Xenobiotic Metabolism Genes and Clubfoot

Idiopathic or isolated clubfoot is a common orthopedic birth defect that affects approximately 135,000 children worldwide. It is characterized by equinus, varus and adductus deformities of the ankle and foot. Correction of clubfoot involves months of serial manipulations, castings and bracing, with surgical correction needed in forty percent of cases. Multifactorial etiology has been suggested in numerous studies with both environmental and genetic factors playing an etiologic role. Maternal smoking during pregnancy is the only common environmental factor that has consistently been shown to increase the risk for clubfoot. Moreover, a positive family history of clubfoot and maternal smoking increases the risk of clubfoot twenty fold. These findings suggest that genetic variation in smoking metabolism genes may increase susceptibility to clubfoot. Based on this reasoning, we interrogated eight candidate genes, chosen based on their involvement in phase 1 and 2 cigarette smoke metabolism. Twenty-two SNPs and two null alleles in eight genes (CYP1A1, CYP1A2, CYP1B1, CYP2A6, EPHX1, NAT2, GSTM1 and GSTT1) were genotyped in a dataset composed of nonHispanic white and Hispanic multiplex and simplex families. Only one SNP in CYP1A1, rs1048943, had significantly altered transmission in the aggregate and multiplex NHW datasets (p=0.003 and p=0.009). Perturbation of CYP1A1 by rs1048943 polymorphism causes an increase in the amount of harmful, adduct forming metabolic intermediates. A significant gene interaction between EPHX1 and NAT2 was also found (p=0.007). This interaction may affect the metabolism of harmful metabolic intermediates. Additionally, marginal interactions were found for other xenobiotic genes and these interactions may play a contributory role in clubfoot. Importantly, for CYP1A2, significant maternal (p=0.03; RR=1.24; 95% CI: 1.04-1.44) and fetal (p=0.01; RR=1.33; 95% CI: 1.13-1.54) genotypic effects were identified suggesting that both maternal and fetal genotypes impact normal limb development. No association was found for maternal smoking status and tobacco metabolism genes. Together, these results suggest that xenobiotic metabolism genes may play a contributory role in the etiology of clubfoot regardless of maternal smoking status and may impact foot development through perturbation of tobacco metabolic pathways.

Distribution of genes encoding MSCRAMMs and Pili in clinical and natural populations of Enterococcus faecium.

Enterococcus faecium has recently emerged as an important cause of nosocomial infections. We previously identified 15 predicted surface proteins with characteristics of MSCRAMMs and/or pili and demonstrated that their genes were frequently present in 30 clinical E. faecium isolates studied; one of these, acm, has been studied in further detail. To determine the prevalence of the other 14 genes among various E. faecium populations, we have now assessed 433 E. faecium isolates, including 264 isolates from human clinical infections, 69 isolates from stools of hospitalized patients, 70 isolates from stools of community volunteers, and 30 isolates from animal-related sources. A variable distribution of the 14 genes was detected, with their presence ranging from 51% to 98% of isolates. While 81% of clinical isolates carried 13 or 14 of the 14 genes tested, none of the community group isolates and only 13% of animal isolates carried 13 or 14 genes. The presence of these genes was most frequent in endocarditis isolates, with 11 genes present in all isolates, followed by isolates from other clinical sources. The number of genes significantly associated with clinical versus fecal or animal origin (P = 0.04 to <0.0001) varied from 10 to 13, depending on whether comparisons were made against individual clinical subgroups (endocarditis, blood, and other clinical isolates) or against all clinical isolates combined as one group. The strong association of these genes with clinical isolates raises the possibility that their preservation/acquisition has favored the adaptation of E. faecium to nosocomial environments and/or patients.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Transcriptional analysis of liver-specific expression and differential interleukin-6 response of rat kininogen genes

Analyses of rat T1 kininogen gene/chloramphenicol acetyltransferase (T1K/CAT) constructs revealed two regions important for tissue-specific and induced regulation of T1 kininogen.^ Although the T1 kininogen gene is inducible by inflammatory cytokines, a highly homologous K kininogen gene is minimally responsive. Moreover, the basal expression of a KK/CAT construct was 5- to 7-fold higher than that of the analogous T1K/CAT construct. To examine the molecular basis of this differential regulation, a series of promoter swapping experiments was carried out. Our transfection results showed that at least two regions in the K kininogen gene are important for its high basal expression: a distal 19-bp region (C box) constituted a binding site for CCAAT/enhancer binding protein (C/EBP) family proteins and a proximal 66-bp region contained two adjacent binding sites for hepatocyte nuclear factor-3 (HNF-3). The distal HNF-3 binding site from the K kininogen promoter demonstrated a stronger affinity than that from the T1 kininogen promoter. Since C/EBP and HNF-3 are highly enriched in the liver and known to enhance transcription of liver-specific genes, differential binding affinities of these factors accounted for the higher basal expression of the K kininogen gene.^ In contrast to the K kininogen C box, the T1 kininogen C box does not bind C/EBP presumably due to their two-nucleotide divergence. This sequence divergence, however, converts it to a consensus binding sequence for two IL-6-inducible transcription factors--IL-6 response element binding protein and acute-phase response factor. To functionally determine whether C box sequences are important for their differential acute-phase response, T1 and K kininogen C boxes were swapped and analyzed after transfection into Hep3B cells. Our results showed that the T1 kininogen C box is indeed one of the IL-6 response elements in T1 kininogen promoter. Furthermore, its function can be modulated by a 5$\sp\prime$-adjacent C/EBP-binding site (B box) whose mutation significantly reduced the overall induced activity. Moreover, this B box is the target site for binding and transactivation of another IL-6 inducible transcription factor C/EBP$\delta.$ Evolutionary divergence of a few critical nucleotides can either lead to subtle changes in the binding affinities of a given transcription factor or convert a binding sequence for a constitutive factor to a site recognized by an inducible factor. (Abstract shortened by UMI.) ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

Regulation of the expression of {\it mutS, mutL}, and {\it mutH\/} mismatch repair genes in {\it Escherichia coli\/} K-12

Post-replication DNA mismatch repair plays crucial roles in mutation avoidance and maintenance of chromosome stability in both prokaryotes and eukaryotes. In humans, deficiency in this repair system leads to a predisposition for certain cancers. The biochemistry of this repair system has been best studied in a model bacterium Escherichia coli. In this thesis, regulation of expression of mutS, mutL and mutH genes, whose products mediate methyl-directed mismatch (MDM) repair in E. coli, is investigated. One-step affinity purification schemes were developed to purify E. coli MutS, MutL and MutH proteins fused to a His-6-affinity tag. His-6-MutS exhibited the same mismatch binding activity and specificity as the native MutS protein. Purified His-6-MutS, -MutL and -MutH proteins were used to develop quantitative Western blotting assays for amounts of MutS, MuL and MutH proteins under various conditions. It was found that the three proteins were present in relatively low amounts in exponentially growing cells and MutS and MutH were diminished in stationary-phase cells. Further studies indicated that the drop in the amounts of MutS and MutH proteins in stationary-phase cells was mediated through RpoS, a key global regulator of stationary-phase transition. In both exponential- and stationary-phase cells, MutS amount was also negatively regulated by the Hfq (HF-I) global regulator, which is required for RpoS translation, through an RpoS-independent mechanism. $\beta$-galactosidase assays of mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally, and RNase T2 protection assays revealed that Hfq destabilizes mutS transcripts in exponentially growing cells. To study the relation between regulation of MDM repair and mutagenesis, amounts of MutS, MutL and MutH were measured in starved cells undergoing adaptive mutagenesis. It was found that MutS amount dropped drastically, MutH amount dropped slightly, whereas MutL amount remained essentially constant in starved cells. Overexpression of MutL did not reverse the drop in the amounts of MutS or MutH protein. These results ruled out several explanations for a phenomenon in which overexpression of MutL, but not MutS, reversed adaptive mutagenesis. The findings further suggested that functional MutL is limiting during adaptive mutagenesis. The implications of regulation of the MDM repair are discussed in the context of mutagenesis, pathogenesis and tumorigenesis. ^

Evaluation of candidate genes for susceptibility to spina bifida

Neural tube defects (NTDs) are the most common severely disabling birth defects in the United States, with a frequency of approximately 1–2 of every 1,000 births. This text includes the identification and evaluation of candidate susceptibility genes that confer risk for the development of neural tube defects (NTDs). The project focused on isolated meningomyelocele, also termed spina bifida (SB). ^ Spina bifida is a complex disease with multifactorial inheritance, therefore the subject population (consisting of North American Caucasians and Hispanics of Mexicali-American descent) was composed of 459 simplex SB families who were tested for genetic associations utilizing the transmission disequilibrium test (TDT), a nonparametric linkage technique. Three categories of candidate genes were studied, including (1) human equivalents of genes determined in mouse models to cause NTDs, (2) HOX and PAX genes, and (3) the MTHFR gene involved in the metabolic pathway of folate. ^ The C677T variant of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene was the first mutation in this gene to be implicated as a risk factor for NTDs. Our evaluation of the MTHFR gene provides evidence that maternal C677T homozygosity is a risk factor for upper level spina bifida defects in Hispanics [OR = 2.3, P = 0.02]. This observed risk factor is of great importance due to the high prevalence of this homozygous genotype in the Hispanic population. Additionally, maternal C677T/A1298C compound heterozygosity is a risk factor for upper level spina bifida defects in non-Hispanic whites [OR = 3.6, P = 0.03]. ^ For TDT analysis, our total population of 1128 subjects were genotyped for 54 markers from within and/or flanking the 20 candidate genes/gene regions of interest. Significant TDT findings were obtained for 3 of the 54 analyzed markers: d20s101 flanking the PAX1 gene (P = 0.019), d1s228 within the PAX7 gene (P = 0.011), and d2s110 within the PAX8 gene (P = 0.013). These results were followed-up by testing the genes directly for mutations utilizing single-strand conformational analysis (SSCA) and direct sequencing. Multiple variations were detected in each of these PAX genes; however, these variations were not passed from parent to child in phase with the positively transmitted alleles. Therefore, these variations do not contribute to the susceptibility of spina bifida, but rather are previously unreported single nucleotide polymorphisms. ^

Tp53-dependent repression of cell cycle target genes: Global and mechanistic analysis

Most studies of p53 function have focused on genes transactivated by p53. It is less widely appreciated that p53 can repress target genes to affect a particular cellular response. There is evidence that repression is important for p53-induced apoptosis and cell cycle arrest. It is less clear if repression is important for other p53 functions. A comprehensive knowledge of the genes repressed by p53 and the cellular processes they affect is currently lacking. We used an expression profiling strategy to identify p53-responsive genes following adenoviral p53 gene transfer (Ad-p53) in PC3 prostate cancer cells. A total of 111 genes represented on the Affymetrix U133A microarray were repressed more than two fold (p ≤ 0.05) by p53. An objective assessment of array data quality was carried out using RT-PCR of 20 randomly selected genes. We estimate a confirmation rate of >95.5% for the complete data set. Functional over-representation analysis was used to identify cellular processes potentially affected by p53-mediated repression. Cell cycle regulatory genes exhibited significant enrichment (p ≤ 5E-28) within the repressed targets. Several of these genes are repressed in a p53-dependent manner following DNA damage, but preceding cell cycle arrest. These findings identify novel p53-repressed targets and indicate that p53-induced cell cycle arrest is a function of not only the transactivation of cell cycle inhibitors (e.g., p21), but also the repression of targets that act at each phase of the cell cycle. The mechanism of repression of this set of p53 targets was investigated. Most of the repressed genes identified here do not harbor consensus p53 DNA binding sites but do contain binding sites for E2F transcription factors. We demonstrate a role for E2F/RB repressor complexes in our system. Importantly, p53 is found at the promoter of CDC25A. CDC25A protein is rapidly degraded in response to DNA damage. Our group has demonstrated for the first time that CDC25A is also repressed at the transcript level by p53. This work has important implications for understanding the DNA damage cell cycle checkpoint response and the link between E2F/RB complexes and p53 in the repression of target genes. ^

Identification of diabetic retinopathy genes through a genome-wide association study among Mexican-Americans from Starr County, Texas

To identify genetic susceptibility loci for severe diabetic retinopathy, 286 Mexican-Americans with type 2 diabetes from Starr County, Texas completed detailed physical and ophthalmologic examinations including fundus photography for diabetic retinopathy grading. 103 individuals with moderate-to-severe non-proliferative diabetic retinopathy or proliferative diabetic retinopathy were defined as cases for this study. DNA samples extracted from study subjects were genotyped using the Affymetrix GeneChip® Human Mapping 100K Set, which includes 116,204 single nucleotide polymorphisms (SNPs) across the whole genome. Single-marker allelic tests and 2- to 8-SNP sliding-window Haplotype Trend Regression implemented in HelixTreeTM were first performed with these direct genotypes to identify genes/regions contributing to the risk of severe diabetic retinopathy. An additional 1,885,781 HapMap Phase II SNPs were imputed from the direct genotypes to expand the genomic coverage for a more detailed exploration of genetic susceptibility to diabetic retinopathy. The average estimated allelic dosage and imputed genotypes with the highest posterior probabilities were subsequently analyzed for associations using logistic regression and Fisher's Exact allelic tests, respectively. To move beyond these SNP-based approaches, 104,572 directly genotyped and 333,375 well-imputed SNPs were used to construct genetic distance matrices based on 262 retinopathy candidate genes and their 112 related biological pathways. Multivariate distance matrix regression was then used to test hypotheses with genes and pathways as the units of inference in the context of susceptibility to diabetic retinopathy. This study provides a framework for genome-wide association analyses, and implicated several genes involved in the regulation of oxidative stress, inflammatory processes, histidine metabolism, and pancreatic cancer pathways associated with severe diabetic retinopathy. Many of these loci have not previously been implicated in either diabetic retinopathy or diabetes. In summary, CDC73, IL12RB2, and SULF1 had the best evidence as candidates to influence diabetic retinopathy, possibly through novel biological mechanisms related to VEGF-mediated signaling pathway or inflammatory processes. While this study uncovered some genes for diabetic retinopathy, a comprehensive picture of the genetic architecture of diabetic retinopathy has not yet been achieved. Once fully understood, the genetics and biology of diabetic retinopathy will contribute to better strategies for diagnosis, treatment and prevention of this disease.^

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^

Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^