Early-life atomic-bomb irradiation accelerates immunological aging and elevates immune-related intracellular reactive oxygen species.

Reactive oxygen species (ROS) play an important role in immune responses; however, their excessive production and accumulation increases the risk of inflammation-related diseases. Although irradiation is known to accelerate immunological aging, the underlying mechanism is still unclear. To determine the possible involvement of ROS in this mechanism, we examined 10,023 samples obtained from 3752 atomic-bomb survivors in Hiroshima and Nagasaki, who participated in repeated biennial examinations from 2008 to 2016, for the effects of aging and radiation exposure on intracellular ROS (H2 O2 and O2 •- ) levels, percentages of T-cell subsets, and the effects of radiation exposure on the relationship between cell percentages and intracellular ROS levels in T-cell subsets. The cell percentages and intracellular ROS levels in T-cell subsets were measured using flow cytometry, with both fluorescently labeled antibodies and the fluorescent reagents, carboxy-DCFDA and hydroethidine. The percentages of naïve CD4+ and CD8+ T cells decreased with increasing age and radiation dose, while the intracellular O2 •- levels in central and effector memory CD8+ T cells increased. Additionally, when divided into three groups based on the percentages of naïve CD4+ T cells, intracellular O2 •- levels of central and effector memory CD8+ T cells were significantly elevated with the lowest radiation dose group in the naïve CD4+ T cells. Thus, the radiation exposure-induced decrease in the naïve CD4+ T cell pool size may reflect decreased immune function, resulting in increased intracellular ROS levels in central and effector memory CD8+ T cells, and increased intracellular oxidative stress.

Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells.

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated in vitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded in vivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

Maternal Baseline Characteristics and Perinatal Outcomes: The Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study.

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.

Machine learning approaches to predict gestational age in normal and complicated pregnancies via urinary metabolomics analysis.

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 2741 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.

Study Profile of the Tohoku Medical Megabank Community-Based Cohort Study.

BACKGROUND: We established a community-based cohort study to assess the long-term impact of the Great East Japan Earthquake on disaster victims and gene-environment interactions on the incidence of major diseases, such as cancer and cardiovascular diseases. METHODS: We asked participants to join our cohort in the health check-up settings and assessment center based settings. Inclusion criteria were aged 20 years or over and living in Miyagi or Iwate Prefecture. We obtained information on lifestyle, effect of disaster, blood, and urine information (Type 1 survey), and some detailed measurements (Type 2 survey), such as carotid echography and calcaneal ultrasound bone mineral density. All participants agreed to measure genome information and to distribute their information widely. RESULTS: As a result, 87,865 gave their informed consent to join our study. Participation rate at health check-up site was about 70%. The participants in the Type 1 survey were more likely to have psychological distress than those in the Type 2 survey, and women were more likely to have psychological distress than men. Additionally, coastal residents were more likely to have higher degrees of psychological distress than inland residents, regardless of sex. CONCLUSION: This cohort comprised a large sample size and it contains information on the natural disaster, genome information, and metabolome information. This cohort also had several detailed measurements. Using this cohort enabled us to clarify the long-term effect of the disaster and also to establish personalized prevention based on genome, metabolome, and other omics information.

Genome-wide association study identifies new loci for albuminuria in the Japanese population.

BACKGROUND: Urinary albumin excretion (UAE) is a risk factor for cardiovascular diseases, metabolic syndrome, chronic kidney disease, etc. Only a few genome-wide association studies (GWAS) for UAE have been conducted in the European population, but not in the Asian population. Here we conducted GWAS and identified several candidate genes harboring single nucleotide polymorphisms (SNPs) responsible for UAE in the Japanese population. METHODS: We conducted GWAS for UAE in 7805 individuals of Asian ancestry from health-survey data collected by Tohoku Medical Megabank Organization (ToMMo) and Iwate Tohoku Medical Megabank Organization (IMM). The SNP genotype data were obtained with a SNP microarray. After imputation using a haplotype panel consisting of 2000 genome sequencing, 4,962,728 SNP markers were used for the GWAS. RESULTS: Eighteen SNPs at 14 loci (GRM7, EXOC1/NMU, LPA, STEAP1B/RAPGEF5, SEMA3D, PRKAG2, TRIQK, SERTM1, TPT1-AS1, OR5AU1, TSHR, FMN1/RYR3, COPRS, and BRD1) were associated with UAE in the Japanese individuals. A locus with particularly strong associations was observed on TSHR, chromosome 14 [rs116622332 (p = 3.99 × 10-10)]. CONCLUSION: In this study, we successfully identified UAE-associated variant loci in the Japanese population. Further study is required to confirm this association.

Oral Microbiome Analysis in Prospective Genome Cohort Studies of the Tohoku Medical Megabank Project.

A baseline oral microbiome study of the Tohoku Medical Megabank Organization (TMM) was planned to characterize the profile of the oral microbiome in the Japanese population. The study also aimed to clarify risk factors for multifactorial diseases by integrated analysis of the oral microbiome and host genome/omics information. From 2013 to 2016, we collected three types of oral biospecimens, saliva, supragingival plaque, and tongue swab, from a total of 25,101 participants who had a dental examination in TMM. In this study, we used two independent cohorts; the Community-Based Cohort and Birth and Three-Generation Cohort as discovery and validation cohorts, respectively, and we selected participants examined by a single dentist. We found through the 16S ribosomal RNA gene sequencing analysis of 834 participants of the Community-Based Cohort Study that there are differences in the microbial composition and community structure between saliva and plaque. The species diversities in both saliva and plaque were increased in correlation with the severity of periodontal disease. These results were nicely reproduced in the analysis of 455 participants of the Birth and Three-Generation Cohort Study. In addition, strong positive and negative associations of microbial taxa in both plaque and saliva with periodontitis-associated biofilm formation were detected by co-occurrence network analysis. The classes Actinobacteria and Bacilli, including oral health-associated bacterial species, showed a positive correlation in saliva. These results revealed differences in microbial composition and community structure between saliva and plaque and a correlation between microbial species and the severity of periodontal disease. We expect that the large database of the oral microbiome in the TMM biobank will help in the discovery of novel targets for the treatment and prevention of oral diseases, as well as for the discovery of therapeutic and/or preventive targets of systemic diseases.

A single nucleotide polymorphism (-250 A/C) of the GFAP gene is associated with brain structures and cerebral blood flow.

AIM: Glial fibrillary acidic protein (GFAP), the intermediate filament protein expressed in astrocytes, plays a key role in many aspects of brain function through communication with neurons or blood vessels. A common single nucleotide polymorphism (SNP), GFAP -250 C/A (rs2070935), is associated with the transcriptional regulation of GFAP, which can potentially result in the genotype-specific brain structure. This study aimed to verify the biological effects of the GFAP variants on brain structure and function. METHODS: We investigated the associations between the GFAP variants and magnetic resonance imaging findings, including gray and white matter volumes, white matter integrity, and resting arterial blood flow, from 1212 healthy Japanese subjects. RESULTS: The GFAP -250 C/A genotype was significantly associated with total gray matter volume, total white matter volume, average mean diffusivity, and mean cerebral blood flow. In voxel-by-voxel analyses, the GFAP genotype showed significant associations with the regional gray and white matter volumes in the inferior frontal lobe and corpus callosum, the regional mean diffusivity in the left posterior region, and the regional cerebral blood flow throughout the brain. CONCLUSION: This study revealed a common SNP that is significantly associated with multiple global brain structure parameters.

Construction of JRG (Japanese reference genome) with single-molecule real-time sequencing.

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.

Estimating carrier frequencies of newborn screening disorders using a whole-genome reference panel of 3552 Japanese individuals.

Incidence rates of Mendelian diseases vary among ethnic groups, and frequencies of variant types of causative genes also vary among human populations. In this study, we examined to what extent we can predict population frequencies of recessive disorders from genomic data, and explored better strategies for variant interpretation and classification. We used a whole-genome reference panel from 3552 general Japanese individuals constructed by the Tohoku Medical Megabank Organization (ToMMo). Focusing on 32 genes for 17 congenital metabolic disorders included in newborn screening (NBS) in Japan, we identified reported and predicted pathogenic variants through variant annotation, interpretation, and multiple ways of classifications. The estimated carrier frequencies were compared with those from the Japanese NBS data based on 1,949,987 newborns from a previous study. The estimated carrier frequency based on genomic data with a recent guideline of variant interpretation for the PAH gene, in which defects cause hyperphenylalaninemia (HPA) and phenylketonuria (PKU), provided a closer estimate to that by the observed incidence than the other methods. In contrast, the estimated carrier frequencies for SLC25A13, which causes citrin deficiency, were much higher compared with the incidence rate. The results varied greatly among the 11 NBS diseases with single responsible genes; the possible reasons for departures from the carrier frequencies by reported incidence rates were discussed. Of note, (1) the number of pathogenic variants increases by including additional lines of evidence, (2) common variants with mild effects also contribute to the actual frequency of patients, and (3) penetrance of each variant remains unclear.

Maternity Log study: a longitudinal lifelog monitoring and multiomics analysis for the early prediction of complicated pregnancy.

PURPOSE: A prospective cohort study for pregnant women, the Maternity Log study, was designed to construct a time-course high-resolution reference catalogue of bioinformatic data in pregnancy and explore the associations between genomic and environmental factors and the onset of pregnancy complications, such as hypertensive disorders of pregnancy, gestational diabetes mellitus and preterm labour, using continuous lifestyle monitoring combined with multiomics data on the genome, transcriptome, proteome, metabolome and microbiome. PARTICIPANTS: Pregnant women were recruited at the timing of first routine antenatal visits at Tohoku University Hospital, Sendai, Japan, between September 2015 and November 2016. Of the eligible women who were invited, 65.4% agreed to participate, and a total of 302 women were enrolled. The inclusion criteria were age ≥20 years and the ability to access the internet using a smartphone in the Japanese language. FINDINGS TO DATE: Study participants uploaded daily general health information including quality of sleep, condition of bowel movements and the presence of nausea, pain and uterine contractions. Participants also collected physiological data, such as body weight, blood pressure, heart rate and body temperature, using multiple home healthcare devices. The mean upload rate for each lifelog item was ranging from 67.4% (fetal movement) to 85.3% (physical activity), and the total number of data points was over 6 million. Biospecimens, including maternal plasma, serum, urine, saliva, dental plaque and cord blood, were collected for multiomics analysis. FUTURE PLANS: Lifelog and multiomics data will be used to construct a time-course high-resolution reference catalogue of pregnancy. The reference catalogue will allow us to discover relationships among multidimensional phenotypes and novel risk markers in pregnancy for the future personalised early prediction of pregnancy complications.

Genome analyses for the Tohoku Medical Megabank Project towards establishment of personalized healthcare.

Personalized healthcare (PHC) based on an individual's genetic make-up is one of the most advanced, yet feasible, forms of medical care. The Tohoku Medical Megabank (TMM) Project aims to combine population genomics, medical genetics and prospective cohort studies to develop a critical infrastructure for the establishment of PHC. To date, a TMM CommCohort (adult general population) and a TMM BirThree Cohort (birth+three-generation families) have conducted recruitments and baseline surveys. Genome analyses as part of the TMM Project will aid in the development of a high-fidelity whole-genome Japanese reference panel, in designing custom single-nucleotide polymorphism (SNP) arrays specific to Japanese, and in estimation of the biological significance of genetic variations through linked investigations of the cohorts. Whole-genome sequencing from >3,500 unrelated Japanese and establishment of a Japanese reference genome sequence from long-read data have been done. We next aim to obtain genotype data for all TMM cohort participants (>150,000) using our custom SNP arrays. These data will help identify disease-associated genomic signatures in the Japanese population, while genomic data from TMM BirThree Cohort participants will be used to improve the reference genome panel. Follow-up of the cohort participants will allow us to test the genetic markers and, consequently, contribute to the realization of PHC.

Long non-coding RNA-dependent mechanism to regulate heme biosynthesis and erythrocyte development.

In addition to serving as a prosthetic group for enzymes and a hemoglobin structural component, heme is a crucial homeostatic regulator of erythroid cell development and function. While lncRNAs modulate diverse physiological and pathological cellular processes, their involvement in heme-dependent mechanisms is largely unexplored. In this study, we elucidated a lncRNA (UCA1)-mediated mechanism that regulates heme metabolism in human erythroid cells. We discovered that UCA1 expression is dynamically regulated during human erythroid maturation, with a maximal expression in proerythroblasts. UCA1 depletion predominantly impairs heme biosynthesis and arrests erythroid differentiation at the proerythroblast stage. Mechanistic analysis revealed that UCA1 physically interacts with the RNA-binding protein PTBP1, and UCA1 functions as an RNA scaffold to recruit PTBP1 to ALAS2 mRNA, which stabilizes ALAS2 mRNA. These results define a lncRNA-mediated posttranscriptional mechanism that provides a new dimension into how the fundamental heme biosynthetic process is regulated as a determinant of erythrocyte development.

Regional genetic differences among Japanese populations and performance of genotype imputation using whole-genome reference panel of the Tohoku Medical Megabank Project.

BACKGROUND: Genotype imputation from single-nucleotide polymorphism (SNP) genotype data using a haplotype reference panel consisting of thousands of unrelated individuals from populations of interest can help to identify strongly associated variants in genome-wide association studies. The Tohoku Medical Megabank (TMM) project was established to support the development of precision medicine, together with the whole-genome sequencing of 1070 human genomes from individuals in the Miyagi region (Northeast Japan) and the construction of the 1070 Japanese genome reference panel (1KJPN). Here, we investigated the performance of 1KJPN for genotype imputation of Japanese samples not included in the TMM project and compared it with other population reference panels. RESULTS: We found that the 1KJPN population was more similar to other Japanese populations, Nagahama (south-central Japan) and Aki (Shikoku Island), than to East Asian populations in the 1000 Genomes Project other than JPT, suggesting that the large-scale collection (more than 1000) of Japanese genomes from the Miyagi region covered many of the genetic variations of Japanese in mainland Japan. Moreover, 1KJPN outperformed the phase 3 reference panel of the 1000 Genomes Project (1KGPp3) for Japanese samples, and IKJPN showed similar imputation rates for the TMM and other Japanese samples for SNPs with minor allele frequencies (MAFs) higher than 1%. CONCLUSIONS: 1KJPN covered most of the variants found in the samples from areas of the Japanese mainland outside the Miyagi region, implying 1KJPN is representative of the Japanese population's genomes. 1KJPN and successive reference panels are useful genome reference panels for the mainland Japanese population. Importantly, the addition of whole genome sequences not included in the 1KJPN panel improved imputation efficiencies for SNPs with MAFs under 1% for samples from most regions of the Japanese archipelago.

Omics research project on prospective cohort studies from the Tohoku Medical Megabank Project.

Population-based prospective cohort studies are indispensable for modern medical research as they provide important knowledge on the influences of many kinds of genetic and environmental factors on the cause of disease. Although traditional cohort studies are mainly conducted using questionnaires and physical examinations, modern cohort studies incorporate omics and genomic approaches to obtain comprehensive physical information, including genetic information. Here, we report the design and midterm results of multi-omics analysis on population-based prospective cohort studies from the Tohoku Medical Megabank (TMM) Project. We have incorporated genomic and metabolomic studies in the TMM cohort study as both metabolome and genome analyses are suitable for high-throughput analysis of large-scale cohort samples. Moreover, an association study between the metabolome and genome show that metabolites are an important intermediate phenotype connecting genetic and lifestyle factors to physical and pathologic phenotypes. We apply our metabolome and genome analyses to large-scale cohort samples in the following studies.

Polymorphisms in the microglial marker molecule CX3CR1 affect the blood volume of the human brain.

AIM: CX3CR1, a G-protein-coupled receptor, is involved in various inflammatory processes. Two non-synonymous single nucleotide polymorphisms, V249I (rs3732379) and T280M (rs3732378), are located in the sixth and seventh transmembrane domains of the CX3CR1 protein, respectively. Previous studies have indicated significant associations between T280M and leukocyte functional characteristics, including adhesion, signaling, and chemotaxis, while the function of V249I is unclear. In the brain, microglia are the only proven and widely accepted CX3CR1-expressing cells. This study aimed to specify whether there were specific brain regions on which these two single nucleotide polymorphisms exert their biological impacts through their functional effects on microglia. METHODS: Associations between the single nucleotide polymorphisms and brain characteristics, including gray and white matter volumes, white matter integrity, resting arterial blood volume, and cerebral blood flow, were evaluated among 1300 healthy Japanese individuals. RESULTS: The major allele carriers (V249 and T280) were significantly associated with an increased total arterial blood volume of the whole brain, especially around the bilateral precuneus, left posterior cingulate cortex, and left posterior parietal cortex. There were no significant associations between the genotypes and other brain structural indicators. CONCLUSION: This finding suggests that the CX3CR1 variants may affect arterial structures in the brain, possibly via interactions between microglia and brain microvascular endothelial cells.

Evaluation of reported pathogenic variants and their frequencies in a Japanese population based on a whole-genome reference panel of 2049 individuals.

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.

The orphan nuclear receptor TR4 regulates erythroid cell proliferation and maturation.

The orphan nuclear receptors TR4 (NR2C2) and TR2 (NR2C1) are the DNA-binding subunits of the macromolecular complex, direct repeat erythroid-definitive, which has been shown to repress ε- and γ-globin transcription during adult definitive erythropoiesis. Previous studies implied that TR2 and TR4 act largely in a redundant manner during erythroid differentiation; however, during the course of routine genetic studies, we observed multiple variably penetrant phenotypes in the Tr4 mutants, suggesting that indirect effects of the mutation might be masked by multiple modifying genes. To test this hypothesis, Tr4+/- mutant mice were bred into a congenic C57BL/6 background and their phenotypes were reexamined. Surprisingly, we found that homozygous Tr4 null mutant mice expired early during embryogenesis, around embryonic day 7.0, and well before erythropoiesis commences. We further found that Tr4+/- erythroid cells failed to fully differentiate and exhibited diminished proliferative capacity. Analysis of Tr4+/- mutant erythroid cells revealed that reduced TR4 abundance resulted in decreased expression of genes required for heme biosynthesis and erythroid differentiation (Alad and Alas2), but led to significantly increased expression of the proliferation inhibitory factor, cyclin dependent kinase inhibitor (Cdkn1c) These studies support a vital role for TR4 in promoting erythroid maturation and proliferation, and demonstrate that TR4 and TR2 execute distinct, individual functions during embryogenesis and erythroid differentiation.