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Background The Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study was launched in 2013 to evaluate the complex interactions of genetic and environmental factors in multifactorial diseases. The present study describes the maternal baseline profile and perinatal data of participating mothers and infants. Methods Expectant mothers living in Miyagi prefecture were recruited from obstetric facilities or affiliated centers between 2013 and 2017. Three sets of self-administered questionnaires were collected, and the medical records were reviewed to obtain precise information about each antenatal visit and each delivery. Biospecimens, including blood, urine, umbilical cord blood, and breast milk, were collected for the study biobank. The baseline maternal sociodemographic characteristics, results of screening tests, and obstetric outcomes were analyzed according to the maternal age group. Results A total of 23 406 pregnancies involving 23 730 fetuses resulted in 23 143 live births. Younger maternal participants had a tendency toward a higher incidence of threatened abortion and threatened premature labor, while older age groups exhibited a significantly higher rate of low lying placenta, placenta previa, gestational diabetes and hypertensive disorders of pregnancy. Conclusions The present study clearly shows the distribution of maternal baseline characteristics and the range of perinatal outcomes according to maternal age group. This cohort study can provide strategic information for creating breakthroughs in the pathophysiology of perinatal, developmental, and noncommunicable diseases by collaborative data visiting or sharing.

Ethnic-specific SNP arrays are becoming more important to increase the power of genome-wide association studies in diverse population. In the Tohoku Medical Megabank Project, we have been developing a series of Japonica Arrays (JPA) for genotyping participants based on reference panels constructed from whole-genome sequence data of the Japanese population. Here, we designed a novel version of the SNP array for the Japanese population, called Japonica Array NEO (JPA NEO), comprising a total of 666,883 markers. Among them, 654,246 tag SNPs of autosomes and X chromosome were selected from an expanded reference panel of 3,552 Japanese, 3.5KJPNv2, using pairwise r2 of linkage disequilibrium measures. Additionally, 28,298 markers were included for the evaluation of previously identified disease risk markers from the literature and databases, and those present in the Japanese population were extracted using the reference panel. Through genotyping 286 Japanese samples, we found that the imputation quality r2 and INFO score in the minor allele frequency bin >2.5–5% were >0.9 and >0.8, respectively, and >12 million markers were imputed with an INFO score >0.8. From these results, JPA NEO is a promising tool for genotyping the Japanese population with genome-wide coverage, contributing to the development of genetic risk scores., Graphical Abstract

Background Some patients with inflammatory bowel disease (IBD) who were under mesalamine treatment develop adverse reactions called “mesalamine allergy,” which includes high fever and worsening diarrhea. Currently, there is no method to predict mesalamine allergy. Pharmacogenomic approaches may help identify these patients. Here we analyzed the genetic background of mesalamine intolerance in the first genome-wide association study of Japanese patients with IBD. Methods Two independent pharmacogenetic IBD cohorts were analyzed: the MENDEL (n = 1523; as a discovery set) and the Tohoku (n = 788; as a replication set) cohorts. Genome-wide association studies were performed in each population, followed by a meta-analysis. In addition, we constructed a polygenic risk score model and combined genetic and clinical factors to model mesalamine intolerance. Results In the combined cohort, mesalamine-induced fever and/or diarrhea was significantly more frequent in ulcerative colitis vs Crohn’s disease. The genome-wide association studies and meta-analysis identified one significant association between rs144384547 (upstream of RGS17) and mesalamine-induced fever and diarrhea (P = 7.21e-09; odds ratio = 11.2). The estimated heritability of mesalamine allergy was 25.4%, suggesting a significant correlation with the genetic background. Furthermore, a polygenic risk score model was built to predict mesalamine allergy (P = 2.95e-2). The combined genetic/clinical prediction model yielded a higher area under the curve than did the polygenic risk score or clinical model alone (area under the curve, 0.89; sensitivity, 71.4%; specificity, 90.8%). Conclusions Mesalamine allergy was more common in ulcerative colitis than in Crohn’s disease. We identified a novel genetic association with and developed a combined clinical/genetic model for this adverse event.

Background: Multi-phenotype analysis of genetically correlated phenotypes can increase the statistical power to detect loci associated with multiple traits, leading to the discovery of novel loci. This is the first study to date to comprehensively analyze the shared genetic effects within different hemostatic traits, and between these and their associated disease outcomes. Objectives: To discover novel genetic associations by combining summary data of correlated hemostatic traits and disease events. Methods: Summary statistics from genome wide-association studies (GWAS) from seven hemostatic traits (factor VII [FVII], factor VIII [FVIII], von Willebrand factor [VWF] factor XI [FXI], fibrinogen, tissue plasminogen activator [tPA], plasminogen activator inhibitor 1 [PAI-1]) and three major cardiovascular (CV) events (venous thromboembolism [VTE], coronary artery disease [CAD], ischemic stroke [IS]), were combined in 27 multi-trait combinations using metaUSAT. Genetic correlations between phenotypes were calculated using Linkage Disequilibrium Score Regression (LDSC). Newly associated loci were investigated for colocalization. We considered a significance threshold of 1.85 × 10−9 obtained after applying Bonferroni correction for the number of multi-trait combinations performed (n = 27). Results: Across the 27 multi-trait analyses, we found 4 novel pleiotropic loci (XXYLT1, KNG1, SUGP1/MAU2, TBL2/MLXIPL) that were not significant in the original individual datasets, were not described in previous GWAS for the individual traits, and that presented a common associated variant between the studied phenotypes. Conclusions: The discovery of four novel loci contributes to the understanding of the relationship between hemostasis and CV events and elucidate common genetic factors between these traits.

Human lymphoblastoid cell lines (LCLs) are valuable for the functional analyses of diseases. We have established more than 4200 LCLs as one of the resources of an integrated biobank. While oxidative and inflammatory stresses play critical roles in the onset and progression of various diseases, the responsiveness of LCLs, especially that of biobank-made LCLs, to these stresses has not been established. To address how LCLs respond to these stresses, in this study, we performed RNA sequencing of eleven human LCLs that were treated with an electrophile, diethyl maleate (DEM) and/or an inflammatory mediator, lipopolysaccharide (LPS). We found that over two thousand genes, including those regulated by a master regulator of the electrophilic/oxidative stress response, NRF2, were upregulated in LCLs treated with DEM, while approximately three hundred genes, including inflammation-related genes, were upregulated in LPS-treated LCLs. Of the LPS-induced genes, a subset of proinflammatory genes was repressed by DEM, supporting the notion that DEM suppresses the expression of proinflammatory genes through NRF2 activation. Conversely, a part of DEM-induced gene was repressed by LPS, suggesting reciprocal interference between electrophilic and inflammatory stress-mediated pathways. These data clearly demonstrate that LCLs maintain, by and large, responsive pathways against oxidative and inflammatory stresses and further endorse the usefulness of the LCL supply from the biobank.

We performed a metabolome genome-wide association study for the Japanese population in the prospective cohort study of Tohoku Medical Megabank. By combining whole-genome sequencing and nontarget metabolome analyses, we identified a large number of novel associations between genetic variants and plasma metabolites. Of the identified metabolite-associated genes, approximately half have already been shown to be involved in various diseases. We identified metabolite-associated genes involved in the metabolism of xenobiotics, some of which are from intestinal microorganisms, indicating that the identified genetic variants also markedly influence the interaction between the host and symbiotic bacteria. We also identified five associations that appeared to be female-specific. A number of rare variants that influence metabolite levels were also found, and combinations of common and rare variants influenced the metabolite levels more profoundly. These results support our contention that metabolic phenotyping provides important insights into how genetic and environmental factors provoke human diseases., By combining whole genome sequencing and metabolomics, Koshiba et al. identify a large number of associations between genetic variants and plasma metabolites. They find that some genetic variants affect the interaction between the host and symbiotic bacteria. This study provides insights into how genetic and environmental factors interact to shape human metabolism.

The overwhelming majority of participants in current genetic studies are of European ancestry. To elucidate disease biology in the East Asian population, we conducted a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 320 independent signals in 276 loci for 27 diseases, with 25 novel loci (P < 9.58 × 10-9). East Asian-specific missense variants were identified as candidate causal variants for three novel loci, and we successfully replicated two of them by analyzing independent Japanese cohorts; p.R220W of ATG16L2 (associated with coronary artery disease) and p.V326A of POT1 (associated with lung cancer). We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, and identified 378 significant enrichments across nine diseases (false discovery rate < 0.05) (for example, NKX3-1 for prostate cancer). This large-scale GWAS in a Japanese population provides insights into the etiology of complex diseases and highlights the importance of performing GWAS in non-European populations.

In order to assess the long-term impact of the Great East Japan Earthquake on the oral health of disaster victims and to evaluate gene-environmental interactions in the development of major oral diseases and oral-systemic associations, the oral part of two large-scale genome cohort studies by the Tohoku Medical Megabank Organization (ToMMo), including the Community-based cohort (CommCohort) study and the Birth and Three-Generation cohort (BirThree) study, have been conducted. The study population comprised 32,185 subjects, including 16,886 participants in the CommCohort study and 15,299 participants in the BirThree cohort study, recruited from 2013 to 2017. The oral studies consist of a questionnaire regarding oral hygiene behavior, clinical examinations by dentists, and oral plaque and saliva sampling for microbiome analyses, which were carried out at seven community support centers in Miyagi prefecture. The median age of all participants was 55.0 years, and 66.1% of participants were women. Almost all participants reported that they brushed their teeth more than once a day. The median number of present teeth was 27.0, and the decayed, missing and filled tooth number was 16.0, with a significant difference according to age and sex. The median periodontal pocket and clinical attachment level was 2.48 mm and 4.00 mm, respectively. Periodontal parameters increased significantly according to age, except for the accumulation of dental calculus. The oral part of these extensive cross-sectional studies provides a unique and important platform for future studies on oral health and diseases that elicit through interactions with systemic diseases, lifestyles, life events and genetic backgrounds, and contributes to researches clarifying the long-term effects of disasters on oral health.

Background and Aims Mosaic chromosomal alterations [mCAs] increase the risk for haematopoietic malignancies and may be risk factors for several other diseases. Inflammatory bowel diseases [IBDs], including Crohn’s disease [CD] and ulcerative colitis [UC], are associated with mCAs, and patients may be at risk for haematopoietic malignancy development and/or modification of IBD phenotypes. In the present study, we screened patients with IBD for the presence of mCAs and explored the possible pathophysiological and genetic risk factors for mCAs. Methods We analysed mCAs in peripheral blood from 3339 patients with IBD and investigated the clinical and genetic risk factors for mCAs. Results CD and exposure to thiopurines before the age of 20 years were identified as novel independent risk factors for mCAs [odds ratio = 2.15 and 5.68, p = 1.17e-2 and 1.60e-3, respectively]. In contrast, there were no significant associations of disease duration, anti-tumour necrosis factor alpha antibodies, or other clinical factors with mCAs. Gene ontology enrichment analysis revealed that genes specifically located in the mCAs in patients with CD were significantly associated with factors related to mucosal immune responses. A genome-wide association study revealed that ERBIN, CD96, and AC068672.2 were significantly associated with mCAs in patients with CD [p = 1.56e-8, 1.65e-8, and 4.92e-8, respectively]. Conclusions The difference in mCAs between patients with CD and UC supports the higher incidence of haematopoietic malignancies in CD. Caution should be exercised when using thiopurines in young patients with IBD, particularly CD, in light of possible chromosomal alterations.

Examining plasma metabolic profiling during pregnancy and postpartum could help clinicians understand the risk factors for postpartum depression (PPD) development. This analysis targeted paired plasma metabolites in mid-late gestational and 1 month postpartum periods in women with (n = 209) or without (n = 222) PPD. Gas chromatogram-mass spectrometry was used to analyze plasma metabolites at these two time points. Among the 170 objected plasma metabolites, principal component analysis distinguished pregnancy and postpartum metabolites but failed to discriminate women with and without PPD. Compared to women without PPD, those with PPD exhibited 37 metabolites with disparate changes during pregnancy and the 1-month postpartum period and an enriched citrate cycle. Machine learning and multivariate statistical analysis identified two or three compounds that could be potential biomarkers for PPD prediction during pregnancy. Our findings suggest metabolic disturbances in women with depression and may help to elucidate metabolic processes associated with PPD development.

Pharmacogenomic (PGx) testing results provide valuable information on drug selection and appropriate dosing, maximization of efficacy, and minimization of adverse effects. Although the number of large-scale, next-generation-sequencing-based PGx studies has recently increased, little is known about the risks and benefits of returning PGx results to ostensibly healthy individuals in research settings.Single-nucleotide variants of three actionable PGx genes, namely,Whereas the majority of participants were unfamiliar with the term PGx, and none had undergone PGx testing before the study, more than 80% of the participants felt that they could acquire basic PGx knowledge sufficient to understand their genomic results and were satisfied with their potential benefit and use in future prescriptions. On the other hand, some felt that the PGx concepts or terminology was difficult to fully understand and suggested that in-person return of the results was desirable.These results collectively suggest possible benefits of returning preemptive PGx information to ostensibly healthy cohort participants in a research setting.

The genetic variants of NUDT15 have been verified to induce adverse events (AEs) of thiopurines. Codon 139 variants are frequently observed in Asians, while multiple variants are seen in codon 18 which also cause AEs including the European ancestry. The purpose of this study is to establish a technique capable of the simple genotyping of NUDT15 codon 18 and to evaluate its efficacy. A high-resolution melt (HRM) technique is performed to simply determine genotypes. The accuracy of HRM analysis was evaluated with DNAs from 1245 Japanese patients with inflammatory bowel diseases. Subsequently, another group of 572 patients was analyzed to verify the method. The diplotypes and the frequency of their AEs were estimated on the basis of codon 18 and 139 genotypes. The HRM analysis enabled the correct identification of the three main genotypes, ref/ref, ref/ins, and ref/V18I, in 1236 of 1241 cases. All rare genotypes including ref/del were identified as the impossible-to-determine group, the proper diagnosis rate was 99.6%. In the verification test using other samples, the diagnosis rate was 99.7%. By estimating diplotypes using both codon 18 and 139 genotypes, 2.74% and 2.13% of Japanese patients with Arg/Arg and Arg/Cys of codon 139 have a lower enzymatic activity of NUDT15 and a higher risk for adverse responses than those estimated by codon 139 genotypes alone. Our study showed that HRM method enables simple genotyping of complicated codon 18 variants essential to haplotype estimation of the NUDT15.

Genome-wide association studies (GWAS) have identified ~170 genetic loci associated with prostate cancer (PCa) risk, but most of them were identified in European populations. We here performed a GWAS and replication study using a large Japanese cohort (9, 906 cases and 83, 943 male controls) to identify novel susceptibility loci associated with PCa risk. We found 12 novel loci for PCa including rs1125927 (TMEM17, P = 3.95 × 10−16), rs73862213 (GATA2, P = 5.87 × 10−23), rs77911174 (ZMIZ1, P = 5.28 × 10−20), and rs138708 (SUN2, P = 1.13 × 10−15), seven of which had crucially low minor allele frequency in European population. Furthermore, we stratified the polygenic risk for Japanese PCa patients by using 82 SNPs, which were significantly associated with Japanese PCa risk in our study, and found that early onset cases and cases with family history of PCa were enriched in the genetically high-risk population. Our study provides important insight into genetic mechanisms of PCa and facilitates PCa risk stratification in Japanese population., 前立腺がんの若年発症を予測するゲノム診断手法を開発 --前立腺がんのゲノムワイド関連解析からゲノム医療へ--. 京都大学プレスリリース. 2019-10-08.

In recent genome analyses, population-specific reference panels have indicated important. However, reference panels based on short-read sequencing data do not sufficiently cover long insertions. Therefore, the nature of long insertions has not been well documented. Here, we assembled a Japanese genome using single-molecule real-time sequencing data and characterized insertions found in the assembled genome. We identified 3691 insertions ranging from 100 bps to ~10,000 bps in the assembled genome relative to the international reference sequence (GRCh38). To validate and characterize these insertions, we mapped short-reads from 1070 Japanese individuals and 728 individuals from eight other populations to insertions integrated into GRCh38. With this result, we constructed JRGv1 (Japanese Reference Genome version 1) by integrating the 903 verified insertions, totaling 1,086,173 bases, shared by at least two Japanese individuals into GRCh38. We also constructed decoyJRGv1 by concatenating 3559 verified insertions, totaling 2,536,870 bases, shared by at least two Japanese individuals or by six other assemblies. This assembly improved the alignment ratio by 0.4% on average. These results demonstrate the importance of refining the reference assembly and creating a population-specific reference genome. JRGv1 and decoyJRGv1 are available at the JRG website., Reference genome: Reading longer sequences improves Japanese reference Researchers in Japan have assembled a Japanese reference genome, which includes sequences missing from the international reference genome, as well as others specific to East Asian populations. A team led by Masao Nagasaki and Masayuki Yamamoto sequenced a Japanese individual using a method, which produces longer sequences than previous technologies. Using this approach, they identified thousands of sequences spanning 2.5 million bases, which were absent in the international reference genome. Many of these were sequences able to move within the genome. They showed that the majority of these sequences are also present in early humans and chimpanzees, demonstrating that their absence from the current reference is due to deletions or limitations of earlier sequencing methodologies. In addition to providing a population-specific reference, these findings demonstrate the importance of continually improving the international reference genome.

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.

To elucidate the differences in ocular biometric parameters by generation and gender and to identify axial length (AL)-associated genetic variants in Japanese individuals, we analyzed Tohoku Medical Megabank Organization (ToMMo) Eye Study data.We designed the ToMMo Eye Study, examined AL variations, and conducted genome-wide association studies (GWASs).In total, 33 483 participants aged18 years who were recruited into the community-based cohort (CommCohort) and the birth and three-generation cohort (BirThree Cohort) of the ToMMo Eye Study were examined.Each participant was screened with an interview, ophthalmic examinations, and a microarray analysis. The GWASs were performed in 22 379 participants in the CommCohort (discovery stage) and 11 104 participants in the BirThree Cohort (replication stage). We evaluated the associations of single nucleotide polymorphisms (SNPs) with AL using a genome-wide significance threshold (5 × 10We identified the association of SNPs with AL and distributions of AL in right and left eyes and individuals of different sexes and ages.In the discovery stage, the mean AL of the right eye (23.99 mm) was significantly greater than that of the left eye (23.95 mm). This difference was reproducible across sexes and ages. The GWASs revealed 703 and 215 AL-associated SNPs with genome-wide significance in the discovery and validation stages, respectively, and many of the SNPs in the discovery stage were replicated in the validation stage. Validated SNPs and their associated loci were meta-analyzed for statistical significance (Our large-scale GWASs exploiting ToMMo Eye Study data identified 31 loci linked to variations in AL, 7 of which are newly reported in this article. The results revealed genetic heterogeneity and similarity in SNPs related to ethnic variations in AL.

The elucidation of dynamic metabolomic changes during gestation is particularly important for the development of methods to evaluate pregnancy status or achieve earlier detection of pregnancy-related complications. Some studies have constructed models to evaluate pregnancy status and predict gestational age using omics data from blood biospecimens; however, less invasive methods are desired. Here we propose a model to predict gestational age, using urinary metabolite information. In our prospective cohort study, we collected 2,741 urine samples from 187 healthy pregnant women, 23 patients with hypertensive disorders of pregnancy, and 14 patients with spontaneous preterm birth. Using gas chromatography-tandem mass spectrometry, we identified 184 urinary metabolites that showed dynamic systematic changes in healthy pregnant women according to gestational age. A model to predict gestational age during normal pregnancy progression was constructed; the correlation coefficient between actual and predicted weeks of gestation was 0.86. The predicted gestational ages of cases with hypertensive disorders of pregnancy exhibited significant progression, compared with actual gestational ages. This is the first study to predict gestational age in normal and complicated pregnancies by using urinary metabolite information. Minimally invasive urinary metabolomics might facilitate changes in the prediction of gestational age in various clinical settings.

Certain large genome cohort studies attempt to return the individual genomic results to the participants; however, the implementation process and psychosocial impacts remain largely unknown. The Tohoku Medical Megabank Project has conducted large genome cohort studies of general residents. To implement the disclosure of individual genomic results, we extracted the potential challenges and obstacles. Major challenges include the determination of genes/disorders based on the current medical system in Japan, the storage of results, prevention of misunderstanding, and collaboration of medical professionals. To overcome these challenges, we plan to conduct multilayer pilot studies, which deal with different disorders/genes. We finally chose familial hypercholesterolemia (FH) as a target disease for the first pilot study. Of the 665 eligible candidates, 33.5% were interested in the pilot study and provided consent after an educational "genetics workshop" on the basic genetics and medical facts of FH. The genetics professionals disclosed the results to the participants. All positive participants were referred to medical care, and a serial questionnaire revealed no significant psychosocial distress after the disclosure. Return of genomic results to research participants was implemented using a well-prepared protocol. To further elucidate the impact of different disorders, we will perform multilayer pilot studies with different disorders, including actionable pharmacogenomics and hereditary tumor syndromes.

Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading concerning the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive motor neuron disease that affects people of all ethnicities. Approximately 90% of ALS cases are sporadic and thought to have multifactorial pathogenesis. To understand the genetics of sporadic ALS, we conducted a genome-wide association study using 1,173 sporadic ALS cases and 8,925 controls in a Japanese population. A combined meta-analysis of our Japanese cohort with individuals of European ancestry revealed a significant association at the ACSL5 locus (top SNP p = 2.97 × 10−8). We validated the association with ACSL5 in a replication study with a Chinese population and an independent Japanese population (1941 ALS cases, 3821 controls; top SNP p = 1.82 × 10−4). In the combined meta-analysis, the intronic ACSL5 SNP rs3736947 showed the strongest association (p = 7.81 × 10−11). Using a gene-based analysis of the full multi-ethnic dataset, we uncovered additional genes significantly associated with ALS: ERGIC1, RAPGEF5, FNBP1, and ATXN3. These results advance our understanding of the genetic basis of sporadic ALS., Gen Sobue, Masao Nagasaki and colleagues report a genome-wide association study for amyotrophic lateral sclerosis (ALS) in a large, multi-ethnic cohort comprising Japanese, Chinese, and European ancestry populations. They find a significant association to variants within the ACSL5 gene and identify novel associations with 4 additional genes using a gene-based approach.

Simple Summary Pancreatic cancer (PC) is among the most lethal malignancies due to an often delayed and difficult initial diagnosis. Therefore, the development of a novel, early stage, diagnostic PC marker in liquid biopsies is of great significance. In this study, we analyzed the differential glycomic profiling of extracellular vesicles (EVs) derived from serum using lectin microarray. The glyco-candidates of PC-specific EVs were quantified using a high-sensitive exosome-counting system, ExoCounter. An absolute quantification system for altered glycan-containing EVs elevated in PC serum was established. EVs recognized by O-glycan-binding lectins ABA or ACA were identified as candidate markers by lectin microarray. Quantitative analyses using ExoCounter revealed that the ABA- or ACA-positive EVs were significantly increased in the serum of PC patients. These specific EVs with O-glycans can act as potential biomarkers in a liquid biopsy for PC patients screening. Abstract Pancreatic cancer (PC) is among the most lethal malignancies due to an often delayed and difficult initial diagnosis. Therefore, the development of a novel, early stage, diagnostic PC marker in liquid biopsies is of great significance. In this study, we analyzed the differential glycomic profiling of extracellular vesicles (EVs) derived from serum (two cohorts including 117 PC patients and 98 normal controls) using lectin microarray. The glyco-candidates of PC-specific EVs were quantified using a high-sensitive exosome-counting system, ExoCounter. An absolute quantification system for altered glycan-containing EVs elevated in PC serum was established. EVs recognized by O-glycan-binding lectins ABA or ACA were identified as candidate markers by lectin microarray. Quantitative analyses using ExoCounter revealed that the ABA- or ACA-positive EVs were significantly increased in the culture of PC cell lines or in the serum of PC patients including carbohydrate antigen 19-9 negative patients with high area under curve values. The elevated numbers of EVs in PC serum returned to normal levels after pancreatectomy. Histological examination confirmed that the tumors stained with ABA/ACA. These specific EVs with O-glycans recognized by ABA/ACA are elevated in PC sera and can act as potential biomarkers in a liquid biopsy for PC patients screening.

Genetic polymorphisms contribute to inter-individual variability in the metabolism of multiple clinical drugs, including warfarin, thiopurines, primaquine, and aminoglycosides. A rapid and sensitive clinical assessment of various genome biomarkers is, therefore, required to predict the individual responsiveness of each patient to these drugs. In this study, we developed a novel genotyping method for the detection of nine pharmacogene variants that are important in the prediction of drug efficiency and toxicity. This genotyping method uses competitive allele-specific PCR and a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) that can unambiguously determine the presence or absence of the gene variant by displaying visible blue lines on the chromatographic printed-array strip. Notably, the results of our STH-PAS method were in 100% agreement with those obtained using standard Sanger sequencing and KASP assay genotyping methods for CYP4F2 gene deletion. Moreover, the results were obtained within 90 min, including the PCR amplification and signal detection processes. The sensitive and rapid nature of this novel method make it ideal for clinical genetic testing to predict drug efficacy and toxicity, and in doing so will aid in the development of individualized medicine and better patient care.

BACKGROUND Although circulating exosomes in blood play crucial roles in cancer development and progression, difficulties in quantifying exosomes hamper their application for reliable clinical testing. By combining the properties of nanobeads with optical disc technology, we have developed a novel device named the ExoCounter to determine the exact number of exosomes in the sera of patients with various types of cancer. METHOD In this system, individual exosomes were captured in the groove of an optical disc coated with antibodies against exosome surface antigens. The captured exosomes were labeled with antibody-conjugated magnetic nanobeads, and the number of the labeled exosomes was counted with an optical disc drive. RESULTS We showed that the ExoCounter could detect specific exosomes derived from cells or human serum without any enrichment procedures. The detection sensitivity and linearity with this system were higher than those with conventional detection methods such as ELISA or flow cytometry. In addition to the ubiquitous exosome markers CD9 and CD63, the cancer-related antigens CD147, carcinoembryonic antigen, and human epidermal growth factor receptor 2 (HER2) were also used to quantify cancer cell line-derived exosomes. Furthermore, analyses of a cross-sectional cohort of sera samples revealed that HER2-positive exosomes were significantly increased in patients with breast cancer or ovarian cancer compared with healthy individuals and those with noncancer diseases. CONCLUSIONS The ExoCounter system exhibits high performance in the direct detection of exosomes in cell culture and human sera. This method may enable reliable analysis of liquid biopsies.

Genetic variations within cytochrome P450 2B6 (CYP2B6) contribute to inter-individual variation in the metabolism of clinically important drugs, including cyclophosphamide, bupropion, methadone and efavirenz (EFZ). In this study, we performed an in vitro analysis of 40 CYP2B6 allelic variant proteins including seven novel variants identified in 1070 Japanese individuals. Wild-type and 39 variant proteins were heterologously expressed in 293FT cells to estimate the kinetic parameters (Km, Vmax, and CLint) of EFZ 8-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin (7-ETC) O-deethylation activities. The concentrations of CYP2B6 variant holo-enzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild-type and 28 variants showed a peak at 450 nm. The kinetic parameters were measured for the wild-type and 24 variant proteins. The values for the remaining 15 variants could not be determined because the enzymatic activity was not detected at the highest substrate concentration used. Compared to wild-type, six variants showed significantly decreased EFZ 8-hydroxylation CLint values, while these values were significantly increased in another six variants, including CYP2B6.6. Although 7-ETC O-deethylation CLint values of CYP2B6 variants did not differ significantly from that of CYP2B6.1, the CLint ratios obtained for 7-ETC O-deethylation were highly correlated with EFZ 8-hydroxylation. Furthermore, three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2B6 variants. Our findings could provide evidence of the specific metabolic activities of the CYP2B6 proteins encoded by these variant alleles.

Clarifying allele frequencies of disease-related genetic variants in a population is important in genomic medicine; however, such data is not yet available for the Japanese population. To estimate frequencies of actionable pathogenic variants in the Japanese population, we examined the reported pathological variants in genes recommended by the American College of Medical Genetics and Genomics (ACMG) in our reference panel of genomic variations, 2KJPN, which was created by whole-genome sequencing of 2049 individuals of the resident cohort of the Tohoku Medical Megabank Project. We searched for pathogenic variants in 2KJPN for 57 autosomal ACMG-recommended genes responsible for 26 diseases and then examined their frequencies. By referring to public databases of pathogenic variations, we identified 143 reported pathogenic variants in 2KJPN for the 57 ACMG recommended genes based on a classification system. At the individual level, 21% of the individuals were found to have at least one reported pathogenic allele. We then conducted a literature survey to review the variants and to check for evidence of pathogenicity. Our results suggest that a substantial number of people have reported pathogenic alleles for the ACMG genes, and reviewing variants is indispensable for constructing the information infrastructure of genomic medicine for the Japanese population.

Background With the goal of realizing genome-based personalized healthcare, we have developed a biobank that integrates personal health, genome, and omics data along with biospecimens donated by volunteers of 150,000. Such a large-scale of data integration involves obvious risks of privacy violation. The research use of personal genome and health information is a topic of global discussion with regard to the protection of privacy while promoting scientific advancement. The present paper reports on our plans, current attempts, and accomplishments in addressing security problems involved in data sharing to ensure donor privacy while promoting scientific advancement. Methods Biospecimens and data have been collected in prospective cohort studies with the comprehensive agreement. The sample size of 150,000 participants was required for multiple researches including genome-wide screening of gene by environment interactions, haplotype phasing, and parametric linkage analysis. Results We established the T ohoku M edical M egabank (TMM) data sharing policy: a privacy protection rule that requires physical, personnel, and technological safeguards against privacy violation regarding the use and sharing of data. The proposed policy refers to that of NCBI and that of the Sanger Institute. The proposed policy classifies shared data according to the strength of re-identification risks. Local committees organized by TMM evaluate re-identification risk and assign a security category to a dataset. Every dataset is stored in an assigned segment of a supercomputer in accordance with its security category. A security manager should be designated to handle all security problems at individual data use locations. The proposed policy requires closed networks and IP-VPN remote connections. Conclusion The mission of the biobank is to distribute biological resources most productively. This mission motivated us to collect biospecimens and health data and simultaneously analyze genome/omics data in-house. The biobank also has the mission of improving the quality and quantity of the contents of the biobank. This motivated us to request users to share the results of their research as feedback to the biobank. The TMM data sharing policy has tackled every security problem originating with the missions. We believe our current implementation to be the best way to protect privacy in data sharing. Electronic supplementary material The online version of this article (doi:10.1186/s12911-017-0494-5) contains supplementary material, which is available to authorized users.

INTRODUCTORY PARAGRAPHThe overwhelming majority of participants in current genetic studies are of European ancestry1–3, limiting our genetic understanding of complex disease in non-European populations. To address this, we aimed to elucidate polygenic disease biology in the East Asian population by conducting a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 383 independent signals in 331 loci for 30 diseases, among which 45 loci were novel (P< 5 × 10−8). Compared with known variants, novel variants have lower frequency in European populations but comparable frequency in East Asian populations, suggesting the advantage of this study in discovering these novel variants. Three novel signals were in linkage disequilibrium (r2> 0.6) with missense variants which are monomorphic in European populations (1000 Genomes Project) including rs11235604(p.R220W ofATG16L2, a autophagy-related gene) associated with coronary artery disease. We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, andidentified 378 significant enrichments across nine diseases (FDR < 0.05) (e.g. NF-κB for immune-related diseases). This large-scale GWAS in a Japanese population provides insights into the etiology of common complex diseases and highlights the importance of performing GWAS in non-European populations.

Human height is a representative phenotype to elucidate genetic architecture. However, the majority of large studies have been performed in European population. To investigate the rare and low-frequency variants associated with height, we construct a reference panel (N = 3,541) for genotype imputation by integrating the whole-genome sequence data from 1,037 Japanese with that of the 1000 Genomes Project, and perform a genome-wide association study in 191,787 Japanese. We report 573 height-associated variants, including 22 rare and 42 low-frequency variants. These 64 variants explain 1.7% of the phenotypic variance. Furthermore, a gene-based analysis identifies two genes with multiple height-increasing rare and low-frequency nonsynonymous variants (SLC27A3 and CYP26B1; PSKAT-O, Thousands of genetic loci are known to associate with human height, but these are mainly based on studies in European ancestry populations. Here, Akiyama et al. construct a genotype reference panel for the Japanese population followed by GWAS and report 573 height associated variants in 191,787 Japanese.

Background Genome cohort studies are used to analyze interactions between genetic and environmental factors, providing valuable information for personalized healthcare. Large-scale and long-term cohort studies require a number of specially trained personnel, of whom those involved in obtaining informed consent play a vital role, especially during the initial phase of such studies. The Japanese Society of Human Genetics (JSHG) previously established a certification system for genome medical research coordinators (GMRCs) responsible for obtaining written consent via face-to-face explanation. Meanwhile, in the Tohoku Medical Megabank Organization (ToMMo), GMRCs are expected to play important roles not only in obtaining informed consent and conducting various assessments, but also in communicating with participants throughout the long-term follow-up. Based on the JSHG program, we therefore developed a specific education and training program for ToMMo GMRCs consisting of 17 lectures, one practical training session on the informed consent procedure, and written and interview examinations. Re-education workshops aimed at self-improvement are also carried out following certification. In this study, we evaluated the education and training program in terms of overall understanding, usefulness, and satisfaction using an anonymous questionnaire. Methods An anonymous questionnaire addressing each aspect of the education and training program (understanding, usefulness, and satisfaction) was distributed among 152 qualified ToMMo GMRCs. Responses were received from 94 participants (61.8%). Results There was a significant association between the level of overall understanding of lectures and medical qualification (nurse or clinical laboratory technologist), but not with age or educational background. The level of understanding and overall usefulness were lower in sessions related to genetics and epidemiology than those dealing with ToMMo practices. In the re-education workshops, GMRCs showed a preference for and hoped to learn more about both background knowledge and research progress in the ToMMo. Conclusions The results of our questionnaire suggest that not all ToMMo GMRCs are able to understand everything during the initial education and training program, especially in terms of genomic medicine. Continuous re-education is therefore vital in improving knowledge, skills and motivation, and preparing GMRCs for a specialist role in community-based personalized healthcare. Electronic supplementary material The online version of this article (10.1186/s12909-019-1725-5) contains supplementary material, which is available to authorized users.