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Dentinogenesis imperfecta (DGI) type II is one of the most common dominantly inherited dentin defects, in which both the primary and permanent teeth are affected. Here, we report a Japanese family with autosomal-dominant DGI type II, including both molecular genetic defects and pathogenesis with histological analysis. Mutation analysis revealed a mutation (c.53T>A, p.V18D, g.1192T>A) involving the second nucleotide of the first codon within exon 3 of the dentin sialophosphoprotein (DSPP) gene. This mutation has previously been reported in a Korean family. Thus far, 24 allelic DSPP mutations have been reported, and this is the seventh mutation involving the DSPP V18 residue. Among those, only one other was shown to be caused by a de novo mutation, and that mutation also affected the V18 amino acid residue. The DSPP V18 residue is highly conserved among other mammalian species. These findings thus suggest that the V18 amino acid might be a sensitive mutational hot spot, playing a critical role in the pathogenesis of DGI.

Periodontitis, which is a widespread and major dental disease, is a multifactorial, lifestyle-related disease and has been analyzed for gene polymorphism. We examined the frequency of the polymorphisms of the pro-inflammatory cytokine genes IL-1 A (-889) and IL-1 B (+3953) in relation to periodontitis in the Japanese population. We also examined whether polymorphism of TLR2 (Arg677Trp) and TLR4 (Asp299Gly), which are receptors recognized by periodontopathic bacteria, may also be associated with periodontitis. The subjects were 92 Japanese individuals, among whom 43 had periodontitis and 49 were healthy controls. We isolated genomic DNA from lingual mucosal cells and tested them for single nucleotide polymorphisms by polymerase chain reaction-restriction fragment length polymorphism. The incidence of polymorphisms was analyzed statistically by Fisher's exact test, and the sensitivity and specificity of the gene polymorphisms were calculated. The purpose was to determine whether such polymorphisms might be effectively used in the diagnosis of periodontitis. However, we found no evidence that the gene polymorphism of IL-1A (p = 0.082), IL-1 B (p = 0.180), TLR2 (p = 1.000) or TLR4 (p = 1.000) and overall gene polymorphism in any of the genes (p = 0.752) correlate with periodontitis. The sensitivity (14.0%) and specificity (83.7%) of the mutations found in all of the genes were low. Therefore, we advise against using the analyses of polymorphism of these genes to detect periodontitis in the Japanese population.

Fasciculation and elongation of protein zeta-1 (FEZ1) is a binding partner of Disrupted-In-Schizophrenia 1 (DISC1). Because the DISC1 gene is shown to be a causative gene for psychosis in a Scottish family, the FEZ1 gene may well have importance in mental disease. A previous association study that analyzed polymorphisms of the FEZ1 gene in Japanese patients with schizophrenia and control subjects found significant association of the Asp123Glu polymorphism with schizophrenia. In the present study, we examined two polymorphic markers, rs559668 and rs597570 (Asp123Glu), in the FEZ1 gene to confirm the association in 1920 Japanese patients with schizophrenia and 1920 control subjects. The power to detect an association was more than 0.98. However, we did not detect genotypic associations of either of these two single nucleotide polymorphisms with schizophrenia (p=1 and 0.79, respectively). We concluded that the missense mutation Asp123Glu of the FEZ1 gene is unlikely to play a substantial role in the genetic susceptibility to schizophrenia.

Aim: The indispensable role of interleukin-6 receptor (IL-6R) in regulating IL-6 responses has been clearly established. We have previously reported that IL6R polymorphisms strongly influenced the serum levels of soluble IL-6R. In this study, we investigated the association between these genetic variations and periodontitis., Material and Methods: Among the seven novel IL6R single-nucleotide polymorphisms (SNPs) reported, we genotyped two important sites: the +48892 A/C in exon 9 and the -183 G/A in the promoter region. The SNP in exon 9 results in Asp-->Ala substitution in the proteolytic cleavage site of IL-6Ralpha. In total, 212 periodontitis cases and 210 healthy controls were genotyped using polymerase chain reaction, restriction fragment length polymorphisms and direct sequencing methods., Results: Analysis of the genotype distribution of the +48892 A/C SNP in periodontitis patients and in controls revealed a suggestive association with aggressive (p = 0.04) and chronic periodontitis (p = 0.04). In addition, the carriage rate for the A allele was significantly higher in chronic periodontitis patients [p = 0.02, odds ratio (OR) = 2.25]. No association was found in the -183 G/A SNP. The two markers were in linkage disequilibrium (LD) (|D'| = 0.53)., Conclusion: The IL6R+48892 A/C polymorphism could act as a risk factor for periodontitis; however, further association and biological studies are needed.

Coronary vasospasm appears to play a significant role in the etiology of myocardial ischemia in patients with hypertrophic cardiomyopathy (HCM). Furthermore, the management of patients with coexistent HCM and coronary spastic angina (CSA) presents a therapeutic challenge. The purpose of this study was to examine the Glu298Asp variant of the endothelial nitric oxide synthase (eNOS) gene to determine whether this polymorphism was associated with susceptibility to CSA in patients with HCM. The eNOS gene polymorphism (Glu298Asp) was genotyped in 150 HCM patients by the TaqMan chemical method. Patients were classified into group A (n=12) if they had CSA provoked by intracoronary acetylcholine, and group B (n=138) if they did not. In group A, the frequency of Glu/Glu, Glu/Asp, and Asp/Asp genotypes was 5 (41.7%), 6 (50%), and 1 (8.3%), respectively. In group B, it was 119 (86.2%), 17 (12.3%), and 2 (1.5%), respectively. The frequency of the Asp298 variant was significantly higher in group A than in group B (P<0.001). Multivariate logistic regression analysis showed that the Asp298 variant was a significant risk factor for CSA (odds ratio 11.8; P<0.001) that was independent of age, gender, smoking status or body mass index. Significantly more drugs were used by the patients in group A than those in group B and the patients with the Asp298 variant were treated with significantly more drugs than those without it. In conclusion, the Asp298 variant of the eNOS gene may be associated with CSA in HCM patients. HCM patients with CSA or the Asp298 variant may need more drugs to relieve their symptoms.

SN-38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-beta-d-glucuronide by hepatic UDP-glucuronosyltransferase (UGT) 1A1. Recent studies have revealed that other UGT1A isoforms, UGT1A7 and UGT1A9, also participate in SN-38 glucuronidation. Although several genetic polymorphisms are reported for UGT1A1 and UGT1A7 that affect the SN-38 glucuronidation activities, no such polymorphisms have been identified for UGT1A9. In the present study, UGT1A9 exon 1 and its flanking regions were sequenced from 61 Japanese cancer patients who were all treated with irinotecan. A novel nonsynonymous single nucleotide polymorphism was identified in UGT1A9 exon 1, heterozygous 766G>A resulting in the amino acid substitution of D256N. The wild-type and D256N UGT1A9s were transiently expressed at similar protein levels in COS-1 cells, and their membrane fractions were characterized in vitro for the glucuronidation activities toward SN-38. The apparent Km values were 19.3 and 44.4 microM, and the Vmax values were 2.94 and 0.24 pmol/min/mg of membrane protein for the wild-type and D256N variant, respectively. The SN-38 glucuronidation efficiency (normalized Vmax/Km) of D256N was less than 5% that of wild-type UGT1A9. These results clearly indicate that the D256N variant is essentially nonfunctional with regard to SN-38 glucuronidation. These findings highlight the importance of further studies into the potential influence of UGT1A9 D256N variant to irinotecan metabolism in vivo.

Objective: The gene responsible for hereditary hemochromatosis close to the human leukocyte antigen A locus was previously identified and designated as HFE. This study was performed to evaluate the clinical significance of two mutations, C282Y and H63D of HFE, in Japanese patients with hepatic iron overload., Patients and Methods: We examined C282Y and H63D in 11 patients with primary hemochromatosis, 94 patients with chronic hepatitis C, 54 patients with miscellaneous liver diseases, and 151 healthy volunteers. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by polymerase chain reaction. Restriction enzyme analysis was performed using SnaBI for C282Y and BclI for H63D. Direct sequence analysis was then performed when products suggested the presence of a mutation., Results: All the subjects studied were free from C282Y. None of the patients with hemochromatosis had H63D. One patient with chronic hepatitis C was homozygous, and 4 patients were heterozygous for H63D. Two patients with alcoholic liver disease were heterozygous for H63D. The prevalence of chromosomes with H63D was 6/188 (3.2%) in patients with chronic hepatitis C, 2/108 (1.9%) in patients with miscellaneous liver diseases, and 8/302 (2.6%) in healthy volunteers. These differences were not significant., Conclusion: Our results suggested that neither C282Y nor H63D in HFE affect Japanese patients with hemochromatosis or chronic hepatitis C.

Recently a significant association of a missense mutation (Glu298Asp) of the endothelial nitric oxide synthase (NOS3) gene with late-onset Alzheimer's disease (LOAD) was reported. We tried to replicate this finding in a Japanese sample of 121 patients with LOAD, 51 with early-onset AD (EOAD), and 165 medical controls. However, the genotype and allelic distributions for the Glu298Asp polymorphism were similar for these three groups, suggesting that the Glu298Asp polymorphism of the NOS3 gene has no relevance to the development of AD in Japanese.

The frequency of aspartate at residue 9 (Asp-9) of HLA-C molecules was investigated among 75 Japanese patients with psoriasis vulgaris and 50 healthy controls. We developed a technique of polymerase chain reaction sequence-specific primer (PCR-SSP) amplification of genomic DNA for HLA-C alleles with a codon for Asp-9. The specificity of amplification was confirmed by direct sequencing of the amplified products and amplification from total RNA (RT-PCR). Asp-9 was positive in all individuals with Cw6 and/or Cw7, but negative in the others, indicating that Asp-9 was specific to Cw6 and Cw7 antigens in our subjects. The frequency of Asp-9 was significantly increased in the patient group (48% vs. 20%; P < 0.002). The frequency of alanine at residue 73 (Ala-73), which was positive for Cw4, Cw6, Cw7, and some C blanks, was also increased in our previous study (81% vs. 48%; P < 0.0001). Asp-9 is located on a beta sheet of alpha 1 domain of HLA-C molecule and influences the peptide binding of the C pocket of the groove together with Ala-73. Both Asp-9 and Ala-73 could contribute to the disease susceptibility to psoriasis vulgaris in the immune responses.

Familial male-limited precocious puberty (FMPP) is an autosomal dominant disorder characterized by marked elevation of serum testosterone despite low levels of gonadotropin. Recently, a single point mutation in the LH/hCG receptor (LH/CGR) gene was found in FMPP families that constitutively activates the LH/CGR, causing Leydig cell activation and precocious puberty. Among the Japanese population, only four sporadic cases of male-limited precocious puberty have been reported. In the current study, we examined one of the four reported Japanese patients with sporadic male-limited precocious puberty and found the same mutation as that in the FMPP families. Genomic DNA was isolated, and the polymerase chain reaction (PCR) was performed to amplify a fragment of LH/CGR DNA encoding amino acid residues that include transmembrane helixes 5 and 6. Sequencing of the PCR products revealed a heterozygous adenosine-guanine transition at nucleotide 1733 in codon 578. The mutation encodes an aspartic acid578-glycine substitution in transmembrane helix 6. The mutant LH/CGR, created by site-directed mutagenesis in vitro, exhibited constitutively higher cAMP levels in transfected COS-7 cells than the wild-type LH/CGR, as described previously; however, basal inositol phosphate levels were not increased by transfection with complementary DNA for the mutant receptor. The concentration and affinity of [125I]hCG-binding sites were similar in cells transfected with the mutant and wild-type LH/CGR complementary DNAs, indicating that the mutant did not alter the production of receptor or its ability to bind human LH/CG. The sporadic occurrence of this case was confirmed by further studies. The mutation creates a recognition site for the restriction endonuclease MspI. Restriction digestion was positive for the mutant not digested by MspI, indicating that the patient's mutant allele was not inherited from his parents. DNA analysis of the patient and the parents, using microsatellite repeat markers, was compatible with biological paternity and maternity. We conclude that the aspartic acid578-->glycine mutation in the LH/CGR has arisen in the Japanese population and is the cause of a sporadic case of male-limited precocious puberty.