Large scale genotyping study for asthma in the Japanese population
© Noguchi et al; licensee BioMed Central Ltd. 2009
Received: 21 August 2008
Accepted: 31 March 2009
Published: 31 March 2009
Asthma is a complex phenotype that is influenced by both genetic and environmental factors. Genome-wide linkage and association studies have been performed to identify susceptibility genes for asthma. These studies identified new genes and pathways implicated in this disease, many of which were previously unknown.
To perform a large-scale genotyping study to identify asthma-susceptibility genes in the Japanese population.
We performed a large-scale, three-stage association study on 288 atopic asthmatics and 1032 controls, by using multiplex PCR-Invader assay methods at 82,935 single nucleotide polymorphisms (SNPs) (1st stage). SNPs that were strongly associated with asthma were further genotyped in samples from asthmatic families (216 families, 762 members, 2nd stage), 541 independent patients, and 744 controls (3rd stage).
SNPs located in the 5' region of PEX19 (rs2820421) were significantly associated with P < 0.05 through the 1st to the 3rd stage analyses; however, the P values did not reach statistically significant levels (combined, P = 3.8 × 10-5; statistically significant levels with Bonferroni correction, P = 6.57 × 10-7). SNPs on HPCAL1 (rs3771140) and on IL18R1 (rs3213733) were associated with asthma in the 1st and 2nd stage analyses, but the associations were not observed in the 3rd stage analysis.
No association attained genome-wide significance, but several loci for possible association emerged. Future studies are required to validate these results for the prevention and treatment of asthma.
Asthma is the most common chronic disorder in children, and asthma exacerbation is an important cause of childhood morbidity and hospitalization. Currently, approximately 300 million people worldwide have asthma, and this disease claims the lives of 180,000 people every year .
Asthma and atopy are complex phenotypes that are influenced by both genetic and environmental factors. Twin studies have supported the role of a strong genetic contribution with a heritability of 0.71 , and asthma shows a familial risk higher than that of many common diseases such as stroke, epilepsy, and most types of cancer . Atopy is characterized by increased levels of immunoglobulin E (IgE) against common environmental allergens, and is considered the strongest predisposing factor for asthma. Majority of children with asthma develop specific IgE against house dust mites, and dust mite allergy is strongly associated with asthma [4, 5], however, only a small subset of subjects with dust mite allergy develops asthma . These data suggest that other factors are involved in the development of asthma, and genome-wide linkage and association studies have been used to find novel asthma genes and their associated pathways . Using family and case-referent panels of European populations and based on the genome-wide association study conducted on asthma, Moffatt et al. identified that the cis-acting single nucleotide polymorphisms (SNPs) in ORMDL3 were associated with asthma , and the results were replicated in independent populations .
In order to identify novel asthma susceptibility genes, we performed a large-scale, 3-stage association study using the Japanese population. No association attained genome-wide significance, but several loci for possible association emerged. Further studies are required to validate these results in the future.
Subjects and genotyping results
Clinical details of the asthmatic families
No. of Families
No. of children
No. of affected children
Mean age (yeas ± SD)
10.9 ± 2.4
Log(total IgE) (IU/ml ± SD)
2.8 ± 0.6
No. of parents
Mean age (yeas ± SD)
40.7 ± 7.5
Log(total IgE) (IU/ml ± SD)
2.0 ± 0.7
No of SNPs
Childhood atopic Asthma: 288
General Japanese population: 1032
χ2 test (allelic)
Family with childhood atopic asthma (216 families, 762 members)
Childhood atopic Asthma: 541
Non atopic control: 752
χ2 test (allelic)
Large-scale genotyping using 82,935 randomly selected gene-based SNPs was carried out using the high-throughput multiplex PCR-Invader assay method as described previously . The population frequency of asthma in Japan was 0.065 , and the statistical power of the 1st stage analysis was 0.93 and 0.44 at the alpha level of 0.001 and 0.000001, respectively if the relative risk for asthma in those persons carrying a putative risk allele is 2 and the high risk allele frequency is 0.3 compared with that in persons without the allele. Therefore, our sample size may not be enough to detect a low risk allele.
The SNPs genotyped in the 1st stage analysis were those identified in the JSNP project . There are 2 approaches to the construction of SNP databases: one is genome-wide screening, and the other is gene-based screening. Although SNPs around genes are likely to be functional SNPs, it should be noted that SNPs outside genes have also been found to be associated with diseases.
In the present study, the distribution of allelic frequencies was largely even, with an average minor allele frequency of 24%. The SNPs with problematic genotyping in the 1st stage were flagged, and we excluded these SNPs from the analysis (n = 4683). Moreover, Hardy-Weinberg equilibrium was calculated using the χ2 test with 2 degrees of freedom on the basis of the observed and expected genotype frequencies; SNPs with P < 0.001 (n = 2160) were excluded from the analysis. After the exclusions, 76,092 autosomal SNPs were available for analysis. The significance of the differences in the allele frequencies in case-control comparisons was determined by the χ2 test with 1 degree of freedom. The distribution of the observed P values was as follows: P < 0.0001, 20 SNPs (0.0263%); P < 0.001, 146 SNPs (0.192%); and P < 0.01, 1111 SNPs (1.46%). The genomic inflation factor of the study population was calculated using the method described by Devlin et al  and was found to be 1.13. A previous study has reported that the inclusion of different proportions of individuals from different regions of Japan in case and control groups can lead to an exaggerated number of false-positive results when the sample sizes are large, and it has recommended the exclusion of subjects belonging to the Ryukyu (southern island of Japan) cluster . The patients of the present study were from the Kanto and Kinki regions, and the controls were from the Kinki region alone. On the basis of the results of a previous simulation study, we can state that the subjects from the Kanto region are not genetically different from those from the Kinki region ; moreover, we did not include cases or controls from the Ryukyu island. However, we cannot exclude the possibility that population stratification exists in our case-control samples.
Association results of three SNPs
Our results revealed a few candidate genes of pediatric asthma. Though the P values did not reach statistically significant levels, SNPs in the 5' region of PEX19 were consistently associated with asthma in the 1st to the 3rd stage analyses. On the contrary, SNPs on hippocalcin-like 1 (HPCAL1, rs3771140) and on interleukin (IL)18R1 (rs3213733) were associated with asthma in the 1st and 2nd stage analyses, but the associations were not observed in the 3rd stage analysis.
PEX19 is a human ortholog of the Saccharomyces cerevisiae gene, Pex19p, which encodes an oleic acid-inducible, farnesylated protein essential for peroxisome biogenesis . Peroxisomes function to rid cells of toxic substances, such as hydrogen peroxide, or other metabolites and are essential for human survival. It has been reported that, in mice cells, Pex19p interacts with p19ARF in the cell cytoplasm and excludes p19ARF from the nucleus, leading to a concurrent inactivation of p53 function . p19ARF is encoded by the cyclin-dependent kinase inhibitor 2a (Cdkn2a), and the human ortholog of Cdkn2a (CDKN2A) has been extensively examined in relation to cancer and aging . Down regulation of Pex19p by its antisense expression resulted in increased levels of p19ARF, increased p53 function, and a p53/p21WAF1-mediated senescence . p19ARF proteins regulate p53 pathways, and the disruption of these proteins results in aberrant cell cycle regulation and perturbation of apoptotic response . Recently, it has been shown that p19Arf overexpression resulted in impaired transition from CD4(-)CD8(-) (double negative stage) to CD4(+)CD8(+) (double positive stage), leading to impaired thymocyte expansion and development . Functions of other 3 genes, WDR42A, PEA15 and COPA for the immune systems are currently not well understood.
IL-18 was initially identified as a potent interferon gamma (IFNγ)-inducing factor, and was later shown to have the potential to induce IL-4 production. Therefore, IL-18 can induce both IFNγ and IL-4 responses depending on its cytokine environment . Recently, polymorphisms in IL18 receptor 1 (IL18R1) have been reported to be associated with asthma and bronchial hyperresponsiveness in the European population [23, 24]. Therefore, IL18R1 is a good candidate for asthma, and further replication studies are required to determine the causal variants.
In the present study, we did not detect statistically significant associations of asthma with SNPs. This may be because of the limited statistical power, considering the sample size and extent of multiple testing. Our statistical power in the 3rd-stage analysis was 95%, but the powers of the 1st- and 2nd-stage analyses were 0.95 and 0.85 with a genotypic relative risk of 2.0, and 0.41 and 0.45 with a genotypic relative risk of 1.5, respectively. Another reason is population stratification in the 1st-stage analysis. Although we collected the case and control samples from geographic regions wherein people are considered to be genetically similar, population stratification may exists in our case-control samples, leading to the inflated test statistics.
In summary, we performed a large scale genotyping study to identify the susceptibility genes for pediatric asthma. Although no SNPs attained genome-wide significance, we identified several loci with a possible association with asthma. Further studies are required to validate these results for the prevention and treatment of asthma.
We thank Drs. Satoko Nakahara, Tetsuo Nogami, Michiharu Inudou, for collecting samples and thank all family members who participated in the study. This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (18018003).
- Braman SS: The global burden of asthma. Chest. 2006, 130: 4S-12S. 10.1378/chest.130.1_suppl.4S.View ArticlePubMedGoogle Scholar
- Nystad W, Roysamb E, Magnus P, Tambs K, Harris JR: A comparison of genetic and environmental variance structures for asthma, hay fever and eczema with symptoms of the same diseases: a study of Norwegian twins. Int J Epidemiol. 2005, 34: 1302-1309. 10.1093/ije/dyi061.View ArticlePubMedGoogle Scholar
- Hemminki K, Li X, Sundquist K, Sundquist J: Familial risks for asthma among twins and other siblings based on hospitalizations in Sweden. Clin Exp Allergy. 2007, 37: 1320-1325. 10.1111/j.1365-2222.2007.02737.x.View ArticlePubMedGoogle Scholar
- Sears MR, Herbison GP, Holdaway MD, Hewitt CJ, Flannery EM, Silva PA: The relative risks of sensitivity to grass pollen, house dust mite and cat dander in the development of childhood asthma. Clin Exp Allergy. 1989, 19: 419-424. 10.1111/j.1365-2222.1989.tb02408.x.View ArticlePubMedGoogle Scholar
- Shibasaki M, Tajima K, Morikawa A, Mitsuhashi M, Sumazaki R, Tokuyama K: Relation between frequency of asthma and IgE antibody levels against Dermatophagoides farinae and total serum IgE levels in schoolchildren. J Allergy Clin Immunol. 1988, 82: 86-94. 10.1016/0091-6749(88)90056-5.View ArticlePubMedGoogle Scholar
- Shibasaki M, Noguchi E, Takeda K, Takita H: Distribution of IgE and IgG antibody levels against house dust mites in schoolchildren, and their relation with asthma. J Asthma. 1997, 34: 235-242. 10.3109/02770909709068194.View ArticlePubMedGoogle Scholar
- Vercelli D: Discovering susceptibility genes for asthma and allergy. Nat Rev Immunol. 2008, 8: 169-182. 10.1038/nri2257.View ArticlePubMedGoogle Scholar
- Moffatt MF, Kabesch M, Liang L, Dixon AL, Strachan D, Heath S, Depner M, von Berg A, Bufe A, Rietschel E, Heinzmann A, Simma B, Frischer T, Willis-Owen SA, Wong KC, Illig T, Vogelberg C, Weiland SK, von Mutius E, Abecasis GR, Farrall M, Gut IG, Lathrop GM, Cookson WO: Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma. Nature. 2007, 448: 470-473. 10.1038/nature06014.View ArticlePubMedGoogle Scholar
- Hirota T, Harada M, Sakashita M, Doi S, Miyatake A, Fujita K, Enomoto T, Ebisawa M, Yoshihara S, Noguchi E, Saito H, Nakamura Y, Tamari M: Genetic polymorphism regulating ORM1-like 3 (Saccharomyces cerevisiae) expression is associated with childhood atopic asthma in a Japanese population. J Allergy Clin Immunol. 2008, 121: 769-770. 10.1016/j.jaci.2007.09.038.View ArticlePubMedGoogle Scholar
- Noguchi E, Yokouchi Y, Zhang J, Shibuya K, Shibuya A, Bannai M, Tokunaga K, Doi H, Tamari M, Shimizu M, Shirakawa T, Shibasaki M, Ichikawa K, Arinami T: Positional identification of an asthma susceptibility gene on human chromosome 5q33. Am J Respir Crit Care Med. 2005, 172: 183-188. 10.1164/rccm.200409-1223OC.View ArticlePubMedGoogle Scholar
- Haga H, Yamada R, Ohnishi Y, Nakamura Y, Tanaka T: Gene-based SNP discovery as part of the Japanese Millennium Genome Project: identification of 190,562 genetic variations in the human genome. Single-nucleotide polymorphism. J Hum Genet. 2002, 47: 605-610. 10.1007/s100380200092.View ArticlePubMedGoogle Scholar
- Morikawa A: Asthma: what we learn from each other's problems? The present state of asthma in Japan and Japanese Pediatric Guidelines for the Treatment and Management of Asthma (JPGTMA). Paediatr Respir Rev. 2006, 7 (Suppl 1): S121-122. 10.1016/j.prrv.2006.04.153.View ArticlePubMedGoogle Scholar
- Devlin B, Roeder K, Wasserman L: Genomic control, a new approach to genetic-based association studies. Theor Popul Biol. 2001, 60: 155-166. 10.1006/tpbi.2001.1542.View ArticlePubMedGoogle Scholar
- Yamaguchi-Kabata Y, Nakazono K, Takahashi A, Saito S, Hosono N, Kubo M, Nakamura Y, Kamatani N: Japanese population structure, based on SNP genotypes from 7003 individuals compared to other ethnic groups: effects on population-based association studies. Am J Hum Genet. 2008, 83: 445-456. 10.1016/j.ajhg.2008.08.019.PubMed CentralView ArticlePubMedGoogle Scholar
- Martin ER, Monks SA, Warren LL, Kaplan NL: A test for linkage and association in general pedigrees: the pedigree disequilibrium test. Am J Hum Genet. 2000, 67: 146-154. 10.1086/302957.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirov G, Jones I, McCandless F, Craddock N, Owen MJ: Family-based association studies of bipolar disorder with candidate genes involved in dopamine neurotransmission: DBH, DAT1, COMT, DRD2, DRD3 and DRD5. Mol Psychiatry. 1999, 4: 558-565. 10.1038/sj.mp.4000565.View ArticlePubMedGoogle Scholar
- Gotte K, Girzalsky W, Linkert M, Baumgart E, Kammerer S, Kunau WH, Erdmann R: Pex19p, a farnesylated protein essential for peroxisome biogenesis. Mol Cell Biol. 1998, 18: 616-628.PubMed CentralView ArticlePubMedGoogle Scholar
- Sugihara T, Kaul SC, Kato J, Reddel RR, Nomura H, Wadhwa R: Pex19p dampens the p19ARF-p53-p21WAF1 tumor suppressor pathway. J Biol Chem. 2001, 276: 18649-18652. 10.1074/jbc.C100011200.View ArticlePubMedGoogle Scholar
- Kim WY, Sharpless NE: The regulation of INK4/ARF in cancer and aging. Cell. 2006, 127: 265-275. 10.1016/j.cell.2006.10.003.View ArticlePubMedGoogle Scholar
- Sharpless NE, DePinho RA: The INK4A/ARF locus and its two gene products. Curr Opin Genet Dev. 1999, 9: 22-30. 10.1016/S0959-437X(99)80004-5.View ArticlePubMedGoogle Scholar
- Miyazaki M, Miyazaki K, Itoi M, Katoh Y, Guo Y, Kanno R, Katoh-Fukui Y, Honda H, Amagai T, van Lohuizen M, Kawamoto H, Kanno M: Thymocyte Proliferation Induced by Pre-T Cell Receptor Signaling Is Maintained through Polycomb Gene Product Bmi-1-Mediated Cdkn2a Repression. Immunity. 2008, 28: 231-245. 10.1016/j.immuni.2007.12.013.View ArticlePubMedGoogle Scholar
- Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H: Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev. 2001, 12: 53-72. 10.1016/S1359-6101(00)00015-0.View ArticlePubMedGoogle Scholar
- Reijmerink NE, Postma DS, Bruinenberg M, Nolte IM, Meyers DA, Bleecker ER, Koppelman GH: Association of IL1RL1, IL18R1, and IL18RAP gene cluster polymorphisms with asthma and atopy. J Allergy Clin Immunol. 2008, 122: 651-654. 10.1016/j.jaci.2008.06.030.View ArticlePubMedGoogle Scholar
- Zhu G, Whyte MK, Vestbo J, Carlsen K, Carlsen KH, Lenney W, Silverman M, Helms P, Pillai SG: Interleukin 18 receptor 1 gene polymorphisms are associated with asthma. Eur J Hum Genet. 2008, 16: 1083-1090. 10.1038/ejhg.2008.67.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.