The molecular pathophysiology underlying lumbar spondylosis development remains unclear. To identify genetic factors associated with lumbar spondylosis, we conducted a genome-wide association study using 83 severe lumbar spondylosis cases and 182 healthy controls and identified 65 candidate disease-associated single nucleotide polymorphisms (SNPs). Replication analysis in 510 case and 911 control subjects from five independent Japanese cohorts identified rs2054564, located in intron 7 of ADAMTS17, as a disease-associated SNP with a genome-wide significance threshold (P = 1.17 × 10-11, odds ratio = 1.92). This association was significant even after adjustment of age, sex, and body mass index (P = 7.52 × 10-11). A replication study in a Korean cohort, including 123 case and 319 control subjects, also verified the significant association of this SNP with severe lumbar spondylosis. Immunohistochemistry revealed that fibrillin-1 (FBN1) and ADAMTS17 were co-expressed in the annulus fibrosus of intervertebral discs (IVDs). ADAMTS17 overexpression in MG63 cells promoted extracellular microfibrils biogenesis, suggesting the potential role of ADAMTS17 in IVD function through interaction with fibrillin fibers. Finally, we provided evidence of FBN1 involvement in IVD function by showing that lumbar IVDs in patients with Marfan syndrome, caused by heterozygous FBN1 gene mutation, were significantly more degenerated. We identified a common SNP variant, located in ADAMTS17, associated with susceptibility to lumbar spondylosis and demonstrated the potential role of the ADAMTS17-fibrillin network in IVDs in lumbar spondylosis development.

Primary biliary cholangitis (PBC) is a rare autoimmune disease with a clear predisposition for human leukocyte antigen (HLA)-DR/DQ-associated loss of immune tolerance for the E2 component of the pyruvate dehydrogenase complex. Three-field-resolution HLA imputation of 1,670 Japanese PBC patients and 2,328 healthy controls was conducted using Japanese population-specific HLA reference panels. Eighteen previously reported Japanese PBC-associated HLA alleles were confirmed and extended to 3-field-resolution, including HLA-DRB1*08:03 to HLA-DRB1*08:03:02, HLA-DQB1*03:01 to HLA-DQB1*03:01:01, HLA-DQB1*04:01 to HLA-DQB1*04:01:01 and HLA-DQB1*06:04 to HLA-DQB1*06:04:01. In addition, additional significant novel HLA alleles were identified, including 3 novel susceptible HLA-DQA1 alleles: HLA-DQA1*03:03:01, HLA-DQA1*04:01:01, HLA-DQA1*01:04:01 and 1 novel protective HLA-DQA1 allele, HLA-DQA1*05:05:01. In addition, PBC patients carrying HLA-DRB1*15:01:01 and HLA-DQA1*03:03:01 would have a higher predisposition toward developing concomitant autoimmune hepatitis (AIH). Further, late-stage and symptomatic PBC shared the same susceptible HLA alleles of HLA-A*26:01:01, HLA-DRB1*09:01:02 and HLA-DQB1*03:03:02. Lastly, HLA-DPB1*05:01:01 was identified as a potential risk HLA allele for development of hepatocellular carcinoma (HCC) in PBC patients. In conclusion, we have extended the current knowledge of HLA allele associations to 3-field resolution and identified novel HLA allele associations with predisposition risk, staging, symptomatic state, and AIH and HCC events for Japanese PBC patients.

Primary biliary cholangitis (PBC) is a chronic, progressive cholestatic liver disease in which intrahepatic bile ducts are destroyed by an autoimmune reaction. Our previous genome-wide association study (GWAS) identified chromosome 11q23.1 as a susceptibility gene locus for PBC in the Japanese population. Here, high-density association mapping based on single nucleotide polymorphism (SNP) imputation and in silico/in vitro functional analyses identified rs1944919 as the primary functional variant. Expression-quantitative trait loci analyses showed that the PBC susceptibility allele of rs1944919 was significantly associated with increased COLCA1/COLCA2 expression levels. Additionally, the effects of rs1944919 on COLCA1/COLCA2 expression levels were confirmed using genotype knock-in versions of cell lines constructed using the CRISPR/Cas9 system and differed between rs1944919-G/G clones and -T/T clones. To our knowledge, this is the first study to demonstrate the contribution of COLCA1/COLCA2 to PBC susceptibility.

Genome-wide association studies have been performed to identify common genetic variants associated with hepatitis B (HB). However, little is known about copy number variations (CNVs) in HB. In this study, we performed a genome-wide CNV analysis between 1830 healthy controls and 1031 patients with HB infection after quality control. Using signal calling by the Axiom Analysis Suite and CNV detection by PennCNV software, we obtained a total of 4494 CNVs across all individuals. The genes with CNVs that were found only in the HB patients were associated with the immune system, such as antigen processing. A gene-level CNV association test revealed statistically significant CNVs in the contactin 6 (CNTN6) gene. Moreover, we also performed gene-level CNV association tests in disease subgroups, including hepatocellular carcinoma patients, liver cirrhosis patients, and HBV carriers, including asymptomatic carriers and patients with HBV-derived chronic hepatitis. Our findings from germline cells suggested that patient-specific CNVs may be inherent genetic risk factors for HB.

We have performed a genome-wide association study (GWAS) including 473 Japanese HBV (hepatitis B virus)-positive HCC (hepatocellular carcinoma) patients and 516 HBV carriers including chronic hepatitis and asymptomatic carrier individuals to identify new host genetic factors associated with HBV-derived HCC in Japanese and other East Asian populations. We identified 65 SNPs with P values < 10-4 located within the HLA class I region and three SNPs were genotyped in three independent population-based replication sets. Meta-analysis confirmed the association of the three SNPs (rs2523961: OR = 1.73, P = 7.50 × 10-12; rs1110446: OR = 1.79, P = 1.66 × 10-13; and rs3094137: OR = 1.73, P = 7.09 × 10-9). We then performed two-field HLA genotype imputation for six HLA loci using genotyping data to investigate the association between HLA alleles and HCC. HLA allele association testing revealed that HLA-A * 33:03 (OR = 1.97, P = 4.58 × 10-4) was significantly associated with disease progression to HCC. Conditioning analysis of each of the three SNPs on the HLA class I region abolished the association of HLA-A*33:03 with disease progression to HCC. However, conditioning the HLA allele could not eliminate the association of the three SNPs, suggesting that additional genetic factors may exist in the HLA class I region.

Panic disorder (PD) is a severe and chronic psychiatric disorder with genetic components underlying in its etiology. The Phosphodiesterase 4B (PDE4B) gene has been reported to be associated with several psychiatric disorders. Several studies indicated that PDE4B may be involved in the regulation of anxiety and depression. Therefore, we investigate the association of PDE4B with PD in the Japanese population.

The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance) were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM) and specific (concentration of unexpected amplicons <2 nM) amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites) in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.

The recent development of high-resolution DNA microarrays, in which hundreds of thousands of single nucleotide polymorphisms (SNPs) are genotyped, enables the rapid identification of susceptibility genes for complex diseases. Clusters of these SNPs may show runs of homozygosity (ROHs) that can be analyzed for association with disease. An analysis of patients whose parents were first cousins enables the search for autozygous segments in their offspring. Here, using the Affymetrix® Genome-Wide Human SNP Array 5.0 to determine ROHs, we genotyped 9 individuals with schizophrenia (SCZ) whose parents were first cousins. We identified overlapping ROHs on chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 19, 20, and 21 in at least 3 individuals. Only the locus on chromosome 5 has been reported previously. The ROHs on chromosome 5q23.3-q31.1 include the candidate genes histidine triad nucleotide binding protein 1 (HINT1) and acyl-CoA synthetase long-chain family member 6 (ACSL6). Other overlapping ROHs may contain novel rare recessive variants that affect SCZ specifically in our samples, given the highly heterozygous nature of SCZ. Analysis of patients whose parents are first cousins may provide new insights for the genetic analysis of psychiatric diseases.

Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance.

Feeding and the circadian system regulate lipid absorption and metabolism, and the expression of enzymes involved in lipid metabolism is believed to be directly controlled by the clock system. To investigate the interaction between the lipid metabolism system and the circadian system, we analyzed the effect of a CLOCK/BMAL1 heterodimer on the transcriptional regulation of PPAR-controlled genes through PPAR response elements (PPREs). Transcription of acyl-CoA oxidase, cellular retinol binding protein II (CRBPII), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was altered by CLOCK/BMAL1, and transcriptional activity via PPRE by PPARs/RXRalpha was enhanced by CLOCK/BMAL1 and/or by PPARs ligand/activators. We also found that CLOCK/BMAL1-mediated transcription of period (PER) and cryptochrome (CRY) was modulated by PPARalpha/RXRalpha. These results suggest that there may be crosstalk between the PPARs/RXRalpha-regulated system and the CLOCK/BMAL1-regulated system.

Hepatitis B virus (HBV), a highly persistent pathogen causing hepatocellular carcinoma (HCC), takes full advantage of host machinery, presenting therapeutic targets. Here we aimed to identify novel druggable host cellular factors using the reporter HBV we have recently generated. In an RNAi screen of G protein-coupled receptors (GPCRs), GPCR39 (GPR39) appeared as the top hit to facilitate HBV proliferation. Lentiviral overexpression of active GPR39 proteins and an agonist enhanced HBV replication and transcriptional activities of viral promoters, inducing the expression of CCAAT/enhancer binding protein (CEBP)-β (CEBPB). Meanwhile, GPR39 was uncovered to activate the heat shock response, upregulating the expression of proviral heat shock proteins (HSPs). In addition, glioma-associated oncogene homologue signaling, a recently reported target of GPR39, was suggested to inhibit HBV replication and eventually suppress expression of CEBPB and HSPs. Thus, GPR39 provirally governed intracellular circuits simultaneously affecting the carcinopathogenetic gene functions. GPR39 and the regulated signaling networks would serve as antiviral targets, and strategies with selective inhibitors of GPR39 functions can develop host-targeted antiviral therapies preventing HCC.

People in the Solomon Islands today are considered to have derived from Asian- and Papuan-related ancestors. Papuan-related ancestors colonized Near Oceania about 47,000 years ago, and Asian-related ancestors were Austronesian (AN)-speaking population, called Lapita, who migrated from Southeast Asia about 3,500 years ago. These two ancestral populations admixed in Near Oceania before the expansion of Lapita people into Remote Oceania. To understand the impact of the admixture on the adaptation of AN-speaking Melanesians in Near Oceania, we performed the genome-wide single nucleotide polymorphism (SNP) analysis of 21 individuals from Munda, the main town of the New Georgia Islands in the western Solomon Islands. Population samples from Munda were genetically similar to other Solomon Island population samples. The analysis of genetic contribution from the two different ancestries to the Munda genome revealed significantly higher proportions of Asian- and Papuan-related ancestries in the region containing the annexin A1 (ANXA1) gene (Asian component > 82.6%) and in the human leukocyte antigen (HLA) class II region (Papuan component > 85.4%), respectively. These regions were suspected to have undergone natural selection since the time of admixture. Our results suggest that admixture had affected adaptation of AN-speaking Melanesians in the Solomon Islands.

The therapeutic use of interferon (IFN) is known to cause depression that frequently interrupts treatment. To identify genetic variants associated with IFN-induced depression, we conducted a genome-wide association study (GWAS) of 224 Japanese chronic hepatitis C patients receiving IFN-based therapy in a multicenter prospective study and stratified them into two groups according to the Beck Depression Inventory, Second Edition (BDI-II) score. In the GWAS stage, we selected 42 candidate single nucleotide polymorphisms (SNPs) to perform replication analysis in an independent set of 160 subjects. The SNP rs1863918 in strong linkage disequilibrium with SNPs located around the Zinc finger 354C (ZNF354C) gene on chromosome 5 showed a significant association when the results of GWAS and replication were combined (odds ratio = 2.55, P = 7.89×10-8 in the allele frequency model), suggesting that the rs1863918 T allele was associated with IFN-induced depression. Furthermore, logistic regression analysis showed that rs1863918 T allele, a history of depression, and younger age were independent predictive factors for IFN-induced depression. Interestingly, western blotting and immunofluorescence showed that ZNF354C was highly expressed in the hippocampus in mice, a region implicated in the pathology of psychiatric symptoms. In conclusion, we identified rs1863918 as significantly associated with IFN-induced depression, and revealed that the candidate gene ZNF354C is highly expressed in the hippocampus of mice. Our data might be useful for elucidating the pathogenic mechanisms of depression induced by drugs including IFN.

Phosphatidylcholine (PC) and its hydrolysates are considered to stimulate intestinal lipid absorption, however, their exact effects on lipoproteins and apolipoprotein (apo) metabolism remain ambiguous. This study aimed to further differentiate the effects of them using fully differentiated enterocyte-like Caco-2 cells. Lipid micelles (oleic acid 0.6, cholesterol 0.05, monooleylglycerol 0.2, taurocholate 2 in mmol/l) with or without choline, PC, and lysoPC (0.2 mmol/l each) were applied apically to Caco-2 cells. (3)H-oleic acid and (14)C-cholesterol were added to the micelles when necessary. Secreted lipoproteins were analyzed by a HPLC method. LysoPC had the most potent promoting effect on lipid uptake, and lipoprotein and apolipoprotein B-48 secretion among the molecules tested. LysoPC doubled the output of cholesterol and triglyceride as the lipoprotein component, but PC did not. On the other hand, PC only increased the secretion of apoA-IV in the presence of lipid micelles. These findings confirm that the alteration of PC by PLA(2) hydrolysis is intrinsically involved in the intestinal lipid absorption process and suggest that PC and its hydrolysis are coordinately associated with not only lipid absorption efficiency but also lipoprotein output and metabolism.

Numerous genome-wide association studies (GWAS) have been performed to identify susceptibility genes to various human complex diseases. However, in many cases, neither a functional variant nor a disease susceptibility gene have been clarified. Here, we show an efficient approach for identification of a functional variant in a primary biliary cholangitis (PBC)-susceptible region, chromosome 17q12-21 (ORMDL3-GSDMB-ZPBP2-IKZF3). High-density association mapping was carried out based on SNP imputation analysis by using the whole-genome sequence data from a reference panel of 1,070 Japanese individuals (1KJPN), together with genotype data from our previous GWAS (PBC patients: n = 1,389; healthy controls: n = 1,508). Among 23 single nucleotide polymorphisms (SNPs) with P < 1.0 × 10-8, rs12946510 was identified as the functional variant that influences gene expression via alteration of Forkhead box protein O1 (FOXO1) binding affinity in vitro. Moreover, expression-quantitative trait locus (e-QTL) analyses showed that the PBC susceptibility allele of rs12946510 was significantly associated with lower endogenous expression of ORMDL3 and GSDMB in whole blood and spleen. This study not only identified the functional variant in chr.17q12-21 and its molecular mechanism through which it conferred susceptibility to PBC, but it also illustrated an efficient systematic approach for post-GWAS analysis that is applicable to other complex diseases.

COVID-19, a novel coronavirus-related illness, has spread worldwide. Patients with apparently mild/moderate symptoms can suddenly develop severe pneumonia. Therefore, almost all COVID-19 patients require hospitalization, which can reduce limited medical resources in addition to overwhelming medical facilities. To identify predictive markers for the development of severe pneumonia, a comprehensive analysis of serum chemokines and cytokines was conducted using serial serum samples from COVID-19 patients. The expression profiles were analyzed along the time axis. Serum samples of common diseases were enrolled from a BioBank to confirm the usefulness of predictive markers. Five factors, IFN-λ3, IL-6, IP-10, CXCL9, and CCL17, were identified as predicting the onset of severe/critical symptoms. The factors were classified into two categories. Category A included IFN-λ3, IL-6, IP-10, and CXCL9, and their values surged and decreased rapidly before the onset of severe pneumonia. Category B included CCL17, which provided complete separation between the mild/moderate and the severe/critical groups at an early phase of SARS-CoV-2 infection. The five markers provided a high predictive value (area under the receiver operating characteristic curve (AUROC): 0.9-1.0, p < 0.001). Low expression of CCL17 was specifically observed in pre-severe COVID-19 patients compared with other common diseases, and the predictive ability of CCL17 was confirmed in validation samples of COVID-19. The factors identified could be promising prognostic markers to distinguish between mild/moderate and severe/critical patients, enabling triage at an early phase of infection, thus avoiding overwhelming medical facilities.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The relentless spread and pathogenicity of the virus have become a global public health emergency. One of the striking features of this pandemic is the pronounced impact on specific regions and ethnic groups. In particular, compared with East Asia, where the virus first emerged, SARS-CoV-2 has caused high rates of morbidity and mortality in Europe. This has not been experienced in past global viral infections, such as influenza, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) and is unique to SARS-CoV-2. For this reason, we investigated the involvement of genetic factors associated with SARS-CoV-2 infection with a focus on angiotensin-converting enzyme (ACE)-related genes, because ACE2 is a receptor for SARS-CoV-2. We found that the ACE1 II genotype frequency in a population was significantly negatively correlated with the number of SARS-CoV-2 cases. Similarly, the ACE1 II genotype was negatively correlated with the number of deaths due to SARS-CoV-2 infection. These data suggest that the ACE1 II genotype may influence the prevalence and clinical outcome of COVID-19 and serve as a predictive marker for COVID-19 risk and severity.

Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intrahepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening.

Homo sapiens have experienced admixture many times in the last few thousand years. To examine how admixture affects local adaptation, we investigated genomes of modern Polynesians, who are shaped through admixture between Austronesian-speaking people from Southeast Asia (Asian-related ancestors) and indigenous people in Near Oceania (Papuan-related ancestors).