【作者】 尚旭亚；

【导师】 黄昌林；

【作者基本信息】 第四军医大学 ， 外科学， 2011， 博士

【摘要】 引言:辅肌动蛋白是肌动蛋白结合蛋白家族的成员。两个钙敏感的α-辅肌动蛋白亚型(ACTN2和ACTN3)表达在骨骼肌。它们是肌节Z线的主要结构部分,起到锚合肌动蛋白细肌丝的作用。在人类,ACTN2表达在骨骼肌和心肌纤维,而ACTN3仅仅表达在骨骼肌快速糖酵解的IIx纤维。此外,α-辅肌动蛋白-2和α-辅肌动蛋白-3相互作用参与肌肉蛋白一系列的信号传递和代谢作用。因为一个未成熟的终止密码子造成基因ACTN3的16号外显子第1747位核苷酸存在C→T的转换,即精氨酸密码子被取代,造成基因ACTN3的多态性。XX等位基因不能产生α-辅肌动蛋白-3,但是没有和任何疾病表型有关联。先前的研究表明除了人类没有别的哺乳动物存在这种多态性,各种人种都有这种基因ACTN3多态性。有大约19%的高加索人是缺失ACTN3的,在世界上不同人种X等位基因频率分布超过15%。α-辅肌动蛋白有很高的保守性,最近的研究显示ACTN3的缺失是人类进化的选择,它影响肌肉的代谢。近来有很多的研究显示ACTN3影响人的运动能力。对精英高加索运动员研究证实,在精英爆发力和力量型运动员中纯合子RR频率分布明显高于对照。XX基因型明显和女性耐力运动员相关,但是和男性耐力运动员没有关联。相似的发现被报道证明这种相关性,大量的横断面研究支持, R-等位基因和肌肉高速收缩呈正相关。此外,IIx纤维中ACTN3蛋白含量高于IIa型纤维,在RR基因型中IIx(快肌糖酵解)纤维的比例大于XX基因型的。在运动中踝关节扭伤是最常见的骨骼肌肉关节损伤之一。研究显示几个内在和外在的原因和急性踝关节扭伤(AAS)有关。最近的文献表明腱和韧带组织的损伤与基因遗传相关。编码一些腱和韧带组织的细胞外基质蛋白质类的基因变异与骨骼肌肉软组织损伤存在关联。研究表明在运动积极的人群中基因TNC, COL5A1和MMP3与肌腱慢性病变有关。基因TNC的变异和跟腱断裂有关,基因COL1A1, COL12A1和COL5A1的变异与十字韧带断裂和肩关节脱位有关。但是引起AAS的基因因素目前还未见有报道。肌肉力量较弱和肌力不平衡是踝关节扭伤的原因之一,因此提高肌肉力量和瞬间爆发力应该能减少这种损伤。仅仅表达在快速糖酵解肌纤维Z线的基因ACTN3和力量/爆发力表型存在关联。在高加索人种,ACTN3 R577X与运动成绩的相关性已清楚地在精英级运动员中观察到,但涉及非运动员的研究却提供了许多混淆的结果。ACTN3 R577X多态性和骨骼肌生理功能之间的相关性在中国非运动员中没有被调查过。根据这一假设,ACTN3 R577X多态性对中国汉族人运动能力的影响与对白种人的影响是一致的,即ACTN3 RR纯合子比XX纯合子有较高的基线肌肉力量/强度。我们还假设,XX型个体将有更好的耐力相比RR纯合子携带者。为了检验这一假设,并确定在中国非运动员的ACTN3 R577X多态性与身体和性能表型的关联,我们对452个年轻男士兵的身体组成特征,力量、爆发力和耐力进行相关分析。在高加索人种,ACTN3 R577X与耐力表型的相关性已清楚地在精英级运动员中观察到,但涉及中国运动员的研究却未见报道。我们假设中国汉族耐力型运动员XX基因型频率分布较高而RR基因型频率分布较低相比普通对照。为验证这个假设,我们分析中国耐力运动员和基因ACTN3的关联。目前,ACTN3 R577X与肌肉力量/爆发力的相关性已清楚地在很多研究中观察到。就我们所知道的,还没有ACTN3 R577X基因多态性与急性踝关节扭伤是否相关的研究报道。因此,本研究的主要目的是探讨中国汉族男性军人急性踝关节扭伤患者与ACTN3 R577X基因多态性的相关性。目的:先前的研究表明基因ACTN3 577R等位基因和爆发力、力量能力有显著的关联,而577X等位基因则有利于耐力能力。首先,我们检测中国汉族男性军人ACTN3基因型和等位基因的频率分布,并分析ACTN3基因和中国汉族男性军人运动能力的关联。其次,我们测试XX基因型是否在中国耐力运动员中有较高的频率分布相对于普通对照组。最后,本实验将探讨ACTN3基因多态性和急性踝关节扭伤表型的关联性。方法:通过研究452个中国年轻男性军人,我们检测ACTN3基因型和等位基因的频率分布,分析ACTN3基因和运动能力的关联。通过研究250个中国省级和国家级水平的耐力运动员和450个普通对照,我们检测ACTN3基因型和等位基因的频率分布,分析ACTN3基因和运动能力的关联。142个经临床或者手术诊断的急性踝关节扭伤患者和280个从没有过踝关节损伤史的自愿者进行对照研究,分析其相关性。用荧光定量PCR法检测ACTN3基因的多态性。结果:1. ACTN3基因型频率分布(RR 39.8%, RX 43.4%, XX 16.8%)和ACTN3等位基因频率分布(R 61.5%, X 38.5%)明显和高加索人种分布一致。我们仅仅发现ACTN3基因和握力有关联。XX型的个体的握力要明显低于RR型的个体的握力(P = 0.007),但是XX型的个体和RX型的个体握力没有差别(P = 0.121),RR型的个体和RX型的个体握力也没有差别(P = 0.117)。我们没有发现ACTN3基因多态性和短跑、耐力有关联。2.我们的结果显示在女性耐力运动员中ACTN3 XX基因型(21.2 vs. 15.8%; P = 0.02)和X等位基因(51.3 vs. 41.1%; P = 0.019)的分布频率明显高于对照,但男性运动员没有差异。另外,ACTN3 XX基因型频率分布在顶尖的女性耐力运动员中是28.6%,明显高于其他对照。3.急性踝关节扭伤组和对照组的ACTN3频率分布差异显著,ACTN3 RR基因型频率分布(23.2 vs. 35%; P = 0.011)和R等位基因型频率分布(47.9 vs. 58.9%; P = 0.002)。AAS组ACTN3 RR基因型频率明显低于对照组(23.2% vs 35%; OR = 1.8; 95%可信区间:1.1-2.8; P = 0.014)。ACTN3 RR基因型频率随着AAS损伤等级的提高有一个下降的线性趋势。结论:①我们的结果显示ACTN3基因仅仅和中国年轻男性士兵的握力有关联。②我们的结果显示ACTN3 R577X多态性和中国女性耐力运动员有关联,但是和中国男性耐力运动员没有关联。③本研究结果显示在中国汉族军人急性踝关节扭伤患者和ACTN3 R577X多态性有关联。这是第一次发现携带ACTN3 RR基因型的个体有降低AAS的风险。更多还原

【Abstract】 Introduction: The alpha-actinins are a family of actin-binding proteins. Two calcium-insensitiveα-actinins isoforms (ACTN2 and ACTN3) are expressed in skeletal muscle. They constitute the major structural components of the sarcomeric Z-line involved in anchoring together the actin-containing thin filaments. In humans, ACTN2 is found in skeletal and cardiac muscle fibers, while the expression ofα-actinin-3 is restricted to a subset of fast glycolytic skeletal muscle typeⅡx fibres. Moreover,α-actinin-2 andα-actinin-3 also interact with many muscle proteins carry out a variety of signaling and metabolic functions.The R577X SNP originated as a result of a C-to-T transition at position 1747 in exon 16 that replaces an arginine codon 577(577R) for a premature stop codon (577X). The X-allele fail to produceα-actinin-3 protein, but without association with any disease phenotype. A previous study has suggested that no other mammal has polymorphism for null alleles of ACTN3 except human. This allele is found in every human population tested. There are approximate 19% of Caucasian (combined male and female) who are ACTN3 deficient, and the X-allele frequency is greater than 15% in several of the world’s general populations. Theα-actinin has been shown to be highly conserved, and recent evidence has proposed that absence of ACTN3 gene function shows positive selection in humans owing to its effect on muscle metabolism. Recently, there are more and more studies indicating that the ACTN3 genotype impact on human exercise performance phenotypes. The research of the R577X polymorphism in elite Caucasian athletes demonstrated RR homozygotes are over-represented in male and female elite sprint and power athletes relative to those in controls. The XX genotype was significantly associated with female elite endurance athletic performance, without the same effect on male endurance athletes. Similar findings have been replicated to have confirmed these associations and a number of cross-sectional studies have also supported that there is a positive association between the R-allele and high velocity muscle contractions. Furthermore, recent study has shown that the proportions of type IIx (fast-twitch glycolytic) fibers was greater in the RR than those in the XX genotype group, and ACTN3 protein content is higher in type IIx compared with type IIa fibers.Ankle sprains are one of the more common sites of musculoskeletal injuries during physical activity. Several extrinsic and intrinsic factors have been shown to be associated with these injuries. Recent reviews indicate that a genetic component has been implicated in those tendons and ligaments injuries. Sequence variants within genes that encode for several tendon and/or ligament extracellular matrix proteins have been shown to be associated with specific musculoskeletal soft tissues injuries. Polymorphisms within the TNC, COL5A1 and MMP3 genes have been associated with chronic Achilles tendinopathy in a physically active population. The variant within the TNC gene also appears to co-segregate with Achilles tendon ruptures, while sequence variants within the COL1A1, COL12A1 and COL5A1 genes have been shown to be associated with cruciate ligament ruptures and/or shoulder dislocations. However, the exact mechanisms which cause these acute ankle injuries are unknown. Muscle weakness/imbalance has been shown to be associated with ankle sprains, and therefore increased muscle strength/power may improve ankle sprains. The ACTN3 gene, which is only expressed in the Z line of fast glycolytic muscle fiber was found to associate with power/strength performance.In Caucasian, the associations of ACTN3 R577X with athletic performance have been clearly observed in elite-level athletes, but studies involving non-athletes have provided many confounded results (Moran et al., 2007; Santiago et al., 2009). The functional relationships between ACTN3 R577X polymorphism and physical functioning of skeletal muscle in Chinese non-athletes have not been examined. Under the hypothesis that the influence of the ACTN3 R577X polymorphism on athletic performance in Chinese is in accordance with the influence in Caucasian, it would be predicted that the ACTN3 homozygotes (RR) would have higher baseline muscle power/strength compared with the homozygotes (XX). We also hypothesized that the XX genotype would have more endurance compared with the homozygotes (RR). To test this hypothesis, and determine the association of the ACTN3 R577X polymorphism with physical- and performance-related phenotypes in a Chinese non-athlete population, we executed an association analysis using 452 Chinese young male soldiers for body composition-, strength/power- and endurance-related traits.In Caucasian, the associations of ACTN3 R577X with endurance performance have been clearly observed in elite-level athletes, but studies involving Chinese athletes have not been examined. We hypothesized that the frequency of ACTN3 XX genotype is higher and that of RR is lower among Han Chinese athletes. To test this hypothesis, we sought to determine the association of the ACTN3 R577X polymorphism with endurance athletes in China.At present, the associations of ACTN3 with muscle strength/power performance have been clearly observed in many studies. To the best of our knowledge, however, no information is available concerning the association between ACTN3 gene polymorphisms and acute ankle sprains. Therefore, the principal aim of this study was to determine the genetic association between the ACTN3 gene and acute ankle sprains in a young Chinese Han population.Objective:Previous studies have shown highly significant association between ACTN3 genotype and sprint/power performance, while the nonfunctional allele (577X) was believed to provide an advantage for endurance performance. First, we examined the distribution of ACTN3 genotypes and alleles and analyzed the association between ACTN3 genotypes and athletic performance in Chinese young male soldiers. Second, in this study we tested whether XX genotype was over-represented in Chinese endurance athletes compared to the general population.Third, the aim of this study was also to investigate if the ACTN3 gene polymorphism is associated with acute ankle sprains in Chinese young soldiers.Methods:In a study of 452 Chinese young male soldiers, we examined the distribution of ACTN3 genotypes and alleles and analyzed the association between ACTN3 genotypes and athletic performance.In a study of 250 Chinese endurance athletes of provincial or national competitive standard and 450 controls, we examined the distribution of ACTN3 genotypes and alleles and analyzed the association between ACTN3 genotypes and athletic performance.One hundred and forty-two participants with clinically and surgically diagnosed acute ankle sprains(AAS), as well as 280 physically active controls participants (CON) without any history of ankle sprains were included in this case-control genetic association study.The ACTN3 allelic variant and genotype was analyzed by Q-PCR.Results:1. The frequencies of the ACTN3 R577X genotype (RR 39.8%, RX 43.4%, and XX 16.8%) and R577X allele (R 61.5%, X 38.5%) were significantly consistent with the frequencies of Caucasian. We only observed a significant association between ACTN3 R577X genotypes and grip strength. Subjects with the XX genotype displayed significantly lower handgrip strength compared with individuals with RR (P = 0.007) genotypes, but the difference between XX and RX means (P = 0.121) and that between RR and RX means (P = 0.117) was not significant. We did not observe a predominant association between the ACTN3 R577X genotypes and sprint phenotypes and endurance phenotypes.2. We proved that the ACTN3 XX genotype (21.2 vs. 15.8%; P = 0.02) and X allele (51.3 vs. 41.1%; P = 0.019) were significantly over-represented in female endurance athletes compared to controls, while no genotype-related differences were observed in male endurance athletes. Besides, the frequency of the ACTN3 XX genotype (28.6%) was the highest in a group of highly elite athletes compared with other groups, which supported the hypothesis that the absence ofα-actinin-3 provided some sort of advantage for endurance athletes.3. The distribution of the ACTN3 gene in the AAS and controls showed significant differences with both the frequencies of the ACTN3 RR genotype (23.2 vs. 35%; P = 0.011) and R allele (47.9 vs. 58.9%; P = 0.002). The RR genotype of the ACTN3 was significantly low-represented in the AAS group compared with the controls (23.2% vs 35%; odds ratio = 1.8; 95% confidence interval, 1.1-2.8; P = 0.014). Compared with healthy controls, there was a decreasing linear trend (P = 0.01) in the 577RR genotype with increasing AAS grade.Conclusions:①Our results indicate that ACTN3 R577X polymorphism is only associated with grip strength in Chinese young male soldiers.②Our results indicated that ACTN3 R577X polymorphism was associated with endurance performance in female athletes but not male athletes in China.③The ACTN3 R577X is associated with AAS in Chinese participants in this study. This is the first study to suggest that individual with a RR genotype are at decreased risk of AAS. Further studies are required to explore and replicate these findings.更多还原