Genome-wide association meta-analysis of cortical bone mineral density unravels allelic heterogeneity at the RANKL locus and potential pleiotropic effects on bone

Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMD_C) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMD_C, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMD_C associations that had p<1×10⁻⁵ in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMD_C in all cohorts (overall p = 2×10⁻¹⁴, n = 5739). Each minor allele was associated with a decrease in BMD_C of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm³ per C allele, p = 2×10⁻⁶; females −2.79 mg/cm³ per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/RANKL/OPG pathway may be involved in skeletal development.     

Interferon-gamma release assay (modified QuantiFERON) as a potential marker of infection for Leishmania donovani, a proof of concept study

Background

In areas endemic for visceral leishmaniasis (VL), a large number of infected individuals mount a protective cellular immune response and remain asymptomatic carriers. We propose an interferon-gamma release assay (IFN-γRA) as a novel marker for latent L. donovani infection.

Methods and Findings

We modified a commercial kit (QuantiFERON) evaluating five different leishmania-specific antigens; H2B, H2B-PSA2, H2B-Lepp12, crude soluble antigen (CSA) and soluble leishmania antigen (SLA) from L. donovani with the aim to detect the cell-mediated immune response in VL. We evaluated the assay on venous blood samples of active VL patients (n = 13), cured VL patients (n = 15), non-endemic healthy controls (n = 11) and healthy endemic controls (n = 19). The assay based on SLA had a sensitivity of 80% (95% CI = 54.81–92.95) and specificity of 100% (95% CI = 74.12–100).

Conclusion

Our findings suggest that a whole-blood SLA-based QuantiFERON assay can be used to measure the cell-mediated immune response in L. donovani infection. The positive IFN-γ response to stimulation with leishmania antigen in patients with active VL was contradictory to the conventional finding of a non-proliferative antigen-specific response of peripheral blood mononuclear cells and needs further research.     

Changes in body condition of hibernating bats support the thrifty female hypothesis and predict consequences for populations with white-nose syndrome

White-nose syndrome (WNS) is a new disease of bats that has devastated populations in eastern North America. Infection with the fungus, Geomyces destructans, is thought to increase the time bats spend out of torpor during hibernation, leading to starvation. Little is known about hibernation in healthy, free-ranging bats and more data are needed to help predict consequences of WNS. Trade-offs presumably exist between the energetic benefits and physiological/ecological costs of torpor, leading to the prediction that the relative importance of spring energy reserves should affect an individual''s use of torpor and depletion of energy reserves during winter. Myotis lucifugus mate during fall and winter but females do not become pregnant until after spring emergence. Thus, female reproductive success depends on spring fat reserves while male reproductive success does not. Consequently, females should be “thrifty” in their use of fat compared to males. We measured body condition index (BCI; mass/forearm length) of 432 M. lucifugus in Manitoba, Canada during the winter of 2009/2010. Bats were captured during the fall mating period (n = 200), early hibernation (n = 125), and late hibernation (n = 128). Adult females entered hibernation with greater fat reserves and consumed those reserves more slowly than adult males and young of the year. Consequently, adult females may be more likely than males or young of the year to survive the disruption of energy balance associated with WNS, although surviving females may not have sufficient reserves to support reproduction.     

Expensive egos: narcissistic males have higher cortisol

Background

Narcissism is characterized by grandiosity, low empathy, and entitlement. There has been limited research regarding the hormonal correlates of narcissism, despite the potential health implications. This study examined the role of participant narcissism and sex on basal cortisol concentrations in an undergraduate population.

Methods and Findings

Participants were 106 undergraduate students (79 females, 27 males, mean age 20.1 years) from one Midwestern and one Southwestern American university. Narcissism was assessed using the Narcissistic Personality Inventory, and basal cortisol concentrations were collected from saliva samples in a laboratory setting. Regression analyses examined the effect of narcissism and sex on cortisol (log). There were no sex differences in basal cortisol, F(1,97) = .20, p = .65, and narcissism scores, F(1,97) = .00, p = .99. Stepwise linear regression models of sex and narcissism and their interaction predicting cortisol concentrations showed no main effects when including covariates, but a significant interaction, β = .27, p = .04. Narcissism was not related to cortisol in females, but significantly predicted cortisol in males. Examining the effect of unhealthy versus healthy narcissism on cortisol found that unhealthy narcissism was marginally related to cortisol in females, β = .27, p = .06, but significantly predicted higher basal cortisol in males, β = .72, p = .01, even when controlling for potential confounds. No relationship was found between sex, narcissism, or their interaction on self-reported stress.

Conclusions

Our findings suggest that the HPA axis is chronically activated in males with unhealthy narcissism. This constant activation of the HPA axis may have important health implications.     

The spectrum of central nervous system infections in an adult referral hospital in hanoi, Vietnam

Objectives

To determine prospectively the causative pathogens of central nervous system (CNS) infections in patients admitted to a tertiary referral hospital in Hanoi, Vietnam.

Methods

From May 2007 to December 2008, cerebrospinal fluid (CSF) samples from 352 adults with suspected meningitis or encephalitis underwent routine testing, staining (Gram, Ziehl-Nielsen, India ink), bacterial culture and polymerase chain reaction targeting Neisseria meningitidis, Streptococcus pneumoniae, S. suis, Haemophilus influenzae type b, Herpes simplex virus (HSV), Varicella Zoster virus (VZV), enterovirus, and 16S ribosomal RNA. Blood cultures and clinically indicated radiology were also performed. Patients were classified as having confirmed or suspected bacterial (BM), tuberculous (TBM), cryptococcal (CRM), eosinophilic (EOM) meningitis, aseptic encephalitis/meningitis (AEM), neurocysticercosis and others.

Results

352 (male: 66%) patients were recruited: median age 34 years (range 13–85). 95/352 (27.3%) diagnoses were laboratory confirmed and one by cranial radiology: BM (n = 62), TBM (n = 9), AEM (n = 19), CRM (n = 5), and neurocysticercosis (n = 1, cranial radiology). S. suis predominated as the cause of BM [48/62 (77.4%)]; Listeria monocytogenese (n = 1), S. pasteurianus (n = 1) and N. meningitidis (n = 2) were infrequent. AEM viruses were: HSV (n = 12), VZV (n = 5) and enterovirus (n = 2). 5 patients had EOM. Of 262/352 (74.4%) patients with full clinical data, 209 (79.8%) were hospital referrals and 186 (71%) had been on antimicrobials. 21 (8%) patients died: TBM (15.2%), AEM (10%), and BM (2.8%).

Conclusions

Most infections lacked microbiological confirmation. S. suis was the most common cause of BM in this setting. Improved diagnostics are needed for meningoencephalitic syndromes to inform treatment and prevention strategies.     

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10⁻⁸–1.2×10⁻⁴³). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10⁻⁴). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10⁻³, n = 22,044), increased triglycerides (p = 2.6×10⁻¹⁴, n = 93,440), increased waist-to-hip ratio (p = 1.8×10⁻⁵, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10⁻³, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10⁻¹³, n = 96,748) and decreased BMI (p = 1.4×10⁻⁴, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.     

The Guinea-Bissau family of Mycobacterium tuberculosis complex revisited

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048).     

Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates

Background

Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are opportunistic pathogens and serve as a large reservoir of staphylococcal cassette chromosome mec (SCCmec). Characterization of SCCmec in MR-CoNS can generate useful information on the mobilization and evolution of this element.

Methodology/Principal Findings

Non-repetitive MR-CoNS clinical isolates (n = 84; 39 S. epidermidis, 19 S. haemolyticus, 9 S. hominis, 6 S. capitis, 4 S. warneri, 2 S. cohnii, 2 S. saprophyticus, 1 S. kloosii, 1 S. simulans and 1 S. massiliensis) were collected. All isolates could grow on plates with 4 mg/L cefoxitin and all had mecA as detected by PCR. Strain typing using RAPD and ERIC-PCR revealed that almost all isolates were of different strains. SCCmec typing was performed using multiplex PCR published previously. For isolates in which SCCmec could not be typed, the mec complex classes were determined by additional PCR and the ccr genes were amplified with published or newly-designed primers and then sequenced. SCCmec types were assigned for 63 isolates by multiplex PCR and were assigned for 14 other isolates by PCR targeting mec and ccr. Among 77 isolates with determined SCCmec types, 54 had a single type, including type III (n = 19), IV (n = 14), V (n = 10), II (n = 2), I (n = 1), VIII (n = 1) and five unnamed types (n = 7), while 23 isolates had two types, III+V (n = 12), II+V (n = 8), II+IV (n = 2) or IV+V (n = 1). The five unnamed types were assigned UT1 (class A mec, ccrA1/ccrB4), UT2 (class C1 mec, ccrA4/ccrB4), UT3 (class A mec, ccrA5/ccrB3), UT4 (class C2 mec, ccrA2/ccrB2 plus ccrC1) and UT5 (class A mec, ccrA1/ccrB1 plus ccrC1).

Conclusions/Significance

SCCmec types III, IV and V were prevalent in MR-CoNS and many isolates could harbor more than one type. Several new types of SCCmec were identified, highlighting the great genetic diversity and the need of developing classification schemes for SCCmec in MR-CoNS.     

Multi-Locus Variable Number of Tandem Repeat Analysis for Rapid and Accurate Typing of Virulent Multidrug Resistant Escherichia coli Clones

One hundred E. coli isolates from Norway (n = 37), Sweden (n = 24), UK (n = 20) and Spain (n = 19), producing CTX-M-type - (n = 84), or SHV-12 (n = 4) extended spectrum β-lactamases, or the plasmid mediated AmpC, CMY-2 (n = 12), were typed using multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes (MTCs). A strong agreement between the MLST profile and MLVA typing results was observed, in which all ST131-isolates (n = 39) and most of the STC-648 (n = 10), STC-38 (n = 9), STC-10 (n = 9), STC-405 (n = 8) and STC-23 (n = 6) isolates were clustered distinctly into MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate tool for genotyping isolates of globally disseminated virulent multidrug resistant E. coli lineages, including ST131.     

Hepatitis C virus in Vietnam: high prevalence of infection in dialysis and multi-transfused patients involving diverse and novel virus variants

Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2–3% of the world''s population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n = 8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n = 475) and genotype (n = 282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n = 26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n = 556/1000), dialysis patients (26.6%, n = 153/575) commercial sex workers (CSWs; 8.7%, n = 87/1000), and recipients of multiple blood transfusions (6.0%, n = 32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11–43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85–2.34), p = 0.001], time from first transfusion [OR: 1.07 (1.01–1.13), p = 0.023], duration of dialysis [OR: 1.31 (1.19–1.43), p<0.001] and male gender [OR: 1.60 (1.06–2.41), p = 0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1%). HCV genotype 1 predominated in Vietnam (60%, n = 169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion.     

Clinical, biological and genetic analysis of anorchia in 26 boys

Background

Anorchia is defined as the absence of testes in a 46,XY individual with a male phenotype. The cause is unknown.

Methods

We evaluated the clinical and biological presentation, and family histories of 26 boys with anorchia, and sequenced their SRY, NR5A1, INSL3, MAMLD1 genes and the T222P variant for LGR8.

Results

No patient had any associated congenital anomaly. At birth, testes were palpable bilaterally or unilaterally in 13 cases and not in 7; one patient presented with bilateral testicular torsion immediately after birth. The basal plasma concentrations of anti-Müllerian hormone (AMH, n = 15), inhibin B (n = 7) and testosterone (n = 19) were very low or undetectable in all the patients evaluated, as were the increases in testosterone after human chorionic gonadotropin (hCG, n = 12). The basal plasma concentrations of follicle stimulating hormone (FSH) were increased in 20/25, as was that of luteinising hormone in 10/22 cases. Family members of 7/26 cases had histories of primary ovarian failure in the mother (n = 2), or sister 46,XX, together with fetal malformations of the only boy with microphallus and secondary foot edema (n = 1), secondary infertility in the father (n = 2), or cryptorchidism in first cousins (n = 2). The sequences of all the genes studied were normal.

Conclusion

Undetectable plasma concentrations of AMH and inhibin B and an elevated plasma FSH, together with 46,XY complement are sufficient for diagnosis of anorchia. The hCG test is unnecessary. NR5A1 and other genes implicated in gonadal development and testicle descent were not mutated, which suggests that other genes involved in these developments contribute to the phenotypes.     

Elevated peripheral visfatin levels in narcoleptic patients

Objective

Narcolepsy is a severe sleep disorder that is characterized by excessive daytime sleepiness, cataplexies and a tendency towards obesity. Recent discoveries indicate that the major pathophysiology is a loss of hypocretin (orexin) producing neurons due to immunologically mediated degeneration. Visfatin is a recently described proinflammatory adipokine. It is identical to the immune modulating pre-B-cell colony enhancing factor (PBEF). Our study examines the hypothesis that visfatin levels are altered in narcoleptic patients.

Methods

For the analysis, a total of n = 54 patients (n = 18 males and n = 36 females) with the diagnosis of narcolepsy according to DSM-IV and the International Classification of Sleep Disorders were examined (BMI mean 30.3±5.5, age mean 52.5±16.1 years). As a control group 39 unrelated (n = 12 males and n = 27 females) healthy volunteers with no sleep disorder according to DSM-IV were included (BMI mean 28.5±4.6, age mean 51.1±13.6 years). Peripheral visfatin levels were measured using a commercial enzyme immunoassay kit with a measurement range from 0.1–1000 ng/ml. Narcolepsy symptoms, severity and frequency of symptoms as well as the total duration of various aspects of the symptomatology were assessed by unstructured and structured clinical interviews in including the Stanford Center for Narcolepsy Sleep Inventory.

Results

Circulating visfatin was found to be significantly increased in HLA DR2 positive narcoleptic patients compared to controls.

Conclusion

Taken together, our results add to the evidence of disturbed immunological regulation in patients with narcolepsy.     

Despotism and risk of infanticide influence grizzly bear den-site selection

Given documented social dominance and intraspecific predation in bear populations, the ideal despotic distribution model and sex hypothesis of sexual segregation predict adult female grizzly bears (Ursus arctos) will avoid areas occupied by adult males to reduce risk of infanticide. Under ideal despotic distribution, juveniles should similarly avoid adult males to reduce predation risk. Den-site selection and use is an important component of grizzly bear ecology and may be influenced by multiple factors, including risk from conspecifics. To test the role of predation risk and the sex hypothesis of sexual segregation, we compared adult female (n = 142), adult male (n = 36), and juvenile (n = 35) den locations in Denali National Park and Preserve, Alaska, USA. We measured elevation, aspect, slope, and dominant land cover for each den site, and used maximum entropy modeling to determine which variables best predicted den sites. We identified the global model as the best-fitting model for adult female (area under curve (AUC) = 0.926) and elevation as the best predictive variable for adult male (AUC = 0.880) den sites. The model containing land cover and elevation best-predicted juvenile (AUC = 0.841) den sites. Adult females spatially segregated from adult males, with dens characterized by higher elevations ( = 1,412 m, SE = 52) and steeper slopes ( = 21.9°, SE = 1.1) than adult male (elevation:  = 1,209 m, SE = 76; slope:  = 15.6°, SE = 1.9) den sites. Juveniles used a broad range of landscape attributes but did not avoid adult male denning areas. Observed spatial segregation by adult females supports the sex hypothesis of sexual segregation and we suggest is a mechanism to reduce risk of infanticide. Den site selection of adult males is likely related to distribution of food resources during spring.     

Alcohol consumption, genetic variants in alcohol deydrogenases, and risk of cardiovascular diseases: a prospective study and meta-analysis

Objective

First, to investigate and compare associations between alcohol consumption and variants in alcohol dehydrogenase (ADH) genes with incidence of cardiovascular diseases (CVD) in a large German cohort. Second, to quantitatively summarize available evidence of prospective studies on polymorphisms in ADH1B and ADH1C and CVD-risk.

Methods

We conducted a case-cohort study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort including a randomly drawn subcohort (n = 2175) and incident cases of myocardial infarction (MI; n = 230) or stroke (n = 208). Mean follow-up time was 8.2±2.2 years. The association between alcohol consumption, ADH1B or ADH1C genotypes, and CVD-risk was assessed using Cox proportional hazards regression. Additionally, we report results on associations of variants in ADH1B and ADH1C with ischemic heart disease and stroke in the context of a meta-analysis of previously published prospective studies published up to November 2011.

Results

Compared to individuals who drank >0 to 6 g alcohol/d, we observed a reduced risk of MI among females consuming >12 g alcohol/d (HR = 0.31; 95% CI: 0.10–0.97) and among males consuming >24 to 60 g/d (HR = 0.57; 95% CI: 0.33–0.98) or >60 g alcohol/d (HR = 0.30; 95% CI: 0.12–0.78). Stroke risk was not significantly related to alcohol consumption >6 g/d, but we observed an increased risk of stroke in men reporting no alcohol consumption. Individuals with the slow-coding ADH1B*1/1 genotype reported higher median alcohol consumption. Yet, polymorphisms in ADH1B or ADH1C were not significantly associated with risk of CVD in our data and after pooling results of eligible prospective studies [ADH1B*1/1: RR = 1.35 (95% CI: 0.98–1.88; p for heterogeneity: 0.364); ADH1C*2/2: RR = 1.07 (95% CI: 0.90–1.27; p for heterogeneity: 0.098)].

Conclusion

The well described association between alcohol consumption and CVD-risk is not reflected by ADH polymorphisms, which modify the rate of ethanol oxidation.     

Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptide-microarray

Background

Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals.

Methodology

A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100).

Findings

The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II–III, type I–III or type I–II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers.

Conclusions

Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution.     

Genetic determinants of serum testosterone concentrations in men

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone''s high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10⁻⁴¹ and rs6258, p = 2.3×10⁻²²). Subjects with ≥3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10⁻¹⁶). The rs6258 polymorphism in exon 4 of SHBG affected SHBG''s affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.     

Genetic association study identifies HSPB7 as a risk gene for idiopathic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06×10⁻⁶, OR = 0.67 [95% CI 0.57–0.79] for the minor allele T). Three more SNPs showed p < 2.21×10⁻⁵. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n = 564, n = 981 controls, p = 2.07×10⁻³, OR = 0.79 [95% CI 0.67–0.92]), France 1 (n = 433 cases, n = 395 controls, p = 3.73×10⁻³, OR = 0.74 [95% CI 0.60–0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26×10⁻⁴, OR = 0.63 [95% CI 0.50–0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28×10⁻¹³, OR = 0.72 [95% CI 0.65–0.78]). None of the other three SNPs showed significant results in the replication stage.This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.     

Mycobacterium tuberculosis Population in Northwestern Russia: An Update from Russian-EU/Latvian Border Region

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA −15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia.     

Identifying insects with incomplete DNA barcode libraries, African fruit flies (Diptera: Tephritidae) as a test case

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods.     

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.