Yuji Hidaka

Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor)

SummaryHPRTAnn Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased Km for both substrates, a normal Vmax, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRTAnn Arbor and determined its complete nucleotide sequence. A single nucleotide change (T→G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5′-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.

Crossovers within a short DNA sequence indicate a long evolutionary history of the APRT*J mutation

SummaryAdenine phosphoribosyltransferase (APRT) deficiency causing 2,8-dihydroxyadenine urolithiasis and renal failure is present at a high frequency among the Japanese but not other ethnic groups. A special type of mutant allele, designated APRT*J, with a nucleotide substitution at codon 136 from ATG (Met) to ACG (Thr) is carried by approximately 79% of all Japanese 2,8-dihydroxyadenine urolithiasis patients. We analyzed mutant alleles of 39 APRT deficient patients using a specific oligonucleotide hybridization method after in vitro amplification of a part of the genomic APRT sequence. We found that 24 had only APRT*J alleles. Determination of the haplotypes of 194 APRT alleles from control Japanese subjects and of the 48 different APRT*J alleles indicated that normal alleles occur in four major haplotypes, whereas all APRT*J alleles occur in only two. These results suggest that all APRT*J alleles have a single origin and that this mutant sequence has been maintained for a long period, as calculated from the frequency of the recombinant alleles.

Effects of febuxostat on serum urate level in Japanese hyperuricemia patients

Abstract Objective. We assessed the efficacy and adverse effects of febuxostat in male hyperuricemia patients. Subjects and methods. This was a 12-week, multicenter, open-label, uncontrolled study. The enrolled subjects were 89 hyperuricemic male patients (12 overexcretors, 56 normal excretors, and 21 underexcretors). The endpoint was percent change in serum urate level. Results. The concentration of urate in serum before and 12 weeks after beginning administration of febuxostat in the overexcretors was 9.34 ± 1.48 and 5.59 ± 1.17 mg/dl, respectively, while those were 8.59 ± 1.24 and 5.41 ± 1.35 mg/dl, respectively, in the normal excretors, and 8.29 ± 1.01and 5.11 ± 1.71 mg/dl, respectively, in the underexcretors. After 12 weeks, the rate of change in serum urate after beginning administration of febuxostat was − 0.384 ± 0.186 in the overexcretors, − 0.368 ± 0.128 in the normal excretors, and − 0.365 ± 0.217 in the underexcretors, with no significant differences among them. A common adverse event related to febuxostat was gout flare. Conclusion. Febuxostat effectively reduced the concentration of urate in serum in hyperuricemic patients regardless of the level of uric acid excreted in urine without severe adverse effects.

Stepwise dose increase of febuxostat is comparable with colchicine prophylaxis for the prevention of gout flares during the initial phase of urate-lowering therapy: results from FORTUNE-1, a prospective, multicentre randomised study

Objectives To determine whether febuxostat with stepwise dose increase is as useful as colchicine prophylaxis in reducing gout flares during the initial introduction of urate-lowering therapy in patients with gout in comparison with febuxostat with no dose titration. Methods In this prospective, multicentre, randomised open-label comparative study, patients were randomised to group A (stepwise dose increase of febuxostat from 10 to 40 mg/day), group B (fixed-dose febuxostat 40 mg/day plus colchicine 0.5 mg/day) or group C (fixed-dose febuxostat 40 mg/day) and observed for 12 weeks. Gout flare was defined as non-steroidal anti-inflammatory drug use for gout symptoms. Results A total of 255 patients were randomised, and 241 patients were treated. Among the treated patients, gout flares were experienced by 20/96 (20.8%) in group A, 18/95 (18.9%) in group B and 18/50 (36.0%) in group C. The incidence of flare was significantly lower in groups A and B than that in group C (P=0.047 and P=0.024, respectively), although the differences were not significant after correction for multiple comparisons. No significant difference was noted between the incidence of gout flare in groups A and B. Conclusions Our data suggested that stepwise dose increase of febuxostat and low-dose colchicine prophylaxis effectively reduced gout flares in comparison with fixed-dose febuxostat alone. Stepwise dose increase of febuxostat may be an effective alternative to low-dose colchicine prophylaxis during the introduction of urate-lowering therapy. Trial registration number UMIN 000008414.

Human adenine phosphoribosyltransferase (APRT) deficiency: single mutant allele common to the Japanese.

Adenine phosphoribosyltransferase (APRT) is a purine salvage enzyme, which catalyzes the conversion of adenine to adenylic acid in the presence of phosphoribosylpyrophosphate (PRPP). In a complete APRT deficient subject, accumulated adenine is oxidized to 2, 8-dihydroxyadenine (2, 8-DHA) by xanthine oxidase. 2, 8-DHA excreted into urine precipitates and then forms a urinary stone.1 Partial deficiency develops no clinical symptoms. APRT deficiency is a common genetic disorder caused by a defective APRT gene on chromosome 16 and inherited in an autosomal recessive manner. The APRT gene, which is approximately 2.6 kb in length, consists of five exons and four introns. The frequency of heterozygotes has been estimated to be 0.4% – 1.2%.

55 HUMAN ADENINE PHOSPHORIBOSYLTRANSFERASE (APRT) DEFICIENCY: A SINGLE MUTANT ALLELE COMMON TO THE JAPANESE

Human adenine phosphoribosyltransferase (APRT) deficiency is a relatively common genetic disorder caused by a defective APRT gene, resulting in 2,8-dihydroxyadenine urolithiasis. The kinetic properties of APRT from APRT-deficient Japanese subjects showed similar abnormalities suggesting a distinct “Japanese-type” mutation. Nucleotide sequence analysis of APRT genomic DNA from WR2, a Japanese-type homozygote, identified a T to C substitution in exon 5 on both alleles (Met136→Thr). RNaae mapping analysis revealed that six other Japanese-type homozygotes carried the same mutation on at least one allele. After amplifying the region of the APRT genomic DNA that contains the Japanese-type mutation by the polymerase chain reaction (PCR), dot-blot analysis was performed using normal and mutated allele specific oligonucleotide (ASO) probes. This method demonstrated that all seven Japanese-type homozygotes carried the same mutation on both alleles. The dot-blots clearly distinguish normal, Japanese-type heterozygotes and Japanese-type homozygotes. PCR of genomic sequences and ASO hybridization is a useful tool for detection of common mutations in defined populations, such as “Japanese-type” APRT deficiency.