PIK3CA c.1035T>A, p.Asn345Lys

Reasoning

The ClinGen Brain Malformations VCEP permits PS3 Supporting when functional evidence follows the SVI/BMVCEP quality and minimum-validation-control recommendations. PMID:22949682 directly studied PIK3CA p.N345K in full-length p110alpha/p85alpha complexes using lipid kinase assays, HDX-MS, and protein-lipid FRET. N345K increased basal lipid kinase activity relative to wild type and was the second most potent activator in the 11-mutant panel, after H1047R; the study also demonstrated variant-specific conformational and membrane-associated effects. PMID:17376864 independently reported N345K as a strong transforming mutant with constitutive Akt/TOR signaling and elevated in-vitro lipid kinase activity. These variant-specific damaging functional results support PS3 at Supporting strength. The available reports do not establish the VCEP requirements for escalation above Supporting: the applicable publication-level positive and negative validation controls and the 8-34 or 35-plus variant thresholds are not documented in the reviewed evidence.

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

Evidence considered

• ClinGen Brain Malformations VCEP Supplementary Document 1, Document 1 (PS3), rules (2)-(4): any publication within the spreadsheet may support PS3 Supporting; evidence above Supporting requires positive and negative validation controls, with 8-34 variants for Moderate and 35+ variants for Strong.
• Burke et al., PMID:22949682, Results, "Lipid Kinase Activity of Cancer-Linked Mutations"; 11-mutant panel including N345K: all cancer-linked mutations increased basal lipid kinase activity compared with WT, with H1047R most potent followed by N345K and E545K; N345K was also evaluated by HDX-MS and protein-lipid FRET.
• Gymnopoulos et al., PMID:17376864, Results, "Rare Cancer-Derived Mutants of p110alpha Induce Oncogenic Transformation in CEF"; N345K was a strong transforming mutant, activated downstream Akt/TOR signaling without growth factors, and showed elevated in-vitro lipid kinase activity.

Still missing

• No direct c.1035T>A, p.Asn345Lys, or p.N345K entry was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched; this is a gap in the governing file's variant-specific entries, not evidence that functional evidence is absent.
• Positive and negative validation-control classifications and the VCEP's 8-34 or 35-plus variant escalation thresholds are not documented for the cited publications, so PS3 is not escalated above Supporting.

Sources

Reasoning

PM1_Supporting is met. The authoritative vcep_materials.json domain_tables entry for PIK3CA lists kinase domains spanning amino acids 322-483 and 797-1068. The variant changes residue 345, which lies within the approved 322-483 kinase-domain interval. The VCEP specifies that a supporting point may be awarded for a variant in an exon within an approved Table 4 functional domain; hotspot recurrence is not required for this domain route.

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.

Evidence considered

• vcep_materials.json domain_tables for PIK3CA lists kinase domains at AA 322-483 and AA 797-1068; residue 345 is within AA 322-483.
• ClinGen Brain Malformations VCEP specification v1.1, Table 4, PIK3CA row: kinase domains, AA 322-483 and AA 797-1068.
• The VCEP PM1 specification permits a supporting point for a variant located in an exon within one of the approved Table 4 functional domains.

Sources

Reasoning

PS1 is not met. The governing rule requires the same amino acid change as a previously established pathogenic variant, and the VCEP adds that the comparator must meet BMVCEP pathogenic criteria independently. The reviewed evidence identifies PIK3CA p.N345K itself in functional and structural literature, but does not identify a distinct nucleotide change producing p.Asn345Lys that was established as pathogenic under BMVCEP criteria. The VCEP full-text file clingen_brainmalform_acmg_specifications_v1_1.pdf was searched for c.1035T>A, p.Asn345Lys, and p.N345K and contained no variant-specific table entry.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change

Evidence considered

• The CSPEC defines PS1 as the same amino acid change as a previously established pathogenic variant and requires the previously established variant to meet BMVCEP pathogenic criteria independently.
• ClinVar reports this variant as Pathogenic from three clinical laboratories, but the case evidence records no expert-panel submission and no validated pathogenic comparator at the same amino acid residue.
• PMID:20593314, PMID:17376864, and PMID:22949682 explicitly discuss N345K, but they do not establish a distinct nucleotide-change comparator as pathogenic under BMVCEP criteria.
• The governing VCEP full-text file was searched for c.1035T>A, p.Asn345Lys, and p.N345K; no matching variant-specific entry was found.

Still missing

• No entry for c.1035T>A, p.Asn345Lys, or p.N345K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf; the available ClinVar record is not a BMVCEP pathogenic determination.

Sources

Reasoning

The VCEP BA1 threshold is an allele frequency greater than 0.0926%. The variant was absent from gnomAD v2.1 and gnomAD v4.1, with no reported allele count or frequency, so the threshold is not met. The instructed search of clingen_brainmalform_acmg_specifications_v1_1.pdf found no variant-specific entry under c.1035T>A, p.Asn345Lys, or p.N345K; the governing criterion-level rule therefore comes from the VCEP criterion specification rather than a pre-assigned variant row.

VCEP rule

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

Evidence considered

• VCEP BA1 rule: allele frequency >0.0926%.
• gnomAD v2.1 exome result for NC_000003.11:g.178921553T>A: absent; no allele count or frequency reported.
• gnomAD v4.1 result for NC_000003.12:g.179203765T>A: absent; no allele count or frequency reported.
• The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer datasets also reported the variant as absent.

Still missing

• No c.1035T>A, p.Asn345Lys, or p.N345K entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

Sources

Reasoning

The VCEP BS1 threshold is an allele frequency greater than 0.0185%. The variant was absent from gnomAD v2.1 and gnomAD v4.1, with no reported allele count or frequency, so the threshold is not met. The instructed search of clingen_brainmalform_acmg_specifications_v1_1.pdf found no variant-specific entry under c.1035T>A, p.Asn345Lys, or p.N345K.

VCEP rule

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

Evidence considered

• VCEP BS1 rule: allele frequency >0.0185%.
• gnomAD v2.1 exome result for NC_000003.11:g.178921553T>A: absent; no allele count or frequency reported.
• gnomAD v4.1 result for NC_000003.12:g.179203765T>A: absent; no allele count or frequency reported.
• The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer datasets also reported the variant as absent.

Still missing

• No c.1035T>A, p.Asn345Lys, or p.N345K entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

Sources

Reasoning

The VCEP requires at least three homozygotes in gnomAD or at least three heterozygotes in well-phenotyped family members. The variant is absent from gnomAD v2.1 and v4.1, providing no homozygous observations, and the case contains no well-phenotyped family-member observations; therefore BS2 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

• VCEP BS2 rule requires at least three gnomAD homozygotes or at least three heterozygous observations in well-phenotyped family members.
• gnomAD v2.1 exome and gnomAD v4.1 results are absent, with no homozygous observations reported.
• No qualifying well-phenotyped family-member heterozygote observations are present in the case evidence.

Still missing

• No c.1035T>A, p.Asn345Lys, or p.N345K entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

Sources

Reasoning

The ClinGen Brain Malformations VCEP defines BS3 as well-established in-vitro or in-vivo functional studies showing no damaging effect, subject to the SVI/BMVCEP quality and minimum-validation-control recommendations. The variant-specific studies do not show a benign or function-preserving result: PMID:22949682 found increased basal lipid kinase activity for N345K relative to wild type and ranked it second among the tested activators, while PMID:17376864 reported strong transformation, constitutive Akt/TOR signaling, and elevated lipid kinase activity. Therefore the available functional evidence contradicts, rather than satisfies, BS3.

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 BMVCEP for quality metrics and minimum validation controls required

Evidence considered

• ClinGen Brain Malformations VCEP BS3 rule: BS3 requires well-established functional studies showing no damaging effect and directs use of SVI/BMVCEP quality metrics and minimum validation controls.
• Burke et al., PMID:22949682, Results, "Lipid Kinase Activity of Cancer-Linked Mutations": N345K increased basal lipid kinase activity compared with WT and was the second most potent activator tested.
• Gymnopoulos et al., PMID:17376864, Results, "Rare Cancer-Derived Mutants of p110alpha Induce Oncogenic Transformation in CEF": N345K induced strong oncogenic transformation, constitutively activated Akt/TOR signaling, and showed elevated lipid kinase activity.

Still missing

• No direct c.1035T>A, p.Asn345Lys, or p.N345K entry was found when clingen_brainmalform_acmg_specifications_v1_1.pdf.txt was searched; this is a gap in the governing file's variant-specific entries, not evidence that benign functional evidence is absent.

Sources

Reasoning

The VCEP PS2 rule requires detectable variant evidence in a proband with disease and absence from parental samples with confirmed maternity and paternity for Criteria 1; PS2_Strong additionally requires detectable variant evidence in affected tissue and absence or lower allele fraction in another tissue for Criteria 2. The available case evidence contains no proband-level allele fraction, parental testing, maternity/paternity confirmation, affected-tissue comparison, or phenotype-linked de novo observation for NM_006218.4:c.1035T>A (p.Asn345Lys), so PS2 cannot be evaluated.

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 (e.g. if present in 5% of brain tissue but absent from the blood or skin this point can be awarded). ... 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 v1.1, PS2 row: Criteria 1 requires a detectable allelic fraction in a proband with disease and absence from parental samples with confirmed maternity and paternity; Criteria 2 concerns detectable variant in affected tissue with absence or lower allelic fraction in another tissue.
• The case evidence records no proband-level allele fraction, parental testing, maternity or paternity confirmation, affected-tissue comparison, or phenotype-linked de novo observation for NM_006218.4:c.1035T>A (p.Asn345Lys).

Still missing

• No proband-level detectable allele fraction and disease-linked clinical record were available.
• No parental samples or confirmed maternity and paternity testing were available.
• No affected-tissue versus other-tissue allele-fraction comparison was available.
• No entry for c.1035T>A, p.Asn345Lys, or p.N345K 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 phenotype points and PM2 before PS4 can be applied. The variant is absent from gnomAD v2.1 and v4.1, supporting the PM2 prerequisite, and COSMIC reports 293 somatic occurrences. However, the available COSMIC result does not identify which occurrences are separate eligible primary tumors, exclude normal tissue, or document patient phenotype; the VCEP permits a maximum of nine tumor samples and requires each eligible occurrence to be counted separately. Therefore the PS4 point total and strength cannot be determined reliably from the available evidence. The VCEP full-text file was searched for c.1035T>A, p.Asn345Lys, and p.N345K, but contains no variant-specific table row.

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 ... Presence of this variant in a tumor sample (databases such as COSMIC can be used) ... 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, Table 2A: presence of the variant in a tumor sample, including databases such as COSMIC, contributes 0.25 points; separate primary tumors count separately, but no more than 9 tumor samples may be counted for one variant.
• ClinGen Brain Malformations VCEP specification, Table 2B: PS4_Supporting is 0.5-1.25 points, PS4_Moderate is 1.5-3.25 points, PS4_Strong is 3.5-15.75 points, and PS4_VeryStrong is at least 16 points.
• COSMIC COSV55873276 reports PIK3CA c.1035T>A / p.N345K 293 times, but the available record provides only an aggregate somatic count and does not establish the eligible separate-primary-tumor count required by Table 2A.
• gnomAD v2.1 and gnomAD v4.1 both report the variant as absent, satisfying the available population prerequisite described by the PS4 rule.

Still missing

• Eligible COSMIC tumor-sample identities, normal-tissue exclusion, and non-duplicate primary-tumor count are unavailable; the VCEP caps counted tumor samples at nine.
• No variant-specific entry for c.1035T>A, p.Asn345Lys, or p.N345K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources

Reasoning

The VCEP PM2 rule requires the variant to be absent or rare in controls in an ethnically matched cohort population sample. The variant is absent from gnomAD v2.1, gnomAD v4.1, and the queried non-cancer subsets, but the available case evidence does not identify the patient's ancestry or establish that any queried cohort is ethnically matched. The population observation supports rarity, but the VCEP's ethnic-matching requirement cannot be verified, so PM2 is not assessed.

VCEP rule

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

Evidence considered

• VCEP PM2 rule requires absence or rarity from controls in an ethnically matched cohort population sample, with a sample size of at least one.
• gnomAD v2.1 exome and gnomAD v4.1 report the variant as absent.
• The queried gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer datasets also report the variant as absent.
• No patient ancestry or explicit ethnic-match designation is provided in the available case evidence.

Still missing

• Patient ancestry and an explicitly ethnically matched control cohort are not documented.
• No c.1035T>A, p.Asn345Lys, or p.N345K entry was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

Sources

Reasoning

PM5 cannot be assessed from the available evidence. The governing rule requires a different missense change at the same amino acid residue that was determined to be pathogenic, and the VCEP requires that comparator to meet BMVCEP pathogenic criteria independently. The collected PM5 candidate file contains zero same-residue candidates; its retrieval also reports an HTTP 429 error, so a complete comparator search cannot be confirmed. The VCEP full-text file clingen_brainmalform_acmg_specifications_v1_1.pdf was searched for c.1035T>A, p.Asn345Lys, and p.N345K and contained no variant-specific table entry.

Rule

Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before

Evidence considered

• The CSPEC PM5 rule requires a different missense change at the same amino acid residue that was determined pathogenic, independently of PM5.
• pm5_candidates.json reports same_residue_candidates: 0, expert_panel_plp_candidates: 0, and non_expert_majority_plp_candidates: 0, with an HTTP 429 retrieval error.
• The reviewed papers discuss N345K itself but do not supply a qualifying alternate missense change at residue 345 established as pathogenic under BMVCEP criteria.
• The governing VCEP full-text file was searched for c.1035T>A, p.Asn345Lys, and p.N345K; no matching variant-specific entry was found.

Still missing

• pm5_candidates.json reports zero same-residue candidates but also an HTTP 429 retrieval error; an independently complete search for alternate missense changes at residue 345 is needed.
• No entry for c.1035T>A, p.Asn345Lys, or p.N345K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.

Sources

Reasoning

PP2 cannot be assessed. The VCEP rule requires a missense-constraint z-score computed in ExAC/gnomAD and specifies a threshold greater than 3.09 for PIK3CA. The case bundle contains no PIK3CA ExAC/gnomAD missense-constraint z-score, so the required threshold comparison cannot be performed.

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 applies to PIK3CA and requires awarding PP2 only when the ExAC/gnomAD missense-constraint z-score is greater than 3.09.
• No ExAC/gnomAD PIK3CA missense-constraint z-score is provided in the case evidence.

Still missing

• Pik3CA ExAC/gnomAD missense-constraint z-score is not present in the available case evidence.

Sources

Reasoning

The VCEP marks BP2 as applicable as originally described; the original ACMG/AMP wording requires the variant to be observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or in cis with a pathogenic variant in any inheritance pattern. The available case evidence does not document a phase-tested pathogenic PIK3CA variant in cis or trans with NM_006218.4:c.1035T>A (p.Asn345Lys), and the literature extracts reviewed for this group provide no clinical phase or allelic observation. The governing VCEP full-text file was searched for c.1035T>A, p.Asn345Lys, and p.N345K, with no variant-specific entry found.

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

• vcep_materials.json, BP2 entry: applicability is "Applicable as originally described" and the governing rule is "Observed in cis or trans with a known pathogenic variant in the same gene."
• The allelic evidence bundle contains no documented phase-tested cis/trans relationship, parental/offspring phase result, or affected-proband observation pairing this variant with another known pathogenic PIK3CA variant.
• The paper-extract findings reviewed for this group contain functional observations but no clinical case, segregation, or phase data for p.N345K.
• The governing VCEP full-text file was searched for c.1035T>A, p.Asn345Lys, and p.N345K; no variant-specific entry was found.

Still missing

• No phase or cis/trans relationship to a known pathogenic PIK3CA variant is documented in the available case evidence.
• No entry for c.1035T>A, p.Asn345Lys, or p.N345K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt; this is a search result, not evidence that allelic evidence is absent.

Sources

Reasoning

The VCEP applies BP5 as originally described: the variant must be observed in a case with an alternate molecular basis for disease in a different gene. The available evidence identifies PIK3CA-related conditions and cancer records, but provides no patient-level case demonstrating this exact variant together with an alternate disease-causing variant in a different gene. The criterion therefore cannot be evaluated from the available evidence. The VCEP full-text file was searched for c.1035T>A, p.Asn345Lys, and p.N345K, but contains no variant-specific table row.

Rule

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

Evidence considered

• ClinGen Brain Malformations VCEP specification, BP5: the VCEP recommendation is observation in a case with an alternate molecular basis for disease in a different gene.
• ClinVar variation 376050 lists multiple PIK3CA-related and cancer conditions but does not provide a patient-level alternate molecular diagnosis in a different gene.
• COSMIC reports somatic occurrences of the PIK3CA variant but does not provide the required patient-level alternate molecular basis in a different gene.

Still missing

• A patient-level report documenting the exact variant and an alternate molecular basis in a different gene is unavailable.
• No variant-specific entry for c.1035T>A, p.Asn345Lys, or p.N345K was found in clingen_brainmalform_acmg_specifications_v1_1.pdf.txt.

Sources