The Definitive Haplotype Database (D-HaploDB) is a web-accessible resource of genome-wide definitive haplotypes determined from a collection of Japanese complete hydatidiform moles (CHMs), each of which carries a genome derived from a single sperm. Currently, the database contains genotypes for 281 439 common SNPs from 74 CHMs which were determined by a high-throughput array-based oligonucleotide hybridization technique. The database also presents maps of haplotype blocks and linkage disequilibrium bins together with tagSNPs that might prove useful for association studies of disease genes. Cryptic relatedness among the samples in this study is unlikely, because the formation of a CHM is a maternal event of rare sporadic occurrence, and its genotype is that of the incoming sperm. This is demonstrated by the absence of long extended shared haplotypes (ESHs). The D-HaploDB is freely accessible via the Internet at http://orca.gen.kyushu-u.ac.jp.

The majority of complete hydatidiform moles (CHMs) harbor duplicated haploid genomes that originate from sperm. This makes CHMs more advantageous than conventional diploid cells for determining haplotypes of SNPs and copy-number variations (CNVs), because all of the genetic variants in a CHM genome are homozygous. Here we report SNP and CNV haplotype structures determined by analysis of 100 CHMs from Japanese subjects via high-density DNA arrays. The obtained haplotype map should be useful as a reference for the haplotype structure of Asian populations. We resolved common CNV regions (merged CNV segments across the examined samples) into CNV events (clusters of CNV segments) on the basis of mutual overlap and found that the haplotype backgrounds of different CNV events within the same CNV region were predominantly similar, perhaps because of inherent structural instability.

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10(-9), odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4(+) and CD19(+) peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset.

The functional single nucleotide polymorphism (SNP) in the MDM2 promoter region, SNP309, is known to be associated with various diseases, particularly cancer. Although many studies have been performed to demonstrate the mechanism of allele-specific expression (ASE) on SNP309, they have only utilized in vitro techniques. It is unknown whether ASE of MDM2 is ascribed solely to SNP309, in vivo.

Complete hydatidiform moles (CHMs) are tissues carrying duplicated haploid genomes derived from single sperms, and detecting copy number variations (CNVs) in CHMs is assumed to be sensitive and straightforward methods. We genotyped 108 CHM genomes using Affymetrix SNP 6.0 (GEO#: GSE18642) and Illumina 1 M-duo (GEO#: GSE54948). After quality control, we obtained 84 definitive haplotype consisting of 1.7 million SNPs and 2339 CNV regions. The results are presented in the database of our web site (http://orca.gen.kyushu-u.ac.jp/cgi-bin/gbrowse/humanBuild37D4_1/).

The haplotype map constructed by the HapMap Project is a valuable resource in the genetic studies of disease genes, population structure, and evolution. In the Project, Caucasian and African haplotypes are fairly accurately inferred, based mainly on the rules of Mendelian inheritance using the genotypes of trios. However, the Asian haplotypes are inferred from the genotypes of unrelated individuals based on population genetics, and are less accurate. Thus, the effects of this inaccuracy on downstream analyses needs to be assessed. We determined true Japanese haplotypes by genotyping 100 complete hydatidiform moles (CHM), each carrying a genome derived from a single sperm, using Affymetrix 500 K Arrays. We then assessed how inferred haplotypes can differ from true haplotypes, by phasing pseudo-individualized true haplotypes using the programs PHASE, fastPHASE, and Beagle. We found that, at various genomic regions, especially the MHC locus, the expansion of extended haplotype homozygosity (EHH), which is a measure of positive selection, is obscured when inferred Asian haplotype data is used to detect the expansion. We then mapped the genome using a new statistic, XDiHH, which directly detects the difference between the true and inferred haplotypes, in the determination of EHH expansion. We also show that the true haplotype data presented here is useful to assess and improve the accuracy of phasing of Asian genotypes.

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

Retinopathy of prematurity (ROP) is a complex disease with a genetic predisposition, but little is known about its genetic background. It has a clinical resemblance to familial exudative vitreoretinopathy (FEVR), a hereditary disease characterized by defects in the development of retinal vessels. Several studies have suggested that mutations in the causative genes for FEVR may account for a proportion of advanced ROP, but conflicting data have also been reported for some variants. To address the possibility of genetic involvement of FEVR genes in ROP, we performed comprehensive sequence analyses of 53 Japanese patients with advanced ROP for the FEVR-causing genes.