全基因组外显子测序发现多主棒孢霉致病基因CARD9

发布时间：2018-05-07 22:28

本文选题：暗色丝霉菌病 + 多主棒孢霉　；参考：《安徽医科大学》2016年博士论文

【摘要】：研究背景随着广谱抗生素、抗肿瘤药物等的应用,真菌感染的发病率不断提高,人类生命受到严重危害。真菌病根据病变部位不同可分成浅部真菌病及深部真菌病两大类。浅部真菌病可侵犯毛发、指(趾)甲及皮肤,而深部真菌病则可以侵袭心、脑、肾、肺、血液等多个器官及系统,预后差、死亡率高。暗色丝孢霉病是由一组条件致病的暗色真菌引起的浅表组织、皮肤、皮下组织、角膜,甚至系统性感染的真菌病。截至1995年,致病菌已发现50多个属,100多个种。一些为病原性真菌,一些为条件致病性真菌,如链格孢霉、弯孢霉、出芽短梗孢、毛壳菌、奔马赭霉、草本支孢等,成为近年来新发现的一些对人类致病的菌种。本课题组在临床诊疗中诊断了一例长期误诊的暗色丝孢霉病病人,该病人经真菌培养及分子生物学鉴定确诊为多主棒孢霉。多主棒孢霉为条件致病菌,广泛分布于自然界。该患者无糖尿病、无长期服用激素及免疫抑制剂的服药史,平素身体健康,自由职业者,入院各项检查结果无明显异常,否认发病前有过外伤史,上述特征均表明此患者可能存在某种免疫缺陷,即先天基因缺陷,因此,我们将通过全基因组外显子测序的方法,寻找该患者的可能致病基因。目的对患者感染的真菌进行分离鉴定,并运用全基因组外显子测序技术寻找该致病菌的致病基因,在分子水平上阐明该致病真菌导致感染的发病机制。方法(1)第一部分报告1例由多主棒孢霉菌感染所致的暗色丝孢霉病病例,并对多主棒孢霉菌进行分离鉴定。患者女,37岁,面部红斑斑块4年,无外伤史。曾经就诊于多家医院,按“药疹、结节病”等治疗,均无效果,应用糖皮质激素治疗后皮损加重。查体:面部双颊、额部暗红色浸润性斑块,右眼内见脓性分泌物,下颌部见脓性分泌物。皮肤分泌物真菌镜检见细长分隔菌丝,未见硬壳小体;真菌培养可见暗褐色絮状菌落形成;组织病理学检查示真皮内混合性炎症细胞浸润,过碘酸雪夫染色(PAS染色)阳性。经过转录间隔区(ITS)测序分析显示该菌株为多主棒孢霉菌。诊断:多主棒孢霉菌所致皮下暗色丝孢霉病。治疗:给予两性霉素B系统应用,住院14天好转出院。(2)第二部分利用患者外周血全基因组DNA进行全基因组外显子测序,通过逐步滤过公共数据库,利用Sanger测序在患者及对照中对候选基因的突变位点进行测序验证,获得易感基因CARD9基因。结果(1)经真菌培养、ITS测序结果显示该菌为多主棒孢霉菌。本病例为多主棒孢霉菌所致的暗色丝孢霉病,经两性霉素B治疗后病情好转;(2)经全基因组外显子测序技术检测,得到了949个全基因组外显子测序样本的SNPs、indels的数据集合;(3)考虑到引起疾病的变异可能是罕见变异,存在于公共数据库中的可能性极小,同时可能与既往真菌易感基因相重叠。通过与生物信息数据库逐步滤过后,发现在CARD9基因上一个插入突变即c.191-192Ins TGCT,p.L64fs X59,该突变导致了其下游的第59个氨基酸变为终止密码子,且proven软件预测为有害突变;(4)通过Sanger测序对该样本的该位点进行测序验证,证实c.191-192Ins TGCT,p.L64fs X59确实存在。结论(1)通过全基因组外显子测序发现了一个中国汉族多主棒孢霉菌感染所致的暗色丝孢霉病患者的致病基因,证实了全基因组外显子测序鉴定单基因病致病基因的有效性。(2)通过直接测序方法检测出患者CARD9基因插入突变c.191-192Ins TGCT,p.L64fs X59,丰富了CARD9基因突变的临床表型信息,为将来的遗传咨询、产前诊断及基因治疗奠定了理论基础。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R756

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