Endothelin-1 regulation is entangled in a complex web of epigenetic mechanisms in diabetes

Endothelial cells (ECs) are primary targets of glucose-induced tissue damage. As a result of hyperglycemia, endothelin-1 (ET-1) is upregulated in organs affected by chronic diabetic complications. The objective of the present study was to identify novel transcriptional mechanisms that influence ET-1 regulation in diabetes. We carried out the investigation in microvascular ECs using multiple approaches. ECs were incubated with 5 mM glucose (NG) or 25 mM glucose (HG) and analyses for DNA methylation, histone methylation, or long non-coding RNA- mediated regulation of ET-1 mRNA were then performed. DNA methylation array analyses demonstrated the presence of hypomethylation in the proximal promoter and 5\u27 UTR/first exon regions of EDN1 following HG culture. Further, globally blocking DNA methylation or histone methylation significantly increased ET-1 mRNA expressions in both NG and HG-treated HRECs. While, knocking down the pathogenetic lncRNAs ANRIL, MALAT1, and ZFAS1 subsequently prevented the glucose-induced upregulation of ET-1 transcripts. Based on our past and present findings, we present a novel paradigm that reveals a complex web of epigenetic mechanisms regulating glucose-induced transcription of ET-1. Improving our understanding of such processes may lead to better targeted therapies

Effects of Oral, Vaginal, and Transdermal Hormonal Contraception on Serum Levels of Coenzyme Q10, Vitamin E, and Total Antioxidant Activity

The use of the transdermal contraceptive patch is associated with greater bioavailability of ethinyl estradiol (EE) compared with contraceptive vaginal ring or oral contraceptives (OC). We compared the influences of three contraceptive methods (OC, vaginal ring, and transdermal patch) on serum levels of coenzyme Q10, α-tocopherol, γ-tocopherol and total antioxidant capacity in premenopausal women. Blood samples from 30 premenopausal women who used hormonal contraception for at least 4 months were collected. Forty subjects who did not use any contraception were studied as control. Serum levels of coenzyme Q10, α-tocopherol and γ-tocopherol were measured by high-pressure liquid chromatography. Serum samples were also assayed for total antioxidant capacity (TAOC). Serum levels of coenzyme Q10 and α-tocopherol were found to be significantly lower (P < .05) in all three contraceptive users compared with controls. Contraceptive patch users had the lowest levels of coenzyme Q10 levels compared with normal subjects. Serum TAOC levels were significantly lower (P < .05) among the contraceptive user groups. Alterations in coenzyme Q10 and α-tocopherol induced by hormonal contraception and the potential effect(s) of exogenous ovarian hormones should be taken into consideration in future antioxidant research

Examining the clinical use of hemochromatosis genetic testing

BACKGROUND: Hereditary hemochromatosis leads to an increased lifetime risk for end-organ damage due to excess iron deposition. Guidelines recommend that genetic testing be performed in patients with clinical suspicion of iron overload accompanied by elevated serum ferritin and transferrin saturation levels. OBJECTIVE: To evaluate guideline adherence and the clinical and economic impact of HFE genetic testing. METHODS: The electronic charts of patients submitted for HFE testing in 2012 were reviewed for genetic testing results, biochemical markers of iron overload and clinical history of phlebotomy. RESULTS: A total of 664 samples were sent for testing, with clinical, biochemical and phlebotomy data available for 160 patients. A positive C282Y homozygote or C282Y/H63D compound heterozygote test result was observed in 18% of patients. Patients with an at-risk HFE genotype had significantly higher iron saturation, serum iron and hemoglobin (P\u3c0.001), without higher ferritin or liver enzyme levels. Fifty percent of patients referred for testing did not have biochemical evidence of iron overload (transferrin saturation \u3e45% and ferritin level \u3e300μg/L). Patients were four times more likely to undergo phlebotomy if they were gene test positive (RR 4.29 [95% CI 2.35 to 7.83]; P\u3c0.00001). DISCUSSION: One-half of patients referred for testing did not exhibit biochemical evidence of iron overload. Many patients with biochemical evidence of iron overload, but with negative genetic test results, did not undergo phlebotomy. A requisition to determine clinical indication for testing may reduce the use of the HFE genetic test. Finally, improvement of current genetic test characteristics would improve rationale for the test. CONCLUSION: A significant proportion of hemochromatosis genetic testing does not adhere to current guidelines and would not alter patient management

Population structure and diversity of the needle pathogen Dothistroma pini suggests human-mediated movement in Europe

Dothistroma needle blight (DNB) is an important disease of Pinus species that can be caused by one of two distinct but closely related pathogens; Dothistroma septosporum and Dothistroma pini. Dothistroma septosporum has a wide geographic distribution and is relatively well-known. In contrast, D. pini is known only from the United States and Europe, and there is a distinct lack of knowledge regarding its population structure and genetic diversity. The recent development of 16 microsatellite markers for D. pini provided an opportunity to investigate the diversity, structure, and mode of reproduction for populations collected over a period of 12 years, on eight different hosts in Europe. In total, 345 isolates from Belgium, the Czech Republic, France, Hungary, Romania, Western Russia, Serbia, Slovakia, Slovenia, Spain, Switzerland, and Ukraine were screened using microsatellite and species-specific mating type markers. A total of 109 unique multilocus haplotypes were identified and structure analyses suggested that the populations are influenced by location rather than host species. Populations from France and Spain displayed the highest levels of genetic diversity followed by the population in Ukraine. Both mating types were detected in most countries, with the exception of Hungary, Russia and Slovenia. Evidence for sexual recombination was supported only in the population from Spain. The observed population structure and several shared haplotypes between non-bordering countries provides good evidence that the movement of D. pini in Europe has been strongly influenced by human activity in Europe

Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients\u27 age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (\u3c6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (\u3c6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement. © 2010 Sadikovic et al

Correction: Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes (PLoS ONE (2010) 5:12 (e15687) DOI: 10.1371/journal.pone.0015687)

The middle initial of the third author is missing. The third author’s complete name is: Ayman W. El-Hattab. The correct citation is: Sadikovic B, Wang J, El-Hattab AW, Landsverk M, Douglas G, Brundage EK, et al. (2010) Sequence Homology at the Breakpoint and Clinical Phenotype of Mitochondrial DNA Deletion Syndromes. PLoS ONE 5(12): e15687. https://doi.org/10.1371/journal.pone.0015687

MRI plaque imaging reveals high-risk carotid plaques especially in diabetic patients irrespective of the degree of stenosis

<p>Abstract</p> <p>Background</p> <p>Plaque imaging based on magnetic resonance imaging (MRI) represents a new modality for risk assessment in atherosclerosis. It allows classification of carotid plaques in high-risk and low-risk lesion types (I-VIII). Type 2 diabetes mellitus (DM 2) represents a known risk factor for atherosclerosis, but its specific influence on plaque vulnerability is not fully understood. This study investigates whether MRI-plaque imaging can reveal differences in carotid plaque features of diabetic patients compared to nondiabetics.</p> <p>Methods</p> <p>191 patients with moderate to high-grade carotid artery stenosis were enrolled after written informed consent was obtained. Each patient underwent MRI-plaque imaging using a 1.5-T scanner with phased-array carotid coils. The carotid plaques were classified as lesion types I-VIII according to the MRI-modified AHA criteria. For 36 patients histology data was available.</p> <p>Results</p> <p>Eleven patients were excluded because of insufficient MR-image quality. DM 2 was diagnosed in 51 patients (28.3%). Concordance between histology and MRI-classification was 91.7% (33/36) and showed a Cohen's kappa value of 0.81 with a 95% CI of 0.98-1.15. MRI-defined high-risk lesion types were overrepresented in diabetic patients (n = 29; 56.8%). Multiple logistic regression analysis revealed association between DM 2 and MRI-defined high-risk lesion types (OR 2.59; 95% CI [1.15-5.81]), independent of the degree of stenosis.</p> <p>Conclusion</p> <p>DM 2 seems to represent a predictor for the development of vulnerable carotid plaques irrespective of the degree of stenosis and other risk factors. MRI-plaque imaging represents a new tool for risk stratification of diabetic patients.</p> <p>See Commentary: <url>http://www.biomedcentral.com/1741-7015/8/78/abstract</url></p

Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy

Background: Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. Methods: Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts.Results: RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. Conclusion: These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma. © 2010 Sadikovic et al; licensee BioMed Central Ltd

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5′ and 3′ of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants

Frameshift mutations at the C-terminus of HIST1H1E result in a specific DNA hypomethylation signature

BACKGROUND: We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. RESULTS: Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This "episignature" was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. CONCLUSIONS: We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis