VLR-based adaptive immunity.

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

GWAS meta-analysis of kidney function traits in Japanese populations.

BACKGROUND: Genetic epidemiological evidence for the kidney function traits in East Asian population including Japanese remain still relatively unclarified. Especially, the number of GWASs for kidney traits reported still remains limited, and the sample size of each independent study is relatively small. Given the genetic variability between ancestries/ethnicities, implementation of GWAS with sufficiently large sample sizes in specific population of Japanese is considered meaningful. METHODS: We conducted the GWAS meta-analyses of kidney traits by leveraging the GWAS summary data of the representative large genome cohort studies with about 200,000 Japanese participants (n = 202,406 for estimated glomerular filtration rate [eGFR] and n = 200,845 for serum creatinine [SCr]). RESULTS: In the present GWAS meta-analysis, we identified 110 loci with 169 variants significantly associated with eGFR (on chromosomes 1-13 and 15-22; p < 5×10-8), whereas we also identified 112 loci with 176 variants significantly associated with SCr (on chromosomes 1-22; p < 5×10-8), of which one locus (more than 1Mb distant from known loci) with one variant (CD36 rs146148222 on chromosome 7) for SCr was considered as the truly novel finding. CONCLUSIONS: The present GWAS meta-analysis of largest genome cohort studies in Japanese provided some original genomic loci associated with kidney function in Japanese, which may contribute to the possible development of personalized prevention of kidney diseases based on genomic information in the near future.

Study Profile of the Tsuruoka Metabolomics Cohort Study (TMCS).

The Tsuruoka Metabolomics Cohort Study (TMCS) is an ongoing population-based cohort study being conducted in the rural area of Yamagata Prefecture, Japan. This study aimed to enhance the precision prevention of multi-factorial, complex diseases, including non-communicable and aging-associated diseases, by improving risk stratification and prediction measures. At baseline, 11,002 participants aged 35-74 years were recruited in Tsuruoka City, Yamagata Prefecture, Japan, between 2012 and 2015, with an ongoing follow-up survey. Participants underwent various measurements, examinations, tests, and questionnaires on their health, lifestyle, and social factors. This study used an integrative approach with deep molecular profiling to identify potential biomarkers linked to phenotypes that underpin disease pathophysiology and provide better mechanistic insights into social health determinants. The TMCS incorporates multi-omics data, including genetic and metabolomic analyses of 10,933 participants and comprehensive data collection ranging from physical, psychological, behavioral, and social to biological data. The metabolome is used as a phenotypic probe because it is sensitive to changes in physiological and external conditions. The TMCS focuses on collecting outcomes for cardiovascular disease, cancer incidence and mortality, disability, functional decline due to aging and disease sequelae, and the variation in health status within the body represented by omics analysis that lies between exposure and disease. It contains several sub-studies on aging, heated tobacco products, and women's health. This study is notable for its robust design, high participation rate (89%), and long-term repeated surveys. Moreover, it contributes to precision prevention in Japan and East Asia as a well-established multi-omics platform.

Development and validation of genome-wide polygenic risk scores for predicting breast cancer incidence in Japanese females: a population-based case-cohort study.

PURPOSE: This study aimed to develop an ancestry-specific polygenic risk scores (PRSs) for the prediction of breast cancer events in Japanese females and validate it in a longitudinal cohort study. METHODS: Using publicly available summary statistics of female breast cancer genome-wide association study (GWAS) of Japanese and European ancestries, we, respectively, developed 31 candidate genome-wide PRSs using pruning and thresholding (P + T) and LDpred methods with varying parameters. Among the candidate PRS models, the best model was selected using a case-cohort dataset (63 breast cancer cases and 2213 sub-cohorts of Japanese females during a median follow-up of 11.9 years) according to the maximal predictive ability by Harrell's C-statistics. The best-performing PRS for each derivation GWAS was evaluated in another independent case-cohort dataset (260 breast cancer cases and 7845 sub-cohorts of Japanese females during a median follow-up of 16.9 years). RESULTS: For the best PRS model involving 46,861 single nucleotide polymorphisms (SNPs; P + T method with PT = 0.05 and R2 = 0.2) derived from Japanese-ancestry GWAS, the Harrell's C-statistic was 0.598 ± 0.018 in the evaluation dataset. The age-adjusted hazard ratio for breast cancer in females with the highest PRS quintile compared with those in the lowest PRS quintile was 2.47 (95% confidence intervals, 1.64-3.70). The PRS constructed using Japanese-ancestry GWAS demonstrated better predictive performance for breast cancer in Japanese females than that using European-ancestry GWAS (Harrell's C-statistics 0.598 versus 0.586). CONCLUSION: This study developed a breast cancer PRS for Japanese females and demonstrated the usefulness of the PRS for breast cancer risk stratification.

Body mass index and colorectal cancer risk: A Mendelian randomization study.

Traditional observational studies have reported a positive association between higher body mass index (BMI) and the risk of colorectal cancer (CRC). However, evidence from other approaches to pursue the causal relationship between BMI and CRC is sparse. A two-sample Mendelian randomization (MR) study was undertaken using 68 single nucleotide polymorphisms (SNPs) from the Japanese genome-wide association study (GWAS) and 654 SNPs from the GWAS catalogue for BMI as sets of instrumental variables. For the analysis of SNP-BMI associations, we undertook a meta-analysis with 36 303 participants in the Japanese Consortium of Genetic Epidemiology studies (J-CGE), comprising normal populations. For the analysis of SNP-CRC associations, we utilized 7636 CRC cases and 37 141 controls from five studies in Japan, and undertook a meta-analysis. Mendelian randomization analysis of inverse-variance weighted method indicated that a one-unit (kg/m2 ) increase in genetically predicted BMI was associated with an odds ratio of 1.13 (95% confidence interval, 1.06-1.20; P value <.001) for CRC using the set of 68 SNPs, and an odds ratio of 1.07 (1.03-1.11, 0.001) for CRC using the set of 654 SNPs. Sensitivity analyses robustly showed increased odds ratios for CRC for every one-unit increase in genetically predicted BMI. Our MR analyses strongly support the evidence that higher BMI influences the risk of CRC. Although Asians are generally leaner than Europeans and North Americans, avoiding higher BMI seems to be important for the prevention of CRC in Asian populations.

The immune system of jawless vertebrates: insights into the prototype of the adaptive immune system.

Jawless vertebrates diverged from an ancestor of jawed vertebrates approximately 550 million years ago. They mount adaptive immune responses to repetitive antigenic challenges, despite lacking major histocompatibility complex molecules, immunoglobulins, T cell receptors, and recombination-activating genes. Instead of B cell and T cell receptors, agnathan lymphocytes express unique antigen receptors named variable lymphocyte receptors (VLRs), which generate diversity through a gene conversion-like mechanism. Although gnathostome antigen receptors and VLRs are structurally unrelated, jawed and jawless vertebrates share essential features of lymphocyte-based adaptive immunity, including the expression of a single type of receptor on each lymphocyte, clonal expansion of antigen-stimulated lymphocytes, and the dichotomy of cellular and humoral immunity, indicating that the backbone of the adaptive immune system was established in a common ancestor of all vertebrates. Furthermore, recent evidence indicates that, unlike previously thought, agnathans have a unique classical pathway of complement activation where VLRB molecules act as antibodies instead of immunoglobulins. It seems likely that the last common ancestor of all vertebrates had an adaptive immune system resembling that of jawless vertebrates, suggesting that, as opposed to jawed vertebrates, agnathans have retained the prototype of vertebrate adaptive immunity.

Ancient BCMA-like Genes Herald B Cell Regulation in Lampreys.

The TNF superfamily ligands BAFF and APRIL interact with three receptors, BAFFR, BCMA, and TACI, to play discrete and crucial roles in regulating B cell selection and homeostasis in mammals. The interactions between these ligands and receptors are both specific and redundant: BAFFR binds BAFF, whereas BCMA and TACI bind to either BAFF or APRIL. In a previous phylogenetic inquiry, we identified and characterized a BAFF-like gene in lampreys, which, with hagfish, are the only extant jawless vertebrates, both of which have B-like and T-like lymphocytes. To gain insight into lymphocyte regulation in jawless vertebrates, in this study we identified two BCMA-like genes in lampreys, BCMAL1 and BCMAL2, which were found to be preferentially expressed by B-like lymphocytes. In vitro analyses indicated that the lamprey BAFF-like protein can bind to a BCMA-like receptor Ig fusion protein and to both BCMAL1- and BCMAL2-transfected cells. Discriminating regulatory roles for the two BCMA-like molecules are suggested by their differential expression before and after activation of the B-like lymphocytes in lampreys. Our composite results imply that BAFF-based mechanisms for B cell regulation evolved before the divergence of jawed and jawless vertebrates.

Evaluation of clinical formalin-fixed paraffin-embedded tissue quality for targeted-bisulfite sequencing.

Formalin-fixed paraffin-embedded (FFPE) tissues are promising biological resources for genetic research. Recent improvements in DNA extraction from FFPE samples allowed the use of these tissues for multiple sequencing methods. However, fundamental research addressing the application of FFPE-derived DNA for targeted-bisulfite sequencing (TB-seq) is lacking. Here, we evaluated the suitability of FFPE-derived DNA for TB-seq. We conducted TB-seq using FFPE-derived DNA and corresponding fresh frozen (FF) tissues of patients with kidney cancer and compared the quality of DNA, libraries, and TB-seq statistics between the two preservation methods. The approximately 600-bp average fragment size of the FFPE-derived DNA was significantly shorter than that of the FF-derived DNA. The sequencing libraries constructed using FFPE-derived DNA and the mapping ratio were approximately 10 times and 10% lower, respectively, than those constructed using FF-derived DNA. In the mapped data of FFPE-derived DNA, duplicated reads accounted for > 60% of the obtained sequence reads, with lower mean on-target coverage. Therefore, the standard TB-seq protocol is inadequate for obtaining high-quality data for epigenetic analysis from FFPE-derived DNA, and technical improvements are necessary for enabling the use of archived FFPE resources.

The interaction between ABCA1 polymorphism and physical activity on the HDL-cholesterol levels in a Japanese population.

Few studies have investigated the interactions between HDL-C-related SNPs identified by genome-wide association (GWA) study and physical activity (PA) on HDL-C. First, we conducted a sex-stratified GWA study in a discovery sample (2,231 men and 2,431 women) and replication sample (2,599 men and 3,109 women) to identify SNPs influencing log-transformed HDL-C in Japanese participants in the baseline survey of the Japan Multi-Institutional Collaborative Cohort Study. We also replicated previously reported HDL-C-related SNPs in a combined (discovery plus replication) sample (4,830 men and 5,540 women). We then analyzed the interactions of the HDL-C-related SNPs with PA on HDL-C. The sex-stratified GWA analyses identified 11 and 10 HDL-C-related SNPs in men and women as targets for an interaction analysis. Among these, only one interaction of ABCA1 rs1883025 with PA was statistically significant in men, after Bonferroni correction [P-interaction = 0.001 (α = 0.05/21 = 0.002)]. The per-major-allele (C allele) increase in log-transformed HDL-C was lost in men with low PA (ß = 0.008) compared with those with medium (ß = 0.032) or high PA (ß = 0.034). These findings suggest that the benefit of carrying a C allele of ABCA1 rs1883025 on enhancing HDL-C may be attenuated in inactive men.

Elephant shark genome provides unique insights into gnathostome evolution.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

Comparative genomics of the NKG2D ligand gene family.

NKG2D ligands (NKG2DLs) are a group of stress-inducible major histocompatibility complex (MHC) class I-like molecules that act as a danger signal alerting the immune system to the presence of abnormal cells. In mammals, two families of NKG2DL genes have been identified: the MIC gene family encoded in the MHC region and the ULBP gene family encoded outside the MHC region in most species. Some mammals have a third family of NKG2DL-like class I genes which we named MILL (MHC class I-like located near the leukocyte receptor complex). Despite the fact that MILL genes are more closely related to MIC genes than ULBP genes are to MIC genes, MILL molecules do not function as NKG2DLs, and their function remains unknown. With the progress of mammalian genome projects, information on the MIC, ULBP, and MILL gene families became available in many mammalian species. Here, we summarize such information and discuss the origin and evolution of the NKG2DL gene family from the viewpoint of host-pathogen coevolution.

Characterization of Lamprey BAFF-like Gene: Evolutionary Implications.

BAFF (TNF superfamily [TNFSF] 13B/Blys) and APRIL (TNFSF13) are important regulatory factors for lymphocyte activation and survival in mammals. A BAFF/APRIL-like relative called BAFF- and APRIL-like molecule (BALM) has also been identified in cartilaginous and bony fishes, and we report in this study a BAFF-like gene in lampreys. Our phylogenetic analysis of these genes and a related TNFSF12 gene called TNF-like weak inducer of apoptosis (TWEAK) suggest that, whereas an ancestral homolog of BAFF and APRIL was already present in a common ancestor of jawed and jawless vertebrates, TWEAK evolved early on in the jawed vertebrate lineage. Like mammalian BAFF and APRIL, the lamprey BAFF-like gene is expressed in T-like, B-like, and innate immune cells. The predicted protein encoded by this BAFF-like gene in lampreys exhibits higher sequence similarity with mammalian BAFF than APRIL. Correspondingly, we find BAFF orthologs in all of the jawed vertebrate representatives that we examined, although APRIL and/or BALM orthologs are not identifiable in certain jawed vertebrates. For example, BALM is not identifiable in tetrapods, and APRIL is not identifiable in several bony fishes or in birds, the latter of which also lack a TWEAK-like gene. Our analysis further suggests that a hybrid molecule called TWE-PRIL, which is a product of an in-genomic fusion between APRIL and TWEAK genes evolved early in mammalian evolution.

Evolution of two prototypic T cell lineages.

Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA(+) and VLRC(+) lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αß or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity.

Copy number and sequence variation of leucine-rich repeat modules suggests distinct functional constraints operating on variable lymphocyte receptors expressed by agnathan T cell-like and B cell-like lymphocytes.

Unlike jawed vertebrates that use T cell and B cell receptors for antigen recognition, jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLR) as antigen receptors. VLRs generate high levels of diversity by assembling variable leucine-rich repeat (LRR) modules. Of the three VLRs thus far identified, VLRB is expressed on B cell-like lymphocytes and functions as antibodies, whereas VLRA and VLRC are expressed on T cell-like lymphocytes and function as membrane-bound receptors. In the present study, we show that the copy number of LRRV modules in lamprey and hagfish VLRB transcripts follows a binominal distribution with the success rates of 15.5 and 22.4 %, respectively. By contrast, the copy number distribution of LRRV modules in VLRA and VLRC transcripts deviates from the binominal distribution mainly because transcripts with two or less LRRV modules occur infrequently. Notably, the second LRRV module shows distinctive sequence signatures in VLRA and VLRC, but not in VLRB transcripts. These observations suggest that distinct functional constraints operate on VLRs expressed by agnathan T cell-like and B cell-like lymphocytes.

Genetic architecture of alcohol consumption identified by a genotype-stratified GWAS and impact on esophageal cancer risk in Japanese people.

An East Asian-specific variant on aldehyde dehydrogenase 2 (ALDH2 rs671, G>A) is the major genetic determinant of alcohol consumption. We performed an rs671 genotype-stratified genome-wide association study meta-analysis of alcohol consumption in 175,672 Japanese individuals to explore gene-gene interactions with rs671 behind drinking behavior. The analysis identified three genome-wide significant loci (GCKR, KLB, and ADH1B) in wild-type homozygotes and six (GCKR, ADH1B, ALDH1B1, ALDH1A1, ALDH2, and GOT2) in heterozygotes, with five showing genome-wide significant interaction with rs671. Genetic correlation analyses revealed ancestry-specific genetic architecture in heterozygotes. Of the discovered loci, four (GCKR, ADH1B, ALDH1A1, and ALDH2) were suggested to interact with rs671 in the risk of esophageal cancer, a representative alcohol-related disease. Our results identify the genotype-specific genetic architecture of alcohol consumption and reveal its potential impact on alcohol-related disease risk.

Identification of a third variable lymphocyte receptor in the lamprey.

Jawless vertebrates such as lamprey and hagfish lack T-cell and B-cell receptors; instead, they have unique antigen receptors known as variable lymphocyte receptors (VLRs). VLRs generate diversity by recombining highly diverse leucine-rich repeat modules and are expressed clonally on lymphocyte-like cells (LLCs). Thus far, two types of receptors, VLRA and VLRB, have been identified in lampreys and hagfish. Recent evidence indicates that VLRA and VLRB are expressed on distinct populations of LLCs that resemble T cells and B cells of jawed vertebrates, respectively. Here we identified a third VLR, designated VLRC, in the lamprey. None of the approximately 100 VLRC cDNA clones subjected to sequencing had an identical sequence, indicating that VLRC can generate sufficient diversity to function as antigen receptors. Notably, the C-terminal cap of VLRC exhibits only limited diversity and has important structural differences relative to VLRA and VLRB. Single-cell PCR analysis identified LLCs that rearranged VLRC but not VLRA or VLRB, suggesting the presence of a unique population of LLCs that express only VLRC.

Integrated analysis of human DNA methylation, gene expression, and genomic variation in iMETHYL database using kernel tensor decomposition-based unsupervised feature extraction.

Integrating gene expression, DNA methylation, and genomic variants simultaneously without location coincidence (i.e., irrespective of distance from each other) or pairwise coincidence (i.e., direct identification of triplets of gene expression, DNA methylation, and genomic variants, and not integration of pairwise coincidences) is difficult. In this study, we integrated gene expression, DNA methylation, and genome variants from the iMETHYL database using the recently proposed kernel tensor decomposition-based unsupervised feature extraction method with limited computational resources (i.e., short CPU time and small memory requirements). Our methods do not require prior knowledge of the subjects because they are fully unsupervised in that unsupervised tensor decomposition is used. The selected genes and genomic variants were significantly targeted by transcription factors that were biologically enriched in KEGG pathway terms as well as in the intra-related regulatory network. The proposed method is promising for integrated analyses of gene expression, methylation, and genomic variants with limited computational resources.

Anti-Helicobacter pylori antibody status is associated with cancer mortality: A longitudinal analysis from the Japanese DAIKO prospective cohort study.

Paradoxically, patients with advanced stomach cancer who are Helicobacter pylori-positive (HP+) have a higher survival rate than those who are HP-. This finding suggests that HP infection has beneficial effects for cancer treatment. The present study examines whether HP+ individuals have a lower likelihood of death from cancer than those who are HP-. Prospective cohort data (n = 4,982 subjects enrolled in the DAIKO study between 2008-2010) were used to assess whether anti-HP antibody status was associated with cancer incidence. The median age in the primary registry was 53 years-old (range 35-69 years-old). Over the 8-year observation period there were 234 (4.7%) cancer cases in the cohort and 88 (1.8%) all-cause deaths. Urine anti-HP antibody data was available for all but one participant (n = 4,981; 99.98%). The number of HP+ and HP- individuals was 1,825 (37%) and 3,156 (63%), respectively. Anti-HP antibody distribution per birth year revealed that earlier birth year was associated with higher HP+ rates. With a birth year-matched cohort (n = 3,376), all-cancer incidence was significantly higher in HP+ individuals than those who were HP- (p = 0.00328), whereas there was no significant difference in the cancer death rate between HP+ and HP- individuals (p = 0.888). Cox regression analysis for prognostic factors revealed that the hazards ratio of HP+ was 1.59-fold (95%CI 1.17-2.26) higher than HP- in all-cancer incidence. Potential systemic effects of HP+ status may contribute to reduced likelihood of death for patients after an initial diagnosis of cancer.

East Asian-specific and cross-ancestry genome-wide meta-analyses provide mechanistic insights into peptic ulcer disease.

Peptic ulcer disease (PUD) refers to acid-induced injury of the digestive tract, occurring mainly in the stomach (gastric ulcer (GU)) or duodenum (duodenal ulcer (DU)). In the present study, we conducted a large-scale, cross-ancestry meta-analysis of PUD combining genome-wide association studies with Japanese and European studies (52,032 cases and 905,344 controls), and discovered 25 new loci highly concordant across ancestries. An examination of GU and DU genetic architecture demonstrated that GUs shared the same risk loci as DUs, although with smaller genetic effect sizes and higher polygenicity than DUs, indicating higher heterogeneity of GUs. Helicobacter pylori (HP)-stratified analysis found an HP-related host genetic locus. Integrative analyses using bulk and single-cell transcriptome profiles highlighted the genetic factors of PUD being enriched in the highly expressed genes in stomach tissues, especially in somatostatin-producing D cells. Our results provide genetic evidence that gastrointestinal cell differentiations and hormone regulations are critical in PUD etiology.