Matt Strub Will Present His Research on Wednesday, 2/11/15

Matt’s Abstract

DISCOVERY OF DRUGS TO RESCUE ΔF508-CFTR USING A GENOMIC SIGNATURE APPROACH
Matthew D. Strub and Paul B. McCray, Jr.
Background: Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the CFTR gene. The most common CFTR mutation, ΔF508, causes protein misfolding, resulting in proteosomal degradation. However, if ΔF508-CFTR is allowed to traffick to the cell membrane, anion channel function may be partially restored. The McCray Lab previously reported that transfection with a miR-138 mimic or knockdown of SIN3A in primary CF airway epithelia increases ΔF508-CFTR mRNA and protein levels, and partially restores cAMP-stimulated Cl- conductance.
Objective: We hypothesized that a genomic signature approach can be used to identify new bioactive molecules affecting ΔF508-CFTR rescue.
The Connectivity Map (CMAP): CMAP is a catalog of gene expression profiles from cells treated with a variety of bioactive molecules and has pattern-matching software to mine data. CMAP queries using miR-138 mimic and SIN3A DsiRNA gene expression signatures identified 27 molecules that mimicked miR-138 and SIN3A DsiRNA treatments. The molecules were screened in vitro for efficacy in improving ΔF508-CFTR trafficking, maturation, and Cl- current. The McCray Lab reported the identification of 4 molecules that partially restored ΔF508-CFTR, highlighting the utility of a genomic signature approach in drug discovery.
LINCS: CMAP has greatly expanded into the Library of Integrated Network-based Cellular Signatures (LINCS). Previously generated gene sets were used to iteratively query LINCS and 125 candidate molecules were selected for further testing. Functional screens performed in CFBE-(ΔF508/ΔF508) cells identified 7/125 compounds that partially rescued ΔF508.
Conclusion: Querying LINCS with relevant genomic signatures offers a novel method to identify new candidates for rescuing ΔF508-CFTR. Further analysis of these molecules and their derivatives are ongoing. We are also generating additional genomic signatures representing ΔF508 rescue for use in LINCS queries. These results represent an important step forward from our proof-of-concept CMAP studies and highlight the utility of LINCS in drug discovery for CF.

Stephanie Haase and Xue Xiao Will Present Their Research on Wednesday, 1/14/2015

Stephanie’s Abstract

Exploring Clock Neuron Activity using the ArcLight fluorescent voltage sensor
Molecular clocks control rhythmic fluctuations in behavior, transcription, and physiology on approximately 24 hour cycles.  These circadian rhythms persist in the absence of external light cues and are driven by special clock neurons in both Drosophila and mammals.  The interactions of these clock neurons as a network are not fully understood.  Previously, changes in clock neuron function that affected circadian rhythmicity were studied primarily through behavioral assays.  ArcLight is a powerful tool that will allow characterization of changes to clock neuron function at the cellular level.  ArcLight, a fluorescent voltage sensing protein, has allowed the use of optical electrophysiology on clock neurons that traditional electrophysiology cannot access.  By expressing ArcLight protein in these neurons, we are able to identify a putative daily rhythm of activity for a subset of these neurons.  We are currently working on identifying daily rhythms of other clock neurons using ArcLight and plan to characterize behavioral mutants at the cellular level in the future.
Xue’s Abstract 
Rescuing the Renal Phenotype in Murine Model of C3 Glomerulonephritis by Delivering Soluble CR1Using the PiggyBac Transposon System
 
C3 glomerulopathy (C3G) encompasses two prototypical diseases, dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Both diseases are characterized by fluid-phase dysregulation of the alternative pathway (AP) of complement that leads to C3 deposition in the renal glomerulus. Because disease triggers are unknown and specific treatments are lacking, progress to end-stage renal failure is the common final outcome in 50% of patients.  Soluble complement receptor 1 (sCR1) is a potent regulator of complement.  In short-term studies it is capable of rescuing the renal phenotype in a murine model of C3GN, the Cfh-/-/huCR1-Tg. To determine whether long-term expression of sCR1 could affect renal outcome, we used hydrodynamic tail vein injection and the PiggyBac transposon system to introduce a sCR1 construct into the same mouse model.  Injected mice expressed sCR1 (LHR A-C), had elevated circulating C3 and showed arrested glomerular deposition of C3. These data suggest that long-term expression of sCR1 corrects the C3GN phenotype in these animals and may be a viable treatment solution for patients with this disease.

Spring 2015 Presentation Schedule

Date Presenters Location
1/14/2015 Xue Xiao 2-501 BSB
Stephanie Haase
2/11/2015 Matthew Strub 2-501 BSB
Byron Williams
3/11/2015 Patricia Braun 2-501 BSB
Emily Petruccelli
4/8/2015 Johnny Cruz Corchado 2-501 BSB
Juan Santana
5/13/2015 Ralph Hazlewood 2-501 BSB
Samuel Trammell

Genetics Winter Party!

The Genetics Social Activities Committee would like to thank everyone for making the Genetics Winter Party a blast!

GWP5GWP4GWP3GWP1

Congratulations to our Ugly Sweater Contest winners!

GWP2

1st place – Autumn

2nd place – Nikale and Brian

And a special thank you to those who helped with set up and take down!

 

Lisa Harney and Patrick Lansdon Will Present their Research Wednesday, 12/9/2014

Lisa’s Abstract

THE ROLE OF COPY NUMBER VARIATION IN NON-SYNDROMIC CLEFT LIP AND PALATE

LA Harney1,3, BW Darbro1,3, A Long2, J Standley1, JC Murray1,3, JR Manak1,2,3

1Department of Pediatrics, The University of Iowa

2Department of Biology, The University of Iowa

3Interdiciplinary Genetics Program, The University of Iowa

Orofacial clefting is a common congenital abnormality with clefts of the lip and/or palate (CL/P) affecting approximately 1 in 700 live births. About 70% of CL/P cases are estimated to be non-syndromic (NS) and do not exhibit cognitive or multiple congenital abnormalities. Although numerous genetic studies have been performed, no large-scale studies have examined the contribution of amplified and deleted regions of the genome, known as copy number variations (CNVs), to CL/P. We performed array-based genomic hybridization on a NSCL/P cohort from the Philippines to identify CNVs associated with clefting. After using bioinformatic quality controls to minimize false-positives, we analyzed 84 NSCL/P cases and processed a replication cohort of 854 NSCL/P cases for further analysis. We used an analysis pipeline to identify CNVs that overlapped with exons of genes in regions sharing 50% or less overlap with segmental duplications and common CNVs annotated in the Database of Genomic Variants. Analysis of CNVs in the cohort of 84 NSCL/P cases identified 358 genes in amplified regions and 36 genes in deleted regions. 21 of these genes have been previously linked to clefting including SKI, CDH1, CHD7, PAX6, OFD1 and TGFBR3. We are conducting a trio study using the losses identified in the small cohort to determine if the CNVs are de novo or familial. CNV analysis of the replication cohort is currently underway, and we will perform expression analysis of genes within the altered copy number regions and alter their dose in zebrafish to determine their role in CL/P. In the future we plan to extend this analysis to intronic and intergenic CNVs in hopes to define how CNVs contribute to NSCL/P and identify novel, causative variants for the disease.

Patrick’s Abstract

Understanding the mechanism of nutritional therapies for inherited seizure disorders in Drosophila

Epilepsy is one of the most common neurologic problems in the world. A significant portion of epileptic individuals are diagnosed with refractory epilepsy, and will not respond to anti-epileptic drugs. Nutritional therapies, such as the high-fat, low-carbohydrate ketogenic diet, show great promise to prevent or treat refractory epilepsy inexpensively and without serious side effects. However, the mechanism behind the beneficial effects of certain diets remains unknown. In the Kitamoto lab, we use the fruit fly Drosophila melanogaster as a model organism to study the effects of diet on neurological phenotypes displayed by mutants for the voltage-gated sodium (Nav) channel. Nav channels have been implicated in various human seizure disorders. In particular, mutations in the human SCN1A gene encoding a Nav channel have been associated with multiple seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Dravet Syndrome. Shudderer (Shu), a gain-of-function mutant for the Drosophila Nav channel gene, is characterized by seizure-like behavioral defects such as spontaneous leg jerking and twitching. We have recently shown that food containing milk whey drastically suppresses these neurological phenotypes of Shu.
Here we find that the same dietary therapy which improved Shu’s phenotypes can significantly improve the seizure-like phenotypes of Drosophila Nav channel mutants, bang senseless (bss1, bss2) and a Drosophila knock-in model of human GEFS+ (dGEFS+). Further, we found that the rescue effect of milk whey can be extended to the seizure-prone mutants, easily shocked (eas) and slamdance (sda) which lack altered Nav channel function. These results suggest milk whey has a broad effect among Drosophila seizure-prone mutants. Ongoing research aims to further characterize the effects of milk whey on these and other seizure-prone mutants and identify the specific component in milk whey that improves their seizure-like phenotypes.

Congratulations Alex Wagner!

The Genetics Social Activities Committee would like to congratulate Dr. Alex Wagner on the successful defense of his thesis and completion of his Doctorate!

Alex is from the Braun and Stone labs in the Biomedical Engineering and Ophthalmology and Visual Sciences departments.  His thesis seminar, entitled “Computational methods for identification of disease-associated variations in exome sequencing”, was on Wednesday, November 26th.  Alex has accepted a postdoctoral appointment at The Genome Institute at Washington University in St. Louis, where we expect he will continue to excel.

We asked Alex a few questions about his experience in the Genetics program here at Iowa:

Q. What is the most valuable/favorite thing that you have learned/experienced here at Iowa?

A. The Old Capitol building is definitely worth checking out.

 

Q. What was your favorite class to take?

A. Knowledge Discovery with Prof. Nick Street.

 

Q. What was your favorite class to TA?

A. Bioinformatics Tools and Techniques with Terry Braun

 

Q. Do you have any advice about preparing for defense?

A. Don’t sweat it. If you’ve made it this far, you’ve already succeeded.

 

Again, congratulations Alex and good luck at The Genome Institute!

The Genetics Website Committee Will be Hosting a Q&A and Eric Monson will Present his Research on Wednesday, 11/12/14

Eric’s Abstract

Assessment of Whole Exome Sequence Data in Attempted Suicide

In this study, we present the first large-scale sequencing project designed to assess the role of functional genetic variation within the human exome in the risk for suicidal behavior. Our analysis takes advantage of recently-developed variant collapsing methods to determine whether suicide attempters have elevated rates of functional mutational burden as compared to non-attempters. To do this, we generated whole exome sequencing data for 387 bipolar subjects with a history of a moderate or serious suicide attempt and 631 bipolar subjects with no history of suicide attempt. Additional sequencing targets for core regulatory regions of approximately 1500 genes predicted to be involved with synaptic function were also included in the data. Functional variant sets were assessed in groups defined by gene-loci and pathways using mutational burden and sequence kernel association tests. No signals survived correction for multiple testing. Our suggestive findings implicate glutamatergic signaling, as did our previous genome wide association study. This study demonstrates a first look at the potential power behind whole exome sequencing in the investigation of functional coding and regulatory variation contributing to the complex phenotype of suicidal behavior and the promise such techniques might afford as large scale next generation sequencing efforts continue to expand.

Sophia Gaynor and Hannah Seberg Will Present Their Research Wednesday, 10/8/2014

Sophia’s Abstract

NEXT-GENERATION SEQUENCING IN ATTEMPTED SUICIDE

S. Gaynor1, E. Monson1, M. Breen1, K. Novak1, J.B. Potash1, V.L. Willour1

1 University of Iowa, Department of Psychiatry

Suicidal behavior is a complex phenotype with an estimated heritability of 30-50%. While this heritability is partly dependent on the presence of psychiatric disorders, other evidence implicates an independent heritable factor. In order to assess the genetic basis of this independent factor, we are conducting a next-generation targeted sequencing project on 38 candidate genes and two candidate regions in 500 bipolar (BP) subjects that have attempted suicide and 500 BP subjects that have not attempted suicide. The candidate genes and regions were chosen based on hypotheses generated by our lab and evidence from the suicide literature. The target regions for sequencing include all exons of all alternative transcripts, intron-exon boundaries, alternative promoter regions, and any putative regulatory elements identified by ENCODE, including 10kb upstream and downstream of each gene. We currently have completed the sequencing for all of our samples and have data analyzed for 505 of these samples, including 254 BP attempters and 251 BP non-attempters. For these first 505 samples, we found 14,159 unique variant sites following quality control filtering. We performed both individual variant tests and gene burden tests on these variant sites. Our top findings from the individual variant testing include an intergenic region of 2p25 (p=1.20×10-4) and an intronic region of LRRTM4 (p=7.16×10-4). For gene burden testing, our top results based on p-value are DLG3 (p=1.07×10-2) and TMEM132A (p=1.3×10-2). Our top results based on odds ratio are NLGN4X (OR=0.191) and GRIN2B (OR=5.02). We are currently in the process of analyzing the remaining samples, and the addition of these samples will provide more power to identify significant variant or gene associations. The identification of variants associated with suicidal behavior in these candidate genes and regions will help elucidate the biological basis of this complex phenotype.

Hannah’s Abstract

TFAP2A and MITF work in parallel to activate melanocyte differentiation genes
Hannah Seberg1, Eric Van Otterloo2, Gregory Bonde2, Robert Cornell1,2

1Interdisciplinary Program in Genetics, 2Department of Anatomy and Cell Biology

Transcription factor activator protein 2 alpha (TFAP2A) is widely expressed in the neural crest and multiple neural crest-derived cell types, including melanocytes. Mutations in tfap2a cause pigmentation phenotypes in humans, mice, and zebrafish. However, it is unclear how TFAP2A activity relates to that of lineage-specific Micropthalmia-associated transcription factor (MITF), which directly regulates melanocyte differentiation effectors such as melanin synthesis genes. This issue is complicated by the redundant expression of Tfap2 paralogs. In zebrafish melanocytes, tfap2e is highly expressed along with lower levels of tfap2a and tfap2c. To study the role of multiple paralogs in melanocyte development, we created a tfap2e mutant using zinc finger nucleases. Whereas the number of melanocytes in tfap2a mutants is reduced by about 66%, tfap2e mutants have no discernable phenotype. However, tfap2a/e double mutants display about 50% reduction of melanocytes, suggesting partially redundant functions for tfap2a and tfap2e. We next assessed the genetic interaction between tfap2a and mitfa. Single heterozygous embryos are phenotypically normal, while tfap2a;mitfa double heterozygotes have fewer melanocytes. These data indicate that TFAP2A and MITF interact genetically, but the mechanism of this interaction is unknown. To test the model that TFAP2A and MITF co-activate melanocyte differentiation genes, we identified genes that are likely to be direct targets of TFAP2A. First, we generated a profile of genes that are significantly downregulated in trunks of tfap2a null zebrafish embryos. We then conducted anti-TFAP2A ChIP-seq in human primary melanocytes to create a profile of TFAP2A-bound loci. Genes at the intersection of these profiles include several melanin synthesis genes, such as DCT, PMEL, and OCA2. Many of these genes are also known to be direct targets of MITF. These results provide evidence that TFAP2A and MITF work in parallel to promote melanocyte differentiation, and show that the widely-expressed transcription factor TFAP2A can directly regulate expression of lineage-specific targets.

Michael Molumby and Salleh Ehaideb Will Present Their Research Wednesday, 9/10/14

Michael’s Abstract

Branching out by sticking together: gamma-protocadherins regulate dendrite arborization

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 the survival of subsets of neurons.  We have shown that the g-Pcdhs promiscuously form cis-tetramers that interact strictly homophilically in trans (Schreiner and Weiner, PNAS, 2010). The g-Pcdh cluster could thus generate 104-105distinct adhesive interfaces, providing CNS cells with molecular identities. We recently showed that the γ-Pcdhs promote cortical dendrite arborization by inhibiting a FAK/PKC signaling pathway (Garrett, et al., Neuron, 2012).

Here we provide further evidence for cortical dendrite arborization mediated by γ-Pcdhs homophilic matching. We demonstrate that Emx-Cre driven overexpression of a single γ-Pcdh isoform (A1 or C3) in the cortex increases dendrite arborization compared to control mice. We hypothesize that this increase is a result of increased matching of γ-Pcdhs tetramers in trans-interactions from the over expression of a single γ-Pcdhs isoform (γ-Pcdh-A1/C3) in the cortex.  To further support our hypothesis, we are pursuing experiments to disrupt trans-interactions of g-Pcdhs in vitro and in vivo to examine the effect on dendrite arborization.

Salleh’s Abstract

 Elucidating the mechanism of epilepsy in flies

Prickle spiny-legs (Pksple) is one of two adult isoforms encoded by the prickle gene, and plays a role in the non-canonical WNT signaling/planar cell polarity (PCP) pathway in flies. We previously reported that pksple mutants are seizure-prone, and that mutations in prickle orthologues are associated with myoclonic (muscle jerk) seizures in both flies and humans. Using a seizure stimulation paradigm, we find that the pksple heterozygous flies have a lowered seizure threshold compared to control flies, with increases in spiking activity after electric shock, similar to what is observed for other seizure-prone flies. Such seizure activity can be ameliorated by treatment with valproic acid, a human anti-epileptic drug which has been shown to be effective in treating human patients with PRICKLE mutations. Since these patients also suffer from ataxia, or uncontrolled gait, we developed an assay to assess whether our pksple flies had similar phenotypes. Notably, both pksple homozygotes and heterozygotes exhibited a statistically significant loss of coordinated gate compared to controls, with loss of both functional pksple copies resulting in a more severe ataxia than loss of just one copy. These data further underscore the striking parallels between the prickle-associated myoclonic epilepsy syndromes observed in flies and humans.

Presentation Schedule: Fall 2014

Date Presenters Location
9/10/2014 Michael Molumby 1-107 BSB
  Salleh Ehaideb  
10/8/2014 Hannah Seberg 1-107 BSB
  Sophia Gaynor   
11/12/2014 Jessica Ponce   1-107 BSB
  Eric Monson  
12/10/2014 Patrick Lansdon  1-107 BSB
  Lisa Harney  
     
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