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ROLE OF THE PRKCA GENE AND OF MICRORNAS IN THE SUSCEPTIBILITY TO MULTIPLE SCLEROSIS
No full textMultiple sclerosis (MS) is a multifactorial neurological disorder characterized by chronic inflammation, demyelination, and axonal damage, probably caused by an altered immune response against myelin antigens. Despite extensive studies, the genetic and environmental determinants of MS are still poorly understood. In this thesis, we analyzed the role of the PRKCA gene that encodes the protein kinase C alpha, and has been involved in MS both by linkage and association studies in different populations (Finns, Canadians, and UK population), as well as the possible involvement of microRNAs (miRNAs) in the pathogenesis of MS.
As far as PRKCA is concerned, a case-control association study performed on the Italian population allowed us to identify two association signals in the PRKCA gene: a protective allele mapping in the promoter region (P=0.033; OR=0.12, 95% CI=0.015-0.94) and a risk haplotype, partially overlapping the predisposing haplotypes observed both in Finns and Canadians within PRKCA intron 3 (P=7.4*10-4; OR=1.57, 95% CI=1.24-1.99). We identified and characterized, both in vitro and in vivo, the functional variants underlying these association signals, and demonstrated that higher PRKCA expression levels play a protective role towards MS predisposition. Moreover, we describe here for the first time so-far unknown PRKCA transcript isoforms, which are differentially expressed in MS cases and controls, and eventually lead to the synthesis of aberrant proteins, potentially dangerous for the cells. Furthermore, we characterized the MIR634 gene, mapping within the PRKCA locus, and demonstrated its involvement in the regulation of PRKCA expression.
As far as miRNAs are concerned, we monitored the differential expression of several immunity-related miRNAs in peripheral blood mononuclear cells of MS patients and healthy controls, and identified miR-155 as the most upregulated miRNA in MS cases (fold change=3.30; P=0.013). Interestingly, this miRNA was previously reported to be up-regulated also in MS brain lesions. The role of miR-155 in MS susceptibility was also investigated by genotyping four single nucleotide polymorphisms mapping in the miR-155 genomic region. A three-SNP haplotype resulted associated with the disease status (P=0.035; OR=1.36, 95% CI=1.05-1.77), suggesting that this locus strongly deserves further investigations.
In conclusion, this thesis work points to a possible role of post-transcriptional regulation mechanisms in the pathogenesis of MS, particularly supporting the uprising hypothesis that in MS a dysregulation of the splicing may play a pivotal role in MS
Differential expression of microRNAs in peripheral blood mononuclear cells of Multiple Sclerosis patients
No full textMicroRNAs (miRNAs) are a class of short (~22 nucleotides) single-stranded RNAs that modulate the expression of multiple target mRNAs by inducing either translational repression or mRNA degradation. MiRNAs have emerged has key post-transcriptional regulators of diverse biological processes and their deregulation has been implicated in several complex human diseases, including neurodegenerative and inflammatory disorders.
Multiple sclerosis (MS) is a multifactorial disease of the central nervous system characterized by chronic inflammation, demyelination, axonal damage, and progressive neurological dysfunction. In spite of extensive research, the molecular events involved in the initiation and progression of MS are still poorly understood. To be relevant for MS pathogenesis, candidate genes would be expected to be expressed either in tissues relevant for immune response, or in tissues affected by the disease process. Several miRNAs are known to be expressed specifically in the immune system and miRNA-dependent alterations in gene expression in hematopoietic cells are critical for mounting an appropriate immune response. Moreover, deregulation of hematopoietic-specific miRNA expression may result in defects in both central and peripheral tolerance.
Therefore, we sought to explore the possible involvement of miRNAs in MS by monitoring for differential expression of specific miRNAs in peripheral blood mononuclear cells (PBMCs) of MS patients and healthy controls. We selected a set of 22 candidate miRNAs that are expressed in the immune system and/or are transcribed from previously reported MS susceptibility loci. The differential expression of candidate miRNAs in cases versus controls was evaluated using a microbead-based technology. In a pilot experiment, performed on PBMC RNA of relapsing-remitting MS patients and controls, 4 miRNAs resulted >3 folds up-regulated in MS vs controls, whereas only the miR-150 resulted down-regulated (2-fold decrease). Interestingly, two of the most up-regulated miRNAs, mir-155 and mir-146a, have been reported to be altered also in rheumatoid arthritis and systemic lupus erythematosus, suggesting shared pathogenic mechanisms between different chronic inflammatory diseases. This is, to our knowledge, the first evidence that the expression of specific miRNAs is altered in MS
Differential expression of microRNAs in peripheral blood mononuclear cells of multiple sclerosis patients
No full textMicroRNAs (miRNAs) are a class of short single-stranded RNAs that act as post-transcriptional regulators of gene expression and represent a major group of regulators potentially associated with human multifactorial diseases.
Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system characterized by chronic inflammation, demyelination, and axonal damage. As miRNA-dependent alterations in gene expression in hematopoietic cells are critical for mounting an appropriate immune response, their deregulation may result in defects in immune tolerance.
In this frame, we sought to explore the possible involvement of miRNAs in MS pathogenesis by monitoring the differential expression of 22 miRNAs (expressed in the immune system or transcribed from previously
reported MS susceptibility loci) in peripheral blood mononuclear cells of MS patients and healthy controls by using a microbead-based technology. Four miRNAs resulted >3 folds up-regulated in MS vs controls, whereas only 1 resulted down-regulated. Interestingly, 2 of the most up-regulated miRNAs (mir-155, mir-146a) have been reported to be altered also in rheumatoid arthritis and systemic lupus erythematosus, suggesting shared pathogenic mechanisms with MS. To confirm mir-155 and mir-146a up-regulation, qPCR experiments will be performed. Moreover, the role of these miRNAs in MS susceptibility will be investigated by genotyping single nucleotide polymorphisms mapping in mir-155 and mir-146a regions
The Protein Kinase C Alpha (PRKCA) gene is associated with multiple sclerosis in the Italian population
No full textMultiple sclerosis (MS) is a chronic neurological disorder characterized by multicentric inflammation, demyelination, and axonal damage.
The Protein Kinase C Alpha (PRKCA) gene was found to be associated with MS in Finnish, Canadian, and UK populations, in which specific risk haplotypes were identified. Moreover, PRKCA transcript levels were shown to be higher in CD4- mononuclear cells of MS patients carrying the risk haplotypes, suggesting a contribution of PRKCA regulatory mechanisms in the pathogenesis of MS.
In this study, we analyzed the role of PRKCA in MS susceptibility in a cohort of 358 cases and 662 controls from continental Italy. An association analysis was performed genotyping 3 microsatellites and 20 single nucleotide polymorphisms (SNPs) covering the whole gene. A significant association with 2 microsatellites, mapping respectively in the promoter region and in intron 2, was found (P=0.032 and P=0.027). The 5’ regulatory region of the PRKCA gene was hence further investigated by sequencing the first 424 bp of the promoter and the entire exon 1 in all MS cases and controls. However, no genetic variants specific for MS cases were identified.
Analysis of individual SNPs did not show any evidence for association; however, a haplotype of 7 SNPs, spanning a genomic region of 43 kb of PRKCA intron 3, resulted strongly associated with the disease status (P=7.4*10-4; OR=1.54, 95% CI=1.22-1.95). This haplotype, which partially overlaps the risk haplotypes observed both in Finns and Canadians, includes an alternative exon characterizing a shorter PRKCA protein isoform. Since no information were available on the tissue distribution of this isoform, its expression pattern was analyzed and compared to the expression profile of PRKCA in a panel of 20 human tissues by RT-PCR: the shorter isoform showed a more marked tissue-specific expression. The possible differential expression of the 2 transcripts between MS patients and controls is currently underway by qRT-PCR on RNA extracted from peripheral blood mononuclear cells.
In conclusion, our study provides a strong replication evidence that haplotypes characterizing PRKCA increase the risk of MS; a metanalysis including all studies investigating the role of PRKCA as a MS susceptibility gene reported so far showed the considerable OR of 1.52 (95% CI=1.36-1.69; P=2.2*10-14)
Identification of the first alu-mediated large deletion involving the f5 gene in a compound heterozygous patient with severe factor v deficiency
No full textFactor V (FV) deficiency is a rare autosomal recessive haemorrhagic disorder associated with moderate to severe bleeding symptoms. Conventional mutational screening leads to a complete molecular genetic diagnosis only in about 80-90% of cases. Large gene rearrangements, which could explain at least part of the "missing alleles" have not been reported so far in FV-deficient patients. In this work, we investigated a family with hereditary FV deficiency, in which the proband is compound heterozygous for a 205-Kb deletion, involving the first seven exons of F5, and the entire selectin P, L, and E genes, and for a novel splicing mutation (IVS12+5G>A). The deletion breakpoints, determined by using a combination of semi-quantitative real-time PCR and long PCR assays, occurred within AluY repeat sequences, suggesting an Alu-mediated unequal homologous recombination as the mechanism responsible for the deletion. The in vitro characterisation of the IVS12+5G>A mutation demonstrated that this mutation causes the skipping of exon 12 and the activation of a cryptic splice site. Low levels of residual wild-type splicing were also detectable, in agreement with the notion that the complete absence of FV may be not compatible with life. © Schattauer 2011
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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