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UTILITY OF PERIPHERAL miRNA EXPRESSION PROFILES AS BIOMARKERS AND PATHOLOGICAL INDICATORS OF NEUROPSYCHIATRIC DISEASE
Abstracts
S915 n
Ashish Kumar ,1, Sobha Puppala1, Ellen Quillen1, Jennifer L. Neary2, Joanne E. Curran3, Ravindranath Duggirala3, David C. Glahn4, John Blangero3, Dianne Cruz5, Mark Z. Kos3, Melanie A. Carless1
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.240
1
Texas Biomedical Research Institute Avagen Health 3 University of Texas Rio Grande Valley 4 Yale University 5 Duke Kathleen Price Bryan Brain Bank and Biorepository 2
Background: We recently assessed blood-based miRNA expression profiles for association with age in a cohort of 895 Mexican Americans. Of 2,747 miRNAs that were tested (including SNP-derived and novel miRNAs in addition to those reported in miRBase 21), we identified significant associations for 46 known miRNAs (po1.82 10-5) and chronological age. miRNA over-representation analysis through the miEAA tool identified Alzheimer's disease as the top-ranked disease category (p=5.31 10–34) over-represented in our data set. Several published studies have identified multiple miRNAs associated with Alzheimer's disease, including miR-34a-5p, which has been proposed as a biomarker for diagnosis and was significantly associated with age in our study (p=1.14 10–11). In addition, miR-34a-5p has been implicated as either a peripheral biomarker, or within brain tissue, in unipolar and bipolar depression and schizophrenia. Published cell studies implicate miR-34a in neuronal differentiation and maturation and animal studies indicate involvement in Alzheimer's disease pathology and related cognitive deficits, as well as in learning and emotion. Methods: We conducted in vivo studies to test for correlations between blood and brain miR-34a-5p expression across seven brain regions in four baboons. Then, to investigate the consequences of increased miRNA expression on gene expression, we transfected four neuronal and glial cell lines (CHP-212, HCN-2, SVG-P12 and A172) with a miR-34a-5p mimic. We performed functional annotation of genes that were at least 2-fold up- or down-regulated in each of the cell lines using DAVID. Results: Our in vivo studies using baboons as a model organism show moderate to strong correlation of miR-34a5p expression across blood and amygdala (r = 0.69), dorsal anterior cingulate cortex (r= 0.95), hypothalamus (r = 0.77), lateral orbitofrontal cortex (r= 0.81) and medial orbitofrontal cortex (0.56), further strengthening its potential role in neuropsychiatric disease. Our in vitro studies identified several functional categories and gene ontologies that were consistent across two or more cell lines, including phosphoprotein, alternative splicing, protein binding and cell cycle. Discussion: Through a combination of in vivo, in vitro and in silico studies, we strengthen the argument for miR-34a5p as an important player in neuropsychiatric research. Our findings suggest important downstream consequences of induced overexpression of miR-34a-5p. We are currently trying to develop a novel unbiased in vitro method to detect miRNA-mRNA binding interactions that would be broadly applicable to any cell type using miR-34a-5p as a model.
SU52. UTILITY OF PERIPHERAL miRNA EXPRESSION PROFILES AS BIOMARKERS AND PATHOLOGICAL INDICATORS OF NEUROPSYCHIATRIC DISEASE n
Melanie Carless ,1, Sobha Puppala1, Ellen Quillen1, Jennifer Neary2, Peter Fox3, Rene Olvera3, Joanne Curran4, Ravindranath Duggirala4, Cun Li5, David Glahn6, John Blangero4, Dianne Cruz7, Peter Nathanielsz5 1
Texas Biomedical Research Institute Avagen Health 3 University of Texas Health Science Center at San Antonio 4 University of Texas Rio Grande Valley 5 University of Wyoming 6 Yale University 7 Duke University 2
Background: Given the inaccessibility of brain tissue for large cohort-based and longitudinal studies, analysis of peripheral blood as a surrogate tissue has become a mainstay within the neuropsychiatric field for analysis of gene and miRNA expression and DNA methylation. Although the importance of peripheral biomarkers as diagnostic and prognostic indicators is well recognized, translation of findings into meaningful pathology is more difficult to achieve. Lack of validation of peripheral biomarkers in brain tissue may be confounded by smaller sample sizes, investigation of inappropriate brain regions, or post-mortem complications. Methods: To shed some light on the utility of blood-based biomarkers to inform upon pathology of disease, we investigated correlations between miRNA expression levels in blood and brain tissue from 14 cortical and subcortical regions within two groups of baboons (n = 4, n= 6; 7 brain regions each). To better understand how blood-brain correlations might relate to human biology and disease, we investigated a subset of 212 miRNAs whose peripheral blood expression was at least moderately correlated (r Z0.5) with 50% or more of the brain regions examined in a family-based cohort of Mexican Americans (n = 896) for whom we have extensive neurocognitive and neuroanatomical phenotypes as well as peripheral blood miRNA profiles. We tested 145 human peripheral blood-expressed miRNAs for association against 34 cortical and 10 subcortical structures (using magnetic resonance imaging data), and 24 neurocognitive phenotypes. Due to high inter-relatedness and potentially unknown correlations across many of the phenotypes assessed, we corrected only for the number of miRNAs tested, setting the significance threshold at po3.45 10-4. Results: The overall correlation (r) of miRNA expression profiles between blood and 14 different brain regions in baboons ranged from 0.62–0.66. We identified 444 miRNAs that were expressed in 90% of all samples analyzed and of these, 212 showed at least moderate correlation (r Z0.5)
S916
Abstracts
between blood and 50% or more of the brain regions examined. Within this subset of baboon-defined correlated miRNAs, we identified 145 that were also expressed in at least 90% of our human cohort and tested these for association with neurological traits. We identified 34 significant associations between miRNAs and neurological traits. Of these, one of the most interesting findings was association of peripheral miR-144-3p expression with amygdala (p = 8.50 10-5) and hippocampus (p = 5.86 10-5) volumes, as well as thickness of the fusiform gyrus (p = 1.98 10-5). In our baboon study, peripheral expression of this miRNA was highly correlated with expression in the amygdala (r = 1.00) and hippocampus (r= 0.66), expression was not measured in the fusiform gyrus. Discussion: Prior studies have shown that overexpression of miR-144-3p in the basolateral amygdala of mice rescued impaired fear extinction, and also that expression of miR144-3p is increased in the hippocampus of rats following chronic treatment of both lithium and valproate mood stabilizers. Our studies highlight the utility of using peripheral blood as a surrogate tissue for miRNA profiling and indicate that disease associations identified using peripheral blood may point toward pathological processes within the brain, particularly for miRNAs whose expression is correlated between blood and brain.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.241
SU53. GENOME-WIDE DNA METHYLATION ANALYSIS IN THE SALIVA OF CRACK COCAINE DEPENDENTS
with pvalueo0.05, the Differentially Methylated Regions (DMRs, at least seven CpG sites in 300 bases, adjusted p-valueo0.05) were identified using bumphunter. DMRs located in gene promoters were analyzed using Functional Epigenetic Modules (FEM) package to identify gene-gene interaction networks potentially involved with crack cocaine use. Results: Comparison between crack cocaine dependents and controls revealed 3,857 DMRs which were involved with metabolic processes related to nucleic acid, ncRNA and rRNA processing, gene expression and protein-DNA complex assembly. The gene-gene interaction analysis revealed three networks: one containing 11 genes, which were enriched for biological processes related to mitochondrial function; a second network composed by 36 genes which was enriched mainly for glycosylphosphatidylinositol (GPI) activity and extracellular membrane interaction biological processes; and a third network with 22 genes that was enriched for synapse related processes (GABaergic, serotononergic and cholinergic synapses) and, interestingly, biological processes related to visual perception. Discussion: These results suggest that the use of saliva may help to understand the mechanisms underlying the pathology of drug abuse. Changes in DNA methylation found in the saliva revealed perturbation in pathways related to the reward system, often found affected in drug dependents. Moreover, the enrichment analysis of the networks replicated our previous study performed in peripheral blood where molecular functions related to protein activity were associated with cocaine and crack dependency.
Disclosure: Nothing to disclose.
n
Caroline Camilo ,1, Mariana Maschietto2, André Brooking Negrão1, Marcelo Ribeiro3, Ronaldo Laranjeira3, Helena Brentani1, Homero Vallada1
http://dx.doi.org/10.1016/j.euroneuro.2017.08.242
1
University of São Paulo Medical School Brazilian Bioscience National Laboratory, National Center for Research in Energy and Materials 3 Federal University of São Paulo 2
Background: DNA methylation has been increasingly recognized in the etiology of drug dependence. Due to the difficulties in assessing brain tissue in living humans, peripheral cells such as blood and saliva have been explored as alternatives to explore the involvement of DNA methylation in drug dependency. Here, we characterized DNA methylation changes in the saliva from crack cocaine users compared to health controls. Methods: DNA was extracted from saliva of 128 crack cocaine dependents and 109 health controls (males, age 34.8 + /- 9.9 years). Bisulfite converted DNA was hybridized in the Illumina Infinium MethylationEPIC BeadChip arrays. Cellular composition heterogeneity was corrected using RefFreeEwas followed identification of differentially CpG sites using limma (DMSs). Using DMSs
SU54. A PILOT METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING: HYPERMETHYLATION OF THE DEATH-ASSOCIATED PROTEIN 3 (DAP3) AND MIR2110 IN FIBROMYALGIA ASSOCIATED WITH CHILD ABUSE HISTORY AMONG AFRICAN AMERICAN FEMALES n
Hyunhwa Lee ,1, Annette Mullis1, Sijung Yun2, Katrina Isla1, Jonica Estrada1, Nada Lukkahatai3, Leorey Saligan4, Brian Walitt4 1
University of Nevada, Las Vegas Yotta Biomed, LLC 3 Johns Hopkins University 4 National Institutes of Health 2
Background: Epigenetics, such as DNA methylation, has been hypothesized as one possible mechanism to explain the association between adverse childhood experiences and later health problems. The goal of this study was to identify genome-wide DNA methylation markers from peripheral
S915 n
Ashish Kumar ,1, Sobha Puppala1, Ellen Quillen1, Jennifer L. Neary2, Joanne E. Curran3, Ravindranath Duggirala3, David C. Glahn4, John Blangero3, Dianne Cruz5, Mark Z. Kos3, Melanie A. Carless1
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.240
1
Texas Biomedical Research Institute Avagen Health 3 University of Texas Rio Grande Valley 4 Yale University 5 Duke Kathleen Price Bryan Brain Bank and Biorepository 2
Background: We recently assessed blood-based miRNA expression profiles for association with age in a cohort of 895 Mexican Americans. Of 2,747 miRNAs that were tested (including SNP-derived and novel miRNAs in addition to those reported in miRBase 21), we identified significant associations for 46 known miRNAs (po1.82 10-5) and chronological age. miRNA over-representation analysis through the miEAA tool identified Alzheimer's disease as the top-ranked disease category (p=5.31 10–34) over-represented in our data set. Several published studies have identified multiple miRNAs associated with Alzheimer's disease, including miR-34a-5p, which has been proposed as a biomarker for diagnosis and was significantly associated with age in our study (p=1.14 10–11). In addition, miR-34a-5p has been implicated as either a peripheral biomarker, or within brain tissue, in unipolar and bipolar depression and schizophrenia. Published cell studies implicate miR-34a in neuronal differentiation and maturation and animal studies indicate involvement in Alzheimer's disease pathology and related cognitive deficits, as well as in learning and emotion. Methods: We conducted in vivo studies to test for correlations between blood and brain miR-34a-5p expression across seven brain regions in four baboons. Then, to investigate the consequences of increased miRNA expression on gene expression, we transfected four neuronal and glial cell lines (CHP-212, HCN-2, SVG-P12 and A172) with a miR-34a-5p mimic. We performed functional annotation of genes that were at least 2-fold up- or down-regulated in each of the cell lines using DAVID. Results: Our in vivo studies using baboons as a model organism show moderate to strong correlation of miR-34a5p expression across blood and amygdala (r = 0.69), dorsal anterior cingulate cortex (r= 0.95), hypothalamus (r = 0.77), lateral orbitofrontal cortex (r= 0.81) and medial orbitofrontal cortex (0.56), further strengthening its potential role in neuropsychiatric disease. Our in vitro studies identified several functional categories and gene ontologies that were consistent across two or more cell lines, including phosphoprotein, alternative splicing, protein binding and cell cycle. Discussion: Through a combination of in vivo, in vitro and in silico studies, we strengthen the argument for miR-34a5p as an important player in neuropsychiatric research. Our findings suggest important downstream consequences of induced overexpression of miR-34a-5p. We are currently trying to develop a novel unbiased in vitro method to detect miRNA-mRNA binding interactions that would be broadly applicable to any cell type using miR-34a-5p as a model.
SU52. UTILITY OF PERIPHERAL miRNA EXPRESSION PROFILES AS BIOMARKERS AND PATHOLOGICAL INDICATORS OF NEUROPSYCHIATRIC DISEASE n
Melanie Carless ,1, Sobha Puppala1, Ellen Quillen1, Jennifer Neary2, Peter Fox3, Rene Olvera3, Joanne Curran4, Ravindranath Duggirala4, Cun Li5, David Glahn6, John Blangero4, Dianne Cruz7, Peter Nathanielsz5 1
Texas Biomedical Research Institute Avagen Health 3 University of Texas Health Science Center at San Antonio 4 University of Texas Rio Grande Valley 5 University of Wyoming 6 Yale University 7 Duke University 2
Background: Given the inaccessibility of brain tissue for large cohort-based and longitudinal studies, analysis of peripheral blood as a surrogate tissue has become a mainstay within the neuropsychiatric field for analysis of gene and miRNA expression and DNA methylation. Although the importance of peripheral biomarkers as diagnostic and prognostic indicators is well recognized, translation of findings into meaningful pathology is more difficult to achieve. Lack of validation of peripheral biomarkers in brain tissue may be confounded by smaller sample sizes, investigation of inappropriate brain regions, or post-mortem complications. Methods: To shed some light on the utility of blood-based biomarkers to inform upon pathology of disease, we investigated correlations between miRNA expression levels in blood and brain tissue from 14 cortical and subcortical regions within two groups of baboons (n = 4, n= 6; 7 brain regions each). To better understand how blood-brain correlations might relate to human biology and disease, we investigated a subset of 212 miRNAs whose peripheral blood expression was at least moderately correlated (r Z0.5) with 50% or more of the brain regions examined in a family-based cohort of Mexican Americans (n = 896) for whom we have extensive neurocognitive and neuroanatomical phenotypes as well as peripheral blood miRNA profiles. We tested 145 human peripheral blood-expressed miRNAs for association against 34 cortical and 10 subcortical structures (using magnetic resonance imaging data), and 24 neurocognitive phenotypes. Due to high inter-relatedness and potentially unknown correlations across many of the phenotypes assessed, we corrected only for the number of miRNAs tested, setting the significance threshold at po3.45 10-4. Results: The overall correlation (r) of miRNA expression profiles between blood and 14 different brain regions in baboons ranged from 0.62–0.66. We identified 444 miRNAs that were expressed in 90% of all samples analyzed and of these, 212 showed at least moderate correlation (r Z0.5)
S916
Abstracts
between blood and 50% or more of the brain regions examined. Within this subset of baboon-defined correlated miRNAs, we identified 145 that were also expressed in at least 90% of our human cohort and tested these for association with neurological traits. We identified 34 significant associations between miRNAs and neurological traits. Of these, one of the most interesting findings was association of peripheral miR-144-3p expression with amygdala (p = 8.50 10-5) and hippocampus (p = 5.86 10-5) volumes, as well as thickness of the fusiform gyrus (p = 1.98 10-5). In our baboon study, peripheral expression of this miRNA was highly correlated with expression in the amygdala (r = 1.00) and hippocampus (r= 0.66), expression was not measured in the fusiform gyrus. Discussion: Prior studies have shown that overexpression of miR-144-3p in the basolateral amygdala of mice rescued impaired fear extinction, and also that expression of miR144-3p is increased in the hippocampus of rats following chronic treatment of both lithium and valproate mood stabilizers. Our studies highlight the utility of using peripheral blood as a surrogate tissue for miRNA profiling and indicate that disease associations identified using peripheral blood may point toward pathological processes within the brain, particularly for miRNAs whose expression is correlated between blood and brain.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.241
SU53. GENOME-WIDE DNA METHYLATION ANALYSIS IN THE SALIVA OF CRACK COCAINE DEPENDENTS
with pvalueo0.05, the Differentially Methylated Regions (DMRs, at least seven CpG sites in 300 bases, adjusted p-valueo0.05) were identified using bumphunter. DMRs located in gene promoters were analyzed using Functional Epigenetic Modules (FEM) package to identify gene-gene interaction networks potentially involved with crack cocaine use. Results: Comparison between crack cocaine dependents and controls revealed 3,857 DMRs which were involved with metabolic processes related to nucleic acid, ncRNA and rRNA processing, gene expression and protein-DNA complex assembly. The gene-gene interaction analysis revealed three networks: one containing 11 genes, which were enriched for biological processes related to mitochondrial function; a second network composed by 36 genes which was enriched mainly for glycosylphosphatidylinositol (GPI) activity and extracellular membrane interaction biological processes; and a third network with 22 genes that was enriched for synapse related processes (GABaergic, serotononergic and cholinergic synapses) and, interestingly, biological processes related to visual perception. Discussion: These results suggest that the use of saliva may help to understand the mechanisms underlying the pathology of drug abuse. Changes in DNA methylation found in the saliva revealed perturbation in pathways related to the reward system, often found affected in drug dependents. Moreover, the enrichment analysis of the networks replicated our previous study performed in peripheral blood where molecular functions related to protein activity were associated with cocaine and crack dependency.
Disclosure: Nothing to disclose.
n
Caroline Camilo ,1, Mariana Maschietto2, André Brooking Negrão1, Marcelo Ribeiro3, Ronaldo Laranjeira3, Helena Brentani1, Homero Vallada1
http://dx.doi.org/10.1016/j.euroneuro.2017.08.242
1
University of São Paulo Medical School Brazilian Bioscience National Laboratory, National Center for Research in Energy and Materials 3 Federal University of São Paulo 2
Background: DNA methylation has been increasingly recognized in the etiology of drug dependence. Due to the difficulties in assessing brain tissue in living humans, peripheral cells such as blood and saliva have been explored as alternatives to explore the involvement of DNA methylation in drug dependency. Here, we characterized DNA methylation changes in the saliva from crack cocaine users compared to health controls. Methods: DNA was extracted from saliva of 128 crack cocaine dependents and 109 health controls (males, age 34.8 + /- 9.9 years). Bisulfite converted DNA was hybridized in the Illumina Infinium MethylationEPIC BeadChip arrays. Cellular composition heterogeneity was corrected using RefFreeEwas followed identification of differentially CpG sites using limma (DMSs). Using DMSs
SU54. A PILOT METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING: HYPERMETHYLATION OF THE DEATH-ASSOCIATED PROTEIN 3 (DAP3) AND MIR2110 IN FIBROMYALGIA ASSOCIATED WITH CHILD ABUSE HISTORY AMONG AFRICAN AMERICAN FEMALES n
Hyunhwa Lee ,1, Annette Mullis1, Sijung Yun2, Katrina Isla1, Jonica Estrada1, Nada Lukkahatai3, Leorey Saligan4, Brian Walitt4 1
University of Nevada, Las Vegas Yotta Biomed, LLC 3 Johns Hopkins University 4 National Institutes of Health 2
Background: Epigenetics, such as DNA methylation, has been hypothesized as one possible mechanism to explain the association between adverse childhood experiences and later health problems. The goal of this study was to identify genome-wide DNA methylation markers from peripheral