Structural Characterization of the Highly Restricted Down Syndrome Critical Region on 21q22.13: New KCNJ6 and DSCR4 Transcript Isoforms

Down syndrome (DS) is caused by trisomy of chromosome 21 and it is the most common genetic cause of intellectual disability (ID) in humans. Subjects with DS show a typical phenotype marked by facial dysmorphisms and ID. Partial trisomy 21 (PT21) is a rare genotype characterized by the duplication of a delimited chromosome 21 (Hsa21) portion and it may or may not be associated with DS diagnosis. The highly restricted Down syndrome critical region (HR-DSCR) is a region of Hsa21 present in three copies in all individuals with PT21 and a diagnosis of DS. This region, located on distal 21q22.13, is 34 kbp long and does not include characterized genes. The HR-DSCR is annotated as an intergenic region between KCNJ6-201 transcript encoding for potassium inwardly rectifying channel subfamily J member 6 and DSCR4-201 transcript encoding Down syndrome critical region 4. Two transcripts recently identified by massive RNA-sequencing (RNA-Seq) and automatically annotated on Ensembl database reveal that the HR-DSCR seems to be partially crossed by KCNJ6-202 and DSCR4-202 isoforms. KCNJ6-202 shares the coding sequence with KCNJ6-201 which is involved in many physiological processes, including heart rate in cardiac cells and circuit activity in neuronal cells. DSCR4-202 transcript has the first two exons in common with DSCR4-201, the only experimentally verified gene uniquely present in Hominidae. In this study, we performed in silico and in vitro analyses of the HR-DSCR. Bioinformatic data, obtained using Sequence Read Archive (SRA) and SRA-BLAST software, were confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sanger sequencing on a panel of human tissues. Our data demonstrate that the HR-DSCR cannot be defined as an intergenic region. Further studies are needed to investigate the functional role of the new transcripts, likely involved in DS phenotypes.

Human intelectin-1 (ITLN1) genetic variation and intestinal expression

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan β-D-galactofuranose or protein–protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

RESEARCH ON POLYMORPHIC VARIANTS CANDIDATE GENES FOR PSORIASIS

В статье рассмотрены результаты исследования полиморфных вариантов генов предрасположенности к псориазу PSORS1C1, POU5F1, IL23R. Исследованы 192 образца ДНК, из них 116 образцов (77 больных псориазом и 39 человек, без признаков данной патологии) соответствовали стандартным требованиям к пробам ДНК. Полученные результаты сравнивались с референсной версией генома человека CRch 37. Распределение частот генотипов полиморфных вариантов трех генов соответствовали уравнению Харди-Вайнберга. Установлены 17 полиморфных вариантов локуса PSORS1C1, один вариант полиморфизма гена POU5F1 и два варианта полиморфизма гена IL23R, представленных в международной базе данных Ensembl. Впервые, выявлен еще один полиморфный вариант гена IL23R, ранее не аннотированный в базе данных Ensembl. The article discusses the results of the study of polymorphic variants of the PSORS1C1, POU5F1, IL23R genes which predisposition to psoriasis. 192 DNA samples were examined, in which 116 samples (77 patients with psoriasis and 39 people without signs of this pathology) met the standard requirements for DNA samples. Obtained results were compared with the reference version of the human genome CRch 37. The distribution of the genotype frequencies of the polymorphic variants of the three genes corresponded to the Hardy-Weinberg equation. Identified 17 polymorphic variants of the PSORS1C1 locus, one variant of the POU5F1 gene polymorphism and two variants of the IL23R gene polymorphism, which presented in the international database Ensembl. For the first time, identified another polymorphic variant of the IL23R gene, which had not been previously annotated in the Ensembl database.

Long Non-coding RNA 332443 Inhibits Preadipocyte Differentiation by Targeting Runx1 and p38-MAPK and ERK1/2-MAPK Signaling Pathways

Long non-coding RNAs (lncRNAs) have emerged as integral regulators of pathophysiological processes, but their specific roles and mechanisms in adipose tissue development remain largely unknown. Here, through microarray analysis, co-expression, and tissue specific analysis of adipocyte tissues after fasting for 72 h, we found that Lnc-FR332443 expression was dramatically decreased, as well as the expression of Runx1. The UCSC database and Ensembl database indicated that Lnc-FR332443 is the antisense lncRNA of Runx1. Lnc-FR332443 and Runx1 are highly enriched in adipose tissue and downregulated during adipogenic differentiation. Adipose tissue-specific knockdown of Lnc-FR332443 increased fat mass in vivo, and specific knockdown of Lnc-FR332443 in 3T3-L1 preadipocytes promoted adipogenic differentiation. In this process, Runx1 expression was decreased when Lnc-FR332443 was downregulated in adipocytes or 3T3-L1 preadipocytes, and vice versa, when Lnc-FR332443 was upregulated, the expression of Runx1 was increased. However, overexpression of Runx1 decreased the expression of the adipocyte cell marker genes PPARγ, C/EBPα and FABP4 significantly, while not affected the expression of Lnc-FR332443. Mechanistically, Lnc-FR332443 positively regulates Runx1 expression in mouse adipocytes and suppresses adipocyte differentiation by attenuating the phosphorylation of MAPK-p38 and MAPK-ERK1/2 expression. Thus, this study indicated that Lnc-FR332443 inhibits adipogenesis and which might be a drug target for the prevention and treatment of obesity.

Single Nucleotide Polymorphims (snps) Identification of Inhibin Sub Unit-α (inha) Gene on Madura Bulls

INHA gene is a gene that is suggested to have role in reproductive system. Single Nucleotide Polymorphisms (SNPs) of Madura Bulls were identified in this study. Polymerase Chain Reactions (PCR) was used to amplify INHA gene region and MEGA 7 program was utilized to align the amplified region sequences with sequence from Ensembl database. Four SNPs found in INHA and they are located at the first exon. Two SNPs were misssense mutations that causing the substitution of amino acid leucine21 by proline, and amino acid valine63 by methionine and the other two SNPs were synonymous mutation. One of the synonymous SNPs was a novel mutation. Based on those identified SNPs, they could be suggested as potential candidate markers of reproduction traits for Madura bulls. Moreover, through heterozygosity value from the observed bulls, it was indicated that the genotype was varied in population. Therefore a molecular selection program could be designed to determine the Madura superior bull.

Bioinformatics of Nile tilapia (Oreochromis niloticus) lymphocyte cytosolic protein 1 (lcp1) gene

Bioinformatics analysis of lymphocyte cytosolic protein 1 (lcp1) gene in tilapia (Oreochromis niloticus) which is a model organism in experimental studies were completed in this study. For this purpose, characterization and identification of lcp1 has been completed and ensembl database has been used to design the structure of lcp1 gene. In addition, the chromosome region of tilapia lcp1 and other genes in the same region with lcp1were determined. The chromosome of these genes were detected in zebrafish and human which are identical orthologos of tilapia. Conserved gene synteny designed manually according to these chromosomal regions. In addition, amino acid sequences synthesized by lcp1 gene in some vertebrates were determined using some bioinformatics databases such as UNIPROT, ENSEMBL and NCBI before determine the phylogenetic relationship between these organisms and tilapia. Sequence similarity-identity rate of tilapia lcp1 gene with zebrafish, rainbow trout, human, mouse and platyfish lcp1/Lcp1 was calculated using BLOSUM62 matrix algorithm. This study is of great importance for the completion of in silico analysis of lcp1 gene in tilapia because it is an aquatic model organism and it has an important place among economic aquaculture species. However this study provides the basic pioneering information for the future studies on molecular stress response in fish.

In silico screening and identification of deleterious missense SNPs along with their effects on CD-209 gene: An insight to CD-209 related-diseases

DC-SIGN receptor articulated by macrophages and dendritic cells is encoded by CD209 gene and plays a role to activate and proliferate the T-lymphocytes in response of virus attack. The dysfunctional activity of DC-SIGN receptor because of missense SNPs can lead to cause dengue haemorrhage fever, HIV-1 infection etc. Out of 11 transcripts of CD209, all missense SNPs of canonical transcript were retrieved from Ensembl database and evaluated by their deleteriousness by using Polyphen-2, PMut, SIFT, MutPred, PROVEAN and PhD-SNP together with stimulation of its complete 3D structure. 10 nsSNPs were chosen depending on both the significance value of nsSNP and their prediction among SNPs evaluating servers which are based on different algorithms. Moreover, the position and native role of 10 nsSNPs in wild 3D model has been described which assist to acknowledge their importance. This study urges the researcher’s community to experimentally validate these SNPs and their association in causing the diseases like dengue fever, Tuberculosis etc.

PRODUCTION OF POLYCLONAL ANTIBODIES FOR DETECTING HUMAN TYPE 2 TRANSGLUTAMINASE VARIANTS IN COLON CANCER

Type 2 transglutaminase (TGM2) is a multifunctional ubiquitous protein, involving in protein cross-linking, apoptosis, and cell differentiation. Recently, some reports have suggested that TGM2 expression is a potential prognostic marker, and often associates with advanced stages of disease, metastatic spread, as well as drug resistance in many cancer cell lines although its primary function is unknown. To elucidate the role of TGM2 in cancer, the expression profile of the TGM2 need to be examined. In this study, new polyclonal antibodies detecting four TGM2 variants encoding protein from ENSEMBL database are produced and their specificities are confirmed by western blot analysis with E.coli overexpressing TGM2-002 protein, HEK293T cells overexpressing TGM2-S protein and human colon cancer samples. Western blot data showed that these antibodies could detect not only TGM2-002 in E. coli overexpressing TGM2-002 but also smaller molecular weight protein (about 62 kDa) in HEK293T overexpressing TGM2-S cells which was further confirmed by MALDI-TOF analysis. We found that both TGM2-1 and TGM2-4 antibodies could detect the full length TGM2-002 protein in colon cancer samples. Furthermore, in normal sample, we found that majority of TGM2-002 protein existed in membrane fraction but not in total lysate whereas TGM2-002 protein was found in both total lysate and membrane fraction in colon cancer samples.

Mutations in NPHS1 and PLCE1 (NPHS3) in two Vietnamese patients with conginetal nephrotic syndrome

Congenital nephrotic syndrome (CNS), a genetic disease caused by the mutations in genes on autosomes,is usually occurs in the first three months after birth. The mutations in genes that encode for the structural andfunctional proteins of podocytes lead to loss of the function of glomerular filtration. So far, a number of genesrelated to the disease have been identified such as: NPHS1, NPHS2, PLCE1 (NPHS3), ACTN4, CD2AP, INF2and WT1. In this article, we amplified all of exons in NPHS1 and PLCE1 genes of two Vietnamese patientswith CNS and members of patients’ family. PCR products were purified and sequenced directly on automaticsequencer ABI 3500 Bio System (USA). The sequencing results were compared with the sequences of NPHS1and PLCE1 genes published in the Ensembl database (ENSG00000161270 and ENSG00000138193,respectively) by using BioEdit software to detect mutations. We identified two mutations: c.2398C>T(p.Arg800Cys, exon 18), c.3315A>G (p.Ser1105Ser, exon 26) in NPHS1 gene and two mutations: c.5330 C>T(p.Thr1777Ile, exon 23), c.5780A>G (p.His1927Arg, exon 25) in PLCE1 gene in study patients. These twopatients carried simultaneously the mutations in the NPHS1 and PLCE1 genes with serious phenotype. Theresults of our study might be evidences for the role of mutations in NPHS1 and PLCE1 genes in thedevelopment of disease in patients. These are useful information in identifying the cause of disease and providethe genetic counseling to the patients’ family.

PSX-26 The identification of novel SNPs significant for reproductive abilities in sheep using GWAS

Reproductive ability of ewes is one of the most important traits in sheep breeding. Hence the mechanisms underlying this complex trait are poorly understood till now. To study the genetic architecture of litter size and search for SNPs associated with Total lambs born (TLB) we conducted genome-wide association study of Volgograd sheep. A total of 48 sheep after the second lambing were collected: 24 of them had 1 lamb, and 24 others had two lambs. All the samples were genotyped using the OvineSNP50 Genotyping BeadChip according to the manufacturer’s protocol. Then they were filtered using the parameters –hwe = 10–3, --maf = 0.05, --geno = 0.1, --indep-pairwise 50 5 0.5. After the quality control 46249 SNPs of 54241 were retained in the working dataset for the GWAS. A total of 14 significant SNPs located on chromosomes 1, 2, 4, 7, 15, 18, and 19 reached the genome-wide significance level. More than 50% of SNPs are located in the introns of genes, the rest are located in regulatory regions of genes and intergenic regions. We searched for candidate genes located near the significant SNPs using the Ensembl database. A total of 14 genes were selected. Gene annotation was completed using Panther. The detailed information about those significant SNPs and the possible genes associated with TLB are presented in Table. These genes take part in cellular processes, biological regulation and regulation of metabolic processes, some of them are involved in the formation of the immune system and biological adhesion. The data obtained allowed us to determine a number of SNPs that are significant for the formation of such complex reproductive trait as Total lamb born (TLB) in sheep and could be potentially applied in sheep breeding programs.