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

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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.