Genetics Winter Party!

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

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Congratulations to our Ugly Sweater Contest winners!

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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  
     

Allison Cox and Hung-Lin Chen Will Present their Research Thursday, 8/7/14

Allison’s Abstract

Whole Exome Analysis of Individuals and Families with Chronic Recurrent Multifocal Osteomyelitis (CRMO)

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, autoinflammatory bone disease presenting in infancy and childhood. While CRMO is characterized by painful bone lesions, it is often comorbid with psoriasis or Crohn disease and many patients have close relatives with either of the more common disorders. Some syndromic cases of CRMO are caused by truncation mutations in the interleukin-1 receptor agonist (IL-1RA) gene and these children respond very well to treatment with recombinant IL-1RA. However, most cases are non-syndromic with an unknown genetic cause, although many patients respond to TNF-α blocking agents, implicating the pathway in the disease. Using whole exome sequencing paired with genetic analyses based on inheritance patterns in several affected families, I hope to determine a component of CRMO’s underlying genetics. Preliminary results implicate a shared pathway in the pathogenesis of the disease. I am currently sequencing a candidate gene in a large cohort of CRMO samples from our laboratory.

Hung-Lin’s Abstract

Effect of diet on genetically inherited seizure-like behavior in Drosophila
Hung-Lin Chen,1 Patrick Lansdon,1 Junko Kasuya,2 Toshihiro Kitamoto1,2
1Interdisciplinary Genetics Ph.D. Program and 2Department of Anesthesia

Nutritional therapies have the great potential to prevent or treat various neurological disorders, such as epilepsy, in an effective, natural, and economical manner. However, their exact therapeutic values have not been rigorously evaluated in most cases and the molecular mechanisms responsible for their beneficial effects remain elusive. The goal of this study is to unravel the fundamental molecular underpinnings of nutritional therapies for genetically inherited neurological dysfunctions using the fruit fly Drosophila melanogaster as a model organism. Shudderer (Shu) is a classical Drosophila mutant that is characterized by strong spontaneous jerking and twitching. Our molecular analysis revealed that Shu carries a gain-of-function mutation in the voltage-gated sodium channel gene, paralytic. Here we report that feeding Shu mutants a modified diet results in drastic improvement of their seizure-like phenotypes. Shu needs to be fed the “therapeutic” diet during the larval stage in order to receive a maximum benefit from the food, suggesting that the diet has a therapeutic effect on Shu by affecting development of the nervous system. These findings are significant because they provide an unprecedented opportunity to employ versatile experimental tools available for Drosophila and investigate the mechanisms underlying the diet-dependent improvement of inherited behavioral abnormalities under strictly controlled genetic and environmental conditions.

University of Iowa Alumni Igor and Patricia Schneider Will Meet with Genetics Students 7/24/14

Danielle Herrig and Autumn Marsden will Present their Research Thursday, 7/10/14

Danielle’s Abstract

SPECIATION GENETICS IN DROSPHILA: INSIGHTS FROM GENES, GENOMES, AND TRANSCRIPTOMES (YEAR 3)

Speciation typically occurs when a single species splits into two populations in which gene flow is severely reduced. Over time, the two populations accumulate genetic differences that eventually produce two independent species. While hybridization between two species, and thus the potential for gene exchange, has traditionally been viewed as a reproductive mistake, recent studies suggest that it is not as rare as once believed. Previous studies suggest that although regions of the mitochondrial and nuclear genomes can be exchanged independently or together, the X chromosome often plays a large role in speciation and gene exchange is typically barred on the X. One way in which the X chromosome may play a large role in speciation is though X-linked trans-regulatory elements (TREs) effecting the expression of autosomal genes through interactions with autosomal cis-regulatory elements (CREs). Indeed, whole-genome analyses of gene expression in our lab indicate that X-linked genes are more differentially expressed between species while autosomal genes are preferentially misexpressed in hybrid males. Because there is only one copy of the X chromosome producing X-linked TREs in hybrid males, we hypothesize that the hemizygosity of the X chromosome leads to greater levels of autosomal misexpression in the heterogametic sex. To investigate the effects of the uni-parental origin of the X chromosome on autosomal misexpression, we will analyze whole-genome expression in attached-X stocks of Drosophila yakuba, D. santomea, and their hybrids wherein the X chromosomes are inherited in a uni-parental manner in females. It is my expectation that if the hemizygosity of the X chromosome contributes to autosomal misexpression in males, attached-X F1 hybrid females will mimic results from F1 hybrid males.

Autumn’s Abstract

Calcium-dependent Naked-Dishevelled Interaction Modulates Wnt Signalling Outputs

AN Marsden1,2, SW Derry1, TA Westfall1, DC Slusarski1

1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics

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 is not completely understood, especially since they share receptors and cellular effectors. My project focuses on two such shared components that also bind each other, Dvl and Naked (Nkd). Previously we demonstrated that Nkd is required for zebrafish dorsal forerunner cell (DFC) migration, Kupffer’s vesicle formation and proper organ laterality. Moreover, we identified calcium fluxes in the DFCs and determined that the EF-hand motif in Nkd weakly binds calcium. Using a combination of biochemical and functional assays, we show calcium-induced conformational changes in the Nkd-Dvl complex and identify a requirement for the Nkd EF-hand in cell polarity but not in β-catenin transcriptional outputs. We predict that Nkd and Dvl form a cooperative calcium binding pocket, which allows for conformational changes or subcellular localization to direct Wnt/PCP output. We identified a region in Dvl that may coordinate ion binding. We have mutated this novel Dvl calcium binding site, and performed biochemical, genetic, and functional studies. To determine the impact upon Wnt signaling output, I utilize gene knockdown and rescue in the zebrafish DFCs, in a tissue that hosts converging Wnt signals. I also determined the subcellular localization of Nkd and Dvl components within the cells known to have calcium fluxes and cells that are quiescent. Our data suggests that calcium-induced secondary structure changes in the Nkd-Dvl complex serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.

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