Raghu Metpally

Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illuminas TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.

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.

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.

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

Background Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression. Methodology/Principal Findings Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects. Conclusions/Significance This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

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.

Rare Variant Pathogenicity Triage and Inclusion of Synonymous Variants Improves Analysis of Disease Associations

Many G protein-coupled receptors (GPCRs) lack common variants that lead to reproducible genome-wide disease associations. Here we used rare variant approaches to assess the disease associations of 85 orphan or understudied GPCRs in an unselected cohort of 51,289 individuals. Rare loss-of-function variants, missense variants predicted to be pathogenic or likely pathogenic, and a subset of rare synonymous variants were used as independent data sets for sequence kernel association testing (SKAT). Strong, phenome-wide disease associations shared by two or more variant categories were found for 39% of the GPCRs. Validating the bioinformatics and SKAT analyses, functional characterization of rare missense and synonymous variants of GPR39, a Family A GPCR, showed altered expression and/or Zn2+-mediated signaling for members of both variant classes. Results support the utility of rare variant analyses for identifying disease associations for genes that lack common variants, while also highlighting the functional importance of rare synonymous variants. Author summary Rare variant approaches have emerged as a viable way to identify disease associations for genes without clinically important common variants. Rare synonymous variants are generally considered benign. We demonstrate that rare synonymous variants represent a potentially important dataset for deriving disease associations, here applied to analysis of a set of orphan or understudied GPCRs. Synonymous variants yielded disease associations in common with loss-of-function or missense variants in the same gene. We rationalize their associations with disease by confirming their impact on expression and agonist activation of a representative example, GPR39. This study highlights the importance of rare synonymous variants in human physiology, and argues for their routine inclusion in any comprehensive analysis of genomic variants as potential causes of disease.

Exome Sequencing–Based Screening for BRCA1/2 Expected Pathogenic Variants Among Adult Biobank Participants

Key Points Question Can population-level genomic screening identify those at risk for disease? Findings In this cross-sectional study of an unselected population cohort of 50 726 adults who underwent exome sequencing, pathogenic and likely pathogenic BRCA1 and BRCA2 variants were found in a higher proportion of patients than was previously reported. Meaning Current methods to identify BRCA1/2 variant carriers may not be sufficient as a screening tool; population genomic screening for hereditary breast and ovarian cancer may better identify patients at high risk and provide an intervention opportunity to reduce mortality and morbidity.

Sa1685 Circulating Micro-RNAs Pattern in Relationship to Hepatic Fibrosis Progression Among Patients With Chronic Hepatitis C

been identified as a dominant stimulator for ECM production in HSC. Our results indicated that pre-treatment with 0.75μM of CYD0692, blocked TGF β1-induced FN expression in LX-2 andHSC-T6 cells; and accordingly, decreased the downstream factors of TGFβsignaling, such as Phospho-Smad2/3 and phospho-ERK. Conclusion. In comparison with oridonin, its novel derivative CYD0692 has demonstrated to be a more potent and safer anti-fibrogenic agent for the hepatic fibrosis.

Abstract 4148: MicroRNA biomarkers prognostic for disease-free and overall survival in stage I and II lung adenocarcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Approximately 50% of all non-small cell lung carcinoma (NSCLC) cases in the United States are adenocarcinoma histology. While definitive lung cancer resection offers a chance for cure in stage I and II NSCLC, up to 57% of patients will have disease relapse or metastatic disease within 5 years. A majority of the relapsed cases will take place within 2 years of definitive surgical resection. Thus, pathologic staging alone does not stratify patients for prognosis sufficiently. Identifying patients with aggressive versus indolent tumors would have critical implications for directing treatment and designing future clinical trials. We propose using microRNA (miRNA) for discovery and validation of prognostic biomarkers for stage I and II lung adenocarcinoma. Methods: For the Discovery Cohort, we identified treatment-naive AJCC 7th edition stage I and II lung adenocarcinoma patient samples from 26 disease-free survivors (DFS) and overall survival (OS) at least 47 months and 14 patients with DFS and overall survival less than 23 months. Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples. Differential miRNA array expression profiling between these two groups was assessed in the Discovery cohort on an 8x15 Agilent microarray printed with 886 miRNAs. Target miRNAs were selected based on minimum of 2-fold change in expression and Welch t-test p-value cut-off of 47 months and N=12 with DFS and OS 47 months and N=4 DFS/OS <23 months) is ongoing. Conclusion: We have validated miRNAs prognostic for DFS and OS in stage I and II lung adenocarcinoma in at least one cohort of 89 patients so far. A two miRNA classifier is currently being evaluated and we anticipate results for accuracy prediction on the Independent cohort at the time of the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4148. doi:1538-7445.AM2012-4148

Abstract B198: Integrated genomic analysis of breast cancer CNS metastasis to the CNS.

With improvements in systemic therapies, the brain is becoming an increasingly common sanctuary site of metastatic disease in patients with breast cancer. There have been limited improvements in the treatment of brain metastases and current treatment paradigms focus on resection and radiation therapy. As cancer dissemination to the brain currently leads to fatal outcomes developing new therapeutic approaches is paramount. We performed a deep integrated genomic and epigenomic analysis to catalogue the compendium of common and rare events that underlie breast cancer CNS metastasis. Our samples represent breast cancer CNS metastasis, primary breast cancer, non-neoplastic brain and non-neoplastic breast tissue. We performed copy number analysis using array-based comparative genomic hybridization (aCGH, Agilent SurePrint G3 Human High-Resolution Discovery Microarray Kit 1×1M platform); gene expression profiling (GEP) (Agilent, Human GE 4×44 v2 Microarray platform); and DNA methylation analysis (Illumina, HumanMethylation 27 BeadChip). Here we report frequent large chromosomal gains in 1q, 8q, 10p, 21q, and 22q. Frequent large chromosomal deletions were also seen on 8p, and 17p. Focal gains and losses occurred frequently on chromosomes 14, 17 and 22. The PAM50 breast cancer intrinsic classifier was used to classify CNS metastasis tumors. The most common molecular subtypes identified were Luminal B, Her2+/ER negative and Basal-like tumors. Although breast CNS metastasis tumors retained features of their original intrinsic subtype, additional changes were acquired. In addition, DNA methylation analysis of the most variable methylated genes demonstrated epigenetic similarities among the individual molecular subtypes and that overall CNS metastasis is associated with hypermethylation. On average 108 genes had alterations by copy number, gene expression and DNA methylation. Among these are candidate tumor suppressor genes SERPINF1 and SFRP1. Pathway enrichment using Ingenuity Systems revealed a large number of enrichment maps concentrated around cell cycle and G2/M transition. This enrichment contained important cancer genes such AURKA, AURKB and FOXM1. Current studies are ongoing to validate novel candidates genes using cross platform and independent sample cohorts. Characterization of such candidates is expected to lead to novel therapeutic approaches and an enhanced understanding of the biology mediating breast CNS metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B198.