中国南方地区GNE肌病基因突变分析及临床特点研究

发布时间：2018-03-06 20:43

本文选题：GNE肌病　切入点：DMRV　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：GNE肌病,旧称遗传性包涵体肌病(hereditary inclusion body myopathy, hIBM)、 DMRV (distal myopathy with rimmed vacuoles,伴镶边空泡的远端肌病)或Nonaka肌病,是一种罕见的常染色体隐性遗传的远端肌病。由GNE基因(UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, GenBank NM_005476)突变导致。GNE基因包含12个外显子,编码催化唾液酸合成的限速酶。GNE肌病临床上主要20-40岁起病,大多数病人在起病后10-20年丧失独自行走能力。起病表现为小腿前群肌无力和萎缩,因足背屈无力导致足下垂和步态异常。病情呈缓慢进展,至疾病后期,下肢近端、上肢及中轴肌肉亦可受累。整个病程中股四头肌始终相对回避。血清肌酸激酶(creatine kinase, CK)水平正常范围或轻至中度升高。肌电图呈肌源性损害。肌肉病理特点是肌纤维萎缩和在萎缩的肌纤维中出现镶边空泡,可见肌间质增生。肌纤维变性、坏死、再生少见,无明显炎性细胞浸润。电镜下可见肌细胞胞浆和肌细胞核内直径8-10nm的管丝样包涵体。目前GNE肌病报道最多的国家是日本和中东犹太人。国内共报道GNE肌病患者53例,其中49例来自中国北方的北京和山东,4例来自台湾。而中国南方尚未有大样本量的报道。本研究选取中国南方的DMRV患者进行GNE基因分析,回顾总结基因确诊患者的临床、病理和肌肉MRI资料,并分析基因型和临床表型的关系,以期了解中国南方GNE肌病患者的特点,比较与中国北方患者之间的不同,并进一步扩大中国人群GNE肌病样本量,探讨与日本、中东地区之间的差异。第一部分中国南方地区GNE肌病患者基因突变分析目的：了解中国南方地区GNE基因突变的特点,与北方地区相比较。并进一步探讨与日本、中东地区之间GNE基因突变的差异。方法：选取复旦大学附属华山医院神经内科神经肌病组2005年2月至2014年3月收治的临床病理特点符合入选标准的无血缘关系的散发病例共30例,入选标准如下：(1)青少年晚期或成年期起病的下肢远端肌无力和肌萎缩,以小腿前群受累为主；(2)随疾病进展,股四头肌相对回避；(3)CK水平正常或轻至中度升高；(4)肌电图呈肌源性损害；(5)肌肉病理需排除炎性肌病、肌营养不良、代谢性肌病等肌肉疾病。从入选病人的外周静脉血白细胞或肌肉组织中提取基因组DNA,采用聚合酶链式反应(PCR)方法特异性扩增GNE基因的第1-12号外显子并进行测序,与正常序列进行比对分析,明确突变位点,分析是否发生氨基酸改变。结果：1.本研究纳入的30例患者中,基因诊断明确的GNE肌病患者共24例,其中8例为纯合子(33.3%),16例为复杂杂合子(66.7%)。24例患者均为汉族,分别来自中国南方的江西、浙江、安徽、上海和江苏。其中23例于我院活检,1例来自门诊。2.共发现23个GNE基因突变位点,包括18个错义突变(78.3%)：p.C13S. p.F66V、p.I106F、p.R162C、p.D176V、p.Y186H、p.K245E、p.R246W、p.R246Q、 p.K353E、p.L508S、p.H509Y、p.A524V、p.F562S、p.V572L、p.A591V、p.A638P、 p.G652E;2个无义突变(8.7%):p.Y186X, p.Q436X;2个缺失突变(8.7%)：p.D515fsX2 (c.1543-1544delGA)、p.A600fsX43 (c.1798de1G);1个复杂双突变(4.3%):p.E329fsX43 (c.985de1G+987-988GATT)。分别位于GNE基因的第2、3、4、6、8、g、10、11、12号外显子。其中已知突变10个(43.5%),新发突变13个(56.5%):p.F66V、p.I106F、p.Y186H、p.Y186X、p.K245E、p.E329fsX43、 p.K353E、p.D515fsX2、p.F562S、p.A600fsX43、p.A591V、p.A638P、p.G652E。而中国其他报道中,53例患者共发现18个突变位点,与本研究相同的突变位点仅6个。3.在23个突变中,最常见的三个突变为p.D176V(25.0%)、p.R246Q(12.5%)和p.K353E(10.4%)。而中国北方报道的最常见的三个突变为p.L508S (19%)、 p.A631V (15%)和p.D176V(14%),仅p.D176V为同一突变,但突变频率相差较大。以上特点本研究与中国北方的报道有一定差别。结论：本研究是针对中国南方GNE肌病患者的首个较大样本量的研究,首次在中国GNE肌病患者中发现了缺失突变、复杂双突变及位于GNE基因第6号和第8号外显子的突变。与中国的其他报道相比,本研究突变位点的分布范围更加分散,突变形式更加多样。本研究突变热点与中国其他报道也不尽相同。除中国其他研究中纳入了家族性患者之外,二者的差别也可能与患者所在的地域不同有关。第二部分中国南方地区GNE肌病患者临床特点及基因型-临床表型关系分析目的：对基因诊断明确的24例GNE肌病患者的临床表现、肌肉MRI和病理特点进行回顾总结,并分析基因型与临床表型的关系。方法：收集24例经GNE基因诊断明确的患者的临床资料、血液生化指标、肌电图等检查结果。根据Mercuri提出的T1WI观察肌肉脂肪化的评估标准对10例有肌肉MRI资料患者的肌肉MRI进行臀部和下肢共18块肌肉的脂肪化评分。回顾23例有肌肉病理资料的病理特点,主要分析HE染色和Gomori染色。结合第一部分,进行基因型与临床表型之间关系的分析。结果：1.24例患者中男女比例为13：11。发病年龄15-52岁,平均33.2±8.8岁(中国其他报道：20.5±8.1岁,p0.0001)。病程(起病至就诊间隔时间)1-17年,平均5.04±4.4年。4例有父母近亲婚配史(16.7%)。17例(70.8%)以单肢或双下肢无力/足下垂起病。体格检查中,抬头肌受累者(抬头肌力5级)12人(50.0%)。下肢肌无力全部为远端重于近端,胫前肌重于腓肠肌。所有患者股四头肌均相对回避(100%)。24例患者肌酸激酶(CK)水平165-1826 U/L(正常值：38-174 U/L),平均值为477.8±380.9 U/L,中位数为351.0 U/L。所有患者肌电图均示肌源性损害。2.GNE肌病患者的肌肉MRI检查发现,大腿肌群以后群受累明显,大腿前群肌肉中,股四头肌外侧头、股中间肌、股直肌相对回避,而股四头肌内侧头轻度受累；大腿外侧群的缝匠肌轻度受累；大腿后群肌肉整体受累严重,但其中股薄肌相对回避为中度受累；小腿前后群肌肉均为中度到重度受累,以胫前肌、比目鱼肌和腓肠肌内侧头受累最为严重。3.23例患者肌肉病理特点符合已报道的典型特征：可见细胞大小不等；炎性细胞浸润、变性、坏死和再生均少见,即使出现程度亦较轻；可见成簇萎缩肌纤维和脂肪结缔组织增生；在15例(65.2%)患者中发现镶边空泡。4.p.D176V为本研究中最常见的突变位点,对比发现携带该突变的患者发病年龄明显更晚(36.8±10.4 vs.29.6±5.2,p=0.0432),抬头肌力明显更易受累(75%vs.25%,p=0.014),股四头肌力轻度受累比例更高(50% vs.25%)。由于样本量的限制,部分数值的比较在统计学上并无差异,但是可以看出一定趋势。而携带缺失突变和无义突变的患者在临床表现上与其他患者相比无明显差异。结论：由于中国其他报道大部分来自中国北方,因此一定程度上可代表中国北方GNE肌病患者的特点。中国南方与中国北方的GNE肌病患者相比,发病年龄明显更晚。临床症状上与GNE肌病典型表现一致。肌肉病理特点与典型GNE肌病相符。镶边空泡的出现并非诊断GNE肌病的必须条件,其出现与否与活检部位的选择和疾病的进展程度有关。肌肉MRI检查是一种对GNE肌病进行鉴别诊断的很好的方法,T1WI可观察肌肉脂肪化程度。含p.D176V突变的患者发病年龄更晚,抬头肌力更易受累。
[Abstract]:GNE myopathy, formerly known as hereditary inclusion body myopathy (hereditary inclusion body myopathy, hIBM), DMRV (distal myopathy with rimmed vacuoles distal myopathy with rimmed vacuoles, or Nonaka) is a kind of distal myopathy myopathy, a rare autosomal recessive gene. By GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, GenBank NM_005476) mutation result.GNE gene contains 12 exons, encoding sialic acid catalytic synthesis rate limiting enzyme.GNE myopathy clinically 20-40 years of onset, most patients walking alone in the 10-20 years after the onset of loss of ability. The initial presentation is the anterior group weakness and atrophy, because of foot dorsiflexion weakness leads foot drop and abnormal gait. The disease is slow progress, to the late stage of the disease, the proximal end of the lower limbs, upper limbs and medial muscle were also involved. In the whole course of the stock four muscles always relative serum muscle avoidance. Acid kinase (creatine kinase, CK) normal levels or mild to moderate elevations. EMG showed myogenic lesions. Pathological features of muscle is the muscle fiber atrophy and rimmed vacuoles appeared in atrophic muscle fibers, visible muscle interstitial hyperplasia. Muscle fiber degeneration, necrosis, regeneration is rare, no obvious inflammatory cell infiltration. Under the electron microscope tube filament like skeletal muscle cells in the cytoplasm and nucleus of muscle diameter of 8-10nm inclusion body. At present, GNE reported myopathy in most countries are Japan and the Middle East. The Jews reported 53 cases of GNE patients, including 49 cases of Chinese from northern Beijing and Shandong, 4 cases from Taiwan and South Chinese. Not yet a big the quantity of samples is reported. This study selected China south in DMRV GNE gene analysis, gene diagnosis clinical review of patients, pathology and muscle MRI data, and analyze the relationship between genotype and clinical phenotype, in order to understand China Southern GNE myopathy patients, compared with patients with different China between the north, and to further expand the China population GNE myopathy sample, and discuss differences between Japan and the Middle East. The southern part of the region Chinese GNE myopathy gene mutation analysis objective: To explore the characteristics of GNE gene mutations in South Chinese, compared with the northern region and further discussion with Japan, the mutation of GNE gene in the Middle East. The differences between methods: clinical and pathological features selected from Huashan Hospital Affiliated to Fudan University of Neurology from February 2005 to March 2014 were patients eligible unrelated sporadic cases were 30 cases, the criteria are as follows: (1) late adolescence or adult onset of lower extremity weakness and to the front of the lower leg muscle atrophy, group involvement; (2) with the progression of the disease, the stock four muscles relative avoidance; (3) the level of CK Normal or mild to moderate elevations; (4) EMG showed myogenic damage; (5) muscle pathology excluded inflammatory myopathy, muscular dystrophy, metabolic myopathy and muscular diseases. The genome DNA was extracted from patients with peripheral blood white cells or muscle tissues by polymerase chain reaction (PCR) methods the specific amplification of the GNE gene in exon 1-12 was sequenced and compared with the normal sequence, specific mutations, analysis whether the amino acid change. Results: 30 cases of 1. patients enrolled in the study, a total of 24 cases of GNE patients diagnosed muscle genes, of which 8 cases were homozygous (33.3%), 16 cases of complex heterozygous (66.7%).24 patients were Han, China were from Jiangxi in the south, Zhejiang, Anhui, Shanghai and Jiangsu. Among the 23 patients in our hospital 1 cases from outpatient biopsy,.2. found 23 GNE gene mutations, including 18. Mutation (78.3%):p.C13S. p.F66V, p.I106F, p.R162C, p.D176V, p.Y186H, p.K245E, p.R246W, p.R246Q, p.K353E, p.L508S, p.H509Y, p.A524V, p.F562S, p.V572L, p.A591V, p.A638P, p.G652E; 2 nonsense mutations (8.7%): p.Y186X, p.Q436X; 2 deletion mutation (8.7%):p.D515fsX2 (c.1543-1544delGA) (c.1798de1G, p.A600fsX43); 1 (4.3%): complex double mutant p.E329fsX43 (c.985de1G+987-988GATT). Located in the GNE gene in 2,3,4,6,8, G, 10,11,12 in exon 10. The known mutations (43.5%), a new mutation in 13 (56.5%): p.F66V, p.I106F, p.Y186H, p.Y186X, p.K245E, p.E329fsX43 p.K353E, p.D515fsX2, p.F562S, p.A600fsX43, p.A591V, p.A638P, p.G652E., and other reports Chinese, 53 cases were found 18 mutations, with the same study mutation only 6.3. mutations in 23, three of the most common mutations in p.D176V (25%), p.R246 Q (12.5%) and p.K353E (10.4%). Three of the most common mutations in the north and Chinese reported by p.L508S (19%), p.A631V (15%) and p.D176V (14%), only p.D176V for the same mutation, but the mutation frequency is larger. There is some difference between the above characteristics of the research and Chinese North reported. Conclusion this study is for the first big sample Chinese South GNE myopathy patients, for the first time in China GNE myopathy patients found the deletion mutation of complex double mutations and mutation in GNE gene exon eighth and sixth. Compared with other reported China, the mutation site distribution is more dispersed mutation, more diverse forms. This research hot spot mutations and other reports Chinese are not the same. In addition to other studies Chinese included familial patients, the difference between the two patients may be different from the geographical location of the second part China south. The clinical phenotype and clinical characteristics of GNE gene in patients with myopathy - side area analysis objective: to clear the gene diagnosis of 24 cases of GNE patients with clinical manifestations of muscle myopathy, MRI and pathological features were analyzed retrospectively, and analyze the relationship between the genotype and phenotype. Methods: to collect the clinical data of 24 cases of patients diagnosed by GNE gene the blood biochemical index, EMG examination results. According to the observation of muscle fat Mercuri proposed T1WI evaluation criteria of 10 cases with fat score data of patients with muscle MRI muscle MRI hip and lower limb muscles were 18. Review the clinicopathologic characteristics of 23 cases of muscle pathology, HE staining and Gomori analysis staining. Combined with the first part, analyze the relationship between genotype and phenotype. Results: 1.24 patients with male to female ratio of 13:11. age of 15-52 years, an average of 33.2 + 8.8 years (in In other reports: 20.5 + 8.1, P0.0001). (the duration of onset to treatment time interval) for 1-17 years, an average of 5.04 + 4.4 years.4 cases parents have consanguineous marriage history (16.7%).17 (70.8%) cases with single limb or lower extremity weakness / drop onset. Physical examination, muscle involvement (look up lift the head strength of grade 5) 12 (50%). All of the lower limb muscle weakness in proximal distal, anterior tibial muscle weight in gastrocnemius muscle. All patients with femoral head four muscles were relative avoidance (100%).24 patients with creatine kinase (CK) level 165-1826 U/L (normal value: 38-174 U/L), the average value is 477.8 + 380.9 U/L, found the median muscle MRI examination 351 U/L. all patients were EMG showed myogenic changes in.2.GNE patients with myopathy, thigh muscles after group obviously affected, former thigh muscles, unit four biceps lateral head, femoral muscle, rectus femoris is avoided, and the femoral head four medial gastrocnemius mild thigh group involvement; Sartorius muscle overall mild involvement; severe involvement of thigh, but the gracilis muscle was moderate relative to avoid involvement; leg muscles before and after were moderate to severe involvement in the tibialis anterior muscle, the characteristics of serious.3.23 patients muscle pathology for soleus and medial gastrocnemius muscle involvement has been reported in accordance with the typical characteristics visible cell size; inflammatory cell infiltration, degeneration, necrosis and regeneration are rare, even if the degree is lighter; visible clusters of atrophic fibers and fat connective tissue hyperplasia; in 15 cases (65.2%) were found in the air bubble edge.4.p.D176V was the most common mutation sites in this study, found that patients carrying the age of onset of the mutation significantly later (36.8 + 10.4 vs.29.6 + 5.2, p=0.0432), the rise of strength was more involved (75%vs.25%, p=0.014), higher proportion of mild muscle involvement in femoral head four (50% vs.25%) due. Sample size difference was no difference part of the value, but you can see a certain trend. While carrying a deletion mutation and nonsense mutation in patients with clinical manifestations and other patients showed no significant difference. Conclusion: because other reports Chinese mostly from the northern part of China, so to a certain extent on behalf of the North China GNE patients were compared. Chinese South and North China GNE patients, age of onset was significantly more late. Clinical symptoms and GNE myopathy consistent. Typical pathological features of GNE myopathy with rimmed vacuoles. Typical consistent appearance must not condition diagnosis of GNE myopathy. The progress of its presence and biopsy site selection and disease. Muscle MRI examination is a good method for differential diagnosis of GNE myopathy, T1WI can observe the degree of muscle fat containing p.D176V mutation. The age of the patient is more late, and the muscle strength is more vulnerable.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R746

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