前交叉韧带断裂易感基因的遗传学特征

doi:10.3969/j.issn.2095-4344.0235

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
候选基因(candidate gene)：也译成后选基因，定位于染色体部位，这一类基因在疾病中表达情况尚不明确，其蛋白产物也正在探讨中。它们参与生物体的表型表达，连锁分析提示其与基因组某一部位有关联。这些基因可能是结构基因，调节基因或是在生化代谢途径中影响性状表达的基因。
易感基因：所谓疾病易感性是指由遗传决定的易于患某种或某类疾病的倾向性。具有疾病易感性的人一定具有特定的遗传特征，简单地说就是带有某种疾病地易感基因组型。

摘要
背景：前交叉韧带断裂在年轻运动员中很常见，其发生使患者处于更大的继发骨关节炎的风险中。
目的：回顾了前交叉韧带断裂的相关的遗传研究，针对前交叉韧带断裂易感基因方面最近进展进行综述。
方法：以(gene OR genetic OR genetics)和(cruciate ligament OR ACL)在PubMed数据库检索2017年1月之前列出的文献，纳入人前交叉韧带断裂遗传相关的研究54篇进行分析。
结果与结论：70%的前交叉韧带断裂是由于非接触机制所导致的，多方面的证据证明了前交叉韧带断裂的发生是有相当大的遗传因素在其中起到作用。目前的病例-对照研究中，已报道多个骨与关节发育相关的候选基因中均存在与前交叉韧带断裂相关的多态性位点。但候选基因检测的结果只能作为评估人群，尤其是运动员个体的前交叉韧带断裂多因素风险模型的一部分。还需要了解前交叉韧带断裂易感性多态位点在不同人群中的分布差异，从而排除遗传研究可能的不同群体的选择偏倚。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
ORCID: 0000-0002-3774-6418(严沫琦)

关键词: 前交叉韧带断裂, 膝关节, 病因, 遗传学, 易感基因, 组织构建

Abstract:

BACKGROUND: Anterior cruciate ligament rupture (ACLR) is commonly seen in young athletes, which has a great possibility to induce secondary osteoarthritis.
OBJECTIVE: To review ACLR-related genetic researches and summarize the research advances in ACLR susceptibility genes.
METHODS: PubMed database retrieval was conducted for the literature published before January 2017, using search strategy “gene OR genetic OR genetics and cruciate ligament OR ACL”. Finally, 54 articles addressing human ACLR heredity were included.
RESULTS AND CONCLUSION: Totally 70% of ACLR is a result of non-contact mechanism, and genetic factors have been shown to play a role in the occurrence of ACLR. Case-control studies have reported that several ACLR-related genetic loci have been identified in the candidate susceptibility genes, which are involved in bone and joint biology. However, the results are limited in individual assessment for athletes. Thereafter, a further study on the distribution of polymorphic locus of ACLR susceptibility genes in different populations is needed, to reduce selection bias.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Anterior Cruciate Ligament, Knee Joint, Genetics, Genes, Tissue Engineering

中图分类号:

R318

严沫琦，韩峰，管喆恒，王一帆，陆霁航，杨惠林，孙烨. 前交叉韧带断裂易感基因的遗传学特征[J]. 中国组织工程研究, 2018, 22(20): 3275-3280.

Yan Mo-qi, Han Feng, Guan Zhe-heng, Wang Yi-fan, Lu Ji-hang, Yang Hui-lin, Sun Ye. Genetic characteristics of anterior cruciate ligament rupture susceptibility genes[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(20): 3275-3280.

图/表（结果） 1

2.1 COL5A1 COL5A1基因编码了低丰度原纤维胶原蛋白中的一个α链，通过荧光原位杂交技术，人类 COL5A1 基因已经被定位于9q34.3。COL5A1 基因由 66个外显子组成，COL5A1第一个内含子的最小尺寸为600 kb，是目前最大的内含子。原纤维胶原分子是由一种或多种类型的纤维胶原蛋白α链组成的三聚体。V型胶原一般存在于含有Ⅰ型胶原的组织中，起到调节由Ⅰ型和V型胶原组成的异型纤维的组装的作用。COL5A1在韧带和肌腱的形态发育中起关键作用^[8-9]，研究发现COL5A1基因的突变会导致严重的韧带和肌腱疾病，如Ehlers-Danlos综合征^[8]，跟腱炎和前交叉韧带断裂等^[10-11]。动物研究表明，在Col5a1基因敲除的小鼠中，前交叉韧带的最大负荷和刚度均明显降低，导致小鼠关节不稳定^[9]。而在韧带和肌腱中V型胶原的定向敲除也同样会导致严重的纤维组织的异常，这在为关节提供稳定性的前交叉韧带中，主要表现为原纤维直径的增加和原纤维数量减少，从而改变胶原原纤维结

构，导致关节松弛和早发性骨关节炎^[8]。

以上结果说明V型胶原在肌腱和韧带结构和功能中起着关键作用，表明V型胶原的调节功能障碍与小鼠及患者异常关节表型是相关的。Posthumus等^[11]报道了COL5A1基因，特别是BstUI限制性片段长度多态性(rs12722)和前交叉韧带断裂之间的关联。该研究中纳入了129例前交叉韧带断裂的患者和216例正常对照研究结果显示rs12722的CC基因型在前交叉韧带断裂的女性参与者中是低表达的(27.4% vs.5.6%；优势比=6.6； 95% 可信区间，1.5-29.7；P=0.006)。

2.2 COL12A1 COL12A1基因位于第6号染色体，编码Ⅻ型胶原的α1链。Ⅻ型胶原是与Ⅰ型胶原结合的同源三聚体，其被认为是起到用来修饰Ⅰ型胶原原纤维与周围基质之间的相互作用的功能。在之前已报道的一个研究中，有人提出在COL12A1基因中多态性rs970547的AA基因型可能改变胶原原纤维的生物力学特性，从而可能增加前交叉韧带断裂的风险^[12]。此外，有研究证实当与AG+GG基因型相比时，rs970547的AA基因型明显增加患者膝关节的松弛度，临床上膝关节的松弛最终将导致前交叉韧带的断裂。并且，September等^[13]观察到rs970547的AA基因型在具有韧带或肌腱损伤家族史的女性患者中有过表达的倾向(AA VS GA+GG，P=0.06)。然而，在Ficek等^[14]的另一项研究中，他们否认rs970547与前交叉韧带断裂之间的关联。这些有关COL12A1基因中的rs970547位点的不一致的研究结果，也是作者认为需要对前交叉韧带断裂患者的遗传易感性进行不同人群大样本重复验证的重要的原因。有趣的是，O'Connell等^[15]的研究发现COL5A1 rs12722 T/ C位点和COL12A1 rs970547 A/G位点在前交叉韧带断裂的发病风险中存在显著的相互作用，这突出了基因- 基因相互作用在前交叉韧带断裂患者群体病因中的意义。

2.3 COL1A1 COL1A1 基因位于17号染色体q21上，基因大小约18 kb。其在大多数结缔组织中均存在，并且在骨，肌腱和韧带组织中极为丰富。Ⅰ型胶原蛋白是肌腱和韧带的主要组成成分，约占肌腱总质量的60％至80%^[16-17]。在几个不同的研究中，COL1A1基因的突变被发现与前交叉韧带破裂有关^[18-20]。 COL1A1基因第一个内含子中的Sp1结合位点是增强COL1A1基因转录活性及调节Ⅰ型胶原的α1链产物所必需的^[21]。在Posthumus等^[19]和Khoschnau等^[20]报导的关于Sp1结合位点多态性rs1800012的研究中，一致得出结论，rs1800012位点的TT基因型在前交叉韧带断裂患者中是低表达的，这一结果表明TT基因型对前交叉韧带断裂具有保护作用。

在COL1A1 基因近端启动子区域的rs1107946位点，与Sp1多态性位点连锁，已有报道 rs1107946位点与rs1800012位点在患者骨密度的调节中存在显著的相互作用^[22-23]。此外，体外实验也发现，在成骨细胞中rs1107946位点能明显调节COL1A1基因的表达^[24]。因此，Ficek等^[18]研究发现在足球运动员中，前交叉韧带断裂与COL1A1基因的这两个不同多态性位点(rs1800012和rs1107946)存在显著的相关性，并证实了COL1A1 G-T单体型(rs1800012和rs1107946)的患者，其前交叉韧带断裂的发病风险显著降低，而这可能是通过对COL1A1基因转录调控来达到的。

2.4 COL3A1 COL3A1基因编码Ⅲ型胶原的原α1链，Ⅲ型胶原是一种存在于可延伸结缔组织中的原纤维胶原^[25]，而Ⅲ型胶原的弹力较低，主要形成细的纤维束，是不成熟、不稳定的胶原，在伤口愈合过程中起作用。COL3A1一般在伤口愈合过程的早期不受调节，之后COL3A1基因保持数周的高水平表达，并且在愈合过程中逐渐被Ⅰ型胶原替代，导致组织强度增加^[26]。COL3A1基因的突变与Ehlers-Danlos综合征Ⅳ型和盆腔器官脱垂(POP)有关^[27-28]。COL3A1基因的rs1800255的AA基因型与POP显著相关，POP是女性常见的结缔组织疾病^[28]。Stepien-Slodkowska等^[29]的研究揭示了滑雪者人群中，COL3A1基因的rs1800255多态性位点与该人群的前交叉韧带断裂发病存在显著的关联。在具有该位点AA基因型的滑雪者，其发生前交叉韧带断裂的风险比具有AG+GG基因型的滑雪者高5倍，这也提示COL3A1是前交叉韧带断裂遗传研究中潜在的候选基因。

2.5 基质金属蛋白酶基因基质金属蛋白酶家族的蛋白质参与了众多正常生理过程以及疾病过程(如关节炎和肌腱疾病^[30])中细胞外基质的分解。在白种人群中，已经有研究证实基质金属蛋白酶3基因内的常见多态性位点与慢性跟腱附着点病变有关。有研究发现在前交叉韧带断裂患者中，前交叉韧带组织中检测到基质金属蛋白酶1和基质金属蛋白酶3的mRNA过表达^[31]，提示基质金属蛋白酶基因有可能作为前交叉韧带断裂风险相关的候选基因。Malila等^[32]报道了在基质金属蛋白酶3基因启动子区中的一个具有调节基质金属蛋白酶3蛋白表达水平的多态性位点与前交叉韧带的损伤相关。基质金属蛋白酶的蛋白产物及活性是至关重要的，因为细胞外基质稳态可被基质金属蛋白酶信号通路的异常所打破^[33]。有研究发现基质金属蛋白酶12基因多态性位点rs2276109与非接触性的前交叉韧带断裂风险显著相关^[34]。而基质金属蛋白酶12的mRNA仅在膝关节滑膜中检测到，这也暗示了细胞外基质合成分解之间的平衡可能在前交叉韧带断裂中具有重要的的意义。

2.6 蛋白聚糖蛋白聚糖(例如双糖链蛋白聚糖，核心蛋白聚糖和聚集蛋白聚糖)占韧带干质量的3%^[35-36]。蛋白聚糖在调节细胞外基质重塑和胶原原纤维分泌后进入细胞外空间，并与胶原网络，韧带和腱的主要结构部件相互作用的过程中起着重要作用，蛋白聚糖促进了韧带的结构完整性^[37-38]。在富含亮氨酸的小蛋白聚糖(SLRPs)、双糖链蛋白聚糖或核心蛋白聚糖缺陷的小鼠中，具有与人类经典Ehlers-Danlos综合征相似的临床表型，其胶原原纤维组织异常表现是由纤维生成过程的异常导致的^[39-40]。Young等^[35]最近研究了断裂的前交叉韧带组织的细胞外基质含量，与未破裂的对照组相比，在断裂的人前交叉韧带组织中观察到显著较低的蛋白聚糖和糖胺聚糖(GAG)水平^[35]。提示前交叉韧带的断裂伴随着蛋白聚糖的低表达。核心蛋白聚糖是一种富含亮氨酸的小蛋白聚糖(SLRP)，它是韧带和肌腱中最丰富的蛋白聚糖^[36^，^41]，肌腱中双糖聚糖与核心蛋白聚糖的缺乏，显著改变动态力学平衡，导致了肌腱最大荷载的显著降低^[39]。在SLRP敲除的肌腱中^[41]，胶原纤维排列分布错乱，与正常纤维结构存在明显差异。在Mannion等^[42]进行的一项病例-对照研究中，发现DCN基因中rs516115多态性位点和聚合蛋白聚糖(ACAN)基因中rs1516797多态性位点均与前交叉韧带断裂有关。ACAN蛋白在韧带与软骨相连接的区域中表达水平较高，已有的证据显示，ACAN基因突变能导致家族性的剥脱性骨软骨炎和骨骼系统发育不良^[43-44]。这些证据进一步表明蛋白多糖编码基因在前交叉韧带断裂的遗传易感性中起着不可或缺的作用。

2.7 血管内皮生长因子A 与血管生成相关的信号级联瀑布受到多种生长因子的调节，该信号通路在受到机械负荷过程中细胞外基质的重塑中起到重要作用。有研究发现血管生成细胞因子和生长因子的水平在体外受到机械载荷的肌腱成纤维细胞中表达以及断裂肌腱和韧带中均有显著升高^[45-47]。例如，在损伤的韧带和肌腱的组织有学者观察到血管内皮生长因子蛋白水平的显著增加^[46-47]。之前有猜想认为，编码血管生成相关信号通路的基因多态性的差异可能是不同个体韧带对机械负荷承受能力差异的原因，这种差异导致了不同人群韧带与肌腱组织的完整性和功能的差异。由血管内皮生长因子A编码的血管内皮生长因子同源蛋白被认为具有最强的血管生成能力，其通过与KDR基因编码的KDR受体结合，起到激活血管生成信号通路的关键调节作用。血管内皮生长因子基因拷贝的缺失会导致小鼠胚胎的死亡^[48]，并且血管内皮生长因子已牵涉几种病理状态，包括类风湿关节炎^[49]。Rahim等^[50]发表了血管发生相关信号基因的多个多态性位点和前交叉韧带断裂之间的遗传易感性研究。血管内皮生长因子A rs699947 CC基因型(OR：1.92，95%CI：1.17-3.17)在非接触性前交叉韧带断裂患者中显著过表达，而血管内皮生长因子A rs1570360 GA基因型在正常对照组中过表达(OR：1.70，95%CI：1.16-2.50)。此外，KDR基因 rs2071559 GA基因型在女性对照组中是过表达的(OR：2.16，95%CI：1.11-4.22)。以前有体外研究显示血管内皮生长因子蛋白的表达受血管内皮生长因子A基因多态性功能位点的影响^[51]。位于血管内皮生长因子A基因启动子区域的的功能位点rs699947的C等位基因(-2578 C> A)能明显增强血管内皮生长因子蛋白的表达，而血管内皮生长因子蛋白表达的增加同时也会导致基质金属蛋白酶表达水平的上调^[52-53]，这最终可能影响韧带及肌腱组织的细胞外基质平衡，从而带来韧带生物力学特性的改变。他们也推测KDR基因多态性位点rs2071559(604 G>A)的G等位基因可能导致其启动子区结合位点结构的改变，从而减少KDR基因转录^[54]，导致血管生成信号通路的异常。

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