Molecular basis of argininosuccinate synthetase deficiency in citrullinemia

瓜氨酸血症精氨琥珀酸合成酶缺乏的分子基础

• 批准号: 04670167
• 负责人: KOBAYASHI Keiko
• 金额: $ 1.34万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670167/
• 关键词: Citrullinemia; Argininosuccinate synthetase deficiency; Urea cycle enzyme deficiency; Tissue specific abnormality; Homozygosity mapping; Mutation analysis; Abnormal splicing; DNA diagnosis; シリトルン血症; RFLP解析

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

瓜氨酸血症是一种常染色体隐性遗传病，由精氨酸琥珀酸合成酶缺乏引起，精氨酸琥珀酸合成酶是肝脏尿素循环的一个成员。这种酶缺陷存在于典型新生儿瓜氨酸血症（I型和III型）的所有组织或细胞中。此前，在新生儿瓜氨酸血症中发现了9个错义突变、4个与mRNA外显子缺失相关的突变和1个剪接突变（Kobayashi等人）。J Biol Chem 1990; Mol Biol Med 1991)。14例患者除3例日本等位基因外，主要为美国人。此外，利用mRNA逆转录、cDNA扩增和cDNA克隆测序对11例日本瓜氨酸血症患者的突变进行了表征。本文描述了5个新的错义突变（A118T、A192V、R273C、G280R和R363L）和1个新的mRNA插入突变。3个等位基因有R304W突变，8个等位基因有DELTAEx7突变，缺失了mRNA的第7外显子。为了进一步确定引起DELTAEx7突变的ASS基因异常并建立DNA诊断，我们分离并测序了一个包含7号外显子6和7侧翼区域的噬菌体克隆。DELTAEx7突变导致3'侧和6内含子剪接位点内的ccagGT向ccggGT转变（IVS6^<-2>突变），并导致MspI限制位点的产生。29个日本经典瓜氨酸血症等位基因的DNA诊断显示，15个等位基因有IVS6^<-2>突变，4个等位基因有R304W突变，这两种突变出现在日本患者66%的突变等位基因中（Kobayashi et al.manuscript in preparation）。我们还描述了另一种类型的瓜氨酸血症。II型瓜氨酸血症在日本大多数成人型瓜氨酸血症患者中发现，而ASS缺乏症特别在肝脏中发现。既往研究表明，肝脏ASS活性降低是由于具有正常动力学性质的酶蛋白减少所致，肝脏ASS mRNA的数量、翻译活性和大体结构未见明显异常。在目前的工作中，我们通过测序分析表明，两名II型瓜氨酸血症患者的ASS mRNA没有突变。我们还报道了通过使用位于ASS基因位点内的三个DNA多态性对II型瓜氨酸血症的近亲家族进行RFLP分析。尽管有近亲父母，但该患者不是ASS基因的纯合单倍型。对16例近亲亲属患者的RFLP分析表明，其中5例患者的3个DNA探针中有一个存在杂合模式，杂合单倍型的频率与对照频率无明显差异。这些结果表明，II型瓜氨酸血症的主要缺陷不在ASS基因位点内（Kobayashi等）。Am J Hum Genet 1993)。少

项目成果

小林 圭子: "尿素サイクル異常症(シトルリン血症)" current Laboratory Medicine(最新検査). 9. 430-434 (1992)

Keiko Kobayashi：“尿素循环障碍（瓜氨酸血症）”当前检验医学 9. 430-434 (1992)。

Keiko Kobayashi: "Urea cycle enzyme deficiency : Citrullinemia." Curr.Lab.Med.9. 430-434 (1992)

小林惠子：“尿素循环酶缺乏症：瓜氨酸血症。”

Keiko Kobayashi: "Citrullinemia." Rinsho-iden-igaku (T.Furusho et al., eds). 412-416 (1993)

小林惠子：“瓜氨酸血症。”

Masahisa Horiuchi: "Carnitine administration to juvenile visceral steatosis mice corrects the suppressed expression of urea cycle enzymes by normalizing their transcription." J.Biol.Chem.267. 5032-5035 (1992)

Masahisa Horiuchi：“对幼年内脏脂肪变性小鼠施用肉碱，可以通过使尿素循环酶的转录正常化来纠正其受抑制的表达。”

Takeshi Ohno: "Argininosuccinate synthetase gene expression of Ieukemias:Potential diagnostic marker for blastic crisis of chronic myelocytic leukemia." Leuk.Res.16. 475-483 (1992)

Takeshi Ohno：“白血病的精氨琥珀酸合成酶基因表达：慢性粒细胞白血病急变的潜在诊断标志物。”