Using principal component (PC) analysis, we studied the genetic constitution of 3,112 individuals from Europe as portrayed by more than 270,000 single nucleotide polymorphisms (SNPs) genotyped with the Illumina Infinium platform. In cohorts where the sample size was >100, one hundred randomly chosen samples were used for analysis to minimize the sample size effect, resulting in a total of 1,564 samples. This analysis revealed that the genetic structure of the European population correlates closely with geography. The first two PCs highlight the genetic diversity corresponding to the northwest to southeast gradient and position the populations according to their approximate geographic origin. The resulting genetic map forms a triangular structure with a) Finland, b) the Baltic region, Poland and Western Russia, and c) Italy as its vertexes, and with d) Central- and Western Europe in its centre. Inter- and intra- population genetic differences were quantified by the inflation factor lambda (λ) (ranging from 1.00 to 4.21), fixation index (Fst) (ranging from 0.000 to 0.023), and by the number of markers exhibiting significant allele frequency differences in pair-wise population comparisons. The estimated lambda was used to assess the real diminishing impact to association statistics when two distinct populations are merged directly in an analysis. When the PC analysis was confined to the 1,019 Estonian individuals (0.1% of the Estonian population), a fine structure emerged that correlated with the geography of individual counties. With at least two cohorts available from several countries, genetic substructures were investigated in Czech, Finnish, German, Estonian and Italian populations. Together with previously published data, our results allow the creation of a comprehensive European genetic map that will greatly facilitate inter-population genetic studies including genome wide association studies (GWAS).

Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (K d ) of 2.5 nM. This was comparable with our measured K d of LOX binding to tropoelastin (1.9 nM) and type I collagen (5.2 nM), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity.Lysyl oxidase (LOX), 1 a copper-dependent amine oxidase, catalyzes the oxidative deamination of peptidyl lysine residues in collagen and elastin molecules to ␦-aminoadipic-␤-semialdehyde or allysine, which can then spontaneously condense with neighboring amino groups or other peptidyl aldehydes to form covalent cross-links in fibrillar collagens and elastin (1, 2). The covalent cross-linking of these extracellular matrix (ECM) proteins by LOX is essential to the formation of insoluble collagen and elastic fibers and for normal mammalian development (3, 4). In many human pathologies, including cardiovascular disease, fibrosis, and cancer, LOX expression and activity have been demonstrated to be altered as compared with normal conditions (1, 2).The human LOX protein is synthesized as a prepro-enzyme of 417 amino acids with an N-terminal signal peptide sequence for secretion. During protein trafficking in the Golgi, a copper atom is incorporated at the copper-binding site (residues 286 -296) (5-7), which allows the formation of a lysyl-tyrosylquinone cofactor derived from Tyr-355 and Lys-320 residues (8). After secretion into the extracellular space, proteolytic cleavage between residues Gly-168 and Asp-169 removes the N-terminal propeptide, yielding an active enzyme of 30 kDa (9 -11). This processing is mainly accomplished by the procollagen C-proteinase bone morphogenic protein-1 (BMP-1), and to a lesser degree, by mammalian Tolloid-like 1 protein (12, 13). However, it is not know...

Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.

Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction obstructions, and despite their high prevalence, we have a poor understanding of their etiology and scarcity of genetic models. The eight-protein exocyst complex regulates polarized exocytosis of intracellular vesicles in a large variety of cell types. Here we report generation of a conditional knockout mouse for Sec10, a central component of the exocyst, which is the first conditional allele for any exocyst gene. Inactivation of Sec10 in ureteric bud-derived cells using Ksp1.3-Cre mice resulted in severe bilateral hydronephrosis and complete anuria in newborns, with death occurring 6–14 hours after birth. Sec10FL/FL;Ksp-Cre embryos developed ureteropelvic junction obstructions between E17.5 and E18.5 as a result of degeneration of the urothelium and subsequent overgrowth by surrounding mesenchymal cells. The urothelial cell layer that lines the urinary tract must maintain a hydrophobic luminal barrier again urine while remaining highly stretchable. This barrier is largely established by production of uroplakin proteins that are transported to the apical surface to establish large plaques. By E16.5, Sec10FL/FL;Ksp-Cre ureter and pelvic urothelium showed decreased uroplakin-3 protein at the luminal surface, and complete absence of uroplakin-3 by E17.5. Affected urothelium at the UPJ showed irregular barriers that exposed the smooth muscle layer to urine, suggesting this may trigger the surrounding mesenchymal cells to overgrow the lumen. Findings from this novel mouse model show Sec10 is critical for the development of the urothelium in ureters, and provides experimental evidence that failure of this urothelial barrier may contribute to human congenital urinary tract obstructions.

Recently, associations were found between several autoimmune diseases and functional variants of interleukin‐23 receptor (IL23R) gene; here, we studied the possible association of nine polymorphisms of IL23R with ankylosing spondylitis (AS) and with Sjögren syndrome (SS). In our study, we genotyped groups of patients with AS (n = 206), SS (n = 156) and healthy controls (n = 235) for rs11805303, rs10889677, rs1004819, rs2201841, rs11209032, rs11209026, rs10489629, rs7517847 and rs7530511 variants using PCR‐RFLP methods. We observed significant increase in the carriage of the T allele of rs11805303 and the A allele of rs1004189 in the AS group compared with the controls. For the rs10889677 variant, the prevalence of the AA genotype and for the rs2201841, the CC genotype showed a more than two‐fold increase in the AS group compared with the controls. By contrast, the GA heterozygous genotype of rs11209026 variant showed a significant decrease in AS patients compared with controls. Haplotype analysis revealed association of four IL23R haplotypes with AS. There was no difference in the distribution of any of the examined IL23R variants between controls and SS patients. In conclusion, we confirmed the susceptibility or protective associations of IL23R polymorphisms with AS in a Hungarian population and first demonstrated the involvement of the rs11805303 intronic single nucleotide polymorphisms, which was tested so far only for other autoimmune diseases.

No abstract

Genome-wide association studies identified many loci associated with the two forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC). Components of the interleukin-23 signalling pathway, such as IL23R, JAK2 and STAT3, have been implicated in both diseases. In addition, emerging evidence supports the role of IL23-driven Th17 cells in inflammation. Here, we studied the susceptibility nature of three components of IL23 signalling and Th17 cell differentiation: JAK2 rs10758669, STAT3 rs744166 and CCR6 rs2301436 initially associated with CD in Hungarian CD and UC patients. A total of 616 unrelated subjects with either form of IBD and 496 healthy controls were genotyped with PCR-RFLP methods. We also tested the genetic interactions of JAK2, STAT3 and CCR6 polymorphisms in a pairwise fashion with regard to disease risk. We could confirm the susceptibility of STAT3 rs744166 TT homozygotes for UC (OR: 1.483, 95% CI: 1.103-1.992, P = 0.009). Data on genetic interaction reveals that the above JAK2 and STAT3 risk alleles contribute to CD susceptibility in combination with each other (OR: 2.218; 95% CI: 1.097-4.487; P = 0.024), while the JAK2 variant shows a tendency to confer UC risk only on a wild-type STAT3 background (OR: 1.997, 95%CI: 0.994-4.009, P = 0.049). Our results may help in understanding how these natural variants contribute to development of IBD through their genetic association.

One requirement for establishing polarity within a cell is the asymmetric trafficking of intracellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight-protein exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for proteins required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst genes are conserved in mammals, in which the specifics of exocyst-mediated trafficking are highly cell-type-dependent. Some exocyst members bind to certain Rab GTPases on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establishment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front-back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.