The spinocerebellar ataxias are a group of diseases characterized by loss of motor coordination. Spinocerebellar ataxia types 2 and 7 are monogenic, autosomal dominant, late-onset neurodegenerative diseases characterized by ataxia with no effective treatments in the clinic. The most striking feature of these diseases is the degeneration of Purkinje neurons of the cerebellum. Spinocerebellar ataxia type 7 is also characterized by vision loss due to degeneration of the retinal photoreceptors. In this work, we tested the hypothesis that reducing mutant gene expression by RNAi would alleviate disease phenotypes in these two spinocerebellar ataxias. For spinocerebellar ataxia type 7 (SCA7), we designed and tested RNAi sequences that could reduce the expression of both wildtype and mutant ataxin-7, an approach that would be applicable to all SCA7 patients. We found that AAV1-mediated delivery of a candidate RNAi sequence to the Purkinje neurons of SCA7 mice resulted in long-term sustained reduction of both wildtype and mutant ataxin-7 and resulted in significant improvements in ataxic and neuropathological phenotypes. We also delivered the RNAi sequence (AAV1-mediated) to reduce the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina and evaluated retinal function long-term. We observed a preservation of normal retinal function and no adverse toxicity with reduction of wildtype and mutant ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 therapy. To identify therapies for spinocerebellar ataxia type 2 (SCA2), we designed and tested several RNAi sequences to reduce the expression of both wildtype and mutant ataxin-2 in vitro and in vivo. We found that reduction of wildtype ataxin-2 expression in the mouse cerebellum was tolerated 4 months post injection without inducing behavioral deficits or cerebellar pathology. Additionally, we tested other sequences for improved silencing efficacy, and identified a potent RNAi sequence that significantly reduced the expression of both mutant and wildtype ataxin-2 in the cerebellum of a SCA2 mouse model. Ongoing work will establish if long-term reduction of both mutant and wildtype ataxin-2 will provide therapeutic benefit in the SCA2 mouse setting, and the safety of this sequence in normal cerebella.

Cutaneous wound healing is an inherent biological process to every injury, surgical procedure, and cases of trauma. Wound healing is loosely categorized into three overlapping phases: inflammation, epithelialization, and maturation. Dysregulation of any of these steps leads to poor wound healing, as do conditions such as diabetes, obesity, and advanced age. We recently identified the clefting syndrome, Van der Woude (VWS), as another condition resulting in poor healing. VWS and the allelic syndrome, popliteal pterygium (PPS) are caused by mutations in Interferon Regulatory Factor 6 (IRF6). In the mouse, removal of Irf6 leads to major developmental defects, including limb, craniofacial and cutaneous anomalies. As embryonic development and wound healing share common biological processes, we hypothesized that Irf6 is critical to the regulation of differentiation, proliferation, and migration of keratinocytes, processes essential to wound healing. Upon in vitro differentiation, wild type murine keratinocytes exhibited increased Irf6 expression. Fittingly, Irf6-/- keratinocytes exhibited a defect in terminal differentiation and increased capacity for long-term proliferation. We also determined that a craniofacial enhancer of Irf6 is functional in differentiating keratinocytes in vitro and in vivo. As cellular migration is a critical process to wound epithelialization, we tested the role of Irf6 in keratinocyte migration. We report a significant delay in closing an in vitro scratch wound in the absence of Irf6. Analysis of time-lapse microscopy revealed that this delay was due to a reduction in keratinocyte velocity. We report a substrate non-specific reduction in adhesion in Irf6-/- keratinocytes compared to wild type cells. In addition,Irf6-/- keratinocytes exhibited longer and more prominent actin stress fibers that were rescued by the addition of the ROCK inhibitor, Y27632. The active form of RhoA, a GTPase regulating stress fiber formation upstream of ROCK was increased in Irf6-/- keratinocytes, suggesting a role for Irf6 in regulating RhoA-dependent stress fibers. We further identified Arhgap29, a GTPase activating protein deactivator of RhoA, as a downstream effector of Irf6 in skin and keratinocytes. Finally, we used a conditional strategy to delete Irf6 in adult murine epidermis to study in vivo wound healing. We report a significantly smaller macroscopically visible wound that was not confirmed by wound stereological analysis. We also utilized an embryonic model to exclusively test epithelialization in the absence of Irf6. Our results indicate a trend toward faster migration in Irf6-/- e10.5 wounds, yet the difference in wound closure after 6 hours of healing was not significant. Thus we conclude that Irf6 regulates the balance between keratinocyte proliferation and differentiation. Furthermore, it regulates stress fiber formation and speed of migrating keratinocytes in vitro, while Irf6 is dispensable for cutaneous wound epithelialization in vivo.

A primary goal of evolutionary biology is to elucidate the factors necessary for a single interbreeding species to become two independent species. Observations and data collected and recorded since the 6th century B.C. have added to our comprehension of the “the origin of species—that mystery of mysteries” (DARWIN 1859). To continue to add to our knowledge of how speciation occurs and how species interact, it is crucial to determine 1) how different categories of genes evolve as species diverge, 2) what happens to hybrids of two species, and 3) if genetic exchange is allowed between species, where it is located. In the first research aim of my dissertation, I look for population genetic trends and signatures of gene flow in a minimally studied set of Drosophila sister species using sequences of 26 nuclear and mitochondrial regions in 29 isofemale lines of D. subobscura and D. madierensis. Standard population genetic tests revealed that the X chromosome evolves faster than the autosomes in these species. We also find evidence of genetic exchange for some autosomal genes while both the sex chromosomes and mitochondrial genomes remain distinct between species. In the second research aim of my dissertation, I assess the rates of gene expression evolution for sex-biased genes located on the X chromosome and autosomes. We find that gene expression evolves faster in males than females and find evidence of faster-X evolution that is exclusive to genes expressed at higher levels in males. The X chromosome has previously been shown to have a disproportionately large influence on hybrid male sterility compared to autosomes. I investigate this trend and find that the sex chromosomes have a large influence on autosomal expression levels in hybrid males and hybrid females. Specifically, uniparental inheritance of the X chromosome results in greater differences between reciprocal hybrids and higher levels of hybrid misexpression.

Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder due to deficiency of the fatty aldehyde dehydrogenase (FALDH) component of fatty alcohol:NAD+ oxidoreductase (FAO). We investigated the enzymatic defect in SLS in order to elucidate the role of FALDH in fatty aldehyde and fatty alcohol metabolism. Genetic studies were performed to investigate carrier detection for SLS. Cultured skin fibroblasts from normal controls, SLS obligate heterozygotes, and SLS homozygotes were assayed for FAO and FALDH activities using 18-carbon substrates. In SLS homozygotes, mean FAO and FALDH activities were 8% of normal, and there was no overlap between the homozygote and heterozygote ranges. We demonstrated that mean FAO activity in SLS obligate heterozygotes was 78 ± 16% of normal (mean ± SD); whereas mean FALDH activity was 60 ± 15% of normal. Using both FAO and FALDH assays in concert, none of the SL8 obligate heterozygotes (n=11) had both FAO and FALDH activities which overlapped the normal range (n=12). Therefore, we conclude that measurement of FAO and FALDH activities in cultured skin fibroblasts using 18-carbon substrates is useful for SLS carrier detection. Prenatal diagnosis was undertaken to monitor FAO and FALDH activities in four pregnancies at-risk for SLS. Enzymatic results in one case using cultured amniocytes obtained during the second trimester predicted an affected SLS fetus, which was confirmed at termination of the pregnancy. Another at-risk fetus was predicted to be affected with SLS using cultured chorionic villi cells obtained during the first trimester, and fetal skin fibroblasts obtained after termination of the pregnancy confirmed a profound deficiency of FAO and FALDH activities. Two other fetuses were correctly predicted to be unaffected. These results demonstrate that SLS can be diagnosed prenatally during either the first or second trimester of pregnancy using enzymatic methods. In order to better understand the role of FALDH in fatty alcohol oxidation, we determined the subcellular localization of FALDH in human liver, a human cultured hepatocyte cell line (HepG2), and human cultured skin fibroblasts. Differential centrifugation and density gradient centrifugation in Nycodenz were utilized to separate subcellular organelles. Organelle-specific enzyme markers confirmed the subcellular separations that were attained. FALDH activity was primarily localized to the microsomes in human liver, a cultured HepG2 cells, and human cultured skin fibroblasts. FALDH was solubilized from human liver microsomes and purified 167-fold by column chromatography. Purified FALDH had a subunit molecular weight estimated by SDS-PAGE to be 54,000 daltons. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of purified FALDH indicated that the enzyme formed large, polymeric protein aggregates with a molecular weight greater than 500,000 daltons. FALDH was NAD+-dependent, had optimal catalytic activity at pH 9.8, and was thermolabile at 47°C. Km and Vmax values were determined for saturated and unsaturated aliphatic aldehydes ranging from 2 to 24 carbons in length, as well as dihydrophytal, a 20-carbon branched chain aldehyde. FALDH was active towards long-chain fatty aldehydes greater than 6 carbons in length. FALDH was sensitive to inhibition by disulfiram, iodoacetamide, iodoacetate, a,p-dibromoacetophenone, and p-chloromercuribenzoate, but it was unaffected by magnesium ions. These investigations represent the first purification and characterization of human microsomal FALDH.

Endocrine disorders are substantial contributors to neonatal morbidity and mortality and, of these, congenital hypothyroidism (CH) is the most common (Kumar et al. 2009). CH is a common and preventable cause of mental retardation with an incidence of approximately 1 in 2,350 live births (Hinton et al. 2010). In Iowa, the Iowa Neonatal Metabolic Screening program (INMSP) uses thyroid stimulating hormone (TSH) to screen for CH at birth. However, TSH is highly variable among healthy newborns as well as adults, leading to false positive results in some cases. Previous studies have observed that adult TSH variability is under strong genetic regulation with estimated heritability of up to 65% (Panicker et al. 2008). Additionally, there have been multiple studies examining genetic factors associated with adult TSH levels. TSH heritability has never been estimated in the neonatal period and we aimed to determine the heritability in neonates and compare it to heritability estimates in adults. We examined 381 twin pairs obtained from the INMSP. Heritability was estimated using multilevel mixed-effects linear regression adjusting for factors affecting TSH levels; gestational age, gender, weight and age at time of sample collection. We estimated neonatal TSH heritability to be 58% with a P-value of 2x10-5, which mirrors adult heritability estimates, and provides direct evidence for a strong genetic contribution to TSH variability at birth. We next examined genetic factors that may contribute to the observed heritability. Genetic contribution to TSH variation has been studied extensively in adults, but not in neonates. We genotyped a population of Iowa neonates; term (n=827) and preterm (n=815), for 45 single nucleotide polymorphisms (SNPs) that we selected based on reported genetic associations with adult TSH levels from the literature, as well as other candidate genes. TSH values were obtained from the INMSP. Analysis of variance was performed to identify genetic associations with TSH concentrations. The strongest association identified was rs4704397 in the PDE8B gene (p=1.3x10-4), followed by rs965513 (p=6.4x10-4) on chromosome 9 upstream of the FOXE1 gene. Both of these SNPs met statistical significance after correction for multiple testing. Our results demonstrated for the first time two genetic associations with neonatal TSH levels that replicate findings with adult TSH levels, and these findings could have clinical implications for the early prediction of risk for adult diseases and conditions associated with thyroid hormone levels. Finally, we aimed to identify the etiologic variants that may be responsible for the observed associations by fine mapping adjacent enhancer elements. This was done in two stages; first we sequenced 58 term neonates with TSH levels at both ends of the normal distribution to identify associated variants, then we replicated the findings in an additional population of 306 term neonates. None of the observed variants were statistically significant for an association with TSH levels, however, one of the rare variants (rs112053411) identified was detected in three neonates, all in the upper distribution of TSH levels including one infant that was later diagnosed with CH. This rare variant is worth pursuing in a larger study population and in functional studies. Together this work has advanced our knowledge of the genetic basis of TSH variation in the neonatal period, as well as provided insight into the early prediction of risk for adulthood thyroid related diseases through shared genetic associations between adults and neonates.

Suicidal behavior, a complex phenotype encompassing attempted and completed suicide, has been shown through epidemiological studies to have a heritable component. Two major factors contribute to this heritability. The first is a preexisting psychiatric disorder, such as bipolar disorder or major depression. These disorders are heritable themselves, and individuals with these disorders are at a higher risk of suicide than the general population. The second contributing factor is thought to be independent of psychiatric disorders and more specific to suicide. In order to investigate this independent factor, our lab previously conducted a genome-wide association study (GWAS) of attempted suicide in bipolar disorder. This GWAS investigated common variation across the genome and implicated a number of genes and regions in attempted suicide. The first goal of this dissertation was to build upon the results of this GWAS by investigating both common and rare genetic variation through next-generation sequencing techniques. We did this by first conducting a whole-exome sequencing study to investigate rare genetic variation within coding regions across the genome. We did not identify any study-wide significant variation through this study, suggesting that coding variation with both moderately large effect size and moderately low frequency is likely not a major contributor to the heritability of suicidal behavior. We next used a targeted sequencing approach to investigate several candidate genes and regions that had been implicated through our attempted suicide GWAS and the literature. This dissertation outlines four different candidate gene studies investigating variation in two genes (SAT1 and FKBP5), a set of 16 glutamatergic signaling genes, and one candidate region (2p25). Three of these studies were unable to identify any replicable significant variation, suggesting a need for larger sample sizes and more extensive sequencing coverage. However, the 2p25 study identified three variants that were significantly associated with attempted suicide in males. This finding suggests a sex-specific association of this region with suicide and highlights the need for sex-specific analyses within psychiatric genetics. The second goal of this dissertation was to functionally assess our most promising findings from the sequencing studies. We did this using the CRISPR-Cas9 genome editing system to delete a segment of our implicated 2p25 region within cell lines. We were able to show that deletion of this segment affected gene expression across the genome, indicating our region does have regulatory potential. This functional assessment represents the first time such an approach has been taken in the field of suicide genetics. Through the work outlined in this dissertation, we have produced sequencing data across the genome and even more extensive data for a number of candidate genes and regions. Additionally, we performed a functional assessment of one of our implicated regions. Functional characterization of GWAS and sequencing results will help us to better understand the basic biology of disease and, through this better understanding, improve treatment for complex disorders like suicidal behavior.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Transcript abundance measured for any gene on a microarray can be considered as a quantitative trait. If the transcription profile of a sufficient number of individuals from a segregating progeny is generated by microarrays, it allows mapping of genomic regions regulating variation in transcript abundance using traditional methods of QTL analysis. We generated transcript level profiles of wood forming tissue (differentiating xylem) collected from 91 individuals from a <I>E. grandis</I> x <I>E. globulus</I> F1 hybrid x <I>E. grandis</I> backcross population, using microarrays containing 2608 cDNAs. Least-square means estimates of transcript abundance were generated for each individual and cDNA, and mapped as QTLs in two single-tree linkage maps (hybrid paternal and <I>E. grandis</I> maternal) using composite interval mapping. QTLs were identified for 811 genes in the <I>Eucalyptus</I> hybrid map, displaying in most cases a simple genetic architecture, with a single QTL controlling up to 70% of the transcript level variation. A more complex genetic architecture was detected in one third of the genes, where up to five QTLs could be identified for three genes. QTL hotspots were identified in both maps, typically for genes encoding several enzymes of specific metabolic pathways, suggesting coordinated genetic regulation. Transcript level QTLs were co-localized to QTLs detected previously in this family for wood quality and growth traits and candidate genes were identified by the analysis of correlation between gene expression and phenotypic variation.

Understanding the genetic architecture of complex traits requires identification of the underlying genes and characterization of gene-by-gene and genotype by environment interactions (GEI). Behaviors that mediate interactions between organisms and their environment are complex traits that are especially sensitive to environmental conditions. Drosophila melanogaster presents an opportunity to systematically dissect epistasis and GEI, since large numbers of genetically identical individuals can be reared under defined environmental conditions. The olfactory system of Drosophila and its behavioral response to odorants have been well characterized. Previous studies on olfactory behavior have shown that the genetic architecture of this model behavior depends on epistatic networks of pleiotropic genes. I have used P-element mutagenesis in a co-isogenic background to identify genes that contribute to olfactory behavior. I have demonstrated that the effects of the transposon insertions are often dependent on developmental stage and that hypomorphic mutations in developmental genes can elicit profound adult behavioral deficits. I also assessed epistasis among these genes by constructing all possible double heterozygotes and measuring avoidance responses at two odorant concentrations. I observed enhancer and suppressor effects among subsets of these genes, and surprisingly, these epistatic interactions shifted with changes in the concentration of the olfactory stimulus. I then assessed variation in olfactory behavior in a population of 41 wild-derived inbred lines and asked to what extent different larval rearing environments would influence adult olfactory behavior and whether GEI is a minor or major contributing source of phenotypic variation. My results show that about 50% of phenotypic variation in adult olfactory behavior is attributable to GEI. In contrast, transcriptional analysis revealed that only 20 genes show GEI at the level of gene expression (FDR<0.05), some of which are associated with physiological responses to environmental chemicals. Quantitative complementation tests with piggyBac-tagged mutants for two of these genes (CG9664 and Transferrin 1) demonstrate that genes that show transcriptional GEI are candidate genes for olfactory behavior, and that GEI at the level of gene expression is correlated with GEI at the level of phenotype.