HIGH-THROUGHPUT BEHAVIORAL ASSAY TO INVESTIGATE SEIZURE SENSITIVITY IN ZEBRAFISH IMPLICATES SYNE1 AND ZFHX3 IN EPILEPSY
TD Fuller1,2, AN Marsden1,2, Das T1, JM Hermosillo1, TA Westfall1, DC Slusarski1
1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics
Epilepsy, which affects ~1% of the population, is caused by abnormal synchronous neural activity in the central nervous system (CNS). While there is a significant genetic contribution to epilepsy, the underlying causes for the majority of genetic cases remain unknown. The NIH Undiagnosed Diseases Project (UDP) utilized exome sequencing to identify genetic variants in patients affected by various conditions with undefined etiology, including epilepsy. Confirming the functional relevance of the candidate genes identified by exome sequencing in a timely manner is crucial to translating exome data into clinically useful information. To this end, I developed a high throughput version of a seizure-sensitivity assay in zebrafish (Danio rerio) to rapidly evaluate candidate genes found by exome sequencing. This assay uses pentylenetetrazol (PTZ) to induce seizures in zebrafish larvae and the motility tracking software of the Zebrabox (Viewpoint Life Sciences) is utilized to record each larva’s total movement (cm). This generates massive data sets. Therefore, I developed an open access software, SEIZR (Studying Epilepsy In Zebrafish using R), to rapidly and efficiently analyze the data. My project focuses on characterizing the functional role of fifteen genes in the NIH UDP for which mutations have been associated with epilepsy, and for which zebrafish orthologues have been identified. Using SEIZR, I characterized all fifteen candidate genes in the context of seizure sensitization. Here, I show the findings of two genes, syne1b and zfhx3, both of which result in seizure sensitization when knocked down in the zebrafish. I show that each of these genes is expressed in regions of the brain involved in motor control during critical times of neural development. Further, I find syne1b knockdown results in axon defects in the retina.
EVALUATING RECOMBINATION REGULATION: LESSONS FROM HYBRIDS OF DROSOPHILA
N Pettie1, J Comeron1,2 , and A Llopart1,2
1 Interdisciplinary Graduate Program in Genetics, University of Iowa, 2 Department of Biology, University of Iowa
Meiotic recombination is a polygenic, highly regulated, evolutionarily conserved process that increases genetic diversity and ensures proper chromosome segregation. Despite the almost universal presence of recombination among eukaryotes, the molecular processes responsible for the number and distribution of recombination events across genomes are highly variable. The mechanisms responsible for hotspot localization of meiotic recombination, for instance, are not conserved. Recombination localization in most mammals including humans and mice is regulated by PRDM9, which targets specific sequences of DNA motifs. In canids, however, there is no active PRDM9 and recombination is highly correlated with CpG content, which is also the case in yeast. Recombination in Drosophila appears to be controlled differently than in mammals or yeast. Recombination rates and distribution in Drosophila are influenced by many epigenetic factors, such as age, nutrition, temperature, and transcription. Here, we use the term epigenetic to describe any factor that changes the ‘phenotype’ of recombination, including gene expression changes, while maintaining equivalent genetic material. We will generate high-resolution recombination maps from two closely related species of Drosophila, D. yakuba and D. santomea, to study short-term evolutionary differences between species. We will also generate high-resolution recombination maps in their hybrids to evaluate the hypothesis that the independent evolution of this polygenic ‘phenotype’ of recombination in the two lineages has led to incompatible interactions in hybrids and ultimately distorted genetic maps.
Genetic therapeutic strategies for the BBS1 M390R mutation
Bardet-Biedl syndrome (BBS) is a rare ciliopathy caused by mutations in a number of cilia related genes. Common features of BBS include retinopathy, male infertility, polycystic kidney disease, hypogonadism, polydactyly, obesity, and several others. Currently, there are no efficacious treatments for BBS, illustrating a critical need to develop therapeutic strategies. The most common genetic cause of BBS is the M390R mutation in BBS1, accounting for approximately 20% of all genetically diagnosed BBS cases. BBS1 is normally part of a multi-protein complex called the BBsome, a complex that is important for trafficking proteins to cilia and the cell membrane. Due to the accessibility of the eyes and testes, the retinal degeneration and male infertility phenotypes of BBS are strong candidates for treatment via gene therapy and gene correction. Previous work in mice focused at using gene therapy for the treatment of retinal degeneration in BBS mice has met some challenges, as overexpression of BBS1 causes toxicity and further loss of photoreceptor cells. Our lab has previously shown that postnatal correction of BBsome proteins can halt photoreceptor death and restore male fertility in mouse models of BBS. Continued work is underway using naked DNA, viral vectors, and CRISPR-Cas9 mediated gene correction to ameliorate the phenotypes of BBS in mouse models.
The role of the anti-sigma factor RsiV in lysozyme sensing and stress response in Bacillus subtilis and Clostridium difficile
*Ana N Castro1, Lincoln T Lewerke1, Ben D Cortes2, Jessica L Hastie1, Craig D Ellermeier1
1Microbiology Department, College of Medicine, University of Iowa, Iowa City, IA; 2Biology Department, Benedictine College, Atchison, KS. *Anaemail@example.com
ECF σ factors are alternative σ factors that allow many bacteria to sense and respond to changes in the environment. σV, an ECF σ factor, is found in low GC Gram-positive bacteria, induces resistance to lysozyme and is important for virulence in pathogens. In the absence of lysozyme, σV is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded by regulated intramembrane proteolysis (RIP). RIP is initiated by signal peptidase cleavage of RsiV at site-1 resulting in the release and activation of σV. We demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme. By altering the signal peptide, we find that the spacing between the cleavage site and the transmembrane is critical to RsiV avoiding signal peptidase cleavage in the absence of lysozyme. The lab previously determined the X-ray crystal structure of the extracellular domain of RsiV in complex with lysozyme. This structure revealed that RsiV does not bind near the signal peptidase cleavage site. This has led to our model in which binding of lysozyme to RsiV triggers a conformational change, which allows signal peptidase to recognize the cleavage site. Currently, we are determining the structure of full-length RsiV in both the absence and presence of lysozyme. Together this will provide information on the changes that occur to RsiV upon binding lysozyme that lead to σV activation. This will broaden our knowledge on a novel role for signal peptidase and could lead to novel drug targets.
Patient-specific iPSCs to investigate pathophysiology and develop treatments for RPGR-associated XLRP
Giacalone J.C. 1, Burnight E.R. 1,Sharma T.P., Wiley L.A.1, Ochoa D.1, Collins, M.M.1, Mullins R.F.1, Tucker B.A.1, and Stone E.M.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA
Purpose: Retinitis Pigmentosa (RP) is a heterogeneous disease that causes death of the light sensing photoreceptor cells of the outer neural retina and affects as many as 80,000 individuals in the US alone. X-linked RP (XLRP) is responsible for some of the most severe and earliest onset cases. The majority of XLRP cases are caused by mutations in the gene RPGR. The retinal-predominant isoform of RPGR, known as ORF15, contains a long repetitive sequence, which harbors the majority of the pathogenic mutations that cause RPGR-associated XLRP. The purpose of this study was to: 1) model RPGR-associated XLRP using patient-specific induced pluripotent stem cells (iPSCs) and 2) develop CRISPR/Cas9-based genome editing strategies for correction of disease-causing mutations.
Methods: Patient-specific iPSCs were generated from dermal fibroblasts ofpatients with molecularly confirmed RPGR-associated XLRP. Pluripotency was confirmed using the TaqMan Scorecard Assay. CRISPR/Cas9 constructs were generated to target patient-specific mutations. Gene targeting constructs and homology directed repair constructs were introduced to iPSCs via NEON transfection. Correction was confirmed via T7E1 assay and Sanger sequencing. Patient-specific iPSC-derived 3D retinal eyecups were generated and characterized via Western blot, immunocytochemistry and confocal microscopy.
Results: Seven iPSC lines were generated with varying mutations, disease severity, and disease phenotypes. Genome editing of patient-specific iPSCs was achieved with transfection efficiencies of 30 percent, and the resulting modified iPSC clones were isolated and expanded via reporter selection. Patient-specific iPSC-derived retinal eyecups were generated and displayed the retinal progenitor and photoreceptor-specific markers PAX6, OTX2, RCVRN, CRX and NRL.
Conclusions: With the advent of iPSC technology, we are now able to generate retinal cells from male patients with mutations in RPGR ORF15. We have shown that genome editing via the CRISPR/Cas9 system can successfully correct patient-specific mutations in iPSCs, which will serve as a valuable tool for characterizing the observed disease phenotype.
Time heals all wounds, but not without IRF6 and ARHGAP29
T Reeb, M Dunnwald
Chronic wounds affect 6.5 million people in the US, yet little is known about the genetic and molecular mechanisms regulating wound healing. Wound closure requires the concerted action of cellular proliferation, differentiation, and migration. Interferon Regulatory Factor 6 (IRF6) has been shown to regulate all of these processes, with murine embryos deficient for Irf6 displaying impaired wound healing. Additionally, IRF6-deficient keratinocytes have been shown to display both a decrease in the expression of Rho GTPase Activating Protein 29 (ARHGAP29) as well as an increase in stress fibers. ARHGAP29 is a Rho GTPase Activating Protein with a high affinity for RhoA. RhoA is a Rho GTPase which has been shown to play key roles in wound healing and the regulation of stress fibers. However, despite all that we know about IRF6 and RHOA, little is known about how IRF6 regulates ARHGAP29 and the role of ARHGAP29 in cellular migration, cellular adhesion, and wound healing. We hypothesize that ARHGAP29 is transcriptionally regulated by IRF6 and functionally regulates Rho GTPases. Perturbing this system will result in impaired wound healing. We plan to test this hypothesis by performing full thickness excisional wounds on Arhgap29 mutant mice. To test whether Irf6 regulatesArhgap29, a rescue experiments will be performed to determine if overexpression of Arhgap29 can alleviate the phenotypes observed in Irf6-deficient keratinocytes. By further understanding the roles of IRF6 and ARHGAP29 in wound healing, it would provide positive impacts including the ability to predict wound healing complications, the generation of novel therapies as a means preventing such complications, the potential to gain insights into the role of ARHGAP29 and IRF6 in other disorders (such as cleft lip and palate), and ultimately, the improvement of patient outcomes.
DUAL ROLES OF CYCLIN C IN HEART DISEASE
Ponce1,2, D. Hall2, I. Martin2, C. Grueter1,
Interdisciplinary Graduate Program in Genetics, Department of Internal Medicine, University of Iowa.
Cardiovascular disease is the leading cause of death worldwide. The damage inflicted on the myocardium during myocardial infarction (MI) results from (1) hypoxia during ischemia and (2) oxidative damage upon subsequent reperfusion. Despite extensive investigation, the pathophysiology of myocardial injury in response to ischemia is not fully understood. Cyclin C is a coactivator of the Mediator kinase subcomplex which regulates transcription of genes involved in cardiac metabolism, energy homeostasis and responsiveness of the heart to stress. Recent studies have shown Cyclin C to function independent of mediator in regulating stress-induced mitochondrial hyper-fission in yeast in response to oxidative damage. In humans, the constant electrical and mechanical activities of the heart require a continuous energy supply met by a rich stockpile of mitochondria. Additionally, mitochondrial dysfunction increases the pathogenesis in response to ischemia injury. Although studies have shown the effects of mitochondrial dysfunction in heart disease, there is a current gap in knowledge to understand the functional role of Cyclin C in cardiac mitochondria. We hypothesize that injury in response to IR depends on the translocation of Cylinc C from the nucleus to mitochondria where it regulates mitochondrial dynamics. Preliminary data demonstrates Cyclin C translocation in response to stress in cardiomyocytes isolated from adult mouse and neonatal rats. The overall goal of this project is to define the mechanisms whereby Cyclin C regulates metabolism, energy homeostasis in heart disease via two functions: regulating mitochondrial dynamics, as well as regulating transcription of crucial mitochondrial genes. These studies will provide new insights into the regulation of cardiac energy metabolism and may yield novel therapeutic strategies for modulating these processes in the settings of heart disease.
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.