Aminoacyl-tRNA synthetases: Probing the molecular basis of catalysis and discrimination

Aminoacyl-tRNA synthetases (RSs) are responsible for the essential connection of amino acids with trinucleotide sequences of tRNA's. The RS family constitutes two structurally dissimilar groups of proteins, class I and class II. Methionyl-tRNA synthetase (MetRS) and isoleucyl-tRNA synthetase (IleRS), both members of class I, were the focus of this work. Both enzymes are zinc-containing proteins; show a high degree of amino acid specificity; and edit activated noncognate amino acids, thereby ensuring the fidelity of the genetic code. The goals of this work were to further delineate the molecular basis of catalysis and discrimination in these enzymes by mapping active site geometries using high-resolution nuclear magnetic resonance spectroscopy (NMR).^ Internuclear distances obtained from transferred nuclear Overhauser effects were used to define the conformations of Mg($\alpha$,$\beta$-methylene)ATP bound to E. coli MetRS and E. coli IleRS in multiple complexes. Identical conformations were found for the bound ATP. Thus, the predicted structural homology between IleRS and MetRS is supported by consensus enzyme-bound nucleotide conformations. The conformation of the bound nucleotide is not sensitive to occupation of the amino acid site of MetRS or IleRS. Therefore, conformational changes known to occur in the synthetases upon ligand binding appear not to alter the bound conformation of the adenosine portion of the nucleotide. Nuclear Overhauser effects on the substrate amino acid L-selenomethionine were also used to evaluate the enzyme-bound conformation of the cognate amino acid. The amino acid assumes a conformation which is consistent with a proposed editing mechanism.^ The E. coli MetRS was shown to catalyze amino acid $\alpha$-proton exchange in the presence of deuterium oxide of all cognate amino acids. It is proposed that the enzyme-bound zinc coordinates the $\alpha$-carboxylate of the amino acid, rendering the $\alpha$-proton more acidic. An enzymic base is responsible for exchange of the $\alpha$-proton. This proposal suggests that the enzyme-bound zinc may have a role in amino acid discrimination in MetRS. However, the role of this exchange reaction in catalysis remains unknown. ^

The genetic basis of thoracic aortic aneurysms and dissections: Genetic heterogeneity and mapping of TAAD1 and TAAD2 loci

Thoracic aortic aneurysms leading to aortic dissections (TAAD) are a major cause of morbidity and mortality in the United States. TAAD is a complication of some known genetic disorders, such as Marfan syndrome and Turner syndrome, but the majority of familial cases are not due to a known genetic syndrome. Previous studies by our group have established that nonsyndromic, familial TAAD is inherited in an autosomal dominant manner with decreased penetrance and variable expression. Using one large family with multiple members with TAAD for the genome wide scan, a major locus for familial TAAD was mapped to 5q13–14 (TAAD1). Nine out of 15 families studied were linked to this locus, establishing that TAAD1 was a major locus, and that there was genetic heterogeneity for the condition. Mapping of TAAD2 locus was accomplished using a single large family with multiple members with TAAD not linked to known loci of aneurysm formation. This established a second novel locus for familial TAAD on 3p24–25 (LOD score of 4.3), termed the TAAD2 locus. Two putative loci with suggestive LOD scores were mapped on 4q and 12q through a genome scan carried out using three families. TAAD phenotype in 12 families did not segregate with known loci, indicating further genetic heterogeneity. An STS-tagged BAC based contig was constructed for 7.8Mb and 25Mb critical interval of TAAD1 and TAAD2 respectively and characterized to identify the defective gene. The hypothesis that the defective genes responsible for the TAAD1 and TAAD2 encoded extracellular matrix (ECM) proteins, the major components of the elastic fiber system in the aortic media was tested. Four genes encoding ECM proteins, versican, thrombospondin-3, CRTL1, on TAAD1 and FBLN2 at TAAD2 were sequenced, but no disease-causing mutations were identified. Studies to identify the defective gene are initiated through the positional candidate gene approach using combination of bioinformatics and expression studies. The identification of the TAAD susceptibility genes will allow for presymptomatic diagnosis of individuals at risk for this life threatening disease. The identification of the molecular defects that contribute to TAAD will also further our understanding of the proteins that provide structural integrity to the aortic wall. ^

The genetic contribution of the major histocompatibility complex to bleomycin-induced lung injury

Pulmonary fibrosis (PF) is the result of a variety of environmental and cancer treatment related insults and is characterized by excessive deposition of collagen. Gas exchange in the alveoli is impaired as the normal lung becomes dense and collapsed leading to a loss of lung volume. It is now accepted that lung injury and fibrosis are in part genetically regulated. ^ Bleomycin is a chemotherapeutic agent used for testicular cancer and lymphomas that induces significant pulmonary toxicity. We delivered bleomycin to mice subcutaneously via a miniosmotic pump in order to elicit lung injury (LI) and quantified the %LI morphometrically using video imaging software. We previously identified a quantitative trait loci, Blmpf-1(LOD=17.4), in the Major Histocompatibility Complex (MHC), but the exact genetic components involved have remained unknown. ^ In the current studies, Blmpf-1 was narrowed to an interval spanning 31.9-32.9Mb on Chromosome 17 using MHC Congenic mice. This region includes the MHC Class II and III genes, and is flanked by the TNF-alpha super locus and MHC Class I genes. Knockout mice of MHC Class I genes (B2mko), MHC Class II genes (Cl2ko), and TNF-alpha (TNF-/-) and its receptors (p55-/-, p75-/-, and p55/p75-/-) were treated with bleomycin in order to ascertain the role of these genes in the pathogenesis of lung injury. ^ Cl2ko mice had significantly better survival and %LI when compared to treated background BL/6 (B6, P<.05). In contrast, B2mko showed no differences in survival or %LI compared to B6. This suggests that the MHC Class II locus contains susceptibility genes for bleomycin-induced lung injury. ^ TNF-alpha, a Class III gene, was examined and it was found that TNF-/- and p55-/- mice had higher %LI and lower survival when compared to B6 (P<.05). In contrast, p75-/- mice had significantly reduced %LI when compared to TNF-/-, p55-/-, and B6 mice as well as higher survival (P<.01). These data contradict the current paradigm that TNF-alpha is a profibrotic mediator of lung injury and suggest a novel and distinct role for the p55 and p75 receptors in mediating lung injury. ^

The molecular and genetic characterization of the MADS box transcription factor {\it Drosophila\/} MEF2

The myocyte enhancer factor (MEF)-2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates, but the precise position of these regulators within the genetic hierarchy leading to myogenesis is unclear. The MEF2 proteins bind to a conserved A/T-rich DNA sequence present in numerous muscle-specific genes, and they are expressed in the cells of the developing somites and in the embryonic heart at the onset of muscle formation in mammals. The MEF2 genes belong to the MADS box family of transcription factors, which control specific programs of gene expression in species ranging from yeast to humans. Each MEF2 family member contains two highly conserved protein motifs, the MADS domain and the MEF2-specific domain, which together provide the MEF2 factors with their unique DNA binding and dimerization properties. In an effort to further define the function of the MEF2 proteins, and to evaluate the degree of conservation shared among these factors and the phylogenetic pathways that they regulate, we sought to identify MEF2 family members in other species. In Drosophila, a homolog of the vertebrate MEF2 genes was identified and termed D-mef2. The D-MEF2 protein binds to the consensus MEF2 element and can activate transcription through tandem copies of that site. During Drosophila embryogenesis, D-MEF2 is specific to the mesoderm germ layer of the developing embryo and becomes expressed in all muscle cell types within the embryo. The role of D-mef2 in Drosophila embryogenesis was examined by generating a loss-of-function mutation in the D-mef2 gene. In embryos homozygous for this mutant allele, somatic, cardiac, and visceral muscles fail to differentiate, but precursors of these myogenic lineages are normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2 and suggest that this program has been conserved from Drosophila to mammals. ^

Genetic analysis of factors regulating mesenchymal cell fates

Formation of cartilage and bone involves sequential processes in which undifferentiated mesenchyme aggregates into primordial condensations which subsequently grow and differentiate, resulting in morphogenesis of the adult skeleton. While much has been learned about the structural molecules which comprise cartilage and bone, little is known about the nuclear factors which regulate chondrogenesis and osteogenesis. MHox is a homeobox-containing gene which is expressed in the mesenchyme of facial, limb, and vertebral skeletal precursors during mouse embryogenesis. MHox expression has been shown to require epithelial-derived signals, suggesting that MHox may regulate the epithelial-mesenchymal interactions required for skeletal organogenesis. To determine the functions of MHox, we generated a loss-of-function mutation in the MHox gene. Mice homozygous for a mutant MHox allele exhibit defects of skeletogenesis, involving the loss or malformation of craniofacial, limb and vertebral skeletal structures. The affected skeletal elements are derived from the cranial neural crest, as well as somitic and lateral mesoderm. Analysis of the mutant phenotype during ontogeny demonstrated a defect in the formation or growth of chondrogenic and osteogenic precursors. These findings provide evidence that MHox regulates the formation of preskeletal condensations from undifferentiated mesenchyme. In addition, generation of mice doubly mutant for the MHox and S8 homeobox genes reveal that these two genes interact to control formation of the limb and craniofacial skeleton. Mice carrying mutant alleles for S8 and MHox exhibit an exaggeration of the craniofacial and limb phenotypes observed in the MHox mutant mouse. Thus, MHox and S8 are components of a combinatorial genetic code controlling generation of the skeleton of the skull and limbs. ^

Genetic analysis of tumor progression susceptibility in the mouse skin model

In the field of chemical carcinogenesis the use of animal models has proved to be a useful tool in dissecting the multistage process of tumor formation. In this regard the outbred SENCAR mouse has been the strain of choice in the analysis of skin carcinogenesis given its high sensitivity to the chemically induced acquisition of premalignant lesions, papillomas, and the later progression of these lesions into squamous cell carcinomas (SCC).^ The derivation of an inbred strain from the SENCAR stock called SSIN, that in spite of a high sensitivity to the development of papillomas lack the ability to transform these premalignant lesions into SCC, suggested that tumor promotion and progression were under the genetic control of different sets of genes.^ In the present study the nature of susceptibility to tumor progression was investigated. Analysis of F1 hybrids between the outbred SENCAR and SSIN mice suggested that there is at least one dominant gene responsible for susceptibility to tumor progression.^ Later development of another inbred strain from the outbred SENCAR stock, that had sensitivity to both tumor promotion and progression, allowed the formulation of a more accurate genetic model. Using this newly derived line, SENCAR B/Pt. and SSIN it was determined that there is one dominant tumor progression susceptibility gene. Linkage analysis showed that this gene maps to mouse chromosome 14 and it was possible to narrow the region to a 16 cM interval.^ In order to better characterize the nature of the progression susceptibility differences between these two strains, their proliferative pattern was investigated. It was found that SENCAR B/Pt, have an enlarged proliferative compartment with overexpression of cyclin D1, p16 and p21. Further studies showed an aberrant overexpression of TGF-$\beta$ in the susceptible strain, an increase in apoptosis, p53 protein accumulation and early loss of connexin 26. These results taken together suggest that papillomas in the SENCAR B/Pt. mice have higher proliferation and may have an increase in genomic instability, these two factors would contribute to a higher sensitivity to tumor progression. ^

GENETIC ORGANIZATION OF THE STRUCTURAL GENES FOR NITRATE REDUCTASE (TN5)

Nitrate reductase in Escherichia coli is a membrane-bound anaerobic enzyme that is repressed by oxygen and induced by nitrate. The genetic organization of the structural genes for the two larger subunits of nitrate reductase ((alpha) and (beta)) was determined by immunoprecipitation analysis of the formation of these proteins in nitrate reductase-deficient mutants resulting from transposon Tn5 mutagenesis. The results suggested that the genes encoding the (alpha) and (beta) subunits (narG and H) were arranged in an operon with transcription in the direction promoter(--->)(alpha)(--->)(beta). Segments of the chromosome containing the Tn5 inserts from several of the mutants were cloned into plasmid pBR322 and the positions of the transposons determined by restriction mapping. The Tn5 insertion sites were localized on two contiguous EcoRI fragments spanning about 6.6 kilobases of DNA. The narI gene (proposed to encode the (gamma) subunit) was positioned immediately downstream from the (beta)-gene (narH) by Southern analysis of Tn10 insertions into the narI locus. A Tn10 insertion into the narK locus, proposed to encode a nitrate-sensitive repressor of other anaerobic enzymes, was located about 1.5 kilobases upstream from the narGHI operon promoter. The narL locus, proposed to encode a nitrate-sensitive positive regulator of the narGHI operon and known to be genetically linked to the other nar genes, was demonstrated to lie outside a 19.3-kilobase region of the chromosome which encompasses the other nar genes. The physical limit of the narGHI promoter was defined by studying the effect of Tn5 insertions into a hybrid plasmid containing the functional operon. The points of origin of the coding regions for the (alpha) and (beta) genes were deduced by alignment of the chromosomal map of Tn5 insertion sites with the sizes of (alpha) and (beta) subunit fragments produced by plasmids carrying these Tn5 inserts in the nar operon. The coding region for the (alpha) subunit (143,000 daltons) begins about 250 nucleotides downstream from the deduced limit of the promoter region and includes about 4.0 kilobases of DNA; the region encoding (beta) (60,000 daltons) lies immediately downstream from the (alpha)-gene and is approximately 1.6 kilobases in length. The adjacent region encoding the (gamma) subunit (19,000 daltons) is approximately 0.5 kilobase in length. ^

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. ^

The sequence comparison index: A novel method for comparing proteins and proteomes

Historically morphological features were used as the primary means to classify organisms. However, the age of molecular genetics has allowed us to approach this field from the perspective of the organism's genetic code. Early work used highly conserved sequences, such as ribosomal RNA. The increasing number of complete genomes in the public data repositories provides the opportunity to look not only at a single gene, but at organisms' entire parts list. ^ Here the Sequence Comparison Index (SCI) and the Organism Comparison Index (OCI), algorithms and methods to compare proteins and proteomes, are presented. The complete proteomes of 104 sequenced organisms were compared. Over 280 million full Smith-Waterman alignments were performed on sequence pairs which had a reasonable expectation of being related. From these alignments a whole proteome phylogenetic tree was constructed. This method was also used to compare the small subunit (SSU) rRNA from each organism and a tree constructed from these results. The SSU rRNA tree by the SCI/OCI method looks very much like accepted SSU rRNA trees from sources such as the Ribosomal Database Project, thus validating the method. The SCI/OCI proteome tree showed a number of small but significant differences when compared to the SSU rRNA tree and proteome trees constructed by other methods. Horizontal gene transfer does not appear to affect the SCI/OCI trees until the transferred genes make up a large portion of the proteome. ^ As part of this work, the Database of Related Local Alignments (DaRLA) was created and contains over 81 million rows of sequence alignment information. DaRLA, while primarily used to build the whole proteome trees, can also be applied shared gene content analysis, gene order analysis, and creating individual protein trees. ^ Finally, the standard BLAST method for analyzing shared gene content was compared to the SCI method using 4 spirochetes. The SCI system performed flawlessly, finding all proteins from one organism against itself and finding all the ribosomal proteins between organisms. The BLAST system missed some proteins from its respective organism and failed to detect small ribosomal proteins between organisms. ^

Genetic variability and the hypothalamic control of energy balance

Background. Obesity is a major health problem throughout the industrialized world. Despite numerous attempts to curtail the rapid growth of obesity, its incidence continues to rise. Therefore, it is crucial to better understand the etiology of obesity beyond the concept of energy balance.^ Aims. The first aim of this study was to first investigate the relationship between eating behaviors and body size. The second goal was to identify genetic variation associated with eating behaviors. Thirdly, this study aimed to examine the joint relationships between eating behavior, body size and genetic variation.^ Methods. This study utilized baseline data ascertained in young adults from the Training Interventions and Genetics of Exercise (TIGER) Study. Variables assessed included eating behavior (Emotional Eating Scale, Eating Attitudes Test-26, and the Block98 Food Frequency Questionnaire), body size (body mass index, waist and hip circumference, waist/hip ratio, and percent body fat), genetic variation in genes implicated related to the hypothalamic control of energy balance, and appropriate covariates (age, gender, race/ethnicity, smoking status, and physical activity. For the genetic association analyses, genotypes were collapsed by minor allele frequency, and haplotypes were estimated for each gene. Additionally, Bayesian networks were constructed in order to determine the relationships between genetic variation, eating behavior and body size.^ Results. We report that the EAT-26 score, Caloric intake, percent fat, fiber intake, HEAT index, and daily servings of vegetables, meats, grains, and fats were significantly associated with at least one body size measure. Multiple SNPs in 17 genes and haplotypes from 12 genes were tested for their association with body size. Variation within both DRD4 and HTR2A was found to be associated with EAT-26 score. In addition, variation in the ghrelin gene (GHRL) was significantly associated with daily Caloric intake. A significant interaction between daily servings of grains and the HEAT index and variation within the leptin receptor gene (LEPR) was shown to influence body size.^ Conclusion. This study has shown that there is a substantial genetic component to eating behavior and that genetic variation interacts with eating behavior to influence body size.^

A GENETIC MARKER STUDY OF NON - INSULIN DEPENDENT DIABETICS IN THE MEXICAN-AMERICAN COMMUNITY FROM STARR COUNTY, TEXAS (PLASMA GLUCOSE, HEMOGLOBIN ANALYSIS)

Diabetes mellitus occurs in two forms, insulin-dependent (IDDM, formerly called juvenile type) and non-insulin dependent (NIDDM, formerly called adult type). Prevalence figures from around the world for NIDDM, show that all societies and all races are affected; although uncommon in some populations (.4%), it is common (10%) or very common (40%) in others (Tables 1 and 2).^ In Mexican-Americans in particular, the prevalence rates (7-10%) are intermediate to those in Caucasians (1-2%) and Amerindians (35%). Information about the distribution of the disease and identification of high risk groups for developing glucose intolerance or its vascular manifestations by the study of genetic markers will help to clarify and solve some of the problems from the public health and the genetic point of view.^ This research was designed to examine two general areas in relation to NIDDM. The first aims to determine the prevalence of polymorphic genetic markers in two groups distinguished by the presence or absence of diabetes and to observe if there are any genetic marker-disease association (univariate analysis using two by two tables and logistic regression to study the individual and joint effects of the different variables). The second deals with the effect of genetic differences on the variation in fasting plasma glucose and percent glycosylated hemoglobin (HbAl) (analysis of Covariance for each marker, using age and sex as covariates).^ The results from the first analysis were not statistically significant at the corrected p value of 0.003 given the number of tests that were performed. From the analysis of covariance of all the markers studied, only Duffy and Phosphoglucomutase were statistically significant but poor predictors, given that the amount they explain in terms of variation in glycosylated hemoglobin is very small.^ Trying to determine the polygenic component of chronic disease is not an easy task. This study confirms the fact that a larger and random or representative sample is needed to be able to detect differences in the prevalence of a marker for association studies and in the genetic contribution to the variation in glucose and glycosylated hemoglobin. The importance that ethnic homogeneity in the groups studied and standardization in the methodology will have on the results has been stressed. ^

GENETIC PREDICTORS OF HYPERGLYCEMIA DUE TO HYDROCHLOROTHIAZIDE THERAPY

Response to pharmacological treatment is variable among individuals. Some patients respond favorably to a drug while others develop adverse reactions. Early investigations showed evidence of variation in genes that code for drug receptors, drug transporters, and drug metabolizing enzymes; and pharmacogenetics appeared as the science that studies the relationship between drug response and genetic variation. Thiazide diuretics are the recommended first-line monotherapy for hypertension (i.e. SBP>140 or DBP>90). Even so, diuretics are associated with adverse metabolic side effects, such as hyperglycemia, which increase the risk of developing type 2 diabetes. Published approaches testing variation in candidate genes (e.g. the renin-angiotensin-aldosteron system (RAAS) and salt–sensitivity genes) have met with only limited success. We conducted the first genome wide association study to identify genes influencing hyperglycemia as an adverse effect of thiazide diuretics in non-Hispanic White hypertensive patients participating in the Genetic Epidemiology of Responses to Antihypertensives (GERA) and Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) clinical trials. No SNP reached the a priori defined threshold of statistical significance (p<5x10-8). We detected 50 SNPs in 9 genomic regions with suggestive p-values (p<1x10-5). Two of them, rs6870564 (p-value=3.28 X 10-6) and rs7702121 (p-value=5.09 X 10-6), were located close to biologic candidate genes, MYO and MGAT1, and one SNP in a genomic region in chromosome 6, rs7762018 (p-value=4.59 X 10-6) has been previously related to Insulin-Dependent Diabetes Mellitus (IDDM8). I conclude that 1) there are unlikely to be common SNPs with large effects on the adverse metabolic effects to hydrochlorothiazide treatment and 2) larger sample sizes are needed for pharmacogenetic studies of inter-individual variation in response to commonly prescribed medication.

Genetic variants within the Wnt/ B-catenin signaling pathway as indicators of bladder cancer clinical outcomes

The genetic factors that influence bladder cancer clinical outcomes are largely unknown. In this clinical outcomes study, I assessed genetic variations in the Wnt/β-catenin stem-cell pathway genes for association with recurrence and progression. A total of 230 SNPS in 40 genes from the Wnt/β-catenin pathway were genotyped in 419 histologically confirmed non-muscle invasive bladder cancer cases. Several significant associations were observed in the clinical outcomes analysis. Under the dominant model WNT8B: rs4919464 (HR: 1.55, 95% CI: 1.17-2.06, P=2.2x10-3) and WNT8B: rs3793771 (HR: 1.54, 95% CI: 1.09-1.62, P=4.6x10-3 ) were statistically significantly associated with an increase risk of recurrence while two other variants, APC2: rs11668593 (HR: 2.50, 95% CI: 1.43-4.35, P=1.2x10-3) and LRP5 : rs312778 (HR: 1.81, 95% CI: 1.23-2.65, P=2.7x10-3), were significantly associated with recurrence risk under the recessive model of inheritance. Four SNPs in the recessive model were associated with an increased risk of progression (AXIN2: rs1544427, LRP5: rs312778, AXIN1: rs370681, AXIN1: rs2301522). LRP5: rs312778 had the most significant increased risk of progression with a 2.68 (95% CI: 1.52-4.72, P=6.4x10-4)-fold increased risk. Stratification analysis based on treatment regimen (transurethral resection (TUR) and Bacillus Calmette-Guérin (BCG)) was also performed. Individuals with at least one variant in AXIN2: rs2007085 were found to have a 2.09 (95% CI: 1.24-3.52, P=5.4x10-3) -fold increased risk of recurrence in those that received TUR only, and no statistically significant effect was seen in those that received BCG. Individuals who received TUR with at least one variant in LEF1: rs10516550 were found to have a 2.26 (95% CI: 1.22-4.18, P=9.7x10-3)-fold increase risk of recurrence and no statistically significant effect was found in individuals who received BCG. Also, the recessive model of LRP6: rs2302684 in TUR only treatment was shown to have a 1.95 (95%CI: 1.18-3.21, P=8.8x10 -3)-fold increased risk of recurrence, and a suggested protective effect associated with a (HR: 0.83, 95% CI: 0.51-1.37, P=0.468) decreased risk of recurrence. Together, these findings implicate the Wnt/β-catenin stem-cell pathway as playing a role in bladder cancer clinical outcomes and have important implications for personalization of future treatment regimens. ^