本文選題：GNE肌病　切入點(diǎn)：DMRV　出處：《復(fù)旦大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：GNE肌病,舊稱遺傳性包涵體肌病(hereditary inclusion body myopathy, hIBM)、 DMRV (distal myopathy with rimmed vacuoles,伴鑲邊空泡的遠(yuǎn)端肌病)或Nonaka肌病,是一種罕見的常染色體隱性遺傳的遠(yuǎn)端肌病。由GNE基因(UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, GenBank NM_005476)突變導(dǎo)致。GNE基因包含12個(gè)外顯子,編碼催化唾液酸合成的限速酶。GNE肌病臨床上主要20-40歲起病,大多數(shù)病人在起病后10-20年喪失獨(dú)自行走能力。起病表現(xiàn)為小腿前群肌無力和萎縮,因足背屈無力導(dǎo)致足下垂和步態(tài)異常。病情呈緩慢進(jìn)展,至疾病后期,下肢近端、上肢及中軸肌肉亦可受累。整個(gè)病程中股四頭肌始終相對(duì)回避。血清肌酸激酶(creatine kinase, CK)水平正常范圍或輕至中度升高。肌電圖呈肌源性損害。肌肉病理特點(diǎn)是肌纖維萎縮和在萎縮的肌纖維中出現(xiàn)鑲邊空泡,可見肌間質(zhì)增生。肌纖維變性、壞死、再生少見,無明顯炎性細(xì)胞浸潤(rùn)。電鏡下可見肌細(xì)胞胞漿和肌細(xì)胞核內(nèi)直徑8-10nm的管絲樣包涵體。目前GNE肌病報(bào)道最多的國(guó)家是日本和中東猶太人。國(guó)內(nèi)共報(bào)道GNE肌病患者53例,其中49例來自中國(guó)北方的北京和山東,4例來自臺(tái)灣。而中國(guó)南方尚未有大樣本量的報(bào)道。本研究選取中國(guó)南方的DMRV患者進(jìn)行GNE基因分析,回顧總結(jié)基因確診患者的臨床、病理和肌肉MRI資料,并分析基因型和臨床表型的關(guān)系,以期了解中國(guó)南方GNE肌病患者的特點(diǎn),比較與中國(guó)北方患者之間的不同,并進(jìn)一步擴(kuò)大中國(guó)人群GNE肌病樣本量,探討與日本、中東地區(qū)之間的差異。第一部分中國(guó)南方地區(qū)GNE肌病患者基因突變分析目的：了解中國(guó)南方地區(qū)GNE基因突變的特點(diǎn),與北方地區(qū)相比較。并進(jìn)一步探討與日本、中東地區(qū)之間GNE基因突變的差異。方法：選取復(fù)旦大學(xué)附屬華山醫(yī)院神經(jīng)內(nèi)科神經(jīng)肌病組2005年2月至2014年3月收治的臨床病理特點(diǎn)符合入選標(biāo)準(zhǔn)的無血緣關(guān)系的散發(fā)病例共30例,入選標(biāo)準(zhǔn)如下：(1)青少年晚期或成年期起病的下肢遠(yuǎn)端肌無力和肌萎縮,以小腿前群受累為主；(2)隨疾病進(jìn)展,股四頭肌相對(duì)回避；(3)CK水平正�；蜉p至中度升高；(4)肌電圖呈肌源性損害；(5)肌肉病理需排除炎性肌病、肌營(yíng)養(yǎng)不良、代謝性肌病等肌肉疾病。從入選病人的外周靜脈血白細(xì)胞或肌肉組織中提取基因組DNA,采用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)方法特異性擴(kuò)增GNE基因的第1-12號(hào)外顯子并進(jìn)行測(cè)序,與正常序列進(jìn)行比對(duì)分析,明確突變位點(diǎn),分析是否發(fā)生氨基酸改變。結(jié)果：1.本研究納入的30例患者中,基因診斷明確的GNE肌病患者共24例,其中8例為純合子(33.3%),16例為復(fù)雜雜合子(66.7%)。24例患者均為漢族,分別來自中國(guó)南方的江西、浙江、安徽、上海和江蘇。其中23例于我院活檢,1例來自門診。2.共發(fā)現(xiàn)23個(gè)GNE基因突變位點(diǎn),包括18個(gè)錯(cuò)義突變(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個(gè)無義突變(8.7%):p.Y186X, p.Q436X;2個(gè)缺失突變(8.7%)：p.D515fsX2 (c.1543-1544delGA)、p.A600fsX43 (c.1798de1G);1個(gè)復(fù)雜雙突變(4.3%):p.E329fsX43 (c.985de1G+987-988GATT)。分別位于GNE基因的第2、3、4、6、8、g、10、11、12號(hào)外顯子。其中已知突變10個(gè)(43.5%),新發(fā)突變13個(gè)(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。而中國(guó)其他報(bào)道中,53例患者共發(fā)現(xiàn)18個(gè)突變位點(diǎn),與本研究相同的突變位點(diǎn)僅6個(gè)。3.在23個(gè)突變中,最常見的三個(gè)突變?yōu)閜.D176V(25.0%)、p.R246Q(12.5%)和p.K353E(10.4%)。而中國(guó)北方報(bào)道的最常見的三個(gè)突變?yōu)閜.L508S (19%)、 p.A631V (15%)和p.D176V(14%),僅p.D176V為同一突變,但突變頻率相差較大。以上特點(diǎn)本研究與中國(guó)北方的報(bào)道有一定差別。結(jié)論：本研究是針對(duì)中國(guó)南方GNE肌病患者的首個(gè)較大樣本量的研究,首次在中國(guó)GNE肌病患者中發(fā)現(xiàn)了缺失突變、復(fù)雜雙突變及位于GNE基因第6號(hào)和第8號(hào)外顯子的突變。與中國(guó)的其他報(bào)道相比,本研究突變位點(diǎn)的分布范圍更加分散,突變形式更加多樣。本研究突變熱點(diǎn)與中國(guó)其他報(bào)道也不盡相同。除中國(guó)其他研究中納入了家族性患者之外,二者的差別也可能與患者所在的地域不同有關(guān)。第二部分中國(guó)南方地區(qū)GNE肌病患者臨床特點(diǎn)及基因型-臨床表型關(guān)系分析目的：對(duì)基因診斷明確的24例GNE肌病患者的臨床表現(xiàn)、肌肉MRI和病理特點(diǎn)進(jìn)行回顧總結(jié),并分析基因型與臨床表型的關(guān)系。方法：收集24例經(jīng)GNE基因診斷明確的患者的臨床資料、血液生化指標(biāo)、肌電圖等檢查結(jié)果。根據(jù)Mercuri提出的T1WI觀察肌肉脂肪化的評(píng)估標(biāo)準(zhǔn)對(duì)10例有肌肉MRI資料患者的肌肉MRI進(jìn)行臀部和下肢共18塊肌肉的脂肪化評(píng)分�；仡�23例有肌肉病理資料的病理特點(diǎn),主要分析HE染色和Gomori染色。結(jié)合第一部分,進(jìn)行基因型與臨床表型之間關(guān)系的分析。結(jié)果：1.24例患者中男女比例為13：11。發(fā)病年齡15-52歲,平均33.2±8.8歲(中國(guó)其他報(bào)道：20.5±8.1歲,p0.0001)。病程(起病至就診間隔時(shí)間)1-17年,平均5.04±4.4年。4例有父母近親婚配史(16.7%)。17例(70.8%)以單肢或雙下肢無力/足下垂起病。體格檢查中,抬頭肌受累者(抬頭肌力5級(jí))12人(50.0%)。下肢肌無力全部為遠(yuǎn)端重于近端,脛前肌重于腓腸肌。所有患者股四頭肌均相對(duì)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),平均值為477.8±380.9 U/L,中位數(shù)為351.0 U/L。所有患者肌電圖均示肌源性損害。2.GNE肌病患者的肌肉MRI檢查發(fā)現(xiàn),大腿肌群以后群受累明顯,大腿前群肌肉中,股四頭肌外側(cè)頭、股中間肌、股直肌相對(duì)回避,而股四頭肌內(nèi)側(cè)頭輕度受累；大腿外側(cè)群的縫匠肌輕度受累；大腿后群肌肉整體受累嚴(yán)重,但其中股薄肌相對(duì)回避為中度受累；小腿前后群肌肉均為中度到重度受累,以脛前肌、比目魚肌和腓腸肌內(nèi)側(cè)頭受累最為嚴(yán)重。3.23例患者肌肉病理特點(diǎn)符合已報(bào)道的典型特征：可見細(xì)胞大小不等；炎性細(xì)胞浸潤(rùn)、變性、壞死和再生均少見,即使出現(xiàn)程度亦較輕；可見成簇萎縮肌纖維和脂肪結(jié)締組織增生；在15例(65.2%)患者中發(fā)現(xiàn)鑲邊空泡。4.p.D176V為本研究中最常見的突變位點(diǎn),對(duì)比發(fā)現(xiàn)攜帶該突變的患者發(fā)病年齡明顯更晚(36.8±10.4 vs.29.6±5.2,p=0.0432),抬頭肌力明顯更易受累(75%vs.25%,p=0.014),股四頭肌力輕度受累比例更高(50% vs.25%)。由于樣本量的限制,部分?jǐn)?shù)值的比較在統(tǒng)計(jì)學(xué)上并無差異,但是可以看出一定趨勢(shì)。而攜帶缺失突變和無義突變的患者在臨床表現(xiàn)上與其他患者相比無明顯差異。結(jié)論：由于中國(guó)其他報(bào)道大部分來自中國(guó)北方,因此一定程度上可代表中國(guó)北方GNE肌病患者的特點(diǎn)。中國(guó)南方與中國(guó)北方的GNE肌病患者相比,發(fā)病年齡明顯更晚。臨床癥狀上與GNE肌病典型表現(xiàn)一致。肌肉病理特點(diǎn)與典型GNE肌病相符。鑲邊空泡的出現(xiàn)并非診斷GNE肌病的必須條件,其出現(xiàn)與否與活檢部位的選擇和疾病的進(jìn)展程度有關(guān)。肌肉MRI檢查是一種對(duì)GNE肌病進(jìn)行鑒別診斷的很好的方法,T1WI可觀察肌肉脂肪化程度。含p.D176V突變的患者發(fā)病年齡更晚,抬頭肌力更易受累。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R746

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