线粒体脑肌病临床与全基因研究

发布时间：2018-05-11 22:24

本文选题：Leigh综合征 + mtDNA　；参考：《山西医科大学》2017年硕士论文

【摘要】：线粒体脑肌病(mitochondrial encephalomyopathy)是由于线粒体DNA(mitochondrial DNA,mt DNA)或核DNA(nucleus DNA,n DNA)缺陷导致线粒体结构和功能障碍、ATP合成不足所致的遗传代谢性疾病,即为原发性线粒体疾病。目前明确的综合征包括线粒体脑肌病伴高乳酸血症和卒中样发作(mitochondrial encephalomyopathy with lactic acidemia and stroke-like episodes,MELAS)、KSS综合征(Kearns-Sayre syndrome,KSS)、慢性进行性眼外肌瘫痪(chronic progressive external ophthalmoplegia,CPEO)、Leigh综合征(Leigh syndrome,LS)等多种综合征。线粒体疾病的诊断需要结合其临床表现、家族史、实验室检查、影像学资料、肌肉组织化学染色、线粒体呼吸链酶活性测定、mt DNA及n DNA的检测等信息。每一种方法对于线粒体的功能评估都有着一定的局限性,需要综合多方面的信息进行系统的分析。Leigh综合征(Leigh syndrome,LS)也称亚急性坏死性脑脊髓病,是与线粒体氧化磷酸化(OXPHOS)缺陷相关的进行性加重的神经退行性疾病。主要受累人群是婴幼儿和学龄前儿童,临床症状主要是运动迟缓、智力低下、进行性认知功能减退、运动障碍、肌张力减退、共济失调、脑干功能异常等。分为早发型LS和晚发型LS,暂时还不能从年龄上明确的将其分开。本文通过分析我院经治的四例线粒体脑肌病患者的临床资料,行骨骼肌活检术及线粒体全基因检测,明确全基因检测在该病诊断中的价值。通过汇总分析已发表的Leigh综合征(LS)病例,总结LS临床症状和呼吸链酶复合物基因突变的发生率、发病年龄与基因突变类型之间的相关性。第一部分线粒体脑肌病四例:临床、病理与基因分析目的:分析四例临床疑诊为线粒体病患者的临床资料,行骨骼肌活检术及线粒体全基因检测,明确全基因检测在该病诊断中的价值。方法:四例临床表现疑似线粒体病的患者行肌肉活检术及冰冻酶组织化学染色。对四例患者留取3ml外周血行线粒体全基因测序,应用Sanger测序、二代测序、MLPA、CNV等方法对mt DNA和n DNA进行检测。结果:1.四例患者累及骨骼肌均表现为不同程度的四肢肌肉无力及运动不耐受,症状呈波动性且进行性加重;神经系统损害表现为智力低下、癫痫等;其他系统表现如肾衰竭、代谢性酸中毒、视力下降、神经性耳聋。CK值正常或轻度增高,多在1000U/L以下。两例患者乳酸运动试验阳性,其它两例患者血气中乳酸值增高。2.两例患者头颅MRS提示受损部位可见倒置的乳酸峰。3.肌肉活检发现三例患者MGT染色上存在破碎红纤维(RRFs),百分比为10%--80%,COX染色提示酶活性部分缺失。一例患者肌肉活检未发现特异性RRF,但结合临床表现、影像学检查及基因检测确诊为线粒体脑病。4.线粒体全基因组测定发现一例患者为线粒体细胞色素B基因大片段缺失变异,两例患者是线粒体核基因点突变,一例患者未发现明确致病性突变。结论:1.线粒体脑肌病的基因型、临床表型具有高度异质性。2.临床表现、影像学检查、肌肉活检、基因检测是线粒体脑肌病诊断重要依据。第二部分385例Leigh综合征患者临床表现、基因突变分析目的:通过文献资料回顾性分析评估Leigh综合征(Leigh syndrome,LS)的临床表现,总结LS临床症状及呼吸链酶复合物基因突变的发生率、发病年龄与基因突变类型之间的相关性。方法:采用R2.15.3软件进行Meta分析。首先对每个临床症状的发生率进行logit转换,然后以样本量为权重计算合并率,最后得到合并的“发生率”及其95%CI。通过Homogeneity test(Q检验)进行异质性检验(检验水准为α=0.1),再结合I2定量判断异质性的大小。若P0.10且I250%,提示研究结果之间存在异质性,采用随机效应模型进行Meta分析;反之采用固定效应模型进行Meta分析。发表偏倚采用Eggers’test检验。最后通过排除最低质量的文献后重新计算合并率来进行敏感性分析。Meta分析的检验水准为α=0.05。用Mann-Whitney秩和检验和Kruskal-Wallis秩和检验进行不同基因突变间发病年龄差异性分析。用多个率之间双重比较的Pearson卡方检验进行常见临床症状之间发生率的差异性分析,取P0.05有统计学差异。结果:共纳入5篇文献汇总了385例Leigh综合征。临床症状合并率的Meta分析结果为乳酸值增高62%、发育迟缓60%、肌张力减退47%、癫痫33%、眼肌受损与呼吸功能受损28%、肌肉无力27%、喂养困难26%、共济失调20%。不同临床症状之间发生率有统计学意义,乳酸值增高可能是最早期表现,应注意早发现早诊断。对LS患者进行全基因测序,mt DNA与n DNA两种基因发病年龄差异有统计学意义,mt DNA基因突变较n DNA年龄大。n DNA基因突变位点发病年龄有统计学意义,PDHA1基因发病年龄较晚,SLC19A3基因发病年龄最早。呼吸链酶复合物基因突变发生率Meta分析结果提示复合物I型34%、复合物IV 20%、复合物I+IV 15%、复合物V 9%。结论:LS临床表现多种多样,乳酸值增高可能是最早期表现。发病年龄与基因突变类型有关,呼吸链酶复合物基因突变是LS最常见原因,常见为复合物Ⅰ基因突变。
[Abstract]:Mitochondrial encephalomyopathy (mitochondrial encephalomyopathy) is due to mitochondrial DNA (mitochondrial DNA, MT DNA) or nuclear DNA (nucleus DNA, n DNA) defects resulting in mitochondrial structure and dysfunction, the genetic metabolic disease caused by deficiency of synthesis, that is, primary myelopathy. The present clear syndrome includes mitochondrial encephalomyopathy Hyperlactoemia and stroke like seizures (mitochondrial encephalomyopathy with lactic acidemia and stroke-like episodes, MELAS), KSS syndrome (Kearns-Sayre syndrome), chronic progressive extraocular paralysis, etc. The diagnosis of body disease needs to be combined with its clinical manifestations, family history, laboratory examination, imaging data, muscle histochemical staining, mitochondrial respiratory chain enzyme activity determination, MT DNA and N DNA detection. Each method has certain limitations for the evaluation of mitochondrial function, which requires a comprehensive and multiple information system. The analysis of.Leigh syndrome (Leigh syndrome, LS), also known as subacute necrotizing encephalomyelitis, is a progressive neurodegenerative disease associated with mitochondrial oxidative phosphorylation (OXPHOS) defects. The main affected population is infants and preschool children, and the main clinical symptoms are slow movement, mental retardation, progressive cognitive impairment, and progressive cognitive impairment. Dynamic disorders, myosmosis, ataxia, abnormal brain stem function, etc., which are divided into early onset LS and late onset LS, which can not be separated from age. In this paper, the clinical data of four patients with mitochondrial encephalomyopathy treated by our hospital were analyzed, and the skeletal muscle biopsy and mitochondrial total gene detection were performed, and the whole gene detection was determined. The value of the diagnosis of the disease. By summarizing and analyzing the published Leigh syndrome (LS) cases, the incidence of LS clinical symptoms and gene mutations in the respiratory chain enzyme complex, the correlation between the age of the disease and the type of gene mutation. The first part of the mitochondrial encephalomyopathy in four cases: clinical, pathological and genetic analysis: analysis of four cases of suspected clinical diagnosis The clinical data of patients with mitochondrial disease, skeletal muscle biopsy and mitochondrial total gene detection were used to determine the value of full gene detection in the diagnosis of the disease. Methods: four patients with suspected mitochondrial diseases were performed muscle biopsy and frozen enzyme histochemical staining. Four patients with 3ml peripheral blood were sequenced by mitochondrial whole gene and should be sequenced. Sanger sequencing, two generation sequencing, MLPA, CNV and other methods were used to detect MT DNA and N DNA. Results: 1. and four patients were involved in skeletal muscle weakness and exercise intolerance to varying degrees, symptoms were fluctuating and progressive; nervous system impairment was manifested as retardation, epilepsy, and other systems such as renal failure, instead of renal failure. The.CK value of metabolic acidosis, visual acuity, neurogenic deafness was normal or slightly higher, more than 1000U/L. Two cases of lactic acid exercise test were positive, two cases of other patients had increased lactate value.2., two cases of cranium MRS suggested that the damaged area of lactic acid peak.3. muscle biopsy showed that three patients were stained with broken red on MGT staining. Fiber (RRFs), the percentage of 10%--80%, COX staining suggested that the active part of the enzyme was missing. One case of muscle biopsy did not find specific RRF, but combined with clinical manifestations, imaging examination and gene detection confirmed mitochondrial encephalopathy.4. mitochondrial genome determination found that a patient was a large deletion mutation of mitochondrial cytochrome B gene, two The patient was a point mutation of mitochondrial nuclear gene, one patient did not find a clear pathogenicity mutation. Conclusion: 1. the genotype of mitochondrial encephalomyopathy, clinical phenotype with highly heterogeneous.2. clinical phenotype, imaging examination, muscle biopsy, gene detection is the basis for the diagnosis of mitochondrial encephalomyopathy. The second part of the patients with 385 cases of Leigh syndrome is clinical. Expression, gene mutation analysis objective: To evaluate the clinical manifestation of Leigh syndrome (Leigh syndrome, LS) through literature review, summarize the incidence of LS clinical symptoms and gene mutation of respiratory chain enzyme complex, the correlation between the age of the disease and the type of gene mutation. Methods: R2.15.3 software is used for Meta analysis. First, each of them is analyzed. The incidence of clinical symptoms was converted by logit, then the combination rate was calculated with the weight of samples. Finally, the combined "incidence" and its 95%CI. were tested by Homogeneity test (Q test) for heterogeneity test (the test level was alpha =0.1), and then I2 was used to determine the size of heterogeneity. If P0.10 and I250%, the results suggested the existence of the results. Heterogeneity, Meta analysis using random effect model, Meta analysis using fixed effect model, Eggers' test test for publication bias. Finally, the level of sensitivity analysis for sensitivity analysis of.Meta analysis by eliminating the minimum quality literature and re calculating the combination rate for.Meta analysis is the Mann-Whitney rank sum test and Kruskal-W of alpha =0.05.. Allis rank sum test was used to analyze the age difference between different gene mutations. The difference analysis of the incidence of common clinical symptoms was carried out with a double comparison of multiple rates of Pearson chi square test. The results were statistically different from P0.05. Results: a total of 385 cases of Leigh syndrome were collected in 5 articles. The Meta analysis of the clinical symptom merger rate was analyzed. The results showed that lactic acid increased by 62%, growth retardation 60%, muscular dystonia 47%, epilepsy 33%, ocular muscle damage and respiratory impairment 28%, muscle weakness 27%, and feeding difficulty 26%. The incidence of ataxia 20%. in different clinical symptoms was statistically significant, and the increase of lactic acid may be the earliest manifestation. Attention should be paid to early detection and early diagnosis. All LS patients should be diagnosed as early as possible. Gene sequencing, the age difference between the two genes of MT DNA and N DNA is statistically significant. The MT DNA gene mutation is more significant than the n DNA age.N DNA gene mutation site, the PDHA1 gene is late, and the age of the SLC19A3 gene is the earliest. The results of the mutation rate of the respiratory chain enzyme complex suggest the compound I 34%, compound IV 20%, complex I+IV 15% and complex V 9%. conclusion: the clinical manifestations of LS are varied, and the increase of lactic acid may be the most early manifestation. The age of the disease is related to the type of gene mutation, and the mutation of the respiratory chain enzyme complex is the most common cause of LS, and the common mutation of the complex I gene is common.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R746

【参考文献】