Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.     

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex

The membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113 degrees C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an alpha-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement.     

The Iho670 Fibers of Ignicoccus hospitalis: a New Type of Archaeal Cell Surface Appendage

Ignicoccus hospitalis forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 μm), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle.The existence of surface appendages on archaeal cells has been known for a long time (6; for a recent review, see reference 15); functional studies defined archaeal flagella as motility organelles (e.g., see references 1 and 2) and archaeal fimbriae as adhesins (3, 7, 22). Two other types of very special archaeal cell surface appendages are the hami formed by the SM1 euryarchaeum (13) and the cannulae produced by Pyrodictium occultum (16, 21). In the case of hami, their function is obvious: they are 1- to 3-μm-long filamentous structures with regular spikes, ending in a hook to resemble in ultrastructure a barbed wire, which ends in a grappling hook (13). Thereby, SM1 cells adhere to each other (and structures in their biotope); SM1, indeed can be harvested from polyethylene nets placed into its natural habitat (cold sulfidic springs) (8). The function of the cannulae is still not known: they are extracellular tubes 25 nm wide with an inner diameter of ca. 20 nm and are composed of five related proteins (12). Cannulae enter the periplasmic space but do not reach into the cytoplasm; they have different lengths and connect the highly irregular Pyrodictium cells to form nets easily visible by naked eye. Here, we describe a new type of archaeal cell surface appendage, the fibers which we have detected on cells of the crenarchaeum Ignicoccus hospitalis KIN4/I^T.I. hospitalis originally was described to possess up to nine flagellum-like appendages, anchored at one pole into the cell (17). In repeated experiments designed to examine possible motility of I. hospitalis, however, we never observed such behavior. For those experiments, we used our thermomicroscope, allowing analyses of cells under anaerobic conditions at 90°C (9). To study these cell surface appendages in more detail, cells were grown at 90°C (in medium described in reference 17) either in 120-ml serum bottles filled with 20 ml medium, an 80:20 H₂-CO₂ gas phase, and with shaking at 100 rpm or in a 300-liter fermentor filled with 250 liters as described previously (10). In initial experiments, the cell surface appendages were removed from cells by shearing, differential centrifugation, and a final CsCl gradient centrifugation (similar to the protocol established for the preparation of flagella from Pyrococcus furiosus [14]). Yields, using this method, however, were very low (data not shown). A breakthrough was our finding that the cell surface appendages are very brittle and that the majority of fibers were removed from the cell surface by normal lab handling (compare Fig. 1A and B). Such manipulations include especially sampling of cells using the syringe-needle method (to transfer the strictly anaerobic archaea) and centrifugations to concentrate cells. Therefore, another strategy was used to isolate the cell surface appendages. To aliquots of the supernatant of the harvest (after overnight centrifugation at 16,000 × g) of cells grown to stationary phase in a 300-liter fermentor—containing the majority of fibers, broken off during the harvest centrifugation—NaCl was added to a 5.8% final concentration and polyethylene glycol 6000 (PEG 6000; Fluka, Sigma-Aldrich, Steinheim, Germany) was added to a final concentration of 10.5%. Precipitation was carried out overnight at 6°C, followed by centrifugation (30 min at 10,000 × g). The pelleted material obtained from a 10-liter aliquot of centrifugation supernatant was dissolved in 8 ml of Millipore-purified water (aqua bidest). After addition of 3.6 g of CsCl, the sample was centrifuged for 48 h (SW60 Ti rotor, 250,000 × g, 4°C in Beckman Optima LE-80K centrifuge) and the resulting band (Fig. ​(Fig.1C)1C) was dialyzed extensively against 5 mM MES buffer (1 mM MgSO₄·7H₂O with 1 mM dithiothreitol; pH 6.0). Yields did increase dramatically: the PEG 6000 precipitation method resulted in at least 100-fold more fiber material than the method used initially. (Shearing from 10 liters of cells which had been concentrated by centrifugation resulted in 10 to 50 μg of fiber protein.) In repeated experiments, the total yield of fiber protein, precipitated from 10 liters of centrifugation supernatant by PEG-NaCl addition, varied from 35 to 45 mg. Since the cell yield of 10 liters of culture was 0.75 g, we estimate that the fiber protein constitutes at least 5% of the cellular protein.Open in a separate windowFIG. 1.Ultrastructural and biochemical analyses of Ignicoccus hospitalis fibers. (A) A 5-μl aliquot of a fermentor culture, grown to stationary phase, was applied onto a carbon-coated grid, negatively stained with 2% uranyl acetate, and analyzed by TEM. The sample was collected using a wide-bore pipette. (B) An aliquot of the same culture as that in panel A was analyzed by the TEM procedure as described for panel A. The sample was collected with a syringe and needle. (C) CsCl gradient (volume, 10 ml) of I. hospitalis fibers concentrated by PEG precipitation from 10 liters of supernatant of a 16,000 × g centrifugation (used to harvest cells from the fully grown fermentor culture). (D) TEM analysis of the CsCl gradient-purified fibers, prepared and negatively stained as for panel A. (E to G) SDS-PAGE analyses of CsCl gradient-purified fibers (12.5% gel, silver stained). (E) Broad-range protein marker (NEB). Sizes in kDa are indicated to the left. (F) Heat-denatured fibers (0.5 μg treated for 15 min at 100°C). (G) Partially heat-denatured fibers (5 μg treated for 3 min at 100°C). (H) Amino acid sequence of the fiber protein. The sequence given in boldface and underlined was obtained by N-terminal protein sequencing and used to identify Igni_0670 as the structural gene coding for the I. hospitalis fibers (NCBI reference sequence NC_009776.1). The size bars in panels A, B, and D are 1 μm each.Biochemical and bioinformatic analyses of the fiber protein indicated the following. The material obtained after PEG 6000 precipitation and CsCl gradient centrifugation consisted of mainly one protein with a mass of ca. 33 kDa, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. ​(Fig.1F).1F). Protein samples were resolved by SDS-PAGE with 12.5% polyacrylamide (11); proteins were stained with Coomassie brilliant blue G250 or via silver staining (4). The same preparation, if not completely denatured by heat treatment, resulted in protein bands of ca. 33, 60, and 120 kDa (with weaker bands of ca. 90 and 180 kDa) (Fig. ​(Fig.1G).1G). We take this to indicate that these bands represent fiber protein oligomers. The fiber protein seems not to be glycosylated, as indicated by the lack of a positive reaction (data not shown) with periodic acid-Schiff staining (24), although we note that there is no absolute correlation between the presence or absence of glycosylation with a positive or negative periodic acid-Schiff staining. A lack of glycosylation would differentiate the fiber protein from most other archaeal cell surface appendages described as flagellins, for which such a modification is the rule rather than an exception (23). N-terminal sequencing by Edman degradation (performed by the central protein analytic facility of the Biology Department of the University of Regensburg) determined the N terminus over a length of 23 amino acids (Fig. ​(Fig.1H).1H). Since the genome sequence of I. hospitalis is known (18), identification of the fiber protein was possible: it is encoded by I. hospitalis gene Igni_0670. In order to differentiate fiber proteins potentially occurring in other Ignicoccus species (and to comply with the generally accepted rule to name proteins with a three-letter code), we propose to name the fiber protein of I. hospitalis Iho670. The Iho670 protein is processed since the first 7 amino acids are not found in the mature fiber protein. The two programs Flafind and SOSUI indeed predict such a short signal peptide, while other programs (like signalP) indicate a much longer signal peptide of 38 amino acids: similar difficulties with prediction of signal peptides had been observed earlier for archaeal cell surface proteins, e.g., for the Mth60 fimbrin and the I. hospitalis outer membrane protein Ihomp1 (5, 22; see also reference 3 for a detailed discussion of archaeal signal peptides). A signal peptidase processing the Iho670 protein at the correct site (i.e., after amino acid 7) very recently was identified in I. hospitalis (S.-V. Albers, personal communication). The fiber protein Iho670 shows no homologies to other proteins in its amino acid sequence, especially to the archaeal cell surface appendage proteins identified up to now: archaeal flagellins, the archaeal fimbrin Mth60, the hamus protein, and the three cannula proteins. The Igni_0670 gene might be argued to be part of an operon, because Igni_0668 to Igni_0677 are predicted to be transcribed counterclockwise from the I. hospitalis genome, with a maximum intergenic space of 73 nucleotides (18). Predictions of the proteins encoded by these genes, however, do not favor this possibility, because Igni_0668, Igni_0669, Igni_0670, and Igni_0672 code for hypothetical proteins; Igni_0671 and Igni_0673 code for flavin adenine dinucleotide-dependent pyridine nucleotide disulfide oxidoreductases; Igni_0674 codes for an NiFe hydrogenase maturation protein; Igni_0675 encodes a nonspecific serine/threonine protein kinase; Igni_0676 encodes a protein homologous to eukaryotic initiation factor 1A; and Igni_0677 encodes a 30S ribosomal protein, S6e. Obviously, there is no functional context between the encoded proteins.The results of our ultrastructural analyses of the fibers can be summarized as follows. Transmission electron microscopic (TEM) analyses of the purified Iho670 fibers indicate that they can be up to 20 μm long, with a diameter of 14 nm. TEM analyses of I. hospitalis cells growing on carbon-coated grids (see reference 14 for technical details) confirmed these data. Obviously, these cell surface appendages are very long and brittle; therefore, we were not able to decide how many fibers are synthesized per cell: we estimate this number to be at least 20. For TEM, a drop of cell-suspension was placed on a carbon-coated 200-mesh copper grid (Plano, Wetzlar, Germany). These samples were either unidirectionally shadowed with platinum and carbon at 15° (CFE 50; Cressington Ltd., Watford, United Kingdom) or negatively stained for 1 min with 2% uranyl acetate. All TEM micrographs were recorded using a slow-scan charge-coupled device camera (TEM 1000; TVIPS-Tietz, Gauting, Germany) attached to a CM 12 transmission electron microsope (FEI, Eindhoven, The Netherlands).It turned out that scanning electron microscopic (SEM) analyses of the coccoid cells with emanating fibers is extremely difficult. This is due to the fact that the outer membrane of I. hospitalis is a very delicate structure, being destroyed on nearly every cell during standard fixation and processing steps for TEM and SEM. We have proven that under the same conditions, other archaeal cells and their appendages are well preserved and can be nicely visualized (13, 14, 17, 19, 20). TEM analyses of I. hospitalis cells need special precautions to conserve the labile membranes, like growth in cellulose capillaries and high-pressure freezing and freeze-substitution (19); a simple glutaraldehyde fixation and dehydration at room-temperature will destroy, especially, the outer membrane. For SEM analyses, a protocol preserving the outer membrane is not yet available.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.     

Evolution of translational initiation: new insights from the archaea

Recent in silico and experimental data have shed new light on the mechanism and components of translational initiation in archaea. The available data about the structure of archaeal mRNAs, mRNA/ribosome interaction and archaeal translation initiation factors are reviewed and analyzed in the conceptual framework of the evolution of translational initiation. A model of the initiation step of translation in the Last Universal Common Ancestor of extant cells is presented and discussed.     

A tale of two polymers: new insights into helical filaments

Many proteins function as helical polymers within the cell. Two intensively studied examples are eukaryotic actin and bacterial RecA, which belong to two different protein superfamilies. However, most other members of these superfamilies do not polymerize into helical filaments. General features of polymorphism, cooperativity and allostery that emerge from studies of eukaryotic actin and bacterial RecA raise more general issues about how conserved these filamentous structures have been during evolution.     

Embryonic development of mouse external genitalia: insights into a unique mode of organogenesis

The mammalian external genitalia are specialized appendages for efficient copulation, internal fertilization and display marked morphological variation among species. In this paper, we described the embryonic development of mouse genital tubercle (GT), an anlage of the external genitalia utilizing the scanning electron microscope (SEM) analysis. It has been shown that the Distal Urethral Epithelium (DUE) may fulfill an essential role in the outgrowth control of the GT. Our present SEM analysis revealed a small distal protrusion at the tip of the GT of normal embryos as well as some morphological differences between male and female embryonic external genitalia. Previous analysis shows that the teratogenic dose of Retinoic Acid (RA) induces a drastic marformation of the urethral plate, but not gross abnormalities for GT outgrowth. Interestingly, a small distal protrusion at the tip of GT was clearly observed also after RA treatement. Furthermore, we showed that treatment with anti-androgen flutamide resulted in the demasculinization of the GT in males. The unique character of GT development and the sexual dimorphism are discussed.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo β-oxidation but are first subjected to fatty acid α-oxidation in which the terminal carboxyl-group is released as CO₂. The mechanism of α-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid α-oxidation, and the complete α-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid α-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo beta-oxidation but are first subjected to fatty acid alpha-oxidation in which the terminal carboxyl-group is released as CO(2). The mechanism of alpha-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid alpha-oxidation, and the complete alpha-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid alpha-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

Location, location, location: new insights into O-GalNAc protein glycosylation

O-GalNAc glycosylation of proteins confers essential structural, protective and signaling roles in eumetazoans. Addition of O-glycans onto proteins is an extremely complex process that regulates both sites of attachment and the types of oligosaccharides added. Twenty distinct polypeptide GalNAc-transferases (GalNAc-Ts) initiate O-glycosylation and fine-tuning their expression provides a mechanism for regulating this action. Recently, a new mode of regulation has emerged where activation of Src kinase selectively redistributes Golgi-localized GalNAc-Ts to the ER. This relocalization results in a strong increase in the density of O-glycan decoration. In this review, we discuss how different mechanisms can regulate the number and the types of O-glycans decorating proteins. In addition, we speculate how Src-dependent relocation of GalNAc-Ts could play an important role in cancerous cellular transformation.     

Repeat-protein folding: new insights into origins of cooperativity, stability, and topology

Although our understanding of globular protein folding continues to advance, the irregular tertiary structures and high cooperativity of globular proteins complicates energetic dissection. Recently, proteins with regular, repetitive tertiary structures have been identified that sidestep limitations imposed by globular protein architecture. Here we review recent studies of repeat-protein folding. These studies uniquely advance our understanding of both the energetics and kinetics of protein folding. Equilibrium studies provide detailed maps of local stabilities, access to energy landscapes, insights into cooperativity, determination of nearest-neighbor interaction parameters using statistical thermodynamics, relationships between consensus sequences and repeat-protein stability. Kinetic studies provide insight into the influence of short-range topology on folding rates, the degree to which folding proceeds by parallel (versus localized) pathways, and the factors that select among multiple potential pathways. The recent application of force spectroscopy to repeat-protein unfolding is providing a unique route to test and extend many of these findings.     

From rags to enriched: metagenomic insights into ammonia-oxidizing archaea following ammonia enrichment of a denuded oligotrophic soil ecosystem

Stored topsoil acts as a microbial inoculant for ecological restoration of land after disturbance, but the altered circumstances frequently create unfavourable conditions for microbial survival. Nitrogen cycling is a critical indicator for ecological success and this study aimed to investigate the cornerstone taxa driving the process. Previous in silico studies investigating stored topsoil discovered persistent archaeal taxa with the potential for re-establishing ecological activity. Ammonia oxidization is the limiting step in nitrification and as such, ammonia-oxidizing archaea (AOA) can be considered one of the gatekeepers for the re-establishment of the nitrogen cycle in disturbed soils. Semi-arid soil samples were enriched with ammonium sulfate to promote the selective enrichment of ammonia oxidizers for targeted genomic recovery, and to investigate the microbial response of the microcosm to nitrogen input. Ammonia addition produced an increase in AOA population, particularly within the genus Candidatus Nitrosotalea, from which metagenome-assembled genomes (MAGs) were successfully recovered. The Ca. Nitrosotalea archaeon candidates' ability to survive in extreme conditions and rapidly respond to ammonia input makes it a potential bioprospecting target for application in ecological restoration of semi-arid soils and the recovered MAGs provide a metabolic blueprint for developing potential strategies towards isolation of these acclimated candidates.     

Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution

The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris'', a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris'' genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.     

Genetics of venous thrombosis: insights from a new genome wide association study

Background

Venous Thrombosis (VT) is a common multifactorial disease associated with a major public health burden. Genetics factors are known to contribute to the susceptibility of the disease but how many genes are involved and their contribution to VT risk still remain obscure. We aimed to identify genetic variants associated with VT risk.

Methodology/Principal Findings

We conducted a genome-wide association study (GWAS) based on 551,141 SNPs genotyped in 1,542 cases and 1,110 controls. Twelve SNPs reached the genome-wide significance level of 2.0×10⁻⁸ and encompassed four known VT-associated loci, ABO, F5, F11 and FGG. By means of haplotype analyses, we also provided novel arguments in favor of a role of HIVEP1, PROCR and STAB2, three loci recently hypothesized to participate in the susceptibility to VT. However, no novel VT-associated loci came out of our GWAS. Using a recently proposed statistical methodology, we also showed that common variants could explain about 35% of the genetic variance underlying VT susceptibility among which 3% could be attributable to the main identified VT loci. This analysis additionally suggested that the common variants left to be identified are not uniformly distributed across the genome and that chromosome 20, itself, could contribute to ∼7% of the total genetic variance.

Conclusions/Significance

This study might also provide a valuable source of information to expand our understanding of biological mechanisms regulating quantitative biomarkers for VT.