Tomokazu Suzuki

Molecular genotyping of N-acetylation polymorphism to predict phenotype

SummaryN-acetylation polymorphism is one of the representative pharmacogenetic traits that underlie interindividual and interethnic differences in response to xenobiotics. To develop a practical genotyping method to predict acetylator phenotype, we studied the conditions for accurate phenotyping, and identified the phenotype in 51 Japanese. Then we performed Southern blot analysis of genomic DNA from these subjects using 32P-labeled cDNA for polymorphic N-acetyltransferase in the liver, and found that four N-acetyltransferase alleles generated six genotypes. The present genotyping method predicted the rapid, intermediate, and slow acetylators correctly in 48 of 51 overall subjects (96%) and in all of 4 slow acetylators.

An improved method for genotyping of N -acetyltransferase polymorphism by polymerase chain reaction

SummaryPolymorphic N-acetyltransferase in human liver catalyzes N-acetylation of various arylamine-containing drugs and environmental chemicals. To accelerate the pharmacogenetic and ecogenetic studies of N-acetyltransferase polymorphism, we have developed a rapid and simple method for genotyping using a polymerase chain reaction based restriction fragment length polymorphism. This method distinguishes four kinds of allele of the N-acetyltransferase gene using a single polymerase chain reaction starting with a set of primers, followed by successive Asp718, BamHI and TaqI digestions, and then running the samples on a single electrophoresis lane. This method allows us to determine ten different genotypes easily and reliably.

A novel therapeutic trial of homogentisic aciduria in a murine model of alkaptonuria

AbstractAlkaptonuria is a rare autosomal recessive disorder characterized by homogentisic aciduria, ochronosis, and arthritis. Although a deficiency of homogentisic acid 1,2-dioxygenase has recently been confirmed at the molecular level, no effective treatment regimen has yet been developed for this disorder. In the present study, 2(-2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), a potent inhibitor of p-hydroxyphenylpyruvate dioxygenase (which catalyzes the formation of homogentisic acid from p-hydroxyphenylpyruvic acid) was adopted as a possible therapeutic agent for alkaptonuria. NTBC dose-dependently reduced the urinary output of homogentisic acid in a murine model of alkaptonuria that had been created with ethylnitrosourea. These findings suggest that NTBC may be the first potent pharmacotherapeutic agent for alkaptonuria.

Molecular analysis of the transferrin gene in a patient with hereditary hypotransferrinemia

AbstractWe previously reported a patient with hereditary hypotransferrinemia who suffered from severe anemia and growth retardation and was diagnosed on the basis of an extremely low level of serum transferrin (TF). By an isoelectric focusing analysis, we found that the patient and his father shared a variant TF protein with an abnormal isoelectric point. The study suggested that the patient was a compound heterozygote with a variant allele, encoding the mutant TF, of paternal origin and a null allele of maternal origin. In the present study, we investigated the TF gene of the patient and his family. We showed that the patient and his father shared a variant TF gene bearing a GAA to AAA transition at codon 394. This nucleotide substitution causes a nonconservative amino acid change from glutamate to lysine in amino acid residue 375 of the TF protein. This single amino acid mutation is predicted to cause a conformational change in the coiled region of the carboxyl-terminal iron-binding lobe. As for the maternal null allele, no mutation was found in either the coding region or the exon-intron boundaries, suggesting an abnormality in the transcription or stability of mRNA of maternal allele origin.

Strong linkage disequilibrium and haplotype analysis in Japanese pedigrees with Machado-Joseph disease

To identify the markers tightly linked to Machado-Joseph disease (MJD) and to investigate whether a limited number of ancestral chromosomes are shared by Japanese MJD pedigrees, a detailed linkage analysis employing D14S55, D14S48, D14S67, D14S291, D14S280, AFM343vf1, D14S81, D14S265, D14S62, and D14S65 was performed. The results of multipoint linkage analysis as well as detection of critical recombination events indicate that the gene for MJD is localized in a 4-cM region between D14S280-D14S81. We found strong linkage disequilibria at AFM343vf1 and D14S81, and association of a few common haplotypes with MJD. These results indicate that there is an obvious founder effect in Japanese MJD and suggest the possibility of the existence of predisposing haplotypes which are prone to expansions of CAG repeats.

Genetic polymorphism of N-acetyltransferase 2 in patients with esophageal cancer

OBJECTIVE:N-Acetylation polymorphism is a representative genetic trait related to an individuals susceptibility to several cancers. However, there remains a controversy and no consensus concerning whether there is a true association between esophageal cancer and N-acetylation polymorphism.METHODS:To analyze the distribution of N-acetyltransferase 2 polymorphism in Japanese patients with esophageal squamous cell cancer, a molecular genotyping method using a polymerase chain reaction-based restriction fragment length polymorphism was used.RESULTS:Based on an analysis of 71 Japanese patients with esophageal squamous cell cancer and 329 healthy control subjects, the distribution of the slow acetylator phenotype was significantly higher in esophageal cancer patients than in the controls (19.7% and 9.4%, respectively, p = 0.040). The odds ratio of esophageal cancer for the slow phenotype was 2.55 (95% CI = 1.15–5.65, p = 0.023) compared with the rapid type. Furthermore, a significant difference between the distribution of acetylator phenotype and the incidence of lymph node metastasis and lymphatic involvement was found based on the clinicopathological features of these cancers. Esophageal cancer patients with a higher smoking exposure history tended to have the rapid acetylator phenotype.CONCLUSION:These results suggest that N-acetylation polymorphism may be implicated as a genetic trait affecting an individuals susceptibility and biological behavior of esophageal squamous cell cancer.

A convenient method for genotyping of humanO 6-methylguanine-DNA methyltransferase polymorphism

SummaryO6-methylguanine-DNA methyltransferase (MGMT) is one of the DNA repair enzymes in mammals. We previously screened the variant alleles for the MGMT gene in the general population, and found three variants (V1, V2, V3), two of which caused amino acid substitutions (Leu84Phe for V1, and Trp65Cys for V2). In order to accelerate the ecogenetic and pharmacogenetic studies on MGMT polymorphism, we therefore developed a new PCR-based RFLP method for genotyping. The present method has some advantages over the initial PCR-single strand conformation polymorphism (SSCP) method, particularly regarding its simplicity, rapidity and specificity.

Cancer Mortality in Low Radon Spa Area

Recently lower mortality for cancers of all sites was reported among inhabitants in the Misasa spa area, where there is a high radon background. To clarify the effects of radon exposure on cancer mortality, the effects of a hot spring itself on cancer mortality was investigated in the Beppu spa area, which has only a low radon background, and adjacent control areas. For females, the mortalities for cancers of all sites, liver and lung were higher in Beppu than those for all Japan on the basis of the standardized mortality ratio (SMR), while the SMR for all cancers was lower in adjacent areas. For the male inhabitants in hoth areas the cancer mortalities of all sites were not significantly different from those of all Japan. When we directly compared the most typical spa areas in Beppu and an adjacent control area, a Poisson regression analysis did not show that the relative risk of dying from cancer of all sites was decreased in the spa areas. These results are thus consistent with the view that the lower cancer mortality in the Misasa spa area might be related to exposure to low levels of radon .

Hyperornithinemia, hyperammonemia and homocitrullinuria : a case report and study of ornithine metabolism using in vivo deuterium labelling

Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH syndrome) is a rare inborn error of the urea cycle metabolism, clinically characterized by mental retardation, lethargy, seizures and ataxia [l]. Since the first report by Shih et al. [2], over 30 cases have been described [l-4], and an autosomal recessive inheritance has been suggested [1,5]. Although the primary metabolic defect of this syndrome remains unknown, an impairment of the transport of ornithine from cytoplasm into mitochondria in liver cells has been suggested as the basic mechanism [ 1,3,6]. In the present paper, we report an additional patient with this syndrome and a study on the kinetics of deuterium-labelled ornithine in vivo in the disorder.

Generalized Resistance to Thyroid Hormone--Identification of a Novel c-erbAβ Thyroid Hormone Receptor Variant(Leu450) in a Japanese Family and Analysis of Its Secondary Structure by the Chou and Fasman Method

SummaryGeneralized resistance to thyroid hormone (GRTH) is characterized by elevated circulating levels of thyroid hormone in the presence of a eumetabolic state and failure to respond to triiodothyronine. Various point mutations in the c-erbAβ thyroid hormone receptor gene are known to be responsible for different phenotypes of GRTH. We herein report a new c-erbAβ variant in a Japanese family. The variant consisting of a cytosine to adenine base substitution at nucleotide position 1650 altered phenylalanine to leucine in codon 450 in the T3-binding domain of c-erbAβ. This base substitution was found in one allele of the 2 affected members of the family. The in vitro translation products of this mutant c-erbAβ gene demonstrated a significantly reduced T3-binding affinity. The secondary structure of this mutant thyroid hormone receptor predicted by the Chou and Fasman method included a new turn in the α helix structure in the T3-binding domain. We also discuss the secondary structures of the previously reported mutant receptors.