Metabolic features of treatment-refractory major depressive disorder with suicidal ideation.

Peripheral blood metabolomics was used to gain chemical insight into the biology of treatment-refractory Major Depressive Disorder with suicidal ideation, and to identify individualized differences for personalized care. The study cohort consisted of 99 patients with treatment-refractory major depressive disorder and suicidal ideation (trMDD-SI n = 52 females and 47 males) and 94 age- and sex-matched healthy controls (n = 48 females and 46 males). The median age was 29 years (IQR 22-42). Targeted, broad-spectrum metabolomics measured 448 metabolites. Fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) were measured as biomarkers of mitochondrial dysfunction. The diagnostic accuracy of plasma metabolomics was over 90% (95%CI: 0.80-1.0) by area under the receiver operator characteristic (AUROC) curve analysis. Over 55% of the metabolic impact in males and 75% in females came from abnormalities in lipids. Modified purines and pyrimidines from tRNA, rRNA, and mRNA turnover were increased in the trMDD-SI group. FGF21 was increased in both males and females. Increased lactate, glutamate, and saccharopine, and decreased cystine provided evidence of reductive stress. Seventy-five percent of the metabolomic abnormalities found were individualized. Personalized deficiencies in CoQ10, flavin adenine dinucleotide (FAD), citrulline, lutein, carnitine, or folate were found. Pathways regulated by mitochondrial function dominated the metabolic signature. Peripheral blood metabolomics identified mitochondrial dysfunction and reductive stress as common denominators in suicidal ideation associated with treatment-refractory major depressive disorder. Individualized metabolic differences were found that may help with personalized management.

High-resolution analysis of the human placental DNA methylome in early gestation.

BACKGROUND/OBJECTIVE: We communicate high-read-depth bisulfite sequencing analysis of the chorionic villus (CV) DNA methylome from samples obtained between 11 and 13 weeks gestation and samples of gestationally age-matched maternal blood cells (MBC). METHODS: This was achieved through solution-phase targeted region capture (84 Mb) of bisulfite converted human DNA. RESULTS: We identified biphasic distribution of methylation in CV and MBC genomes. We found greater numbers of intermediate methylated sites (20%-80% methylated) in CV and greater number of high methylation sites in MBC and investigated distributions of these in promoters, introns, exons, CpG islands, CpG islands shores, and enhancers. We identified differentially methylated sites distinguishing CV and MBC. These are less likely to occur in CpG islands (CGIs), particularly those that exist outside promoters, exons, and introns. We found that gene promoter and gene body methylation patterns are associated with mRNA transcriptional profiles in CV. Despite the relative hypomethylation of CV genomes, we found that these contain DM regions that are more likely to be hypermethylated in CV relative to MBC. CONCLUSIONS: Our data provide novel insight into the structure and organization of the CV epigenome, which may inform future studies of placental biology and noninvasive prenatal phenotyping.

Rat Urinary Osteopontin and Neutrophil Gelatinase-Associated Lipocalin Improve Certainty of Detecting Drug-Induced Kidney Injury.

Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.

Targeted DNA Methylation Screen in the Mouse Mammary Genome Reveals a Parity-Induced Hypermethylation of Igf1r That Persists Long after Parturition.

The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically.

Discovery of epigenetic biomarkers for the noninvasive diagnosis of fetal disease.

OBJECTIVES: The primary goal of this study was to identify CpG sites in the human genome that are differentially methylated in DNA obtained from chorionic villus sampling (CVS) samples and gestational age-matched maternal blood cell (MBC) samples. METHODS: We used the HumanMethylation27 DNA Analysis BeadChip to characterize DNA methylation in samples of CVS and MBC. We then selected a subset of differentially methylated CpG sites on chromsome 13 and subjected them to analysis by mass spectrometry using the Epityper platform. RESULTS: We identified 718 tissue-specific differentially methylated regions (DMRs) between MBC and CVS; 563 of these were hypermethylated in MBC and hypomethylated in CVS, whereas 155 sites were hypomethylated in MBC and hypermethylated in CVS. Further analysis of 13 DMRs on chromosome 13 by Epityper confirmed the microarray data and provided us with additional data about the methylation patterns of surrounding CpG sites. CONCLUSIONS: Analysis of the resulting data identified a large number of cytosine-guanine dinucleotides that are potential biomarkers for the selective amplification of fetal DNA from maternal plasma and the subsequent noninvasive detection of trisomy 13.

A microarray-based approach for the identification of epigenetic biomarkers for the noninvasive diagnosis of fetal disease.

OBJECTIVES: We describe a novel microarray-based approach for the high-throughput discovery of epigenetic biomarkers for use in the noninvasive detection of fetal genetic disease. METHODS: We combined a 215 060-probe custom oligonucleotide microarray with a comprehensive library preparation method and novel statistical tools to compare DNA methylation patterns in chorionic villus samples (CVS) with gestational age-matched maternal blood cell (MBC) samples. Our custom microarray was designed to provide high-resolution coverage across human chromosomes 13, 18 and 21. RESULTS: We identified 6311 MspI/HpaII sites across all three chromosomes that displayed tissue-specific differential CpG methylation patterns. To maximize the probability of identifying biomarkers that have clinical utility we filtered our data to identify MspI/HpaII sites that are within 150 bp of a highly polymorphic single nucleotide polymorphism (SNP) so that its allelic ratio may be determined for the detection of fetal aneuploidy. Our microarray design and the computational tools used for data analysis are available for download as is the entire data set. CONCLUSIONS: This high-resolution analysis of DNA methylation patterns in the human placenta during the first trimester of pregnancy identifies numerous potential biomarkers for the diagnosis of fetal aneuploidy on chromosomes 13, 18 and 21.

Spontaneous hibernomas in Sprague-Dawley rats.

Hibernomas are rare neoplasms originating in brown adipose tissue of humans and other animal species, including laboratory animals. Background incidence values for these tumors in all common strains of laboratory rats are generally accepted as being <0.1%. Between April 2000 and April 2007, however, sixty-two hibernomas (an overall prevalence of 3.52%) were observed in a total of 1760 Sprague-Dawley rats assigned to three carcinogenesis bioassays at two separate research laboratories. All rats were obtained from Charles River's breeding facilities in either Portage, Michigan, or Raleigh, North Carolina. Tumors (twenty-nine benign and thirty-three malignant) were randomly distributed among test article-treated and control groups and were considered to be spontaneous. Most tumors originated in the thoracic cavity, and they were usually described as soft, mottled to tan masses with nodular to lobulated profiles. Immunohistochemical procedures for uncoupling protein 1 (UCP1) confirmed brown adipose tissue as the site of origin rather than white fat. The marked increase in hibernomas in our studies suggests that greater numbers of spontaneous hibernomas may be sporadically encountered in future carcinogenesis studies with Sprague-Dawley rats. The increased potential for hibernomas to arise as spontaneous neoplasms has important implications in studies involving peroxisome proliferators-activated receptor (PPAR) drugs, lipophilic environmental chemicals (e.g., polychlorinated biphenyls), and other molecules or physiologic processes (e.g., beta-adrenergic stimulation) that may target brown fat adipocytes.

Spontaneous renal tubular hyperplastic and neoplastic lesions in three Sprague-Dawley rats from a 90-day toxicity study.

Multiple renal tubular cell adenomas and atypical tubular hyperplasia were diagnosed in 2 high-dose and 1 mid-dose female Sprague-Dawley (Crl:CD (SD)IGS BR) rats from a 90-day toxicity study of an amino acid found in green tea. The tumors were bilateral multicentric adenomas accompanied by atypical foci of renal tubular hyperplasia in both kidneys of the 3 animals. Toxic tubular changes that typically accompany renal carcinogenesis were not seen in any of the other animals of the study, suggesting rather, an underlying germline mutation of a tumor suppressor gene in these three rats. The histological appearance of these tumors and short latency was reminiscent of the spontaneous lesions reported to arise in Sprague-Dawley rats in the Nihon rat model. Nihon rats develop kidney tumors as a result of a spontaneous mutation in the rat homologue of the Birt-Hogg-Dubé gene (Bhd). Frozen samples of liver from two tumor-bearing rats were assayed for germline alterations in the Bhd gene. The entire coding region (exons 3-13) of the Bhd gene was sequenced, and a guanine (nt106G) to adenine (nt106A) polymorphism was detected resulting in a glycine to arginine (G36R) substitution in both tumor-bearing animals. In the study animals, the frequency of the A-allele (adenine) was determined to be 27% (19/70). Interestingly, rats obtained from two other sources (n = 17) only carried the nt106G-allele, consistent with the published rat sequence for this gene. Genetic fingerprinting of microsatellite loci indicated that the rats had a shared genetic background. Laser capture microdissection (LCM) of the tumor cells demonstrated a loss of heterozygosity in the Bhd gene in neoplastic cells of one of the two animals. Taken together, these data suggest that the tumors observed in these animals arose spontaneously as a result of a shared genetic susceptibility leading to the development of renal tubular neoplasms.

Comparative gene expression analysis of ovarian carcinoma and normal ovarian epithelium by serial analysis of gene expression.

Despite the poor prognosis of ovarian cancer and the importance of early diagnosis, there are no reliable noninvasive biomarkers for detection in the early stages of disease. Therefore, to identify novel ovarian cancer markers with potential utility in early-stage screening protocols, we have undertaken an unbiased and comprehensive analysis of gene expression in primary ovarian tumors and normal human ovarian surface epithelium (HOSE) using Serial Analysis of Gene Expression (SAGE). Specifically, we have generated SAGE libraries from three serous adenocarcinomas of the ovary and, using novel statistical tools, have compared these to SAGE data derived from two pools of normal HOSE. Significantly, in contrast to previous SAGE-based studies, our normal SAGE libraries are not derived from cultured cell lines. We have also compared our data with publicly available SAGE data obtained from primary tumors and "normal" HOSE-derived cell lines. We have thus identified several known and novel genes whose expressions are elevated in ovarian cancer. These include but are not limited to CLDN3, WFDC2, FOLR1, COL18A1, CCND1, and FLJ12988. Furthermore, we found marked differences in gene expression patterns in primary HOSE tissue compared with cultured HOSE. The use of HOSE tissue as a control for these experiments, along with hierarchical clustering analysis, identified several potentially novel biomarkers of ovarian cancer, including TACC3, CD9, GNAI2, AHCY, CCT3, and HMGA1. In summary, these data identify several genes whose elevated expressions have not been observed previously in ovarian cancer, confirm the validity of several existing markers, and provide a foundation for future studies in the understanding and management of this disease.

A DNA sequence polymorphism in the endoglin gene is not associated with intracranial aneurysm or aneurysmal subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Endoglin is a member of the transforming growth factor beta family of proteins and plays a central role in vascular growth and development. There have been conflicting reports that polymorphic variation in the endoglin gene is a risk factor for intracranial aneurysms (IAs). We sought to further investigate the intron 7 5'-TCCCCC-3' endoglin polymorphism as a risk factor for IA and subarachnoid hemorrhage (SAH) in a population of patients from western Pennsylvania. METHODS: We genotyped 98 IA patients and 191 unaffected controls for a length polymorphism in intron 7 using PCR-based methods. RESULTS: The endoglin polymorphism was not associated with IA or the incidence of aneurysm rupture and SAH. No association was found when data were stratified by smoking and hypertension. CONCLUSION: These data, from a population recruited in western Pennsylvania, support recent findings in Japanese and German populations that, despite earlier observation to the contrary, endoglin is not associated with IA. Furthermore, our study extends previous observations by demonstrating no association between endoglin and either IA or SAH regardless of whether data were stratified for modifiable risk factors such as smoking and hypertension.

Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease.

To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up- or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor alpha, IL-1beta, and IFN-gamma) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (+/+) mice but not detected in that of EGR1 null (-/-) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.