Hironori Iwasaki

Association of a Haplotype (196Phe/532Ser) in the Interleukin-1-Receptor- Associated Kinase (IRAK1) Gene With Low Radial Bone Mineral Density in Two Independent Populations

Osteoporosis, a multifactorial common disease, is believed to result from the interplay of multiple environmental and genetic determinants, including factors that regulate bone mineral density. Interleukin‐1 (IL‐1) is one of the most potent bone‐resorbing factors, and interleukin‐1‐associated kinase 1 (IRAK1) is an essential effector of the IL‐1 receptor signaling cascade. In genetic studies of two independent populations of postmenopausal women (cohort A: 220 individuals and cohort T: 126 individuals) from separated geographical regions of Japan, we found that radial bone mineral density levels had similar associations with IRAK1 genotypes in both populations. Two amino acid‐substituting variations in the gene, encoding Phe196Ser and Ser532Leu, were in complete linkage disequilibrium (D′ = 1.0000, r2 = 1.0000, χ2 = 192.000, p = 1.2 × 10−43), and we found two exclusive haplotypes (196F/532S, frequency 0.74; 196S/532L, frequency 0.26) of the IRAK1 gene among our test subjects. In both populations, a significant association with decreased radial bone mineral density was identified with haplotype 196F/532S (in cohort A: r = 0.21, p = 0.0017; in cohort T: r = 0.23, p = 0.011). Radial bone mineral density was lowest among 196F/532S homozygotes, highest among 196S/532L homozygotes, and intermediate among heterozygotes. Accelerated bone loss also correlated with the 196F/532S haplotype in a 5‐year follow‐up. These results suggest that variation of IRAK1 may be an important determinant of postmenopausal osteoporosis, in part through the mechanism of accelerated postmenopausal bone loss.

Association of a Trp16Ser variation in the gonadotropin releasing hormone signal peptide with bone mineral density, revealed by SNP-dependent PCR typing

Osteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone mineral density (BMD). Among those factors, adequate estrogen is essential for achievement of peak bone mass as well as for postmenopausal maintenance of skeletal homeostasis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus is the primary determinant in the hypothalamic-pituitary-gonadal feedback system. In genetic studies of 384 postmenopausal Japanese women, we found a significant association between BMD and an amino acid variation (Trp16Ser) located within the signal peptide of GnRH (r = 0.143, P = 0.005). These results were achieved by genotyping all subjects using a newly developed SNP-dependent PCR method. This automated, high-throughput, and inexpensive procedure is suitable for typing large numbers of samples. BMD was lowest among 16Ser/Ser homozygotes, highest among 16Trp/Trp homozygotes, and intermediate among heterozygotes. A case-control study involving 125 osteoporosis patients and 92 healthy controls revealed a significant association between the presence of a 16Ser GnRH allele and affected status (chi(2) = 4.74, P = 0.041). The results suggested that variation of the GnRH signal peptide may be an important risk factor for postmenopausal osteoporosis.

Thirteen single-nucleotide polymorphisms in the human osteopontin gene identified by sequencing of the entire gene in Japanese individuals

AbstractOsteopontin (OPN) is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts. We systematically surveyed the entire structure of the OPN gene for single-nucleotide polymorphisms (SNPs) by directly sequencing 48 alleles derived from 24 unrelated Japanese individuals. We identified 13 SNPs in the OPN gene. Ten polymorphisms were identified in introns 1, 3, and 5; 2 in the coding region of exons 6 and 7; and 1 in the 3′ untranslated region of exon 7. Allele frequencies for some of the polymorphisms were significantly different from those reported in the United States National Center for Biotechnology Information (NCBI) dbSNP database. These polymorphisms will be useful in genetic studies to evaluate the role of OPN proteins in bone metabolism.

Association of the -381T/C promoter variation of the brain natriuretic peptide gene with low bone-mineral density and rapid postmenopausal bone loss

AbstractOsteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone-mineral density (BMD). Recent quantitative trait locus analysis in human suggested a possible involvement of chromosomal region 1p36.2−p36.3 for determination of BMD. The brain natriuretic peptide (BNP, also named NPPB) gene lies within this candidate region for BMD determination. Overexpression of the BNP resulted in skeletal overgrowth in transgenic mice. Association analysis between nucleotide variations of the BNP gene and radial BMD in 378 Japanese postmenopausal women revealed a significant association of the −381T/C variation of the BNP gene with radial BMD (r = 0.17, P = 0.01). Homozygous T-allele carriers had the lowest BMD values (0.395 ± 0.056g/cm2), homozygous C-allele carriers had the highest (0.429 ± 0.051g/cm2), and heterozygous individuals had intermediate radial BMD values (0.405 ± 0.048g/cm2), indicating a dosage effect. Accelerated bone loss also correlated with the −381 T allele in a 5-year follow-up study (r = 0.21, P = 0.017). These results suggest that variation of BNP may be an important determinant of postmenopausal osteoporosis, in part through the mechanism of accelerated postmenopausal bone loss.

Association of a single nucleotide variant in the human tumour necrosis factor alpha promoter region with decreased bone mineral density

Background : Tumour necrosis factor alpha (TNF f ) has come to be regarded as a potential osteoporotic factor, because it has stimulatory effects on cells of the osteoclast lineage and has been implicated in the pathogenesis of bone loss associated with oestrogen deficiency. We recently described genetic linkage between the TNF f locus and human osteoporosis by sib-pair analysis. However, the molecular mechanism by which this locus regulates bone mineral density (BMD) remains unknown. Aim : We investigated whether the observed linkage reflects a sequence variation which might affect expression of the TNF f gene or alter the function of TNF f protein. Subjects and methods : We examined three single-nucleotide polymorphisms (SNPs) of the TNF f gene in a group of 390 postmenopausal Japanese women living in northern Japan. Minor-allele frequencies for the three SNPs (-1031C, -863A and -857T) in this population were 0.16, 0.13 and 0.20, respectively. Results : Among the three SNPs examined, we observed a significant correlation only between the presence of a T allele at nt -1031 and decreased BMD, by analysis of variance. Among the three genotypic groups at nt -1031, mean BMD values were significantly higher in the T-negative genotype (C/C homozygotes; mean SD = 0.342 - 0.052 g cm -2 ), compared with T-positive genotypes (T/T homozygotes, 0.309 - 0.062 g cm -2; p = 0.0253 and T/C heterozygotes, 0.305 - 0.062 g cm -2 ; p = 0.0164). Conclusions : Given the lines of evidence from different genetic studies, we suggest that TNF f may play a role in pathogenesis of osteoporosis.

Linkage disequilibrium and haplotype analysis among ten single-nucleotide polymorphisms of interleukin 11 identified by sequencing of the gene

AbstractInterleukin 11 (IL11) is a member of the interleukin 6 (IL6)-related cytokine subfamily, which stimulates T cell-dependent development of immunoglobulin-producing B cells. IL11 is also an important paracrine regulator of bone metabolism that induces formation of osteoclasts. In the work reported here, we sequenced the entire IL11 structural gene of 48 alleles in a Japanese test population. These experiments identified ten single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies. One polymorphism was identified upstream of exon 1, one in exon 3, four in intron 4 and four in the 3′ untranslated region (3′UTR) of exon 5. Based on the genotype data, we constructed six haplotypes in the tested population. Two-way comparisons of SNPs revealed two combinations in complete linkage disequilibrium, one with SNPs at nucleotide positions 2753, 3644, 5154, and 5568, and another with SNPs at positions 3686, 5141, and 5734. These results will be useful in disease-association studies where a contribution of the human IL11 gene has been suspected, especially in disorders affecting immune response and bone metabolism.

Five novel single-nucleotide polymorphisms of human interferon gamma identified by sequencing the entire gene

AbstractInterferon gamma (IFNG) plays important roles in the regulation of bone remodelling. We describe here six single-nucleotide polymorphisms (SNPs) in the IFNG gene, five of which are novel, and their allelic frequencies in the Japanese population, as determined by sequencing 48 alleles of the entire gene. Four of these polymorphisms were identified inside the third intron, at nucleotide (nt) positions 2459 (A/G), 2671 (T/C), 3177 (T/G), and 3273 (G/A). In exon 4, SNPs were identified at nt positions 5199 (A/T) and 5272 (A/G). These polymorphic sites will be useful for genetic studies of disorders that affect the inflammatory process or calcium metabolism.

Linkage disequilibrium and haplotype analysis among four novel single-nucleotide polymorphisms in the human leukemia inhibitory factor (LIF ) gene

AbstractLeukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite the possible importance of LIF as a therapeutic target, little is known about the bioregulation of the human LIF gene. We here sequenced the entire structure of the LIF gene of 48 alleles in the Japanese population. These experiments identified four single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies from a 48-allele sequence in the Japanese population. All four SNPs found in the LIF gene were located within exon 3, that is, a C/T at nucleotide (nt) position 3951, a C/G at nt position 4376, an A/C at nt position 4442, and a G/A at nt position 5961 (nucleotide numbering starts from the ATG start codon). Based on the genotypic data, we constructed four major haplotypes in the tested population. Two-way comparisons of SNPs revealed complete linkage disequilibrium between SNPs at positions 3951, 4376, and 4442. These results may prove to be useful as genetic markers for population-based disease-association studies in osteoporosis.

Novel single nucleotide polymorphisms of the human colony-stimulating factor 2 (CSF2) gene identified by sequencing the entire gene

AbstractWe describe three single nucleotide polymorphisms (SNPs) of the human colony-stimulating factor 2 (CSF2) gene and their allelic frequencies, as determined by direct sequencing of 48 alleles of the entire CSF2 gene. Three polymorphisms were identified, at nucleotide positions 1816 (T/C), 2284 (C/T), and 3079 (G/A). These polymorphisms will be useful in genetic studies not only of hematologic disorders but also of disorders of bone metabolism.

Three TNF α single nucleotide polymorphisms in the Japanese population

Background : Tumour necrosis factor-alpha (TNF f ) is an essential regulator of immune responses and is implicated to relate to several types of disease susceptibilities. Population information on polymorphisms is essential for the study of genetic diseases. Aim : To obtain accurate information about single nucleotide polymorphisms (SNPs) in the TNF f gene in the Japanese population. Subjects and Methods : The entire TNF f gene was screened for SNPs by directly sequencing 48 chromosomes derived from 24 unrelated Japanese individuals. Allele frequencies of each polymorphism were determined and compared with those previously reported in other populations. Results : Three SNPs, -308G/A at nt -308, IVS1 + 125G/A at nt 492 and IVS3 + 104G/A at nt 1359 were observed, of which one (IVS3 + 104G/A at nt 1359) was novel. In addition, allele frequencies of -308G/A were remarkably different from those presented in the NCBI dbSNP, indicating a significant ethnic difference. Conclusions : The polymorphisms and allele frequencies obtained in this study will be useful for genetic studies of common diseases such as osteoporosis and rheumatoid arthritis in the Japanese population.