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

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

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

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

神経細胞死を抑制する新たな分子を発見 --脳卒中やアルツハイマー病への応用に期待--. 京都大学プレスリリース. 2021-06-30.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 partial nuclear genome of the Jomons who lived 3000 years ago in Fukushima, Japan

The Jomon period of the Japanese Archipelago, characterized by cord-marked ‘jomon’ potteries, has yielded abundant human skeletal remains. However, the genetic origins of the Jomon people and their relationships with modern populations have not been clarified. We determined a total of 115 million base pair nuclear genome sequences from two Jomon individuals (male and female each) from the Sanganji Shell Mound (dated 3000 years before present) with the Jomon-characteristic mitochondrial DNA haplogroup N9b, and compared these nuclear genome sequences with those of worldwide populations. We found that the Jomon population lineage is best considered to have diverged before diversification of present-day East Eurasian populations, with no evidence of gene flow events between the Jomon and other continental populations. This suggests that the Sanganji Jomon people descended from an early phase of population dispersals in East Asia. We also estimated that the modern mainland Japanese inherited <20% of Jomon peoples’ genomes. Our findings, based on the first analysis of Jomon nuclear genome sequence data, firmly demonstrate that the modern mainland Japanese resulted from genetic admixture of the indigenous Jomon people and later migrants