The Genetics Social Activities Committee would like to congratulate Dr. Ralph Hazelwood on the successful defense of his thesis and completion of his Doctorate!
Ralph is from the Fingert lab in the Visual Sciences department. His thesis seminar, entitled “Molecular genetics of optic nerve disease using patients with cavitary optic disc anomaly”, was on Monday, March 9th, 2015. Ralph has accepted a postdoctoral appointment at Vanderbilt, where we expect he will continue to excel.
We asked Ralph 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. Building networks outside of my department and field was invaluable because it exposed me to different areas of research and allowed me to make great contacts and actually build friendships with well-known and senior investigators at great institutions and markets.
Q. What was your favorite class to TA?
A. Human Molecular Genetics
Q. Do you have any advice about preparing for defense?
A. Tons of advice but the most important ones are: don’t wait until the last minute to write your thesis even if your committee hasn’t given the ok. It will help you gather your thoughts and help you to start making a story which will help in the long run. The Grad college template for submission is no easy task so the earlier your start the better sanity you will have. Also, the defense is literally just that, you have to defend the work you did so be prepared to talk about why you did the particular experiment(s) and why you came to that particular conclusion(s) and the caveats based on the data. But most importantly, you pretty much know more about your project than anyone else and so your thesis committee wouldn’t allow you to schedule a defense if they didn’t feel you were ready. Finally, think about big picture impacts and/or similar genetics mechanisms or disease processes to your project which should show you have a grasp of the field and genetics in general
Again, congratulations Ralph and good luck at Vanderbilt!
Genome-wide analysis of differential DNA methylation due to social defeat stress mouse model
Early life stress is a significant risk factor for anxiety and depressive disorders, and DNA methylation (DNAm) plays an important role in the intervening pathways. The extent to which DNAm is altered across the genome in these disorders is unknown. We sought to identify the genes and regulatory sequences altered by stress in a mouse model of the disorders. Using a social defeat stress paradigm, we assessed genome-wide DNAm changes in mouse brain. We used two cohorts of stressed mice (N=7 and 7) and non-stressed controls (N=8 and 12), for discovery and validation, respectively. DNA was extracted from the dentate gyrus using a punch technique. DNA from the first cohort was examined in the discovery phase using the target enrichment system Methyl-Seq, which captures ~220,000 CpG-rich regulatory regions of the genome. DNA from the second cohort was used for validation with bisulfite pyrosequencing. Analysis of the Methyl-Seq data was done using GENESPRING and MethylKit. Thirty-three regions were significant in the discovery phase. Of these, 31 did not validate. In the remaining two regions, we detected differentially methylated loci between stressed and control groups. These loci represent an intronic region of Drosha (encoding a microRNA processing protein) and an intergenic region on chromosome X (IntchX). However when these two regions were further interrogated with a new cohort of mice (N=16 stressed and non-stressed), only IntchX replicated. IntchX included eight differentially methylated CpGs over 150 bps within an evolutionary conserved region. Because only one differentially methylated region due to social defeat stress region was replicated, further analysis of the Methyl-Seq data with follow-up bisulfite pyrosequencing will be performed to elucidate additional regions. Moreover, the nucleus accumbens, the brain region essential to reward, will be investigated for DNAm differences induced from the social defeat stress paradigm.
Sleep Abnormalities in a Drosophila Model of Human GEFS+
Despite an established link between epilepsy and sleep behavior, it remains unclear exactly how specific epileptogenic mutations affect sleep and how sleep influences epileptic seizures. Drosophila is an attractive model for studying the underlying mechanisms of this seizure/sleep relationship as it is routinely used to examine the genetic basis of seizure susceptibility and sleep behavior. Sun et al (2012) recently created a knock-in fly model of human Generalized Epilepsy with Febrile Seizures Plus (GEFS+), a wide spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a mutation in the voltage-gated sodium channel (Nav) gene, mimicking a disease-causing human Nav mutation (SCN1AK1270T) and display a semidominant heat-induced seizure phenotype as a result of abnormal electrophysiology in inhibitory GABAergic neurons. We found that GEFS+ mutation also dominantly modifies sleep behavior, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. This sleep profile was observed regardless of sex, mating status, and genetic background. Mutants’ exaggerated sleep was more resistant to carbamazepine (CBZ), a drug that reduces Drosophila GABAA receptor activity, and could be suppressed by either constant or acute scotophase light. We further observed that GEFS+ flies have normal circadian rhythm in free-running dark conditions, but significantly lack homeostatic rebound following sleep deprivation. Intriguingly, sleep deprivation treatment increased the heat-induced seizure susceptibility of control flies, but reduced the seizure severity of GEFS+ mutants. Ongoing experiments are addressing the potential significance of GABAergic inhibition on wake-promoting PDF+ neurons in GEFS+ mutant sleep and the impact of seizing on subsequent sleep behavior. Our findings thus far have characterized the sleep architecture of Drosophila harboring a human GEFS+ mutation and provided unique insight into the relationship between sleep and epilepsy.
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.
|1/14/2015||Xue Xiao||2-501 BSB|
|2/11/2015||Matthew Strub||2-501 BSB|
|3/11/2015||Patricia Braun||2-501 BSB|
|4/8/2015||Johnny Cruz Corchado||2-501 BSB|
|5/13/2015||Ralph Hazlewood||2-501 BSB|
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.
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.
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
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.
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.
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.