Citrin缺陷致新生儿肝内胆汁淤积症患儿生化改变研究

目的探讨citrin缺陷致新生儿肝内胆汁淤积症（NICCD）患儿生化改变特征。方法对经基因学确诊且平均月龄为3个月的26例NICCD患儿进行常规生化检查、气相色谱质谱检查及串联质谱检查并分析。结果 NICCD患儿血清总胆红素、直接胆红素、谷草转氨酶、谷丙转氨酶及乳酸水平升高均占100%,血氨及甲胎蛋白水平升高均占95.2%,胆汁酸水平升高占90.0%,低蛋白水平占84.0%,血脂水平升高占50%。半乳糖增高占78.3%,4-羟基苯乳酸增高占52.2%。C14增高占84.7%,C16增高占71.4%,瓜氨酸增高占66.7%。结论 NICCD患儿存在糖,氨基酸及脂肪酸代谢异常,以半乳糖、瓜氨酸及长链酰基肉碱增高明显。     

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.     

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.     

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。     

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...     

A new Caucasian case of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD): a clinical, molecular, and functional study

Citrin is the liver-specific isoform of the mitochondrial aspartate/glutamate carrier (AGC2). AGC2 deficiency is an autosomal recessive disorder with two age related phenotypes: neonatal intrahepatic cholestasis (NICCD, OMIM#605814) and adult-onset type II citrullinemia (CTLN2, OMIM#603471). NICCD arises within the first few weeks of life resulting in prolonged cholestasis and metabolic abnormalities including aminoacidemia and galactosuria. Usually symptoms disappear within the first year of life, thus making a diagnosis difficult after this time. In this study we report a new Caucasian case of NICCD, a seven week old Romanian boy with prolonged jaundice. Sequencing of the AGC2 gene showed a novel homozygous missense double-nucleotide (doublet) mutation, which produces the change of the glycine at position 437 into glutamate. Functional studies, carried out on the recombinant mutant protein, for the first time demonstrated, that NICCD is caused by a reduced transport activity of AGC2. The presence of AGC2 deficiency in other ethnic groups besides Asian population suggests further consideration for NICCD diagnosis of any neonate with an unexplained cholestasis; a prompt diagnosis is crucial to resolve the metabolic decompensation with an appropriate dietary treatment.     

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

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.     

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。     

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。     

Infantile citrullinemia caused by citrin deficiency with increased dibasic amino acids

Thyroid nodules can be found in up to 50% of inhabitants of iodine-deficient areas and are classified as hot or cold thyroid nodules according to their scintigraphic characteristics. Studies of hot thyroid nodules with comparable mutation detection methods and screening at least exon 10 of the TSH receptor reported frequencies for somatic TSH-receptor mutations ranging from 20 to 82% in patients with similar iodine supply. We have recently screened 75 hot thyroid nodules for somatic TSH-receptor mutations with the more sensitive DGGE method and found somatic TSH-receptor mutations in 57% and Gsalpha mutations in 3%. As 50% of the mutation-negative nodules from female patients are of monoclonal origin when tested for X-chromosome inactivation somatic mutations in other genes are likely to cause the development of hot thyroid nodules. Scintigraphically nonsuppressible areas have been identified in up to 40% of euthyroid goiters in iodine-deficient areas. We recently identified somatic TSH-receptor mutations in microscopic autonomous areas with increased 125T uptake in euthyroid goiters studied by autoradiography 20 years ago. These constitutively activating somatic TSH-receptor mutations in minute autoradiographically hot areas of euthyroid goiters are very likely starting foci which most likely lead to toxic thyroid nodules in iodine-deficient goiters. Therefore iodine deficiency does not only lead to euthyroid goiters but also to thyroid autonomy. The latter is also suggested by epidemiologic studies. Similar mechanisms induced by iodine deficiency and the subsequent hyperplasia, mutagenesis, and selection of cell clones could also lead to cold thyroid nodules by somatic mutations that only initiate growth but not hyperfunction of the affected thyroid epithelial cell. Somatic ras mutations have frequently been detected in histologically characterized thyroid adenomas or adenomatous nodules. However, they seem to be rare in cold thyroid nodules. Since the majority of these latter nodules and 60% of the cold thyroid nodules are monoclonal other somatic mutations are likely in these nodules.     

Severe intrahepatic cholestasis caused by amiodarone toxicity after withdrawal of the drug: a case report and review of the literature

Cholestasis has been reported as a rare presentation among patients with severe liver injury secondary to amiodarone hepatic toxicity. We report an unusual case of amiodarone-induced cholestatic hepatotoxicity occurring after amiodarone had been discontinued and the initial abnormal liver function findings had improved. The patient, without jaundice at the initial presentation, developed severe jaundice about 4 months after withdrawal of amiodarone. Light and transmission electron microscopic examination of a specimen secured by computed tomographically guided liver biopsy was consistent with amiodarone hepatic toxicity as the cause of intrahepatic cholestasis. An abdominal ultrasound, endoscopic retrograde cholangiography, and dimethyl iminodiacetic acid and computed tomographic scans of the abdomen all failed to demonstrate any other causes for jaundice other than amiodarone toxicity. Thus, amiodarone hepatic toxicity may occur after drug withdrawal even if results of liver function tests improve. Histopathologic examination of a liver biopsy specimen is of value for diagnosis and prognosis. The liver biopsy findings, clinical course, and liver function test results are discussed, and the English-language literature on amiodarone cholestatic hepatotoxicity is reviewed.     

Novel diagnostic approach to citrin deficiency: analysis of citrin protein in lymphocytes

Citrin deficiency induces two clinical features; namely neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and adult-onset type II citrullinemia. Hypercitrullinemia is the most characteristic feature, whereas there are non-citrullinemic individuals. Diagnosis of citrin deficiency is performed by genetic analysis, although the 12 known mutations in the alleles are not detected in about 15% of cases. Thus, we aimed to examine citrin protein in lymphocytes isolated from peripheral blood as an alternative diagnostic method. We examined 38 children having an episode of cholestatic liver dysfunction, 8 heterozygotes, and 11 healthy individuals. All subjects were evaluated for citrin protein by Western blotting and for the 12 known mutations by gene analysis. Citrin protein was detected in 15 of 38 children with cholestatic liver dysfunction. Fourteen of them were negative for 12 known mutations in both alleles, whereas one patient was found to have a known mutation in one allele. Citrin protein was absent in 23 of the 38 patients. Among these 23, gene analysis diagnosed citrin deficiency in 19, whereas 2 patients were later revealed to be NICCD with novel mutations. In the remaining 2 patients, who exhibit the clinical features of NICCD, a known mutation was detected in one allele but no mutation was identified in another allele. Citrin protein was also detected in the 8 heterozygotes and 11 healthy individuals. We disclosed that citrin was deficient in lymphocytes among patients with citrin deficiency. Analysis of citrin is useful to diagnose citrin deficiency even in patients without known mutations or hypercitrullinemia.     

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.     

105例妊娠期肝内胆汁淤积症临床分析

目的探讨妊娠期肝内胆汁淤积症(ICP)对母儿的不良影响.方法对105例ICP病人进行回顾性分析.结果与孕28w以后出现搔瘁的患者比较,孕28w以前出现搔瘁的患者的胆汁酸、转氨酶及胆红素值显著性升高(P＜0.05),孕妇各种妊娠结局、围生儿结局的发生率及羊水粪染率显著性升高(P＜0.05).结论ICP易引起早产、胎儿宫内窘迫、胎儿突死官内及母亲产后出血,病人发病时间越早,围产儿预后越差,母亲产后出血发生率及羊水粪染率越高.