Gene analysis of patients with HHH syndrome and application of the readthrough effect on nonsense mutations of antibiotics for therapy

HHH综合征患者基因分析及抗生素无义突变通读效应在治疗中的应用

• 批准号: 12670638
• 负责人: TSUJINO Seiichi
• 金额: $ 2.05万
• 依托单位: National Center of Neurology and Psychiatry
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670638/
• 关键词: HHH syndrome; mitochondria; ornithine transporter; nonsense mutation; exon skippin; antibiotics; aminoglycoside; ORNT1

HHH syndrome is a metabolic disorder resulting in various neurological symptoms. The ORNT1 gene encoding mitochondrial ornithine transporter is responsible for this disorder. First, we clarified the structure of the ORNT1 gene, which was interrupted by five introns. Then, we identified five novel mutations, R179X, G27E, insAAC, P126R, and R275X in eight unrelated in Japanese patients, and a mutation, del446G in two Palestinian brothers. Especially, R179X was observed in four Japanese patients, and it was thought to be fairly frequent in Japanese patients.Exon skipping is occasionally associated with premature nonsense codons amid exonic sequences. On the other hand, it is known that aminoglycoside antibiotics allow the reading of full coding sequences through premature termination codons, and this phenomenon may be applied for therapy of human genetic diseases with premature terminal codons. We showed that aminoglycoside antibiotics ameliorate exon skipping due to a premature termination codon, R179X, in the ORNT1 gene, in cultured skin fibroblasts from a patient.By searching an on-line database, we identified ORNT2, a homologu of ORNT1, on chromosome 5. ORNT2 has no introns in the open reading frame (ORF). In the coding region, homology to ORNT1 is 87.7% for the nucleotide sequence, and 87.4% for the amino acid sequence. Multiple dot blot analysis with human RNA showed that ORNT2 is expressed adipose tissue, small intestine and testis, but only minimally in liver. In contrast, ORNT1 is expressed predominantly in liver, where the urea cycle mainly operates. When ORNT2 was expressed in fibroblasts from a patient with HHH syndrome, incorporation of ^<14>C-ornithine into protein was increased, suggesting that ORNT2 also functions as a mitochondrial ornithine transporter, but the function appeared to be less than that of ORNT1.

HHH综合征是一种代谢紊乱，导致各种神经系统症状。编码线粒体鸟氨酸转运蛋白的ORNT1基因是导致这种疾病的原因。首先，我们明确了ORNT1基因的结构，该基因被五个内含子打断。然后，我们在8名无关的日本患者中发现了5个新的突变，R179X、G27E、insAAC、P126R和R275X，在两名巴勒斯坦兄弟中发现了一个突变，del446G。特别是在4名日本患者中观察到R179X，认为它在日本患者中相当常见。外显子跳跃有时与外显子序列中过早无义密码子有关。另一方面，氨基糖苷类抗生素可以通过过早终止密码子读取完整的编码序列，这一现象可能应用于人类过早终止密码子遗传病的治疗。我们发现氨基糖苷类抗生素改善了由于ORNT1基因中一个过早终止密码子R179X而导致的外显子跳跃，在培养的患者皮肤成纤维细胞中。通过在线数据库检索，我们在5号染色体上鉴定出ORNT1的同源物ORNT2。ORNT2在开放阅读框（ORF）中没有内含子。在编码区，与ORNT1核苷酸序列同源性为87.7%，氨基酸序列同源性为87.4%。人RNA多点印迹分析显示，ORNT2在脂肪组织、小肠和睾丸中均有表达，但在肝脏中表达较少。相比之下，ORNT1主要在肝脏中表达，肝脏是尿素循环的主要操作部位。当ORNT2在HHH综合征患者的成纤维细胞中表达时，蛋白质中^<14> c -鸟氨酸的掺入增加，这表明ORNT2也作为线粒体鸟氨酸转运体起作用，但其功能似乎不如ORNT1。

项目成果

辻野精一: "ミトコンドリアオルニチントランスポーター欠損症の分子遺伝学に関する研究"日本臨牀. 59. 2278-2284 (2001)

Seiichi Tsujino：“线粒体鸟氨酸转运蛋白缺陷的分子遗传学研究”Nihon Rinsho. 59. 2278-2284 (2001)。

Miyamoto et al.: "Diagnosis Japanese patients with HHH syndrome by molecular genetic analysis : a common mutation, R179X"J Hum Genet. 46. 260-261 (2001)

Miyamoto 等人：“通过分子遗传学分析诊断日本 HHH 综合征患者：常见突变 R179X”J Hum Genet。

Tsujino et al.: "Three novel mutations (G27E, insAAC, R179X)in the ORNT1 gene of Japanese patients with HHH syndrome"Ann Neurol. 47. 625-631 (2000)

Tsujino 等人：“日本 HHH 综合征患者 ORNT1 基因中的三种新突变（G27E、insAAC、R179X）”Ann Neurol。

Miyamoto et al.: "A novel mutation, P126R, in a Japanese patient with HHH syndrome"Pediatr Neurol. 26. 65-67 (2001)

Miyamoto 等人：“日本 HHH 综合征患者的一种新突变 P126R”Pediatr Neurol。

辻野精一: "ミトコンドリアオルニチントランスポーター欠損症"日本臨牀増刊「ミトコンドリアとミトコンドリア病」. 60. 779-782 (2002)

Seiichi Tsujino：“线粒体鸟氨酸转运蛋白缺乏症”日本临床出版社特别版“线粒体和线粒体疾病”60。779-782（2002）。