SIRT1/HERC4 Locus Associated With Bisphosphonate‐Induced Osteonecrosis of the Jaw: An Exome‐Wide Association Analysis

Osteonecrosis of the jaw (ONJ) is a rare, but serious drug side effect, mainly associated with the use of intravenous (iv) bisphosphonates (BPs). The purpose of this study was to identify genetic variants associated with ONJ in patients of European ancestry treated with iv BPs using whole‐exome sequencing (WES). The WES phase 1 included 44 multiple myeloma patients (22 ONJ cases and 22 controls) and WES phase 2 included 17 ONJ patients with solid tumors. Multivariable logistic regression analysis was performed to estimate the odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age, sex, and principal components for ancestry. Meta‐analysis of WES phase 1 and 2 was performed to estimate the combined ORs. In silico analyses were then performed to identify expression quantitative loci (eQTL) single‐nucleotide polymorphisms (SNPs) that are in high linkage disequilibrium (LD) with the top SNPs. The associations of the potentially functional SNPs were replicated and validated in an independent case‐control study of 48 patients of European ancestry treated with iv BPs (19 ONJ cases and 29 controls). The top SNPs in the exome‐wide association meta‐analysis were two SNPs on chromosome 10: SIRT1 SNP rs7896005 and HERC4 SNP rs3758392 with identical OR of 0.07 (0.01–0.46; p = 3.83 × 10^?5). In the in silico functional analyses, two promoter region SNPs (rs7894483 and rs3758391) were identified to be in high LD with the index SNPs and are eQTLs for SIRT1 gene in whole blood in the GTEx database. The ORs were 0.30 (0.10–0.88), 0.26 (0.12–0.55), and 0.26 (0.12–0.55) for the WES top SNP rs7896005 and two promoter SNPs rs7894483 and rs3758391, respectively, in the replication sample. In summary, we identified the SIRT1/HERC4 locus on chromosome 10 to be associated with iv BP‐induced ONJ and two promoter SNPs that might be the potential genetic markers for this association. © 2017 The Authors.Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Transfection of cytochrome P4504B1 into the cornea increases angiogenic activity of the limbal vessels

Injury to the ocular surface induces the production of the corneal epithelial-derived 12-hydroxyeicosatetrienoic acid (12-HETrE), which exhibits stereospecific potent inflammatory and angiogenic properties and is formed by a cytochrome P450 (P450) enzyme, CYP4B1. We have cloned the rabbit corneal CYP4B1 into the expression plasmid pIRES2-enhanced green fluorescent protein (EGFP) and examined the effect of CYP4B1 overexpression on corneal inflammation in vivo and limbal vessel sprouting ex vivo. Cultured rabbit corneal epithelial cells transfected with pIRES2-EGFP-CYP4B1 metabolized arachidonic acid to 12-HETrE at a rate five times higher than that of pIRES2-EGFP-transfected cells (3.53 +/- 0.08 versus 0.62 +/- 0.10 nmol/h/10(6) cells; mean +/- S.E.M., n = 6, p < 0.05), indicating a functional expression of the CYP4B1. Injection of either plasmid into the rabbit cornea resulted in EGFP fluorescence in the corneal epithelium. However, corneal neovascularization, as measured by the length of penetrating blood vessels, was significantly greater in the corneas of eyes transfected with the pIRES2-CYP4B1 compared with pIRES2-EGFP. Corneal-limbal explants from eyes transfected with pIRES2-CYP4B1 showed a marked angiogenic activity (46 +/- 10 versus 12 +/- 3 mm capillary length, n = 6, p < 0.05), which correlated with increased levels of 12-HETrE, the CYP4B1-derived angiogenic 12-hydroxyeicosanoid (0.93 +/- 0.18 versus 0.15 +/- 0.02 pmol/explant, n = 6, p < 0.05), and was inhibited (76 +/- 5%) by the P450 inhibitor 17-octadecynoic acid. The results further implicate the corneal CYP4B1 as a component of the inflammatory and angiogenic cascade initiated by injury to the ocular surface and raise the possibility of a new therapeutic target for preventing corneal neovascularization, namely, the CYP4B1-12-HETrE system.     

Afferent arteriolar dilation to 11, 12-EET analogs involves PP2A activity and Ca2+-activated K+ Channels

The epoxygenase metabolite, 11, 12-epoxyeicosatrienoic acid (11, 12-EET), has renal vascular actions. 11, 12-EET analogs have been developed to determine the structure activity relationship for 11, 12-EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11, 12-EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large-conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically table 11, 12-EET analogs: 11, 12-EET-N-methylsulfonimide (11, 12-EET-SI), 11-nonyloxy-undec-8(Z)-enoic acid (11, 12-ether-EET-8-ZE), and 11, 12-trans-oxidoeicosa-8(Z)-eonoic acid (11, 12-tetra-EET-8-ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11, 12-EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11, 12-EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11, 12-EET analogs were abolished by K+ channel or PP2A inhibition. 11, 12-EET analogs activated renal myocyte large-conductance KCa channels. 11, 12-EET analogs increased cAMP by 2-fold and PP2A activity increased 3-8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11, 12-EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11, 12-EET utilizes PP2A dependent pathways to activate large-conductance KCa channels and dilate the afferent arteriole.     

Rat strain-dependent variations in brain metabolites detected by in vivo (1) H NMR spectroscopy at 16.4T

Localized in vivo (1) H NMR spectroscopy is playing an increasing role in preclinical studies, because of its ability to quantify the concentrations of up to 20 metabolites in rat brain. To assess the differences between often-used rat strains, the neurochemical profiles of Sprague-Dawley, Wistar and Fischer rats were determined at ultrashort TE at 16.4 T. To ascertain high-qualitative quantification, a first experiment examined the dependence of the measuring time on the quantification results and precision by precisely the number of averages between 16 and 320. It was shown that most metabolites can be quantified accurately within a short scan time, yielding Cramér-Rao lower bounds below 20% and stable concentrations for 16 metabolites with as few as 32 or 64 averages in the thalamus and hippocampus, respectively. Interstrain differences in metabolite concentrations were shown to be moderate, with taurine varying significantly between Sprague-Dawley and Wistar rats, and slightly more distinct differences from Fischer rats, including variations in glutamate and myo-inositol. The high spectral quality and quantification precision of all data again demonstrated the potential of in vivo (1)H NMR spectroscopy at ultrahigh field.     

LCT 13910 C/T polymorphism, serum calcium, and bone mineral density in postmenopausal women

Summary  LCT 13910 CC genotype is associated with lactose intolerance, a condition often resulting in reduced milk intake. Women with the CC genotype were found to have decreased serum calcium and reduced bone mineral density. Introduction  The CC genotype of the 13910 C/T polymorphism of the LCT gene is linked to lactose intolerance and low calcium intake. Methods  We studied 595 postmenopausal women, including 267 osteoporotic, 200 osteopenic, and 128 healthy subjects. Genotyping, osteodensitometry, and laboratory measurements were carried out. Results  Frequency of aversion to milk consumption was 20% for CC genotype and 10% for TT + TC genotypes (p = 0.03). The albumin-adjusted serum calcium was 2.325 ± 0.09 mmol/L for CC genotype and 2.360 ± 0.16 mmol/L for TT + TC genotypes (p = 0.031). Bone mineral density (BMD; Z score) was lower in the CC than TT + TC genotypes, respectively, at the radius (0.105 ± 1.42 vs 0.406 ± 1.32; p = 0.038), at the total hip (−0.471 ± 1.08 vs −0.170 ± 1.09; p = 0.041), and at the Ward’s triangle (−0.334 ± 0.87 vs −0.123 ± 0.82; p = 0.044). Conclusion  LCT 13910 C/T polymorphism is associated with decreased serum calcium level and lower BMD in postmenopausal women. Péter Lakatos and Gábor Speer contributed equally to this work.     

The ubiquitin-proteasome pathway in health and disease of the nervous system

In recent years, proteolysis by the ubiquitin-proteasome pathway has attained prominence as a new molecular mechanism that regulates many vital functions of the nervous system, including development of synaptic connections and synaptic plasticity. Here, we review the latest findings on the role of proteolysis in sculpting the nervous system through control of axonal growth, axonal and dendritic pruning, and regulation of synaptic size and number. We also discuss how protein degradation functions in synaptic plasticity and the roles of local proteolysis in neuronal compartments. In addition, we describe how proteolysis is associated with Alzheimer's disease and ataxia. Furthermore, we highlight the recent approaches that exploit components of the ubiquitin-proteasome pathway for amelioration of these diseases in animal models.