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

目的 探讨citrin缺陷导致的新生儿肝内胆汁淤积症(neonatalintrahepatic cholestasis caused by citrin deficiency,NICCD)患儿的临床表现、实验室检查特点、SLC25A13基因突变情况及预后.方法 对26例NICCD患儿进行常规实验室检查、血氨基酸谱和酰基肉碱谱、尿有机酸和SLC25A13基因分析,并随访2年.结果 NICCD患儿出生体重偏低,平均黄疸出现年龄29 d.实验室检查改变包括肝功能异常、高胆红素血症、低蛋白血症、甲胎蛋白升高、凝血酶原时间延长及低血糖、高氨血症.串联质谱分析发现多数患儿有瓜氨酸等氨基酸特异性升高.尿气相色谱质谱有机酸分析有尿4-羟基苯乳酸和4-羟基苯丙酮酸升高.SLC25A13基因分析共发现12种致病突变,其中G386V,R467X,K453R,1192-1193delT为新突变.26例患儿的突变总检出率84.6%,851del4、1638ins23及IVS6+5G＞A为热点突变,突变率分别占总突变的40.9%、20.5%和11.4%.26例NICCD患儿中5例(19.2%)预后不良,4例死亡,1例接受肝移植.NICCD患儿的基因型与临床表型相关性不明显.结论 851del4、1638ins23及IVS6+5G＞A突变为中国人SLC25A13基因的热点突变,部分NICCD患儿可能预后不良.     

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.     

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。     

应用高分辨率熔解曲线分析快速筛查和诊断citrin缺陷导致的新生儿肝内胆汁淤积症

目的 探讨高分辨率熔解曲线(high-resolution melting,HRM)分析技术用于citrin缺陷导致的新生儿肝内胆汁淤积症(neonatal intrahepatic cholestasis caused by citrin deficiency,NICCD)筛查和诊断的可行性.方法 根据中国人群SLC25A13基因的热点突变类型(851del4、1638ins23、IVS6+5G＞A和IVS16ins3kb)设计特异性HRM扩增引物,挑选经测序证实的50名正常对照和20例NICCD患儿,建立和完善HRM检测条件.用优化后的HRM检测方法对171例临床疑似的NICCD患者进行HRM筛查.若受检样本的熔解曲线与阳性质控样品相吻合,则进行DNA测序分析.结果 优化后的HRM方法能准确地对50名正常对照及20例NICCD患儿进行基因分型,其灵敏度和特异性均为100％ (70/70).重复实验表明,相同基因型的不同样品HRM熔解曲线完全吻合,重复性好.在171例疑似患儿中,有7例患儿熔解曲线与阳性质控样品基因型相符,其HRM基因分型为:1例851del4纯合突变,1例IVS6+5G＞A杂合突变,3例851del4杂合突变,1例[IVS6+5G＞ A]+[851del4],1例[1638ins23+IVS16ins3kb]+[1638ins23].DNA测序证实了HRM基因分型,准确率为100％.结论 HRM技术具有高通量、操作简便、结果准确、重复性好等优点,可对临床疑似的NICCD患儿进行基因筛查和诊断.     

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。     

新生儿肝内胆汁淤积症的质谱分析和SLC25A13基因检测

目的 对新生儿肝内胆汁淤积症(NICCD)患儿进行血液生化、血串联质谱、尿气相色谱质谱技术分析,同时进行SLC25A13基因突变分析,了解有无citrin缺陷,为NICCD的鉴别诊断提供思路.方法 对我院就诊的127例特发性胆汁淤积症患儿进行肝功能、总胆汁酸、甲胎蛋白、凝血功能等生化指标检测,结合血串联质谱、尿气相质谱...     

婴儿肝内胆汁淤积症SLC25A13基因突变分析

目的探讨SLC25A13基因突变在中国婴儿肝内胆汁淤积症患儿中的检出率,初步了解突变患儿血生化及氨基酸谱特征,肝脏活组织病理变化。方法2003年12月至2006年12月就诊于复旦大学附属儿科医院的婴儿肝内胆汁淤积症患儿,满足本研究入选条件者共115例进行了血氨基酸质谱分析,伴血浆瓜氨酸明显升高的患儿进行SLC25A13基因全部外显子及邻近序列测序,不伴血浆瓜氨酸明显升高的患儿进行SLC25A13基因常见突变851del4（突变Ⅰ）及突变1638ins23（突变Ⅲ）筛查,突变851del4采用实时荧光定量PCR双标记探针法检测,突变1638ins23采用PCR产物直接电泳法检测,对仅检出单个位点突变的筛查对象,继续进行其余已报道的10种突变位点检测。检测结果仍为单个杂合突变的对象进行SLC25A13基因所有外显子区及其邻近序列分析。对确诊突变患儿的临床表现、血生化及血氨基酸特征等进行分析。结果5例伴血瓜氨酸明显升高的患儿共检出突变4例,其中纯合突变851del4/851del41例,复合杂合突变851del4/1638ins231例,杂合突变851del42例;110例不伴血浆瓜氨酸明显升高患儿共检出突变6例,其中纯合突变851del4/851del41例,复合杂合突变851del4/1638ins231例,杂合突变851del44例。115例婴儿肝内胆汁淤积症患儿共检出SLC25A13基因突变10例,占8.7%。突变患儿血生化改变包括胆红素、γ-谷氨酰转移酶以及碱性磷酸酶等明显升高,AST升高较ALT明显。血串联质谱发现5例突变患儿有特征性氨基酸瓜氨酸、苏氨酸及蛋氨酸升高,另5例突变患儿并无血氨基酸改变。10例患儿中有7例行肝脏活组织病理学检查,4例有显著的脂肪变性。结论SLC25A13基因突变是中国婴儿肝内胆汁淤积症的重要原因之一。肝脏活组织病理、血生化及氨基酸谱等检查对诊断SLC25A13基因突变患儿有重要意义,但最终仍需通过基因检测确诊。     

16例Citrin蛋白缺陷所致的婴儿肝内胆汁淤积症的 SLC25A13基因变异分析

目的:探讨16例Citrin蛋白缺陷所致的婴儿肝内胆汁淤积症(neonatal intrahepatic cholestasis caused by citrin deficiency,NICCD) SLC25A13基因的变异特点。 方法:应用高通量测序法对目标基因的编码外显子和侧翼区域进行捕获...     

16例Citrin蛋白缺陷所致的婴儿肝内胆汁淤积症的SLC25A13基因变异分析CSCD

目的探讨16例Citrin蛋白缺陷所致的婴儿肝内胆汁淤积症(neonatal intrahepatic cholestasis caused by citrin deficiency,NICCD)SLC25A13基因的变异特点。方法应用高通量测序法对目标基因的编码外显子和侧翼区域进行捕获,对变异位点进行Sanger测序验证和致病性分析。结果在16例NICCD患儿中,共发现致病变异15种,其中6种既往未见报道,包括IVS14-9A>G、c.1640G>A、c.762 T>A、c.736delG、c.1098delT、c.851G>A。结论通过高通量测序发现6种新变异,丰富了SLC25A13基因的变异谱,为患儿家系的遗传咨询和产前诊断提供了依据。     

Neonatal intrahepatic cholestasis caused by citrin deficiency in Korean infants

Citrin is a liver-type mitochondrial aspartate-glutamate carrier encoded by the SLC25A13 gene, and its deficiency causes adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Here, the authors investigated clinical findings in Korean infants with NICCD and performed mutation analysis on the SLC25A13 gene. Of 47 patients with neonatal cholestasis, three infants had multiple aminoacidemia (involving citrulline, methionine, and arginine) and galactosemia, and thus were diagnosed as having NICCD. Two of these three showed failure to thrive. The laboratory findings showed hypoproteinemia and hyperammonemia, and liver biopsies revealed micro-macrovesicular fatty liver and cholestasis. The three patients each harbored compound heterozygous 1,638-1,660 dup/ S225X mutation, compound heterozygous 851del4/S225X mutation, and heterozygous 1,638-1,660 dup mutation, respectively. With nutritional manipulation, liver functions were normalized and catch-up growth was achieved. NICCD should be considered in the differential diagnosis of cholestatic jaundice in Korean infants.     

Mitochondrial aspartate glutamate carrier (citrin) deficiency as the cause of adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD)

 By using homozygosity mapping and positional cloning, we have shown that adult-onset type II citrullinemia (CTLN2) is caused by mutations of the SLC25A13 gene, which is localized on chromosome 7q21.3 and encodes a mitochondrial solute carrier protein named citrin. So far, we have reported nine mutations, most of which cause loss of citrin, and we have established several methods for DNA diagnosis. These methods have shown that more than 90% of the patients diagnosed as suffering from CTLN2 by enzymatic analysis carry SLC25A13 mutations in both alleles, indicating that CTLN2 is caused by citrin deficiency. Furthermore, by using the same DNA diagnosis methods, we discovered that 70 neonates or infants suffering from a particular type of neonatal hepatitis carry the same SLC25A13 mutations. Since the symptoms of the neonates are different from those of the more severe CTLN2 and usually ameliorate without special treatment, we designated the neonatal disease neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). We conclude that citrin deficiency causes NICCD in neonates and CTLN2 in adults through the additional effects of genetic or environmental modifiers. Since the function of citrin, together with that of an isoform, aralar, was found to be as a mitochondrial aspartate glutamate carrier, the various symptoms of NICCD and CTLN2 may be understood as caused by defective aspartate export from the mitochondria to the cytosol and defects in the malate aspartate shuttle. It is, however, still difficult to understand the cause of the hepatic deficiency of argininosuccinate synthetase protein in CTLN2. Received: March 20, 2002 / Accepted: March 28, 2002