Ivana Malenica

Profiles of Extracellular miRNA in Cerebrospinal Fluid and Serum from Patients with Alzheimer's and Parkinson's Diseases Correlate with Disease Status and Features of Pathology

The discovery and reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue- such as the inability to biopsy or test tissue from the central nervous system directly. RNAs originating from hard to access tissues, such as neurons within the brain and spinal cord, have the potential to get to the periphery where they can be detected non-invasively. The formation and extracellular release of microvesicles and RNA binding proteins have been found to carry RNA from cells of the central nervous system to the periphery and protect the RNA from degradation. Extracellular miRNAs detectable in peripheral circulation can provide information about cellular changes associated with human health and disease. In order to associate miRNA signals present in cell-free peripheral biofluids with neurodegenerative disease status of patients with Alzheimers and Parkinsons diseases, we assessed the miRNA content in cerebrospinal fluid and serum from postmortem subjects with full neuropathology evaluations. We profiled the miRNA content from 69 patients with Alzheimers disease, 67 with Parkinsons disease and 78 neurologically normal controls using next generation small RNA sequencing (NGS). We report the average abundance of each detected miRNA in cerebrospinal fluid and in serum and describe 13 novel miRNAs that were identified. We correlated changes in miRNA expression with aspects of disease severity such as Braak stage, dementia status, plaque and tangle densities, and the presence and severity of Lewy body pathology. Many of the differentially expressed miRNAs detected in peripheral cell-free cerebrospinal fluid and serum were previously reported in the literature to be deregulated in brain tissue from patients with neurodegenerative disease. These data indicate that extracellular miRNAs detectable in the cerebrospinal fluid and serum are reflective of cell-based changes in pathology and can be used to assess disease progression and therapeutic efficacy.

High-throughput sequencing reveals altered expression of hepatic microRNAs in nonalcoholic fatty liver disease-related fibrosis.

Recent evidence suggests that microRNAs (miRNAs), small, noncoding RNA molecules that regulate gene expression, may play a role in the regulation of metabolic disorders, including nonalcoholic fatty liver disease (NAFLD). To identify miRNAs that mediate NAFLD-related fibrosis, we used high-throughput sequencing to assess miRNAs obtained from liver biopsies of 15 individuals without NAFLD fibrosis (F0) and 15 individuals with severe NAFLD fibrosis or cirrhosis (F3-F4), matched for age, sex, body mass index, type 2 diabetes status, hemoglobin A1c, and use of diabetes medications. We used DESeq2 and Kruskal-Wallis test to identify miRNAs that were differentially expressed between NAFLD patients with or without fibrosis, adjusting for multiple testing using Bonferroni correction. We identified a total of 75 miRNAs showing statistically significant evidence (adjusted P value <0.05) for differential expression between the 2 groups, including 30 upregulated and 45 downregulated miRNAs. Quantitative reverse-transcription polymerase chain reaction analysis of selected miRNAs identified by sequencing validated 9 of 11 of the top differentially expressed miRNAs. We performed functional enrichment analysis of dysregulated miRNAs and identified several potential gene targets related to NAFLD-related fibrosis including hepatic fibrosis, hepatic stellate cell activation, transforming growth factor beta signaling, and apoptosis signaling. We identified forkhead box O3 and F-box WD repeat domain containing 7, E3 ubiquitin protein ligase (FBXW7) as potential targets of miR-182, and found that levels of forkhead box O3, but not FBXW7, were significantly decreased in fibrotic samples. These findings support a role for hepatic miRNAs in the pathogenesis of NAFLD-related fibrosis and yield possible new insight into the molecular mechanisms underlying the initiation and progression of liver fibrosis and cirrhosis.

Comparison of Analysis Tools for miRNA High Throughput Sequencing Using Nerve Crush as a Model

Recent advances in sample preparation and analysis for next generation sequencing have made it possible to profile and discover new miRNAs in a high throughput manner. In the case of neurological disease and injury, these types of experiments have been more limited. Possibly because tissues such as the brain and spinal cord are inaccessible for direct sampling in living patients, and indirect sampling of blood and cerebrospinal fluid are affected by low amounts of RNA. We used a mouse model to examine changes in miRNA expression in response to acute nerve crush. We assayed miRNA from both muscle tissue and blood plasma. We examined how the depth of coverage (the number of mapped reads) changed the number of detectable miRNAs in each sample type. We also found that samples with very low starting amounts of RNA (mouse plasma) made high depth of mature miRNA coverage more difficult to obtain. Each tissue must be assessed independently for the depth of coverage required to adequately power detection of differential expression, weighed against the cost of sequencing that sample to the adequate depth. We explored the changes in total mapped reads and differential expression results generated by three different software packages: miRDeep2, miRNAKey, and miRExpress and two different analysis packages, DESeq and EdgeR. We also examine the accuracy of using miRDeep2 to predict novel miRNAs and subsequently detect them in the samples using qRT-PCR.

Meeting report: Discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

microRNA changes in liver tissue associated with fibrosis progression in patients with hepatitis C

Accumulating evidence indicates that microRNAs play a role in a number of disease processes including the pathogenesis of liver fibrosis in hepatitis C infection. Our goal is to add to the accruing information regarding microRNA deregulation in liver fibrosis to increase our understanding of the underlying mechanisms of pathology and progression.

A Frame-Shift Mutation in CAV1 Is Associated with a Severe Neonatal Progeroid and Lipodystrophy Syndrome.

A 3-year-old female patient presenting with an unknown syndrome of a neonatal progeroid appearance, lipodystrophy, pulmonary hypertension, cutis marmorata, feeding disorder and failure to thrive was investigated by whole-genome sequencing. This revealed a de novo, heterozygous, frame-shift mutation in the Caveolin1 gene (CAV1) (p.Phe160X). Mutations in CAV1, encoding the main component of the caveolae in plasma membranes, cause Berardinelli-Seip congenital lipodystrophy type 3 (BSCL). Although BSCL is recessive, heterozygous carriers either show a reduced phenotype of partial lipodystrophy, pulmonary hypertension, or no phenotype. To investigate the pathogenic mechanisms underlying this syndrome in more depth, we performed next generation RNA sequencing of peripheral blood, which showed several dysregulated pathways in the patient that might be related to the phenotypic progeroid features (apoptosis, DNA repair/replication, mitochondrial). Secondly, we found a significant down-regulation of known Cav1 interaction partners, verifying the dysfunction of CAV1. Other known progeroid genes and lipodystrophy genes were also dysregulated. Next, western blotting of lysates of cultured fibroblasts showed that the patient shows a significantly decreased expression of wild-type CAV1 protein, demonstrating a loss-of-function mutation, though her phenotype is more severe that other heterozygotes with similar mutations. This phenotypic variety could be explained by differences in genetic background. Indications for this are supported by additional rare variants we found in AGPAT2 and LPIN1 lipodystrophy genes. CAV1, AGPAT2 and LPIN1 all play an important role in triacylglycerol (TAG) biosynthesis in adipose tissue, and the defective function in different parts of this pathway, though not all to the same extend, could contribute to a more severe lipoatrophic phenotype in this patient. In conclusion, we report, for the first time, an association of CAV1 dysfunction with a syndrome of severe premature aging and lipodystrophy. This may contribute to a better understanding of the aging process and pathogenic mechanisms that contribute to premature aging.

Characterization of X chromosome inactivation using integrated analysis of whole-exome and mRNA sequencing

In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patients exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation.

A De Novo Mutation in TEAD1 Causes Non–X-Linked Aicardi Syndrome

PURPOSE Aicardi syndrome (AIC) is a congenital neurodevelopmental disorder characterized by infantile spasms, agenesis of the corpus callosum, and chorioretinal lacunae. Variation in phenotype and disease severity is well documented, but chorioretinal lacunae represent the most constant pathological feature. Aicardi syndrome is believed to be an X-linked-dominant disorder occurring almost exclusively in females, although 46, XY males with AIC have been described. The purpose of this study is to identify genetic factors and pathways involved in AIC. METHODS We performed exome/genome sequencing of 10 children diagnosed with AIC and their parents and performed RNA sequencing on blood samples from nine cases, their parents, and unrelated controls. RESULTS We identified a de novo mutation in autosomal gene TEAD1, expressed in the retina and brain, in a patient with AIC. Mutations in TEAD1 have previously been associated with Sveinssons chorioretinal atrophy, characterized by chorioretinal degeneration. This demonstrates that TEAD1 mutations can lead to different chorioretinal complications. In addition, we found that altered expression of genes associated with synaptic plasticity, neuronal development, retinal development, and cell cycle control/apoptosis is an important underlying potential pathogenic mechanism shared among cases. Last, we found a case with skewed X inactivation, supporting the idea that nonrandom X inactivation might be important in AIC. CONCLUSIONS We expand the phenotype of TEAD1 mutations, demonstrate its importance in chorioretinal complications, and propose the first putative pathogenic mechanisms underlying AIC. Our data suggest that AIC is a genetically heterogeneous disease and is not restricted to the X chromosome, and that TEAD1 mutations may be present in male patients.

Serum MicroRNAs Reflect Injury Severity in a Large Animal Model of Thoracic Spinal Cord Injury

Therapeutic development for spinal cord injury is hindered by the difficulty in conducting clinical trials, which to date have relied solely on functional outcome measures for patient enrollment, stratification, and evaluation. Biological biomarkers that accurately classify injury severity and predict neurologic outcome would represent a paradigm shift in the way spinal cord injury clinical trials could be conducted. MicroRNAs have emerged as attractive biomarker candidates due to their stability in biological fluids, their phylogenetic similarities, and their tissue specificity. Here we characterized a porcine model of spinal cord injury using a combined behavioural, histological, and molecular approach. We performed next-generation sequencing on microRNAs in serum samples collected before injury and then at 1, 3, and 5 days post injury. We identified 58, 21, 9, and 7 altered miRNA after severe, moderate, and mild spinal cord injury, and SHAM surgery, respectively. These data were combined with behavioural and histological analysis. Overall miRNA expression at 1 and 3 days post injury strongly correlates with outcome measures at 12 weeks post injury. The data presented here indicate that serum miRNAs are promising candidates as biomarkers for the evaluation of injury severity for spinal cord injury or other forms of traumatic, acute, neurologic injury.

Differentially methylated loci in NAFLD cirrhosis are associated with key signaling pathways

Altered DNA methylation events contribute to the pathogenesis and progression of metabolic disorders, including nonalcoholic fatty liver disease (NAFLD). Investigations of global DNA methylation patterns in liver biopsies representing severe NAFLD fibrosis have been limited. We used the HumanMethylation 450K BeadChip to analyze genome-wide methylation in patients with biopsy-proven grade 3/4 NAFLD fibrosis/cirrhosis (N = 14) and age- and sex-matched controls with normal histology (N = 15). We identified 208 CpG islands (CGIs), including 99 hypomethylated and 109 hypermethylated CGIs, showing statistically significant evidence (adjusted P value < 0.05) for differential methylation between cirrhotic and normal samples. Comparison of β values for each CGI to the read count of its corresponding gene obtained from RNA-sequencing analysis revealed negative correlation (adjusted P value < 0.05) for 34 transcripts. These findings provide supporting evidence for a role for CpG methylation in the pathogenesis of NAFLD-related cirrhosis, including confirmation of previously reported differentially methylated CGIs, and contribute new insight into the molecular mechanisms underlying the initiation and progression of liver fibrosis and cirrhosis.