And Alox5ap Mef2a Gene Mutation And Study On Relationship Between Coronary Atherosclerotic Heart Disease

BACKGROUD Coronary artery disease has a complex aetiology, involving multiple genetic and environmental influences and interactions. Our views of the pathophysiology of CAD and MI have evolved over time. More researches focus on identification of the role of inflammation and immune system or dysfunction or abnormal development of the endothelium on the cause of CAD The mutations of myocyte enhancer factor (MEF2A) and arachidonate 5-lipoxygenase-activating protein (ALOXSAP) gene have been more implicated in the susceptibility to coronary atherosclerotic heart disease (CAD). They may contribute to CAD by the common and/or different mechanisms respectively. 5—lipoxygenase activating protein (FLAP) are essential for the cellular synthesis of leukotrienes. FLAP that appears to serve as an arachidonic acid binding and transfer protein thereby facilitating 5-LO enzyme activity. And many studies suggest links between the 5-LO pathway and atherogenesis. Mutation of ALOX5AP gene may affect the synthsis of LTs whose biological effects associated with the pathophysiology of inflammatory disorders. In this study, we sought to identify variations in the all the exons of MEF2A and ALOX5AP gene and investigate their association with CAD in Chinese Han population. In addition, we also carried out the functional analysis of 7aa-deletion mutation to identify their probable mechanism which is involved in CAD.METHODS Case-control design was applied in this study. We recruited independent patients with unequivocal diagnosis of CAD based on cardiac angiography (CAG):Single-strand conformation polymorphism (SSCP) and DNA sequence analyses were used to identidy mutations in MEF2A and ALOX5AP. The nuclear localization signal of MEF2A mutation was identified by expression of green fluorescent protein (GFP)/MEF2 fusion proteins in the transfected 293T/Hela cells. And, the effect of mutation of MEF2A on transcriptional activation activity was analysed using the dual luciferase reporter gene assay.RESULTS 1) No mutation was detected in exon 7 of MEF2A gene in all the 899 samples. 2) We found 9 variations in all exons that one variation in exon 9 and the others in exon 11 of MEF2A gene in the 1000 samples. There are 3 SNPs (N297N,P435P, G451G ) which didn't alter the amino acid sequence of the MEF2A protein, and 6 variations which altered the amino acid sequence of the MEF2A protein in exon 11, which included polyglutamine tandem repeats varied from 4 and 15, the proline tandem repeats (P431/432 deletion or P433/434 deletion),△7aa-del (21-bp deletion),△3aa(PQQ 430) insertion and one missense mutation (P435S). Statistical analysis showed that the shorter CAG repeat lengths (4～8 repeats), carried by approximately 7.64% of alleles in CAD patients in Chinese, has a significant association with CAD [p=0.037, OR=1.664 (95%CI, 1.026-2.699)]. 3) Further genetic analysis of△7aa-deletion mutation showed that there may be several possible models for the 7-aa deletion. The wild MEF2A/GFP proteins were strictly expressed in nuclear, and, the considerable proportion of△7aa-deletion/GFP fluorescence were located in the nucleus. Western blot analysis showed that both wild type and△7aa-deletion mutant proteins were successfully expressed in transfected cells. And both types of MEF2A proteins activated transcription of the HRC promoter, as well as, the ANF promoter. Moreover,△7aa-deletion didn't reduce the transcriptional activation activity of MEF2A protein. And two alternatively MEF2A proteins express same transcriptional activation ability. 4) We didn't identify any mutations in ALOX5AP coding regions.CONCLUSION 1) Mution in exon 7 of MEF2A gene may not be genetic risk factor leading to CAD in the Chinese Han population. The discrepancy between the foreign and our research may be due to area- and race-specific. 2) The alleles with CAG repeat numbers from 4 to 8 may be regarded as a susceptible molecular marker of risk for CAD. 3) A C-terminal region encompassing aa 434-446 is not necessary for fully localizing GFP/MEF2A in the nucleus of 293T or Hela cell. This area formation maybe due to evolution. 4) The genetic variations in ALOX5AP coding region may not contribute to CAD in the Chinese Han population.