Ryohei Furukawa

Genetic Predisposition to Ischemic Stroke: A Polygenic Risk Score

Background and Purpose— The prediction of genetic predispositions to ischemic stroke (IS) may allow the identification of individuals at elevated risk and thereby prevent IS in clinical practice. Previously developed weighted multilocus genetic risk scores showed limited predictive ability for IS. Here, we investigated the predictive ability of a newer method, polygenic risk score (polyGRS), based on the idea that a few strong signals, as well as several weaker signals, can be collectively informative to determine IS risk. Methods— We genotyped 13 214 Japanese individuals with IS and 26 470 controls (derivation samples) and generated both multilocus genetic risk scores and polyGRS, using the same derivation data set. The predictive abilities of each scoring system were then assessed using 2 independent sets of Japanese samples (KyushuU and JPJM data sets). Results— In both validation data sets, polyGRS was shown to be significantly associated with IS, but weighted multilocus genetic risk scores was not. Comparing the highest with the lowest polyGRS quintile, the odds ratios for IS were 1.75 (95% confidence interval, 1.33–2.31) and 1.99 (95% confidence interval, 1.19–3.33) in the KyushuU and JPJM samples, respectively. Using the KyushuU samples, the addition of polyGRS to a nongenetic risk model resulted in a significant improvement of the predictive ability (net reclassification improvement=0.151; P<0.001). Conclusions— The polyGRS was shown to be superior to weighted multilocus genetic risk scores as an IS prediction model. Thus, together with the nongenetic risk factors, polyGRS will provide valuable information for individual risk assessment and management of modifiable risk factors.

Intraindividual dynamics of transcriptome and genome-wide stability of DNA methylation

Cytosine methylation at CpG dinucleotides is an epigenetic mechanism that affects the gene expression profiles responsible for the functional differences in various cells and tissues. Although gene expression patterns are dynamically altered in response to various stimuli, the intraindividual dynamics of DNA methylation in human cells are yet to be fully understood. Here, we investigated the extent to which DNA methylation contributes to the dynamics of gene expression by collecting 24 blood samples from two individuals over a period of 3 months. Transcriptome and methylome association analyses revealed that only ~2% of dynamic changes in gene expression could be explained by the intraindividual variation of DNA methylation levels in peripheral blood mononuclear cells and purified monocytes. These results showed that DNA methylation levels remain stable for at least several months, suggesting that disease-associated DNA methylation markers are useful for estimating the risk of disease manifestation.

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λadjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12–1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λadjusted = 1.00–1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

Genome-wide identification of inter-individually variable DNA methylation sites improves the efficacy of epigenetic association studies

Epigenome-wide association studies, which searches for blood-based DNA methylation signatures associated with environmental exposures and/or disease susceptibilities, is a promising approach to a better understanding of the molecular aetiology of common diseases. To carry out large-scale epigenome-wide association studies while avoiding false negative detection, an efficient strategy to determine target CpG sites for microarray-based or sequencing-based DNA methylation profiling is essentially needed. Here, we propose and validate a hypothesis that a strategy focusing on CpG sites with high DNA methylation level variability may attain an improved efficacy. Through whole-genome bisulfite sequencing of purified blood cells collected from > 100 apparently healthy subjects, we identified ~2.0 million inter-individually variable CpG sites as potential targets. The efficacy of our strategy was estimated to be 3.7-fold higher than that of the most frequently used strategy. Our catalogue of inter-individually variable CpG sites will accelerate the discovery of clinically relevant DNA methylation biomarkers in future epigenome-wide association studies.Epigenomics: Finding DNA modifications that predict diseasePatterns of chemical modifications on DNA can predict the risk of certain diseases, but the challenge is knowing where to look for them. Atsushi Shimizu from Iwate Medical University in Japan and his colleagues determined the location of these modifications, methyl groups added to DNA’s cytosine bases, in over hundred healthy people and found two million regions that vary widely between individuals. They used previous data to show that these regions are far more likely to change during the course of disease than genomic loci that are unchanged in all subjects. Current methods to look for disease-specific methylation changes can only profile at most 20% of the genome and Shimizu’s work will help focus on the 20% that matter for identifying disease risk and lead to better diagnosis and prognosis.

Reduction of Systematic Bias in Transcriptome Data from Human Peripheral Blood Mononuclear Cells for Transportation and Biobanking

Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. Therefore, to collect high-quality biobank samples for expression analysis, specimens must be transported under stable conditions. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from peripheral blood mononuclear cells (PBMCs) to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at −80°C. We randomly chose 7 samples from 11 samples individually, and systematic bias in expression levels was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA sequencing (RNA-Seq) experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. qRT-PCR experiments for 19 selected transcripts revealed systematic bias in the expression levels of five transcripts. RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, qRT-PCR and RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.

Two macrophage migration inhibitory factors regulate starfish larval immune cell chemotaxis.

Immune cell recruitment is critical step in the inflammatory response and associated diseases. However, the underlying regulatory mechanisms are poorly understood in invertebrates. Mesenchyme cells of the starfish larvae, which allowed Metchnikoff to complete his landmark experiments, are important model for analysis of immune cell migration. The present study investigated the role of macrophage migration inhibitory factor (MIF)—an evolutionarily conserved cytokine that is functionally similar to chemokines—in the larvae of the starfish Patiria (Asterina) pectinifera, which were found to possess two orthologs, ApMIF1 and ApMIF2. ApMIF1 and ApMIF2 clustered with mammalian MIF and its homolog d‐dopachrome tautomerase (DDT), respectively, in the phylogenetic analysis. In contrast to the functional similarity between mammalian MIF and DDT, ApMIF1 knockdown resulted in the excessive recruitment of mesenchyme cells in vivo, whereas ApMIF2 deficiency inhibited the recruitment of these cells to foreign bodies. Mesenchyme cells migrated along a gradient of recombinant ApMIF2 in vitro, whereas recombinant ApMIF1 completely blocked ApMIF2‐induced directed migration. Moreover, the expression patterns of ApMIF1 and ApMIF2 messenger RNA in bacteria‐challenged mesenchyme cells were consistent with in vivo observations of cell behaviors. These results indicate that ApMIF1 and ApMIF2 act as chemotactic inhibitory and stimulatory factors, respectively, and coordinately regulate mesenchyme cell recruitment during the immune response in starfish larvae. This is the first report describing opposing functions for MIF‐ and DDT‐like molecules. Our findings provide novel insight into the mechanisms underlying immune regulation in invertebrates.

Starfish ApDOCK protein essentially functions in larval defense system operated by mesenchyme cells

In larvae of the starfish, Asterina pectinifera, mesenchyme cells operate in the defense system through various behaviors. We have investigated mesenchyme cell dynamics during the immune response by identifying ApDOCK, a new member of the DOCK180 superfamily protein. In 4‐day‐old bipinnaria larvae processed for morpholino oligonucleotide‐mediated knockdown of ApDOCK, injection of inorganic foreign substances revealed that (1) mesenchyme cells fail to undergo either directed migration toward a large oil‐droplet or persistent spreading on the oil‐droplet after contact; (2) neither uptake of micro‐beads nor cell‐to‐cell fusion on the large oil‐droplet differed from that of mesenchyme cells from control larvae. Similar behaviors were also recorded in experiments where bacteria were injected. Under culture conditions, the expression level of ApDOCK mRNA was significantly associated with the immunological behavior of mesenchyme cells. Apparently, the mesenchyme cells from ApDOCK loss‐of‐function larvae exhibited insufficient lamellipodium formation via lack of fibrous form of actin organization at the leading edge. These results suggest that the migratory congregation and persistence of encapsulation of larval mesenchyme cells are intracellularly regulated by ApDOCK protein, and this regulation is associated with organization of cytoskeletal actin.

iMETHYL: an integrative database of human DNA methylation, gene expression, and genomic variation

We launched an integrative multi-omics database, iMETHYL (http://imethyl.iwate-megabank.org). iMETHYL provides whole-DNA methylation (~24 million autosomal CpG sites), whole-genome (~9 million single-nucleotide variants), and whole-transcriptome (>14 000 genes) data for CD4+ T-lymphocytes, monocytes, and neutrophils collected from approximately 100 subjects. These data were obtained from whole-genome bisulfite sequencing, whole-genome sequencing, and whole-transcriptome sequencing, making iMETHYL a comprehensive database.

Characterization of TRPA channels in the starfish Patiria pectinifera : involvement of thermally activated TRPA1 in thermotaxis in marine planktonic larvae

The vast majority of marine invertebrates spend their larval period as pelagic plankton and are exposed to various environmental cues. Here we investigated the thermotaxis behaviors of the bipinnaria larvae of the starfish, Patiria pectinifera, in association with TRPA ion channels that serve as thermal receptors in various animal species. Using a newly developed thermotaxis assay system, we observed that P. pectinifera larvae displayed positive thermotaxis toward high temperatures, including toward temperatures high enough to cause death. In parallel, we identified two TRPA genes, termed PpTRPA1 and PpTRPA basal, from this species. We examined the phylogenetic position, spatial expression, and channel properties of each PpTRPA. Our results revealed the following: (1) The two genes diverged early in animal evolution; (2) PpTRPA1 and PpTRPA basal are expressed in the ciliary band and posterior digestive tract of the larval body, respectively; and (3) PpTRPA1 is activated by heat stimulation as well as by known TRPA1 agonists. Moreover, knockdown and rescue experiments demonstrated that PpTRPA1 is involved in positive thermotaxis in P. pectinifera larvae. This is the first report to reveal that TRPA1 channels regulate the behavioral response of a marine invertebrate to temperature changes during its planktonic larval period.