The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.

High-temperature stress causes abortive male reproductive development in many plant species. Here, we report a putative mechanism of high-temperature injury during anther early development in barley plants (Hordeum vulgare L). Under high-temperature conditions (30 degrees C day/25 degrees C night), cell-proliferation arrest, increased vacuolization, over-development of chloroplasts, and certain abnormalities of the mitochondria, nuclear membrane, and rough endoplasmic reticulum (RER) were observed in developing anther cells, but not in developing ovule cells. Moreover, premature degradation of tapetum cells and premature progression to meiotic prophase in pollen mother cells (PMCs) were also observed. To monitor transcriptional alterations during high-temperature injury, we performed DNA microarray analysis using the 22K Barley1 GeneChip. Expression profiles were captured at four time points during the early development of panicles, and during vegetative growth of seedlings as a control, with or without high-temperature treatment. Abiotic or biotic stress related genes were equally or more dominantly up-regulated in the seedlings exposed to high temperatures compared with the panicles. In contrast, certain genes associated with histones, DNA replication initiation, mitochondria, and ribosomes were specifically repressed in the exposed panicles. In situ hybridization studies indicated that repression locally occurred on the developing anther cells exposed to high temperatures. Microarray analysis also indicated that a series of genes, including a meiosis-specific gene Asy1 and anther-specific lipid transfer protein genes, was prematurely up-regulated at an earlier stage under high-temperature conditions. Real-time quantitative RT-PCR analyses well confirmed the expression differences of certain key genes predicted by the DNA microarrays. These results suggest that high-temperature causes premature progression of anther early development program and fate, such as progression to meiosis of PMCs, cell-proliferation arrest and degradation in anther wall cells, accompanied by comprehensive alterations in transcription.

Several dermatoses, including psoriasis, atopic dermatitis, and rosacea, alter the expression of the innate immune effector human cathelicidin antimicrobial peptide (CAMP). To elucidate the roles of aberrant CAMP in dermatoses, we performed cDNA array analysis in CAMP-stimulated human epidermal keratinocytes, the primary cells responding to innate immune stimuli and a major source of CAMP LL37 in skin. Among LL37-inducible genes, IL-1 cluster genes, particularly IL36G, are of interest because we observed coordinate increases in CAMP and IL-36γ in the lesional skin of psoriasis, whereas virtually no CAMP or IL-36γ was observed in nonlesional skin and normal skin. The production and release of IL-36γ were up to 20–30 ng/ml in differentiated keratinocytes cultured in high-calcium media. G-protein inhibitor pertussis toxin and p38 inhibitor suppressed IL-36γ induction by LL37. As an alarmin, LL37 induces chemokines, including CXCL1, CXCL8/IL8, CXCL10/IP-10, and CCL20/MIP3a, and IL-36 (10–100 ng/ml) augments the production of these chemokines by LL37. Pretreatment with small interfering RNA against IL36γ and IL-36R IL36R/IL1RL2 and IL1RAP suppressed LL37-dependent IL8, CXCL1, CXCL10/IP10, and CCL20 production in keratinocytes, suggesting that the alarmin function of LL37 was partially dependent on IL-36γ and its receptors. Counting on CAMP induction in innate stimuli, such as in infection and wounding, IL-36γ induction by cathelicidin would explain the mechanism of initiation of skin inflammation and occasional exacerbations of psoriasis and skin diseases by general infection.

The Tohoku Medical Megabank Organization constructed the reference panel (referred to as the 1KJPN panel), which contains 420 million single nucleotide polymorphisms (SNPs), from whole-genome sequence data from 1070 Japanese individuals. The 1KJPN panel contains the largest number of haplotypes of Japanese ancestry to date. Here, from the 1KJPN panel, we designed a novel custom-made SNP array, named the Japonica array, which is suitable for whole-genome imputation of Japanese individuals. The array contains 659 253 SNPs, including tag SNPs for imputation, SNPs of Y chromosome and mitochondria, and SNPs related to previously reported genome-wide association studies and pharmacogenomics. The Japonica array provides better imputation performance for Japanese individuals than the existing commercially available SNP arrays with both the 1KJPN panel and the International 1000 genomes project panel. For common SNPs (minor allele frequency (MAF)45%), the genomic coverage of the Japonica array (r 2 40.8) was 96.9%, that is, almost all common SNPs were covered by this array. Nonetheless, the coverage of low-frequency SNPs (0.5%oMAF ⩽ 5%) of the Japonica array reached 67.2%, which is higher than those of the existing arrays. In addition, we confirmed the high quality genotyping performance of the Japonica array using the 288 samples in 1KJPN; the average call rate 99.7% and the average concordance rate 99.7% to the genotypes obtained from high-throughput sequencer. As demonstrated in this study, the creation of custom-made SNP arrays based on a populationspecific reference panel is a practical way to facilitate further association studies through genome-wide genotype imputations.

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Although hydrogen peroxide (H2O2) is better known for its cytotoxic effects, in recent years it has been shown to play a crucial role in eukaryotic signal transduction. In respiratory tract epithelial cells, the dual oxidase (DUOX) proteins 1 and 2 has been identified as the cellular source of H2O2. However, the expression of DUOX1 or DUOX2 has not yet been examined in keratinocytes. In this study, using a DNA microarray, we demonstrated that, of the seven NOX/DUOX family members in normal human epidermal keratinocytes (NHEK), IL-4/IL-13 treatment augments the expression of only DUOX1 mRNA. We next confirmed the IL-4/IL-13 induction of DUOX1 in NHEK at the mRNA and protein level using quantitative real-time PCR and Western blotting, respectively. In addition, we demonstrated that this augmented DUOX1 expression was accompanied by increased H2O2 production, which was significantly suppressed both by diphenyleneiodonium, an inhibitor of NADPH oxidase, and by small interfering RNA against DUOX1. Finally, we demonstrated that the increased expression of DUOX1 in IL-4/IL-13–treated NHEK augments STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B. These results revealed a novel role of IL-4/IL-13–induced DUOX1 expression in making a positive feedback loop for IL-4/IL-13 signaling in keratinocytes.

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