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，由肝脏中作为尿素循环成员的精氨酸琥珀酸合成酶(ASS)缺乏引起，这种酶缺陷存在于典型的新生儿瓜氨酸血症的所有组织或细胞中。对14例新生儿CDNA进行测序扩增，发现14个突变。导致新生儿瓜氨酸血症的突变是非常不同的，到目前为止研究的所有非血缘个体都是复合杂合子。四种哺乳动物、酵母和三种细菌的核苷酸编码序列和推导的氨基酸分析是可用的。人类的九个错义突变中有六个发生在这些有机体中完全保守的氨基酸位置。为了证实这些错义突变和缺失突变，我们利用PCAGGS载体在BMT-10细胞中建立了AsS蛋白的体外表达系统。5个突变的CDNA在该表达系统中被检测，证实所有的突变体都没有ASS活性，没有或低水平的ASS-CRM。我们还描述了一种不同类型的瓜氨酸血症(成年型)，其发生时肝脏的ASS活性降低，但肾脏和其他细胞的活性正常。具有正常动力学性质和热稳定性的肝脏ASS降低伴随着肝脏ASSmRNA的正常水平，没有大体结构异常和翻译活性。ASSmRNA全序列(5‘端20个碱基除外)均未发现突变。到目前为止，我们已经分析了大约80名成年瓜氨酸血症患者，其中有血缘关系的患者比例约为20%。对11例近亲婚姻患者的RFLP分析表明，原发缺陷可能不在ASS基因上。我们现在正在进行进一步的纯合子作图。

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. Fourteen mutations were identified by sequencing amplified CDNA from 14 neonatal patients. Mutations causing neonatal citrullinemia are extremely heterogeneous, and all non-consanguineous individuals studied to date are compound heterozygotes. The nucleotide coding sequence and deduced amino acid analysis are available for four mammalian species, yeast and three bacterial species. Six of nine missense mutations in humans occur in amino acid positions that are completely conserved in these organisms. To confirm these missense and deletion mutations, we established the in vitro expression system for ASS protein using PCAGGS vector in BMT-10 cells. Five mutant CDNA were tested in this expression system and were confirmed that all mutant had no ASS activity and no or low level of ASS-CRM.We also describe a different type of citrullinemia(adult onset form)which occurs in association with decreased ASS activity in the liver but normal activity in the kidney and other cells. Decreased hepatic ASS with normal kinetics properties and heat stability is accompanied by normal levels of ASSmRNA in the liver, no gross structural abnormality, and translational activity. No mutations were found in the whole sequence of ASSmRNA(except 20 base of 5'-end). We have analyzed about 80 patients with adult citrullinemia to date, and the proportion of patients with consanguinity were approximately 20%. RFLP analysis of 11 patients from consanguineous marriage suggested the possibility that the primary defect is not located on the ASS gene. We are now performing further homozygosity mapping.