复发性念珠菌颈部淋巴结炎1例易感基因筛查及免疫学研究

【摘要】 目的 探究1例复发性念珠菌性颈部淋巴结炎患者的遗传学病因及抗真菌免疫功能。方法 采用二代测序技术筛查患者及父母真菌病易感基因，提取患者及6例健康对照者外周血单个核细胞（PBMC）以及中性粒细胞，与白念珠菌进行体外共培养，采用Western印迹检测患者PBMC中胱天蛋白募集域蛋白9（CARD9）表达水平，酶联免疫吸附试验检测肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-6、IL-17A、IL-1β和粒细胞-巨噬细胞集落刺激因子（GM-CSF）的分泌水平，菌落计数法检测中性粒细胞处理后白念珠菌存活率。两组间均数比较采用t检验。结果 患者CARD9基因存在2号外显子c.68C>A（p.S23X）和6号外显子c.820dupG（p.D274Gfs*61）复合杂合突变，两突变分别来自其父母。Western印迹显示，健康对照组PBMC中CARD9蛋白相对表达水平为0.41 ± 0.07，而患者表达缺如。热灭活白念珠菌孢子刺激后，患者PBMC的TNF-α、IL-6、IL-17A、IL-1β和GM-CSF分泌水平均低于健康对照组（P < 0.001）。体外培养30、120 min时，与患者中性粒细胞共培养的白念珠菌存活率（78.00%、74.00%）均高于健康对照（70.91% ± 1.75%、34.55% ± 5.35%），差异有统计学意义（t值分别为3.743、6.988，P <0.05）。结论 1例复发性念珠菌颈部淋巴结炎患者CARD9基因存在复合杂合突变，导致CARD9蛋白表达缺如，该患者存在抗白念珠菌免疫功能缺陷。

Screening of susceptibility genes and an immunological study in a patient with recurrent cervical lymphadenitis caused by Candida albicans

【Abstract】 Objective To explore genetic etiology and antifungal immunity in a patient with recurrent cervical lymphadenitis caused by Candida albicans. Methods Next-generation sequencing was performed to screen susceptibility genes for mycosis in a patient with recurrent cervical lymphadenitis caused by Candida albicans and his parents. Peripheral blood mononuclear cells （PBMCs） and neutrophils were extracted from the patient and 6 healthy controls, and subjected to in vitro co-culture with Candida albicans. Western blot analysis was performed to determine the expression of caspase recruitment domain-containing protein 9 （CARD9） in PBMCs of the patient, enzyme-linked immunosorbent assay to detect levels of tumor necrosis factor-α （TNF-α）, interleukin （IL）-6, IL-17A, IL-1β and granulocyte-macrophage colony-stimulating factor （GM-CSF） in the co-culture medium, and a colony-counting method was used to detect the survival rate of Candida albicans after treatment with neutrophils. Statistical analysis was carried out by using t test for comparisons between two groups. Results Two compound heterozygous mutations were identified in the CARD9 gene of the patient, including c.68C>A （p.S23X） in exon 2 inherited from his father and c.820dupG （p.D274Gfs*61） in exon 6 inherited from his mother. Western blot analysis showed that the relative expression level of CARD9 protein in the PBMCs was 0.41 ± 0.07 in the healthy control group, but CARD9 expression was absent in the patient. After stimulation with heat-inactivated Candida albicans spores, the levels of TNF-α, IL-6, IL-17A, IL-1β and GM-CSF secreted by PBMCs of the patient were significantly lower than those by PBMCs of the healthy controls （all P < 0.001）. After 30- and 120-minute in vitro co-culture with neutrophils, the survival rates of Candida albicans were significantly higher in the patient （78.00%, 74.00%, respectively） than in the healthy controls （70.91% ± 1.75%, 34.55% ± 5.35%, t = 3.743, 6.988, respectively, both P < 0.05）. Conclusion Compound heterozygous mutations were identified in the CARD9 gene of the patient with recurrent cervical lymphadenitis caused by Candida albicans, leading to the absence of CARD9 protein expression, and the deficiency in the immunity against Candida albicans.