Background for Yang Xu’s talk
Genome-Wide Mapping of Genetic Factors of Metabolic Syndrome in Rats
Ralph Hazlewood and Matthew Jorgenson will present their research on Thursday, December 15th, 2011
Background on Ralphs talk:
Identification and Characterization of Genetic Factors Responsible for Cavitary Optic Disk Anomalies
Glaucoma is the second leading cause of irreversible blindness in America and is characterized as an optic neuropathy with visual field loss and progressive degeneration of the optic nerve seen as increased optic disk cupping. Many cases of glaucoma occur in part due to high intraocular pressure (IOP), but glaucoma can occur at any IOP; glaucoma at normal IOP is called normal tension glaucoma (NTG). One way to study NTG is to investigate similar forms of optic nerve disease that also occur in the absence of elevated IOP. Cavitary optic disc anomalies (CODA) are associated with congenital excavation of the optic nerve that in some patients progressively deteriorate resembling the cupping seen in glaucoma. Based on the similarities between NTG and CODA patients, we are searching for the gene that causes autosomal dominant inheritance of CODA in a large family. Prior linkage studies mapped the CODA gene to a 13.5 Mbp segment of chromosome 12q14 (maximum non-parametric linkage score = 21.7). We have examined the linked region for the gene that causes CODA using comparative genome hybridization (CGH) and expression constructs. Study of affected family members with CODA using CGH identified a copy number variation (CNV) within the previously linked locus. A heterozygous triplication of a 6kb segment was found to be co-inherited with CODA in this pedigree and absent in unaffected family members and in normal controls. Subsequent analysis showed a two-fold increase in expression when the 6kb segment is cloned into a reporter vector and transfected into HEK293T cells. We report a CNV within the previously linked region that is co-inherited with CODA in our family. We hypothesize that this CNV leads to dysregulation of the expression of an extracellular protease gene and ultimately to the development of CODA. Future studies include fluorescent in situ hybridization (FISH), Southern blotting to characterize the triplication and generation of transgenic animals.
Background on Matthew’s talk:
Discovery and characterization of new bacterial cell division genes
Cell division is a process essential to all organisms, yet it remains poorly understood. In the bacterium Escherichia coli, cell division is mediated by the formation of the septal ring at the midcell through the coordinated interaction of at least thirty proteins. Two components of this assembly are FtsE and FtsX, which together comprise a putative ATP-binding cassette transporter. While previous work with FtsEX has demonstrated it is important in maintaining the integrity of the septal ring, its exact biochemical function is unknown. To further characterize ftsEX function in cell division, our lab carried out a screen for multicopy suppressors of an ftsEX null mutant. Two of the plasmids returned in this screen carried a gene for an outer membrane lipoprotein named NlpI and various amounts of flanking DNA. Interestingly, an nlpI mutant has a cell division defect at elevated temperature (Ohara et al. 1999), but the precise function of NlpI is not known.
To show that nlpI is indeed responsible for rescue of the ftsEX mutant, I subcloned nlpI into an expression vector and demonstrated that rescue did not require any DNA outside the nlpI gene. Multicopy nlpI was not able to rescue ftsA(Ts), ftsI(Ts),or ftsZ(Ts) but did show weak suppression of an ftsQ(Ts) mutant. I raised polyclonal antibody against NlpI and used it to show that there are ~2,000 molecules of the protein per cell during growth in LB media. Currently I am conducting a structure-function analysis of the protein that focuses on the importance of localization to the outer membrane and a proposed substrate binding pocket. I am also revisiting the reported phenotypes of an nlpI null mutant in hopes that this will provide some more specific insights into the role of the protein in cell division.
Xitiz Chamling and Johnny Cruz Corchado will present their research on 17th November, 2011
Background for Xitiz Chamlings talk:
A novel BBSome interacting protein is an ADP/ATP translocase, SLC25A31
Chamling X.1, Seo S.3, Sheffield V.C.1,2,3
1Department of Pediatrics, Interdisciplinary program of genetics University of Iowa, Iowa City IA 52242 2Department of Pediatrics, Howard Hughes Medical Institute, IA 52242 and 3Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa, IA 52242
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogenitalism and renal defects. At least 15 different BBS genes have been reported. Recent findings have implicated BBS with primary cilia dysfunction, and BBS proteins are thought to be involved in the trafficking of various ciliary proteins. Specifically, the BBSome, a stable complex of seven highly conserved BBS proteins, BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and a novel protein, BBBIP10, is believed to traffic to cilia bringing various cargos along with it. In recent studies, various different interacter proteins including the leptin receptor, Rabin 8, PCM1 and few others have been associated with the BBSome. Due to the pleiotropic nature of the disease and the abundance of proteins in and around cilia where BBS proteins localize, we hypothesized that there are other proteins that interact with the BBSome which have not been identified. To investigate this hypothesis we generated a transgenic (LAP-BBS4) mouse line that expresses LAP tagged BBS4 in multiple different tissues including eye, brain and testis. Using lysates from brain and testis of the transgenic mouse, we pulled down BBSome interacters. Mass spectrometry confirmed one specific interacting protein to be SLC25A31 (solute carrier family 25 member 31), also known as ADP/ATP translocase 4 or Ant4.
Background for Johnny Cruz Corchado’s talk:
Identification of new genetic variants in Dense Deposit Disease using sequence capture of complement and complement related genes
Johnny Cruz Corchado1,2, Bu Fengxiao2, Tara Maga1,2, Richard J. Smith1,2
1Department of Otolaryngology – Head and Neck Surgery, University of Iowa, Iowa City, IA, USA, 2 Interdepartmental PhD Program in Genetics, Department of Otolaryngology, University of Iowa, Iowa City, Iowa City, IA, USA
Dense Deposit Disease (DDD) is a complement mediated disease that leads to renal failure in 50% of affected patients. Its genetics are complex but underlying disease pathogenesis is uncontrolled fluid-phase activation of the alternative pathway (AP) of the complement system. Genetic variability in the complement AP is associated with DDD, but most of the patients do not present a pathological variant in the genes screened. Therefore, we sought to identify novel pathogenic variants by increasing the number of genes screened. In addition, we sought to define a more comprehensive DDD “at risk” haplotype by identifying new polymorphic variants that segregate preferentially with the disease. We completed a focused targeted-sequence capture of 85 complement and complement-related genes that may play a role in DDD. This in-house developed platform called CASCADE includes genes in the three pathways of the complement system, regulators of the complement activity (RCA), and other complement related genes. Herein we report our preliminary findings and experience with 15 DDD patients using CASCADE.
Ben Brett and Dina Ahram will present on Sept 15th in 2-501BSB at noon
Background on Ben Brett’s talk
Background on Dina Ahram’s talk
Clinical and Genetic Investigation of Familial Angle-Closure Glaucoma in the Basset Hound
Primary angle closure glaucoma (PACG) is an optic neuropathy marked by progressive retinal ganglion cell degeneration, cupping of the optic nerve head and subsequent loss of vision in association with a gradual increase in intraocular pressure (IOP). The condition is most commonly caused as a result of the collapse of the irido-corneal angle due to the movement of the root of the iris anteriorly towards the cornea. We have identified a number of Basset Hound pedigrees with characteristic PACG that in many aspects recapitulates the clinical PACG phenotype observed in human patients. The condition in Basset Hounds appears at variable ages of onset, segregates in an autosomal recessive manner and displays complete penetrance. Our goal is to utilize the Basset Hound PACG model in order to characterize and better understand the pathophysiology of PACG and its predisposing risk factors. We are uniquely positioned to achieve our goal through a combination of high density mapping of genetic markers, as well as analysis of multiple quantitative traits, copy number variations and gene expression changes in PACG Basset Hound pedigrees, which may enable us to identify the gene or gene network implicated in PACG. Once identified, we aim to characterize the role of this gene in PACG through molecular and protein functional studies. Ultimately, we anticipate for these studies to provide valuable insight into the pathophysiology and genetics of human PACG.
Ji Wan and Pavitra Narasimha will present their work at student seminar in 2-501 BSB on 25th August 2011
Background on Pavitra Narasimha’s talk :
RNAi therapy for Spinocerebellar ataxia 7 (SCA7)
We are investigating RNA interference (RNAi) therapy for the autosomal dominant polyglutamine (polyQ) expansion disorder spinocerebellar ataxia type 7 (SCA7). Normal individuals have ~10 CAG repeats in ATXN7, while an expansion of >37 CAG repeats causes the disease. SCA7 is characterized by Purkinje cell (PC) degeneration and retinal cone-rod dystrophy (>59 CAG repeats). Using our prior experience in developing artificial miRNAs for directed gene silencing (Boudreau et al., 2009), we designed miR-based vectors for silencing mouse and human ataxin-7 (ATXN7). RNAi triggers were designed for allele-specific and non-allele specific silencing (miS1-S4). For allele-specific silencing, artificial miRNAs were designed targeting a SNP (miSNP) common among South African patients. The miR vector candidates and a scrambled control were tested for knockdown of human-ATXN7-92Q (myc-tagged human ATXN7 with a pathogenic repeat of 92 polyQs) in vitro in HEK293 cells. Significant knockdown of human ATXN7 protein was seen with miS4 and miSNP by western blot. To test knockdown of mouse Atxn7, N2A (neuroblastoma) cells were used. MiS4 showed a significant (P<0.05) ~40% knockdown of Atxn7 mRNA, while miSNP vector did not, demonstrating its allele-specificity. The ATXN7 targeting vectors (miR.Sca7S4 and miR.Sca7SNP) are being tested in vivo for SCA7 therapy.
We have successfully transduced cerebellar PCs by stereotactic injection into the wildtype mouse cerebellum using AAV2/1 viral vectors expressing eGFP. Thus we can use AAV2/1 to target cerebellar PCs for SCA7 therapy.To test for transduction of retinal photoreceptors, we performed sub-retinal injections in wildtype mice eyes with AAV2/1 and AAV2/2 viral vectors expressing eGFP. We were able to successfully transduce the photoreceptor cells of the retina, as seen by eGFP expression. To determine the mRNA expression pattern of Atxn7 in the retina, RNA in situ hybridization was initially performed on the mouse wildtype retina. In situ hybridization is now being done on human retinal sections to confirm ATXN7 mRNA expression. These studies will determine the cellular target for SCA7 RNAi therapy in the retina.
Background on Ji Wan’s talk
Analysis of molecular dynamics of HD disease by mRNA and microRNA profiling of BA4 cortex samples
Background for Elizabeths talk
“Pursuit of etiologic variants for cleft lip and palate following a successful genome wide association study”
Nonsyndromic cleft lip and/or cleft palate (NSCL/P) is a common birth defect with complex etiology reflecting the action of multiple genetic and/or environmental factors. The search for genetic contributors to NSCL/P has been intensive using a variety of approaches including candidate gene, genome wide linkage, and genome wide association. Four genome wide association studies have been published in recent years, identifying several novel associations and confirming previous associations from candidate gene studies. The challenge following genome wide association studies is identifying the disease-causing etiologic variants at each associated locus tagged by presumably non-causal markers. We hypothesize that etiologic variants contributing to NSCL/P will be a combination of common and rare variants, possibly located in noncoding regulatory regions of the genome. I am using two approaches to identify these variants. The first approach could be considered “sequence first” and will leverage next generation sequencing technologies by capturing large intervals surrounding thirteen genes and loci associated with NSCL/P and deep sequencing them in a large set of case-parent triads with NSCL/P. The second approach uses craniofacial enhancers identified by collaborators to prioritize regions of the genome for sequencing. At seminar I will discuss these approaches and present new results identifying potential etiologic variants at a GWAS locus near the transcription factor, VAX1.
Background for Danielle’s talk
Genome-wide CNV analysis of schizophrenia subjects
Schizophrenia is characterized by positive and negative symptoms that include hallucinations, delusions and disorganized thought as well as flat affect and decreased speech. Genomic copy number variation (CNV) has been found to underlie and increase risk for a large number of neuropsychiatric disorders including schizophrenia. This project focuses on identification and characterization of pathogenic CNVs in a sample set of 457 schizophrenia patients and 290 psychiatrically healthy controls. To date, 181 probands and 141 controls have been genotyped using the Affymetrix Genome-Wide Human SNP 6.0 Arrays and analyzed for CNVs with Genotyping Console and CNAG. CNVs were compared between cases and controls and cross-referenced with the Database of Genomic Variants. The proximal arm of chromosome 15 contains canonical segmental duplications that predispose the region to CNV formation, but only recently have disease-associated CNVs been reported between the two most proximal breakpoints (BPs), BP1 and BP2. Here we report three case subjects that have a ~1Mb deletion of the BP1-BP2 region at chromosome 15q11.2. The BP1-BP2 deletion encompasses the four genes CYFIP1, NIPA1, NIPA2 and GCP5. The deletion is present in three probands, two were maternally inherited and none were found in controls. All CNVs of interest were validated by qPCR or NimbleGen 385K whole-genome array CGH. The four genes within the deletion have previous association with Prader Willi syndrome, Angleman syndrome, autism and schizophrenia and are often inherited from a mildly or unaffected parent. CYFIP1 is of primary interest due to its direct interaction with the Fragile X Mental Retardation Protein (FMRP) and its reported role in regulation of neuronal translation during brain development. NIPA1, NIPA2 and GCP5 are also expressed in the brain. It is a distinct possibility that in addition to the 15q11.2 deletion in probands, a “second CNV hit” contributes to disease pathology. Thus, the two probands with maternal inheritance of the deletion were further analyzed for sporadic secondary CNVs. For example, a de novo duplication of four exons of ATXN2 as well as a de novo deletion of the promoter and the first three exons in SYN3, a gene with previous association with schizophrenia, was called in one proband. In the second family the proband had an additional de novo duplication of two exons in two synapse-associated genes CACNA1B and DLGAP2 as well as a de novo intronic duplication of SYN3.
Background for Lily’s talk - 
Evaluating THE ROLE OF Prickle mutations in Human PROGRESSIVE MYOCLONIC EPILEPSY
Epilepsy is a complex genetic brain disorder characterized by seizures that afflicts ~1% of the population. For over 70% of patients, the underlying cause of epilepsy remains unknown and seizures remain uncontrolled in about 25%. Progressive Myoclonic Epilepsy (PME) is a rare epilepsy characterized by neurodegeneration, myoclonus, and generalized seizures. Bassuk et. al identified mutations in Prickle1 and Prickle2 responsible for PME in families and in the general population.
The Prickle genes are expressed in the developing nervous system and mature neurons, involved Planar Cell Polarity and are players in Wnt-mediated signaling; a cascade likely critical in neurodevelopment and neurodegeneration. Studies have shown that Prickle mutations are associated with seizures in Drosophila and mice. In zebrafish, overexpression of mutant Prickle resulted in compromised protein function. My ongoing and future experiments include characterizing the effect of Prickle mutations on protein-protein interactions, neuronal differentiation, calcium activation, and REST-mediated transcriptional repression. To resolve some of these questions, I have established stable lines expressing wild-type and mutant Prickle 1 and Prickle 2. Findings from these studies would clarify molecular mechanisms by which Prickle mutations alter neuronal function and help identify novel therapeutic targets.
BACKGROUND FOR MATHEW FARON’S TALK
Francisella tularensis is a gram negative facultative intracellular pathogen and is the causative agent of tularemia. Though tularemia is not a common disease, its low infective dose and high mortality rate for untreated infections has led to F. tularensis being classified as a class A select agent that could be utilized as a bioterrorism weapon. Francisella research has focused on how it evades the host immune system and survives and replicates within both macrophages and neutrophils. However, little is known about how Francisella adapts and survives within such hostile environments. What has been shown is that Francisella utilizes a 17 gene pathogenic island which allows Francisella to escape from the phagosome and replication within the cytosol. Several regulators have been identified, but the molecular mechanisms are unknown. Our research has focused on characterizing these regulators and their role in virulence. Currently I have identified several environmental signals that significantly alter FPI expression.
Emily Petruccelli and Tryphena Cuffy will be presenting in Student Seminar on June 23rd at noon
Emily Petruccelli
Background information:
In Dr. Toshi Kitamoto’s lab we are interested in understanding how the external environment affects neural plasticity leading to changes in behavior. I am specifically interested in how unconventional steroid hormone action- occurring independent of new mRNA synthesis- affects neuromodulation in complex behaviors. Importantly, drug abuse, as well as many neuropsychiatric diseases, are associated with steroid hormone dysfunction. Unfortunately, the function and mechanism of “nongenomic” steroid actions are not well understand or are highly controversial. To address this gap in our knowledge, I am using the plethora of genetic tools and established behavioral paradigms of the tractable model organism Drosophila.
Recently, the G-protein coupled receptor DopEcR was identified as a novel nonclassical ecdysone receptor in flies. Ecdysone is the major insect molting hormone of insects and is well known for its role in regulating developmental transitions and oogenesis in adult females. However, work in our lab and others have shown that ecdysone also regulates complex adult behaviors such as long-term memory formation, sleep and longevity. To understand how nongenomic signaling of ecdysone impacts neural plasticity and behavior, we identified and examined hypomorphic DopEcR mutants. Interestingly, DopEcR-mediated signaling appears to be important for both associative and non-associative learning as well as for regulating ethanol-induced behavior. I am currently generating a null DopEcR mutant using homologous recombination techniques and further characterizing the incoordinating, sedation and tolerance of mutants exposed to ethanol vapor.
Since both steroid hormone signaling and ethanol-induced behaviors are highly conserved between flies and mammals, this work will advance vertebrate research efforts to understand nongenomic steroid action and better our understanding of neuroendocrinology.
Tryphena Cuffy
Background information 
Exfoliation syndrome (XFS) is a common age-related disorder characterized by the pathological accumulations of fibrillar exfoliative material in the anterior chamber of the eye. Patients with XFS can go on to develop exfoliative glaucoma; potentially as a result of an accumulation of exfoliative material at the drainage structures of the eye. Human eyes with XFS exhibit a striking pattern of Marcel-like iris transillumination defects. The same pattern is recapitulated in mice containing the Lystbg-J mutation. We have found that the molecular basis of the bg-J mutation is a three base pair deletion in the WD40 encoding region of the Lyst gene. In yeast two-hybrid assays others have identified a small number of proteins capable of interacting with the LYST WD40 motif – one of these being CSNK2B. Testing CSNK2B, as a candidate interacting protein, previous GST-pulldown experiments confirmed that wild-type LYST can bind to CSNK2B whereas LYSTbg-J can not. This result suggests that LYST may play a role in regulating activity or localization of CSNK2B. In testing this hypothesis, experiments with primary fibroblasts expressing a GFP-tagged CSNK2B fusion protein suggest that LYST regulates the subcellular localization of CSNK2B. Furthermore, two CSNK2B substrates (PER2 and CDH1) exhibit functional deficits in the presence of the Lystbg-J mutation. Combined, these results implicate CSNK2B and its substrates in the pathogenesis of XFS. In addition, another striking phenotype associated with the bg-J mutation is a dysfunction in the circadian regulation of pressure in the eye – intraocular pressure. Many patients, who exhibit elevated intraocular pressure along with glaucoma, are treated with drugs that lower that intraocular pressure. My goal is to also investigate elevated intraocular pressure in glaucoma patients using the B6-Lystbg-J mouse as a model.
Background for Amber Hohls’ talk :
IDENTIFICATION OF MUTATIONS IN TOPOISOMERASE 2, A PROTEIN REQUIRED FOR SOMATIC PAIRING IN DROSOPHILA
Amber M. Hohl1,2, Morgan Thompson2, Jianhong Wu3, James Morris4, Tao Shih-Hsieh3, Ting Wu2, and Pamela K. Geyer1,5
1Graduate Program in Genetics, University of Iowa, Iowa City IA 2 Department of Genetics, Harvard Medical School, Boston MA; 3Department of Biochemistry, Duke University, Durham NC; 4Department of Biology, Brandeis University, Waltham MA; 5Department of Biochemistry, University of Iowa, Iowa City IA
Homologous chromosomes display associations in many organisms. Many of these interactions lead to changes in gene expression, as exemplified by processes of transvection, mammalian X-inactivation, and imprinting. Drosophila serves as an excellent model to study pairing interactions because homologous chromosomes in all cells are fully aligned. Using a cell culture based assay, Topoisomerase 2 (Top2) was found to be required for chromosome pairing. This gene encodes an ATP dependent homodimeric enzyme that generates double stranded breaks to change DNA topology. Top2 has many known functions, including DNA replication, chromosome segregation and transcription. To expand our understanding of the role of Top2 in chromosome pairing, we conducted a genetic screen to isolate germ line mutations in Top2. Fourteen recessive lethal alleles were identified from a screen of three thousand chromosomes. Molecular analyses of these alleles uncovered single or multiple base pair substitutions within each mutant line. These included changes that generated premature stop codons or missense mutations within previously identified functional domains. Complete loss of Top2 is lethal, with evidence of decreased cell division. Even though flies carrying each missense allele in trans to a deficiency produce inviability, hetero-allelic combinations of several missense alleles produce live flies. Current studies are directed at using the viable hetero-allelic combinations to examine chromosome structure and function during development. Further, these studies indicate that female germline development is particularly sensitive to decreased Top2 activity, implying roles for this enzyme in meiotic pairing. These data suggest that the collection of Top2 mutations will provide a resource for understanding chromosome functions that require homolog interactions.
Background for Heather Brockways’ Talk:
Breaking It Down: Investigating Synaptonemal Complex Disassembly In C. elegans Using RNAi
Heather M Brockway1, Sarit Smolikove1,2
1University of Iowa Interdisciplinary Program in Genetics, 2University of Iowa Department of Biological Sciences
The events in meiotic prophase I are essential for proper chromosome segregation. In prophase I, homologous chromosomes pair, synapse, and form crossovers to recombine the genetic material. Once these events are completed, the homologous chromosomes segregate in preparation for meiosis II, at which time the sister chromatids separate. This process is highly regulated by a series of molecular checkpoints, ensuring that each step proceeds correctly. The synaptonemal complex (SC) is involved in most prophase I events and is a highly conserved protein structure formed between homologous chromosomes during meiosis. The absence of a functional SC leads to missegregation of chromosomes and generates aneuploid gametes. We utilize the model organism C. elegans to elucidate the underlying molecular mechanisms of the SC disassembly pathway. Our research employs RNAi methodology as a means of discovering the proteins associated with SC disassembly. Prior screens conducted in our lab have uncovered a novel mutant possessing meiotic defects specific to SC disassembly. Using this new mutant, we have initiated an RNAi screen to search for enhancers and suppressors of the embryonic lethal phenotype associated with this mutation. The goal of this screen is to identify SC disassembly proteins via their interaction with this new mutant. To date, 4% of the RNAi library has been screened. In this pilot, we identified three suppressors and seven enhancers that were mutant-specific. Eight of these clones have not shown an embryonic lethal phenotype in other screens, including a screen on the wild-type background, thus indicating we are capable of identifying novel clones with this method. Of these eight clones, two are known genes while the rest are uncharacterized ORFs. Cytological analyses are conducted to determine if the changes in embryonic lethality are associated with defects in SC disassembly. Identifying meiotic genes involved in SC disassembly will lead to a better understanding of the molecular mechanisms involved with the disassembly process and chromosome segregation in general.
Emily Petruccelli has volunteered to be the student representative for the website committee (Dr. Tina Tootle and Dr. Sarit Smolikove). Please send any suggestions about the Genetics Dept website to Emily.
