本文選題：暗色絲霉菌病 + 多主棒孢霉　； 參考：《安徽醫(yī)科大學》2016年博士論文

【摘要】：研究背景隨著廣譜抗生素、抗腫瘤藥物等的應用,真菌感染的發(fā)病率不斷提高,人類生命受到嚴重危害。真菌病根據(jù)病變部位不同可分成淺部真菌病及深部真菌病兩大類。淺部真菌病可侵犯毛發(fā)、指(趾)甲及皮膚,而深部真菌病則可以侵襲心、腦、腎、肺、血液等多個器官及系統(tǒng),預后差、死亡率高。暗色絲孢霉病是由一組條件致病的暗色真菌引起的淺表組織、皮膚、皮下組織、角膜,甚至系統(tǒng)性感染的真菌病。截至1995年,致病菌已發(fā)現(xiàn)50多個屬,100多個種。一些為病原性真菌,一些為條件致病性真菌,如鏈格孢霉、彎孢霉、出芽短梗孢、毛殼菌、奔馬赭霉、草本支孢等,成為近年來新發(fā)現(xiàn)的一些對人類致病的菌種。本課題組在臨床診療中診斷了一例長期誤診的暗色絲孢霉病病人,該病人經(jīng)真菌培養(yǎng)及分子生物學鑒定確診為多主棒孢霉。多主棒孢霉為條件致病菌,廣泛分布于自然界。該患者無糖尿病、無長期服用激素及免疫抑制劑的服藥史,平素身體健康,自由職業(yè)者,入院各項檢查結(jié)果無明顯異常,否認發(fā)病前有過外傷史,上述特征均表明此患者可能存在某種免疫缺陷,即先天基因缺陷,因此,我們將通過全基因組外顯子測序的方法,尋找該患者的可能致病基因。目的對患者感染的真菌進行分離鑒定,并運用全基因組外顯子測序技術尋找該致病菌的致病基因,在分子水平上闡明該致病真菌導致感染的發(fā)病機制。方法(1)第一部分報告1例由多主棒孢霉菌感染所致的暗色絲孢霉病病例,并對多主棒孢霉菌進行分離鑒定�；颊吲�,37歲,面部紅斑斑塊4年,無外傷史。曾經(jīng)就診于多家醫(yī)院,按“藥疹、結(jié)節(jié)病”等治療,均無效果,應用糖皮質(zhì)激素治療后皮損加重。查體:面部雙頰、額部暗紅色浸潤性斑塊,右眼內(nèi)見膿性分泌物,下頜部見膿性分泌物。皮膚分泌物真菌鏡檢見細長分隔菌絲,未見硬殼小體;真菌培養(yǎng)可見暗褐色絮狀菌落形成;組織病理學檢查示真皮內(nèi)混合性炎癥細胞浸潤,過碘酸雪夫染色(PAS染色)陽性。經(jīng)過轉(zhuǎn)錄間隔區(qū)(ITS)測序分析顯示該菌株為多主棒孢霉菌。診斷:多主棒孢霉菌所致皮下暗色絲孢霉病。治療:給予兩性霉素B系統(tǒng)應用,住院14天好轉(zhuǎn)出院。(2)第二部分利用患者外周血全基因組DNA進行全基因組外顯子測序,通過逐步濾過公共數(shù)據(jù)庫,利用Sanger測序在患者及對照中對候選基因的突變位點進行測序驗證,獲得易感基因CARD9基因。結(jié)果(1)經(jīng)真菌培養(yǎng)、ITS測序結(jié)果顯示該菌為多主棒孢霉菌。本病例為多主棒孢霉菌所致的暗色絲孢霉病,經(jīng)兩性霉素B治療后病情好轉(zhuǎn);(2)經(jīng)全基因組外顯子測序技術檢測,得到了949個全基因組外顯子測序樣本的SNPs、indels的數(shù)據(jù)集合;(3)考慮到引起疾病的變異可能是罕見變異,存在于公共數(shù)據(jù)庫中的可能性極小,同時可能與既往真菌易感基因相重疊。通過與生物信息數(shù)據(jù)庫逐步濾過后,發(fā)現(xiàn)在CARD9基因上一個插入突變即c.191-192Ins TGCT,p.L64fs X59,該突變導致了其下游的第59個氨基酸變?yōu)榻K止密碼子,且proven軟件預測為有害突變;(4)通過Sanger測序?qū)υ摌颖镜脑撐稽c進行測序驗證,證實c.191-192Ins TGCT,p.L64fs X59確實存在。結(jié)論(1)通過全基因組外顯子測序發(fā)現(xiàn)了一個中國漢族多主棒孢霉菌感染所致的暗色絲孢霉病患者的致病基因,證實了全基因組外顯子測序鑒定單基因病致病基因的有效性。(2)通過直接測序方法檢測出患者CARD9基因插入突變c.191-192Ins TGCT,p.L64fs X59,豐富了CARD9基因突變的臨床表型信息,為將來的遺傳咨詢、產(chǎn)前診斷及基因治療奠定了理論基礎。
[Abstract]:Background: with the application of broad-spectrum antibiotics and antitumor drugs, the incidence of fungal infection is increasing and human life is seriously damaged. Mycosis can be divided into two categories: superficial mycosis and deep mycosis according to different lesion sites. Superficial mycosis can invade hair, finger (toe) and skin, and deep mycosis can invade Multiple organs and systems, such as heart, brain, kidney, lung and blood, have poor prognosis and high mortality. Dark mycosis is a superficial tissue, skin, subcutaneous tissue, cornea, and even systemic fungal disease caused by a group of pathogenic fungi. As of 1995, more than 50 genera and more than 100 species have been found. Some are pathogenic fungi. Some pathogenic fungi, such as cyclosporin, cyclosporin, buds, spore, Trichoderma, ochre gallbladder, herbaceous spore, have become some newly discovered pathogenic bacteria in recent years. The confirmed diagnosis is mycophenolate mycophena. Mycophenolate mycophena is a conditional pathogenic bacteria, widely distributed in nature. The patient has no diabetes, no long-term use of hormone and immunosuppressant medicine history, normal health, freelancers, there are no obvious abnormalities in the examination results and deny the history of trauma before the onset of the disease. The above characteristics all indicate this patient There may be some kind of immune deficiency, that is, congenital gene defect. Therefore, we will find the possible pathogeny gene of the patient by the method of full genome exon sequencing. The pathogenesis of infection caused by this pathogenic fungus. Method (1) the first part reported 1 cases of dark mycporosis caused by mycpora myctis and isolated and identified mycpora mycosis. The female, 37 years old, facial erythema plaque for 4 years, without a history of trauma, had been treated in many hospitals, and had been treated with "drug rash, sarcoidosis" and so on. The skin lesions were aggravated by the use of corticosteroids. Facial double cheeks, dark red infiltrating patches in the forehead, pus secretions in the right eye, and pyogenic secretions in the mandible. The skin secretions were found to see slender septate mycelium and no hard shell bodies; fungal culture could be found in dark brown floc colony formation; histopathological examination showed true Intradermal mixed inflammatory cell infiltration and iodate schyev staining (PAS staining) positive. The strain was analyzed by ITS sequencing analysis. The strain was diagnosed as hypodermous aspora mildew caused by molspa multiple. Treatment: amphotericin B system should be used for 14 days in hospital. (2) second parts of the patients were used outside the hospital. The whole genome DNA of the peripheral blood was sequenced by the whole genome exon. By gradually filtering the public database, the mutation site of the candidate gene was sequenced and verified by Sanger sequencing. The susceptible gene CARD9 gene was obtained. Results (1) the result of fungal culture and ITS sequencing showed that the bacterium was a multi principal mycotic fungus. Amphotericum mildew caused by amphotericin B was improved after amphotericin B treatment; (2) the total genomic exon sequencing technology was detected, and 949 full genome exons sequencing samples were obtained for SNPs, indels data collection; (3) it is considered that the variation in the disease may be rare, and the possibility of existence in the public database is extremely possible. At the same time, it may overlap with previous fungal susceptibility genes. After gradual filtration with the bioinformation database, an insertion mutation in the CARD9 gene, c.191-192Ins TGCT, p.L64fs X59, has been found, which causes the fifty-ninth amino acids in the downstream to become terminated codon and proven software is predicted to be a harmful mutation; (4) Sanger measured by Sanger. The sequence was sequenced to verify that the c.191-192Ins TGCT and p.L64fs X59 existed. Conclusion (1) a whole genome exon sequencing was used to detect the pathogenic gene of the dark mycporosis in a Chinese Han Polyporus infection, and the whole genome exon sequencing was used to identify the pathogenicity of the monogenic disease. (2) CARD9 gene insertion mutation c.191-192Ins TGCT and p.L64fs X59 were detected by direct sequencing, which enriched the clinical phenotypic information of the mutation of CARD9 gene, laying a theoretical basis for future genetic counseling, prenatal diagnosis and gene therapy.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R756

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