Interleukin-4 Gene Polymorphisms in Romanian Patients with Inflammatory Bowel Diseases: Association with Disease Risk and Clinical Features.

1. INTRODUCTION: Multiple cytokines have been studied for their role in the propagation of the inflammatory process related to inflammatory bowel diseases (IBD), but the role of interleukin-4 remains controversial. The aim of this study was to evaluate the role of two IL-4 gene single nucleotide polymorphisms (SNPs) in disease susceptibility and phenotypic expression. 2. MATERIALS AND METHODS: A group of 160 patients with IBD (86CD/74UC) and 160 healthy controls were genotyped for IL-4 rs2243250/-590C/T and rs2070874/-34C/T using real-time polymerase chain reaction with TaqMan assay. 3. RESULTS: The analysis of IBD patients and controls revealed a significantly reduced frequency of the minor allele T of both SNPs in CD patients (p = 0.03, OR 0.55 and p = 0.02, OR 0.52) and for the entire IBD group (p = 0.01, OR 0.57 and p = 0.01, OR 0.55). Haplotype analysis identified the most frequent haplotype (rs2243250/rs2070874 CC) associated with a high risk for developing IBD (either UC or CD) (p = 0.003). IBD patients with extraintestinal manifestations had significantly increased frequency of the minor alleles T. We also found an association between the presence of allele C of rs2070874 and response to antiTNF treatment. 4. CONCLUSIONS: This is the first study to investigate the IL-4 gene's relation to IBD susceptibility conducted in Romania. Both SNPs were found to be associated with disease susceptibility and phenotypic features, such as extraintestinal manifestations and response to antiTNF agents.

Decline of unique Pontocaspian biodiversity in the Black Sea Basin: A review.

The unique aquatic Pontocaspian (PC) biota of the Black Sea Basin (BSB) is in decline. The lack of detailed knowledge on the status and trends of species, populations, and communities hampers a thorough risk assessment and precludes effective conservation. This paper reviews PC biodiversity trends in the BSB (Bulgaria, Romania, Moldova, Ukraine, and Russia) using endemic mollusks as a model group. We aim to assess changes in PC habitats, community structure, and species distribution over the past century and to identify direct anthropogenic threats. The presence/absence data of target mollusk species were assembled from literature, reports, and personal observations. Pontocaspian biodiversity trends in the northwestern BSB coastal regions were established by comparing 20th- and 21st-century occurrences. The direct drivers of habitat and biodiversity change were identified and documented. We found that a pronounced decline of PC species and communities is driven by (a) damming of rivers, (b) habitat modifications that disturbed previous natural salinity gradients and settings in the studied area, (c) pollution and eutrophication, (d) invasive alien species, and (e) climate change. Four out of the 10 studied regions, namely, the Danube Delta-Razim Lake system, Dniester Liman, Dnieper-Bug estuary, and Taganrog Bay-Don Delta, contain favorable ecological conditions for PC communities and still host threatened endemic PC mollusk species. Distribution data are incomplete, but the scale of deterioration of PC species and communities is evident from the assembled data, as are major direct threats. Pontocaspian biodiversity in the BSB is profoundly affected by human activities. Standardized observation and collection data as well as precise definition of PC biota and habitats are necessary for targeted conservation actions. This study will help to set the research and policy agenda required to improve data collection to accommodate effective conservation of the unique PC biota.

Unravelling the Extent of Diversity within the Iberian Medicinal Leeches (Hirudinea: Hirudo) Using Molecules and Morphology.

Until the beginning of the 21st century, the famous medicinal leech was thought to be represented by only one species, Hirudo medicinalis. However, recent publications have demonstrated that under that name, at least five different species of medicinal leeches were hidden. During the last decade, the biogeography of Western-Palaearctic leeches has begun to unravel, untangling their diversity in practically all of Europe, except for its westernmost peninsula, Iberia. Hirudo medicinalis has been repeatedly reported from Iberia, but those records were considered questionable. We discovered H. verbana in northern Spain, constituting its first record in Iberia. Using an integrative approach (combining morpho-anatomical data and molecular analyses using three genes, COI,12S rRNA, and ITS2), two endemic and geographically separated Iberian lineages have been found. One of them is easily distinguished by its distinctive colour-pattern and is described as H. verbana bilineata ssp. nov. We characterized the new subspecies morphologically, ecologically, and genetically. We also established its phylogenetic relationships with other European Hirudo spp. and confirm the presence of H. troctina in Iberia, occurring as far as 43° lat. N. Iberian H. verbana records constitute its westernmost known distribution to date. The provided distribution pattern of H. verbana contributes to a better understanding of the complexity of Iberia as a glacial refugium/cradle for endemisms, harbouring populations with a high degree of genetic structure that began to settle throughout the Pleistocene. Iberian Hirudo populations are declining in recent decades and there is an urgent need to assess their conservation status and to initiate conservation measures to reverse their decline.

The future of endangered crayfish in light of protected areas and habitat fragmentation.

The long-term survival of a species requires, among other things, gene flow between populations. Approaches for the evaluation of fragmentation in the frame of freshwater habitats consider only a small amount of the information that combined demography and geography are currently able to provide. This study addresses two species of Austropotamobius crayfish in the light of population genetics, spatial ecology and protected areas of the Carpathians. Advancing the classical approaches, we defined ecological distances upon the rasterised river network as a surrogate of habitat resistance to migration, quantifying the deviations from the species´ suitability range for a set of relevant geospatial variables in each cell of the network. Molecular analyses revealed the populations of the two Austropotamobius crayfish species are clearly distinct, lacking hybridisation. Comparing pairs of populations, we found, in some cases, a strong disagreement regarding genetic and ecological distances, potentially due to human-mediated translocations or the geophysical phenomena of regressive erosion, which may have led to unexpected colonisation routes. Protected areas were found to offer appropriate local habitat conditions but failed to ensure connectivity. The methodology applied in this study allowed us to quantify the contribution of each geospatial (environmental) variable to the overall effect of fragmentation, and we found that water quality was the most important variable. A multilevel approach proved to reveal a better understanding of drivers behind the distribution patterns, which can lead to more adequate conservation measures.

Mollusc species from the Pontocaspian region - an expert opinion list.

Defining and recording the loss of species diversity is a daunting task, especially if identities of species under threat are not fully resolved. An example is the Pontocaspian biota. The mostly endemic invertebrate faunas that evolved in the Black Sea - Caspian Sea - Aral Sea region and live under variable salinity conditions are undergoing strong change, yet within several groups species boundaries are not well established. Collection efforts in the past decade have failed to produce living material of various species groups whose taxonomic status is unclear. This lack of data precludes an integrated taxonomic assessment to clarify species identities and estimate species richness of Pontocaspian biota combining morphological, ecological, genetic, and distribution data. In this paper, we present an expert-working list of Pontocaspian and invasive mollusc species associated to Pontocaspian habitats. This list is based on published and unpublished data on morphology, ecology, anatomy, and molecular biology. It allows us to (1) document Pontocaspian mollusc species, (2) make species richness estimates, and (3) identify and discuss taxonomic uncertainties. The endemic Pontocaspian mollusc species richness is estimated between 55 and 99 species, but there are several groups that may harbour cryptic species. Even though the conservation status of most of the species is not assessed or data deficient, our observations point to deterioration for many of the Pontocaspian species.

Modelling the invasion history of Sinanodonta woodiana in Europe: Tracking the routes of a sedentary aquatic invader with mobile parasitic larvae.

Understanding the invasive potential of species outside their native range is one of the most pressing questions in applied evolutionary and ecological research. Admixture of genotypes of invasive species from multiple sources has been implicated in successful invasions, by generating novel genetic combinations that facilitate rapid adaptation to new environments. Alternatively, adaptive evolution on standing genetic variation, exposed by phenotypic plasticity and selected by genetic accommodation, can facilitate invasion success. We investigated the population genetic structure of an Asian freshwater mussel with a parasitic dispersal stage, Sinanodonta woodiana, which has been present in Europe since 1979 but which has expanded rapidly in the last decade. Data from a mitochondrial marker and nuclear microsatellites have suggested that all European populations of S. woodiana originate from the River Yangtze basin in China. Only a single haplotype was detected in Europe, in contrast to substantial mitochondrial diversity in native Asian populations. Analysis of microsatellite markers indicated intensive gene flow and confirmed a lower genetic diversity of European populations compared to those from the Yangtze basin, though that difference was not large. Using an Approximate Bayesian Modelling approach, we identified two areas as the probable source of the spread of S. woodiana in Europe, which matched historical records for its establishment. Their populations originated from a single colonization event. Our data do not support alternative explanations for the rapid recent spread of S. woodiana; recent arrival of a novel (cold-tolerant) genotype or continuous propagule pressure. Instead, in situ adaptation, facilitated by repeated admixture, appears to drive the ongoing expansion of S. woodiana. We discuss management consequences of our results.

A new feather mite species of the genus Trouessartia Canestrini, 1899 (Acarina, Trouessartiidae) - an integrative description (morphology and DNA barcoding data).

A new species of the feather mite genus Trouessartia (Trouessartiidae) is described from the Large NiltavaNiltavagrandis (Blyth) (Passeriformes, Muscicapidae) in Northeast India (Meghalaya, Jaintia Hills, Shnongrim village). Trouessartianiltavae Constantinescu, sp. n. is morphologically closely related (no phylogenetic meaning) to T.bulligera Gaud, 1968 from Clytorhynchushamlini (Mayr) (Passeriformes: Monarchidae), sharing in males a unique character within the genus, by having setae e on legs IV hemispheroid, with spine-shaped apex. Males of the new species have the prodorsal shield without ornamentation, the prohysteronotal shield and lobar shield connected, and the terminal cleft parallel sided. Females have the posterior half of the hysteronotal shield ornamented with large ovate lacunae in central area and small elliptical lacunae marginally. To the morphological description of this new feather mite species we added sequence data on the mitochondrial cytochrome c oxidase subunit I gene fragment (COI). The phylogenetic relationships between Trouessartia species are briefly discussed.

A genetic portrait of the South-Eastern Carpathians based on autosomal short tandem repeats loci used in forensics.

OBJECTIVES: This work aimed to describe the genetic landscape of the Balkan Peninsula, as revealed by STR markers commonly used in forensics and spatial methods specifically developed for genetic data. METHODS: We generated and analyzed 16 short tandem repeats (STRs) autosomal genotypes in 287 subjects from ten administrative/geographical regions of Eastern Europe (Romania and the Republic of Moldova). We report estimates of the allele frequencies in these sub-populations, their fixation indexes, and use these results to complement previous spatial analyses of Southern Europe. RESULTS: In seven out of ten analyzed regional samples the heterozygosity, averaged across loci, was lower than expected. The average Fis was 0.011. Among the 16 loci, five returned a significant fixation index Fst. The composite Fst across the 16 loci, among the 10 regional samples, was 0.00417, a figure twice as large as that obtained with the same markers across the entire Northern Mediterranean. The first spatial principal component (sPC1) returned the picture of a Central-European pattern of frequencies for the Carpathians, which extended to the Southern boundary of the Balkan Peninsula. However, the 8 alleles extracted by sPC1 returned a picture of a strong reduction of the migration rate in the Carpathian region, mostly between the inner locations. CONCLUSIONS: Our results revealed an unexpected heterogeneity in the area. We believe that populations from some regions will require treatment as distinct entities when considered in forensic applications.

A new feather mite species of the genus Proterothrix Gaud, 1968 (Acarina, Proctophyllodidae) from the Large Niltava, Niltava grandis (Passeriformes, Muscicapidae) - an integrative description.

A new species of the feather mite genus Proterothrix (Proctophyllodidae: Pterodectinae) is described from the Large Niltava Niltava grandis (Blyth) (Passeriformes, Muscicapidae) in northeast India (Meghalaya, Jaintia Hills, Shnongrim village). Proterothrix chachulae Constantinescu, sp. n. differs from all known species of the genus by having in males the aedeagus with bilobate tip. The morphological description is supplemented with molecular characterisation of a fragment f near the 5` terminus of the mitochondrial COI gene.

ERAP1 and ERAP2 Gene Variations Influence the Risk of Psoriatic Arthritis in Romanian Population.

Psoriatic arthritis (PsA) is a chronic inflammatory disorder that belongs to the group of spondyloarthritis (SpA). It was found that single nucleotide polymorphisms (SNPs) of endoplasmic reticulum aminopeptidase (ERAP1 and ERAP2) genes influence the risk of ankylosing spondylitis, the most common form of SpA and the risk of psoriasis. Our purpose was to investigate the possible association of ERAP1 and ERAP2 gene SNPs with psoriatic arthritis susceptibility in Romanian population. Subsequent analyses included patients' subgroups according to HLA-B27 status. Psoriatic arthritis patients (N = 98) and random healthy controls (N = 139) were genotyped for ERAP1/2 genes SNPs rs30187, rs27044, rs2910686, and rs2248374 by TaqMan Allelic Discrimination Assays. An additional control group (N = 108; 100% HLA-B27 positive) was used for subsequent analyses. The results showed the association of rs2248374 SNP of ERAP2 gene with the risk of PsA, especially for HLA-B27 negative disease (p = 0.02; OR 1.59). ERAP2 haplotype GT (rs2248374/rs2910686) was significantly under-represented in PsA patients than in controls (43 vs. 55%; p = 0.02). The analysis of ERAP1 SNPs in HLA-B27 positive controls and PsA subgroup showed strong evidence of association for rs30187 (p = 0.005; OR 2.73) and for CC rs30187/rs27044 haplotype (47% in patients vs. 70.5% in controls; p = 0.006). In conclusion, we found a significant association of ERAP2 with PsA and HLA-B27 negative PsA, while ERAP1 association was restricted only to HLA-B27 positive disease. To our knowledge, this is the first study that investigated ERAP2 polymorphisms in relation to PsA susceptibility.

Association of Leukotriene C4 Synthase A-444C Polymorphism with Asthma and Asthma Phenotypes in Romanian Population.

INTRODUCTION: Leukotriene C4 synthase (LTC4S) gene -444A/C polymorphism has been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. The aim of this study was to investigate the association between the -444A/C polymorphism of LTC4S gene and asthma, asthma phenotypes (aspirin intolerant/tolerant asthma) and different characteristics of the patients. MATERIAL AND METHODS: We included 106 patients with asthma (60 with aspirin tolerant asthma - ATA, 46 with aspirin intolerant asthma - AIA) and 103 controls. All the subjects were genotyped for LTC4S-444 A/C by Real-Time PCR. We assessed the association of LTC4S promoter polymorphism with asthma and its phenotypes and with clinical and biological characteristics of asthmatic patients. RESULTS: We did not find a significant association between the studied polymorphism and asthma, but the minor allele tended to be more frequent in AIA patients. We found a significant association between the minor allele C and lower levels of serum total immunoglobulin E and eosinophils, suggesting a possible role of -444A/C LTC4S polymorphism as modulating factor of allergic inflammation in asthma. CONCLUSION: The results show that LTC4S -444A/C SNP is not associated with susceptibility to asthma in Romanian patients, but could influence asthma phenotype, namely aspirin intolerant asthma.

Fine-scale population genetics analysis of Platynereis dumerilii (Polychaeta, Nereididae) in the Black Sea: how do local marine currents drive geographical differentiation?

In this study we analyzed at a submeso-geographic scale (2 km) the genetic diversity of two sub-populations of Platynereis dumerilii and correlated this with the physical characteristics of the marine currents along the western Black Sea coast. For this purpose, we developed a set of 13 new polymorphic microsatellite markers and used them to assess the genetic differentiation, as well as the bi-directional migration rates between the studied sub-populations. We also computed the Peclet number (Pe) as an indicator of the relative effect of advection and eddy diffusion on larval dispersion for the specific conditions of the Black Sea study area. The results indicated no genetic structure in P. dumerilii sub-populations which indicates that the longitudinal alongshore currents dominate in the population structuring of this species. This finding is important, because with the average current speeds of 5 cm/sec on the Black Sea coast during May-August we might have expected a certain population structuring to occur. In accordance with the periodical change of direction of the longitudinal current (either form North to South, or form South to North) the gene flow was found to be bi-directional, with the same intensity.

Isolation and characterization of microsatellite loci for Stys's bush-cricket, Isophya stysi, and cross-species amplification in closely related species from the Phaneropteridae family.

Ten microsatellite loci were isolated and characterized for Stys's bush-cricket, Isophya stysi Cejchan (Orthoptera: Tettigoniidae), an endemic Orthoptera species to the Carpathian Basin, using an enriched genomic library procedure. The polymorphism of these loci were tested in two populations of I. stysi, and the number of alleles per locus varied from 4 to 16. The expected and observed heterozygosities ranged from 0.612 to 0.925 and from 0.625 to 1.000, respectively. The interspecific applicability of these microsatellites was evaluated by amplification in 20 related species: Isophya camptoxypha, Isophya sicula, Isophya ciucasi, Isophya pienensis, Isophya harzi, Isophya kraussii, Isophya zubovskii, Isophya rectipennis, Isophya modesta, Isophya longicaudata, Isophya dobrogensis, Isophya hospodar, Isophya speciosa, Isophya modestior, Poecilimon fussii, Poecilimon affinis, Polysarcus denticauda, Barbitistes constrictus, Leptophyes discoidalis, Phaneroptera falcata. All primer pairs for the 10 loci yielded successful amplifications in at least one other taxon from the Isophya genus. This set of microsatellite loci would be useful for genetic studies in I. stysi and other species of the genus Isophya.

Association study in Romanians confirms IL23A gene haplotype block rs2066808/rs11171806 as conferring risk to psoriatic arthritis.

BACKGROUND: The cytokines IL12 and IL23 have been recently implicated in the pathogenesis of psoriatic arthritis (PsA). In this study we investigated the genetic variations in the genes coding for IL12, IL23 and IL23 receptor as a plausible source of susceptibility and modification of clinical symptoms of PsA in Romanian population. METHODS: Twenty five SNPs mapping to IL12A, IL12B, IL23A, IL23R and IL12RB1 genes were genotyped in 94 PsA patients and 161 healthy controls of Romanian ethnicity using the Sequenom genotyping platform. RESULTS: The exonic SNP rs11171806 from IL23A gene was significantly underrepresented in patients versus controls (p=0.03, OR 0.391) and the carriers of rs11171806/rs2066808 AC haplotype had decreased risk for PsA (p=0.03). The two SNPs of the highly conserved gene IL23A are in complete LD in our population. Genetic variants of IL12B gene were associated with polyarticular subtype of PsA. No associations were found between SNPs from IL12A, IL23R and IL12RB1 genes and susceptibility to PsA and its phenotypes. CONCLUSION: We confirm the previously described association of rs2066808 variant with psoriasis and PsA and we show evidence of an extended genomic region inside IL23A gene as carrier of true disease susceptibility factors. These data suggest a role for IL23 in the PsA pathogenesis in Romanians.

Isolation and characterization of new microsatellite markers for the invasive softshell clam, Mya arenaria (L.) (Bivalvia: Myidae).

The invasive softshell clam (Mya arenaria Linnaeus, 1758) is native to the northwestern region of the Atlantic Ocean. This species has been introduced in the northeast Pacific and along the European coasts, due to intense naval transports and aquaculture, and it is now present in all the European seas. In this paper we describe seven new microsatellite loci for Mya arenaria. The isolated loci are polymorphic with a number of alleles per locus between 6 and 14. The observed and expected heterozygosities ranged from 0.417 to 0.951, and from 0.643 to 0.895, with an average of 0.716 and 0.775, respectively. These microsatellite markers should be useful in analyzing this species' genetic diversity, which could explain various processes of its invasion history.

A pilot study of the association of tumor necrosis factor alpha polymorphisms with psoriatic arthritis in the Romanian population.

Tumor necrosis factor alpha (TNF-alpha) is an important pro-inflammatory cytokine implicated in the pathogenesis of psoriatic arthritis. We have performed a case-control association study of three TNF-alpha gene polymorphisms in a group of Romanian psoriatic arthritis patients versus ethnically matched controls. A second group of patients with undifferentiated spondyloarthritis was used in order to look for similarities in the genetic background of the two rheumatic disorders. The -857C/T polymorphism was associated with susceptibility to psoriatic arthritis in our population at the individual level (p = 0.03, OR 1.65, 95% CI 1.05-2.57) and in combined haplotypes with the -238G/A and -308G/A SNPs. Regarding the investigated polymorphisms and derived haplotypes, no potential association was found with the susceptibility to undifferentiated spondyloarthritis in Romanian patients.

Sinanodonta woodiana (Mollusca: Bivalvia: Unionidae): isolation and characterization of the first microsatellite markers.

Sinanodonta woodiana (Lea, 1834) is a large Unionid species with a real invasion success. It colonized Europe, Central America, the Indonesian Islands and recently North America. The species life cycle involves a larval parasitic stage on freshwater fish species which contributes to the spread of the mussel. In this paper we describe, for the first time, eight polymorphic microsatellite loci for the species Sinanodonta woodiana. The genetic screening of individuals confirmed that all loci were highly polymorphic. The number of alleles per locus ranged from 7 to 14 and the observed heterozygosity ranged from 0.650 to 0.950. These loci should prove useful to study the species population genetics which could help to infer important aspects of the invasion process.

Isolation and characterization of the first microsatellite markers for the endangered relict mussel Hypanis colorata (Mollusca: Bivalvia: Cardiidae).

Hypanis colorata (Eichwald, 1829) (Cardiidae: Lymnocardiinae) is a bivalve relict species with a Ponto-Caspian distribution and is under strict protection in Romania, according to national regulations. While the species is depressed in the western Black Sea lagoons from Romania and Ukraine, it is also a successful invader in the middle Dniepr and Volga regions. Establishing a conservation strategy for this species or studying its invasion process requires knowledge about the genetic structure of the species populations. We have isolated and characterized nine polymorphic microsatellite markers in H. colorata. The number of alleles per locus ranged from 4 to 28 and the observed heterozygosity ranged from 0.613 to 1.000. The microsatellites developed in the present study are highly polymorphic and they should be useful for the assessment of genetic variation within this species.

A deletion of a novel heat shock gene on the Y chromosome associated with azoospermia.

Deletions of the Y chromosome are a significant cause of spermatogenic failure. Three major deletion intervals have been defined and termed AZFa, AZFb and AZFc. Here, we report an unusual case of a proximal AZFb deletion that includes the Y chromosome palindromic sequence P4 and a novel heat shock factor (HSFY). This deletion neither include the genes EIF1AY, RPS4Y2 nor copies of the RBMY1 genes. The individual presented with idiopathic azoospermia. We propose that deletions of the testis-specific HSFY gene family may be a cause of unexplained cases of idiopathic male infertility. This deletion would not have been detected using current protocols for Y chromosome microdeletion screens, therefore we recommend that current screening protocols be extended to include this region and other palindrome sequences that contain genes expressed specifically in the testis.