基于人PBLs亚群SLC25A13编码的AGC亚型结构、功能和定位分析的NICCD患儿分子诊断研究

Mutations of human SLC25A13 gene impair the function of Aspartate-Glutamate Carrier isoform 2 (AGC2), leading to the formation of Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD), a genetic disease that remains a serious challenge against the infant health in China. Our recent research findings suggest that, besides AGC2 expressed in human peripheral blood lymphocytes (PBLs), SLC25A13 gene in specific lymphocyte subpopulations might encode AGC3, a novel isoform that harbors the AGC subcellular localization ability and carrier function, and can be used as a novel molecular marker for NICCD diagnosis. This hypothesis, however, needs to be verified by detailed experimental tests. This project was designed to explore the distribution feature of SLC25A13-encoded AGC isoforms, including AGC2 and AGC3, in diverse subpopulations of human PBLs by using such techniques as lymphocyte sorting, cDNA cloning and Western blot; and to compare the structures of the AGC isoforms and to investigate their function and subcellular localization by means of cDNA recombination, immunoblot and confocal microscopy as well; moreover, according to the findings of the above studies, to further identify the insidious SLC25A13 mutations with familial SNP analysis and other methods in NICCD patients, and to explore the pathogenecity of novel SLC25A13 mutations by utilizing the tools such as functional analysis of the corresponding recombinant variants. The project will reveal the expression characteristics of the AGC isoforms in specific subpopulations of human PBLs, provide new sample source and molecular markers for the analysis of SLC25A13 products, and supply new laboratory evidences for the definite diagnosis of NICCD infants, therefore having great theoretical value and scientific significance.

人SLC25A13基因突变影响肝细胞线粒体内膜AGC2功能而形成严重危害我国婴儿健康的遗传病NICCD。我们近期研究结果提示：人PBLs除了表达AGC2，其特定亚群中SLC25A13可能还编码一种具备AGC亚细胞定位能力和载体功能、可用于NICCD诊断的新亚型AGC3，但这一假说需要实验验证。本项目用淋巴细胞分选、cDNA克隆和蛋白印迹等技术研究SLC25A13编码的AGC亚型在人PBLs不同亚群的分布特点；以cDNA重组、免疫印迹和共聚焦显微镜等方法比较AGC亚型结构并探讨其功能和亚细胞定位；在以上研究基础上，借助家系SNP分析等方法识别NICCD患儿SLC25A13隐匿突变并通过重组变异体功能分析等技术研究新突变致病性。本项目将揭示人PBLs特定亚群AGC亚型的表达特征，为SLC25A13产物分析提供新标本来源和分子标志，为NICCD患儿确诊提供新实验依据，具有重要理论价值和科学意义。

人SLC25A13基因突变影响肝细胞线粒体内膜AGC2功能而形成严重危害我国婴儿健康的遗传病NICCD。NSFC面上项目(No.81570793)主要研究SLC25A13编码AGC亚型在人不同PMCs类型中的分布特点，探讨AGC2和AGC3亚型的结构、功能和亚细胞定位特征，进一步识别NICCD患儿SLC25A13新突变并分析其致病性。通过四年研究，本项目取得一系列重要发现：通过Western blotting和线粒体蛋白分离等技术，在人PBLs中检测到清晰的AGC2和AGC3亚型信号，并证实AGC3亚型不表达于线粒体；通过细胞培养和SLC25A13基因cDNA克隆分析，发现单核细胞来源巨噬细胞中SLC25A13基因mRNA结构独特，更适合NICCD分子诊断；通过SLC25A13高频突变筛查和Sanger测序等技术，识别c.1841+3_c.1841+4del,c.177_189del13,c.188delG,c.1620delT,c.1722delA,c.1210G>T,c.889G>T,c.1193T>A,c.742G>A,c.762T>A,c.1352T>A,IVS2+5G>A,IVS14-1G>A,c.1853_1855dupGAT和c.1603_1609dup等SLC25A13新突变15 种,并通过同源基因agc1敲除的酵母模型、RT-PCR和免疫印迹等技术，证实了其致病性；新增NICCD患者196人，构建了一个总数达458人的大规模的NICCD患者研究队列。本项目揭示了人PMCs特定亚群AGC亚型的表达特征，为SLC25A13产物分析提供了新标本来源和分子标志，为NICCD患儿确诊提供了新的实验室依据。

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