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P366. rs1001179 CAT gene polymorphism could explain the inhibition of CAT expression in CD patients

M. Iborra1, F. Rausell2, E. Buso3, I. Moret1, G. Bastida1,2, L. Tortosa1, M. Aguas1, P. Nos1,2, B. Beltrán1,2

1Hospital Universitario la Fe/Instituto de Investigacion Sanitaria, Valencia, Spain; 2CIBERehd, Barcelona, Spain; 3SCSIE-Universitat de València, Valencia, Spain

Aim: We have previously shown a permanent reduction in activity and protein levels of CAT due to down-regulated genetic expression of CAT in Crohn's disease (CD). In contrast, we detected an increased activity, protein levels and genetic expression of Mn-SOD enzyme at onset of disease. The cause for those findings in mRNA expression in both enzymes in CD patients has never been analyzed by evaluation of single nucleotide polymorphisms (SNPs) in their respective genes. We have aimed to analyze several SNPs from CAT and Mn-SOD genes in order to characterize the cause of the alterations in both enzymes function in CD patients.

Methods: Blood samples from healthy subjects (n = 29) and from patients at first flare of CD (n = 29). The following SNPs of CAT and/or Mn-SOD enzymes were analyzed: rs1001179, rs704724, rs7943316, rs1049982, rs525938, rs2268064, rs2300182, rs3758730, rs2284365, rs564250, rs494024, rs12273124, rs475043 for CAT and rs4880, rs5746096, rs2758346, rs2758339, rs5746136 for SOD. PWMC were isolated by Ficoll-Histopaque sedimentation. Genotyping was carried out using the Sequenom MassArray platform. All these markers were genotyped according to manufacturers instructions (Sequenom, San Diego, CA, USA). The SNP assay was designed using Assay Design software. The experimental protocol was carried out according to the specifications provided by Sequenom. The reaction mixture was then spotted on a SpectroCHIP II microarray and subjected to MALDI-TOF mass spectrometry. Successful genotyping assays were defined as those for which 90% of all possible genotyping calls were obtained. With that on account we analyzed a total of 25 SNPs. Statistical significance was evaluated by an ANOVA test using Golden Helix SVS software.

Results: CAT SNP rs1001179 (−262 C>T) was significantly increased in CD patients (p = 0.038) compared to healthy controls. Also three CAT SNPs analyzed showed an increased distribution in CD patients but not significantly (rs475043, rs494024 and 704724; p = 0.083, 0.091 and 0.092 respectively). In contrast, none of the Mn-SOD SNPs selected showed different distribution in CD patients compared to healthy controls.

Conclusion: Our findings suggest that SNPs of CAT gene may contribute to the inhibition of CAT expression that we have previously reported in CD patients. SNP rs1001179 has been described in the literature as a responsible of down regulated expression of CAT enzyme. However, it is necessary that our results are confirmed in larger samples and further studies are conducted to assess the role of CAT SNPs in CD patients.