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.
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.
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.
|9/10/2014||Michael Molumby||1-107 BSB|
|10/8/2014||Hannah Seberg||1-107 BSB|
|11/12/2014||Jessica Ponce||1-107 BSB|
|12/10/2014||Patrick Lansdon||1-107 BSB|
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.
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.
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.
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.
Abstract for Emily Beck
Cyto-nuclear co-intogression in the sister species Drosophila yakuba and D. santomea
Introgression, also called introgressive hybridization, is the effective exchange of genetic information between species through natural hybridization, and can occur when reproductive isolation is incomplete. Previous genetic analysis of the hybrid zone formed by Drosophila yakuba and its sister species D. santomea showed that the mitochondrial genome of the former species had introgressed into the latter and completely replaced its native form. Since mitochondrial proteins work intimately with nuclear-encoded proteins in the oxidative phosphorylation (Oxphos) pathway, we hypothesized that some nuclear genes in Oxphos co-introgressed along with the mitochondrial genome allowing for preservation of function. We analyzed nucleotide variation in the 12 nuclear genes that form cytochrome c oxidase (COX), an Oxphos enzyme complex composed of both nuclear- and mitochondrial-encoded proteins, in 33 Drosophila lines. Using maximum likelihood methods, we detected significant introgression from D. yakuba to D. santomea in only the three nuclear genes composing subunit V of this complex. The detection of introgression in the three proteins that work together in same subunit, interact with one another, and directly with the mitochondrial-encoded core strongly supports coordinated cyto-nuclear co-introgression to allow for optimal COX activity. We aim to investigate the nuclear genes of all the other subunits of Oxphos (88 genes total) for evidence of co-introgression.
Abstract for Fengxiao Bu
CFH-mutation related atypical hemolytic uremic syndrome may be modulated by Coagulation factor X
F. Bu1,2, N. Borsa2, W. Tollefson3, M. Schnieders3, H. Azaiez2, K. Wang4, C. Thomas2,5, C. Nester2,5, R. Smith1,2,5
1) Interdepartmental PhD Program in Genetics, University of Iowa, Iowa City, IA; 2) Molecular Otolaryngology and Renal Research Laboratories, University of Iowa, Iowa City, IA; 3) Department of Biochemistry, University of Iowa, Iowa City, IA, USA; 4) Department of Biostatistics, College of Public Health, University of Iowa, Iowa City, IA; 5) Rare Renal Disease Clinic, Departments of Pediatrics and Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA,
Background: Atypical hemolytic uremic syndrome (aHUS) is a complement-related rare renal disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. Mutations in CFH account for approximately 22% of aHUS cases. In familial cases, CFH-mutation penetrance ranges from 12.5% to 100% suggesting the involvement of other genetic factors/modifiers in the etiology of this disorder.
Methods: Five families carrying the same disease mutation – c.3644G>A, p.Arg1215Gln – in CFH were included in this study. Using targeted genomic enrichment and massively parallel sequencing, all coding exons of genes in complement and coagulation cascades were screened for coding variants. Data were analyzed using a customized local Galaxy pipeline. After filtering for quality and frequency, novel and rare variants were annotated based on computational algorithms and reported studies. For selected variants the predicted functional impact was confirmed in vitro.
Results: A known factor X deficiency variant (F10 c.424G>A, p.Glu142Lys) was identified in a three-generation pedigree. No carriers of both the F10 p.Glu142Lys variant and the CFH p.Arg1215Gln variant developed aHUS, although two persons carrying only the CFH p.Arg1215Gln variant developed disease in early childhood. Protein modeling shows that wild-type Glu142 hydrogen bonds with Cys129, while mutated Lys142 hydrogen bonds with Ser146, a shift that destabilizes an important intra-light-chain interaction between a two-stranded beta-sheet and a small alpha-helical secondary structure element. Consistent with this prediction, recombinant mutant factor X secretion was altered in HEK293 cells and its activity was reduced by 30%.
Conclusion: We have identified a variant in F10, p.Glu142Lys, that may modify CFH-related aHUS perhaps protecting CFH-mutation carriers from developing the phenotype.This finding may explain some instances of incomplete penetrance and offer new therapeutic targets to treat this life-threating disease.
|6/12/2014||Jennifer Teitle: The Versatile PhD||106 BBE|
|6/26/2014||Emily Beck||106 BBE|
|7/10/2014||Danielle Herrig||106 BBE|
|7/24/2014||Igor and Patricia (Genetics Alumni)||106 BBE|
|8/7/2014||Hung-Lin Chen||106 BBE|
The Role of Maternal Wnts and Frizzleds in Dorso-Ventral Axis Specification
Elucidating the molecular mechanisms that transform a symmetric egg into a more complex embryonic body has interested developmental biologists for some time. The asymmetric distribution of determinants within the oocyte is critical to normal developmental processes such as dorsal axis determination and cell migration. In Xenopus laevis in particular, maternal mRNAs involved in the patterning of the secondary body axis are localized to the oocyte vegetal cortex, and upon sperm entry, are subsequently translocated to the future dorsal side. This process, called cortical rotation, is required for the asymmetric activation of the Wnt/β-Catenin signaling pathway on the dorsal side of the blastula. β-catenin, a down-stream wnt effector, regulates the transcriptional activation of dorsal-specific genes at the midblastula transition. Support for the role for β-Catenin signaling in the specification of the dorso-ventral (D/V) axis was garnered from maternal mRNA depletion studies in Xenopus. Recent evidence has suggested that secreted Wnt ligands Wnt11 and Wnt5a may act together in axis formation. However, the function of other maternally expressed Wnts and their cognate receptors (frizzleds) remains uncharacterized. We propose that one or more uncharacterized wnts/fzds act in D/V axis specification. Preliminary analysis of the developmental expression patterns of all wnts and frizzleds (fzds) present in frog, found that wnts 1, 2, 5a, 5b, and 10b and fzds 1, 3, 4, 5, 6, and 7 are expressed in the oocyte, suggestive of a role in D/V patterning. Moreover, maternal fzd1-depleted embryos exhibit a ventralization phenotype and partial defects in dorsal-specific gene expression, while fzd4-depleted embryos exhibit a dorsalization phenotype and an expansion of dorsal specific markers. Further characterization of the genetic factors involved in D/V axis specification could advance the understanding of the pervasive Wnt signaling pathway and provide mechanistic insights into developmental defects.
Long-Range Chromatin Interactions in Fetal and Adult Hematopoietic Stem Cells
HSCs in fetal liver undergo rapid self-renewal divisions, which lead to a massive increase in cell number of the HSC pool. In contrast, the adult bone marrow HSCs have lower self-renewal capacity. Fetal and adult HSCs display differences in their differentiated cell output. Fetal HSCs have erythro-myeloid lineage output while adult HSCs have balanced lineage output. These differences of biological properties between fetal and adult HSCs correlate with distinct gene expression in HSCs. The transcription factor Sox17 is required for the maintenance of fetal, but not adult, HSCs. Ezh2, a core component of polycomb repressive complex 2 (PRC2), is essential for fetal, but not adult, HSCs. In contrast, Bmi1, Gfi1, Etv6, and C/EBPα are required for restriction of self-renewal in adult HSCs. Several studies have shown that the expression of Sox17 is controlled by long-range chromatin interaction. SUZ12, a PRC2 subunit, can occupy the promoter region of Sox17 and repress its expression in ES cells. In addition, the expression of Sox17 is controlled by polycomb protein and H3K27me3 during pancreatic differentiation. The transcription factor SMAD2/3 can bind to the promoter region of Sox17 and is associated with H3K27me3 depletion to activate gene expression. Ezh2 and Bmi1, aother two polycomb proteins, are also involved into HSCs regulation. It is clear that polycomb proteins are key factors of long-range chromatin interaction. In addition, the expression of Gfi1 is regulated by five regulatory regions with Scl/Tal1, Gata2, PU.1, Erg, Meis1, and Runx1 as upstream regulators. These indicate that long-range chromatin interaction is essential to the expression of key regulators in fetal and adult HSCs. We currently use 3C-based technologies to identify the long-range chromatin interactions in both fetal liver and bone marrow HSCs.
Characterizing the NPHP10 Mouse
Nephronophthisis (NPHP) is a recessive kidney disorder that is the leading cause of early onset, end-stage renal failure. Localization to the ciliary-centrosomal complex of many proteins mutated in cystic kidney disease provided a coalescing mechanism for NPHP-related ciliopathies (NPHP-RC). Aside from renal failure and kidney cysts, the two other main phenotypes are retinal and cerebellar degeneration. One such disease with NPHP-RC phenotypes is Bardet-Biedl Syndrome (BBS), with cardinal phenotypes of retinal degeneration, renal abnormalities, obesity, polydactyl, and learning disabilities. An NPHP gene associated with BBS is serologically defined colon cancer antigen 8 (SDCCAG8), later defined as NPHP10. BBS patients with NPHP10 mutations exhibit retinal and renal abnormalities, obesity, and learning disabilities. An NPHP10 knockout (KO) mouse model has been generated using a Sleeping Beauty transposon insertion and these KO mice die shortly after birth. Current work focuses on characterizing the phenotypes of this mouse and determining if death is due to a gross morphological defect or a severe signaling pathway defect.
Global View of Enhancer-Promoter Interactome in Human Cells
Enhancer mapping has been greatly facilitated by various genomic marks associated with it. However, little is available in our toolbox to link enhancers with their target promoters, hampering mechanistic understanding of enhancer-promoter (EP) interaction. We develop and characterize multiple genomic features for distinguishing true EP pairs from non-interacting pairs. We integrate these features into a probabilistic predictor for EP interactions. Multiple validation experiments demonstrate a significant improvement over state-of-the-art approaches. Systematic analyses of EP interactions across eleven cell types reveal several global features of EP interactions: 1) about 60% of EP interactions are tissue-specific; 2) promoters controlled by multiple enhancers have higher tissue specificity but the regulating enhancers are less conserved; 3) cohesin plays a role in mediating tissue-specific EP interactions via chromatin looping in a CTCF independent manner. Our approach presents a systematic and effective strategy to decipher the mechanisms underlying EP communication.