Pharmacogenomics aims to tailor pharmacological treatment to each individual by considering associations between genetic polymorphisms and adverse drug effects (ADEs). With technological advances, pharmacogenomic research has evolved from candidate gene analyses to genome-wide association studies. Here, we integrate deep whole-genome sequencing (WGS) information with drug prescription and ADE data from Estonian electronic health record (EHR) databases to evaluate genome- and pharmacome-wide associations on an unprecedented scale. We leveraged WGS data of 2240 Estonian Biobank participants and imputed all single-nucleotide variants (SNVs) with allele counts over 2 for 13,986 genotyped participants. Overall, we identified 41 (10 novel) loss-of-function and 567 (134 novel) missense variants in 64 very important pharmacogenes. The majority of the detected variants were very rare with frequencies below 0.05%, and 6 of the novel loss-of-function and 99 of the missense variants were only detected as single alleles (allele count = 1). We also validated documented pharmacogenetic associations and detected new independent variants in known gene-drug pairs. Specifically, we found that CTNNA3 was associated with myositis and myopathies among individuals taking nonsteroidal anti-inflammatory oxicams and replicated this finding in an extended cohort of 706 individuals. These findings illustrate that population-based WGS-coupled EHRs are a useful tool for biomarker discovery.

The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes.

MicroRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.

Despite differences in types of infection and causative organisms, pharmacokinetic-pharmacodynamic (PKPD) targets of vancomycin therapy derived from adult studies are suggested for neonates. We aimed to identify doses needed for the attainment of AUC/MIC > 400 and AUC/MIC > 300 in neonates with sepsis and correlate these targets with recommended doses and treatment outcome. Neonates who had Vancomycin therapeutic drug monitoring (TDM) performed between January 1, 2010 and December 31, 2012 were studied. Clinical characteristics, episodes of Gram-positive sepsis with outcomes and all neonatal blood culture isolates in hospital were collected from medical records. To estimate probability of target attainment of AUC/MIC >400 and AUC/MIC >300 a 1000-subject Monte Carlo simulation was performed by calculating AUC using Anderson’s (Anderson et al. 2006) and TDM trough concentrations (Ctrough) based population PK models. Final dataset included 76 patients; 57 with confirmed Gram-positive sepsis. TDM was taken after the 1st to 44th dose. 84.1% of Ctrough were within the range 5–15 mg/L. Currently recommended doses achieved probability of the targets (PTA) of AUC/MIC >400 and AUC/MIC >300 in less than 25% and 40% of cases, respectively. Doses required for 80% PTA of AUC/MIC > 400 for MIC ≥2 mg/L resulted in Ctrough values ≥14 mg/L. Mean AUC/MIC values were similar in treatment failure and success groups. With currently recommended vancomycin dosing the therapeutic target of AUC/MIC > 400 is achieved only by 25% of neonates. Appropriate PKPD targets and respective dosing regimens need to be defined in prospective clinical studies in this population.

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix–receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients’ peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis.

Group B streptococci are common causative agents of early-onset neonatal sepsis (EOS). Pharmacokinetic (PK) data for penicillin G have been described for extremely preterm neonates but have been poorly described for late-preterm and term neonates. Thus, evidence-based dosing recommendations are lacking. We describe the PK of penicillin G in neonates with a gestational age (GA) of ≥32 weeks and a postnatal age of 90% for MICs of ≤2 mg/liter with doses of 25,000 IU/kg q12h. In neonates, regardless of GA, the PK parameters of penicillin G were similar. The dose of 25,000 IU/kg q12h is suggested for treatment of group B streptococcal EOS diagnosed within the first 72 h of life. (This study was registered with the EU Clinical Trials Register under EudraCT number 2012-002836-97.)