Increased prevalence of MEFV exon 10 variants in Japanese patients with adult-onset Still's disease.

Autoinflammatory diseases include a large spectrum of monogenic diseases, e.g. familial Mediterranean fever (FMF), as well as complex genetic trait diseases, e.g. adult-onset Still's disease (AOSD). In populations where FMF is common, an increased MEFV mutation rate is found in patients with rheumatic diseases. The aim of this study was to examine MEFV mutations in Japanese patients with AOSD. Genomic DNA was isolated from 49 AOSD patients and 105 healthy controls, and exons 1, 2, 3 and 10 of the MEFV gene genotyped by direct sequencing. MEFV mutation frequencies in AOSD patients were compared with controls. We found no significant difference in overall allele frequencies of MEFV variants between AOSD patients and controls. However, MEFV exon 10 variants (M694I and G632S) were significantly higher in AOSD patients than controls (6.1 versus 0%). In addition, there was no significant difference between MEFV variant carriers and non-carriers with clinical manifestations, but the monocyclic clinical course of the AOSD disease phenotype was observed less frequently in patients without MEFV variants. AOSD patients had significantly higher frequencies of MEFV exon 10 mutations, suggesting that low-frequency variants of MEFV gene may be one of the susceptibility factors of AOSD.

MEFV gene polymorphisms and TNFRSF1A mutation in patients with inflammatory myopathy with abundant macrophages.

Inflammatory myopathy with abundant macrophages (IMAM) has recently been proposed as a new clinical condition. Although IMAM shares certain similarities with other inflammatory myopathies, the mechanisms responsible for this condition remain unknown. Patients with familial Mediterranean fever (FMF) and tumour necrosis factor receptor-associated periodic syndrome (TRAPS) also often develop myalgia. We therefore investigated the polymorphisms or mutations of MEFV and TNFRSF1A genes in patients with IMAM to identify their potential role in this condition. We analysed the clinical features of nine patients with IMAM and sequenced exons of the MEFV and TNFRSF1A genes. The patients with IMAM had clinical symptoms such as myalgia, muscle weakness, erythema, fever and arthralgia. Although none of the patients were diagnosed with FMF or TRAPS, seven demonstrated MEFV polymorphisms (G304R, R202R, E148Q, E148Q-L110P and P369S-R408Q), and one demonstrated a TNFRSF1A mutation (C43R). These results suggest that MEFV gene polymorphisms and TNFRSF1A mutation are susceptibility and modifier genes in IMAM.

The rs150311303 polymorphism in FcγRIIa enhances IgG binding capacity.

Fc gamma receptor (FcγR) provides an important link between humoral and cellular immune responses. FcγRIIa-H131R polymorphism has been associated with differential binding to IgG subclasses and susceptibility to severe malaria phenotypes among different populations in the malaria endemic world. In this study, the effect of FCGR2A gene polymorphisms on susceptibility to symptomatic malaria among Ghanaian cohort children was investigated. Blood samples from four hundred and 29 (429) healthy Ghanaian children were genotyped for FCGR2A polymorphisms by direct DNA sequencing. Attributable and relative risks to symptomatic malaria were calculated for the polymorphic variants. Two major FCGR2A polymorphisms, rs1801274A/G (FcγRIIa-H131R) and rs150311303 (FcγRIIa-ins170L), were identified in the study population, and assessment of their risks did not show significant association with susceptibility to symptomatic malaria. The functional significance of these polymorphisms was also examined by evaluating their binding abilities to IgG subclasses using flow cytometric analysis of HEK cells transfected with the FcγRIIa haplotype variants. The binding assay revealed the rs150311303, which was observed only among carriers of the FcγRIIa-131RR genotype for the rs1801274 to consistently enhance binding capacities to all IgG subclasses. Thus, of the three FcγRIIa haplotype variants observed in this study population, the FcγRIIa(RL) haplotype variant was observed to have the highest binding ability to IgG1, IgG3 and IgG4.

Surfactant protein C G100S mutation causes familial pulmonary fibrosis in Japanese kindred.

Several mutations in the surfactant protein C (SP-C) gene (SFTPC) have been reported as causing familial pulmonary fibrosis (FPF). However, the genetic background and clinical features of FPF are still not fully understood. We identified one Japanese kindred, in which at least six individuals over three generations were diagnosed with pulmonary fibrosis. We examined the patients radiologically and histopathologically and sequenced their SFTPC and ABCA3 genes. We also established a cell line stably expressing the mutant gene. All the patients had similar radiological and histopathological characteristics. Their histopathological pattern was that of usual interstitial pneumonia, showing numerous fibroblastic foci even in areas without abnormal radiological findings on chest high-resolution computed tomography. No child had respiratory symptoms in the kindred. Sequencing of SFTPC showed a novel heterozygous mutation, c.298G>A (G100S), in the BRICHOS domain of proSP-C, which co-segregated with the disease. However, in the ABCA3 gene, no mutation was found. In vitro expression of the mutant gene revealed that several endoplasmic reticulum stress-related proteins were strongly expressed. The mutation increases endoplasmic reticulum stress and induces apoptotic cell death compared with wild-type SP-C in alveolar type II cells, supporting the significance of this mutation in the pathogenesis of pulmonary fibrosis.

MEFV mutations in Japanese rheumatoid arthritis patients.

OBJECTIVE: Familiar Mediterranean Fever (FMF) is common among Mediterranean populations, while other populations are rarely affected. The aim of this study was to assess the involvement of MEFV gene mutations among Japanese rheumatoid arthritis patients with or without amyloid A (AA) amyloidosis. METHODS: The frequency of the MEFV mutations, which were identified in Japanese FMF patients, was determined in 126 Japanese RA patients and 76 Japanese healthy subjects. RESULTS: The M694I mutation was not observed among RA patients and healthy subjects. Allele frequency of R408Q, P369S, E148Q, L110P mutations account respectively for 3.3%, 3.9%, 23.7%, 9.2% in healthy subjects and 5.6%, 6.7%, 24.2%, 9.5% in RA patients. The overall mutation rate was comparable between the RA patients and healthy subjects, as well as between the RA patients with and without amyloidosis. CONCLUSION: This study shows the high prevalence of mutations of the MEFV genes in Japanese RA patients. However, our data suggest that the MEFV gene mutations may not be a genetic factor affecting the susceptibility of RA or the development of amyloidosis in a Japanese population.

Association of polymorphism in the NeuroD/BETA2 gene with type 1 diabetes in the Japanese.

NeuroD/BETA2, a transcription factor of the insulin gene, also plays an important role in the development of pancreatic beta-cells. Recently, the NeuroD/BETA2 gene has been mapped to the long arm of human chromosome 2 (2q32) where the IDDM7 gene has previously been mapped, implying its involvement in diabetes. To identify mutations in the NeuroD/BETA2 gene that may predispose patients to develop diabetes, we studied the gene in 50 Japanese subjects with diabetes (4 with type 1 and 46 with type 2) by the polymerase chain reaction (PCR) followed by single-strand conformation polymorphism and sequencing analyses. Further analysis was performed in 392 Japanese subjects (60 with type 1 and 158 with type 2 diabetes and 174 healthy control subjects) by mismatch PCR restriction fragment length polymorphism. We found a DNA polymorphism of the NeuroD/BETA2 gene. A nucleotide G-to-A transition results in the substitution of alanine to threonine at codon 45 (Ala45Thr). The frequencies of heterozygotes for the Ala45Thr variant were 9.8% in the control subjects, 9.5% in the patients with type 2 diabetes, and 25.0% in the patients with type 1 diabetes, a significant difference (P = 0.006). Because the variant of the NeuroD/BETA2 gene (Ala45Thr) is associated with type 1 but not type 2 diabetes, it may be implicated in the loss of pancreatic beta-cells in type 1 diabetes.

Structure of the mouse NDRF gene and its regulation during neuronal differentiation of P19 cells.

We have isolated and characterized the mouse gene for NDRF (neuroD-related factor), a basic helix-loop-helix transcription factor implicated in neural development and function. The gene consists of two exons and the entire protein-coding sequence is encoded by a single downstream exon. RNA blot hybridization analysis revealed that NDRF mRNA was detectable at day 4 and increased to a maximal level at day 6 during neuronal differentiation of P19 cells. To elucidate the regulatory mechanisms of the NDRF gene expression during this process, a construct containing the genomic DNA fragment of about 3 kbp upstream of the NDRF coding region fused to a luciferase reporter gene was transfected into P19 cells, and stable transformants were pooled for assay of luciferase activities. When the stable transformants were treated with RA and aggregated to induce neuronal differentiation, the luciferase activities were induced in a temporal expression pattern similar to that of the endogenous NDRF mRNA. Further experiments using a series of deletion and mutation constructs indicated that the 376-bp sequence in the 5'-flanking region of the NDRF gene is important, and that one of the E boxes in the sequence plays a critical role in the regulated expression. Transient transfection experiments also showed that the same E box is required for the transactivation of the NDRF promoter activity by neurogenin 1. These results suggest that the NDRF gene expression is regulated by an E box-binding factor during neuronal differentiation of P19 cells.

Encoding tasks and the processing of perceptual information in young and older adults.

This study examined the degree to which different tasks promote the encoding of the characteristics of a talker's voice in young and older adults, and whether these characteristics encoded in long-term memory facilitate spoken word identification under difficult listening conditions. During the encoding phase, participants were given extensive exposure to the voices of two talkers and performed tasks that focused their attention on either voice characteristics (explicitly or incidentally) or linguistic information. Subsequently, participants identified novel words masked by noise, half of which were spoken by one of the familiar talkers and half by an unfamiliar talker. Young adults identified with greater accuracy words spoken in a familiar voice, whereas older adults benefited from voice familiarity only under instructions that promoted attention to voice characteristics either explicitly or incidentally. Age-related declines in sensory uptake (hearing loss) accounted for most of these task-dependent voice effects.

Lack of association between LTA and LGALS2 polymorphisms and myocardial infarction in Japanese and Korean populations.

To investigate the recently reported associations of polymorphisms in lymphotoxin-alpha (LTA) and galectin-2 (LGALS2) with myocardial infarction (MI), we analyzed a single nucleotide polymorphism of LTA (LTA 252A>G in LTA intron 1) and that of LGALS2 (LGALS2 3279C>T in LGALS2 intron 1) in Japanese and Korean populations. Although significant associations with MI were not observed in either population, we found that LTA 252GG was significantly associated with the severity of the disease for both the Japanese and Korean populations (P=0.017 and P=0.001, respectively). On the other hand, the polymorphism of LGALS2 was not associated with the severity of coronary atherosclerosis. These observations showed that, while the LTA 252GG genotype might modify the development of coronary atherosclerosis, the relation of LTA and LGALS2 to MI itself remained much less certain.

Association of clinical features with HLA in chronic pulmonary thromboembolism.

The aetiology of chronic thromboembolic pulmonary hypertension (CTEPH) is largely unknown and may be heterogeneous, because there are several ethnic differences in the clinical characteristics of CTEPH. Female predominance and a higher ratio of chronic to acute pulmonary thromboembolism have been reported in Japan as compared with the USA. Because such ethnic differences may be controlled by genetic factors, the current study investigated HLA polymorphisms in Japanese patients with CTEPH. HLA typing by serological and/or DNA typing methods was performed (for HLA-A, B, DPB1, DRB1) in 80 patients and 678 controls, and the association of clinical characteristics with HLA alleles was studied. The frequencies of HLA-B*5201 (40 versus 24%) and DPB1*0202 (19 versus 6%) were significantly higher in the patients. HLA-B*5201 positive patients showed a significant female predominance. Total pulmonary vascular resistance and mixed venous oxygen tension were better in the HLA-B*5201 positive patients. In contrast, cardiac index and gas exchange parameters were worse in the HLA-DPB1*0202 positive patients. In the patients carrying HLA-B*5201 and/or -DPB1*0202, the frequency of deep vein thrombosis was significantly lower than the other patients. These observations suggested that both the susceptibility and clinical characteristics of chronic thromboembolic pulmonary hypertension were controlled in part by the HLA-B and -DPB1 loci.

A novel family of TEA domain-containing transcription factors with distinct spatiotemporal expression patterns.

We cloned two novel types of TEA domain-containing transcription factor (ETFR-1 and -2) cDNAs. Amino acid sequences deduced for ETFR-1 and -2 as well as those of other known proteins of the same family, TEF-1 and ETF, exhibited significant identity (63-75% overall) to each other not only in the TEA DNA binding domain but also in the C-terminal regions. Northern blot analysis revealed that both the mRNAs were expressed in embryos as well as in many adult tissues, although their levels of expression varied. The results demonstrate that the mammalian TEA domain-containing transcription factor family consists of at least four distinct members, TEF-1, ETF, ETFR-1, and ETFR-2, which exhibit overlapping but differing spatiotemporal expression patterns, suggesting their redundant yet unique roles involved in not only developmental control but also tissue-specific regulation.

Genotype of alcohol dehydrogenase and aldehyde dehydrogenase loci in Japanese alcohol flushers and nonflushers.

A much higher incidence of alcohol flushing among Orientals in comparison to Caucasians, i.e., greater than 50% vs 5%-10%, has been attributed to racial differences in alcohol-metabolizing enzymes. A large majority of Orientals are "atypical" in alcohol dehydrogenase-2 locus (ADH2), and their livers exhibit significantly higher ADH activity than the livers of most Caucasians. Approximately 50% of Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH2) activity, and elimination of acetaldehyde might be disturbed. We determined by means of hybridization of genomic DNA samples with allele specific oligonucleotide probes, genotypes of the ADH2 and ALDH2 loci in Japanese alcohol flushers and nonflushers. We found that all individuals with homozygous atypical ALDH2(2)/ALDH2(2) and most of those with heterozygous atypical ALDH1(2)/ALDH2(2) were alcohol flushers, while all subjects with homozygous usual ALDH1(2)/ALDH1(2) were nonflushers. Frequency of the atypical ADH2(2) was found to be higher in alcohol flushers than in nonflushers, but the statistical significance was not established in the sample size examined.

Multiplication of the class I alcohol dehydrogenase locus in mammalian evolution.

Chromosomal DNA samples derived from various primates and other mammals (horse, sheep, rabbit, and mouse) were digested with restriction endonuclease and hybridized with a probe of the sixth exon of the human ADH gene, which is highly conserved in the class I alcohol dehydrogenase of these mammalian species. The copy number of the class I ADH gene in each species was estimated from the number of hybridized bands. Primate DNA samples showed three distinct bands in the blots of PstI digest and DraI digest. Moreover, most of the bands from primate DNA showed a similarity in size so as to allow us to assign the ADH1, ADH2, and ADH3 homologues in each species. In contrast, mouse has only one gene, and rabbit, sheep, and horse seem to have only two genes, for the class I ADH, which showed divergent hybridization bands. These results are consistent with the view that the human class I ADH gene cluster has been generated through gene multiplication events which occurred before the Catarrhini branch point in the course of primate evolution.

Identification and characterization of cell-specific enhancer elements for the mouse ETF/Tead2 gene.

We have identified and characterized by transient transfection assays the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF (Embryonic TEA domain-containing factor)/Tead2 gene required for transcriptional activation in ETF/Tead2 gene-expressing cells, such as P19 cells. The 117-bp enhancer contains one GC-rich sequence (5'-GGGGCGGGG-3'), termed the GC box, and two tandemly repeated GA-rich sequences (5'-GGGGGAGGGG-3'), termed the proximal and distal GA elements. Further analyses, including transfection studies and electrophoretic mobility shift assays using a series of deletion and mutation constructs, indicated that Sp1, a putative activator, may be required to predominate over its competition with another unknown putative repressor, termed the GA element-binding factor, for binding to both the GC box, which overlapped with the proximal GA element, and the distal GA element in the 117-bp sequence in order to achieve a full enhancer activity. We also discuss a possible mechanism underlying the cell-specific enhancer activity of the 117-bp sequence.

A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme.

The human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family consists of five known loci (ADH1-ADH5), which have been mapped close together on chromosome 4 (4q21-25). ADH isozymes encoded by these genes are grouped in three distinct classes in terms of their enzymological properties. A moderate structural similarity is observed between the members of different classes. We isolated an additional member of the ADH gene family by means of cross-hybridization with the ADH2 (class I) cDNA probe. cDNA clones corresponding to this gene were derived from PCR-amplified libraries as well. The coding sequence of a 368-amino-acid-long open reading frame was interrupted by introns into eight exons and spanned approximately 17 kilobases on the genome. The gene contains a glucocorticoid response element at the 5' region. The transcript was detected in the stomach and liver. The deduced amino acid sequence of the open reading frame showed about 60% positional identity with known human ADHs. This extent of homology is comparable to interclass similarity in the human ADH family. Thus, the newly identified gene, which is designated ADH6, governs the synthesis of an enzyme that belongs to another class of ADHs presumably with a distinct physiological role.

The human class I alcohol dehydrogenase gene cluster: three genes are tandemly organized in an 80-kb-long segment of the genome.

The class I alcohol dehydrogenases (ADH; EC 1.1.1.1) play a key role in hepatic alcohol catabolism. Three human class I ADH genes, ADH1, ADH2, and ADH3, which encode the alpha, beta, and gamma subunits respectively, have been isolated and mapped on chromosome 4q21-q23. Genomic cloning using a cosmid vector allowed us to obtain an 88-kb-long genomic segment, which was found to include an entire 80 kb of the class I ADH gene cluster. All three genes lie in the same transcriptional orientation and the order of genes is 5'-ADH3-ADH2-ADH1-3'. It may be of some significance that the order of transcriptional activation in the hepatic development, alpha----beta----gamma, is opposite to the order of gene arrangement. Several members of the AluI family and the KpnI (L1) family of interspersed repetitive sequences were mapped in this region. The divergence of insertional sites suggested that gene multiplication of the class I ADH genes had taken place in the earlier stages of human (or primate) evolution.

Studies on anti-ulcer agents. II. Synthesis and anti-ulcer activities of 6-isopropylazulene-1-sodium sulfonate derivatives.

A series of alkylazulene-1-sodium sulfonate derivatives which has an isopropyl group at 6-position were synthesized, and their anti-ulcer activities were examined in Shay pylorus-ligated rats. The values of lipophilicity (log P) as a parameter of these new azulene derivatives were also examined in reference to the structure-activity relationship. The optimum value of log P, which showed maximal anti-ulcer activity, was about -0.46. Among the derivatives of azulene examined, 3-ethyl-6-isopropylazulene-1-sodium sulfonate (compound IXb:XT1-785) exhibited the most potent inhibitory action against Shay ulcer, and its anti-peptic activity was similar to that of 3-ethyl-7-isopropylazulene-1-sodium sulfonate (KT1-32). It also had more activity than guaiazulene sodium sulfonate (GAS). Furthermore, KT1-785 was extremely stable under heating as compared to GAS.