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.

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip.

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease.

Metabolomic disorders: confirmed presence of potentially treatable abnormalities in patients with treatment refractory depression and suicidal behavior.

BACKGROUND: Refractory depression is a devastating condition with significant morbidity, mortality, and societal cost. Approximately 15% of patients with major depressive disorder are refractory to currently available treatments. We hypothesized metabolic abnormalities contributing to treatment refractory depression are associated with distinct findings identifiable in the cerebrospinal fluid (CSF). Our hypothesis was confirmed by a previous small case-controlled study. Here we present a second, larger replication study. METHODS: We conducted a case-controlled, targeted, metabolomic evaluation of 141 adolescent and adult patients with well-characterized history of depression refractory to three maximum-dose, adequate-duration medication treatments, and 36 healthy controls. Plasma, urine, and CSF metabolic profiling were performed by coupled gas chromatography/mass spectrometry, and high-performance liquid chromatography, electrospray ionization, tandem mass spectrometry. RESULTS: Abnormalities were identified in 67 of 141 treatment refractory depression participants. The CSF abnormalities included: low cerebral folate (n = 20), low tetrahydrobiopterin intermediates (n = 11), and borderline low-tetrahydrobiopterin intermediates (n = 20). Serum abnormalities included abnormal acylcarnitine profile (n = 12) and abnormal serum amino acids (n = 20). Eighteen patients presented with two or more abnormal metabolic findings. Sixteen patients with cerebral folate deficiency and seven with low tetrahydrobiopterin intermediates in CSF showed improvement in depression symptom inventories after treatment with folinic acid and sapropterin, respectively. No healthy controls had a metabolite abnormality. CONCLUSIONS: Examination of metabolic disorders in treatment refractory depression identified an unexpectedly large proportion of patients with potentially treatable abnormalities. The etiology of these abnormalities and their potential roles in pathogenesis remain to be determined.

Neonatal necrotizing enterocolitis-associated DNA methylation signatures in the colon are evident in stool samples of affected individuals.

Aim: Neonatal necrotizing enterocolitis (NEC) is a deadly and unpredictable gastrointestinal disease, for which no biomarker exists. We aimed to describe the methylation patterns in stool and colon from infants with NEC. Methods: We performed a high-resolution genome-wide epigenomic analysis using solution-phase hybridization and next-generation sequencing of bisulfite-converted DNA. Results: Our data reveal significant genomic hypermethylation in NEC tissues compared with non-NEC controls. These changes were more pronounced in regions outside CpG islands and gene regulatory elements, suggesting that NEC-specific hypermethylation is not a nonspecific global phenomenon. Conclusions: This study provides evidence of a methylomic signature associated with NEC that is detectable noninvasively and provides a new opportunity for the development of a novel diagnostic method for NEC.

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.

Chromosome 15q13.3 microduplications are associated with treatment refractory major depressive disorder.

Major depressive disorder (MDD) affects approximately 15 million Americans. Approximately 2 million of these are classified as being refractory to treatment (TR-MDD). Because of the lack of available therapies for TR-MDD, and the high risk of suicide, there is interest in identifying new treatment modalities and diagnostic methods. Understanding of the impact of genomic copy number variation in the etiology of a variety of neuropsychiatric phenotypes is increasing. Low copy repeat elements at 15q13.3 facilitate non-allelic homologous recombination, resulting in recurrent copy number variants (CNVs). Numerous reports have described association between microdeletions in this region and a variety of neuropsychiatric phenotypes, with CHRNA7 implicated as a candidate gene. However, the pathogenicity of 15q13.3 duplications is less clear. As part of an ongoing study, in which we have identified a number of metabolomic anomalies in spinal fluid from TR-MDD patients, we also evaluated genomic copy number variation in patients (n = 125) and controls (n = 26) via array-based copy number genomic hybridization (CGH); the case frequency was compared with frequencies reported in a prior study as well as a larger population-sized cohort. We identified five TR-MDD patients with microduplications involving CHRNA7. CHRNA7 duplications are the most common CNVs identified by clinical CGH in this cohort. Therefore, this study provides insight into the potential involvement of CHRNA7 duplications in the etiology of TR-MDD and informs those involved with care of affected individuals.

DNA methylation alters transcriptional rates of differentially expressed genes and contributes to pathophysiology in mice fed a high fat diet.

OBJECTIVE: Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity. METHODS: RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in vivo knockdown of an identified driver gene, Phlda1, were used to validate the results. RESULTS: A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype. CONCLUSIONS: We conclude that a high fat diet alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity.

Comparative evaluation of the Minimally-Invasive Karyotyping (MINK) algorithm for non-invasive prenatal testing.

Minimally Invasive Karyotyping (MINK) was communicated in 2009 as a novel method for the non-invasive detection of fetal copy number anomalies in maternal plasma DNA. The original manuscript illustrated the potential of MINK using a model system in which fragmented genomic DNA obtained from a trisomy 21 male individual was mixed with that of his karyotypically normal mother at dilutions representing fetal fractions found in maternal plasma. Although it has been previously shown that MINK is able to non-invasively detect fetal microdeletions, its utility for aneuploidy detection in maternal plasma has not previously been demonstrated. The current study illustrates the ability of MINK to detect common aneuploidy in early gestation, compares its performance to other published third party methods (and related software packages) for prenatal aneuploidy detection and evaluates the performance of these methods across a range of sequencing read inputs. Plasma samples were obtained from 416 pregnant women between gestational weeks 8.1 and 34.4. Shotgun DNA sequencing was performed and data analyzed using MINK RAPIDR and WISECONDOR. MINK performed with greater accuracy than RAPIDR and WISECONDOR, correctly identifying 60 out of 61 true trisomy cases, and reporting only one false positive in 355 normal pregnancies. Significantly, MINK achieved accurate detection of trisomy 21 using just 2 million aligned input reads, whereas WISECONDOR required 6 million reads and RAPIDR did not achieve complete accuracy at any read input tested. In conclusion, we demonstrate that MINK provides an analysis pipeline for the detection of fetal aneuploidy in samples of maternal plasma DNA.

Neurometabolic Disorders: Potentially Treatable Abnormalities in Patients With Treatment-Refractory Depression and Suicidal Behavior.

OBJECTIVE: Treatment-refractory depression is a devastating condition with significant morbidity, mortality, and societal cost. At least 15% of cases of major depressive disorder remain refractory to treatment. The authors previously identified a young adult with treatment-refractory depression and multiple suicide attempts with an associated severe deficiency of CSF tetrahydrobiopterin, a critical cofactor for monoamine neurotransmitter synthesis. Treatment with sapropterin, a tetrahydrobiopterin analogue, led to dramatic and long-lasting remission of depression. This sentinel case led the authors to hypothesize that the incidence of metabolic abnormalities contributing to treatment-refractory depression is underrecognized. METHOD: The authors conducted a case-control, targeted, metabolomic evaluation of 33 adolescent and young adult patients with well-characterized histories of treatment-refractory depression (at least three maximum-dose, adequate-duration medication treatments), and 16 healthy comparison subjects. Plasma, urine, and CSF metabolic profiling were performed by coupled gas chromatography/mass spectrometry and high-performance liquid chromatography electrospray ionization tandem mass spectrometry. RESULTS: CSF metabolite abnormalities were identified in 21 of the 33 participants with treatment-refractory depression. Cerebral folate deficiency (N=12) was most common, with normal serum folate levels and low CSF 5-methyltetrahydrofolate (5-MTHF) levels. All patients with cerebral folate deficiency, including one with low CSF levels of 5-MTHF and tetrahydrobiopterin intermediates, showed improvement in depression symptom inventories after treatment with folinic acid; the patient with low tetrahydrobiopterin also received sapropterin. None of the healthy comparison subjects had a metabolite abnormality. CONCLUSIONS: Examination of metabolic disorders in treatment-refractory depression identified an unexpectedly large proportion of patients with potentially treatable abnormalities. The etiology of these abnormalities remains to be determined.

Correction: High Levels of Sample-to-Sample Variation Confound Data Analysis for Non-Invasive Prenatal Screening of Fetal Microdeletions.

[This corrects the article DOI: 10.1371/journal.pone.0153182.].

High Levels of Sample-to-Sample Variation Confound Data Analysis for Non-Invasive Prenatal Screening of Fetal Microdeletions.

Our goal was to test the hypothesis that inter-individual genomic copy number variation in control samples is a confounding factor in the non-invasive prenatal detection of fetal microdeletions via the sequence-based analysis of maternal plasma DNA. The database of genomic variants (DGV) was used to determine the "Genomic Variants Frequency" (GVF) for each 50kb region in the human genome. Whole genome sequencing of fifteen karyotypically normal maternal plasma and six CVS DNA controls samples was performed. The coefficient of variation of relative read counts (cv.RTC) for these samples was determined for each 50kb region. Maternal plasma from two pregnancies affected with a chromosome 5p microdeletion was also sequenced, and analyzed using the GCREM algorithm. We found strong correlation between high variance in read counts and GVF amongst controls. Consequently we were unable to confirm the presence of the microdeletion via sequencing of maternal plasma samples obtained from two sequential affected pregnancies. Caution should be exercised when performing NIPT for microdeletions. It is vital to develop our understanding of the factors that impact the sensitivity and specificity of these approaches. In particular, benign copy number variation amongst controls is a major confounder, and their effects should be corrected bioinformatically.

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.

Maternal cell-free DNA-based screening for fetal microdeletion and the importance of careful diagnostic follow-up.

BACKGROUND: Noninvasive prenatal screening (NIPS) by next-generation sequencing of cell-free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21 in high risk pregnancies. NIPS can identify fetal genomic microdeletions; however, sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) without reporting the genomic coordinates or whether the deletion is maternal or fetal. Here we describe a phenotypically normal mother and fetus who tested positive for atypical 22q deletion via maternal plasma cfDNA testing. METHODS: We performed cfDNA sequencing on saved maternal plasma obtained at 11 weeks of gestation from a phenotypically normal woman with a singleton pregnancy whose earlier screening at a commercial laboratory was reported to be positive for a 22q11.2 microdeletion. Fluorescence in situ hybridization and chromosomal microarray diagnostic genetic tests were done postnatally. CONCLUSION: NIPS detected a 22q microdeletion that, upon diagnostic workup, did not include the DiGeorge critical region. Diagnostic prenatal or postnatal testing with chromosomal microarray and appropriate parental studies to determine precise genomic coordinates and inheritance should follow a positive microdeletion NIPS result.

Recent advances of genomic testing in perinatal medicine.

Rapid progress in genomic medicine in recent years has made it possible to diagnose subtle genetic abnormalities in a clinical setting on routine basis. This has allowed for detailed genotype-phenotype correlations and the identification of the genetic basis of many congenital anomalies. In addition to the availability of chromosomal microarray analysis, exome and whole-genome sequencing on pre- and postnatal samples of cell-free DNA has revolutionized the field of prenatal diagnosis. Incorporation of these technologies in perinatal pathology is bound to play a major role in coming years. In this communication, we briefly present the current experience with use of classical chromosome analysis, fluorescence in situ hybridization, and microarray testing, development of whole-genome analysis by next-generation sequencing technology, offer a detailed review of the history and current status of non-invasive prenatal testing using cell-free DNA, and discuss the advents of these new genomic technologies in perinatal medicine.

High resolution non-invasive detection of a fetal microdeletion using the GCREM algorithm.

BACKGROUND/OBJECTIVE: The non-invasive prenatal detection of fetal microdeletions becomes increasingly challenging as the size of the mutation decreases, with current practical lower limits in the range of a few megabases. Our goals were to explore the lower limits of microdeletion size detection via non-invasive prenatal tests using Minimally Invasive Karyotyping (MINK) and introduce/evaluate a novel statistical approach we recently developed called the GC Content Random Effect Model (GCREM). METHODS: Maternal plasma was obtained from a pregnancy affected by a 4.2-Mb fetal microdeletion and three normal controls. Plasma DNA was subjected to capture an 8-Mb sequence spanning the breakpoint region and sequence. Data were analyzed with our published method, MINK, and a new method called GCREM. RESULTS: The 8-Mb capture segment was divided into either 38 or 76 non-overlapping regions of 200 and 100 Kb, respectively. At 200 Kb resolution, using GCREM (but not MINK), we obtained significant adjusted p-values for all 20 regions overlapping the deleted sequence, and non-significant p-values for all 18 reference regions. At 100 Kb resolution, GCREM identified significant adjusted p-values for all but one 100-Kb region located inside the deleted region. CONCLUSION: Targeted sequencing and GCREM analysis may enable cost effective detection of fetal microdeletions and microduplications at high resolution.

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.

Comprehensive analysis of the transcriptional response of human decidual cells to lipopolysaccharide stimulation.

Decidual cells are central to innate immunity at the maternal/fetal interface. We sought to characterize the response of decidual cells to stimulation and then removal of lipopolysaccharide (LPS) using a whole genome approach. Decidual cells were isolated from term unlabored cesarean sections. Cells were stimulated with LPS and RNA isolated both pre-stimulation and 2 and 24 h post-stimulation. Media were changed and RNA extracted 48 h later. Gene expression was measured using Agilent 44K whole genome microarrays. Data were visualized and interpreted using Ingenuity Pathway Analysis (IPA) software and selected (n=5) target gene expression was verified with quantitative real-time PCR. Genes related to immune function were up-regulated at 2 and 24 h after LPS exposure and then generally returned to baseline or were at least substantially reduced after LPS removal. Pathway analysis also revealed that genes involved in lipid metabolism (specifically cholesterol and steroid biosynthesis), iron metabolism, and the plasminogen system were coordinately altered following exposure to LPS. Our novel, preliminary findings provide insight into possible mechanisms via which the host inflammatory response could contribute to preterm birth and warrant further investigation in preterm samples.