Structured RNAs and synteny regions in the pig genome

Background

Annotating mammalian genomes for noncoding RNAs (ncRNAs) is nontrivial since far from all ncRNAs are known and the computational models are resource demanding. Currently, the human genome holds the best mammalian ncRNA annotation, a result of numerous efforts by several groups. However, a more direct strategy is desired for the increasing number of sequenced mammalian genomes of which some, such as the pig, are relevant as disease models and production animals.

Results

We present a comprehensive annotation of structured RNAs in the pig genome. Combining sequence and structure similarity search as well as class specific methods, we obtained a conservative set with a total of 3,391 structured RNA loci of which 1,011 and 2,314, respectively, hold strong sequence and structure similarity to structured RNAs in existing databases. The RNA loci cover 139 cis-regulatory element loci, 58 lncRNA loci, 11 conflicts of annotation, and 3,183 ncRNA genes. The ncRNA genes comprise 359 miRNAs, 8 ribozymes, 185 rRNAs, 638 snoRNAs, 1,030 snRNAs, 810 tRNAs and 153 ncRNA genes not belonging to the here fore mentioned classes. When running the pipeline on a local shuffled version of the genome, we obtained no matches at the highest confidence level. Additional analysis of RNA-seq data from a pooled library from 10 different pig tissues added another 165 miRNA loci, yielding an overall annotation of 3,556 structured RNA loci. This annotation represents our best effort at making an automated annotation. To further enhance the reliability, 571 of the 3,556 structured RNAs were manually curated by methods depending on the RNA class while 1,581 were declared as pseudogenes. We further created a multiple alignment of pig against 20 representative vertebrates, from which RNAz predicted 83,859 de novo RNA loci with conserved RNA structures. 528 of the RNAz predictions overlapped with the homology based annotation or novel miRNAs. We further present a substantial synteny analysis which includes 1,004 lineage specific de novo RNA loci and 4 ncRNA loci in the known annotation specific for Laurasiatheria (pig, cow, dolphin, horse, cat, dog, hedgehog).

Conclusions

We have obtained one of the most comprehensive annotations for structured ncRNAs of a mammalian genome, which is likely to play central roles in both health modelling and production. The core annotation is available in Ensembl 70 and the complete annotation is available at http://rth.dk/resources/rnannotator/susscr102/version1.02.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-459) contains supplementary material, which is available to authorized users.     

CRP genotype and haplotype associations with serum C-reactive protein level and DAS28 in untreated early rheumatoid arthritis patients

Introduction

Single-nucleotide polymorphisms (SNPs) in the CRP gene are implicated in the regulation of the constitutional C-reactive protein (CRP) expression and its response to proinflammatory stimuli. Previous reports suggest that these effects may have an impact on clinical decision-making tools based on CRP, such as the Disease Activity Score in 28 joints (DAS28). We aimed to investigate the possible association between seven CRP SNPs, their haplotypes and the serum levels of CRP, as well as DAS28 scores, in two cohorts of untreated active early rheumatoid arthritis (RA) patients followed during their initial treatment.

Methods

Overall, 315 patients with RA from two randomized controlled trials (the CIMESTRA and OPERA trials) who were naïve to disease-modifying antirheumatic drugs and steroids with disease durations less than 6 months were included. Seven CRP SNPs were investigated: rs11265257, rs1130864, rs1205, rs1800947, rs2808632, rs3093077 and rs876538. The genotype and haplotype associations with CRP and DAS28 levels were evaluated using linear regression analysis adjusted for age, sex and treatment.

Results

The minor allele of rs1205 C > T was associated with decreased CRP levels at baseline (P = 0.03), with the TT genotype having a 50% reduction in CRP from 16.7 to 8.4 mg/L (P = 0.005) compared to homozygosity of the major allele, but no association was observed at year 1 (P = 0.38). The common H2 haplotype, characterized by the T allele of rs1205, was associated with a 26% reduction in CRP at baseline (P = 0.043), although no effect was observed at year 1 (P = 0.466). No other SNP or haplotype was associated with CRP at baseline or at year 1 (P ≥0.09). We observed no associations between SNPs or haplotypes and DAS28 scores at baseline or at year 1 (P ≥0.10).

Conclusion

CRP genotype and haplotype were only marginally associated with serum CRP levels and had no association with the DAS28 score. This study shows that DAS28, the core parameter for inflammatory activity in RA, can be used for clinical decision-making without adjustment for CRP gene variants.

Trial registration

The OPERA study is registered at Clinicaltrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT00660647","term_id":"NCT00660647"}}NCT00660647). The CIMESTRA study is not listed in a clinical trials registry, because patients were included between October 1999 and October 2002.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0475-3) contains supplementary material, which is available to authorized users.     

Maternal Glomerular Filtration Rate in Pregnancy and Fetal Size

BackgroundThe relationship of maternal glomerular filtration rate (GFR) in pregnancy to fetal size needs to be better characterized as it impacts an ongoing debate about confounding effect of maternal GFR in investigations of important environmental contaminants. We aimed to characterize the size of the association between maternal GFR and infant birth weight.ResultsMaternal GFR-CG (β: 0.73 g/ml/min, p = 0.04) and GFR-MDRD (β: 0.83 g/ml/min, p = 0.04) were associated with infant birth weight in models adjusted for maternal weight in kilograms, preeclampsia, and gestational age at delivery (days). Partial correlation coefficients for the association between infant birth weight and GFR were 0.07 for both formulas. Although the birth weight-GFR association was stronger among the women with preeclampsia, the difference from women without preeclampsia was not statistically significant.ConclusionThese data support an association between GFR during pregnancy and infant birth weight, and indicate that GFR may confound selected epidemiologic associations.     

Investigation of SNPs in the porcine desmoglein 1 gene

Background

Two annual surveys, the abattoir and the fallen stock, monitor the presence of scrapie across Europe. A simple comparison between the prevalence estimates in different countries reveals that, in 2003, the abattoir survey appears to detect more scrapie in some countries. This is contrary to evidence suggesting the greater ability of the fallen stock survey to detect the disease. We applied meta-analysis techniques to study this apparent heterogeneity in the behaviour of the surveys across Europe. Furthermore, we conducted a meta-regression analysis to assess the effect of country-specific characteristics on the variability. We have chosen the odds ratios between the two surveys to inform the underlying relationship between them and to allow comparisons between the countries under the meta-regression framework. Baseline risks, those of the slaughtered populations across Europe, and country-specific covariates, available from the European Commission Report, were inputted in the model to explain the heterogeneity.

Results

Our results show the presence of significant heterogeneity in the odds ratios between countries and no reduction in the variability after adjustment for the different risks in the baseline populations. Three countries contributed the most to the overall heterogeneity: Germany, Ireland and The Netherlands. The inclusion of country-specific covariates did not, in general, reduce the variability except for one variable: the proportion of the total adult sheep population sampled as fallen stock by each country. A large residual heterogeneity remained in the model indicating the presence of substantial effect variability between countries.

Conclusion

The meta-analysis approach was useful to assess the level of heterogeneity in the implementation of the surveys and to explore the reasons for the variation between countries.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.