Major histocompatibility complex (Mhc) class Ib gene duplications, organization and expression patterns in mouse strain C57BL/6

<p>Abstract</p> <p>Background</p> <p>The mouse has more than 30 <it>Major histocompatibility complex </it>(<it>Mhc</it>) class Ib genes, most of which exist in the <it>H2 </it>region of chromosome 17 in distinct gene clusters. Although recent progress in <it>Mhc </it>research has revealed the unique roles of several <it>Mhc </it>class Ib genes in the immune and non-immune systems, the functions of many class Ib genes have still to be elucidated. To better understand the roles of class Ib molecules, we have characterized their gene duplication, organization and expression patterns within the <it>H2 </it>region of the mouse strain C57BL/6.</p> <p>Results</p> <p>The genomic organization of the <it>H2-Q</it>, -<it>T </it>and -<it>M </it>regions was analyzed and 21 transcribed <it>Mhc </it>class Ib genes were identified within these regions. Dot-plot and phylogenetic analyses implied that the genes were generated by monogenic and/or multigenic duplicated events. To investigate the adult tissue, embryonic and placental expressions of these genes, we performed RT-PCR gene expression profiling using gene-specific primers. Both tissue-wide and tissue-specific gene expression patterns were obtained that suggest that the variations in the gene expression may depend on the genomic location of the duplicated genes as well as locus specific mechanisms. The genes located in the <it>H2-T </it>region at the centromeric end of the cluster were expressed more widely than those at the telomeric end, which showed tissue-restricted expression in spite of nucleotide sequence similarities among gene paralogs.</p> <p>Conclusion</p> <p>Duplicated <it>Mhc </it>class Ib genes located in the <it>H2-Q</it>, -<it>T </it>and -<it>M </it>regions are differentially expressed in a variety of developing and adult tissues. Our findings form the basis for further functional validation studies of the <it>Mhc </it>class Ib gene expression profiles in specific tissues, such as the brain. The duplicated gene expression results in combination with the genome analysis suggest the possibility of long-range regulation of <it>H2-T </it>gene expression and/or important, but as yet unidentified nucleotide changes in the promoter or enhancer regions of the genes. Since the <it>Mhc </it>genomic region has diversified among mouse strains, it should be a useful model region for comparative analyses of the relationships between duplicated gene organization, evolution and the regulation of expression patterns.</p

Assessment of Artificial MiRNA Architectures for Higher Knockdown Efficiencies without the Undesired Effects in Mice.

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both -MG amiRNA cells and amiRNA low-expression cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs

Evaluation of PTPN22 polymorphisms and Vogt-Koyanagi-Harada disease in Japanese patients

Purpose: Vogt-Koyanagi-Harada (VKH) disease is an autoimmune disorder against melanocytes. Polymorphisms of the protein tyrosine phosphatase non-receptor 22 gene (PTPN22) have recently been reported to be associated with susceptibility to several autoimmune diseases. In this study, genetic susceptibility to VKH disease was investigated by screening for single nucleotide polymorphisms (SNPs) of PTPN22. Methods: A total of 167 Japanese patients with VKH disease and 188 healthy Japanese controls were genotyped by direct sequencing methods for six SNPs (rs3811021, rs1217413, rs1237682, rs3761935, rs3789608, and rs2243471) of PTPN22 including the uncoding exons. Results: The six SNPs in PTPN22 showed no significant association with susceptibility to VKH disease or its ocular, neurologic, or dermatological manifestation. Conclusions: Further studies are needed to clarify the genetic mechanisms underlying VKH disease

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

A Systems Genetics Approach Provides a Bridge from Discovered Genetic Variants to Biological Pathways in Rheumatoid Arthritis

Genome-wide association studies (GWAS) have yielded novel genetic loci underlying common diseases. We propose a systems genetics approach to utilize these discoveries for better understanding of the genetic architecture of rheumatoid arthritis (RA). Current evidence of genetic associations with RA was sought through PubMed and the NHGRI GWAS catalog. The associations of 15 single nucleotide polymorphisms and HLA-DRB1 alleles were confirmed in 1,287 cases and 1,500 controls of Japanese subjects. Among these, HLA-DRB1 alleles and eight SNPs showed significant associations and all but one of the variants had the same direction of effect as identified in the previous studies, indicating that the genetic risk factors underlying RA are shared across populations. By receiver operating characteristic curve analysis, the area under the curve (AUC) for the genetic risk score based on the selected variants was 68.4%. For seropositive RA patients only, the AUC improved to 70.9%, indicating good but suboptimal predictive ability. A simulation study shows that more than 200 additional loci with similar effect size as recent GWAS findings or 20 rare variants with intermediate effects are needed to achieve AUC = 80.0%. We performed the random walk with restart (RWR) algorithm to prioritize genes for future mapping studies. The performance of the algorithm was confirmed by leave-one-out cross-validation. The RWR algorithm pointed to ZAP70 in the first rank, in which mutation causes RA-like autoimmune arthritis in mice. By applying the hierarchical clustering method to a subnetwork comprising RA-associated genes and top-ranked genes by the RWR, we found three functional modules relevant to RA etiology: “leukocyte activation and differentiation”, “pattern-recognition receptor signaling pathway”, and “chemokines and their receptors”

Identification of CD93 expression on hematopoietic stem cells in human neonatal umbilical cord blood cells

臍帯血由来造血幹細胞表面上のCD93の発現を我々が開発したCD93モノクローナル抗体(mNI-11)、既存のCD34抗体、CD45抗体を組み合わせたフローサイトメトリー法で解析した。その結果、臍帯血由来造血幹細胞(CD34+細胞およびCD34+CD45dim+細胞)表面上にはCD93が発現していることが分かった。以上の結果は、CD93が未熟な造血幹細胞の新たな細胞表面マーカーに成り得ると共に、造血幹細胞におけるCD93の免疫学的な機能解析に非常に有益な情報と考えられる。Human CD93 is a heavily O-glycosylated type I transmembrane protein consisting of unique C-type lectin domains (CTLDs) containing glycoprotein. CD93 is mainly expressed on myeloid cells (monocytes and granulocytes) and endothelial cells. However, the expression patterns of CD93 on various other kinds of cells are not well understood. In this study, we found that CD93 was recognized by a CD93 monoclonal antibody (mAb) (mNI-11) that was established in our laboratories and was expressed on a broad hematopoietic stem cell population (CD34+ cells) from human neonatal umbilical cord blood cells (UCBCs), as shown using a two-color flow cytometric analysis. In addition, the CD93 recognized by mNI-11 was also expressed on a narrow hematopoietic stem cell population(CD34+CD45dim+ cells) in which the non-specific reactivity of CD34 mAb from human neonatal UCBCs was excluded using a three-color flow cytometric analysis. Taken together, these results provide the first evidence concerning the identification of CD93 expression on hematopoietic stem cells. These cell populations (CD34+CD93+ and CD34+CD45dim+CD93+ cells) in human neonatal UCBCs are thought to have an important role in cell biology, transplantation, and immature/mature immune responses