Functional variant in complement C3 gene promoter and genetic susceptibility to temporal lobe epilepsy and febrile seizures

Abstract

Background: Human mesial temporal lobe epilepsies (MTLE) represent the most frequent form of partial epilepsies and are frequently preceded by febrile seizures (FS) in infancy and early childhood. Genetic associations of several complement genes including its central component C3 with disorders of the central nervous system, and the existence of C3 dysregulation in the epilepsies and in the MTLE particularly, make it the C3 gene a good candidate for human MTLE.

Methodology/principal findings: A case-control association study of the C3 gene was performed in a first series of 122 patients with MTLE and 196 controls. Four haplotypes (HAP1 to 4) comprising GF100472, a newly discovered dinucleotide repeat polymorphism [(CA)8 to (CA)15] in the C3 promoter region showed significant association after Bonferroni correction, in the subgroup of MTLE patients having a personal history of FS (MTLE-FS+). Replication analysis in independent patients and controls confirmed that the rare HAP4 haplotype comprising the minimal length allele of GF100472 [(CA)8], protected against MTLE-FS+. A fifth haplotype (HAP5) with medium-size (CA)11 allele of GF100472 displayed four times higher frequency in controls than in the first cohort of MTLE-FS+ and showed a protective effect against FS through a high statistical significance in an independent population of 97 pure FS. Consistently, (CA)11 allele by its own protected against pure FS in a second group of 148 FS patients. Reporter gene assays showed that GF100472 significantly influenced C3 promoter activity (the higher the number of repeats, the lower the transcriptional activity). Taken together, the consistent genetic data and the functional analysis presented here indicate that a newly-identified and functional polymorphism in the promoter of the complement C3 gene might participate in the genetic susceptibility to human MTLE with a history of FS, and to pure FS.

Conclusions/significance: The present study provides important data suggesting for the first time the involvement of the complement system in the genetic susceptibility to epileptic seizures and to epilepsy.

Figure 1. Promoter region of the human complement C3 gene.

Schematic diagram of (top) the human complement C3 gene with exon/intron genomic structure, and (bottom) of its promoter region from the CA-repeat polymorphism marker GF100472 to the start codon. Regulatory elements and nucleotide (nt) positions respective to the canonical transcription start site (+1) are indicated.

Figure 2. Luciferase reporter assays.

(A) Comparison of the two alleles of marker GF100472 with the extreme sizes [(CA)₈ and (CA)₁₅] and (B) with either of GF100472 alleles (CA)₈, (CA)₉, (CA)₁₀, (CA)₁₁ and (CA)₁₅. pGL2 vector containing the C3 promoter region with different CA-repeat variants [(CA)₈, (CA)₉, (CA)₁₀, (CA)₁₁ and (CA)₁₅] was cotransfected into HEK293T cells with a β-galactosidase expression vector (pHSV-LacZ) for normalization of transfection efficiencies. In parallel, empty (promoterless) vector (pGL2-basic) was transfected with pHSV-LacZ. Transcriptional activities were determined by quantifying the luciferase activity of cellular extracts prepared 48 h after transfection. Relative luciferase activity of (CA)₈ was defined as 100. Data show the mean ± SD relative activity from three experiments done in triplicate. Statistical significances were determined by two-tailed unpaired t test. (**) indicates p<0.001. (*) indicates p<0.05. (NS) indicates non significant.