Delineating the molecular basis of human genetic diseases: Epigenetic and functional studies

Identifying and characterizing the genes responsible for inherited human diseases will ultimately lead to a more holistic understanding of disease pathogenesis, catalyze new diagnostic and treatment modalities, and provide insights into basic biological processes. This dissertation presents research aimed at delineating the genetic and molecular basis of human diseases through epigenetic and functional studies and can be divided into two independent areas of research. The first area of research describes the development of two high-throughput melting curve based methods to assay DNA methylation, referred to as McMSP and McCOBRA. The goal of this project was to develop DNA methylation methods that can be used to rapidly determine the DNA methylation status at a specific locus in a large number of samples. McMSP and McCOBRA provide several advantages over existing methods, as they are simple, accurate, robust, and high-throughput making them applicable to large-scale DNA methylation studies. McMSP and McCOBRA were then used in an epigenetic study of the complex disease Ankylosing spondylitis (AS). Specifically, I tested the hypothesis that aberrant patterns of DNA methylation in five AS candidate genes contribute to disease susceptibility. While no statistically significant methylation differences were observed between cases and controls, this is the first study to investigate the hypothesis that epigenetic variation contributes to AS susceptibility and therefore provides the conceptual framework for future studies. ^ In the second area of research, I performed experiments to better delimit the function of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), which when mutated causes various forms of inherited blindness such as Leber congenital amaurosis. A yeast two-hybrid screen was performed to identify putative AIPL1-interacting proteins. After screening 2 × 106 bovine retinal cDNA library clones, 6 unique putative AIPL1-interacting proteins were identified. While these 6 AIPL1 protein-protein interactions must be confirmed, their identification is an important step in understanding the functional role of AIPL1 within the retina and will provide insight into the molecular mechanisms underlying inherited blindness. ^

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies.

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.

AN ASSESSMENT OF KNOWLEDGE AND ATTITUDES OF GENETIC COUNSELING SERVICES IN U.S. HTCs

Hemophilia is a hereditary bleeding disorder which requires lifelong specialized care. A network of Hemophilia Treatment Centers (HTCs) exists to meet the medical needs of patients affected by hemophilia. Genetic counseling services are an integral part of the HTC model of care; however, many HTCs do not have genetic counselors on staff. As a result, the duty to provide these services must fall to other healthcare providers within the HTC. To assess the knowledge and attitudes of these providers we developed a 49 question survey that was distributed electronically to hematologists and nurses at U.S. HTCs. The survey consisted of a three sections: demographic information, knowledge of hemophilia genetics, and attitudes towards genetic services. A total of 111 complete responses were received and analyzed. The average knowledge score among all participants was 74.8% with a total of 81 participants receiving a passing score of 70% or above. Thirty participants scored below 70% in the knowledge section. In general, attitude scores were high indicating that the majority of hematologists and nurses in HTCs feel confident in their ability to provide genetic counseling services. Over 90% of participants reported that they have some form of access to genetic counseling services at their center. Hematologists and nurses practicing in U.S. HTCs demonstrate sufficient knowledge of the genetics of hemophilia, and they generally feel confident in their ability to provide genetic counseling services to their patients. While their knowledge is sufficient, the average knowledge score was lower than 75%. Certain questions covering new genetic technologies and testing practices were more commonly missed than questions asking about more basic aspects of hemophilia genetics, such as inheritance and carrier testing. Finally, many clinics report having access to a counselor, but it is oftentimes a hematologist or nurse who is providing genetic counseling services to patients. Given the inconsistency in knowledge among providers coupled with the high confidence in one’s ability to counsel patients, it leaves room to question whether information about the genetics of hemophilia is being communicated to patients in the most appropriate and accurate manner.

ATTITUDES AND PRACTICES OF GENETIC COUNSELORS IN PROVIDING PREDICTIVE TESTING TO MINORS AT RISK FOR LI-FRAUMENI SYNDROME

Li- Fraumeni Syndrome (LFS) is a rare autosomal dominant hereditary cancer syndrome caused by mutations in the TP53 gene that predisposes individuals to a wide variety of cancers, including breast cancer, soft tissue sarcomas, osteosarcomas, brain tumors, and adrenocortical carcinomas. Individuals found to carry germline mutations in TP53 have a 90% lifetime cancer risk, with a 20% chance to develop cancer under the age of 20. Despite the significant risk of childhood cancer, predictive testing for unaffected minors at risk for LFS historically has not been recommended, largely due to the lack of available and effective screening for the types of cancers involved. A recently developed screening protocol suggests an advantage to identifying and screening children at risk for LFS and we therefore hypothesized that this alongside with the availability of new screening modalities may substantiate a shift in recommendations for predictive genetic testing in minors at risk for LFS. We aimed to describe current screening recommendations that genetic counselors provide to this population as well as explore factors that may have influenced genetic counselors attitude and practice in regards to this issue. An online survey was emailed to members of the National Society of Genetic Counselors (NSGC) and the Canadian Association of Genetic Counsellors (CAGC). Of an estimated 1000 eligible participants, 172 completed surveys that were analyzed. Genetic counselors in this study were more likely to support predictive genetic testing for this population as the minor aged (p

An assessment of obesity and hyperphagia in individuals with Smith-Magenis syndrome

Smith-Magenis syndrome (SMS;OMIM# 182290) is a multiple congenital anomalies and mental retardation syndrome caused by a 3.7- Mb deletion on chromosome 17p11.2 or a mutation in the RAI1 gene. Although the majority of the SMS phenotype has been well described, limited studies are available describing growth patterns in SMS. There is some evidence that individuals with SMS develop obesity. Thus, this study aims to characterize the growth and potential influence of hyperphagia in a cohort of individuals with SMS. A retrospective chart review was conducted of 78 individuals with SMS through Baylor College of Medicine (BCM) at Texas Children¡¯s Hospital (TCH.) All documented height and weight measurements were abstracted and Z-scores (SD units) for height-for-age, length-for-age and BMI-for-age were calculated. Mail-out questionnaires were provided to the corresponding parents of the cohort to assess for the presence of hyperphagia through a validated hyperphagia questionnaire (HQ). Analysis of this data demonstrates that by the age ¡Ý 20 years males with SMS have mean BMI¡¯s in the 85th-90th percentile corresponding to an overweight BMI, and females with SMS had mean BMI¡¯s in the 95th -97th percentile corresponding to an obese BMI. Parents indicated that hyperphagia is present in individuals with SMS as 76% of parent¡¯s report having to lock food away from their child. Females¡¯ age ¡Ý 20 years of age had the highest mean behavior, drive and severity scores as well as the highest BMI. Thus, this study concludes that it appears overweight and obesity, as well as hyperphagia, are present in this cohort of SMS individuals. The results of this study will hopefully enable parents and caregivers of children with SMS to take preventative measures in order to control food related behaviors present in their children as well as to prevent overweight and obesity and the associated negative health consequences.

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

Comparison of accuracy captured by different controlled languages in oral pathology diagnoses.

This project was comparing the accuracy of capturing the oral pathology diagnoses among different coding systems. 55 diagnoses were selected for comparison among 5 coding systems. The results of accuracy in capturing oral diagnoses are: AFIP (96.4%), followed by Read 99 (85.5%), SNOMED 98 (74.5%), ICD-9 (43.6%), and CDT-3 (14.5%). It shows that the currently used coding systems, ICD-9 and CDT-3, were inadequate, whereas the AFIP coding system captured the majority of oral diagnoses. In conclusion, the most commonly used medical and dental coding systems lack terms for the diagnosis of oral and dental conditions.

THE ROLE OF MUCOSAL EPITHELIAL CELLS IN HIV-1 INFECTION

The predominant route of human immunodeficiency virus type 1 (HIV-1) transmission is infection across the vaginal mucosa. Epithelial cells, which form the primary barrier of protection against pathogens, are the first cell type at these mucosal tissues to encounter the virus but their role in HIV infection has not been clearly elucidated. Although mucosal epithelial cells express only low levels of the receptors required for successful HIV infection, productive infection does occur at these sites. The present work provides evidence to show that HIV exposure, without the need for productive infection, induces human cervical epithelial cells to produce Thymic Stromal Lymphopoietin (TSLP), an IL7-like cytokine, which potently activated human myeloid dendritic cells (mDC) to cause the homeostatic proliferation of autologous CD4+ T cells that serve as targets for HIV infection. Rhesus macaques inoculated with simian immunodeficiency virus (SIV) or with the simian-human immunodeficiency virus (SHIV) by the vaginal, oral or rectal route exhibited dramatic increases in: TSLP expression, DC and CD4+ T cell numbers, and viral replication, in the vaginal, oral, and rectal tissues, respectively within the first 2 weeks after virus exposure. Evidence obtained showed that HIV-mediated TSLP production by cervical cells is dependent upon the expression of the cell surface salivary agglutinin (SAG) protein gp340. Epithelial cells expressing gp340 exhibited HIV endocytosis and TSLP expression and genetic knockdown of gp340 or use of a gp340-blocking antibody inhibited TSLP expression by HIV. On the other hand, gp340-null epithelial cells failed to endocytose HIV and produce TSLP, but transfection of gp340 resulted in HIV-induced TSLP expression. Finally, HIV-induced TSLP expression was found to be mediated by TLR7/8 signaling and NF-kB activity because silencing these pathways or use of specific inhibitors abrogated TSLP expression in gp340-postive but not in gp340-null epithelial cells. Overall these studies identify TSLP as a key player in the acute phase of HIV-1 infection in permitting HIV to successfully maneuver the hostile vaginal mucosal microenvironment by creating a conducive environment for sustaining the small amount of virus that initially crosses the mucosal barrier allowing it to successfully cause infection and spread to distal compartments of the body

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.

Adolescent Sexual Behavior: Examining Data from Texas and the US

Background: The US has higher rates of teen births and sexually transmitted infections (STI) than other developed countries. Texas youth are disproportionately impacted. Purpose: To review local, state, and national data on teens’ engagement in sexual risk behaviors to inform policy and practice related to teen sexual health. Methods: 2009 middle school and high school Youth Risk Behavior Survey (YRBS) data, and data from All About Youth, a middle school study conducted in a large urban school district in Texas, were analyzed to assess the prevalence of sexual initiation, including the initiation of non-coital sex, and the prevalence of sexual risk behaviors among Texas and US youth. Results: A substantial proportion of middle and high school students are having sex. Sexual initiation begins as early as 6th grade and increases steadily through 12th grade with almost two-thirds of high school seniors being sexually experienced. Many teens are not protecting themselves from unintended pregnancy or STIs – nationally, 80% and 39% of high school students did not use birth control pills or a condom respectively the last time they had sex. Many middle and high school students are engaging in oral and anal sex, two behaviors which increase the risk of contracting an STI and HIV. In Texas, an estimated 689,512 out of 1,327,815 public high school students are sexually experienced – over half (52%) of the total high school population. Texas students surpass their US peers in several sexual risk behaviors including number of lifetime sexual partners, being currently sexually active, and not using effective methods of birth control or dual protection when having sex. They are also less likely to receive HIV/AIDS education in school. Conclusion: Changes in policy and practice, including implementation of evidence-based sex education programs in middle and high schools and increased access to integrated, teen-friendly sexual and reproductive health services, are urgently needed at the state and national levels to address these issues effectively.

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^

Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^

A systematic review of the animal and human literature on the use of vaccines to prevent dental caries

Streptococcus mutans has been identified as the primary etiological agent of human dental caries. Since its identification, there has been research focused on the development of a vaccine to prevent this disease. Preliminary research has been conducted to test both active and passive vaccines for Streptococcus mutans in animals and humans. Although a vaccine for dental caries caused by Streptococcus mutans would most likely be administered to children, no testing of any type of dental caries vaccines has been conducted on children as of yet. The public health imperative for the development of a vaccine is great. Not only will a vaccine reduce the various consequences, but it would also improve quality of life for many individuals. Among the many possible vaccine antigen candidates, researchers have also been focusing on protein antigens, GTFs, and Gbps as possible candidates for a vaccine. There are also many routes of administration under research, with topical, oral, and intranasal showing a lot of promise. This review will provide an overview on the current state of research, present key factors influencing prevalence of caries, and summarize and discuss the results of animal and human studies on caries vaccines against Streptococcus mutans. The progress and obstacles facing the development of a vaccine to fight dental caries will also be discussed. ^

Attitudes About Predictive MEN1 Genetic Testing in Minors

Multiple Endocrine Neoplasia type 1 (MEN1) is a hereditary cancer syndrome characterized by tumors of the endocrine system. Tumors most commonly develop in the parathyroid glands, pituitary gland, and the gastro-entero pancreatic tract. MEN1 is a highly penetrant condition and age of onset is variable. Most patients are diagnosed in early adulthood; however, rare cases of MEN1 present in early childhood. Expert consensus opinion is that predictive genetic testing should be offered at age 5 years, however there are no evidence-based studies that clearly establish that predictive genetic testing at this age would be beneficial since most symptoms do not present until later in life. This study was designed to explore attitudes about the most appropriate age for predictive genetic testing from individuals at risk of having a child with MEN1. Participants who had an MEN1 mutation were invited to complete a survey and were asked to invite their spouses to participate as well. The survey included several validated measures designed to assess participants’ attitudes about predictive testing in minors. Fifty-eight affected participants and twenty-two spouses/partners completed the survey. Most participants felt that MEN1 genetic testing was appropriate in healthy minors. Younger age and increased knowledge of MEN1 genetics and inheritance predicted genetic testing at a younger age. Additionally, participants who saw more positive than negative general outcomes from genetic testing were more likely to favor genetic testing at younger ages. Overall, participants felt genetic testing should be offered at a younger age than most adult onset conditions and most felt the appropriate time for testing was when a child could understand and participate in the testing process. Psychological concerns seemed to be the primary focus of participants who favored later ages for genetic testing, while medical benefits were more commonly cited for younger age. This exploratory study has implications for counseling patients whose children are at risk of developing MEN1 and illustrates issues that are important to patients and their spouses when considering testing in children.