Citrin缺陷导致的新生儿肝内胆汁淤积症患儿SLC25A13基因突变与生化指标的相关性研究*

 目的：分析citrin缺陷导致的新生儿肝内胆汁淤积症(NICCD)患儿SLC25A13基因突变及生化改变特点,并探讨两者相关性。方法：2013年3月至2013年10月在暨南大学附属第一医院以胆汁淤积性肝病就诊的婴儿59例,其中经SLC25A13基因分析确诊的NICCD患儿36例为病例组,排除NICCD且未发现明确病因的23例特发性新生儿胆汁淤积症(INC)患儿为对照组。抽取静脉血提取DNA进行SLC25A13突变检测,并分析所有研究对象的血糖、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、γ-谷氨酰转移酶（GGT）、碱性磷酸酶（ALP）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）等数据资料。结果：NICCD组ALT及LDL-C水平低于对照组。检出SLC25A13基因突变10种,其中851del4、IVS16ins3kb、IVS6+5G>A和1638ins23 突变占全部突变数量的82%。不同性别及年龄段的NICCD患儿其SLC25A13基因突变分布未见不同。SLC25A13基因突变与患儿的血糖、ALT、AST、ALP、TG及HDL-C水平无关联,而与GTT的水平有关联。结论：低LDL-C血症可能是NICCD患儿血脂紊乱的特点。NICCD患儿SLC25A13基因的高频突变类型为851del4、IVS16ins3kb、IVS6+5G>A和1638ins23。本文NICCD患儿的SLC25A13突变分布与GGT水平之间存在相关性,但这一发现的意义有待深入研究。     

五例新生儿肝内胆汁淤积症患儿基因的突变分析CSCD

目的明确5例新生儿肝内胆汁淤积症患儿的分子机制。方法应用新一代测序技术对患儿的SLC25A13基因进行外显子捕获检测,对突变位点进行Sanger测序验证。用PolyPhen-2软件对新突变的致病性进行分析。结果 5例患儿均携带SLC25A13基因的复合突变,共发现8个突变位点,其中2个既往未见报道(c.1357A>G和c.1663dup23)。5例患儿的父母均为突变携带者。结论 SLC25A13基因的突变可能是5例患儿的发病原因,所携带的突变以851del4和1638-1660dup为主。新发现的c.1357A>G和c.1663dup23突变丰富了SLC25A13基因的突变谱。     

Citrin缺陷导致的新生儿肝内胆汁淤积症临床和SLC25A13基因突变的研究

Objectives To investigate the clinical and laboratory features of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and to characterize the molecular basis and prognosis of this disease. Methods Twenty-six patients with NICCD were collected because of idiopathic intrahepatic cholestasis and jaundice. The diagnosis was made by routine laboratory data collection, tandem mass spectrometry (MS-MS) and gas chromatography mass spectrometry (GC-MS) analyses. SLC25A13 gene mutation was analyzed by using polymerase chain reaction (PCR), direct DNA sequencing and restriction fragment length polymorphism analyses. The patients were followed up for nearly 2 years. Results The NICCD patients showed low birth weight and the average onset of jaundice was 29 days. Laboratory data showed liver dysfunction, hyperbilirubinemia, hypoproteinemia, high levels of α-fetoprotein, prolonged prothrombin time, hypoglycemia and hyperammonemia. MS-MS analysis of the blood samples revealed specific elevation of citrulline, methionine, threonine, tyrosine and elevation of free carnitine, short-chain and long-chain acylcarnitines. GC-MS analysis of the urine samples showed elevated 4-hydroxyl phenyllactic acid and 4-hydroxyi phenylpyruvic acid. Twelve different mutations were identified, including 4 novel mutations, i. e. , G386V, R467X, K453R and 1192-1193delT. Forty-four mutated alleles were identified in the 52 alleles (84.6%). Among them, 851del4, 1638ins23 and IVS6+5G＞A mutations were the most frequent mutations, accounting for 40.9%, 20.5% and 11.4% of the total alleles examined respectively.Five of the 26 patients have not been recovered, including 4died and 1 accepted liver transplantation. No obvious relationship was found between the genotype and phenotype in NICCD. Conclusion The 851del4,1638ins23 and IVS6+5G＞A mutations are the hot-spot mutations in Chinese NICCD patients. Some NICCD patients have poor prognosis.     

Citrin缺陷导致的新生儿肝内胆汁淤积症临床和SLC25A13基因突变的研究

Objectives To investigate the clinical and laboratory features of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and to characterize the molecular basis and prognosis of this disease. Methods Twenty-six patients with NICCD were collected because of idiopathic intrahepatic cholestasis and jaundice. The diagnosis was made by routine laboratory data collection, tandem mass spectrometry (MS-MS) and gas chromatography mass spectrometry (GC-MS) analyses. SLC25A13 gene mutation was analyzed by using polymerase chain reaction (PCR), direct DNA sequencing and restriction fragment length polymorphism analyses. The patients were followed up for nearly 2 years. Results The NICCD patients showed low birth weight and the average onset of jaundice was 29 days. Laboratory data showed liver dysfunction, hyperbilirubinemia, hypoproteinemia, high levels of α-fetoprotein, prolonged prothrombin time, hypoglycemia and hyperammonemia. MS-MS analysis of the blood samples revealed specific elevation of citrulline, methionine, threonine, tyrosine and elevation of free carnitine, short-chain and long-chain acylcarnitines. GC-MS analysis of the urine samples showed elevated 4-hydroxyl phenyllactic acid and 4-hydroxyi phenylpyruvic acid. Twelve different mutations were identified, including 4 novel mutations, i. e. , G386V, R467X, K453R and 1192-1193delT. Forty-four mutated alleles were identified in the 52 alleles (84.6%). Among them, 851del4, 1638ins23 and IVS6+5G＞A mutations were the most frequent mutations, accounting for 40.9%, 20.5% and 11.4% of the total alleles examined respectively.Five of the 26 patients have not been recovered, including 4died and 1 accepted liver transplantation. No obvious relationship was found between the genotype and phenotype in NICCD. Conclusion The 851del4,1638ins23 and IVS6+5G＞A mutations are the hot-spot mutations in Chinese NICCD patients. Some NICCD patients have poor prognosis.     

Identification of 13 novel mutations including a retrotransposal insertion in SLC25A13 gene and frequency of 30 mutations found in patients with citrin deficiency

Deficiency of citrin, liver-type mitochondrial aspartate-glutamate carrier, is an autosomal recessive disorder caused by mutations of the SLC25A13 gene on chromosome 7q21.3 and has two phenotypes: neonatal intrahepatic cholestatic hepatitis (NICCD) and adult-onset type II citrullinemia (CTLN2). So far, we have described 19 SLC25A13 mutations. Here, we report 13 novel SLC25A13 mutations (one insertion, two deletion, three splice site, two nonsense, and five missense) in patients with citrin deficiency from Japan, Israel, UK, and Czech Republic. Only R360X was detected in both Japanese and Caucasian. IVS16ins3kb identified in a Japanese CTLN2 family seems to be a retrotransposal insertion, as the inserted sequence (2,667-nt) showed an antisense strand of processed complementary DNA (cDNA) from a gene on chromosome 6 (C6orf68), and the repetitive sequence (17-nt) derived from SLC25A13 was found at both ends of the insert. All together, 30 different mutations found in 334 Japanese, 47 Chinese, 11 Korean, four Vietnamese and seven non-East Asian families have been summarized. In Japan, IVS16ins3kb was relatively frequent in 22 families, in addition to known mutations IVS11 + 1G > A, 851del4, IVS13 + 1G > A, and S225X in 189, 173, 48 and 30 families, respectively; 851del4 and IVS16ins3kb were found in all East Asian patients tested, suggesting that these mutations may have occurred very early in some area of East Asia.     

Frequency and distribution in East Asia of 12 mutations identified in the SLC25A13 gene of Japanese patients with citrin deficiency

Deficiency of citrin, a liver-type mitochondrial aspartate-glutamate carrier (AGC), encoded by the SLC25A13 gene on chromosome 7q21.3, causes autosomal recessive disorders: adult-onset type II citrullinemia (CTLN2) and neonatal hepatitis associated with intrahepatic cholestasis (NICCD). So far, we have described 12 SLC25A13 mutations: 11 were from Japan and one from Israel. Three mutations found in Chinese and Vietnamese patients were the same as those in Japanese patients. In the present study, we identified a novel mutation IVS6+1G>C in a Japanese CTLN2 patient and widely screened 12 SLC25A13 mutations found in Japanese patients in control individuals from East Asia to confirm our preliminary results that the carrier frequency was high in Asian populations. Mutations 851–854del and 1638–1660dup were found in all Asian countries tested, and 851–854del associated with 290-haplotype in microsatellite marker D7S1812 was especially frequent. Other mutations frequently detected were IVS11+1G>A in Japanese and Korean, S225X in Japanese, and IVS6+5G>A in Chinese populations. We found a remarkable difference in carrier rates in China (including Taiwan) between north (1/940) and south (1/48) of the Yangtze River. We detected many carriers in Chinese (64/4169 = 1/65), Japanese (20/1372 = 1/69) and Korean (22/2455 = 1/112) populations, suggesting that over 80,000 East Asians are homozygotes with two mutated SLC25A13 alleles.     

Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency

The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency.