Single nucleotide polymorphisms and type of steroid impact the functional response of the human glucocorticoid receptor.

BACKGROUND: Clinical trials evaluating the use of steroids in septic shock have shown variable outcomes. Our previous studies have implicated human glucocorticoid receptor (hGR) polymorphisms as a possible cause of altered steroid response. To further evaluate this variability, we hypothesized that hGR polymorphisms along with type of steroid influence the functional response. METHODS: Total RNA was isolated from healthy human blood samples and surveyed for the hGR gene. The National Center for Biotechnology Information hGRα sequence was used as a reference, and two unique single nucleotide polymorphisms (SNPs) (A214G and T962C) were selected for evaluation. Functional response was measured using a luciferase reporting assay after transfecting hGR isoforms into tsA201 cells and stimulation with graded concentrations of hydrocortisone (HYD), methylprednisolone (MPS), and dexamethasone (DEX). RESULTS: Each isoform had a unique dose-response curve with the optimal activity depending on concentration and type of steroid. The presence of either SNP A214G or T962C resulted in a decreased response when compared with hGRα when stimulated with HYD (P < 0.01). The same decreased response occurred for the SNPs with DEX stimulation, but at a much lower concentration range than HYD (P < 0.01). However, in the presence of MPS, SNP A214G resulted in greater activity when compared with hGRα (P < 0.01), whereas the presence of T962C resulted in activity equivalent to hGRα. CONCLUSIONS: SNPs, type of steroid, and concentration range impact the functional response of the hGR. A greater understanding of hGR polymorphisms and steroid response may further elucidate mechanisms explaining the variable response seen with patient treatment.

Application of duplex ultrasound imaging in determining in-stent stenosis during surveillance after mesenteric artery revascularization.

OBJECTIVE: Currently, there are no well-established duplex ultrasound (DUS) criteria for the evaluation of the mesenteric arteries after stenting for occlusive disease. Previous studies suggested DUS velocity criteria in the native superior mesenteric artery (SMA) overestimate stenosis in stented arteries, but most studies have not evaluated DUS imaging after SMA stenting longitudinally. This study was undertaken to determine the accuracy of DUS after mesenteric artery revascularization and, in particular, to evaluate the utility of DUS imaging for the detection of in-stent stenosis (ISS) of the SMA. METHODS: A retrospective record review was performed for all patients who underwent SMA stenting for chronic mesenteric ischemia at a single institution from January 2004 to May 2011. RESULTS: Mesenteric artery occlusive disease resulted in 24 patients undergoing mesenteric stenting of the SMA alone (n = 20) or the SMA and celiac artery simultaneously (n = 3). The mean ± standard deviation peak systolic velocity (PSV) in 13 prestent DUS images of the SMA was 464 ± 130 cm/s. Prestenting angiography revealed an average SMA stenosis of 79% ± 14%. After stenting, completion angiography in each case revealed <20% residual stenosis. No significant correlation was identified between SMA PSV and angiographic stenosis before and after stenting (P > .05). Follow-up SMA DUS imaging showed an average PSV of 335 ± 138 cm/s at 0.9 ± 1.5 months, 360 ± 143 cm/s at 4.8 ±2.6 months, and 389 ± 95 cm/s at 14.4 ± 5.1 months. A significant difference existed between the prestent and the first poststent mean SMA PSV (P < .05), but no significant difference existed between each poststenting interval. Eight reinterventions for SMA ISS were performed, with a mean elevated in-stent SMA PSV of 505 ± 74 vs 341 ± 145 cm/s in patients who did not undergo reintervention. Angiography before the eight reinterventions demonstrated an average SMA ISS of 53% ± 25%. In-stent SMA PSV decreased from 505 ± 74 to 398 ± 108 cm/s after the reintervention (P < .05). CONCLUSIONS: Consistent with other reports, our data demonstrate the PSV in successfully stented SMAs remains higher than the PSV threshold of 275 cm/s used for the diagnosis of high-grade native SMA stenosis. In addition, in-stent SMA PSVs did not significantly change over DUS surveillance for patients who did not undergo reintervention. Thus, obtaining a baseline DUS early after mesenteric stenting should be considered to compare future surveillance DUS. An increase above this baseline or an in-stent SMA PSV approaching 500 cm/s should be considered suspicious for ISS, but larger prospective studies will be required to validate these preliminary findings.

Enhanced steroid response of a human glucocorticoid receptor splice variant.

Glucocorticoids remain a recommended therapy in advanced septic shock despite the often unpredictable response, and our understanding of the mechanisms regulating the steroid and stress response remains limited. Since the initial sequencing of the human glucocorticoid receptor α and ß gene (hGRα and hGRß), only three additional splice variants have been identified--all of which have been postulated to contribute to steroid resistance. During a survey of 97 healthy humans' blood, we identified two novel hGR splice isoforms (hGR-S1 and hGR-S1(-349A) retaining intron H between exons 8 and 9. Human GR-S1(-349A) contained a base deletion causing an early termination and a truncated protein of 118 amino acids, whereas hGR-S1 had an early termination occurring within intron H and resulted in a 745-amino acid protein. Both isoforms had decreased transactivation potentials compared with hGRα when tested in the absence of exogenous steroids. However, after treating with exogenous steroids, dose-response studies showed hGR-S1(-349A) had a substantial augmentation in activity at higher concentrations of hydrocortisone and methylprednisolone when compared with hGRα, whereas hGR-S1 did not. Removal of the 3' untranslated region (3'UTR) of the hGR-S1(-349A) mRNA sequence resulted in a loss of the augmented response. The isoform hGR-S1(-349A) augments the response to steroids, and this significant response appears to be critically regulated by the 3'UTR. The identification and evaluation of these unique hGR isoforms helps further the understanding of the complex genetic regulation of the stress and steroid response.

Novel hyperactive glucocorticoid receptor isoform identified within a human population.

Glucocorticoids serve as important therapeutic agents in diseases of inflammation, but clinical use, especially in advanced septic shock, remains controversial because of the unpredictable response. Prior studies correlate human glucocorticoid receptor (hGR) isoforms with a decreased response to steroid therapy. Further analysis of additional hGR isoforms may improve the understanding of the steroid response. Ninety-seven human volunteers' blood samples were surveyed for hGR isoforms. An isoform matching National Center for Biotechnology Informatics (NCBI) hGRα (hGR NCBI) served as a reference. Two isoforms were of particular interest-one isoform had three nonsynonymous single-nucleotide polymorphisms (SNPs) (hGR NS-1), and the second had a single-nucleotide deletion (hGR DL-1) resulting in a truncated protein. Transactivation potentials were measured using a luciferase reporter assay. Human glucocorticoid receptor NS-1 had activity more than twice of hGR NCBI, whereas hGR DL-1 demonstrated less than 10% of the activity of hGR NCBI. Cotransfection of two isoforms revealed that the presence of hGR NS-1 increased transactivation potential, whereas hGR DL-1 decreased activity. Synthetic constructs isolating individual and paired SNPs of hGR NS-1 were created to identify the SNP responsible for hyperactivity. Transactivation studies revealed a SNP within the ligand-binding domain exerted the greatest influence over hyperactivity. In evaluating the response to hydrocortisone, hGR NCBI and hGR NS-1 displayed an increased dose-dependent response, but hGR NS-1 had a response more than twice hGR NCBI. Characterization of the novel hyperactive hGR NS-1 provides insight into a possible mechanism underlying the unpredictable response to steroid treatment.

Identification of a unique library of complex, but ordered, arrays of repetitive elements in the human genome and implication of their potential involvement in pathobiology.

Approximately 2% of the human genome is reported to be occupied by genes. Various forms of repetitive elements (REs), both characterized and uncharacterized, are presumed to make up the vast majority of the rest of the genomes of human and other species. In conjunction with a comprehensive annotation of genes, information regarding components of genome biology, such as gene polymorphisms, non-coding RNAs, and certain REs, is found in human genome databases. However, the genome-wide profile of unique RE arrangements formed by different groups of REs has not been fully characterized yet. In this study, the entire human genome was subjected to an unbiased RE survey to establish a whole-genome profile of REs and their arrangements. Due to the limitation in query size within the bl2seq alignment program (National Center for Biotechnology Information [NCBI]) utilized for the RE survey, the entire NCBI reference human genome was fragmented into 6206 units of 0.5M nucleotides. A number of RE arrangements with varying complexities and patterns were identified throughout the genome. Each chromosome had unique profiles of RE arrangements and density, and high levels of RE density were measured near the centromere regions. Subsequently, 175 complex RE arrangements, which were selected throughout the genome, were subjected to a comparison analysis using five different human genome sequences. Interestingly, three of the five human genome databases shared the exactly same arrangement patterns and sequences for all 175 RE arrangement regions (a total of 12,765,625 nucleotides). The findings from this study demonstrate that a substantial fraction of REs in the human genome are clustered into various forms of ordered structures. Further investigations are needed to examine whether some of these ordered RE arrangements contribute to the human pathobiology as a functional genome unit.