Identification of regulatory variants associated with genetic susceptibility to meningococcal disease

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Biochemical characterization of sporadic/familial hemiplegic migraine mutations

Contains fulltext : 133904.pdf (publisher's version ) (Closed access)Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na(+),K(+)-ATPase alpha2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na(+) and K(+) affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935-940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na(+) and increased K(+) apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na(+),K(+)-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants

Uremic solutes modulate hepatic bile acid handling and induce mitochondrial toxicity

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Uremic solutes modulate hepatic bile acid handling and induce mitochondrial toxicity

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A world of conductors

Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na(+),K(+)-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na(+),K(+)-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed alpha1 isoform and the more restricted alpha2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the alpha2 isoform, we were able to link residues Met(119) and Ser(124) to differences in affinity between the alpha1 and alpha2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups

Evaluation Report

Drug-induced liver injury (DILI) is a common reason for drug withdrawal from the market. An important cause of DILI is drug-induced cholestasis. One of the major players involved in drug-induced cholestasis is the bile salt efflux pump (BSEP; ABCB11). Inhibition of BSEP by drugs potentially leads to cholestasis due to increased (toxic) intrahepatic concentrations of bile acids with subsequent cell injury. In order to investigate the possibilities for in silico prediction of cholestatic effects of drugs, we developed a mechanistic biokinetic model for human liver bile acid handling populated with human in vitro data. For this purpose we considered nine groups of bile acids in the human bile acid pool, i.e. chenodeoxycholic acid, deoxycholic acid, the remaining unconjugated bile acids and the glycine and taurine conjugates of each of the three groups. Michaelis-Menten kinetics of the human uptake transporter Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) and BSEP were measured using NTCP-transduced HEK293 cells and membrane vesicles from BSEP-overexpressing HEK293 cells. For in vitro-in vivo scaling, transporter abundance was determined by LC-MS/MS in these HEK293 cells and vesicles as well as in human liver tissue. Other relevant human kinetic parameters were collected from literature, such as portal bile acid levels and composition, bile acid synthesis and amidation rate. Additional empirical scaling was applied by increasing the excretion rate with a factor 2.4 to reach near physiological steady-state intracellular bile acid concentrations (80 μM) after exposure to portal vein bile acid levels. Simulations showed that intracellular bile acid concentrations increase 1.7 fold in the presence of the BSEP inhibitors and cholestatic drugs cyclosporin A or glibenclamide, at intrahepatic concentrations of 6.6 and 20 μM, respectively. This simplified model provides a tool for a first indication whether drugs at therapeutic concentrations might cause cholestasis by inhibiting BSEP. © 2018 The Author

A missense variant of the ATP1A2 gene is associated with a novel phenotype of progressive sensorineural hearing loss associated with migraine

Contains fulltext : 155167.pdf (publisher's version ) (Closed access)Hereditary sensorineural hearing loss is an extremely clinical and genetic heterogeneous disorder in humans. Especially, syndromic hearing loss is subdivided by combinations of various phenotypes, and each subtype is related to different genes. We present a new form of progressive hearing loss with migraine found to be associated with a variant in the ATP1A2 gene. The ATP1A2 gene has been reported as the major genetic cause of familial migraine by several previous studies. A Korean family presenting progressive hearing loss with migraine was ascertained. The affected members did not show any aura or other neurologic symptoms during migraine attacks, indicating on a novel phenotype of syndromic hearing loss. To identify the causative gene, linkage analysis and whole-exome sequencing were performed. A novel missense variant, c.571G>A (p.(Val191Met)), was identified in the ATP1A2 gene that showed co-segregation with the phenotype in the family. In silico studies suggest that this variant causes a change in hydrophobic interactions and thereby slightly destabilize the A-domain of Na(+)/K(+)-ATPase. However, functional studies failed to show any effect of the p.(Val191Met) substitution on the catalytic rate of this enzyme. We describe a new phenotype of progressive hearing loss with migraine associated with a variant in the ATP1A2 gene. This study suggests that a variant in Na(+)/K(+)-ATPase can be involved in both migraine and hearing loss