Molecular genetics of citrin deficiency and search for the factor(s) and mechanism of adult-onset type II citrullinemia onset

Citrin 缺乏症是一种新发现的疾病实体，包括胆汁淤积性新生儿肝炎 (NICCD) 和成人 II 型瓜氨酸血症 (CTLN2)，是由编码 Citrin 的 SLC25A13 基因突变引起的。 Citrin 缺乏会在生命的最初几个月内导致 NICCD，在大多数情况下，症状通常会在第一年自行消失。经过一段持续一到几十年的表面健康期后，一些 citrin 缺乏症患者会继续出现严重的 CTLN2。从citrin作为位于线粒体内膜的肝型天冬氨酸-谷氨酸载体的功能来看，NICCD和CTLN2的一些不同症状可以通过天冬氨酸从线粒体输出到细胞质和苹果酸-天冬氨酸NADH穿梭的缺陷来理解。然而，阐明CTLN2发病机制仍然很困难。在本研究中，我们鉴定了近20个新的SLC25A13突变并建立了它们的DNA诊断。此外，我们还开发了一种新的诊断方法，通过使用蛋白质印迹分析来检测淋巴细胞中的柠檬酸蛋白。我们不仅在日本，而且在其他国家也发现了柑橘缺乏症患者。到目前为止，我们已诊断出NICCD（203名日本人、40名中国人、6名韩国人、4名越南人、3名白人、3名巴勒斯坦人、2名犹太人、2名以色列人、1名巴基斯坦人）和CTLN2（157名日本人、3名中国人和2名韩国人）患者。 DNA诊断显示，日本人群中携带者频率为1.65（纯合子频率：1/17,000）。我们对日本患者中发现的 12 种已知突变进行群体分析，在东亚发现了许多携带者：韩国的比率为 1/112，中国的比率为 1/65（广东和湖南：1/40，香港：1/48，台湾：1/57），表明超过 100,000 东亚人是柑橘缺乏症的纯合子。另一方面，我们生成了 citrin-KO（基因敲除）小鼠，但这些小鼠未能表现出人类 citrin 缺乏的特征。基于与人类相比，小鼠肝脏中磷酸甘油穿梭活性增强可以补偿柠檬酸功能丧失的假设，我们培育了柠檬酸和线粒体甘油 3-磷酸脱氢酶 (mGPDH) 基因联合破坏的小鼠。由此产生的双敲除小鼠表现出瓜氨酸血症、高氨血症（口服蔗糖后进一步升高）、低血糖和脂肪肝；人类柠檬酸缺乏症的所有特征（提交给 J Biol Chem）。较少的

Citrin deficiency is a newly-established disease entity that encompasses both cholestatic neonatal hepatitis (NICCD) and adult-onse: type II citrullinemia (CTLN2), and results from mutations in the SLC25A13 gene that encodes citrin. Citrin deficiency results in NICCD during the first few months of life, with symptoms usually self-resolving by the first year in most cases. Following an apparently healthy period that can last from one to several decades, some patients with citrin deficiency go on to develop severe CTLN2. From the function of citrin as a liver-type aspartate-glutamate carrier localized in the mitochondrial inner membrane, some of the various symptoms of NICCD and CTLN2 may be understood through the defects of aspartate export from mitochondria to cytosol and of malate-aspartate NADH shuttle. It is, however, still difficult to clarify the mechanism of CTLN2-onset.In the present study, we identified near 20 novel SLC25A13 mutations and established their DNA diagnoses. Furth … More ermore, we also developed a novel diagnostic method to detect citrin protein in lymphocytes by using Western blot analysis. We have found the patients with citrin deficiency not only in Japan but also in other country. So far, we have diagnosed NICCD (203 Japanese, 40 Chinese, 6 Korean, 4 Vietnamese, 3 Caucasian, 3 Palestinian, 2 Jewish, 2 Israeli, 1 Pakistani) and CTLN2 (157 Japanese, 3 Chinese and 2 Korean) patients. DNA diagnosis revealed that the carrier frequency is 1,65 (homozygote frequency: 1/17,000) in the Japanese population. Our population analysis of 12 known mutations identified in Japanese patients detected many carriers in East Asia: the rates are 1/112 in Korea and 1/65 in China (Guangdong and Hunan: 1/40, Hong Kong: 1/48, Taiwan: 1/57), suggesting that over 100,000 East Asians are homozygotes of citrin deficiency. On the other hand, we generated citrin-KO (knockout) mice, but the mice failed to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity in mouse liver compared to that in human can compensate for the loss of citrin function, we generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH). The resulting double-KO mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and fatty liver; all features of human citrin deficiency (submitted to J Biol Chem). Less