Candidate gene identification following linkage: Search for hypertension susceptibility genes

Following up genetic linkage studies to identify the underlying susceptibility gene(s) for complex disease traits is an arduous yet biologically and clinically important task. Complex traits, such as hypertension, are considered polygenic with many genes influencing risk, each with small effects. Chromosome 2 has been consistently identified as a genomic region with genetic linkage evidence suggesting that one or more loci contribute to blood pressure levels and hypertension status. Using combined positional candidate gene methods, the Family Blood Pressure Program has concentrated efforts in investigating this region of chromosome 2 in an effort to identify underlying candidate hypertension susceptibility gene(s). Initial informatics efforts identified the boundaries of the region and the known genes within it. A total of 82 polymorphic sites in eight positional candidate genes were genotyped in a large hypothesis-generating sample consisting of 1640 African Americans, 1339 whites, and 1616 Mexican Americans. To adjust for multiple comparisons, resampling-based false discovery adjustment was applied, extending traditional resampling methods to sibship samples. Following this adjustment for multiple comparisons, SLC4A5, a sodium bicarbonate transporter, was identified as a primary candidate gene for hypertension. Polymorphisms in SLC4A5 were subsequently genotyped and analyzed for validation in two populations of African Americans (N = 461; N = 778) and two of whites (N = 550; N = 967). Again, SNPs within SLC4A5 were significantly associated with blood pressure levels and hypertension status. While not identifying a single causal DNA sequence variation that is significantly associated with blood pressure levels and hypertension status across all samples, the results further implicate SLC4A5 as a candidate hypertension susceptibility gene, validating previous evidence for one or more genes on chromosome 2 that influence hypertension related phenotypes in the population-at-large. The methodology and results reported provide a case study of one approach for following up the results of genetic linkage analyses to identify genes influencing complex traits. ^

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 epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

Repression of {\it neu} oncogene by E1A: Mechanisms and biological functions

The potential effects of the E1A gene products on the promoter activities of neu were investigated. Transcription of the neu oncogene was found to be strongly repressed by the E1A gene products and this requires that conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 140 base pair (bp) DNA fragment in the upstream region of the neu promoter. To further study if this transcriptional repression of neu by E1A can inhibit the transforming ability of the neu transformed cells, the E1A gene was introduced into the neu oncogene transformed B104-1-1 cells and developed B-E1A cell lines that express E1A proteins. These B-E1A stable transfectants have reduced transforming activity compared to the parental B104-1-1 cell line and we conclude that E1A can suppress the transformed phenotypes of the neu oncogene transformed cells via transcriptional repression of neu.^ To study the effects of E1A on metastasis, we first introduced the mutation-activated rat neu oncogene into 3T3 cells and showed that both the neu oncogene transformed NIH3T3 cells and Swiss Webster 3T3 cells exhibited metastatic properties in vitro and in vivo, while their parental 3T3 cells did not. Additionally, the neu-specific monoclonal antibody 7.16.4, which can down regulate neu-encoded p185 protein, effectively reduced the metastatic properties induced by neu. To investigate if E1A can reduce the metastatic potential of neu-transformed cells, we also compared the metastatic properties of B-E1A cell lines and B104-1-1 cell. B-E1A cell lines showed reduced invasiveness and lung colonization than the parental neu transformed B104-1-1 cells. We conclude that E1A gene products also have inhibitory effect on the metastatic phenotypes of the neu oncogene transformed cells.^ The product of human retinoblastoma (RB) susceptibility gene has been shown to complex with E1A gene products and is speculated to regulate gene expression. We therefore investigated in E1A-RB interaction might be involved in the regulation of neu oncogene expression. We found that the RB gene product can decrease the E1A-mediated repression of neu oncogene and the E1A binding region of the RB protein is required for the derepression function. ^

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

Polymorphisms of the DNA repair gene MGMT and risk and progression of head and neck cancer.

Methylating agents are involved in carcinogenesis, and the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) removes methyl group from O(6)-methylguanine. Genetic variation in DNA repair genes has been shown to contribute to susceptibility to squamous cell carcinoma of the head and neck (SCCHN). We hypothesize that MGMT polymorphisms are associated with risk of SCCHN. In a hospital-based case-control study of 721 patients with SCCHN and 1234 cancer-free controls frequency-matched by age, sex and ethnicity, we genotyped four MGMT polymorphisms, two in exon 3, 16195C>T and 16286C>T and two in the promoter region, 45996G>T and 46346C>A. We found that none of these polymorphisms alone had a significant effect on risk of SCCHN. However, when these four polymorphisms were evaluated together by the number of putative risk genotypes (i.e. 16195CC, 16286CC, 45996GT+TT, and 46346CA+AA), a statistically significantly increased risk of SCCHN was associated with the combined genotypes with three to four risk genotypes, compared with those with zero to two risk genotypes (adjusted odds ratio (OR)=1.27; 95% confidence interval (CI)=1.05-1.53). This increased risk was also more pronounced among young subjects (OR=1.81; 95% CI=1.11-2.96), men (OR=1.24; 95% CI=1.00-1.55), ever smokers (OR=1.25; 95%=1.01-1.56), ever drinkers (OR=1.29; 95% CI=1.04-1.60), patients with oropharyngeal cancer (OR=1.45; 95% CI=1.12-1.87), and oropharyngeal cancer with regional lymph node metastasis (OR=1.52; 95% CI=1.16-1.89). In conclusion, our results suggest that any one of MGMT variants may not have a substantial effect on SCCHN risk, but a joint effect of several MGMT variants may contribute to risk and progression of SCCHN, particularly for oropharyngeal cancer, in non-Hispanic whites.

Genetic variation in genes for the xenobiotic-metabolizing enzymes CYP1A1, EPHX1, GSTM1, GSTT1, and GSTP1 and susceptibility to colorectal cancer in Lynch syndrome.

Individuals with Lynch syndrome are predisposed to cancer due to an inherited DNA mismatch repair gene mutation. However, there is significant variability observed in disease expression likely due to the influence of other environmental, lifestyle, or genetic factors. Polymorphisms in genes encoding xenobiotic-metabolizing enzymes may modify cancer risk by influencing the metabolism and clearance of potential carcinogens from the body. In this retrospective analysis, we examined key candidate gene polymorphisms in CYP1A1, EPHX1, GSTT1, GSTM1, and GSTP1 as modifiers of age at onset of colorectal cancer among 257 individuals with Lynch syndrome. We found that subjects heterozygous for CYP1A1 I462V (c.1384A>G) developed colorectal cancer 4 years earlier than those with the homozygous wild-type genotype (median ages, 39 and 43 years, respectively; log-rank test P = 0.018). Furthermore, being heterozygous for the CYP1A1 polymorphisms, I462V and Msp1 (g.6235T>C), was associated with an increased risk for developing colorectal cancer [adjusted hazard ratio for AG relative to AA, 1.78; 95% confidence interval, 1.16-2.74; P = 0.008; hazard ratio for TC relative to TT, 1.53; 95% confidence interval, 1.06-2.22; P = 0.02]. Because homozygous variants for both CYP1A1 polymorphisms were rare, risk estimates were imprecise. None of the other gene polymorphisms examined were associated with an earlier onset age for colorectal cancer. Our results suggest that the I462V and Msp1 polymorphisms in CYP1A1 may be an additional susceptibility factor for disease expression in Lynch syndrome because they modify the age of colorectal cancer onset by up to 4 years.

Mutation analysis of the PVRL1 gene in caucasians with nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, MIM 119530) is perhaps the most common major birth defect. Homozygous PVRL1 loss-of-function mutations result in an autosomal recessive CL/P syndrome, CLPED1, and a PVRL1 nonsense mutation is associated with sporadic nsCL/P in Northern Venezuela. To address the more general role of PVRL1 variation in risk of nsCL/P, we carried out mutation analysis of PVRL1 in North American and Australian nsCL/P cases and population-matched controls. We identified a total of 15 variants, 5 of which were seen in both populations and 1 of which, an in-frame insertion at Glu442, was more frequent in patients than in controls in both populations, though the difference was not statistically significant. Another variant, which is specific to the PVRL1 beta (HIgR) isoform, S447L, was marginally associated with nsCL/P in North American Caucasian patients, but not in Australian patients, and overall variants that affect the beta-isoform were significantly more frequent among North American patients. One Australian patient had a splice junction mutation of PVRL1. Our results suggest that PVRL1 may play a minor role in susceptibility to the occurrence of nsCL/P in some Caucasian populations, and that variation involving the beta (HIgR) isoform might have particular importance for risk of orofacial clefts. Nevertheless, these results underscore the need for studies that involve very large numbers when assessing the possible role of rare variants in risk of complex traits such as nsCL/P.

Increased myocardial susceptibility to repetitive ischemia with high-fat diet-induced obesity.

Obesity and diabetes are frequently associated with cardiovascular disease. When a normal heart is subjected to brief/sublethal repetitive ischemia and reperfusion (I/R), adaptive responses are activated to preserve cardiac structure and function. These responses include but are not limited to alterations in cardiac metabolism, reduced calcium responsiveness, and induction of antioxidant enzymes. In a model of ischemic cardiomyopathy inducible by brief repetitive I/R, we hypothesized that dysregulation of these adaptive responses in diet-induced obese (DIO) mice would contribute to enhanced myocardial injury. DIO C57BL/6J mice were subjected to 15 min of daily repetitive I/R while under short-acting anesthesia, a protocol that results in the development of fibrotic cardiomyopathy. Cardiac lipids and candidate gene expression were analyzed at 3 days, and histology at 5 days of repetitive I/R. Total free fatty acids (FFAs) in the cardiac extracts of DIO mice were significantly elevated, reflecting primarily the dietary fatty acid (FA) composition. Compared with lean controls, cardiac FA oxidation (FAO) capacity of DIO mice was significantly higher, concurrent with increased expression of FA metabolism gene transcripts. Following 15 min of daily repetitive I/R for 3 or 5 days, DIO mice exhibited increased susceptibility to I/R and, in contrast to lean mice, developed microinfarction, which was associated with an exaggerated inflammatory response. Repetitive I/R in DIO mice was associated with more profound significant downregulation of FA metabolism gene transcripts and elevated FFAs and triglycerides. Maladaptive metabolic changes of FA metabolism contribute to enhanced myocardial injury in diet-induced obesity.

Differential gene expression in multistep tumorigenesis using an {\it in vitro\/} human cell model

Using a human terato-carcinoma cell line, PA-1, the functional role of the oncogenes and tumor suppressor gene involved in the multistep process of carcinogenesis have been analyzed. The expression of AP-2 was strongly correlated with the susceptibility to ras transformation. The differential responsiveness to growth factors between stage 1 ras resistant cells and stage 2 ras susceptible cells was observed, indicating that the ability of stage 2 cells to respond to the mutated ras oncogenes in transformation correlated with the ability to be stimulated by certain growth factors. Using differential screening of cDNA libraries, a number of differentially expressed cDNA clones was isolated. One of those, clone 12, is overexpressed in ras transformed stage 3 cells. The amino acid sequence of clone 12 is almost identical to a mouse LLrep3 gene that was growth-regulated, and 78% similar to a yeast ribosomal protein S4. These results suggest that the S4 gene may be involved in regulation of growth. Clone 9 is expressed in stage 1 ras resistant cells (3.5-kb and 3.0-kb transcripts) but the expression of this clone in stage 2 ras susceptible cells and stage 3 ras-transformed cells is greatly diminished. The expression of this cDNA clone was increased to at least five fold in ras resistant cells and nontumorigenic hybrids treated with retinoic acid but not increased in retinoic acid treated ras susceptible cells, ras transformed cells and the tumorigenic segregants. Partial sequence of this clone showed no homology to the sequences in Genbank. These findings suggest that clone 9 could be a suppressor gene or the genes that are involved in the biochemical pathway of tumor suppression or neurogenic differentiation. The apparent pleiotropic effect of the loss of this suppressor gene function support Harris' proposal that tumor suppressor genes regulate differentiation. The tumor suppressor gene may act as negative regulator of tumor growth by controlling gene expression in differentiation. ^

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. ^

Modifier genes and susceptibility to colorectal cancer in individuals with Lynch syndrome

Lynch syndrome, is caused by inherited germ-line mutations in the DNA mismatch repair genes resulting in cancers at an early age, predominantly colorectal (CRC) and endometrial cancers. Though the median age at onset for CRC is about 45 years, disease penetrance varies suggesting that cancer susceptibility may be modified by environmental or other low-penetrance genes. Genetic variation due to polymorphisms in genes encoding metabolic enzymes can influence carcinogenesis by alterations in the expression and activity level of the enzymes. Variation in MTHFR, an important folate metabolizing enzyme can affect DNA methylation and DNA synthesis and variation in xenobiotic-metabolizing enzymes can affect the metabolism and clearance of carcinogens, thus modifying cancer risk. ^ This study examined a retrospective cohort of 257 individuals with Lynch syndrome, for polymorphisms in genes encoding xenobiotic-metabolizing enzymes-- CYP1A1 (I462V and MspI), EPHX1 (H139R and Y113H), GSTP1 (I105V and A114V), GSTM1 and GSTT1 (deletions) and folate metabolizing enzyme--MTHFR (C677T and A1298C). In addition, a series of 786 cases of sporadic CRC were genotyped for CYP1A1 I462V and EPHX1 Y113H to assess gene-gene interaction and gene-environment interaction with smoking in a case-only analysis. ^ Prominent findings of this study were that the presence of an MTHFR C677T variant allele was associated with a 4 year later age at onset for CRC on average and a reduced age-associated risk for developing CRC (Hazard ratio: 0.55; 95% confidence interval: 0.36–0.85) compared to the absence of any variant allele in individuals with Lynch syndrome. Similarly, Lynch syndrome individuals heterozygous for CYP1A1 I462V A>G polymorphism developed CRC an average of 4 years earlier and were at a 78% increased age-associated risk (Hazard ratio for AG relative to AA: 1.78; 95% confidence interval: 1.16-2.74) than those with the homozygous wild-type genotype. Therefore these two polymorphisms may be additional susceptibility factors for CRC in Lynch syndrome. In the case-only analysis, evidence of gene-gene interaction was seen between CYP1A1 I462V and EPHX1 Y113H and between EPHX1 Y113H and smoking suggesting that genetic and environmental factors may interact to increase sporadic CRC risk. Implications of these findings are the ability to identify subsets of high-risk individuals for targeted prevention and intervention. ^

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^

Genome-wide gene-gene interaction analysis for cardiovascular disease

Numerous studies have been carried out to try to better understand the genetic predisposition for cardiovascular disease. Although it is widely believed that multifactorial diseases such as cardiovascular disease is the result from effects of many genes which working alone or interact with other genes, most genetic studies have been focused on identifying of cardiovascular disease susceptibility genes and usually ignore the effects of gene-gene interactions in the analysis. The current study applies a novel linkage disequilibrium based statistic for testing interactions between two linked loci using data from a genome-wide study of cardiovascular disease. A total of 53,394 single nucleotide polymorphisms (SNPs) are tested for pair-wise interactions, and 8,644 interactions are found to be significant with p-values less than 3.5×10-11. Results indicate that known cardiovascular disease susceptibility genes tend not to have many significantly interactions. One SNP in the CACNG1 (calcium channel, voltage-dependent, gamma subunit 1) gene and one SNP in the IL3RA (interleukin 3 receptor, alpha) gene are found to have the most significant pair-wise interactions. Findings from the current study should be replicated in other independent cohort to eliminate potential false positive results.^

Susceptibility to travelers' diarrhea and histo-blood group antigens

Histo-blood group antigens (HBGAs) have been associated with susceptibility to enteric pathogens including noroviruses (NoVs), enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, and Vibrio cholerae. We performed a retrospective cohort study to evaluate the relationship between traveler HBGA phenotypes and susceptibility to travelers' diarrhea (TD) and post-infectious complications. 364 travelers to Guadalajara, Mexico were followed prospectively from June 1 - September 30, 2007 and from June 7–July 28, 2008 for the development of TD and at 6 months for post-infectious irritable bowel syndrome (PIIBS). Noroviruses were detected from illness stool specimens with RT-PCR. Diarrheal stool samples were also assayed for enterotoxigenic and enteroaggregative E. coli, Salmonella species, Shigella species, Vibrio species, Campylobacter jejuni, Yersinia enterocolitica, Aeromonas species, and Plesiomonas species. Diarrheal stools were evaluated for inflammation with fecal leukocytes, mucus, and occult blood. Phenotyping for ABO and Lewis antigens with an ELISA assay and FUT2 gene PCR genotyping for secretor status were performed with saliva. 171 of 364 (47%) subjects developed TD. HBGA typing for the travelers revealed O (62.9%), A (34.6%), B (1.6%), and AB (0.8%) phenotypes. There were 7% nonsecretors and 93% secretors among the travelers. AB phenotypes were more commonly associated with Cryptosporidium species (P=0.04) and ETEC ( P=0.08) as causes of TD. AB and B phenotype individuals were more likely to experience inflammatory diarrhea, particularly mucoid diarrhea ( P=0.02). However, there were relatively few individuals with AB and B phenotypes. GI and GII NoV and Cryptosporidium species infections and PI-IBS were identified only in secretors, but these differences were not statistically significant, (P=1.00), (P=1.00), and (P=0.60), respectively. Additional studies are needed to evaluate whether AB phenotype individuals may be more susceptible to developing TD associated with Cryptosporidium species or ETEC, and whether AB and B phenotype individuals may be more likely to develop inflammatory TD. Further studies are needed to investigate whether nonsecretor travelers may be at less risk for developing infections with NoVs and Cryptosporidium species and PI-IBS.^