Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes.

The impact of microRNA (miRNA) on the genetics of human complex traits, especially in the context of miRNA-target gene networks, has not been fully assessed. Here, we developed a novel analytical method, MIGWAS, to comprehensively evaluate enrichment of genome-wide association study (GWAS) signals in miRNA-target gene networks. We applied the method to the GWAS results of the 18 human complex traits from >1.75 million subjects, and identified significant enrichment in rheumatoid arthritis (RA), kidney function, and adult height (P < 0.05/18 = 0.0028, most significant enrichment in RA with P = 1.7 × 10(-4)). Interestingly, these results were consistent with current literature-based knowledge of the traits on miRNA obtained through the NCBI PubMed database search (adjusted P = 0.024). Our method provided a list of miRNA and target gene pairs with excess genetic association signals, part of which included drug target genes. We identified a miRNA (miR-4728-5p) that downregulates PADI2, a novel RA risk gene considered as a promising therapeutic target (rs761426, adjusted P = 2.3 × 10(-9)). Our study indicated the significant impact of miRNA-target gene networks on the genetics of human complex traits, and provided resources which should contribute to drug discovery and nucleic acid medicine.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4(+)CD4(+) T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4(+)CD4(+) T cells. Moreover, the frequency of memory CXCR4(+)CD4(+) T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4(+)CD4(+) T cells. Clinically, a higher frequency of memory CXCR4(+)CD4(+) T cells predicted a better response to CTLA4-Ig. Memory CXCR4(+)CD4(+) T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA.

MicroRNAs (miRNAs) modulate the post-transcriptional regulation of target genes and are related to biology of complex human traits, but genetic landscape of miRNAs remains largely unknown. Given the strikingly tissue-specific miRNA expression profiles, we here expand a previous method to quantitatively evaluate enrichment of genome-wide association study (GWAS) signals on miRNA-target gene networks (MIGWAS) to further estimate tissue-specific enrichment. Our approach integrates tissue-specific expression profiles of miRNAs (∼1800 miRNAs in 179 cells) with GWAS to test whether polygenic signals enrich in miRNA-target gene networks and whether they fall within specific tissues. We applied MIGWAS to 49 GWASs (nTotal = 3 520 246), and successfully identified biologically relevant tissues. Further, MIGWAS could point miRNAs as candidate biomarkers of the trait. As an illustrative example, we performed differentially expressed miRNA analysis between rheumatoid arthritis (RA) patients and healthy controls (n = 63). We identified novel biomarker miRNAs (e.g. hsa-miR-762) by integrating differentially expressed miRNAs with MIGWAS results for RA, as well as novel associated loci with significant genetic risk (rs56656810 at MIR762 at 16q11; n = 91 482, P = 3.6 × 10-8). Our result highlighted that miRNA-target gene network contributes to human disease genetics in a cell type-specific manner, which could yield an efficient screening of miRNAs as promising biomarkers.

Psoriasis vulgaris (PsV) risk is strongly associated with variation within the major histocompatibility complex (MHC) region, but its genetic architecture has yet to be fully elucidated. Here, we conducted a large-scale fine-mapping study of PsV risk in the MHC region in 9,247 PsV-affected individuals and 13,589 controls of European descent by imputing class I and II human leukocyte antigen (HLA) genes from SNP genotype data. In addition, we imputed sequence variants for MICA, an MHC HLA-like gene that has been associated with PsV, to evaluate association at that locus as well. We observed that HLA-C(∗)06:02 demonstrated the lowest p value for overall PsV risk (p = 1.7 × 10(-364)). Stepwise analysis revealed multiple HLA-C(∗)06:02-independent risk variants in both class I and class II HLA genes for PsV susceptibility (HLA-C(∗)12:03, HLA-B amino acid positions 67 and 9, HLA-A amino acid position 95, and HLA-DQα1 amino acid position 53; p < 5.0 × 10(-8)), but no apparent risk conferred by MICA. We further evaluated risk of two major clinical subtypes of PsV, psoriatic arthritis (PsA; n = 3,038) and cutaneous psoriasis (PsC; n = 3,098). We found that risk heterogeneity between PsA and PsC might be driven by HLA-B amino acid position 45 (Pomnibus = 2.2 × 10(-11)), indicating that different genetic factors underlie the overall risk of PsV and the risk of specific PsV subphenotypes. Our study illustrates the value of high-resolution HLA and MICA imputation for fine mapping causal variants in the MHC.

A genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific.

Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P < 5 × 10(-8). These loci show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis. Our results include seven loci with associations reaching P < 5 × 10(-8) for the first time, near the genes COL8A1-FILIP1L, IER3-DDR1, SLC16A8, TGFBR1, RAD51B, ADAMTS9 and B3GALTL. A genetic risk score combining SNP genotypes from all loci showed similar ability to distinguish cases and controls in all samples examined. Our findings provide new directions for biological, genetic and therapeutic studies of AMD.

White blood cells (WBCs) mediate immune systems and consist of various subtypes with distinct roles. Elucidation of the mechanism that regulates the counts of the WBC subtypes would provide useful insights into both the etiology of the immune system and disease pathogenesis. In this study, we report results of genome-wide association studies (GWAS) and a replication study for the counts of the 5 main WBC subtypes (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) using 14,792 Japanese subjects enrolled in the BioBank Japan Project. We identified 12 significantly associated loci that satisfied the genome-wide significance threshold of P<5.0×10(-8), of which 9 loci were novel (the CDK6 locus for the neutrophil count; the ITGA4, MLZE, STXBP6 loci, and the MHC region for the monocyte count; the SLC45A3-NUCKS1, GATA2, NAALAD2, ERG loci for the basophil count). We further evaluated associations in the identified loci using 15,600 subjects from Caucasian populations. These WBC subtype-related loci demonstrated a variety of patterns of pleiotropic associations within the WBC subtypes, or with total WBC count, platelet count, or red blood cell-related traits (n = 30,454), which suggests unique and common functional roles of these loci in the processes of hematopoiesis. This study should contribute to the understanding of the genetic backgrounds of the WBC subtypes and hematological traits.

Total white blood cell (WBC) and neutrophil counts are lower among individuals of African descent due to the common African-derived "null" variant of the Duffy Antigen Receptor for Chemokines (DARC) gene. Additional common genetic polymorphisms were recently associated with total WBC and WBC sub-type levels in European and Japanese populations. No additional loci that account for WBC variability have been identified in African Americans. In order to address this, we performed a large genome-wide association study (GWAS) of total WBC and cell subtype counts in 16,388 African-American participants from 7 population-based cohorts available in the Continental Origins and Genetic Epidemiology Network. In addition to the DARC locus on chromosome 1q23, we identified two other regions (chromosomes 4q13 and 16q22) associated with WBC in African Americans (P<2.5×10(-8)). The lead SNP (rs9131) on chromosome 4q13 is located in the CXCL2 gene, which encodes a chemotactic cytokine for polymorphonuclear leukocytes. Independent evidence of the novel CXCL2 association with WBC was present in 3,551 Hispanic Americans, 14,767 Japanese, and 19,509 European Americans. The index SNP (rs12149261) on chromosome 16q22 associated with WBC count is located in a large inter-chromosomal segmental duplication encompassing part of the hydrocephalus inducing homolog (HYDIN) gene. We demonstrate that the chromosome 16q22 association finding is most likely due to a genotyping artifact as a consequence of sequence similarity between duplicated regions on chromosomes 16q22 and 1q21. Among the WBC loci recently identified in European or Japanese populations, replication was observed in our African-American meta-analysis for rs445 of CDK6 on chromosome 7q21 and rs4065321 of PSMD3-CSF3 region on chromosome 17q21. In summary, the CXCL2, CDK6, and PSMD3-CSF3 regions are associated with WBC count in African American and other populations. We also demonstrate that large inter-chromosomal duplications can result in false positive associations in GWAS.

We performed a genome-wide association study (GWAS) on levels of serum total protein (TP), albumin (ALB), and non-albumin protein (NAP). We analyzed SNPs on autosomal chromosomes using data from 9,103 Japanese individuals, followed by a replication study of 1,600 additional individuals. We confirmed the previously- reported association of GCKR on chromosome 2p23.3 with serum ALB (rs1260326, P(meta) = 3.1 × 10(-9)), and additionally identified the significant genome-wide association of rs4985726 in TNFRSF13B on 17p11.2 with both TP and NAP (P(meta) = 1.2 × 10(-14) and 7.1 × 10(-24), respectively). For NAP, rs3803800 and rs11552708 in TNFSF13 on 17p13.1 (P(meta) = 7.2 × 10(-15) and 7.5 × 10(-10), respectively) as well as rs10007186 on 4q21.2 near ANXA3 (P(meta) = 1.3 × 10(-9)) also indicated significant associations. Interestingly, TNFRSF13B and TNFSF13 encode a tumor necrosis factor (TNF) receptor and its ligand, which together constitute an important receptor-ligand axis for B-cell homeostasis and immunoglobulin production. Furthermore, three SNPs, rs4985726, rs3803800, and rs11552708 in TNFRSF13B and TNFSF13, were indicated to be associated with serum levels of IgG (P<2.3 × 10(-3)) and IgM (P<0.018), while rs3803800 and rs11552708 were associated with IgA (P<0.013). Rs10007186 in 4q21.2 was associated with serum levels of IgA (P = 0.036), IgM (P = 0.019), and IgE (P = 4.9 × 10(-4)). Our results should add interesting knowledge about the regulation of major serum components.

Understanding natural selection is crucial to unveiling evolution of modern humans. Here, we report natural selection signatures in the Japanese population using 2234 high-depth whole-genome sequence (WGS) data (25.9×). Using rare singletons, we identify signals of very recent selection for the past 2000-3000 years in multiple loci (ADH cluster, MHC region, BRAP-ALDH2, SERHL2). In large-scale genome-wide association study (GWAS) dataset (n = 171,176), variants with selection signatures show enrichment in heterogeneity of derived allele frequency spectra among the geographic regions of Japan, highlighted by two major regional clusters (Hondo and Ryukyu). While the selection signatures do not show enrichment in archaic hominin-derived genome sequences, they overlap with the SNPs associated with the modern human traits. The strongest overlaps are observed for the alcohol or nutrition metabolism-related traits. Our study illustrates the value of high-depth WGS to understand evolution and their relationship with disease risk.

Genome-wide association studies (GWASs) have identified loci for erythrocyte traits in primarily European ancestry populations. We conducted GWAS meta-analyses of six erythrocyte traits in 71,638 individuals from European, East Asian, and African ancestries using a Bayesian approach to account for heterogeneity in allelic effects and variation in the structure of linkage disequilibrium between ethnicities. We identified seven loci for erythrocyte traits including a locus (RBPMS/GTF2E2) associated with mean corpuscular hemoglobin and mean corpuscular volume. Statistical fine-mapping at this locus pointed to RBPMS at this locus and excluded nearby GTF2E2. Using zebrafish morpholino to evaluate loss of function, we observed a strong in vivo erythropoietic effect for RBPMS but not for GTF2E2, supporting the statistical fine-mapping at this locus and demonstrating that RBPMS is a regulator of erythropoiesis. Our findings show the utility of trans-ethnic GWASs for discovery and characterization of genetic loci influencing hematologic traits.

The contribution of FOXP3-expressing naturally occurring regulatory T (Treg) cells to common polygenic autoimmune diseases remains ambiguous. Here, we characterized genome-wide epigenetic profiles (CpG methylation and histone modifications) of human Treg and conventional T (Tconv) cells in naive and activated states. We found that single-nucleotide polymorphisms (SNPs) associated with common autoimmune diseases were predominantly enriched in CpG demethylated regions (DRs) specifically present in naive Treg cells but much less enriched in activation-induced DRs common in Tconv and Treg cells. Naive Treg cell-specific DRs were largely included in Treg cell-specific super-enhancers and closely associated with transcription and other epigenetic changes in naive and effector Treg cells. Thus, naive Treg cell-specific CpG hypomethylation had a key role in controlling Treg cell-specific gene transcription and epigenetic modification. The results suggest possible contribution of altered function or development of natural Treg cells to the susceptibility to common autoimmune diseases.

Novel biomarkers of rheumatoid arthritis (RA), in addition to antibodies against cyclic citrullinated peptides, are required. Metabolome analysis is a promising approach to identify metabolite biomarkers for clinical diagnosis. We adopted a comprehensive non-targeted metabolomics approach combining capillary electrophoresis time-of-flight mass spectrometry (TOFMS) and liquid chromatography TOFMS. We constructed metabolomics profiling of 286 plasma samples of a Japanese population [92 RA patients, 13 systemic lupus erythematosus (SLE) patients and 181 healthy controls). RA case-control association tests showed that seven metabolites exhibited significantly increased levels in RA samples compared with controls (P < 1.0 × 10-4; UTP, ethanolamine phosphate, ATP, GDP, ADP, 6-aminohexanoic acid and taurine), whereas one exhibited a decreased level (xanthine). The plasma levels of these eight metabolites were not significantly different between seropositive and seronegative RA patients (P > 0.05; n = 68 and 24, respectively). The four nucleotide levels (UTP, ATP, GDP and ADP) were significantly higher in the non-treatment patients in comparison between patients with and without treatment (P < 0.014; n = 57 and 35, respectively). Furthermore, we found that none of the four nucleotide levels showed significant differences in SLE case-control association tests (P > 0.2; 13 patients with SLE and the 181 shared controls) and psoriatic arthritis (PsA) case-control association tests (P > 0.11; 42 patients with PsA and 38 healthy controls), indicating disease specificity in RA. In conclusion, our large-scale metabolome analysis demonstrated the increased plasma nucleotide levels in RA patients, which could be used as potential clinical biomarkers of RA, especially for seronegative RA.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10-9, including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.

Food allergy is a growing health problem worldwide because of its increasing prevalence, life-threatening potential, and shortage of effective preventive treatments. In an outbreak of wheat allergy in Japan, thousands of patients had allergic reactions to wheat after using soap containing hydrolyzed wheat protein (HWP).

Despite significant progress in annotating the genome with experimental methods, much of the regulatory noncoding genome remains poorly defined. Here we assert that regulatory elements may be characterized by leveraging local epigenomic signatures where specific transcription factors (TFs) are bound. To link these two features, we introduce IMPACT, a genome annotation strategy that identifies regulatory elements defined by cell-state-specific TF binding profiles, learned from 515 chromatin and sequence annotations. We validate IMPACT using multiple compelling applications. First, IMPACT distinguishes between bound and unbound TF motif sites with high accuracy (average AUPRC 0.81, SE 0.07; across 8 tested TFs) and outperforms state-of-the-art TF binding prediction methods, MocapG, MocapS, and Virtual ChIP-seq. Second, in eight tested cell types, RNA polymerase II IMPACT annotations capture more cis-eQTL variation than sequence-based annotations, such as promoters and TSS windows (25% average increase in enrichment). Third, integration with rheumatoid arthritis (RA) summary statistics from European (N = 38,242) and East Asian (N = 22,515) populations revealed that the top 5% of CD4+ Treg IMPACT regulatory elements capture 85.7% of RA h2, the most comprehensive explanation for RA h2 to date. In comparison, the average RA h2 captured by compared CD4+ T histone marks is 42.3% and by CD4+ T specifically expressed gene sets is 36.4%. Lastly, we find that IMPACT may be used in many different cell types to identify complex trait associated regulatory elements.

Elucidation of natural selection signatures and relationships with phenotype spectra is important to understand adaptive evolution of modern humans. Here, we conducted a genome-wide scan of selection signatures of the Japanese population by estimating locus-specific time to the most recent common ancestor using the ascertained sequentially Markovian coalescent (ASMC), from the biobank-based large-scale genome-wide association study data of 170,882 subjects. We identified 29 genetic loci with selection signatures satisfying the genome-wide significance. The signatures were most evident at the alcohol dehydrogenase (ADH) gene cluster locus at 4q23 (PASMC = 2.2 × 10-36), followed by relatively strong selection at the FAM96A (15q22), MYOF (10q23), 13q21, GRIA2 (4q32), and ASAP2 (2p25) loci (PASMC < 1.0 × 10-10). The additional analysis interrogating extended haplotypes (integrated haplotype score) showed robust concordance of the detected signatures, contributing to fine-mapping of the genes, and provided allelic directional insights into selection pressure (e.g., positive selection for ADH1B-Arg48His and HLA-DPB1*04:01). The phenome-wide selection enrichment analysis with the trait-associated variants identified a variety of the modern human phenotypes involved in the adaptation of Japanese. We observed population-specific evidence of enrichment with the alcohol-related phenotypes, anthropometric and biochemical clinical measurements, and immune-related diseases, differently from the findings in Europeans using the UK Biobank resource. Our study demonstrated population-specific features of the selection signatures in Japanese, highlighting a value of the natural selection study using the nation-wide biobank-scale genome and phenotype data.

The relationship between autoimmune diseases and the gut microbiome has been intensively studied, and several autoimmunity-associated bacterial taxa have been identified. However, much less is known about the roles of the gut virome in autoimmune diseases.