Understanding the genetic foundations of schizophrenia and the resultant symptom manifestations is an important step as we work toward development of new prevention and treatment strategies. This work has sought better understanding of this disease through use of three subject cohorts and two studies using simulated data exploring features of complex disease. First, we probed the symptoms of schizophrenia in subjects of African and European ancestry drawn from the Genetic Association Information Network (GAIN) schizophrenia study and found significant differences between groups, particularly in affective symptoms. The genetic basis of symptom variation was then explored in a selection of candidate genes in two Irish samples, the Irish Study of High Density Schizophrenia Families (ISHDSF) and Irish Case-Control Study of Schizophrenia (ICCSS). We found a significant association of PAH with delusions, GABRB3 with hallucinations, and SNAP25 with both of these symptom factors. AKT1 alleles conferred greater Schneiderian symptoms, but dysbindin, MAOB, and SLC6A4 were not related to any symptom dimensions. Simulated data were used to probe the parameters necessary to detect susceptibility genes as modifiers in a scenario in which two disease groups with incompletely overlapping symptom profiles are examined together. The heterogeneous genetic underpinnings and variable symptom manifestation of schizophrenia make the findings from this study particularly relevant to this disease. Convergent lines of evidence implicating myelin and synaptic dysfunction in schizophrenia prompted us to test related gene networks for association with this disease in two populations, African-ancestry and European-ancestry, from the GAIN study. Some evidence supporting myelin-related genes in the etiology of schizophrenia was presented but only in the African-ancestry group. Epistatic (gene-gene) interactions may confer much greater disease risk than single-gene results would indicate, but their detection is often difficult. The final study included here explored two approaches to family-based epistasis detection under a range of epistatic models. The haplotype relative risk (HRR) approach yields greater power for detection under conditions of dominance, but the Cordell approach is more powerful under most other models. Together, these studies provide a modest advancement in our understanding of schizophrenia and the methodological avenues available for future studies of this disease.

The etiological connection between internalizing and externalizing disorders is poorly understood. This manuscript aims to investigate the roles of genes and then environment in the development of Major Depressive Disorder (MDD) and Antisocial Personality Disorder (ASPD) and the nature of their comorbidity in young adults. Data from a sample of 2,291 young adult male and female twins from the Young Adult Follow Up Study (YAFU) of the Virginia Twin Study of Adolescent Behavioral Development (VTSABD) were used to estimate the proportion of variation in these traits that can be accounted for by additive genetic, shared environmental, and unique environmental factors. In addition to traditional methods of measurement of psychiatric disorders (symptom sum scores), a more novel approach to measurement through item response theory (IRT) was employed. Through both measurement methods, variation in depression was found to be influenced almost entirely by environmental factors, however IRT analysis revealed genetic influences related to specific symptoms that cannot be gleaned from a traditional univariate analysis. While the symptoms of MDD appear to work well together to define the latent construct, symptoms of ASPD are less cohesive. ASPD item loadings are more disparate and the item response models are somewhat unstable compared with the MDD item analyses. Consistent with the literature, results using traditional sum score data indicated that ASPD was influenced by additive genetic and unique environmental effects. IRT analysis, however, did indicate a role for the shared environment in ASPD variation.ASPD/MDD comorbidity analyses suggest that more than one mechanism may reasonably explain the relationship between the disorders. It appears to be more likely that common genetic factors account for some of the observed comorbidity in females than in males. Conversely, the shared environment is that the most likely link between the two disorders in males. The etiological nature of these disorders is complex and analysis may be further complicated in a population spanning a critical developmental period, such as young adulthood. The item response approach has the potential to provide new insight into how these disorders develop and differ between the sexes and different age groups.

The study of telomere and telomerase biology holds substantial promise in uncovering the molecular process of aging and the treatment of cancers. Studies have shown that telomere shortening is directly linked to cellular aging and that telomerase expression is found in over 85% of human cancers, including 95% of all advanced malignancies. Development of effective model systems to elucidate the molecular mechanisms underlying the role of telomeres and telomerase in the processes aging and cancer is of particular importance. While inbred strains of mice have provided a wealth of information for a variety of pathways and diseases, the mouse model poses a challenge for the study of telomere biology due to their extremely large telomere sizes. The telomerase knockout mouse suffers no adverse effect for at least 6 generations, making studies of telomere shortening-associated pathology in single generations of animal very difficult. For this reason, the identification of new vertebrate models for the study of telomeres and telomerase biology is critical. The chicken, Gallus gallus, has been an important model system for the study of embryology and development for decades. The chicken has three classes of telomeres, characterized and differentiated by their size. Class I ranging from 0.5 to 10 kb in size, Class II ranging between 10 to 40 kb in size, and Class Ill ranging between 40 kb to 2 Mb in size. The class I and II chicken telomeres are close enough in size to those of humans, which range between 4 and 15 kb, to allow for chicken studies to elucidate information valuable to the understanding of the molecular process in humans. The chicken telomerase genes have been cloned and characterized, providing a foundation for unlimited study of telomere biology in the chicken. Additional models, including many different fish species, will also hold promise as telomere models for aging and cancer. The development of an additional model of telomere biogenesis and telomerase regulation should provide important insights into the molecular processes surrounding both the development of cancer and organismal aging.

Hemoglobinopathies are some of the most common monogenic disorders in the world, affecting millions of people and representing a growing burden on health systems worldwide. Although the pathophysiology of sickle cell anemia and beta-thalassemia, two of the most common hemoglobinopathies, have been the focus of much research over the last century, patients affected by these diseases still lack a widely applicable and easily available cure. Sickle cell anemia and beta-thalassemia are caused by defects in the structure and production of the beta-globin chains that, along with the alpha-globin chains make up the heterotetrameric hemoglobin molecule. Studies geared towards re-expression of the silenced fetal gamma-globin gene in adult erythroid cells as a therapeutic strategy to alleviate the symptoms of beta-globin deficiencies have met with some success for the treatment of sickle cell anemia but not for beta-thalassemia. A better understanding of normal gamma-globin gene regulation will undoubtedly advance the development of more effective therapeutic strategies. Because many of the potential targets that may be modulated to achieve gamma-globin re-expression also have functions in erythroid cells other than regulating the gamma-globin gene, it is imperative to understand their role in all aspects of erythropoiesis before they are used for therapy. The current study focuses on the role of two Krüppel-like transcription factors, KLF1 and KLF2, which have known roles in the processes of primitive and definitive erythropoiesis as well as globin gene regulation. The regulation of primitive erythropoiesis by KLF1 and KLF2 is studied using the mouse as a model system because it is not possible to study primitive erythropoiesis in humans. Previous studies have shown that KLF1 and KLF2 are essential for and have overlapping roles in primitive erythropoiesis. Simultaneous ablation of KLF1 and KLF2 results in a severely anemic embryonic phenotype that is not evident in KLF1 or KLF2 single knockout embryos. In this study, we show that this anemia is caused by a paucity of blood cells, and exacerbated by diminished beta-like globin gene expression. The anemia phenotype is dose-dependent, and interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining both normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. It was discovered that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation, through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous deficiency of KLF1 and KLF2, than with loss of a single factor. The regulation of human beta-like globin gene expression is studied using a recently developed in vitro system for the production of erythroid cells from umbilical cord blood hematopoietic precursor cells, representing a more “fetal” model of globin gene expression. Previous studies have shown that KLF1 binds to the promoters of the gamma- and beta-globin genes, while KLF2 binds to the promoter of the gamma-globin gene in cord blood-derived erythroid cells. Studies using transgenic mice carrying the entire human beta-globin locus had indicated that KLF1 and KLF2 positively regulate gamma-globin expression in mouse embryonic erythroid cells. We demonstrate in this study that KLF1 appears to have dual roles in the regulation of gamma-globin expression in human cord blood-derived definitive erythroid cells. Partial depletion of KLF1 causes elevated gamma-globin expression, while nearly complete depletion of KLF1 results in a down-regulation of gamma-globin expression. Of particular interest was the observation that KLF2 positively regulates gamma-globin expression in cord blood-derived erythroid cells. Surprisingly, KLF2 also positively regulates beta-globin expression in these cells. If regulation of gamma-globin by KLF2 proves to be a direct effect, KLF2 will join a very small group of factors known to directly activate gamma-globin expression.

Neurodevelopmental disorders (NDs) are a growing public health concern. These complex disorders cause failure of normal brain development, which leads to intellectual disability (ID) or autism in 3% of children. Accurate diagnosis of NDs is difficult due to complex overlapping phenotypes. Moreover, associations between phenotypically similar NDs and their overlapping molecular mechanisms remain unidentified. The chromosome 2q23.1 region is a newly discovered disease region. We have recently identified a novel ND, 2q23.1 deletion syndrome. The phenotype includes severe ID, significantly delayed speech, behavioral problems, seizures and short stature. This syndrome shares characteristics in common with other genetic syndromes, including Smith-Magenis (SMS, RAI1), Pitt-Hopkins (PTH, TCF4), Angelman (AS, UBE3A) and Rett (RTT, MECP2) syndromes, including ID, speech impairment, and seizures, in addition to other autism spectrum disorder (ASD)-associated phenotypes (associated with mutation of MBD1). The methyl-CpG binding domain protein 5 (MBD5) is thought to be the causative gene for the core phenotype seen in del2q23. We propose that MBD5 is a dosage dependent gene, wherein deletion or duplication results in two distinct syndromes. We hypothesize that deletions, mutations, and duplications in MBD5 and its associated overlapping gene networks are responsible for causing the phenotype seen in 2q23.1 disorders. Furthermore, we hypothesize that syndromic neurodevelopmental genes are involved in common biological networks that, when dysregulated, result in the overlapping phenotypes present in many of these neurodevelopmental disorders. We first show that the causative gene for 2q23.1 deletion syndrome is MBD5. We established a consortium of clinical diagnostic and research laboratories to accumulate a large cohort with genetic alterations of chromosome 2q23.1, acquiring 65 subjects with microdeletion or translocation. We sequenced translocation breakpoints, aligned microdeletions to determine the critical region, assessed effects on mRNA expression, and examined medical records, photos, and clinical evaluations. We identified MBD5 as the only locus that defined the critical region. Partial or complete deletion of MBD5 was associated with haploinsufficiency, intellectual disability, epilepsy, and autistic features. Sixteen alterations disrupted MBD5 alone, including partial deletions of noncoding regions not typically captured or considered pathogenic by current diagnostic screening. Expression profiles and clinical characteristics were largely indistinguishable between MBD5-specific alteration and deletion of the entire 2q23.1 interval. We surveyed MBD5 coding polymorphisms among 747 ASD subjects compared to 2,043 non-ASD subjects analyzed by whole-exome sequencing and detected an association with a highly conserved methyl-CpG binding domain missense variant, G79E (p=0.012). Thus, we establish that haploinsufficiency of MBD5 is the primary causal factor in 2q23.1 microdeletion syndrome and that mutations in MBD5 are associated with autism. Secondly, we show that MBD5 is a dosage dependent region, wherein deletion or duplication results in altered gene dosage. We previously established the 2q23.1 microdeletion syndrome and report herein 23 individuals with 2q23.1 duplications, thus establishing a complementary duplication syndrome. The observed phenotype includes intellectual disability, motor delay, language impairments, infantile hypotonia and gross motor delay, behavioral problems, autistic features, dysmorphic facial features (pinnae anomalies, arched eyebrows, prominent nose, small chin, thin upper lip), and minor digital anomalies (fifth finger clinodactyly and large broad first toe). The microduplication size varies among all cases and ranges from 680 kb to 53.7 Mb, encompassing a region that includes MBD5. Phenotypic analyses suggest that 2q23.1 duplication results in a slightly less severe phenotype than the reciprocal deletion. The features associated with a deletion, mutation, or duplication of MBD5 and the gene expression changes observed support MBD5 as a dosage sensitive gene critical for normal development. Dup(2)(q23.1) causes a phenotype similar to del(2)(q23.1) and other NDs, like SMS and autism, suggesting shared molecular pathways. Finally, chromatin-modifying genes play an important role in the genetic etiology of many NDs, including intellectual disability, epilepsy, and autism. Many monogenic NDs are caused by chromatin modifying genes, including 2q23.1 deletion and duplication, SMS, RTT, AS, fragile X syndrome (FXS), and PTH. Many of these disorders have overlapping features that include language, sleep, and behavioral anomalies. Investigation of relative gene expression by quantitative PCR and microarray of cell lines from individuals with disorders due to altered expression of MBD5, RAI1, MECP2, UBE3A, TCF4, and MBD1 revealed molecular signatures that allowed for the generation of a novel neurodevelopmental molecular network supporting the overlapping features across these syndromes. Further, knockdown of MBD5 and RAI1 in SH-SY5Y and HEK293T cell lines expanded the repertoire of genes involved in these pathways and showed that other chromatin modifying genes, as well as developmental genes are dysregulated. Pathway analyses showed that MBD5 and RAI1 function in chromatin remodeling, circadian rhythm, neuronal development, and cell growth/survival pathways. From these studies, precise gene dosage of chromatin modifying genes, such as RAI1 and MBD5 are clearly a requirement for normal neurodevelopment and function. Taken together, these studies have given us insight into the role of MBD5 as a dosage sensitive gene in two NDs. Furthermore, we gained insight of how dosage effects of MBD5 and RAI1 affect molecular pathways that are linked to neuronal and behavioral development. We have unveiled pathways and genes, which are important to normal human development, neurodevelopment and behavior. These findings support further investigations into the relationships among causative neurodevelopmental genes, which will lead to common points of regulation that may be targeted toward therapeutic intervention.

Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial development disorder and is caused by mutations in the TCOF1 gene. The TCOFl protein treacle is a nucleolar protein and may function in ribosome biogenesis.Previously, we identified downstream candidate genes using microarray analysis after manipulating Tcofl levels in a murine neuroblastoma (NB) cell line. The list of genes includes cell cycle genes as well as the transcription factors Cnbp and Tbx2, which are known to affect the cell cycle through the c-myc and p19-Mdm2-p53-p21 pathways respectively. To further characterize the cellular effects of Tcofl, stably transfected NB cell lines with overexpression or knockdown of Tcofl were generated. Growth curves were generated by counting cell numbers. BrdU incorporation and TUNEL assays were used to determine proliferation and apoptosis levels. Western blot analysis was used to detect protein level changes of candidate downstream pathway genes. Bothoverexpression and knockdown of Tcofl are detrimental to cell growth. Overexpression of Tcofl causes increased apoptosis and knockdown of Tcofl causes reduced cell proliferation and increased apoptosis. Western blot analysis shows that Cnbp and Tbx2 protein levels change with Tcofl, c-myc level is decreased in Tcofl knockdown cells and p19 (Cdkn2d), p53 and p21 (Cdkn1a) levels are increased in Tcofl overexpressing cells. Our results suggest that an optimal Tcofl level is required for cell proliferation and survival, and that overexpression and knockdown of Tcofl may affect cell proliferation and apoptosis through the p19-Mdm2-p53-p21 and Cnbp-c-myc pathways respectively.Heterozygous Tcofl knock out mice are neonatal lethal, which circumvents further analysis of the heterozygous and homozygous mice. In this study, we generated Tcofl conditional allele mice with loxP sites flanking exon 1. These mice were crossed with Wntl-Cre transgenic mice to generate a conditional knockout of Tcofl specifically in neural crest (NC) cells. Homozygous conditional knockout mice show craniofacial abnormalities resembling TCS patients. Heterozygous conditional knockout mice are phenotypically normal, which suggests that Tcofl functions in tissues other than NC cells during development. Cnbp expression is decreased in a proportion of the homozygous conditional knockout mouse embryos. Our results suggest that Tcofl may affect craniofacial development through Cnbp by maintaining cell growth.

The Patient-Centered Medical Home (PCMH) is an emerging model of health care designed to provide a simpler, more effective health care experience. The model places heavy emphasis on the concept of every patient having a "personal physician" who is the point of access for all health care needs and concerns. The personal physician integrates all relevant health care information to provide the patient with a holistic picture of his health. The supposed benefits of the PCMH model include an improved patient experience, increased effectiveness of care, increased efficiency of care, greater access to care, among others. Only now is evidence beginning to emerge to substantiate those clams. As evidence continues to emerge supporting the PCMH model, one area that warrants further study is how those directly involved in health care perceive this model. Here, a survey was developed to assess the following information among a population of pediatric physicians: understanding of the PCMH model, agreement with PCMH principles, interest in moving to a PCMH-based practice, and what issues are perceived as barriers to PCMH integration. Results suggest that there is a high degree of familiarity with the PCMH model and a high level of agreement with PCMH principles in this population, but that agreement does not correlate with interest in moving one’s practice toward the PCMH model. Data further indicate that issues regarding payment and associated expenses for PCMH integration are universally perceived barriers. On the other hand, a lack of evidentiary support and compatibility issues with HIPAA are not perceived as barriers. Other issues, such as human resource needs, were more likely to be perceived as barriers in one subpopulation versus another. These data suggest a disconnect between PCMH familiarity and PCMH interest in pediatric physicians. Further, while some issues are perceived as barriers to all pediatric physicians, some issues are more likely to be perceived as barriers in one physician subpopulation versus another, and these differences must be recognized and addressed to help ensure success of the PCMH movement.

Breast cancer is one of the most prevalent and deadly forms of cancer in women and is not restricted by race or ethnicity. Although a wealth of knowledge has been amassed on the biology of breast cancer, including its risk factors, diagnosis, prognosis, prevention, and treatment, it remains a serious health concern and active area of research. Initial response to standard chemotherapeutic and radiotherapeutic regimens is generally strong for many patients, yet breast tumors often recur, leading to more aggressive and resistant tumors. Because recurrence is such a clinical issue, more effective therapeutic approaches are needed to eliminate partial clinical responses and undesirable side effects. Molecular chaperones like the heat shock protein 90 (Hsp90) family are regarded as ubiquitous, highly conserved proteins that mainly respond upon induction of stress or disruption in cellular homeostasis. Chaperones are critically involved in controlling the conformation, stability, function, and degradation of many oncogenic client proteins by assisting in trafficking, remodeling of improperly folded client proteins, and suppression of protein aggregation. Hsp90-mediated folding events are an ATP-dependent process that involves the association with various co-chaperones and stimulators, including Hsp70, Hsp40, HOP, p23, and Aha1 for client stabilization and modification. Notably, Hsp90 seems to be particularly associated with cell signaling clientele, such as nuclear hormone receptors, protein kinases, and many other oncogenic proteins that directly influence the hallmarks of cancer. In order to define the role of chaperones in breast cancer progression, we assessed chaperone expression levels in normal and tumor lines. Based on our initial finding of increased expression of Hsp90 and p23 in immortal and cancer cell lines, it is possible that this phenomenon may be reflected in normal breast tissue as well as breast carcinoma specimens. Indeed, we observed high Hsp90 expression in invasive carcinomas, such that high nuclear expression correlates with a greater TNM stage, while high cytoplasmic Hsp90 correlates with ER negativity, suggesting that elevated Hsp90 may be an indicator or marker of advanced disease. p23 expression also increases appreciably in established breast cancer cell lines with invasive breast tissue specimens displaying an opposite trend. Although p23 does not appear to have any relationship with TNM stage, significant relationships with ER and PR emerged, with higher nuclear p23 correlating to ER positivity and higher total p23 being positively associated with PR presence. Due to the differential expression of Hsp90 in normal, DCIS, and invasive breast carcinomas, we determined the impact on Hsp90 function, finding that total Hsp90 in tumor cells is associated with an increase in both complexed and uncomplexed Hsp90, which is in contrast to a previously reported study. The intrinsic nature of heat shock proteins makes them especially relevant to a cell’s defense against cancer initiation. The preferential accumulation of Hsp90 in cancer cells also forms the basis for the unique sensitivity of tumor cells to Hsp90 inhibition. The Hsp90 specific inhibitor, radicicol, is more potent in terms of blocking ATPase activity than other Hsp90-related compounds like geldanamycin, which is much more toxic. All Hsp90 inhibitors prevent the association of the co-chaperone p23 with Hsp90, resulting in destabilization of the client protein. For these reasons, it may be possible that Hsp90 inhibition would sensitize breast cancer cells to be more responsive to standard chemotherapeutics. We determined that radicicol negatively alters cellular proliferation, and in combination with Adriamycin, elicits a more robust decline in growth and the expression of Hsp90 client proteins. This finding was associated with an increase in senescent cells without a detectable affect on apoptosis. Radicicol in combination with cisplatin or Taxol contributed to an increase in cell death (apoptosis) and differentially altered the expression of client proteins. Finally, ER negative breast cancer cells do not display altered p53 expression upon radicicol and Adriamycin treatment. Blockade of ER activity in ER positive cells with tamoxifen induced significant reductions in proliferation and decreased p53 expression without a corresponding decrease in p21 levels. In conclusion, these results point to the utility of Hsp90 inhibition as a valid form of targeted therapy for breast cancer, and the value of radicicol as a potential adjuvant treatment option in combination with standard chemotherapeutics.

This thesis investigates two separate features of nonhomologous end-joining (NHEJ) DNA repair: end processing, and DNA repair kinase signaling. DNA end processing was investigated in a mouse model of hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), a congenital neurodegenerative disease. SCAN1 is caused by a homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1). To address how the H493R mutation elicits the specific pathologies of SCAN1 and to further elucidate the role of TDP1 in processing DNA end modifications, we generated a Tdp1 knockout mouse and characterized their behavior and specific repair deficiencies in extracts of embryonic fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate (PG) double-strand breaks (DSBs). Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on PG termini on 3' overhangs of DSBs; these remained completely unprocessed. Altogether, these results suggest that for 3'-PG overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate. Furthermore, there is evidence that TDP1 also localizes to mitochondria, and mitochondrial DNA damage should not be excluded from significantly contributing to SCAN1 pathology. The effect of ATM signaling on NHEJ was investigated via a novel vector that allows for inducing I-SceI-mediated DNA DSBs that can then be analyzed for NHEJ repair events by fluorescence- and PCR-based methods. Using highly specific DNA kinase inhibitors and the repair cassette, we showed that inhibiting ATM reduced NHEJ by 80% in a U87 glioma model. Analysis of the PCR products from the NHEJ repair vector by PsiI restriction cleavage allowed for assessment of the fidelity of the NHEJ repair: inhibiting ATM reduced high-fidelity NHEJ by 40%. Together, these results suggest that ATM is critical for NHEJ of I-SceI DSBs and for high-fidelity repair, possibly due to ATM's effects on chromatin architecture surrounding the DSB.

Smith Magenis Syndrome (SMS) is a multiple congenital anomalies/ mental retardation disorder caused by deletion or mutation of the RAI1 gene on chromosome 17p11.2. The majority of patients with SMS phenotypes have a deletion or mutation of RAI1. However, some patients have been observed with SMS-like phenotypes and yet have no deletions or mutations in the RAI1 gene. One possible explanation could be aberrant methylation of RAI1 since RAI1 is present and yet may be silenced. In order to study this possibility, patient cell lines were treated with 5-Aza-2’-deoxycytidine. RNA was extracted and real-time PCR was used to check the RAI1 expression status on the cells. RAI1 is thought to be a transcription factor, but the DNA binding sequence is still unknown. Sequences from ChIP-chip data were compared to identify a consensus sequence. One gene which contained this consensus sequence was the chemokine-like receptor-1 gene (CMKLR1), which was investigated by luciferase assay. CMKLR1 showed upregulation when co-transfected with RAI1.