Molecular basis of argininosuccinate synthetase deficiency in citrullinemia

瓜氨酸血症是一种常染色体隐性遗传病，由肝脏尿素循环成员-此前，在人类新生儿瓜氨酸血症中已鉴定出9种错义突变、4种与mRNA中外显子缺失相关的突变和1种剪接突变（小林等人，J Biol Chem 1990和Mol Biol Med 1991）。这14名患者主要是美国人，只有3个等位基因来自日本。此外，mRNA的逆转录，cDNA的扩增和cDNA克隆的测序被用来表征11例日本瓜氨酸血症患者的突变。本文描述了5个新的错义突变（A118T、A192V、R273C、G280R和R363L）和1个新的插入突变，其中3个等位基因有R304W突变，8个等位基因有DELTAEx7突变，缺失了mRNA的外显子7。 ...更多信息 为了鉴定引起DELTAEx7突变的ASS基因异常并建立DNA诊断方法，我们分离并测序了一个包含外显子7的内含子6和7侧翼区的噬菌体克隆。DELTAEx7突变导致在内含子6的3'-侧和剪接位点内从ccagGT转变为ccggGT（IVS6 ^突变），并导致产生MspI限制性位点。<-2>对29个日本人典型瓜氨酸血症等位基因的DNA诊断表明，15个等位基因有IVS 6 α突变，4个等位基因有R304 W分裂，这两种突变出现在日本患者66%的突变等位基因中（小林等，手稿准备中）。<-2>II型瓜氨酸血症见于日本大多数成人型瓜氨酸血症患者，ASS缺乏症特别见于肝脏。以往的研究表明，肝ASS活性的降低是由酶蛋白的减少与正常的动力学特性，并有没有明显的异常，在数量，翻译活性，和肝ASS mRNA的总体结构。在目前的工作中，我们通过测序分析表明，有没有突变的ASS mRNA从2例II型瓜氨酸血症。我们还报告了一个II型瓜氨酸血症的近亲家庭的RFLP分析，通过使用位于ASS基因位点内的三个DNA多态性。尽管有血缘关系的父母，病人是不是一个纯合子单倍型的ASS基因。对16例患者的RFLP分析表明，16例患者中有5例为三种探针之一的杂合型，其杂合单倍型频率与对照组无明显差异。这些结果表明，II型瓜氨酸血症的主要缺陷不在ASS基因座内（小林等人，美国遗传学杂志，1993年）。少

Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase(ASS) which functions as a member of urea cycle in the liver.This enzyme defect is found in all tissues or cells of the classic neonatal citrullinemia (type I and III). Previously, nine missense mutations, four mutations associated with an absence of an exon in mRNA, and one splicing mutation have been identified in human neonatal citrullinemia (Kobayashi et al. J Biol Chem 1990 and Mol Biol Med 1991). These fourteen patients were mainly American except three alleles from Japan., Furthermore, reverse transcription of mRNA, amplification of cDNA and sequencing of cDNA clones were used to characterize mutations in eleven Japanese citrullinemic patients. In this paper, we describe five new missense mutations (A118T, A192V, R273C, G280R and R363L) and one new insertion mutation in mRNA.Three alleles have R304W mutation and eight alleles have DELTAEx7 mutation deleted exon 7 in mRNA.In order … More to identify the abnormality in ASS gene causing DELTAEx7 mutation and to establish DNA diagnosis, we isolated and sequenced a phage clone which involves intron 6 and 7 flanking regions of exon7. The DELTAEx7 mutation results in a transition from ccagGT to ccggGT at the 3'-side and within the splice site of intron 6 (IVS6^<-2> mutation), and results in the creation of an MspI restriction site. The DNA diagnoses of 29 Japanese alleles with classical citrullinemia show that 15 alleles have IVS6^<-2> mutation and 4 alleles have R304W mitation and that these two mutations appear in 66% of the mutated alleles in Japanese patients (Kobayashi et al.manuscript in preparation).We also describe a different type of citrullinemia. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS deficiency is found specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there were no apparent abnormalities in the amount, translational activity, and gross structure of hepatic ASS mRNA.In the present work, we show by sequencing analysis that there was no mutation in the ASS mRNA from two patients with type II citrullinemia. We also report RFLP analysisi of a consanguineous family with type II citrullinemia, by using three DNA polymorphisms located within the ASS gene locus. In spite of having consanguineous parents, the patient was not a homozygous haplotype for the ASS gene. The RFLP analysisi of 16 affected patients from consanguineous parents showed that 5 of 16 patients had the heterozygous pattern for one of the three DNA probes and that the frequency of the heterozygous haplotype was not different from the control frequency. These results suggest that the primary defect of type II citrullinemia is not within the ASS gene locus (Kobayashi et al. Am J Hum Genet 1993). Less