The angiotensin-converting enzyme insertion/deletion polymorphism and its associations with carotid artery wall thickness and coronary heart disease: The atherosclerosis risk in communities study

Coronary heart disease (CHD) is the leading cause of death in the United States. Recently, renin-angiotensin system (RAS) was found associated with atherosclerosis formation, with angiotensin II inducing vascular smooth muscle cell growth and migration, platelet activation and aggregation, and stimulation of plasminogen activator inhibitor-1. Angiotensin II is converted from angiotensin I by angiotensin I-converting enzyme (ACE) and this enzyme is mainly genetically determined. The ACE gene has been assigned to chromosome 17q23 and an insertion/deletion (I/D)polymorphism has been characterized by the presence/absence of a 287 bp fragment in intron 16 of the gene. The two alleles form three genotypes, namely, DD, ID and II and the DD genotype has been linked to higher plasma ACE levels and cell ACE activity.^ In this study, the association between the ACE I/D polymorphism and carotid artery wall thickness measured by B-mode ultrasound was investigated in a biracial sample, and the association between the gene and incident CHD was investigated in whites and if the gene-CHD association in whites, if any, was due to the gene effect on atherosclerosis. The study participants are from the prospective Atherosclerosis Risk in Communities (ARIC) Study, including adults aged 45 to 65 years. The present dissertation used a matched case-control design for studying the associations of the ACE gene with carotid artery atherosclerosis and an unmatched case-control design for the association of the gene with CHD. A significant recessive effect of the D allele on carotid artery thickness was found in blacks (OR = 3.06, 95% C.I: 1.11-8.47, DD vs. ID and II) adjusting for age, gender, cigarette smoking, LDL-cholesterol and diabetes. No similar associations were found in whites. The ACE I/D polymorphism is significantly associated with coronary heart disease in whites, and while stratifying data by carotid artery wall thickness, the significant associations were only observed in thin-walled subgroups. Assuming a recessive effect of the D allele, odds ratio was 2.84 (95% C.I:1.17-6.90, DD vs. ID and II) and it was 2.30 (95% C.I:1.22-4.35, DD vs. ID vs. II) assuming a codominant effect of the D allele. No significant associations were observed while comparing thick-walled CHD cases with thin-walled controls. Following conclusions could be drawn: (1) The ACE I/D polymorphism is unlikely to confer appreciable increase in the risk of carotid atherosclerosis in US whites, but may increases the risk of carotid atherosclerosis in blacks. (2) ACE I/D polymorphism is a genetic risk factor for incident CHD in US whites and this effect is separate from the chronic process of atherosclerosis development. Finally, the associations observed here are not causal, since the I/D polymorphism is in an intron, where no ACE proteins are encoded. ^

An Sp1/Sp3 binding polymorphism confers methylation protection.

Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and approximately 2-3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

T-786C polymorphism of the endothelial nitric oxide synthase gene and neuralgia-inducing cavitational osteonecrosis of the jaws.

OBJECTIVE: We hypothesized that, similar to idiopathic hip osteonecrosis, the T-786C mutation of the endothelial nitric oxide synthase (eNOS) gene affecting nitric oxide (NO) production was associated with neuralgia-inducing cavitational osteonecrosis of the jaws (NICO). DESIGN: In 22 NICO patients, not having taken bisphosphonates, mutations affecting NO production (eNOS T-786C, stromelysin 5A6A) were measured by polymerase chain reaction. Two healthy normal control subjects were matched per case by race and gender. RESULTS: Homozygosity for the mutant eNOS allele (TT) was present in 6 out of 22 patients (27%) with NICO compared with 0 out of 44 (0%) race and gender-matched control subjects; heterozygosity (TC) was present in 8 patients (36%) versus 15 control subjects (34%); and the wild-type normal genotype (CC) was present in 9 patients (36%) versus 29 controls (66%) (P = .0008). The mutant eNOS T-786C allele was more common in cases (20 out of 44 [45%]) than in control subjects (15 out of 88 [17%]) (P = .0005). The distribution of the stromelysin 5A6A genotype in cases did not differ from control subjects (P = .13). CONCLUSIONS: The eNOS T-786C polymorphism affecting NO production is associated with NICO, may contribute to the pathogenesis of NICO, and may open therapeutic medical approaches to treatment of NICO through provision of L-arginine, the amino-acid precursor of NO.

Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease.

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.

Study of tumor suppressor genes p53 and Rb in acute myelogenous leukemia

Loss of antiproliferative function of p53 by point mutation occurred frequently in various solid tumors. However, the genetic change of p53 by deletion or point mutation was a rare event (6%) in the cells of 49 AML patients analyzed by single-stranded conformation polymorphism and sequencing. Despite infrequent point mutation, abundant levels of p53 protein were detected in 75% of AML patients studied by immunoprecipitation with p53 specific antibodies. Furthermore, p53 protein in most cases had an altered conformation as analyzed by the reactivity to PAb240 which recognizes mutant p53; p53 protein in mitogen stimulated normal lymphocytes also had similar altered conformation. This altered conformation may be another mechanism for inactivation of p53 function in the growth stimulated environment. Some evidence indicated that posttranslational modification by phosphorylation may contribute to the conformational change of p53.^ Retinoblastoma (Rb) gene inactivation by deletion, rearrangement or mutation has also been implicated in many types of solid tumors. Our studies showed that absence or low levels of Rb protein were observed in more than 20% of AML patients at diagnosis, and the low levels of Rb correlated with shorter survival of patients. The absence of Rb protein was due to gene inactivation in some cases and to abnormal regulation of Rb expression in others. ^

Global patterns of DNA polymorphism in noncoding regions in human populations

DNA sequence variation is currently a major source of data for studying human origins, evolution, and demographic history, and for detecting linkage association of complex diseases. In this dissertation, I investigated DNA variation in worldwide populations from two ∼10 kb autosomal regions on 22q11.2 (noncoding) and 1q24 (introns). A total of 75 variant sites were found among 128 human sequences in the 22q11.2 region, yielding an estimate of 0.088% for nucleotide diversity (π), and a total of 52 variant sites were found among 122 human sequences in the 1q24 region with an estimated π value of 0.057%. The data from these two regions and a 10 kb noncoding region on Xq13.3 all show a strong excess of low-frequency variants in comparison to that expected from an equilibrium population, indicating a relatively recent population expansion. The effective population sizes estimated from the three regions were 11,000, 12,700, and 8,600, respectively, which are close to the commonly used value of 10,000. In each of the two autosomal regions, the age of the most recent common ancestor (MRCA) was estimated to be older than 1 million years among all the sequences and ∼600,000 years among non-African sequences, providing first evidence from autosomal noncoding or intronic regions for a genetic history of humans much more ancient than the emergence of modern humans. The ancient genetic history of humans indicates no severe bottleneck during the evolution of humans in the last half million years; otherwise, much of the ancient genetic history would have been lost during a severe bottleneck. This study strongly suggests that both the “out of Africa” and the multiregional models are too simple for explaining the evolution of modern humans. A compilation of genome-wide data revealed that nucleotide diversity is highest in autosomal regions, intermediate in X-linked regions, and lowest in Y-linked regions. The data suggest the existence of background selection or selective sweep on Y-linked loci. In general, the nucleotide diversity in humans is low compared to that in chimpanzee and Drosophila populations. ^

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

An Sp1/Sp3 site polymorphism associated with hypermethylation of the candidate tumor suppressor gene RIL in cancer

Gene silencing due to promoter methylation is an alternative to mutations and deletions, which inactivate tumor suppressor genes (TSG) in cancer. We identified RIL by Methylated CpG Island Amplification technique as a novel aberrantly methylated gene. RIL is expressed in normal tissues and maps to the 5q31 region, frequently deleted in leukemias. We found methylation of RIL in 55/80 (69%) cancer cell lines, with highest methylation in leukemia and colon. We also observed methylation in 46/80 (58%) primary tumors, whereas normal tissues showed substantially lower degrees of methylation. RIL expression was lost in 13/16 cancer cell lines and was restored by demethylating agent. Screening of 38 cell lines and 13 primary cancers by SSCP revealed no mutations in RIL, suggesting that methylation and LOH are the primary inactivation mechanisms. Stable transfection of RIL into colorectal cancer cells resulted in reduction in cell growth, clonogenicity, and increased apoptosis upon UVC treatment, suggesting that RIL is a good candidate TSG. ^ In searching for a cause of RIL hypermethylation, we identified a 12-bp polymorphic sequence around the transcription start site of the gene that creates a long allele containing 3CTC repeat. Evolutionary studies suggested that the long allele appeared late in evolution due to insertion. Using bisulfite sequencing, in cancers heterozygous for RIL, we found that the short allele is 4.4-fold more methylated than the long allele (P = 0.003). EMSA results suggested binding of factor(s) to the inserted region of the long allele, but not to the short. EMSA mutagenesis and competition studies, as well as supershifts using nuclear extracts or recombinant Sp1 strongly indicated that those DNA binding proteins are Sp1 and Sp3. Transient transfections of RIL allele-specific expression constructs showed less than 2-fold differences in luciferase activity, suggesting no major effects of the additional Sp1 site on transcription. However, stable transfection resulted in 3-fold lower levels of transcription from the short allele 60 days post-transfection, consistent with the concept that the polymorphic Sp1 site protects against time-dependent silencing. Thus, an insertional polymorphism in the RIL promoter creates an additional Sp1/Sp3 site, which appears to protect it from silencing and methylation in cancer. ^

G870A polymorphism of cyclin D1 gene influences cancer risk - From epidemiological study to mechanism analysis

Alternate splicing of the cyclin D1 gene gives rise to transcript a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Through testing transcript a and transcript b in a series of human samples, we found that cyclin D1 transcript b is ubiquitously expressed as transcript a but in the lower abundance compared to transcript a. Epidemiological studies have reported that the cyclin D1 gene (CCND1) G870A polymorphism influences the risk for a variety of cancer. In this investigation, we examined the cyclin D1b levels in tumor samples with different genotypes and found that higher levels of cyclin D1b are expressed from the A allele than the G allele. Cyclin D1 is known as a cell cycle regulator facilitating the progression of the cell cycle from G1 to S phase in response to the mitogenic signals. It also interacts with several transcription factors and transcriptional coregulators to modulate their activities. It has been reported that cyclin D1a can substitute for estrogen to activate estrogen receptor α (ERα) mediated transcription and can induce the proliferation of estrogen responsive tissues. However the biological role of cyclin D1b in ERα transcriptional regulation has not been previously explored. In this study, we determined that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription. Cell proliferation assays provided the evidence that cyclin D1b negatively regulates estrogen responsive breast cancer cell growth. Taken together, our findings show that the CCND1 G870A polymorphism is correlated with increased levels of cyclin D1b and that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription providing evidence for the mechanism by which the CCND1 G870A polymorphism may be protective in certain types of breast cancer. ^

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.