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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.

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.

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.

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.

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.

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., LncRNAs modulate diverse physiological cellular processes, however, their involvement in heme-dependent processes are not yet clear. Here the authors reveal the role of lncRNA UCA1 in erythroid cell development.

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.

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 e- 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.

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.

PurposeA 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.ParticipantsPregnant 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 dateStudy 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 plansLifelog 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.

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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-4942-0) contains supplementary material, which is available to authorized users.

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.

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene, leading to the production of abnormally shaped red blood cells. Sickle cells are prone to hemolysis and thereby release free heme into plasma, causing oxidative stress and inflammation that in turn result in damage to multiple organs. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of the antioxidant cell-defense system. Here we show that constitutive Nrf2 activation by ablation of its negative regulator Keap1 (kelch-like ECH-associated protein 1) significantly improves symptoms in SCD model mice. SCD mice exhibit severe liver damage and lung inflammation associated with high expression levels of proinflammatory cytokines and adhesion molecules compared with normal mice. Importantly, these symptoms subsided after Nrf2 activation. Although hemolysis and stress erythropoiesis did not change substantially in the Nrf2-activated SCD mice, Nrf2 promoted the elimination of plasma heme released by sickle cells' hemolysis and thereby reduced oxidative stress and inflammation, demonstrating that Nrf2 activation reduces organ damage and segregates inflammation from prevention of hemolysis in SCD mice. Furthermore, administration of the Nrf2 inducer CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) also relieved inflammation and organ failure in SCD mice. These results support the contention that Nrf2 induction may be an important means to protect organs from the pathophysiology of sickle cell-induced damage.

The orphan nuclear receptors TR2 and TR4 have been shown to play key roles in repressing the embryonic and fetal globin genes in erythroid cells. However, combined germline inactivation of Tr2 and Tr4 leads to periimplantation lethal demise in inbred mice. Hence, we have previously been unable to examine the consequences of their dual loss of function in adult definitive erythroid cells. To circumvent this issue, we generated conditional null mutants in both genes and performed gene inactivation in vitro in adult bone marrow cells. Compound Tr2/Tr4 loss of function led to induced expression of the embryonic εy and βh1 globins (murine counterparts of the human ε- and γ-globin genes). Additionally, TR2/TR4 function is required for terminal erythroid cell maturation. Loss of TR2/TR4 abolished their occupancy on the εy and βh1 gene promoters, and concurrently impaired co-occupancy by interacting corepressors. These data strongly support the hypothesis that the TR2/TR4 core complex is an adult stage-specific, gene-selective repressor of the embryonic globin genes. Detailed mechanistic understanding of the roles of TR2/TR4 and their cofactors in embryonic and fetal globin gene repression may ultimately enhance the discovery of novel therapeutic agents that can effectively inhibit their transcriptional activity and be safely applied to the treatment of β-globinopathies.

The human β-globin locus is comprised of embryonic, fetal, and adult globin genes, each of which is expressed at distinct stages of pre- and postnatal development. Functional defects in globin proteins or expression results in mild to severe anemia, such as in sickle-cell disease or β-thalassemia, but the clinical symptoms of both disorders are ameliorated by persistent expression of the fetal globin genes. Recent genome-wide association studies (GWAS) identified the intergenic region between the HBS1L and MYB loci as a candidate modifier of fetal hemoglobin expression in adults. However, it remains to be clarified whether the enhancer activity within the HBS1L-MYB regulatory domain contributes to the production of fetal hemoglobin in adults. Here we report a new mouse model of hereditary persistence of fetal hemoglobin (HPFH) in which a transgene was randomly inserted into the orthologous murine Hbs1l-Myb locus. This mutant mouse exhibited typically elevated expression of embryonic globins and hematopoietic parameters similar to those observed in human HPFH. These results support the contention that mutation of the HBS1L-MYB genomic domain is responsible for elevated expression of the fetal globin genes, and this model serves as an important means for the analysis of networks that regulate fetal globin gene expression.

Enhanced fetal γ-globin synthesis alleviates symptoms of β-globinopathies such as sickle cell disease and β-thalassemia, but current γ-globin-inducing drugs offer limited beneficial effects. We show here that lysine-specific demethylase 1 (LSD1) inhibition by RNAi in human erythroid cells or by the monoamine oxidase inhibitor tranylcypromine in human erythroid cells or β-type globin-transgenic mice enhances γ-globin expression. LSD1 is thus a promising therapeutic target for γ-globin induction, and tranylcypromine may serve as a lead compound for the development of a new γ-globin inducer.

Relationship between structural variants of enzymes and metabolic phenotypes in human population was investigated based on the association study of metabolite quantitative traits with whole genome sequence data for 512 individuals from a population cohort. We identified five significant associations between metabolites and non-synonymous variants. Four of these non-synonymous variants are located in enzymes involved in metabolic disorders, and structural analyses of these moderate non-synonymous variants demonstrate that they are located in peripheral regions of the catalytic sites or related regulatory domains. In contrast, two individuals with larger changes of metabolite levels were also identified, and these individuals retained rare variants, which caused non-synonymous variants located near the catalytic site. These results are the first demonstrations that variant frequency, structural location, and effect for phenotype correlate with each other in human population, and imply that metabolic individuality and susceptibility for diseases may be elicited from the moderate variants and much more deleterious but rare variants.

Metabolomics is a promising avenue for biomarker discovery. Although the quality of metabolomic analyses, especially global metabolomics (G-Met) using mass spectrometry (MS), largely depends on the instrumentation, potential bottlenecks still exist at several basic levels in the metabolomics workflow. Therefore, we established a precise protocol initially for the G-Met analyses of human blood plasma to overcome some these difficulties. In our protocol, samples are deproteinized in a 96-well plate using an automated liquid-handling system, and conducted either using a UHPLC-QTOF/MS system equipped with a reverse phase column or a LC-FTMS system equipped with a normal phase column. A normalization protocol of G-Met data was also developed to compensate for intra- and inter-batch differences, and the variations were significantly reduced along with our normalization, especially for the UHPLC-QTOF/MS data with a C18 reverse-phase column for positive ions. Secondly, we examined the changes in metabolomic profiles caused by the storage of EDTA-blood specimens to identify quality markers for the evaluation of the specimens’ pre-analytical conditions. Forty quality markers, including lysophospholipids, dipeptides, fatty acids, succinic acid, amino acids, glucose, and uric acid were identified by G-Met for the evaluation of plasma sample quality and established the equation of calculating the quality score. We applied our quality markers to a small-scale study to evaluate the quality of clinical samples. The G-Met protocols and quality markers established here should prove useful for the discovery and development of biomarkers for a wider range of diseases.

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43 kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.

Sickle cell disease (SCD) is a hematologic disorder caused by a missense mutation in the adult β-globin gene. Higher fetal hemoglobin (HbF) levels in red blood cells of SCD patients have been shown to improve morbidity and mortality. We previously found that nuclear receptors TR2 and TR4 repress expression of the human embryonic ε-globin and fetal γ-globin genes in definitive erythroid cells. Because forced expression of TR2/TR4 in murine adult erythroid cells paradoxically enhanced fetal γ-globin gene expression in transgenic mice, we wished to determine if forced TR2/TR4 expression in a SCD model mouse would result in elevated HbF synthesis and thereby alleviate the disease phenotype. In a “humanized” sickle cell model mouse, forced TR2/TR4 expression increased HbF abundance from 7.6% of total hemoglobin to 18.6%, accompanied by increased hematocrit from 23% to 34% and reticulocyte reduction from 61% to 18%, indicating a significant reduction in hemolysis. Moreover, forced TR2/TR4 expression reduced hepatosplenomegaly and liver parenchymal necrosis and inflammation in SCD mice, indicating alleviation of usual pathophysiological characteristics. This article shows that genetic manipulation of nonglobin proteins, or transcription factors regulating globin gene expression, can ameliorate the disease phenotype in a SCD model animal. This proof-of-concept study demonstrates that modulating TR2/TR4 activity in SCD patients may be a promising therapeutic approach to induce persistent HbF accumulation and for treatment of the disease.

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal beta-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic beta-type globin promoters but not to the adult beta-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1 beta, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult beta-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult beta-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic beta-type globin genes through coordinated epigenetic chromatin modifications.

Summary. Alteration and abnormal expression of the c-myc oncogene were investigated in human multiple myeloma. Human myeloma cells were highly purified (more than 95%) from bone marrow aspirates in 14 cases of advanced multiple myelomas and one case of plasma cell leukaemia. Southern blotting revealed that a rearranged configuration of c-myc gene was found in only one case of them, but this was a novel truncation of the gene in its coding exon II: a rearranged 3·4 kb band was detected by digestion with Xba I using c-myc exon II probe, but no rearranged band was found using exon III probe. In this case, the truncated c-myc allele was not transcribed: normal sized (2·4 kb) c-myc mRNA was markedly expressed, but no aberrant mRNA was detected. On the other hand, by Northern blotting, the nine cases, including the case with the rearranged c-myc gene, showed increased expression of normal sized (2·4 kb) c-myc mRNA. Elevated c-myc mRNA expressions were well related to the in vitro proliferation (3H-TdR uptake), but not to IL-6 response. Interestingly, extremely high expressions of c-myc mRNA were detected in two cases of aggressive myelomas, including the case with the rearranged c-myc gene, and in one of plasma cell leukaemia. These two cases of aggressive myelomas were the ones who showed the markedly high 3H-TdR uptakes, and had the common clinical features with the formation of an extramedullary mass and very short survival. These results suggest that the activation of c-myc gene could induce high proliferative activities and the subsequent aggressive transformation of myeloma cells.

The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of, The Tohoku Medical Megabank Organization establishes a biobank with detailed patient health care and genome information. Here the authors analyse whole-genome sequences of 1,070 Japanese individuals, allowing them to catalogue 21 million single-nucleotide variants including 12 million novel ones.

Purpose. Small cell lung cancer (SCLC) is a rapidly growing neoplasm accounting for approximately 20% of patients with lung cancer. Progastrin-releasing peptide (proGRP) is produced in about two-thirds of SCLC tumors and is used as a specific marker for SCLC. Although GRP is known to have a variety of biological functions, only limited information is available concerning expression of proGRP mRNA and protein, and that of the receptor for GRP (GRPR) in SCLC tumors. Methods. In individuals with SCLC, the levels of serum proGRP(31–98) were measured by enzyme-linked immunosorbent assay. Expression of proGRP as well as GRPR mRNA in SCLC tumor tissues was investigated by reverse transcription-nested polymerase chain reaction (PCR) amplification. The proportions of alternatively spliced proGRP mRNA transcripts were analyzed in proGRP-producing tumors by nested and competitive PCR amplification. Finally, production of proGRP protein in SCLC tumor was evaluated by using immunohistochemical staining with a polyclonal human anti-proGRP antibody. Results. ProGRP mRNA transcripts could be detected only in tumor tissues recovered from individuals with high serum proGRP levels. The proportions of mRNA subtypes in each case were nearly the same, revealing type I of 55.4±7.6%, type II with 21-b deletion of 1.8±3.6%, and type III with 19-b deletion of 42.8±4.3%, respectively. ProGRP protein production was demonstrated in tumor tissues exclusively from individuals exhibiting high serum proGRP levels. In contrast, GRPR mRNA transcripts were detectable in cancer cells from two of five proGRP-expressing tumor tissues. Conclusions. ProGRP mRNA expression is closely related with the synthesis of proGRP protein which is eventually released into the blood. It is suggested GRP may function as an autocrine growth factor for cancer cells in a subgroup of SCLC patients through, at least in part, upregulation of GRPR expression.

We recently described an erythroid epsilon-globin gene repressor activity, which we named DRED (direct repeat erythroid-definitive). We show that DRED binds with high affinity to DR1 sites in the human embryonic (epsilon-) and fetal (gamma-) globin gene promoters, but the adult beta-globin promoter has no DR1 element. DRED is a 540 kDa complex; sequence determination showed that it contains the nuclear orphan receptors TR2 and TR4. TR2 and TR4 form a heterodimer that binds to the epsilon and gamma promoter DR1 sites. One mutation in a DR1 site causes elevated gamma-globin transcription in human HPFH (hereditary persistence of fetal hemoglobin) syndrome, and we show that this mutation reduces TR2/TR4 binding in vitro. The two receptor mRNAs are expressed at all stages of murine and human erythropoiesis; their forced transgenic expression reduces endogenous embryonic epsilony-globin transcription. These data suggest that TR2/TR4 forms the core of a larger DRED complex that represses embryonic and fetal globin transcription in definitive erythroid cells, and therefore that inhibition of its activity might be an attractive intervention point for treating sickle cell anemia.

Background Validation of single nucleotide variations in whole-genome sequencing is critical for studying disease-related variations in large populations. A combination of different types of next-generation sequencers for analyzing individual genomes may be an efficient means of validating multiple single nucleotide variations calls simultaneously. Results Here, we analyzed 12 independent Japanese genomes using two next-generation sequencing platforms: the Illumina HiSeq 2500 platform for whole-genome sequencing (average depth 32.4×), and the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109×). Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni 2.5-8 SNP chip data were used as the reference. We compared the variant calls for the 12 samples, and found that the concordance between the two next-generation sequencing platforms varied between 83% and 97%. Conclusions Our results show the versatility and usefulness of the combination of exome sequencing with whole-genome sequencing in studies of human population genetics and demonstrate that combining data from multiple sequencing platforms is an efficient approach to validate and supplement SNP calls. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-673) contains supplementary material, which is available to authorized users.

To globally survey the changes in transcriptional landscape during terminal erythroid differentiation, we performed RNA sequencing (RNA-seq) on primary human CD34(+) cells after ex vivo differentiation from the earliest into the most mature erythroid cell stages. This analysis identified thousands of novel intergenic and intronic transcripts as well as novel alternative transcript isoforms. After rigorous data filtering, 51 (presumptive) novel protein-coding transcripts, 5326 long and 679 small non-coding RNA candidates remained. The analysis also revealed two clear transcriptional trends during terminal erythroid differentiation: first, the complexity of transcript diversity was predominantly achieved by alternative splicing, and second, splicing junctional diversity diminished during erythroid differentiation. Finally, 404 genes that were not known previously to be differentially expressed in erythroid cells were annotated. Analysis of the most extremely differentially expressed transcripts revealed that these gene products were all closely associated with hematopoietic lineage differentiation. Taken together, this study will serve as a comprehensive platform for future in-depth investigation of human erythroid development that, in turn, may reveal new insights into multiple layers of the transcriptional regulatory hierarchy that controls erythropoiesis.

Peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α) and PGC-1β have been shown to be intimately involved in the transcriptional regulation of cellular energy metabolism as well as other biological processes, but both coactivator proteins are expressed in many other tissues and organs in which their function is, in essence, unexplored. Here, we found that both PGC-1 proteins are abundantly expressed in maturing erythroid cells. PGC-1α and PGC-1β compound null mutant (Pgc-1(c)) animals express less β-like globin mRNAs throughout development; consequently, neonatal Pgc-1(c) mice exhibit growth retardation and profound anemia. Flow cytometry shows that the number of mature erythrocytes is markedly reduced in neonatal Pgc-1(c) pups, indicating that erythropoiesis is severely compromised. Furthermore, hematoxylin and eosin staining revealed necrotic cell death and cell loss in Pgc-1(c) livers and spleen. Chromatin immunoprecipitation studies revealed that both PGC-1α and -1β, as well as two nuclear receptors, TR2 and TR4, coordinately bind to the various globin gene promoters. In addition, PGC-1α and -1β can interact with TR4 to potentiate transcriptional activation. These data provide new insights into our understanding of globin gene regulation and raise the interesting possibility that the PGC-1 coactivators can interact with TR4 to elicit differential stage-specific effects on globin gene transcription.

Background Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B′, and PR72. Specific functions for each variable subunit are not well understood. Results Summary figure Two fission yeast genes pbp1+ and pbp2+ homologous to the regulatory subunit B′ were isolated. Physical in vivo interaction of the gene products with the catalytic subunit was demonstrated. A double disruption haploid mutant (Δpbp1Δpbp2) showed growth defect, cell shape and size abnormality, multiseptation and anucleated cell formation due to abnormality in septum positioning. These phenotypes were suppressed by human B′ cDNA, indicating the striking conservation of the B′ function from yeast to human. Over-expression of fission yeast B′ led to growth defects, a loss of cell shape polarity, septal abnormality and anucleated cell formation. Δpbp1Δpbp2 and pbp1 null haploids were hypersensitive to calcineurin inhibitors, cyclosporin A and FK506, with which the mutants underwent arrest at post-anaphase and cell lysis. Double disruption of calcineurin and pbp1+, but not pbp2+, genes led to synthetic lethality. Figure Summary figure . Roles of B′ and its functional interaction with calcineurin in fission yeast. This hypothetical summary scheme is depicted based on the observations in this study and others (Jiang & Hallberg 2000; Sugiura et al. 1998; Yoshida et al. 1994). CAB′ is the trimeric holoenzyme of PP2A containing B′ as a variable regulatory subunit. Download figure to PowerPoint Conclusion The fission yeast B′ subunit of PP2A plays critical roles in cell shape control and septum formation, and shares essential functions with calcineurin for viability, possibly through their roles in cytokinesis and cell wall integrity.

In previous mass spectrometry and coimmune precipitation studies, we identified tripartite motif-containing 28 (TRIM28; also known as transcriptional intermediary factor1β and Krüppel-associated box-associated protein-1) as a cofactor that specifically copurified with an NR2C1/NR2C2 (TR2/TR4) orphan nuclear receptor heterodimer that previous studies had implicated as an embryonic/fetal β-type globin gene repressor. TRIM28 has been characterized as a transcriptional corepressor that can associate with many different transcription factors and can play functional roles in multiple tissues and cell types. Here, we tested the contribution of TRIM28 to globin gene regulation and erythropoiesis using a conditional loss-of-function in vivo model. We discovered that Trim28 genetic loss in the adult mouse leads to defective immature erythropoiesis in the bone marrow and consequently to anemia. We further found that TRIM28 controls erythropoiesis in a cell-autonomous manner by inducibly deleting Trim28 exclusively in hematopoietic cells. Finally, in the absence of TRIM28, we observed increased apoptosis as well as diminished expression of multiple erythroid transcription factors and heme biosynthetic enzymes in immature erythroid cells. Thus, TRIM28 is essential for the cell-autonomous development of immature erythroblasts in the bone marrow.

Measles is frequently complicated with pneumonia that could be fatal in numerous occasions. However, a prompt and precise diagnosis of measles is not easily made particularly in the early stage of the disease, or in immunocompromised individuals because of the lack of typical clinical features or the defect in antigen-specific antibody production. In the present paper, we describe a 27-yr-old male who developed fever, skin rash typical of measles, and diffuse pulmonary infiltrates associated with respiratory failure. Infection of lung cells with measles virus was proved by detection of viral genome ribonucleic acid within alveolar macrophages and lymphocytes recovered by bronchoalveolar lavage using reverse transcription-polymerase chain reaction amplification. These techniques may offer a useful tool to make the swift and precise diagnosis of measles pneumonia, thus allowing appropriate therapeutic approaches to the disease.

A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.

Human erythrocyte Mn(2+)-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn(2+)-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'.

Soluble interleukin-2 receptor-alpha (IL-2Ralpha) has been reported to be increased in the sera of patients with advanced tuberculosis, and levels decline after therapy in accordance with improvement of radiologic findings. We investigated expression of the IL-2Ralpha in bronchoalveolar lavage (BAL) cells in active pulmonary tuberculosis, and evaluated the mechanism Mycobacterium tuberculosis induces in the IL-2Ralpha using the THP-1 mononuclear phagocyte cell line. We found IL-2Ralpha expression to be increased in BAL cells from involved sites of active pulmonary tuberculosis. Expression of the alpha-chain of IL-2Ralpha on peripheral blood monocytes (PBM) was induced by M. tuberculosis by flow cytometry evaluation. Northern analysis demonstrated increased IL-2Ralpha gene expression after stimulation with M. tuberculosis which was further induced by interferon-gamma (IFN-gamma). The IL-2Ralpha promoter containing the nuclear factor kappa B (NF-kappaB) site was transcriptionally induced by M. tuberculosis and this NF-kappaB site could confer inducibility to a heterologous herpes thymidine kinase (TK) promoter by M. tuberculosis. Electrophoretic mobility shift assays (EMSAs) revealed specific binding of nuclear protein to the NF-kappaB site upon induction with M. tuberculosis. Using antibodies against the p50 and p65 subunits of NF-kappaB in EMSAs, the involvement of both p50 and p65 proteins was further demonstrated. Functional expression of the IL-2Ralpha on mononuclear phagocytes in M. tuberculosis infection may play an important immunomodulatory role in the host response.

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.