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。     

直接胆红素增高的黄疸患儿SLC25A13基因热点突变分析

目的建立快速诊断citrin缺陷导致的新生儿肝内胆汁淤积症（NICCD）的方法,初步探讨NICCD在直接胆红素增高的黄疸患儿中所占比例。方法应用聚合酶链反应（PCR）和聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP）对122例表现为直接胆红素增高的黄疸患儿SLC25A13基因（热点突变分析位点包括：851del4、1638ins23、IVS6＋5G〉A、IVS16ins3kb和IVS11＋1G〉A）进行热点突变分析。结果在122例纳入实验研究的患者中,发现6例患儿为851del4纯合子;2例为1638ins23/IVS6＋5G〉A突变;2例为851del4/IVS16ins3kb突变。还有8例患儿只发现了一个SLC25A13基因突变,其相应等位突变尚不清楚,但是后续的实验证实了这8例患儿为NICCD。暂时未发现有IVS11＋1G〉A突变。在这些热点突变类型中以851del4突变所占比例最高,占了71.4%（20/28）,其次为IVS16ins3kb和1638ins23,各占10.7%。NICCD患儿占总受检人数的14.7%。结论 1.SLC25A13基因热点突变分析能快速准确地诊断NICCD。2.SLC25A13基因突变以851del最为常见。3.NICCD在直接胆红素增高的黄疸患儿中占相当高的比例。     

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。     

双胞胎同患Citrin缺乏症一家系临床及基因分析

目的对一对临床拟诊希特林蛋白(Citrin)缺乏症的双胞胎患儿进行临床资料和SLC25A13基因基因突变分析,为临床治疗及产前诊断提供理论依据。方法收集患儿临床资料,结合血串联质谱、尿气相色谱质谱技术分析,同时对患儿及其父母进行SLC25A13基因测序。结果双胞胎患儿血瓜氨酸明显增高,尿4-羟基苯乳酸及4-羟基苯丙酮酸均显著增高,Sanger测序对SLC25A13基因分析,双胞胎患儿均检出已知致病突变c.851-854del GTAT(纯合),突变来自父母双方。结论 Citrin缺陷导致的临床表现和生化改变多种多样且缺乏特异性,至今没有公认的临床或生化诊断标准。需要综合分析临床、生化、代谢组学等多种结果,而SLC25A13基因分析是本病确诊的可靠依据。     

20个希特林缺陷病家系的基因分析及产前诊断CSCD

目的探讨20个希特林缺陷病家系SLC25A13基因的突变特点以及产前诊断的可行性。方法通过高频突变筛查结合直接测序的技术对20例先证者及其父母进行SLC25A13基因突变分析。在确定每个家系基因型后,为先证者母亲再次妊娠的胎儿提供遗传咨询并进行产前诊断。结果 20个希特林缺陷病先证者均检出SLC25A13双等位基因致病性突变,共发现10种致病突变类型,包括3种缺失突变:c.851del4、c.1092;095delT和c.495delA;2种剪接位点突变:IVS6+5G>A和IVS11+1G>A;2种无义突变:c.775C>T （p.Q259X）和c.72T>A（p.Y24X）;1种重复突变:c.1638;660dup;1种插入突变:IVSl6ins3kb;1种错义突变:c.1775A>C（p.Q592P）。20个家系共行24次产前诊断。其中8例胎儿基因型正常,11例为SLC25A13基因突变携带者,5例为SLC25A13双等位基因突变。2例c.851del4/c.851del4纯合突变胎儿的父母选择继续妊娠,其余3例双等位基因突变胎儿的父母选择终止妊娠。结论对希特林缺陷病家系进行SLC25A13基因突变分析,可以为先证者确诊、受影响家庭的遗传咨询和下一胎产前诊断提供实验依据,有效降低缺陷患儿再发风险。     

一个新生儿肝内胆汁淤积症(NICCD)家系的SLC25A13基因突变检测与产前诊断

目的对一个新生儿肝内胆汁淤积症(neonatal intrahepatic cholestasis caused by citrin deficiency,NICCD)家系的SLC25A13基因突变检测和产前诊断。方法收集患者及父母的外周血标本,提取基因组DNA,在明确先证者病因和基因型的基础上,采用Sanger法对家系中1例已孕15周的胎儿进行SLC25A13基因的相应突变位点进行检测和产前诊断。结果 Sanger法DNA测序检测出该家系中先证者父亲、母亲分别携带SLC25A13基因IVS6+5GA、c.851del4杂合突变,先证者SLC25A13基因IVS6+5GA、c.851del4复合杂合突变来源于父母。对先证者母亲的羊水标本进行此两位点的检测,发现羊水标本SLC25A13基因未携带此两位点的突变,基因型与先证者不一致。结论 SLC25A13基因IVS6+5GA、c.851del4 2个突变为Citrin缺陷导致的NICCD的热点突变,Sanger测序技术可有效的为Citrin缺陷导致的NICCD家系提供遗传咨询和产前诊断服务。     

泉州地区SLC25A13基因常见突变携带者频率的研究及意义

目的 改良citrin缺陷病致病基因SLC25A13常见突变851del4的诊断方法,并对泉州地区该基因常见突变的人群携带率进行探讨.方法 于450名健康成人中筛查SLC25A13常见突变851del4、1638-1660dup及IVS6+5G＞A的突变携带者,应用改良SLC25A13基因常见突变851del4的诊断方法,论证本诊断方法的可行性.结果 共发现6例851del4、3例1638-1660dup及3例IVS6+5G＞A突变携带者.其总携带者频率为0.027(12/450),本研究采用的新型诊断方法筛查的851del4突变人群携带者经(eneScan方法得到确认.结论 本研究改良的诊断方法对突变85ldel4诊断明确且简单实用,泉州地区SLC25A13常见突变携带者频率略高于台湾等周边地区,在该地区应存在一定数量的citrin缺陷病患者,对于该类代谢遗传病应予重视,以免发生因误诊或延误诊治而导致的不良后果.     

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.     

Six cases of citrin deficiency in Korea

Citrin deficiency resulting from mutations of the SLC25A13 gene is associated with two major clinical phenotypes; neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and adult-onset type 2 citrullinemia (CTLN2). In Korea, 6 cases of citrin deficiency were diagnosed based on biochemical and molecular findings. Four NICCD patients (2 boys and 2 girls) presented high citrulline levels on a newborn screening test or neonatal cholestasis. They were associated with conjugated hyperbilirubinemia, elevated liver enzymes, hypoalbuminemia, mild hyperammonemia, elevated citrulline, methionine and threonine. All of the hepatic manifestations were resolved spontaneously at the age of 5-9 months. Mutation analysis identified them as compound heterozygotes carrying each of the c.851del4, IVS11+1G>A, IVS13+1G>A, G393S, and IVS16ins3kb mutant alleles. Two adult male CTLN2 patients were identified. They were aged 24 and 37 years, and presented sudden loss of consciousness, hyperammonemia and citrullinemia. They were compound heterozygotes with IVS13+1G>A and IVS16ins3kb, and with c.851del4 and IVS11+1G>A mutant alleles. This report describes the clinical characteristics, biochemical findings and molecular analysis of the SLC25A13 gene of patients with citrin deficiency in Korea.     

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.     

SLC25A13 gene mutations in Taiwanese patients with non-viral hepatocellular carcinoma

Mutations of the SLC25A13 gene, which encodes citrin, result in adult-onset type II citrullinemia (CTLN2). Because CTLN2 has been associated with hepatocellular carcinoma (HCC) and may be involved in hepatocarcinogenesis, the objective of this study was to assess the frequency of SLC25A13 mutations in patients with non-viral HCC. A retrospective review of 154 patients with HCC, who underwent total tumor resection from July 1998 to August 2005, was conducted. After exclusion of 137 patients infected with hepatitis B and/or C viruses, 17 patients were analyzed. Genomic DNA from stored tumor and normal hepatic samples was analyzed for the SLC25A13 gene mutation. In addition, the clinicopathological and histopathological features of patients with and without the SLC25A13 gene mutation were compared. The SLC25A13 mutation was observed in two patients (12%), and the carrier rate was approximately 1 in 8 patients. The IVS6+5G>A mutation was heterozygous in both normal hepatic and tumor tissues for case 1. On the other hand, the c.851del4 mutation was heterozygous in normal tissue but homozygous in tumor tissue for case 2. No significant differences in patient characteristics were observed. Further analyses of patients with SLC25A13 gene mutations may elucidate the relationship between the citrin gene and susceptibility of HCC.