Deciphering Origin and Establishment of Japonesians mainly based on genome sequence data

金沢大学医薬保健研究域医学系ヒトの主要組織適合遺伝子複合体 (HLA)は免疫応答の入り口として自己と非自己の認識を担うとともに、ヒトにおける機能的遺伝子としては最も高度な多型を示す。この多型は自己免疫性疾患や感染症、さらには薬剤副作用と関連する遺伝要因として研究が進んでいる。さらに、その高度な多型性は個人識別、人類集団の起源や形成過程を探る遺伝マーカーとしても有益である。本研究ではHLA遺伝子の多型に注目し、ヤポネシア人成立までの感染症との戦いの歴史といった免疫学的な視点からもヤポネシア人の歴史を探ることを提案したい。古代人のHLA遺伝子型を決定手法の確立これまでに研究代表者が開発してきた NGSを用いたHLA遺伝子の解析手法を、バクテリアゲノムを多く含み、断片化が進んだ古代人DNAを対象にしたものに最適化するために、プローブデザインを再設計ならびにハイブリダイゼーションの条件を再検証した。DNAライブラリとプローブの比率を変更し、ハイブリの時間を2時間に短縮しても、一般的なヒトゲノムDNAのシークエンスキャプチャーであればオンターゲット率は85%に濃縮できるプロトコールとして最適化した。一方、含まれるヒトゲノムが微量の場合はそのオンターゲット率が15%程度の濃縮にとどまる結果となったが、HLAタイピングが可能なリード数を得ることができる条件であることを確認した。計画研究A02で解析が進められた縄文DNA、尻労安部サンプルを対象として上述のHLA遺伝子解析手法によるHLAタイピングを試みた。結果は良好で、縄文DNAサンプルであってもHLA型をクリニカルグレードとなる第4区域で決定することが可能であった。尻労安部のHLAアレル頻度はいずれの遺伝子においても日本列島人集団において10位以内となる高頻度のタイプであった。現代人のHLA遺伝子解析金沢大学で進めている、志賀町など能登地域を対象にしたゲノムコホートの住民DNAサンプルを対象に37のHLA遺伝子ならびに非HLA遺伝子についてシークエンスを実施した。1,187検体について第3区域(6桁)までのタイピングが完了している。また、日本人集団以外の解析として、東北アジアの5民族集団、北部漢民族、満民族、中国在住朝鮮民族(中国、ハルピン)、ブリヤート民族(ロシア、イルクーツク)およびモンゴル民族(モンゴル、ウランバートル)、さらに、アマゾン先住民族の一つであるワオラニ族のHLAタイピングを実施した。研究課題/領域番号:19H05344, 研究期間(年度):2019-04-01 – 2021-03-31出典：研究課題「HLA遺伝子の多様性にもとづくヤポネシア人進化の解明」課題番号19H05344（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PUBLICLY-19H05344/）を加工して作

Amelioration of diabetes in NOD by additive Aire

Tissue-specific autoimmune diseases are assumed to arise through malfunction of two checkpoints for immune tolerance: defective elimination of autoreactive T cells in the thymus and activation of these T cells by corresponding autoantigens in the periphery. However, evidence for this model and the outcome of such alterations in each or both of the tolerance mechanisms have not been sufficiently investigated. We studied these issues by expressing human AIRE (huAIRE) as a modifier of tolerance function in NOD mice wherein the defects of thymic and peripheral tolerance together cause type I diabetes (T1D). Additive huAIRE expression in the thymic stroma had no major impact on the production of diabetogenic T cells in the thymus. In contrast, huAIRE expression in peripheral antigen-presenting cells (APCs) rendered the mice resistant to T1D, while maintaining other tissue-specific autoimmune responses and antibody production against an exogenous protein antigen, because of the loss of Xcr1+ dendritic cells, an essential component for activating diabetogenic T cells in the periphery. These results contrast with our recent demonstration that huAIRE expression in both the thymic stroma and peripheral APCs resulted in the paradoxical development of muscle-specific autoimmunity. Our results reveal that tissue-specific autoimmunity is differentially controlled by a combination of thymic function and peripheral tolerance, which can be manipulated by expression of huAIRE/Aire in each or both of the tolerance mechanisms

Phase-defined complete sequencing of the HLA genes by next-generation sequencing

Background: The human leukocyte antigen (HLA) region, the 3.8-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, mostly autoimmune diseases. Due to the complex nature of HLA genes, there are difficulties in elucidating complete HLA gene sequences especially HLA gene haplotype structures by the conventional sequencing method. We propose a novel, accurate, and cost-effective method for generating phase-defined complete sequencing of HLA genes by using indexed multiplex next generation sequencing. Results: A total of 33 HLA homozygous samples, 11 HLA heterozygous samples, and 3 parents-child families were subjected to phase-defined HLA gene sequencing. We applied long-range PCR to amplify six HLA genes (HLA-A, -C, -B, DRB1, -DQB1, and –DPB1) followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. Paired-end reads (2 × 250 bp) derived from the sequencer were aligned to the six HLA gene segments of UCSC hg19 allowing at most 80 bases mismatch. For HLA homozygous samples, the six amplicons of an individual were pooled and simultaneously sequenced and mapped as an individual-tagging method. The paired-end reads were aligned to corresponding genes of UCSC hg19 and unambiguous, continuous sequences were obtained. For HLA heterozygous samples, each amplicon was separately sequenced and mapped as a genetagging method. After alignments, we detected informative paired-end reads harboring SNVs on both forward and reverse reads that are used to separate two chromosomes and to generate two phase-defined sequences in an individual. Consequently, we were able to determine the phase-defined HLA gene sequences from promoter to 3′- UTR and assign up to 8-digit HLA allele numbers, regardless of whether the alleles are rare or novel. Parent–child trio-based sequencing validated our sequencing and phasing methods. Conclusions: Our protocol generated phased-defined sequences of the entire HLA genes, resulting in high resolution HLA typing and new allele detection

CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. © 2016, Nature Publishing Group. All rights reserved

A mutation in SFTPA1 and pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by scattered fibrotic lesions in the lungs. The pathogenesis and genetic basis of IPF remain poorly understood. Here, we show that a homozygous missense mutation in SFTPA1 caused IPF in a consanguineous Japanese family. The mutation in SFTPA1 disturbed the secretion of SFTPA1 protein. Sftpa1 knock-in (Sftpa1-KI) mice that harbored the same mutation as patients spontaneously developed pulmonary fibrosis that was accelerated by influenza virus infection. Sftpa1-KI mice showed increased necroptosis of alveolar epithelial type II (AEII) cells with phosphorylation of IRE1α leading to JNK-mediated up-regulation of Ripk3. The inhibition of JNK ameliorated pulmonary fibrosis in Sftpa1-KI mice, and overexpression of Ripk3 in Sftpa1-KI mice treated with a JNK inhibitor worsened pulmonary fibrosis. These findings provide new insight into the mechanisms of IPF in which a mutation in SFTPA1 promotes necroptosis of AEII cells through JNK-mediated up-regulation of Ripk3, highlighting the necroptosis pathway as a therapeutic target for IPF

A NEW LOOK AT TRANSCRIPTIONAL REGULATION BY AIRE IN mTECs

The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire’s primary targets while distinguishing them from the secondary targets

The major histocompatibility complex (Mhc) class IIB region has greater genomic structural flexibility and diversity in the quail than the chicken

BACKGROUND: The quail and chicken major histocompatibility complex (Mhc) genomic regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated class I, class IIB, natural killer (NK)-receptor-like, lectin-like and BG genes. Therefore, the elucidation of genetic factors that contribute to the greater Mhc diversity in the quail would help to establish it as a model experimental animal in the investigation of avian Mhc associated diseases. AIMS AND APPROACHES: The main aim here was to characterize the genetic and genomic features of the transcribed major quail MhcIIB (CojaIIB) region that is located between the Tapasin and BRD2 genes, and to compare our findings to the available information for the chicken MhcIIB (BLB). We used four approaches in the study of the quail MhcIIB region, (1) haplotype analyses with polymorphic loci, (2) cloning and sequencing of the RT-PCR CojaIIB products from individuals with different haplotypes, (3) genomic sequencing of the CojaIIB region from the individuals with the different haplotypes, and (4) phylogenetic and duplication analysis to explain the variability of the region between the quail and the chicken. RESULTS: Our results show that the Tapasin-BRD2 segment of the quail Mhc is highly variable in length and in gene transcription intensity and content. Haplotypic sequences were found to vary in length between 4 to 11 kb. Tapasin-BRD2 segments contain one or two major transcribed CojaIIBs that were probably generated by segmental duplications involving c-type lectin-like genes and NK receptor-like genes, gene fusions between two CojaIIBs and transpositions between the major and minor CojaIIB segments. The relative evolutionary speed for generating the MhcIIBs genomic structures from the ancestral BLB2 was estimated to be two times faster in the quail than in the chicken after their separation from a common ancestor. Four types of genomic rearrangement elements (GRE), composed of simple tandem repeats (STR), were identified in the MhcIIB genomic segment located between the Tapasin-BRD2 genes. The GREs have many more STR numbers in the quail than in the chicken that displays strong linkage disequilibrium. CONCLUSION: This study suggests that the Mhc classIIB region has a flexible genomic structure generated by rearrangement elements and rapid SNP accumulation probably as a consequence of the quail adapting to environmental conditions and pathogens during its migratory history after its divergence from the chicken

HLA-DPB1*04:01 allele is associated with non-obstructive azoospermia in Japanese patients

Azoospermia is defined by absence of sperm in the semen and can either be caused by obstruction of the seminal tract (obstructive azoospermia) or by defects in spermatogenesis (non-obstructive azoospermia, NOA). Previous studies reported that specific alleles and single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region were associated with NOA in East Asians. We attempt to expand upon previous findings by genotyping more HLA genes and to replicate SNP associations by focusing on Japanese NOA patients. HLA typing of six genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) was done on 355 NOA patients using SSO-Luminex assay while genotyping of two previously reported SNPs (rs498422 and rs3129878) was done on 443 patients and 544 fertile males using TaqMan assay. Association was assessed with Chi squared and logistic regression tests. We found that HLA-DPB1*04:01 [corrected p value, Pc 7.13 9 10-6 ; odds ratio (OR) 2.52], DRB1*13:02 (Pc 4.93 9 10-4 , OR 1.97), DQB1*06:04 (Pc 8.94 9 10-4 , OR 1.91) and rs3129878 (p value 3.98 9 10-4 ; OR 1.32) showed significant association with NOA, however, these loci are in linkage disequilibrium with each other. The conditional logistic regression tests showed that DPB1*04:01 is independently associated with NOA, confirming the involvement of the HLA region in the etiology of NOA in Japanese patients

The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6β remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b−/− mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b−/− neurons were recovered by ATF6β and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b−/− and Calr+/− mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6β-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis

A new genotype of bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis

Background: Bovine leukemia virus (BLV) is a member of retroviridae family, together with human T cell leukemia virus types 1 and 2 (HTLV-1 and -2) belonging to the genes deltaretrovirus, and infects cattle worldwide. Previous studies have classified the env sequences of BLV provirus from different geographic locations into eight genetic groups. To investigate the genetic variability of BLV in South America, we performed phylogenetic analyses of whole genome and partial env gp51 sequences of BLV strains isolated from Peru, Paraguay and Bolivia, for which no the molecular characteristics of BLV have previously been published, and discovered a novel BLV genotype, genotype-9, in Bolivia. Results: In Peru and Paraguay, 42.3 % (139/328) and over 50 % (76/139) of samples, respectively, were BLV positive. In Bolivia, the BLV infection rate was up to 30 % (156/507) at the individual level. In Argentina, 325/420 samples were BLV positive, with a BLV prevalence of 77.4 % at the individual level and up to 90.9 % at herd level. By contrast, relatively few BLV positive samples were detected in Chile, with a maximum of 29.1 % BLV infection at the individual level. We performed phylogenetic analyses using two different approaches, maximum likelihood (ML) tree and Bayesian inference, using 35 distinct partial env gp51 sequences from BLV strains isolated from Peru, Paraguay, and Bolivia, and 74 known BLV strains, representing eight different BLV genotypes from various geographical locations worldwide. The results indicated that Peruvian and Paraguayan BLV strains were grouped into genotypes-1, -2, and -6, while those from Bolivia were clustered into genotypes-1, -2, and -6, and a new genotype, genotype-9. Interestingly, these results were confirmed using ML phylogenetic analysis of whole genome sequences obtained by next generation sequencing of 25 BLV strains, assigned to four different genotypes (genotypes-1, -2, -6, and -9) from Peru, Paraguay, and Bolivia. Comparative analyses of complete genome sequences clearly showed some specific substitutions, in both structural and non-structural BLV genes, distinguishing the novel genotype-9 from known genotypes. Conclusions: Our results demonstrate widespread BLV infection in South American cattle and the existence of a new BLV genotype-9 in Bolivia. We conclude that at least seven BLV genotypes (genotypes-1, -2, -4, -5, -6, -7, and -9) are circulating in South America.Facultad de Ciencias VeterinariasInstituto de Genética Veterinari