Clinical And Mechanistic Study Of CPTⅡF352C Mutation In Patients With Severe EV71 Infection

Part I Carnitine palmitoyl transferase 2 polymorphism may be associated with Enterovirus71 severe infection in a Chinese populationGenetic polymorphism in the carnitine palmitoyl transferase 2(CPT2) gene has been reported to be a susceptibility factor in a number of syndromes of acute encephalopathy with various infectious diseases, but evidence of its effect on enterovirus 71(EV71)infection is lacking. The goal of this study was to examine the relationship between genetic polymorphism of CPT2 and severity of EV71 infection in a Chinese population.PCR of five exons of the CPT2 gene was carried out to identify single-nucleotide polymorphisms(SNPs) in EV71-infected subjects(n = 333), including mild cases(n =271) and severe cases(n = 62) as well as healthy controls(n = 328). Blood ATP levels were measured within 24 h of admission. The frequency of the A allele of rs1799821(P =0.023) and the G allele of rs2229291(P = 0.009) in the CPT2 gene was higher in patients with severe EV71 infection. The A-G haplotype of rs1799821 and rs2229291 was directly linked to EV71 severe infection risk when compared to all other haplotypes(OR = 2.005,95 % CI = 1.087-3.700, P = 0.024). The blood ATP levels of severe cases were significantly lower than in mild cases(P\ 0.01) and controls(P\ 0.01). A significant negative correlation was observed in haplotype A-G between ATP levels and physical findings in severe cases(P\ 0.05). These findings suggest that CPT2 polymorphism may be associated with severity of EV71 infection and that the A-G haplotype of the CPT2 gene is involved in the inflammatory process of EV71 infection.Part II The impact of exon-4 F352 C variant on the function of carnitine palmitoyltransferase II in patients with severe EV71 encephalitisCarnitine palmitoyltransferase II(CPT II) is one of the rate-limiting enzymes in the fatty acid oxidation process. Its mutation through single nucleotide polymorphism(SNP)is closely related to mitochondrial fatty acid metabolism and adenosine triphosphate(ATP)production. We have demonstrated that there was a significant decrease of blood CPT II activity and ATP production in children with severe EV71-encephalitis who had1055T>G/F352 C variant in exon-4 of CPT II. We hypothesized that the 1055T>G/F352 C variant leads to changes in the function of CPT II. To test the hypothesis, we designed this series of experiments to achieve the following specific aims: Characterize the enzymatic properties of CPT II variants. We will produce and culture WT CPT II and1055T>G/F352 C variant transfected cells, then measure the enzymatic activity and kinetics of CPT II, mitochondrial membrane potential, mitochondrial β-oxidation and determine the ATP concentration at different temperatures after EV71 infection. We founde that for CPT II F352 C loci, mitochondrial membrane potential were obviously decreased under various temperature of GG genotype carriers, and the obvious negative correlation with temperature after EV71 infection; furthermore, ATP levels of GT, GG genotype Vero cell under 39 ℃ and 41 ℃ cultivation temperature has dropped significantly compared with those under the condition of 37 ℃ by intervention of EV71,but TT genotype after EV71 infection with no significant difference; After EV71 infection,CPT Ⅱenzyme activity of the site G allele carriers were significantly lower residual, and with the temperature rise, thermal instability, the higher the temperature, the lower the CPT Ⅱ residual enzyme activity. These findings suggest that the 1055T>G/F352 C variant in exon-4 affects the function of CPT II in patients with severe EV71-encephalitis.The present research item will lay a foundation for revealing the mechanism of severe EV71-encephalitis through the novel perspective that the CPT II variation leads to the decrease of ATP production.