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

HHH综合征是一种导致各种神经系统症状的代谢紊乱。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，我们阐明了ORNT1基因的结构，该基因被5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名日本患者中发现了R179X，并被认为在日本患者中相当频繁。外显子跳过有时与外显子序列中过早的无意义密码子有关。另一方面，已知氨基糖苷类抗生素允许通过提前终止密码子阅读完整的编码序列，这一现象可能被应用于治疗带有提前终止密码子的人类遗传病。我们发现氨基糖苷类抗生素改善了由于ORNT1基因中一个提前终止密码子R179X而导致的外显子跳跃。通过搜索在线数据库，我们在5号染色体上发现了ORNT1的同源基因ORNT2。ORNT2在开放阅读框架(ORF)中没有内含子。在编码区，与ORNT1的核苷酸序列同源性为87.7%，氨基酸序列同源性为87.4%。与人RNA的多重斑点印迹分析表明，ORNT2在脂肪组织、小肠和睾丸中都有表达，但在肝脏中只有少量表达。相反，ORNT1主要在肝脏中表达，在那里尿素循环主要进行操作。当ORNT2在HHH综合征患者的成纤维细胞中表达时，^&lt；14&gt；C-鸟氨酸在蛋白质中的掺入增加，这表明ORNT2也具有线粒体鸟氨酸转运体的功能，但其功能似乎不如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.