论文部分内容阅读
目的:对1型糖尿病（T1DM）患者含NLR家族CARD域蛋白4（NLRC4）基因外显子及外显子内含子交界区的罕见变异进行鉴定，并探讨其对基因功能的影响。方法:选取2017年8月到2020年9月中南大学湘雅二医院代谢内分泌科508例T1DM患者作为病例组，男264例，女244例，年龄［n M（n Q1，n Q3）］为27（11，43）岁。同时选取同期体检科527名健康对照者，男290名，女237名，年龄［n M（n Q1，n Q3）］为47（36，60）岁。对T1DM患者和健康对照者的NLRC4基因的外显子区域进行捕获测序，并进行一代测序验证。构建NLRC4基因野生型和突变型质粒，转染293T细胞，采用免疫印迹试验（WB）检测NLRC4蛋白表达量以及含半胱氨酸的天冬氨酸蛋白水解酶前体（procaspase-1）切割产物含量。在转染野生型或突变型NLRC4质粒的293T细胞中加入放线菌酮（CHX），检测NLRC4蛋白降解情况。使用免疫荧光检测NLRC4蛋白质的定位。酶联免疫吸附试验（ELISA）检测细胞上清白细胞介素（IL）-1β浓度。n 结果:测序结果显示，4例患者及2名健康对照者NLRC4基因的3号外显子存在杂合变异c.208C>T；2例患者4号外显子存在杂合变异c.1564T>C，1例患者4号外显子存在c.1219G>C；这3种变异可能为T1DM的致病性变异。NLRC4野生型和c.208>T、c.1564T>C、c.1219G>C突变型质粒转染的293T细胞中，NLRC4蛋白表达水平、降解速率、定位以及procaspase-1的切割产物含量无明显改变；但转染c.1219G>C、c.208C>T突变型质粒的293T细胞分泌的IL-1β浓度［n M（n Q1，n Q3）］分别为15.25（12.98，17.52）、15.44（13.81，17.07）ng/L，均低于野生型质粒的18.70（16.59，20.81）ng/L（n P=0.020、0.010）。n 结论:NLRC4基因外显子罕见变异c.208C>T、c.1564T>C和c.1219G>C可能不改变蛋白质的表达水平、蛋白质的降解及定位，但c.208C>T和c.1219G>C错义突变可能抑制了炎症因子IL-1β的产生，从而对基因功能产生影响。“,”Objective:To identify rare variants in exon and exon-intron boundary of containing NLR family CARD domain protein 4 (NLRC4) in type 1 diabetes (T1DM) patients, and to explore their effects on gene function.Methods:A total of 508 T1DM patients and 527 healthy controls in the Department of Metabolic Endocrinology, Second Xiangya Hospital of Central South University from August 2017 to September 2020 were selected. The case group included 264 males and 244 females, and the age [n M (n Q1, n Q3)] was [27 (11, 43)] years. The control group included 290 males and 237 females, and their age［n M（n Q1，n Q3）］was [47 (36, 60)] years old. Identification of rare variants in exons of NLRC4 gene in T1DM patients and healthy controls was performed and verified by next-generation sequencing and sanger sequencing. The NLRC4 gene wild-type and mutant plasmids were constructed and transfected into 293T cells. Western blot (WB) was used to detect the expression of NLRC4 protein and cleavage products of pro-cysteinyl aspartate specific proteinase(procaspase-1). Cycloheximide (CHX) was added to 293T cells transfected with wild-type or mutant NLRC4 plasmid to detect the degradation of NLRC4 protein. The localization of NLRC4 protein was detected by immunofluorescence, and the concentration of IL-1β in the cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA).n Results:The sequencing results showed that 4 patients and 2 healthy controls had a heterozygous variant c.208C>T in exon 3 of the NLRC4 gene. Two patient had a heterozygous variant c.1564T>C in exon 4, and 1 patients had c.1219G>C in exon 4. These three variants might be pathogenic variants in T1DM. In 293T cells transfected with NLRC4 wild-type and c.208C>T、c.1564T>C和c.1219G>C mutant plasmids, the expression level, degradation rate, localization of NLRC4 protein and the content of cleavage products of procaspase-1 did not change significantly. However, the concentration of IL-1β secreted by 293T cells transfected with c.1219G>C and c.208C>T plasmid [n M(n Q1, n Q3)] was 15.25 (12.98, 17.52) and 15.44 (13.81, 17.07) ng/L, respectively, which was lower than 18.70 (16.59, 20.81) ng/L of 293T cells transfected wild-type plasmid (n P=0.020, 0.010).n Conclusions:NLRC4 gene rare variants c.208C>T, c.1564T>C and c.1219G>C may not change the protein expression, degradation and localization, but c.208C>T and c.1219G>C may inhibit the secretion of IL-1β. This result suggests that NLRC4 rare variants may have an impact on gene function.