Association of the MMP9 gene with childhood cedar pollen sensitization and pollinosis.

Matrix metalloproteinase 9 (MMP9) gene has been shown to be involved in the pathogenesis of allergic rhinitis (AR) and asthma. Previous studies suggested that single-nucleotide polymorphisms (SNPs) of the MMP9 gene conferred a risk for childhood asthma. However, whether the SNPs confer a risk for AR has not been previously investigated. The objective of this study was to investigate whether SNPs of the MMP9 gene are associated with risk of seasonal AR (pollinosis), perennial AR and allergen sensitization. A total of 670 school children were recruited in Japan and genotyped for functional polymorphism in the promoter (-1590C/T: rs3918242) and three amino-acid substitutions (R297Q: rs17576; P574R: rs2250889; R668Q: rs17577). Serum levels of total and specific IgE were determined. Disease status and other clinical characteristics of the subjects were investigated using a questionnaire. Associations between the MMP9 SNPs and both AR and serum IgE levels were evaluated. -1590C/T showed significant association with cedar pollinosis (corrected P (Pcor)=0.039). R668Q was in strong linkage disequilibrium (LD) with -1590C/T and showed significant association with cedar pollinosis (Pcor=0.023) and serum cedar pollen-specific IgE level (Pcor=0.022). A haplotype associated with -1590T and 668Q showed a significant association with cedar pollinosis, orchard grass pollinosis and cedar pollen-specific IgE (Pcor=0.0012, Pcor=0.0059 and Pcor=0.0041, respectively). R297Q and P574R were in weak LD with the rest of the SNPs and did not show significant association with disease. Compared with wild-type MMP9 protein (279R-574P-668R), a variant enzyme (279R-574P-668Q) that showed association with pollinosis had lower activity. However, lower enzyme activity was not associated with disease risk because another variant (279Q-574R-668R) showed lower enzyme activity but was not associated with pollinosis. The -1590T allele and its corresponding haplotype was associated with higher promoter activity and with pollen-specific IgE levels and pollinosis, suggesting that -1590C/T may have more impact on sensitization and disease development than R668Q. Our results suggest that the MMP9 gene confers susceptibility to cedar pollinosis in Japanese children. The MMP9 gene may be associated with pollinosis through sensitization processes.

International web-based survey of informed consent procedures in genetic epidemiological studies: towards the establishment of a research coordinator accreditation system.

We conducted an international web-based survey to obtain information about the informed consent (IC) procedures used in genetic epidemiological studies of multifactorial diseases. In total, 53 responses from 15 countries were obtained. We found that support personnel such as research coordinators took charge of a large part of the IC procedures, especially in the United States. Although most support personnel had documented and/or verbal confidentiality agreements, and all had undertaken IC training, accreditation examinations were only conducted in 60% of studies. Peripheral blood specimens were reported to be a major source for DNA extraction, whereas noninvasive methods were used in some studies. To undertake high-quality genetic epidemiological studies, participation of a large number of volunteers is essential for independent sets of samples that allow confirmation of results. On the basis of the survey results obtained, support personnel dedicated to completing the IC procedures are reported to be beneficial and indispensable in alleviating the burden on medical doctors, helping participants to make autonomous decisions and promoting genome research. The establishment of a training program and accreditation system for such personnel is warranted, especially in Japan, where medical staff usually finds it difficult to conduct IC procedures in clinical settings due to heavy workloads.

Association study of the C3 gene with adult and childhood asthma.

Bronchial asthma (BA) is a multifactorial disorder, the development of which is affected by both environmental and genetic factors. The complement system plays an important role in immunological response against invading microorganisms. It has been shown that complement-C3-deficient mice have reduced inflammation of asthmatic airways. Previously, we reported the association of four single nuclear proteins (SNPs) in the exons of the C3 gene with childhood and adult BA. The C3 gene, however, is a large gene, and functional SNPs associated with susceptibility to BA have not yet been identified. We analyzed 26 SNPs in the C3 gene and its promoter region to narrow down the regions showing association with childhood and adult BA. Childhood and adult atopic BA patients and healthy child and adult controls were recruited from urban cities in Japan and genotyped. In SNP analysis, an SNP (SNP24, rs11569562) located in intron 31 of the C3 gene was associated with adult BA [corrected P (Pcor) = 0.030]. In linkage disequilibrium (LD) block 4 spanning exons 24-41, the frequency of the CCC haplotype in adult BA was significantly higher than that in adult controls (Pcor = 0.038). Neither the SNP nor the haplotype showing association with adult BA demonstrated a significant association with serum total immunoglobulin E (IgE) level in BA patients and controls. Our results suggest that LD block 4 confers susceptibility to adult BA with mechanisms relevant to the effector phase of allergic inflammation.

Haplotype analysis of the human collectin placenta 1 (hCL-P1) gene.

Collectins are a family of C-type lectins found in vertebrates. These proteins have four regions, a relatively short N-terminal region, a collagen-like region, an alpha-helical coiled coil, and a carbohydrate recognition domain. Collectins are involved in host defense through their ability to bind carbohydrate antigens on microorganisms. Type A scavenger receptors are classical-type scavenger receptors that also have collagen-like domains. We previously described a new scavenger receptor, collectin from placenta [collectin placenta 1 (CL-P1)]. CL-P1 is a type II membrane protein with all four regions. We found that CL-P1 can bind and phagocytize both bacteria and yeast. In addition to that, it reacts with oxidized low-density lipoprotein (LDL) but not with acetylated LDL. These results suggest that CL-P1 might play important roles in host defenses and/or atherosclerosis formation. One rational strategy to study the role of CL-P1 in these pathological conditions would be to perform a haplotype association study using human samples. As a first step for this strategy, we analyzed the haplotype structure of the CL-P1gene. By sequencing the CL-P1 gene in ten Japanese volunteers, we identified five single-nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 29%. To obtain SNPs in the 5'-upstream region of the gene, we screened a total of 20 SNPs described in the database and finally picked up one SNP for the present study. Thus, a total of six SNPs, one in the 5'-upstream region, two in intron 2, one in exon 5, and two in exon 6, were used to analyze the haplotype structure of the gene, with DNAs derived from 54 individuals (108 alleles). The analysis revealed that only two of six SNPs showed significant linkage disequilibrium ( r(2) > 0.5) with each other. This haplotype information may be useful in disease-association studies in which a contribution of the CL-P1 gene has been suspected, especially in immunological disturbance or atherosclerosis. Two SNPs in exon 6, both leading to amino acid substitutions, could be candidates for influencing disease susceptibility.

Linkage and association analyses of the osteoprotegerin gene locus with human osteoporosis.

Osteoprotegerin (OPG), a secreted glycoprotein and a member of the tumor necrosis factor receptor superfamily, is considered to play an important role in the regulation of bone resorption by modifying osteoclast differentiation. Overexpression of OPG in mice has been reported to result in osteopetrosis, whereas targeted disruption of OPG in mice has been associated with osteoporosis. Accordingly, OPG could be a strong candidate gene for susceptibility to human osteoporosis. Here, we analyzed whether OPG is involved in the etiology of osteoporosis using both linkage and association analyses. We recruited 164 sib pairs in Gunma prefecture, which is located in the central part of Honshu (mainland Japan), for a linkage study, and 394 postmenopausal women in Akita prefecture, which is in the northern part of Honshu, for an association study. We identified two microsatellite polymorphisms in the linkage study, and six single-nucleotide polymorphisms (SNPs) in the OPG region for the association study. Although, no evidence of significant linkage between OPG and osteoporosis was found, a possible association of one SNP, located in the promoter region of the gene, was identified. A haplotype analysis with the six SNPs revealed that four major haplotypes account for 71% of the alleles in the Japanese population.

Methylenetetrahydrofolate reductase genotype, vitamin B12, and folate influence plasma homocysteine in hemodialysis patients.

Hyperhomocysteinemia, a well-recognized cardiovascular risk factor, is frequent in hemodialysis (HD) patients. A common polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, C-->T substitution at nucleotide 677, is associated with homocysteine (Hcy) level elevation. We examined whether three factors involved in the methionine cycle could influence plasma Hcy concentrations in HD patients: MTHFR polymorphism; vitamin B12, an essential cofactor; and folate, the substrate. In a cross-sectional study, serum vitamin B12, folate, and plasma Hcy were measured and MTHFR genotyping was performed in 534 HD patients. Effects of MTHFR genotypes, vitamin B12, and folate on plasma Hcy levels were examined in 450 HD patients not administered vitamin B12 or folate. To examine the effect of vitamin B12 on plasma Hcy concentrations, we compared plasma Hcy concentrations in HD patients with and without vitamin B12 supplementation. To examine whether functional vitamin B12 deficiency exists even in HD patients with normal vitamin B12 concentrations, 15 HD patients (serum vitamin B12 concentrations, 250 to 2,100 pg/mL) were treated with vitamin B12 (mecobalamin, 1.5 mg/d) for 8 weeks. Serum concentrations of methylmalonic acid (MMA) and vitamin B12 were measured. Hcy levels were higher and folate levels were lower in patients with the TT and CT genotypes compared with patients with the CC genotype. Analysis of covariance to determine independent predictors of high Hcy levels identified low serum vitamin B12 and folate levels and high albumin (Alb) levels in CC-genotype patients, low folate levels and high Alb levels in CT-genotype patients, and low folate levels in TT-genotype patients. Plasma Hcy levels were lower in CC- and CT-genotype patients with vitamin B12 supplementation than in those without supplementation. Vitamin B12 supplementation for 8 weeks significantly reduced MMA concentrations in HD patients with normal serum vitamin B12 concentrations. These results indicate that MTHFR genotype influences the correlation of Hcy level with vitamin B12 and folate levels in HD patients. Functional vitamin B12 deficiency may exist, even in HD patients with normal vitamin B12 concentrations. The efficacy of vitamin B12 and folate supplementation on plasma Hcy levels may depend on MTHFR genotype.