强直性肌营养不良1型的临床特点和microRNA的差异表达

发布时间：2018-02-22 07:03

本文关键词： 强直性肌营养不良1型 CTG重复序列微小RNA 临床研究　出处：《中国人民解放军医学院》2016年博士论文　论文类型：学位论文

【摘要】：目的：1.分析和总结强直性肌营养不良1型(myotonic dystrophy type 1, DM1)患者骨骼肌和多系统受累特点,提高对该病复杂临床表现的认识水平。2.分析中国北方汉族人群正常DMPK等位基因CTG重复序列拷贝数的分布特点,丰富对中国汉族人DM1发病率的认识。3.验证在DM1中存在异常表达的miRNA,探索其在DM1发病机制中的作用。方法：1.选取2009年3月一2015年12月在我院神经内科门诊经分子诊断确诊为DM1的患者85例,回顾性分析其肌肉受累程度及多系统受累特点。2.采用PCR片段长度分析法对197名中国北方汉族人(62名DM1患者和135名健康体检者)正常DMPK基因CTG重复片段多态性进行分析,并将该分布特点与中国其他地区人群和其他种族人群进行比较。3.选取12例DM1患者的活检股四头肌标本为实验组,12例(年龄和性别匹配)骨科手术患者的正常股四头肌标本为对照组,采用荧光定量PCR技术检测两组标本间miR-196a、miR-182、miR-451、miR-200c, miR-146a, miR-133a、miR-1, miR-206, CELF1, MBNL1, CELF2和MYF5的相对表达水平进行验证；4.选择荧光素酶实验对miR-196a和miR-182与CELF2的调控关系进行验证。结果：1.85例DM1患者(男52例,女33例)中47例有阳性家族史；首发症状以肢体无力最常见(60%)。肌强直、肌无力、肌萎缩的发生率分别为96.50%、92.90%、68.60%,主要以头面诸肌、颈前肌、肢体远端肌受累明显。69例完成了MIRS分级,1级2例、2级7例、3级24例、4级33例、5级3例,患者MIRS分级和病程(r=0.352,P=0.003)及年龄(r=0.242,P=0.045)具有相关性。MIRS分级与起病年龄(r=0.036,P=0.771)之间无明显相关性。MIRS分级≥4的患者与≤3的患者病程中位数(四分位数间距)分别为10.0(2.3,18.8)年和4.0(2.0,10.0),两组间差异具有统计学意义(Z=-2.570,P=0.010)；平均发病年龄分别为37.9士10.5岁和33.3±13.3岁,两组间差异无统计学意义(t=-1.619,P=0.110)；73例完成了多系统受累的评估,其中60例存在多系统受累,病程中位数(四分位数间距)为7.0(2.3,11.0)年；13例不伴多系统受累,病程中位数(四分位数间距)为4.0(2.0,13.5)年,两组间差异具有统计学意义(Z=1.456,P=0.029)。2.62名DM1患者和135名无症状亲属中共检出332个正常DMPK等位基因,所含CTG拷贝数从5到32不等。其中CTG拷贝数为5的频率最高(24.40%),其次为12拷贝(22.90%),13拷贝(18.70%),11拷贝(17.80%)和14拷贝(5.10%),大等位基因(CTG≥19)的出现频率为2.70%。3. miR-196a、miR-182、miR-146a、miR-200c在DM1中低表达；CELF2、MYF5在DM1中高表达；miR-1、miR-133a、miR-206、miR-451、CELF1和MBNL1在DM1中未发现差异表达。TargetScan提示CELF2可作为miR-182和miR-196a勺靶基因,荧光素酶实验显示miR-196a和miR-182可调控CELF2的表达。结论：1.典型的DM1为青中年隐袭起病,阳性家族史有助于诊断。肌强直以舌肌和大鱼际最敏感,叩击这两个部位有助于不典型肌强直症状的检出。患者病程越长,MIRS分级越高,合并多系统损害的可能性越大。骨骼肌以外,白内障发生率最高,对本病有一定的提示作用。2.正常DMPK基因CTG序列在中国北方人群中的分布特点与南方人群略有不同,DM1的流行病学特征需要进一步行多地区、多民族的大样本研究加以明确。3. miR-196a、miR-182可与CELF2的3’UTR区结合,调控CELF2的表达,进而参与DMl的发病机制。
[Abstract]:Objective: to analyze and summarize the 1. myotonic dystrophy type 1 (myotonic dystrophy 1 type, DM1) in skeletal muscle of patients and characteristics of multi system involvement, improve the clinical features of the disease complex distributed cognition level.2. analysis in Han population in North Chinese normal DMPK allele CTG repeat copy number, rich expression to exist in DM1 Chinese Han people know.3. the incidence of DM1 was validated by miRNA, and explore its role in pathogenesis in DM1. Methods: 1. from March 2009 December 2015 in the outpatient department of Neurology in our hospital by the molecular diagnosis of 85 patients with DM1 diagnosed by PCR were retrospectively analyzed, the muscle involvement and fragment length the characteristics of multi system involvement.2. analysis 197 China North Han people of (62 DM1 patients and 135 healthy persons) analysis of normal DMPK gene CTG repeat polymorphism, and the distribution characteristics Compare.3. to select 12 cases of DM1 patients with biopsy of femoral head four muscle specimens as the experimental group and the population in other areas China and other ethnic groups, 12 patients (age and sex matched) of surgical patients in the Department of orthopedics of normal femoral head four muscle specimens as control group, using fluorescence quantitative PCR detection among two groups were miR-196a, miR-182. MiR-451, miR-200c, miR-146a, miR-133a, miR-1, miR-206, CELF1, MBNL1, the relative expression level of CELF2 and MYF5 verification; verification regulation between the 4. choice of luciferase experiment of miR-196a and miR-182 and CELF2. Results: 1.85 cases of DM1 patients (male 52 cases, female 33 cases) in 47 cases with positive family history the symptoms of limb weakness; the most common (60%). Myotonia, muscle weakness, muscle atrophy rates were 96.50%, 92.90%, 68.60%, mainly in the facial muscles, anterior cervical muscle, distal limb muscle affected obvious.69 patients completed the MIRS grade, 1 grade 2 In 2 cases, 7 cases of grade 3, grade 24 cases, 33 cases of grade 4, grade 5 in 3 cases, patients with MIRS grade and duration (r=0.352, P=0.003) and age (r=0.242, P=0.045) associated with.MIRS grade and age of onset (r=0.036, P=0.771) than in patients with no obvious correlation existed between.MIRS grade of more than 4 3 the patients with median (four percentile interval) were 10 (2.3,18.8) and 4 (2.0,10.0), the difference was statistically significant between the two groups (Z=-2.570, P=0.010); the average age was 37.9 + 10.5 and 33.3 + 13.3 years, there was no significant difference between the two groups (t=-1.619, P=0.110); 73 patients completed the assessment of multi system involvement, including 60 cases with multi system involvement, the course of the median (four percentile interval) was 7 (2.3,11.0) years; 13 cases with multi system involvement, the course of the median (four percentile interval) for 4 years (2.0,13.5), the difference was statistically significant between the two groups (Z=1.456, P=0.029.2.62) DM1 patients and 135 asymptomatic relatives were detected in 332 normal alleles of DMPK, containing the CTG copy number from 5 to 32 dollars. The copy number of CTG is the highest frequency of 5 (24.40%), followed by 12 copies (22.90%), 13 copies, 11 copies (18.70%) and 14 (17.80%) copy (5.10%), large allele (CTG = 19) the frequency of 2.70%.3. miR-196a, miR-182, miR-146a, miR-200c, CELF2, DM1 in low expression; high expression of MYF5 in DM1; miR-1, miR-133a, miR-206, miR-451, CELF1 and MBNL1 were found in the differential expression of.TargetScan suggests that the CELF2 and miR-196a miR-182 as the the target gene in DM1, luciferase experiments showed that miR-196a and miR-182 can regulate the expression of CELF2. Conclusion: 1. typical DM1 for young and middle-aged insidious onset, positive family Shi Youzhu in diagnosis. In the thenar muscle and tongue myotonia is most sensitive, tapping the two parts contribute to the atypical symptoms of myotonia The patients were detected. The longer the duration, the higher the grade of MIRS, with the possibility of multi system damage is greater. The skeletal muscle, the highest incidence of cataract, this disease has suggested a role for.2. in normal DMPK gene CTG sequence in the northern Chinese population in the southern population distribution and a slightly different epidemiological characteristics of DM1 the need for further large sample multi area, multi ethnic study to define.3. miR-196a, miR-182 can be combined with the 3 UTR region and CELF2, the regulation of the expression of CELF2 and pathogenesis in DMl.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R746.2

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