Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

Tuberculosis in persons with concomittant diseases in Houston, Texas, 2004 to 2008

The purpose of this study is to evaluate characteristics of tuberculosis (TB) in diabetics and persons infected with HIV from 2004 to 2008 in Houston, Texas. This analysis will allow us to identify demographic trends. Previous studies have shown that in general, there is a higher risk for HIV+ persons to develop active TB, or to re-activate latent TB, as they progress in their HIV infection. In addition, similar to HIV, diabetes mellitus (DM) weakens the immune system so that persons with DM have also been shown to have a tendency to develop TB. This analysis will examine three areas of research: (a) to explore existing TB trends in Houston/Harris County and associated characteristics, (b) to ascertain the common risk factors of DM and HIV that are correlate with TB infections, and (c) from the analysis of the data, to determine if subsequent TB prevention programs are needed for specific subgroups.^

A Bayesian analysis for spatial processes with application to mapping

In geographical epidemiology, maps of disease rates and disease risk provide a spatial perspective for researching disease etiology. For rare diseases or when the population base is small, the rate and risk estimates may be unstable. Empirical Bayesian (EB) methods have been used to spatially smooth the estimates by permitting an area estimate to "borrow strength" from its neighbors. Such EB methods include the use of a Gamma model, of a James-Stein estimator, and of a conditional autoregressive (CAR) process. A fully Bayesian analysis of the CAR process is proposed. One advantage of this fully Bayesian analysis is that it can be implemented simply by using repeated sampling from the posterior densities. Use of a Markov chain Monte Carlo technique such as Gibbs sampler was not necessary. Direct resampling from the posterior densities provides exact small sample inferences instead of the approximate asymptotic analyses of maximum likelihood methods (Clayton & Kaldor, 1987). Further, the proposed CAR model provides for covariates to be included in the model. A simulation demonstrates the effect of sample size on the fully Bayesian analysis of the CAR process. The methods are applied to lip cancer data from Scotland, and the results are compared. ^

Use of pneumococcal vaccine in people with chronic disease in United States

Objective. The risk of complications and deaths related to pneumococcal infections is high among high risk population (i.e. those with chronic diseases such as diabetes or asthma), despite current immunization recommendations. The aim of this study is to evaluate the use of pneumonia vaccine in adults with and without diabetes or asthma by year of age and whether immunization practices conform to policy recommendations. ^ Methods. Data were drawn from 2005 Behavioral Risk Factor Surveillance Study. Age specific estimated counts and proportions of pneumonia vaccination status were computed. The association of socio-demographic factors with vaccination status was estimated from multiple logistic regression and results were presented for adults (18-64yrs) and elderly (65 or older). ^ Results. Overall 12.3% of the adults and 61.5% of elderly reported ever received pneumonia vaccine. 66.8% of diabetics and 72.6% of asthmatics received the vaccine among elderly. 33.4% of diabetics and 21.6% of asthmatics received the vaccine among adults. These numbers are far away from Healthy people 2010 objective coverage rates of 90% for elderly and 60% for high risk adults. Though diabetes was one of the recommendations for the pneumonia vaccine still the status was less than 70% even at older ages. Although asthma was not an indication for pneumonia vaccine, asthmatics still achieved 50% level by an early age of 60 and reached up to 80% at as early as 75 years. In those having both asthma and diabetes, although the curve reaches to 50% level at a very early age of 40yrs, it is not stable until the age of 55 and percentages reached to as high as 90% in older ages. Odds of receiving pneumonia vaccine were high in individuals with diabetes or asthma in both the age groups. But the odds were stronger for diabetics in adults compared to those in the elderly [2.24 CI (2.08-2.42) and 1.32 CI (1.18-1.47)]. The odds were slightly higher in adults than in elderly for asthmatics [1.92 CI (1.80-2.04) and 1.73 CI (1.50-2.00)].The likelihood of vaccination also differed by gender, ethnicity, marital status, income category, having a health insurance, current employment, physician visit in last year, reporting of good to excellent health and flu vaccine status. ^ Conclusion. There is a very high proportion of high risk adults and elderly that remain unvaccinated. Given the proven efficacy and safety of vaccine there is a need for interventions targeting the barriers for under-vaccination with more emphasis on physician knowledge and practice as well as the recipient attitudes.^

INDUCED-PLURIPOTENT STEM-DERIVED NEURONAL PROGENITOR CELLS AS A NOVEL TREATMENT FOR NEURODEGENERATIVE DISEASES

Cellular therapies, as neuronal progenitor (NP) cells grafting, are promising therapies for patients affected with neurodegenerative diseases like Creutzfeldt-Jakob Disease (CJD). At this time there is no effective treatment or cure for CJD. The disease is inevitably fatal and affected people usually die within months of the appearance of the first clinical symptoms. Compelling evidence indicate that the hallmark event in the disease is the conversion of the normal prion protein (termed PrPC) into the disease-associated, misfolded form (called PrPSc). Thus, a reasonable therapeutic target would be to prevent PrP misfolding and prion replication. This strategy has been applied with poor results since at the time of clinical intervention substantial brain damage has been done. It seems that a more effective treatment aimed at patients with established symptoms of CJD would need to stop further brain degeneration or even recover some of the previously lost brain tissue. The most promising possibility to recover brain tissue is the use of NPs that have the potential to replenish the nerve cells lost during the early stages of the disease. Advanced cellular therapies, beside their potential for cell replacement, might be used as biomaterials for drug delivery in order to stimulate cell survival or the resolution the disease. Also, implanted cells can be genetically manipulated to correct abnormalities causing disease or to make them more resistant to the toxic microenvironments present in damaged tissue. In recent years cell engineering has been within the scope of the scientific and general community after the development of technologies able to “de-differentiate” somatic cells into induced-pluripotent stem (IPS) cells. This new tool permits the use of easy-to-reach cells like skin or blood cells as a primary material to obtain embryonic stem-like cells for cellular therapies, evading all ethical issues regarding the use of human embryos as a source of embryonic stem cells. The complete work proposes to implant IPS-derived NP cells into the brain of prion-infected animals to evaluate their therapeutic potential. Since it is well known that the expression of prion protein in the cell membrane is necessary for PrPSc mediated toxicity, we also want to determine if NPs lacking the prion protein have better survival rates once implanted into sick animals. The main objective of this work is to develop implantable neural precursor from IPS coming from animals lacking the prion protein. Specific aim 1: To develop and characterize cellular cultures of IPS cells from prp-/- mice. Fibroblasts from prp-/- animals will be reprogrammed using the four Yamanaka factors. IPS colonies will be selected and characterized by immunohistochemistry for markers of pluripotency. Their developmental capabilities will be evaluated by teratoma and embryoid body formation assays. Specific aim 2: To differentiate IPS cells to a neuronal lineage. IPS cells will be differentiated to a NP stage by the use of defined media culture conditions. NP cells will be characterized by their immunohistochemical profile as well as by their ability to differentiate into neuronal cells. Specific aim 3: Cellular labeling of neuronal progenitors cells for in vitro traceability. In order to track the cells once implanted in the host brain, they will be tagged with different methods such as lipophilic fluorescent tracers and transduction with GFP protein expression.

Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders.

Molluscan preparations have yielded seminal discoveries in neuroscience, but the experimental advantages of this group have not, until now, been complemented by adequate molecular or genomic information for comparisons to genetically defined model organisms in other phyla. The recent sequencing of the transcriptome and genome of Aplysia californica, however, will enable extensive comparative studies at the molecular level. Among other benefits, this will bring the power of individually identifiable and manipulable neurons to bear upon questions of cellular function for evolutionarily conserved genes associated with clinically important neural dysfunction. Because of the slower rate of gene evolution in this molluscan lineage, more homologs of genes associated with human disease are present in Aplysia than in leading model organisms from Arthropoda (Drosophila) or Nematoda (Caenorhabditis elegans). Research has hardly begun in molluscs on the cellular functions of gene products that in humans are associated with neurological diseases. On the other hand, much is known about molecular and cellular mechanisms of long-term neuronal plasticity. Persistent nociceptive sensitization of nociceptors in Aplysia displays many functional similarities to alterations in mammalian nociceptors associated with the clinical problem of chronic pain. Moreover, in Aplysia and mammals the same cell signaling pathways trigger persistent enhancement of excitability and synaptic transmission following noxious stimulation, and these highly conserved pathways are also used to induce memory traces in neural circuits of diverse species. This functional and molecular overlap in distantly related lineages and neuronal types supports the proposal that fundamental plasticity mechanisms important for memory, chronic pain, and other lasting alterations evolved from adaptive responses to peripheral injury in the earliest neurons. Molluscan preparations should become increasingly useful for comparative studies across phyla that can provide insight into cellular functions of clinically important genes.

Determinants of malnutrition in two states in Sudan

Nearly one in three children in the developing world is malnourished. Poor nutrition contributes to one out of two deaths (53%) associated with infectious diseases among children aged under five in developing countries. Using data from the 2005 World Food Program’s (WFP) Livelihood Vulnerability and Nutritional Assessment of Rural Kassala and Red Sea State this study examines the impact of female headed households and maternal education on malnutrition in children 6-59 months old. The dependent variable investigated in this study is moderate to severe wasting or less than -2 weight for height Z-score, also known as global acute malnutrition (GAM). ^ The study population consisted of 450 households in Kassala State and Red Sea State, Sudan. A total of 900 children 6-59 months of age were part of the households sampled from these states and one child per household (773 children) was randomly chosen for the analysis along with the child’s mother. Results of the study found that 18 percent of children between 6-59 months of age had GAM/wasting. Maternal education, main source of water, and income were strongly related to wasting. Gender of head of household was not found to have a significant relationship with GAM/wasting. Mothers with at least primary education were much less likely to have malnourished children, even after controlling for income and environmental conditions. Children in households with unsafe sources of water were 2.6 more likely to have wasting than those with piped in/tube wells as their main source of water. For every increase of 100 dinar in a household, the children in the household are approximately two-thirds times (.662) less likely to be wasted. ^ The results of this study support the alternate hypothesis that there is an association between maternal education on wasting of children 6-59 months old. The results do not, however, support the alternate hypothesis that there is an association between gender of head of household on wasting of children 6-59 months old. Better understanding of the association of wasting and other measures of malnutrition with maternal education levels can program managers and other health officials to target important nutritional and non-nutritional interventions. ^

Involvement of HMGB1 in the repair of DNA adducts and the responses to DNA damage in mammalian cells

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcription, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair (NER) damage recognition proteins XPA and RPA to triplex-directed psoralen DNA interstrand crosslinks (ICLs). Based on this we hypothesized that HMGB1 is enhancing the repair of DNA lesions, and through this role, is affecting DNA damage-induced mutagenesis and cell survival. Because HMGB1 is also a chromatin protein, we further hypothesized that it is acting to facilitate chromatin remodeling at the site of the DNA damage, to allow access of the repair machinery to the DNA lesion. We demonstrated here that HMGB1 could bind to triplex-directed psoralen ICLs in a complex with NER proteins XPC-RAD23B, XPA and RPA, which occurred in the presence or absence of DNA. Supporting these findings, we demonstrated that HMGB1 enhanced repair of triplex-directed psoralen ICLs (by nucleotide incorporation), as well as removal of UVC irradiation-induced DNA lesions from the genome (by radioimmunoassay). We also explored HMGB1's role in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no increase in histone acetylation after UVC irradiation. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. However, HMGB1 also has a role in DNA repair in the absence of chromatin, as shown by measuring UVC-induced nucleotide incorporation on a naked substrate. Upon exploration of HMGB1's effect on several cellular outcomes of DNA damage, we found that mammalian cells lacking HMGB1 were hypersensitive to DNA damage induced by psoralen plus UVA irradiation or UVC radiation, showing less survival and increased mutagenesis. These results reveal a new role for HMGB1 in the error-free repair of DNA lesions in a chromosomal context. As strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases. ^

Gene-gene interaction and gene-pathway analysis of genome-wide association for schizophrenia

Schizophrenia (SZ) is a complex disorder with high heritability and variable phenotypes that has limited success in finding causal genes associated with the disease development. Pathway-based analysis is an effective approach in investigating the molecular mechanism of susceptible genes associated with complex diseases. The etiology of complex diseases could be a network of genetic factors and within the genes, interaction may occur. In this work we argue that some genes might be of small effect that by itself are neither sufficient nor necessary to cause the disease however, their effect may induce slight changes to the gene expression or affect the protein function, therefore, analyzing the gene-gene interaction mechanism within the disease pathway would play crucial role in dissecting the genetic architecture of complex diseases, making the pathway-based analysis a complementary approach to GWAS technique. ^ In this study, we implemented three novel linkage disequilibrium based statistics, the linear combination, the quadratic, and the decorrelation test statistics, to investigate the interaction between linked and unlinked genes in two independent case-control GWAS datasets for SZ including participants of European (EA) and African (AA) ancestries. The EA population included 1,173 cases and 1,378 controls with 729,454 genotyped SNPs, while the AA population included 219 cases and 288 controls with 845,814 genotyped SNPs. We identified 17,186 interacting gene-sets at significant level in EA dataset, and 12,691 gene-sets in AA dataset using the gene-gene interaction method. We also identified 18,846 genes in EA dataset and 19,431 genes in AA dataset that were in the disease pathways. However, few genes were reported of significant association to SZ. ^ Our research determined the pathways characteristics for schizophrenia through the gene-gene interaction and gene-pathway based approaches. Our findings suggest insightful inferences of our methods in studying the molecular mechanisms of common complex diseases.^