老年黄斑变性患者线粒体DNA突变及氧化磷酸化功能检测与分析

Analysis of mitochondrial DNA mutaions and oxidative phosphorylation with age-related macular degeneration

目的 :探讨老年黄斑变性 (age relatedmacularde generation ,AMD)与线粒体DNA(mitochondrialDNA ,mtD NA)突变及氧化磷酸化功能的相关性 .方法 :选择湿性AMD患者 2 6例 ,干性AMD患者 10例及正常对照者 2 0例 .采集外周血细胞 ,抽提DNA ,检测mtDNA缺失和mtDNA 32 4 3点突变 ,测定电子传递链酶复合物Ⅰ和复合物Ⅳ活性 .结果 :湿性AMD组 2 6例中 ,有 4例检出大小分别为 3.6 7,5 .0 0和 5 .2 0kb的mtDNA多重缺失 ;3例检出 5 .0kb和 5 .2kb的双重缺失 ;2例检出 3.6 7kb的单一缺失 .各组均未检出mtDNA32 4 3点突变 .复合物Ⅰ活性均值湿性AMD组、干性AMD组与对照组分别为 (16 .5 3± 7.2 8) ,(17.2 8± 8.0 6 )和 (18.38±5 .5 3)nmol·min-1·mg-1(P >0 .0 5 ) ;复合物Ⅳ活性均值分别为 (70 .2 5± 2 6 .33)、(92 .13± 30 .6 9)和 (99.13± 32 .11)nmol·min-1·mg-1,湿性AMD组活性较其他两组显著降低 (P <0 .0 5 ) .结论 :①湿性AMD患者体内可能存在较高频率的mtDNA多重缺失 ;②AMD发病可能与母系遗传而来的mtD NA 32 4 3点突变无关 ;③湿性AMD组电子传递链酶复合物Ⅳ活性降低 。

AIM: To detect mitochondrial DNA (mtDNA) mutations and to assay the activity of electron transport chain enzyme complex in age related macular degeneration (AMD) and to analyze the pathogenesis of AMD. METHODS: 26 wet AMD cases and 10 dry AMD cases were chosen and compared with 20 normal controls. First, anti coagulated blood samples were collected and then total cellular DNA was extracted. Using polymerase chain reaction (PCR) and restriction fragment long polymorphism (RFLP) technique, mtDNA deletions and mtDNA A→G point mutation at position 3243 were detected. The electron transport chain enzyme complex I and complex IV activities were assayed by spectrophotometry. RESULTS: There were three different mtDNA deletions detected in 4 wet AMD cases, size in 3.67 kb, 5.00 kb and 5.20 kb respectively. Two mtDNA deletions in size 5.0 kb and 5.2 kb were detected in other 3 wet cases. A 3.67 kb mtDNA deletion was detected in 2 wet AMD cases. No A→G mutation at position 3243 was detected among wet AMD, dry AMD and controls. The means of complex I activity among wet AMD, dry AMD and control were 16.53±7.28, 17.28±8.06 and 18.38±5.53 nmol·min -1 ·mg -1 , respectively. There was no significant difference among the three groups ( P >0.05). The means of complex IV activities were 70.25±26.33, 92.13±30.69 and 99.13±32.11 nmol·min -1 ·mg -1 ,respectively ( P <0.01). The enzyme complex IV activity decreased significantly compared with those in the other two groups. CONCLUSION: ①Higher multiple mtDNA deletions were detected in wet AMD patients. ②The maternal A→G mutation at position 3243 mtDNA point mutation may not be related to AMD. ③The electron transport chain enzyme complex IV activity decreases in wet AMD, which may, to some extent, be related to the defect of oxidative phosphorylation.