by a pathogenic variant in a different gene; therefore BP5 cannot be evaluated as met or not met.

Rule

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

Evidence considered

• ClinGen Brain Malformations VCEP BP5 section: BP5 is observed in a case with an alternate molecular basis for disease in a different gene.
• PMID:15520168 reports PIK3CA H1047L in primary breast-cancer samples but does not report an alternate pathogenic molecular basis in a different gene.

Still missing

• No entry for c.3140A>T, p.His1047Leu, or p.H1047L was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; this is not an evidence-absence claim.
• A case report or complete clinical record identifying both this variant and an alternate pathogenic variant in a different gene is not available in the reviewed evidence.

Sources