Genome-wide DNA methylation analysis of high-dose synthetic glucocorticoid administration within buccal samples of oral surgery patients
Patricia Braun1, Aubrey Chan1, Kumi Yuki1, Benjamin Hing1, Lindsey Gaul1, Jonathan Heinzman1, Nick Sparr1, Julian Robles1, Theodosis Chronis1, Mai Tanaka-Sahker1, Kyle Stein2, James Potash1, Gen Shinozaki1
1Department of Psychiatry, University of Iowa Carver College of Medicine, Iowa City, IA
2Department of Oral and Maxillofacial Surgery, University of Iowa College of Dentistry, Iowa City, Iowa\
Background: Glucocorticoids play a major role in regulating the stress response, and an imbalance of glucocorticoids has been implicated in stress-related disorders. Within mouse models, candidate genes have been shown to be differentially methylated in response to glucocorticoid treatment. However, within humans the extent to which glucocorticoids affect DNA methylation (DNAm) across the genome is unknown.
Method: Buccal samples were collected before and after synthetic glucocorticoid treatment in the context of oral surgery. This included 30 minor tooth extraction surgery patients who received 10 mg of dexamethasone, and 12 major jaw surgery patients who received 750-1,000 mg of methylprednisolone. Genome-wide DNAm was assessed with the Infinium HumanMethylationEPIC array. Data were processed and analyzed with the R package RnBeads. Statistical significance was determined using the limma method. The genome-wide significance threshold for this experiment is p<6.03 x 10-8.
Results: Within the minor surgery samples, 10 CpG sites surpassed the genome-wide significance threshold. The most significantly different CpG in the before vs. after treatment comparison was within the insulin-like growth factor 1 receptor (IGF1R; average DNAm: pre-steroid 7%, post-steroid 15%; p=2.72 x 10-10). Within major surgery subjects, no sites attained genome-wide level of significance. The top differentially methylated CpG was within the small nucleolar RNA host gene 16 (SNHG16; average DNAm: pre-steroid 20%, post-steroid 11%; p=4.49 x 10-5).
Conclusion: High-dose synthetic glucocorticoid administration in the setting of oral surgery is significantly associated with DNAm changes within buccal samples. These findings provide initial evidence for an influence of glucocorticoids on DNAm within humans.
INVESTIGATING SIMILAR GENETIC BURDEN AMONG CLUSTERED PRIMARY AND SECONDARY CNVS
Hahn1,2, M. Parida3, H. Major1, & B. Darbro1,2
1 Stead Family Department of Pediatrics, Carver College of Medicine; 2 Interdisciplinary Graduate Program in Genetics, University of Iowa; 3 Department of Biology, University of Iowa
According to a survey conducted by National Institute of Health, nearly 15% of the US population aged 3-18 are affected by developmental disabilities. While a variety of genetic and environmental factors have been implicated as causative factors, approximately 10-20% of cases can be attributed to copy number variations (CNVs). While chromosomal microarray data has provided insight into the genes which are directly affected by CNVs, and led to the identification of a number of unique genomic disorders, the contribution of individual genes within the pathogenic CNV interval to the clinical phenotype is still under investigation. Our lab has taken a novel approach to identify genotype-phenotype associations for CNVs by utilizing protein-protein interaction networks to better ascertain and model the total genetic burden caused by non-benign CNVs.
Preliminary data demonstrated that our method of network smoothing and non-negative matrix factorization clustering leads to robust segregation of patients with known CNV disorders into unique clusters. Several patient clusters contained individuals with one or more CNVs of unclear clinical significance (secondary CNVs) in absentia of the (primary) pathogenic CNV characteristic of the cluster, suggesting that one or more secondary CNVs can cause the same genetic burden as a primary CNV. We are currently in the process of quality controlling our analysis pipeline and performing more granular phenotypic characterization of our patient cohort. Future work in the lab will utilize bioinformatics techniques and programs to identify the underlying gene networks and genetic burden that inform and underlie each chosen patient cluster. Directed examination of the medical records for patients within each of the chosen clusters will be used to evaluate phenotypic consistency between the individuals with primary and secondary CNVs. Together, the gene network, genic burden, and phenotypic information will be used to develop genotype-phenotype associations.
EXPLORING THE ROLE OF THE POSTERIOR DORSAL CIRCADIAN NEURONS (DN1p) IN REGULATING RHYTHMIC BEHAVIORS IN DROSOPHILA.
S Haase1, BC Lear12, X Lu2, A Iyenger2
1 Interdisciplinary Graduate Program in Genetics, 2 Department of Biology; University of Iowa.
Many biological processes exhibit circadian (daily) rhythms driven by internal clocks. In Drosophila, molecular circadian clocks reside in a number of specialized pacemaker neurons in the brain, and different subsets drive distinct peaks of behavioral activity in morning and evening hours. The small lateral ventral neurons (sLNv) and posterior dorsal neurons 1 (DN1p) are thought to drive morning behavior while the lateral dorsal neurons (LNd) are thought to drive evening behavior. We aim to determine how Drosophila circadian pacemaker neuronal subgroups influence daily behavioral outputs individually and as a network. We have used RNA interference (RNAi) to decrease expression of the NARROW ABDOMEN (NA) sodium leak channel in a subset of the DN1p clock neurons, which are primarily implicated in the regulation of morning behavior. Surprisingly, we find that NA knockdown in these cells causes complex alterations to behavioral phase in constant dark conditions, including effects on morning, midday, and evening activity. We have also performed optical electrophysiological experiments using the fluorescent voltage sensor ArcLight in order to examine the relationship between circadian neuron activity patterns and behavioral output. We find that wild-type DN1p neurons exhibit robust oscillations in membrane activity just prior to the lights turning on (morning), but show little activity just prior to the lights turning off (evening). RNAi knockdown of a positive regulator of the NA channel alters membrane voltage patterns in the DN1p, decreasing the amplitude of membrane oscillations and also decreasing the daily rhythm in membrane activity. Interestingly, the decrease in membrane oscillatory activity is most prominent in putative axonal projections, whereas activity is retained in dendritic regions. Taken together, our data indicate that the DN1p clock neurons function in constant dark conditions to regulate not only morning behavior, but also midday and evening behavior. To further characterize the mechanisms involved, we will continue to combine genetic manipulations with the use the ArcLight voltage sensor. However, our preliminary evidence suggests that the ArcLight sensor may respond differently in axonal vs. dendritic regions, perhaps reflecting differential effects of these manipulations on output vs. input.
AN INTEGRATIVE Genomic Signature-Based Approach to DISCOVER Drugs for ΔF508-CFTR Rescue
M Strub1, P McCray1
1 Interdisciplinary Graduate Program in Genetics
Background: The most common cystic fibrosis-causing mutation, termed ΔF508, results in protein misfolding and proteasomal degradation. However, if ΔF508-CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508-CFTR trafficking and function, including small molecules and RNAi manipulation of CFTR interactome genes. A challenge remains to translate interventions into therapies and to understand their mechanisms. One technique for connecting such interventions to small molecule therapies is via mRNA expression profiling. The idea is to identify small molecule-induced expression responses (catalogued in the CMAP/LINCS databases) that are similar to the expression induced by an intervention. In a complementary strategy, one can identify expression that is reversed compared with profiles characterizing diseased vs. healthy individuals.
Objective: To expand upon previous genomic signature approaches to identify compounds for rescue of ΔF508-CFTR trafficking and function.
Strategy: We developed gene sets for CMAP/LINCS queries by: 1) performing meta-analyses of CF rescue and disease signatures, and 2) extracting gene sets from MetaCore-curated pathways related to CFTR processing. To prioritize compounds for screening, gene sets were scored against all CMAP/LINCS drug profiles. We selected 120 candidates for functional testing. These molecules were administered to ΔF508/ΔF508-CFBE cells and transepithelial chloride conductance was measured.
Results: Functional screens identified 38 compounds that partially restored ΔF508-CFTR function, as assessed by cAMP-activated chloride conductance. Over half of these compounds showed significant cooperativity when administered with the FDA-approved corrector Lumacaftor.
Conclusion: Improved CF corrector therapies are greatly needed. This integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508-CFTR function and identify gene networks underlying such rescue. Ongoing work includes: 1) assessing activity of hits in primary human CF epithelial cells, and 2) informatic analysis to identify common themes among the set of active compounds.
Targeted Sequencing and Functional Assessment of the 2p25 Region in Suicide Attempters with Bipolar Disorder
Sophia C. Gaynor1, Marie E. Breen1, Eric T. Monson1, Kelly de Klerk1, Meredith Parsons1, Peter P. Zandi2, James B. Potash1, Virginia L. Willour1
1 Department of Psychiatry, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA.
2 Department of Mental Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, 21205, USA.
We previously conducted a genome-wide association study (GWAS) of the attempted suicide phenotype. This GWAS implicated common variation in the 2p25 region, a 350kb region encompassing four genes (FAM110C, SH3YL1, ACP1, FAM150B). The top 2p25 signals were exclusively in intergenic regions and were largely driven by males. In the current study, we have conducted a targeted next-generation sequencing study of the entire 2p25 region in 476 bipolar suicide attempters (224 males and 252 females) and 473 bipolar non-attempters (222 males and 251 females) in an attempt to identify both common and rare variants that may contribute to the risk for suicidal behavior. Our top gene-level result from this study was FAM150B (p = 0.022), but this result did not survive correction for multiple testing. Our top individual variant from this study was rs300799, an intergenic variant between FAM110C and SH3YL1. This variant was significantly associated with the attempted suicide phenotype in male subjects, with the minor allele present in 22.3% of attempters and 12.3% of non-attempters (p = 4.84 x 10-5, corrected p = 0.035, odds ratio = 2.13). Nearly all of our top individual-variant results from sequencing fell within an 80kb linkage disequilibrium (LD) block in 2p25. Because this 80kb LD block is entirely intergenic, we performed a functional assessment of this region using the CRISPR-Cas genome-editing tool to identify any potential regulatory elements. This functional assessment revealed nominally increased expression of FAM110C (fold change = 1.39, p = 0.084) and FAM150B (fold change = 1.77, p = 0.082) following deletion of a segment of this 80kb region. This study provides further support for a putative role of the 2p25 region in the attempted suicide phenotype.
USING MAXGEL TO STUDY COMPLEMENT REGULATION BY FH FAMILY PROTEINS IN EXTRACELLULAR MATRIX
Xue Xiao1,2, Yuzhou Zhang2, Richard JH Smith1,2
1 Interdepartmental PhD Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
2Molecular Otolaryngology and Renal Research Laboratories, Divisions of Nephrology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
Complement FH family proteins are important regulators of complement system. They are coded by CFH and CFHRs genes located on chromosome 1. Mutations in this region can cause C3 glomerulopathy (C3G). Although deletion in CFHR3-1 is not uncommon in European American population, this genetic change is associated with C3G occurrence. Recent studies came up with the hypothesis that FHRs and FH can compete with each other in regulating complement activity on cell surface. While there is currently no existing suitable system that can be used to study complement regulation on cell surface and it is still unclear how this competition happens. By using MaxGel to mimic the extracellular matrix structure on glomerular basement membrane, we were able to study the complement regulation on cell surface and to reveal the regulation of complement FH family proteins. In this study, we found that complement components, like C3 and FH can bind to MaxGel. By incubating C3b, FB, FD together, C3 convertase formed on MaxGel and the C3 convertase formation was regulated by complement regulators. FH is a negative regulator in complement system; its concentration influenced C3 convertase formation. We also found that FH binding to M axGel was reduced in some C3G patients, especially in patients with CFHR3-1 copy number changes. This study used a new system to mimic the environment on glomerular basement membrane and illustrate the role of factor H related proteins in the control of complement activity.
Exome Sequencing of Three Israeli Families with Keratoconus
Purpose: Keratoconus (KT) is the most common corneal dystrophy with an occurrence rate of 1 in every 2,000 people. Currently, corneal transplantation is the only treatment for KT when visual acuity is no longer correctable by contact lenses. We hypothesize that KT is a genetically heterogeneous disease that is caused by mutations in one of several genes.
Methods: Samples from 3 Israeli KT families (16 samples) were genotyped using genome-wide SNP microarrays. The SNP data was analyzed for regions of autozygosity using PLINK. Three samples with KT (one from each family) were chosen for exome sequencing. The resulting variants were filtered based upon variant quality, predicted function, and population prevalence. The final variant list for each family was annotated with corneal expression (http://genome.uiowa.edu/otdb) to assist in prioritizing potential candidates.
Results: Using the autozygous regions, we have prioritized candidates and areas of the genome on which to focus our investigation. No plausible variations were found in genes previously reported to cause KT. In addition, no single gene with plausible mutations was shared across all three families. However, we identified at least 3 areas of autozygosity that are shared between the 3 families. Using CoNIFER, we also identified regions of the exomes with shared copy number variations.
Conclusion: Further work is needed to identify the causative mutations in these families. We will continue to pursue these through ascertainment of additional families and family members. This will allow us to further narrow the intervals of the genome in search of the causative mutations.
THE ROLE OF MATERNAL WNT SIGNALING IN DORSO-VENTRAL AXIS SPECIFICATION
Melissa Marchal1 and Douglas Houston1
1The Interdisciplinary Program in Genetics and The Department of Biology, University of Iowa, Iowa City, IA, 52242
Dorsoventral (DV) body axis specification in Xenopus laevis requires the proper translocation of maternal mRNAs and proteins. Upon sperm penetration, these vegetally localized maternal determinants are transported to the future dorsal side of the embryo, in a microtubule dependent process called cortical rotation. An outcome of cortical rotation is the asymmetric activation of the Wnt/β-Catenin signaling pathway in dorsal nuclei. Accumulated β-Catenin then acts to transcriptionally activate dorsal-specific genes at the midblastula transition/zygotic genome activation. Although, previous studies have established a role for various Wnts and Wnt signaling components in DV axis determination, the function of other maternally expressed Wnts, their cognate receptors (Frizzleds), and co-receptors (LRP5/6) have not been thoroughly investigated. Moreover, the upstream factors and mechanisms responsible for initiating β-catenin stabilization, as well as their relationship to the timing of LRP6 activation by phosphorylation are poorly understood. In this work, we characterize the function of fzd1 in DV axis formation through a maternal overexpression/loss-of-function approach. We find that fzd 1 and fzd 7 are expressed abundantly in the oocyte, with fzd7 maintaining high expression levels until the tailbud stages. We show that overexpression of fzd1 results in morphogenetic defects and an expansion of dorsal specific markers. Additionally, we find that depletion of maternal fzd1 results in delayed and disrupted gastrulation, a ventralization phenotype, as well as defects in dorsal-specific gene expression. The specificity of these phenotypes were confirmed via maternal rescue experiments. We also show that oocytes depleted of fzd1, have significantly reduced vegetal microtubule arrays, suggesting that Frizzled-dependent signaling may play a role in cortical rotation. Additionally, we present evidence for the dynamic phosphorylation of LRP6 in cleavage stage embryos. We find that both fzd1 depletion and excessive Wnt signaling inhibit this phosphorylation pattern.
Predictive Models of Recombination Rate Variation across the Drosophila melanogaster Genome
Andrew B. Adrian1,†, Johnny Cruz Corchado2,† and Josep M. Comeron1,2,*
In all eukaryotic species examined, meiotic recombination, and crossovers in particular, occur non‐randomly along chromosomes. The cause for this non-random distribution remains poorly understood but some specific DNA sequence motifs have been shown to be enriched near crossover hotspots in a number of species. We present analyses using machine learning algorithms to investigate whether DNA motif distribution across the genome can be used to predict crossover variation in Drosophila melanogaster, a species without hotspots. Our study exposes a combinatorial non-linear influence of motif presence able to account for a significant fraction of the genome-wide variation in crossover rates at all genomic scales investigated, from 20% at 5-kb to almost 70% at 2,500-kb scale. The models are particularly predictive for regions with the highest and lowest crossover rates and remain highly informative after removing sub-telomeric and -centromeric regions known to have strongly reduced crossover rates. Transcriptional activity during early meiosis and differences in motif use between autosomes and the X chromosome add to the predictive power of the models. Moreover, we show that population-specific differences in crossover rates can be partly explained by differences in motif presence. Our results suggest that crossover distribution in Drosophila is influenced by both meiosis-specific chromatin dynamics and very local constitutive open chromatin associated with DNA motifs that prevent nucleosome stabilization. These findings provide new information on the genetic factors influencing variation in recombination rates and a baseline to study epigenetic mechanisms responsible for plastic recombination as response to different biotic and abiotic conditions and stresses.
NPHP10 affects AIMP2 localization and alters its downstream target, p53
Bardet-Biedl Syndrome (BBS) is an autosomal recessive, pleiotropic disorder, with 21 genes currently identified as causative. One of these genes is serologically defined colon cancer antigen 8 (NPHP10 [MIM 613524]), a nephronophthisis-related ciliopathy gene (NPHP10 or BBS16). Patients with mutations in this gene exhibit retinal and renal abnormalities, obesity, and learning disabilities. Currently, little is known about the molecular functions of NPHP10 and how loss of NPHP10 function leads to the observed phenotypes. Our previous work identified strong interactors of NPHP10, consisting mainly of components of the multi-aminoacyl tRNA synthetase complex (MSC). More specifically, 8 out of 9 aminoacyl tRNA synthetases (ARS) as well as aminoacyl-tRNA-synthetase-complex interacting multifunctional protein 2 (AIMP2) interact with NPHP10. We also determined that among the MSC components, NPHP10 directly interacts with AIMP2. AIMP2 has a known non-canonical role in the apoptotic pathway via p53 activation. In response to DNA damage, a subset of AIMP2 localizes to the nucleus and binds to and prevents the ubiquitination of p53. This leads to an upregulation of apoptosis in response to DNA damage. Recently, we developed two NPHP10 knockout IMCD3 cell lines using CRISPR-mediated technology. Using these cell lines, we examined the effect of NPHP10 loss on AIMP2 localization in these cells. Immunofluorescence microscopy and cell fractionation with immunoblotting show that loss of NPHP10 leads to increased AIMP2 in the nucleus of cells. Additionally, we showed that overall p53 levels increased in knock-out cells. Further work focuses on how these changes affect downstream elements of p53 utilizing a luciferase reporter assay.
The g-Protocadherins interact with neuroligin-1 and inhibit its synaptogenic activity
Michael J. Molumby, Rachel M. Anderson, Dillan J. Newbold, Norah K. Koblesky, Andrew M. Garrett, Dietmar Schreiner, Jason J. Radley, Joshua A. Weiner
The a-, b-, and g-Protocadherins (g-Pcdhs) are cadherin superfamily adhesion molecules encoded by clustered gene families. The 22 g-Pcdhs are combinatorially expressed in the brain, and play critical roles in synaptogenesis, dendrite arborization and patterning, and the survival of subsets of neurons. The g-Pcdhs can interact promiscuously with each other, and with other clustered Pcdhs, in cis, but interact strictly homophilically in trans. Mice lacking the g-Pcdhs in the cerebral cortex exhibit severely reduced dendrite arborization (Garrett, et al., Neuron, 2012). Recently we demonstrated that g-Pcdhs promote dendrite arborization in vivo through local neuron-neuron and neuron-astrocyte homophilic interactions (Molumby et al., Cell Reports, 2016). Though -Pcdhs regulate the progression of spinal cord synaptogenesis (Garrett and Weiner, J. Neurosci., 2009), a role for these molecules in cortical dendritic spines and synapses has yet not been examined.
Here, we provide evidence that the g-Pcdhs negatively regulate synapse formation and spine morphogenesis in forebrain neurons. Mice lacking all -Pcdhs in the cortex exhibit significantly increased spine density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 -Pcdh isoforms. We thus asked whether the -Pcdhs might be inhibitory in an artificial synapse co-culture assay. Indeed, we found that multiple g-Pcdhs can, when co-expressed in COS cells, strongly inhibit the ability of the synaptic cell adhesion molecule neuroligin-1 to promote presynaptic differentiation in contacting axons. The g-Pcdhs physically interact in cis with neuroligin-1 on dendrites of cultured neurons, and can co-immunoprecipitate neuroligin-1 both in vitro and in vivo. We present in vitro evidence that the interaction between -Pcdhs and neuroligin-1 disrupts the latter’s binding to its presynaptic partner neurexin1β. Additionally, we show that g-Pcdh overexpression in cultured hippocampal neurons suppresses the increase in spine density observed upon neuroligin-1 overexpression. This work suggests a potential new mechanism by which g-Pcdhs regulate the “choice” between dendrite arbor growth and formation and/or stabilization of dendritic spines and synapses in the developing brain.
Identification of a novel, de novo FGFR1 mutation in an individual with Hartsfield Syndrome
Harney, L.A., Bernabe, H., Nidey, N., Standley, J., Schnieders, M., Murray, J.C.
Mutations in FGFR1 have recently been associated with Hartsfield Syndrome, a disease with the rare combination of holoprocencephaly (HPE) and ectrodactyly (EEC). Here we report a novel, de novo mutation in FGFR1 in a previously unsolved clinical case of multiple congenital anomalies. The proband presented with bilateral cleft lip and palate, malformed auricles and bilateral EEC of his hands and feet at birth. He was later diagnosed with diabetes insipidus, spastic quadriplegia, developmental delay, and agenesis of the corpus callosum. Microarray analysis resulted in an unremarkable copy number variant profile. Although this exact combination of clinical features has not been reported in a patient to date, we noted phenotypic overlap with individuals with Hartsfield Syndrome. Sequencing of FGFR1 identified a novel and previously unreported de novo mutation in exon 11 (p.G487C), resulting in the diagnosis of Hartsfield Syndrome for this individual.
Drosophila vs The World: The Nkd EF-Hand Domain Modulates Divergent Wnt Signaling Outputs
Autumn N. Marsden, Sarah W. Derry, Trudi A. Westfall, Diane C. Slusarski
The Wnt signaling network plays critical roles in development and is implicated in human disease. Wnts comprise a complex signaling network that, upon ligand binding, activates the phosphoprotein Dishevelled (Dvl), leading to distinct outputs including polarized cell movement (known as planar cell polarity, Wnt/PCP) and stabilization of the transcription factor β-catenin (Wnt/β-catenin). The mechanisms that determine a specific output are not completely understood, especially because they share receptors and cellular effectors, such as Naked-cuticle (Nkd), a Dvl-interacting protein. The Nkd protein contains a myristoylation domain and an EF-hand, a putative calcium binding domain. Genetic evidence in Drosophila demonstrates that Nkd acts as a Wnt/β-catenin antagonist, while in contrast, Nkd modulates both branches of Wnt signaling in vertebrates. We hypothesize that the specialized role of Nkd in Drosophila is due to a disrupted EF-hand that cannot not bind calcium. Indeed, this change is unique to Drosophila and is not present in closely related insects. To test the role of the Nkd EF-hand in Wnt signal integration, we created Nkd with a neutralized EF-hand, as well as a Drosophila-like EF-hand, and manipulate Nkd activity in the zebrafish. Using a combination of biochemical and functional assays, we identified a requirement for the Nkd EF-hand in Wnt/PCP but not in Wnt/β-catenin transcriptional outputs. We demonstrate that the Drosophila-like Nkd antagonizes Wnt/β-catenin more robustly than zebrafish Nkd. The EF-hand of Nkd is similar to the EF-hand of a known calcium binding protein, Recoverin, a myristoyl-swtich protein that shuttles between the membrane and the cytoplasm depending on its calcium bound state. Consistently, we observe that NkdWT shows localization changes in the calcium fluxing DFCs versus calcium quiescent cells but not the mutant forms. The Nkd EF-hand may serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.
Assessment of Whole-Exome Sequence Data in Attempted Suicide within a Bipolar Disorder Cohort
Suicidal behavior, which includes both attempted and completed suicide, is the source of tremendous cost and loss of life. Within the United States alone, suicidal behavior accounts for approximately 836,000 emergency room visits and over 41,000 deaths, ranking as the 10th leading cause of death overall and the 2nd leading cause of death for ages 10-34 as reported by the CDC. Efforts to better understand the biological basis and risk factors for suicidal behavior have included a large number of genetic studies due to a significant genetic component to this phenotype. Existing genetic studies have focused primarily on specific candidate genes and/or common variation and have left a significant proportion of the expected heritability of suicidal behavior unexplained. Our current work is focused on the investigation of the contribution of rare functional variation to the risk for suicidal behavior through the use of next-generation sequencing techniques. Specifically, we have performed a comprehensive analysis of 1,018 whole-exome sequences, divided into 387 individuals with bipolar disorder and a history of suicide attempt and 631 individuals with bipolar disorder and no past suicidal behavior. We performed analyses within ~490,000 single variants, across all covered genes, and within 3,621 pathways. No result from any of these analyses survived conservative Bonferroni correction, though several suggestive and potentially important findings were identified within the gene and pathway-based results. In addition, this study generated a large and highly novel dataset that will be of substantial value to future genetic investigations within suicidal behavior. This effort marks the first large-scale effort to identify the contribution of rare functional variation to suicidal behavior and provides the basis for expanded efforts to explain the biological basis of this devastating phenotype.
Genes involved in bone remodeling are mutated in chronic recurrent multifocal osteomyelitis (CRMO)
Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model determined that the Fblim1 ortholog is the most differentially expressed gene, further implicating it in disease pathogenesis. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In two other families, we detected a shared rare mutation in a related gene, PLCG2. FBLIM1 and PLCG2 are both directly involved in β-integrin activation, which was recently implicated in sterile inflammation via Bruton tyrosine kinase signaling. Additional work involves functional validation of the FBLIM1 and PLCG2 mutations, and pathway analysis of exomes from a cohort of individuals with CRMO. Our data suggest CRMO is a disorder of integrin activation and imbalanced bone remodeling.
TFAP2A drives melanocyte gene expression in parallel with MITF
Hannah E. Seberg, Eric Van Otterloo, Stacie K. Loftus, Greg Bonde, Ramakrishna Sompallae, Juan F. Santana, J. Robert Manak, William J. Pavan, Robert A. Cornell
Disruption of the transcription factor network governing melanocyte differentiation contributes to the pathogenesis of pigmentation disorders and melanoma. Transcription factor activator protein 2 alpha (TFAP2A) is expressed in melanoblasts, and mutations in this gene cause pigmentation phenotypes in humans, mice, and zebrafish. However, the the transcriptional targets of TFAP2A, and the extent to which they are shared with MITF, a master regulator of melanocyte biology and melanoma progression, have been unclear. To determine the position of TFAP2A in the melanocyte gene regulatory network, we conducted anti-TFAP2A ChIP-seq to create profiles of TFAP2A-bound loci in melan-a cells and human primary melanocytes. We then used microarray analysis in zebrafish and mouse immortalized melanocytes (melan-a cells) to profile genes that are downregulated in the absence of TFAP2A. Genes at the intersection of the microarray and ChIP-seq profiles are likely direct targets of TFAP2A. These include mc1r and several melanin synthesis or melanosome structural genes, most of which are also thought to be direct targets of MITF. Comparison of our TFAP2A ChIP-seq profile with a published MITF ChIP-seq profile showed that TFAP2A peaks overlap MITF peaks at a large fraction of promoters and enhancers. In reporter assays, deletion of TFAP2A binding sites decreased activity of a minimal TRPM1 promoter, similar to published results for deletion of MITF binding sites from this element. We also observed a genetic interaction between tfap2a and mitfa in zebrafish. The significance of these findings is, first, they show that TFAP2A and MITF work in parallel to promote gene expression in melanocytes. Second, they show that a widely-expressed transcription factor, TFAP2A, co-operates with a more tissue-restricted transcription factor, MITF, to directly regulate expression of lineage-specific melanocyte genes. Third, they suggest that the reduction of TFAP2A expression levels observed in advanced melanoma is a cause and not an effect of melanoma progression, as such a reduction would be expected to decrease the pro-differentiation effect mediated by TFAP2A.
Speciation Genetics in Drosophila: Insights from Male and Female Transcriptomes
Population genetic models predict that the X chromosome will evolve at a faster rate than autosomes (i.e., faster-X evolution). The basis for faster-X evolution is that recessive genes located on the X chromosome can be exposed in males while these genes are hidden if located on the autosomes. Evaluations of protein-coding sequences have indeed shown mixed results with some studies showing an excess of divergence on the X chromosome compared to autosomes, particularly in genes expressed higher in males than in females (i.e., male-biased genes). In addition, whole-genome analyses of gene expression in Drosophila yakuba and D. santomea males indicate that X-linked genes are more differentially expressed between species (i.e., faster-X evolution of gene expression) than autosomal genes. This trend is once again strongest for male-biased genes. However, these studies utilized only males and were therefore limited in their expression profiles. Here, we investigate the whole-genome profiles of D. yakuba and D. santomea males and females to determine the relative rates of evolution for all gene classes. In addition, we explore the implications of sex chromosome inheritance on autosomal gene expression in the hybrids of these species.
THE ROLE OF ENDOGENOUS MICROBIOTA IN SEIZURE-LIKE BEHAVIOR OF SHUDDERER, A DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL MUTANT
It is widely recognized that mutations in genes encoding voltage-gated sodium (Nav) channels contribute to the etiology underlying various seizure disorders. Shudderer (Shu), a gain-of-function mutant for the Drosophila Nav channel gene, exhibits neuronal hyperexcitability and seizure-like behavioral defects, including spontaneous jerking and heat-induced convulsion. Results of microarray analyses indicated that Shu mutants have an increased innate immune response and reduced insulin signaling. Because the endogenous gut microbiota has a substantial impact on the host immune system and insulin-mediated metabolic regulation, we hypothesized that the microbiome plays a role in Shu phenotypes. Intriguingly, removing the gut microbiota of Shu and wild-type (WT) flies using antibiotic-containing or sterile food significantly suppressed Shu behavioral phenotypes while having no effect on WT behavior. Culturing homogenized guts dissected from Shu and WT flies on MRS agar revealed drastic differences in the number of culturable bacteria. Further, we performed high-throughput sequencing of the bacterial 16S ribosomal RNA gene which revealed species-specific differences in the gut microbiome of Shu and WT flies. Because neurological disorders and changes in the microbiota can individually increase oxidative stress in an organism, we examined the antioxidant response in the guts of Shu and WT flies using a GFP reporter, (GST-D-GFP). GST-D-GFP expression was altered in Shu and WT flies fed antibiotics, suggesting a potential involvement of oxidative stress in the antibiotic-dependent rescue of seizure-like behavior. Additional experiments will determine ROS levels in Shu and WT flies and are expected to yield a better understanding of the role of the microbiome in the context of seizure disorders.
Mutation in regulatory protease, CAPN5, results in eye disease
Uveitis (intraocular inflammation) causes significant visual morbidity and blindness in all ages, yet it has proven refractive to current therapies, underscoring the critical need for new clinical approaches. Nevertheless, a major barrier to developing new medicines has been the lack of any specific molecular cause for the disease. To gain insight into the molecular mechanisms driving uveitis, we sought the disease-associated gene in kindreds with a Mendelian form—Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV). Our studies showed the underlying cause of ADNIV was mutations in CAPN5 (calpain-5), making CAPN5 the first nonsyndromic uveitis gene discovered. This discovery provides an unprecedented starting point for investigating the substantial gap in our understanding of the molecular basis of ocular inflammation.
CAPN5 is a calcium-activated, intracellular protease expressed at photoreceptor synapses. Similar to the function of other calpains, we believe that proteolysis by CAPN5 is a tightly controlled, post-translational regulatory mechanism that deploys protein fragments with altered activity. Although it is a member of a relatively large, well-studied family, little is known regarding the CAPN5 natural substrates. One exception to this is CAPN5 autoproteolysis, which uncovers one natural substrate of CAPN5: CAPN5 itself. We aim to identify the sequences in CAPN5 that are the targets of autoproteolysis and also to determine the CAPN5 cleavage site in a substrate we recently identified, platelet-derived growth factor B (PDGFB). PDGFB was previously identified as a protein cleaved into an active angiogenic form by an unknown protease, and PDGFB is active in a number of similar eye diseases, including AMD, PVR and diabetic retinopathy. The insight gained from these studies may eventually be used to design therapies for a variety of previously untreatable eye diseases.
Functional Characterization of Epilepsy Related Genes in Zebrafish
TD Fuller1,2, TA Westfall1, DC Slusarski1
1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics
Statement of Purpose: Epilepsy is a chronic condition of recurrent seizures which affects approximately one percent of the general population. Though several causative genes have already been identified, these account for only a small percentage of all the genetically caused cases of epilepsy. Further, even when genes are identified, we lack tractable animal models to rapidly translate these findings into mechanistic insights and ultimately new anti-epileptic therapies. For this reason, the zebrafish is increasingly being used as a model of epilepsy due to its high genetic and physiologic homology to humans and its seizure-like behavior in response to various pharmacological and genetic manipulations. Using High throughput sequencing, a significant number of gene variants are being identified, yet their role in the disease state remain unknown. My project utilizes the zebrafish and focuses on characterizing the functional role of 15 genes in the NIH Undiagnosed Diseases Program for which mutations have been associated with epilepsy, and for which zebrafish orthologues have been identified. Methods: I isolated the zebrafish orthologues and characterized gene expression patterns by RTPCR and whole mount in situ hybridization techniques. We previously demonstrated that knockdown of Prickle (PK), a gene associated with human epilepsy, sensitizes zebrafish to seizure-inducing drugs through the use of larval motility assays. To facilitate high-throughput in vivo screens, I adapted this approach and developed a code to rapidly and efficiently analyze the generated data sets. This allowed for the characterization of the 15 candidate genes in the context of seizure sensitization. Results: Of the fifteen candidates, I found five: syne1b, sms, ccdc89, wscd1, and nid2a, to result in seizure sensitization when knocked down in the zebrafish. I show that each of these genes is expressed in specific regions in the brain during critical times of neuronal development. Further, I find genes expressed in the retina result in axon defects when knocked down.