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ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ∼385 nm to an estimated value of ∼42 μM. Directionality of the Ric-8BFL-catalyzed Gα(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.

A combined genome-wide association and linkage study was used to identify loci causing variation in cystic fibrosis lung disease severity. We identified a significant association (P = 3.34 × 10(-8)) near EHF and APIP (chr11p13) in p.Phe508del homozygotes (n = 1,978). The association replicated in p.Phe508del homozygotes (P = 0.006) from a separate family based study (n = 557), with P = 1.49 × 10(-9) for the three-study joint meta-analysis. Linkage analysis of 486 sibling pairs from the family based study identified a significant quantitative trait locus on chromosome 20q13.2 (log(10) odds = 5.03). Our findings provide insight into the causes of variation in lung disease severity in cystic fibrosis and suggest new therapeutic targets for this life-limiting disorder.

Motivation: DNA copy number gains and losses are commonly found in tumor tissue, and some of these aberrations play a role in tumor genesis and development. Although high resolution DNA copy number data can be obtained using array-based techniques, no single method is widely used to distinguish between recurrent and sporadic copy number aberrations., Results: Here we introduce Discovering Copy Number Aberrations Manifested In Cancer (DiNAMIC), a novel method for assessing the statistical significance of recurrent copy number aberrations. In contrast to competing procedures, the testing procedure underlying DiNAMIC is carefully motivated, and employs a novel cyclic permutation scheme. Extensive simulation studies show that DiNAMIC controls false positive discoveries in a variety of realistic scenarios. We use DiNAMIC to analyze two publicly available tumor datasets, and our results show that DiNAMIC detects multiple loci that have biological relevance., Availability: Source code implemented in R, as well as text files containing examples and sample datasets are available at http://www.bios.unc.edu/research/genomic_software/DiNAMIC.

Immortalized human lymphoblastoid cell lines have been used to demonstrate that it is possible to use an in vitro model system to identify genetic factors that affect responses to xenobiotics. To extend the application of such studies to investigative toxicology by assessing interindividual and population-wide variability and heritability of chemical-induced toxicity phenotypes, we have used cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH) trios assembled by the HapMap Consortium. Our goal is to aid in the development of predictive in vitro genetics-anchored models of chemical-induced toxicity. Cell lines from the CEPH trios were exposed to three concentrations of 14 environmental chemicals. We assessed ATP production and caspase-3/7 activity 24 h after treatment. Replicate analyses were used to evaluate experimental variability and classify responses. We show that variability of response across the cell lines exists for some, but not all, chemicals, with perfluorooctanoic acid (PFOA) and phenobarbital eliciting the greatest degree of interindividual variability. Although the data for the chemicals used here do not show evidence for broad-sense heritability of toxicity response phenotypes, substantial cell line variation was found, and candidate genetic factors contributing to the variability in response to PFOA were investigated using genome-wide association analysis. The approach of screening chemicals for toxicity in a genetically defined yet diverse in vitro human cell-based system is potentially useful for identification of chemicals that may pose a highest risk, the extent of within-species variability in the population, and genetic loci of interest that potentially contribute to chemical susceptibility.

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short).

Variability in cystic fibrosis (CF) lung disease is partially due to non-CFTR genetic modifiers. Mucin genes are very polymorphic, and mucins play a key role in the pathogenesis of CF lung disease; therefore, mucin genes are strong candidates as genetic modifiers. DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat (VNTR) length polymorphisms for MUC1, MUC2, MUC5AC, and MUC7. VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium (LD) with flanking single nucleotide polymorphisms (SNPs). No strong associations were found for MUC1, MUC2, or MUC7. A significant association was found between the overall distribution of MUC5AC VNTR length and CF lung disease severity (p = 0.025; n = 468 patients); plus, there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease (p = 6.2×10(-4) after Bonferroni correction). There was strong LD between MUC5AC VNTR length modes and flanking SNPs. The severity-associated 6.4 kb VNTR allele of MUC5AC was confirmed to be genetically distinct from the 6.3 kb allele, as it showed significantly stronger association with nearby SNPs. These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients. Our data also show a novel link between the MUC5AC 6.4 kb VNTR allele and severity of CF lung disease. The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains, or is linked to, important functional genetic variation.

In a cross-sectional study, we investigated the relationship of Mini-Nutrition Assessment (MNA) results with chewing ability tests and oral examinations (number of teeth present and functional tooth units (FTUs)). The participants were 612 older people (Mean [SD] age: 68.8 [5.9]). According to the MNA score, 25.1% of participants were categorized as having normal nutrition, 67.2% were categorized as at risk of malnourishment, and 7.7% were categorized as having malnutrition. The mean numbers of teeth present and FTUs were [15.5] and [8.9], respectively. The ANCOVA analyses adjusted for age and gender showed that participants with malnutrition had lower numbers of teeth present (8.8), FTUs (8.4), and chewing ability (6.8) than those with normal nutrition (13.3, 10.4 and 7.8) (p < 0.05). Nutritional status was associated with mean numbers of teeth present, FTUs, and chewing ability. Therefore, it was concluded that retention of natural teeth with appropriate numbers of FTUs by replacing missing teeth with dentures and improving chewing ability will help the reduce risk of malnutrition in older adults.

Purpose: Periodontal diseases have a multifactorial etiology and their risk factors are influenced by modifiable and nonmodifiable factors. The information on periodontal disease and associated risk factors in the Vietnamese population is sparse. Vietnamese dental patients are specific groups with high dental needs and often have greater overall oral health problems. Thus, the purpose of this study was to examine the relationships of periodontal disease to sociodemographic, health-behavioural, and biological characteristics in Vietnamese dental patients., Materials and Methods: A sample of 243 subjects was selected from the National Hospital of Odonto-Stomatology in Hochiminh City. The sociodemographic and health-behavioural characteristics were investigated by a questionnaire. Oral examination including dental and periodontal conditions was conducted. Bacterial load in tongue coating and hemoglobin in saliva were assessed using the BANA test and Perioscreen test., Results: The Perioscreen test showed moderate sensitivity (0.752) and specificity (0.746) to periodontal disease. The binary logistic regression analyses indicated that older subjects (OR = 2.5), or those who did not frequently visit a dentist (OR = 4.1), brushed their teeth only once a day (OR = 2.5), did not use dental floss (OR = 2.9), were past smokers (OR = 3.1), current smokers (OR = 4.1), or had positive BANA test results (OR = 12.0) were more likely to have periodontal disease., Conclusion: The results revealed the periodontal disease was related to age, dental visit behaviour, smoking behaviour, frequency of tooth brushing, use of dental floss, bacterial load in the tongue coating, and hemoglobin in saliva.

Background: Analysis of microarray experiments often involves testing for the overrepresentation of pre-defined sets of genes among lists of genes deemed individually significant. Most popular gene set testing methods assume the independence of genes within each set, an assumption that is seriously violated, as extensive correlation between genes is a well-documented phenomenon., Results: We conducted a meta-analysis of over 200 datasets from the Gene Expression Omnibus in order to demonstrate the practical impact of strong gene correlation patterns that are highly consistent across experiments. We show that a common independence assumption-based gene set testing procedure produces very high false positive rates when applied to data sets for which treatment groups have been randomized, and that gene sets with high internal correlation are more likely to be declared significant. A reanalysis of the same datasets using an array resampling approach properly controls false positive rates, leading to more parsimonious and high-confidence gene set findings, which should facilitate pathway-based interpretation of the microarray data., Conclusions: These findings call into question many of the gene set testing results in the literature and argue strongly for the adoption of resampling based gene set testing criteria in the peer reviewed biomedical literature.

Background: Oral malodor (halitosis or bad breath) might be an important motivation tool for improving oral health in adolescents. There are few studies that report the epidemiology of oral malodor in high school students and the relationships with lifestyle and oral health status. This research was conducted to obtain underlying data for introducing an oral health education program which targeted prevention of oral malodor as a motivation tool for changing oral health behavior in high school students., Methods: A questionnaire, school oral examination, and oral malodor measurement were conducted on senior high school students in a Tokyo metropolitan school in 2007. A total of 474 students (male: 219, female: 255) were used for the analysis., Results: Over 42% of subjects reported that they had experienced anxiety, or were conscious of oral malodor, on at least 1 occasion. The students who had detectable oral malodor comprised 39.6% of subjects. The binary logistic regression analyses showed that whether or not subjects ate breakfast before the oral examination (p < .05), the presence of plaque (p < .01), and presence of a substantive tongue coating (p < .01) were related to the presence of detectable oral malodor., Conclusions: Cleaning the oral cavity and eating breakfast are important to prevent oral malodor in high school students. This study indicated that school health education incorporating prevention of oral malodor as a motivation tool for oral health promotion could be a valuable procedure to include in high school dental health education programs.

Achieving the ability to identify individuals who are susceptible to drug-induced liver injury (DILI) would represent a major advance in personalized medicine. Clayton et al. demonstrated that the pattern of endogenous metabolites in urine could predict susceptibility to acetaminophen-induced liver injury in rats. We designed a clinical study to test this approach in healthy adults who received 4 g of acetaminophen per day for 7 days. Urine metabolite profiles obtained before the start of treatment were not sufficient to distinguish which of the subjects would develop mild liver injury, as indicated by a rise in alanine aminotransferase (ALT) to a level more than twice the baseline value (responders). However, profiles obtained shortly after the start of treatment, but prior to ALT elevation, could distinguish responders from nonresponders. Statistical analyses revealed that predictive metabolites included those derived from the toxic metabolite N-acetyl paraquinone imine (NAPQI), but that the inclusion of endogenous metabolites was required for significant prediction. This "early-intervention pharmaco-metabonomics" approach should now be tested in clinical trials of other potentially hepatotoxic drugs.

Background and Objective: Smoking has been associated with the number of natural teeth a person has and with the likelihood of periodontitis. The purpose of this study was to determine the relationships between the number of teeth present and periodontal diseases with smoking habits in a cohort of Japanese men., Material and Methods: The study group comprised 1088 men, 40-75 years of age. Oral examinations were conducted in dental clinics. Information on smoking status and on oral health behavior was collected from self-administered questionnaires. The relationship between oral health status and smoking status was estimated using adjusted odds ratios., Results: Compared with those whom had never smoked, the odds ratios of having more than eight missing teeth and having periodontitis, among current smokers, were 1.67 and 1.74, respectively. In those who had stopped smoking for 11 years or longer, there was no increase in the odds ratio of having more than eight missing teeth and periodontitis, compared with those whom had never smoked., Conclusion: Smoking has a positive association with missing teeth and periodontitis. However, smoking cessation is beneficial for oral health. The odds of having more than eight missing teeth, or of having periodontitis, in those who had never smoked was similar to that of individuals who reported that they had stopped smoking for 11 years or more.

Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.

Objectives: The purpose of this research was to analyse the relationship between oral health status, oral motor function, daily life situations and self-assessed oral health., Materials and Methods: A cross-sectional survey was conducted on 190 independent elderly volunteers aged 62-99 years from senior citizen centres in Tokyo, Japan. A questionnaire survey and oral examination were conducted on each participant. For the descriptive analyses, Independent t-test and the chi-squared test were used. Following that, using four latent variables, (oral health, oral function, daily life situations and chronic health condition), a structural equation modelling analysis (SEMA) was undertaken., Results: In the descriptive analyses, there were no significant differences between self-assessed oral health and oral health status. However, there were significant differences between self-assessed oral health and oral function and daily life situations. Findings from SEMA revealed that daily life situations and oral function have independent effects on self-assessed oral health and that the relationship between self-assessed oral health and oral health status was weak., Conclusions: Many factors affect self-assessed oral health status. Dental clinicians and researchers should attempt to understand these factors and incorporate them into effective personal and population-based oral health education and oral health promotion programmes.

Background: Although community water fluoridation has been one of the cornerstone strategies for the prevention and control of dental caries, questions are still raised regarding its cost-effectiveness. This study assessed the impact of changing dental needs on the cost savings from community water fluoridation in Australia., Methods: Net costs were estimated as Costs((programme)) minus Costs((averted caries).) Averted costs were estimated as the product of caries increment in non-fluoridated community, effectiveness of fluoridation and the cost of a carious surface. Modelling considered four age-cohorts: 6-20, 21-45, 46-65 and 66+ years and three time points 1970s, 1980s, and 1990s. Cost of a carious surface was estimated by conventional and complex methods. Real discount rates (4, 7 (base) and 10%) were utilized., Results: With base-case assumptions, the average annual cost savings/person, using Australian dollars at the 2005 level, ranged from $56.41 (1970s) to $17.75 (1990s) (conventional method) and from $249.45 (1970s) to $69.86 (1990s) (complex method). Under worst-case assumptions fluoridation remained cost-effective with cost savings ranging from $24.15 (1970s) to $3.87 (1990s) (conventional method) and $107.85 (1970s) and $24.53 (1990s) (complex method). For 66+ years cohort (1990s) fluoridation did not show a cost saving, but costs/person were marginal., Conclusions: Community water fluoridation remains a cost-effective preventive measure in Australia.

Background: Previous research has shown the oxidizing properties and microbiological efficacies of chlorine dioxide (ClO2). Its clinical efficacies on oral malodor have been evaluated and reported only in short duration trials, moreover, no clinical studies have investigated its microbiological efficacies on periodontal and malodorous bacteria. Thus, the aim of this study was to assess the inhibitory effects of a mouthwash containing ClO2 used for 7 days on morning oral malodor and on salivary periodontal and malodorous bacteria., Methods/design: A randomized, double blind, crossover, placebo-controlled trial was conducted among 15 healthy male volunteers, who were divided into 2 groups. Subjects were instructed to rinse with the experimental mouthwash containing ClO2 or the placebo mouthwash, without ClO2, twice per day for 7 days. After a one week washout period, each group then used the opposite mouthwash for 7 days. At baseline and after 7 days, oral malodor was evaluated with Organoleptic measurement (OM), and analyzed the concentrations of hydrogen sulfide (H2S), methyl mercaptan (CH3SH) and dimethyl sulfide ((CH3)2S), the main VSCs of human oral malodor, were assessed by gas chromatography (GC). Clinical outcome variables included plaque and gingival indices, and tongue coating index. The samples of saliva were microbiologically investigated. Quantitative and qualitative analyses were performed using the polymerase chain reaction-Invader method., Results and Discussion: The baseline oral condition in healthy subjects in the 2 groups did not differ significantly. After rinsing with the mouthwash containing ClO2 for 7 days, morning bad breath decreased as measured by the OM and reduced the concentrations of H2S, CH3SH and (CH3)2S measured by GC, were found. Moreover ClO2 mouthwash used over a 7-day period appeared effective in reducing plaque, tongue coating accumulation and the counts of Fusobacterium nucleatum in saliva. Future research is needed to examine long-term effects, as well as effects on periodontal diseases and plaque accumulation in a well-defined sample of halitosis patients and broader population samples., Trial Registration: ClinicalTrials.gov NCT00748943.

Background/aims: Association studies using unrelated individuals have become the most popular design for mapping complex traits. One of the major challenges of association mapping is avoiding spurious association due to population stratification. Principal component analysis (PCA) on genome-wide marker genotypes is one of the most popular population stratification control methods. It implicitly assumes that the markers are in linkage equilibrium, a condition that is rarely satisfied and that we plan to relax., Methods: We carefully examined the impact of linkage disequilibrium (LD) on PCA, and proposed a simple modification of the standard PCA to automatically adjust for the correlations among markers., Results: We demonstrated that LD patterns in genome-wide association datasets can distort the techniques for stratification control, showing 'subpopulations' reflecting localized LD phenomena rather than plausible population structure. We showed that the proposed method effectively removes the artifactual effect of LD patterns, and successfully recovers underlying population structure that is not apparent from standard PCA., Conclusion: PCA is highly influenced by sets of SNPs with high LD, obscuring the true population substructure. Our shrinkage PCA applies to all available markers, regardless of the LD patterns. The proposed method is easier to implement than most existing LD adjusted PCA methods., (Copyright 2010 S. Karger AG, Basel.)

A number of settings arise in which it is of interest to predict Principal Component (PC) scores for new observations using data from an initial sample. In this paper, we demonstrate that naive approaches to PC score prediction can be substantially biased towards 0 in the analysis of large matrices. This phenomenon is largely related to known inconsistency results for sample eigenvalues and eigenvectors as both dimensions of the matrix increase. For the spiked eigenvalue model for random matrices, we expand the generality of these results, and propose bias-adjusted PC score prediction. In addition, we compute the asymptotic correlation coefficient between PC scores from sample and population eigenvectors. Simulation and real data examples from the genetics literature show the improved bias and numerical properties of our estimators.

Background: Improvements in high-throughput technology and its increasing use have led to the generation of many highly complex datasets that often address similar biological questions. Combining information from these studies can increase the reliability and generalizability of results and also yield new insights that guide future research., Results: This paper describes a novel algorithm called BLANKET for symmetric analysis of two experiments that assess informativeness of descriptors. The experiments are required to be related only in that their descriptor sets intersect substantially and their definitions of case and control are consistent. From resulting lists of n descriptors ranked by informativeness, BLANKET determines shortlists of descriptors from each experiment, generally of different lengths p and q. For any pair of shortlists, four numbers are evident: the number of descriptors appearing in both shortlists, in exactly one shortlist, or in neither shortlist. From the associated contingency table, BLANKET computes Right Fisher Exact Test (RFET) values used as scores over a plane of possible pairs of shortlist lengths 12. BLANKET then chooses a pair or pairs with RFET score less than a threshold; the threshold depends upon n and shortlist length limits and represents a quality of intersection achieved by less than 5% of random lists., Conclusions: Researchers seek within a universe of descriptors some minimal subset that collectively and efficiently predicts experimental outcomes. Ideally, any smaller subset should be insufficient for reliable prediction and any larger subset should have little additional accuracy. As a method, BLANKET is easy to conceptualize and presents only moderate computational complexity. Many existing databases could be mined using BLANKET to suggest optimal sets of predictive descriptors.

Context: A subset (approximately 3%-5%) of patients with cystic fibrosis (CF) develops severe liver disease with portal hypertension., Objective: To assess whether any of 9 polymorphisms in 5 candidate genes (alpha(1)-antitrypsin or alpha(1)-antiprotease [SERPINA1], angiotensin-converting enzyme [ACE], glutathione S-transferase [GSTP1], mannose-binding lectin 2 [MBL2], and transforming growth factor beta1 [TGFB1]) are associated with severe liver disease in patients with CF., Design, Setting, and Participants: Two-stage case-control study enrolling patients with CF and severe liver disease with portal hypertension (CFLD) from 63 CF centers in the United States as well as 32 in Canada and 18 outside of North America, with the University of North Carolina at Chapel Hill as the coordinating site. In the initial study, 124 patients with CFLD (enrolled January 1999-December 2004) and 843 control patients without CFLD were studied by genotyping 9 polymorphisms in 5 genes previously studied as modifiers of liver disease in CF. In the second stage, the SERPINA1 Z allele and TGFB1 codon 10 genotype were tested in an additional 136 patients with CFLD (enrolled January 2005-February 2007) and 1088 with no CFLD., Main Outcome Measures: Differences in distribution of genotypes in patients with CFLD vs patients without CFLD., Results: The initial study showed CFLD to be associated with the SERPINA1 Z allele (odds ratio [OR], 4.72; 95% confidence interval [CI], 2.31-9.61; P = 3.3 x 10(-6)) and with TGFB1 codon 10 CC genotype (OR, 1.53; 95% CI, 1.16-2.03; P = 2.8 x 10(-3)). In the replication study, CFLD was associated with the SERPINA1 Z allele (OR, 3.42; 95% CI, 1.54-7.59; P = 1.4 x 10(-3)) but not with TGFB1 codon 10. A combined analysis of the initial and replication studies by logistic regression showed CFLD to be associated with SERPINA1 Z allele (OR, 5.04; 95% CI, 2.88-8.83; P = 1.5 x 10(-8))., Conclusions: The SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk (OR, approximately 5) of developing severe liver disease with portal hypertension.

Aims/hypothesis: Insulin-requiring diabetes affects 25-50% of young adults with cystic fibrosis (CF). Although the cause of diabetes in CF is unknown, recent heritability studies in CF twins and siblings indicate that genetic modifiers play a substantial role. We sought to assess whether genes conferring risk for diabetes in the general population may play a risk modifying role in CF., Methods: We tested whether a family history of type 2 diabetes affected diabetes risk in CF patients in 539 families in the CF Twin and Sibling family-based study. A type 2 diabetes susceptibility gene (transcription factor 7-like 2, or TCF7L2) was evaluated for association with diabetes in CF using 998 patients from the family-based study and 802 unrelated CF patients in an independent case-control study., Results: Family history of type 2 diabetes increased the risk of diabetes in CF (OR 3.1; p = 0.0009). A variant in TCF7L2 associated with type 2 diabetes (the T allele at rs7903146) was associated with diabetes in CF in the family study (p = 0.004) and in the case-control study (p = 0.02; combined p = 0.0002). In the family-based study, variation in TCF7L2 increased the risk of diabetes about three-fold (HR 1.75 per allele, 95% CI 1.3-2.4; p = 0.0006), and decreased the mean age at diabetes diagnosis by 7 years. In CF patients not treated with systemic glucocorticoids, the effect of TCF7L2 was even greater (HR 2.9 per allele, 95% CI 1.7-4.9, p = 0.00011)., Conclusions/interpretation: A genetic variant conferring risk for type 2 diabetes in the general population is a modifier of risk for diabetes in CF.

We propose a statistical framework, named genoCN, to simultaneously dissect copy number states and genotypes using high-density SNP (single nucleotide polymorphism) arrays. There are at least two types of genomic DNA copy number differences: copy number variations (CNVs) and copy number aberrations (CNAs). While CNVs are naturally occurring and inheritable, CNAs are acquired somatic alterations most often observed in tumor tissues only. CNVs tend to be short and more sparsely located in the genome compared with CNAs. GenoCN consists of two components, genoCNV and genoCNA, designed for CNV and CNA studies, respectively. In contrast to most existing methods, genoCN is more flexible in that the model parameters are estimated from the data instead of being decided a priori. GenoCNA also incorporates two important strategies for CNA studies. First, the effects of tissue contamination are explicitly modeled. Second, if SNP arrays are performed for both tumor and normal tissues of one individual, the genotype calls from normal tissue are used to study CNAs in tumor tissue. We evaluated genoCN by applications to 162 HapMap individuals and a brain tumor (glioblastoma) dataset and showed that our method can successfully identify both types of copy number differences and produce high-quality genotype calls.

Background: Accurate prediction of in vivo toxicity from in vitro testing is a challenging problem. Large public-private consortia have been formed with the goal of improving chemical safety assessment by the means of high-throughput screening., Objective: A wealth of available biological data requires new computational approaches to link chemical structure, in vitro data, and potential adverse health effects., Methods and Results: A database containing experimental cytotoxicity values for in vitro half-maximal inhibitory concentration (IC(50)) and in vivo rodent median lethal dose (LD(50)) for more than 300 chemicals was compiled by Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergaenzungsmethoden zum Tierversuch (ZEBET; National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments). The application of conventional quantitative structure-activity relationship (QSAR) modeling approaches to predict mouse or rat acute LD(50) values from chemical descriptors of ZEBET compounds yielded no statistically significant models. The analysis of these data showed no significant correlation between IC(50) and LD(50). However, a linear IC(50) versus LD(50) correlation could be established for a fraction of compounds. To capitalize on this observation, we developed a novel two-step modeling approach as follows. First, all chemicals are partitioned into two groups based on the relationship between IC(50) and LD(50) values: One group comprises compounds with linear IC(50) versus LD(50) relationships, and another group comprises the remaining compounds. Second, we built conventional binary classification QSAR models to predict the group affiliation based on chemical descriptors only. Third, we developed k-nearest neighbor continuous QSAR models for each subclass to predict LD(50) values from chemical descriptors. All models were extensively validated using special protocols., Conclusions: The novelty of this modeling approach is that it uses the relationships between in vivo and in vitro data only to inform the initial construction of the hierarchical two-step QSAR models. Models resulting from this approach employ chemical descriptors only for external prediction of acute rodent toxicity.

Gene expression quantitative trait locus (eQTL) mapping has become a powerful tool in systems biology. While many authors have made important discoveries using this approach, one persistent challenge in eQTL studies is the selection of loci and genes that should receive further biological investigation. In this study we compared eQTL generated from gene expression profiling in the livers of two panels of mouse strains: 41 BXD recombinant inbred and 36 Mouse Diversity Panel (MDP) strains. Cis-eQTL, loci in which the transcript and its maximum QTL are colocated, have been shown to be more reproducible than trans-eQTL, which are not colocated with the transcript. We observed that between 9.9 and 12.1% of cis-eQTL and between 2.0 and 12.6% of trans-eQTL replicated between the two panels depending on the degree of statistical stringency. Notably, a significant eQTL hotspot on distal chromosome 12 observed in the BXD panel was reproduced in the MDP. Furthermore, the shorter linkage disequilibrium in the MDP strains allowed us to considerably narrow the locus and limit the number of candidate genes to a cluster of Serpin genes, which code for extracellular proteases. We conclude that this strategy has some utility in increasing confidence and resolution in eQTL mapping studies; however, due to the high false-positive rate in the MDP, eQTL mapping in inbred strains is best carried out in combination with an eQTL linkage study.

Background: Chronic fatiguing illness remains a poorly understood syndrome of unknown pathogenesis. We attempted to identify biomarkers for chronic fatiguing illness using microarrays to query the transcriptome in peripheral blood leukocytes., Methods: Cases were 44 individuals who were clinically evaluated and found to meet standard international criteria for chronic fatigue syndrome or idiopathic chronic fatigue, and controls were their monozygotic co-twins who were clinically evaluated and never had even one month of impairing fatigue. Biological sampling conditions were standardized and RNA stabilizing media were used. These methodological features provide rigorous control for bias resulting from case-control mismatched ancestry and experimental error. Individual gene expression profiles were assessed using Affymetrix Human Genome U133 Plus 2.0 arrays., Findings: There were no significant differences in gene expression for any transcript., Conclusions: Contrary to our expectations, we were unable to identify a biomarker for chronic fatiguing illness in the transcriptome of peripheral blood leukocytes suggesting that positive findings in prior studies may have resulted from experimental bias.

In quantitative-trait linkage studies using experimental crosses, the conventional normal location-shift model or other parameterizations may be unnecessarily restrictive. We generalize the mapping problem to a genuine nonparametric setup and provide a robust estimation procedure for the situation where the underlying phenotype distributions are completely unspecified. Classical Wilcoxon-Mann-Whitney statistics are employed for point and interval estimation of QTL positions and effects.

Motivation: Gene expression Quantitative Trait Locus (eQTL) mapping measures the association between transcript expression and genotype in order to find genomic locations likely to regulate transcript expression. The availability of both gene expression and high-density genotype data has improved our ability to perform eQTL mapping in inbred mouse and other homozygous populations. However, existing eQTL mapping software does not scale well when the number of transcripts and markers are on the order of 10(5) and 10(5)-10(6), respectively., Results: We propose a new method, FastMap, for fast and efficient eQTL mapping in homozygous inbred populations with binary allele calls. FastMap exploits the discrete nature and structure of the measured single nucleotide polymorphisms (SNPs). In particular, SNPs are organized into a Hamming distance-based tree that minimizes the number of arithmetic operations required to calculate the association of a SNP by making use of the association of its parent SNP in the tree. FastMap's tree can be used to perform both single marker mapping and haplotype association mapping over an m-SNP window. These performance enhancements also permit permutation-based significance testing., Availability: The FastMap program and source code are available at the website: http://cebc.unc.edu/fastmap86.html.

Summary: A large number of websites and applications perform significance testing for gene categories/pathways in microarray data. Many of these packages fail to account for expression correlation between transcripts, with a resultant inflation in Type I error. Array permutation and other resampling-based approaches have been proposed as solutions to this problem. SAFEGUI provides a user-friendly graphical interface for the assessment of categorical significance in microarray studies, while properly accounting for the effects of correlations among genes. SAFEGUI incorporates both permutation and more recently proposed bootstrap algorithms that are demonstrated to be more powerful in detecting differential expression across categories of genes., Availability: http://cebc.unc.edu/software/.

Recent evidence suggests that older adults may be a sensitive population with regard to environmental exposure to toxic compounds. One source of this sensitivity could be an enhanced variability in response. Studies on phenotypic differences have suggested that variation in response does increase with age. However, few reports address the question of variation in gene expression as an underlying cause for increased variability of phenotypic response in the aged. In this study, we utilized global analysis to compare variation in constitutive gene expression in the retinae of young (4 months), middle-aged (11 months), and aged (23 months) Fischer 344 rats. Three hundred and forty transcripts were identified in which variance in expression increased from 4 to 23 months of age, while only 12 transcripts were found for which it decreased. Functional roles for identified genes were clustered in basic biological categories including cell communication, function, metabolism, and response to stimuli. Our data suggest that population stochastically induced variability should be considered in assessing sensitivity due to old age.

Background: The pulmonary phenotype in cystic fibrosis (CF) is variable; thus, environmental and genetic factors likely contribute to clinical heterogeneity. We hypothesized that genetically determined ABO histo-blood group antigen (ABH) differences in glycosylation may lead to differences in microbial binding by airway mucus, and thus predispose to early lung infection and more severe lung disease in a subset of patients with CF., Methods and Principal Findings: Clinical information and DNA was collected on >800 patients with the DeltaF508/DeltaF508 genotype. Patients in the most severe and mildest quartiles for lung phenotype were enrolled. Blood samples underwent lymphocyte transformation and DNA extraction using standard methods. PCR and sequencing were performed using standard techniques to identify the 9 SNPs required to determine ABO blood type, and to identify the four SNPs that account for 90-95% of Lewis status in Caucasians. Allele identification of the one nonsynonymous SNP in FUT2 that accounts for >95% of the incidence of nonsecretor phenotype in Caucasians was completed using an ABI Taqman assay. The overall prevalence of ABO types, and of FUT2 (secretor) and FUT 3 (Lewis) alleles was consistent with that found in the Caucasian population. There was no difference in distribution of ABH type in the severe versus mild patients, or the age of onset of Pseudomonas aeruginosa infection in the severe or mild groups. Multivariate analyses of other clinical phenotypes, including gender, asthma, and meconium ileus demonstrated no differences between groups based on ABH type., Conclusions and Significance: Polymorphisms in the genes encoding ABO blood type, secretor or Lewis genotypes were not shown to associate with severity of CF lung disease, or age of onset of P. aeruginosa infection, nor was there any association with other clinical phenotypes in a group of 808 patients homozygous for the DeltaF508 mutation.

Background: Pyrethroids are neurotoxic pesticides that interact with membrane bound ion channels in neurons and disrupt nerve function. The purpose of this study was to characterize and explore changes in gene expression that occur in the rat frontal cortex, an area of CNS affected by pyrethroids, following an acute low-dose exposure., Results: Rats were acutely exposed to either deltamethrin (0.3 - 3 mg/kg) or permethrin (1 - 100 mg/kg) followed by collection of cortical tissue at 6 hours. The doses used range from those that cause minimal signs of intoxication at the behavioral level to doses well below apparent no effect levels in the whole animal. A statistical framework based on parallel linear (SAM) and isotonic regression (PIR) methods identified 95 and 53 probe sets as dose-responsive. The PIR analysis was most sensitive for detecting transcripts with changes in expression at the NOAEL dose. A sub-set of genes (Camk1g, Ddc, Gpd3, c-fos and Egr1) was then confirmed by qRT-PCR and examined in a time course study. Changes in mRNA levels were typically less than 3-fold in magnitude across all components of the study. The responses observed are consistent with pyrethroids producing increased neuronal excitation in the cortex following a low-dose in vivo exposure. In addition, Significance Analysis of Function and Expression (SAFE) identified significantly enriched gene categories common for both pyrethroids, including some relating to branching morphogenesis. Exposure of primary cortical cell cultures to both compounds resulted in an increase (approximately 25%) in the number of neurite branch points, supporting the results of the SAFE analysis., Conclusion: In the present study, pyrethroids induced changes in gene expression in the frontal cortex near the threshold for decreases in ambulatory motor activity in vivo. The penalized regression methods performed similarly in detecting dose-dependent changes in gene transcription. Finally, SAFE analysis of gene expression data identified branching morphogenesis as a biological process sensitive to pyrethroids and subsequent in vitro experiments confirmed this predicted effect. The novel findings regarding pyrethroid effects on branching morphogenesis indicate these compounds may act as developmental neurotoxicants that affect normal neuronal morphology.

Background: The Australian population is ageing, and a growing proportion of elderly Australians are now living in residential aged care facilities (RACFs). These residents are at high risk of developing dental diseases, have more teeth present now than at any time in the past 50 years and often have difficulty maintaining adequate oral hygiene. Traditionally, dental service provision has been problematic and sporadic for these residents., Methods: A postal survey of a random sample of Victorian general dentists and Directors of Nursing (DONs) of Victorian RACFs was undertaken in 2006 to ascertain the participation of dentists in the provision of dental care and to identify factors impacting on the organization and provision of dental care for residents., Results: The response rate for dentists was 57.3 per cent, and for DONs 64.4 per cent. Half of the dentists reported that they had provided care to residents of aged care facilities in the past 12 months, and they spent an average of one hour per month providing care. Overall, dentists were concerned with their level of undergraduate education and training in various aspects of dentistry for residents of aged care facilities. DONs reported significant difficulty obtaining adequate dental care for their residents. Common problems identified by both dentists and DONs included a preference for dentists to treat residents in their own practice, dentists not willing to go to RACFs and a lack of portable dental equipment for dentists to use., Conclusions: There were low levels of interest and participation from Victorian dentists in providing dental care for residents of aged care facilities. Dentists had a strong preference for treating patients at their own practice, and there were a number of significant barriers that appeared to impact on the provision of dental care in RACFs.

Little is known for certain about the genetics of schizophrenia. The advent of genomewide association has been widely anticipated as a promising means to identify reproducible DNA sequence variation associated with this important and debilitating disorder. A total of 738 cases with DSM-IV schizophrenia (all participants in the CATIE study) and 733 group-matched controls were genotyped for 492,900 single-nucleotide polymorphisms (SNPs) using the Affymetrix 500K two-chip genotyping platform plus a custom 164K fill-in chip. Following multiple quality control steps for both subjects and SNPs, logistic regression analyses were used to assess the evidence for association of all SNPs with schizophrenia. We identified a number of promising SNPs for follow-up studies, although no SNP or multimarker combination of SNPs achieved genomewide statistical significance. Although a few signals coincided with genomic regions previously implicated in schizophrenia, chance could not be excluded. These data do not provide evidence for the involvement of any genomic region with schizophrenia detectable with moderate sample size. However, a planned genomewide association study for response phenotypes and inclusion of individual phenotype and genotype data from this study in meta-analyses hold promise for eventual identification of susceptibility and protective variants.

In modern whole-genome scans, the use of stringent thresholds to control the genome-wide testing error distorts the estimation process, producing estimated effect sizes that may be on average far greater in magnitude than the true effect sizes. We introduce a method, based on the estimate of genetic effect and its standard error as reported by standard statistical software, to correct for this bias in case-control association studies. Our approach is widely applicable, is far easier to implement than competing approaches, and may often be applied to published studies without access to the original data. We evaluate the performance of our approach via extensive simulations for a range of genetic models, minor allele frequencies, and genetic effect sizes. Compared to the naive estimation procedure, our approach reduces the bias and the mean squared error, especially for modest effect sizes. We also develop a principled method to construct confidence intervals for the genetic effect that acknowledges the conditioning on statistical significance. Our approach is described in the specific context of odds ratios and logistic modeling but is more widely applicable. Application to recently published data sets demonstrates the relevance of our approach to modern genome scans.

Motivation: Reductions in genotyping costs have heightened interest in performing whole genome association scans and in the fine mapping of candidate regions. Improvements in study design and analytic techniques will require the simulation of datasets with realistic patterns of linkage disequilibrium and allele frequencies for typed SNPs., Methods: We describe a general approach to simulate genotyped datasets for standard case-control or affected child trio data, by resampling from existing phased datasets. The approach allows for considerable flexibility in disease models, potentially involving a large number of interacting loci. The method is most applicable for diseases caused by common variants that have not been under strong selection, a class specifically targeted by the International HapMap project., Results: Using the three population Phase I/II HapMap data as a testbed for our approach, we have implemented the approach in HAP-SAMPLE, a web-based simulation tool.

The identification of the genes that are differentially expressed in two-sample microarray experiments remains a difficult problem when the number of arrays is very small. We discuss the implications of using ordinary t-statistics and examine other commonly used variants. For oligonucleotide arrays with multiple probes per gene, we introduce a simple model relating the mean and variance of expression, possibly with gene-specific random effects. Parameter estimates from the model have natural shrinkage properties that guard against inappropriately small variance estimates, and the model is used to obtain a differential expression statistic. A limiting value to the positive false discovery rate (pFDR) for ordinary t-tests provides motivation for our use of the data structure to improve variance estimates. Our approach performs well compared to other proposed approaches in terms of the false discovery rate.

Allelic loss is often part of a multistep process leading to tumorigenesis. Analysis of genomic markers highlights regions of elevated allelic loss, which in turn suggests a nearby tumor suppressor. Furthermore, pooling published analyses to combine evidence can increase the power to detect a tumor suppressor gene. If the pattern of loss for each tumor, or allelotype, is known, a stochastic model proposed by Newton et al. (1998, Statistics in Medicine 17, 1425-1445) can be used to analyze the correlated binary data. Many studies report only incomplete allelotypes, augmented with frequencies of allelic loss (FAL) at each marker, in which the number of informative tumors showing allelic loss is provided along with the number of informative tumors. We describe an extension of the allelotype model to handle FAL data, using a hidden Markov model or a normal approximation to compute the likelihood. The FAL model is illustrated using data from a study of colorectal cancer.

Behaviors are often highly heritable, polygenic traits. To investigate molecular mediators of behavior, we analyzed gene expression patterns across seven brain regions (amygdala, basal ganglia, cerebellum, frontal cortex, hippocampus, cingulate cortex, and olfactory bulb) of 10 different inbred mouse strains (129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and FVB/NJ). Extensive variation was observed across both strain and brain region. These data provide potential transcriptional intermediates linking polygenic variation to differences in behavior. For example, mice from different strains had variable performance on the rotarod task, which correlated with the expression of >2000 transcripts in the cerebellum. Correlation with this task was also found in the amygdala and hippocampus, but not in other regions examined, indicating the potential complexity of motor coordination. Thus we can begin to identify expression profiles contributing to behavioral phenotypes through variation in gene expression.

Objective: To explore and describe international oral health attitudes/ behaviours among final year dental students., Methods: Validated translated versions of the Hiroshima University-Dental Behavioural Inventory (HU-DBI) questionnaire were administered to 1,096 final-year dental students in 17 countries. Hierarchical cluster analysis was conducted within the data to detect patterns and groupings., Results: The overall response rate was 72%. The cluster analysis identified two main groups among the countries. Group 1 consisted of twelve countries: one Oceanic (Australia), one Middle-Eastern (Israel), seven European (Northern Ireland, England, Finland, Greece, Germany, Italy, and France) and three Asian (Korea, Thailand and Malaysia) countries. Group 2 consisted of five countries: one South American (Brazil), one European (Belgium) and three Asian (China, Indonesia and Japan) countries. The percentages of 'agree' responses in three HU-DBI questionnaire items were significantly higher in Group 2 than in Group 1. They include: "I worry about the colour of my teeth."; "I have noticed some white sticky deposits on my teeth."; and "I am bothered by the colour of my gums.", Conclusion: Grouping the countries into international clusters yielded useful information for dentistry and dental education.

Molecular mechanisms mediating group A Streptococcus (GAS)-host interactions remain poorly understood but are crucial for diagnostic, therapeutic, and vaccine development. An optimized high-density microarray was used to analyze the transcriptome of GAS during experimental mouse soft tissue infection. The transcriptome of a wild-type serotype M1 GAS strain and an isogenic transcriptional regulator knockout mutant (covR) also were compared. Array datasets were verified by quantitative real-time reverse transcriptase-polymerase chain reaction and in situ immunohistochemistry. The results unambiguously demonstrate that coordinated expression of proven and putative GAS virulence factors is directed toward overwhelming innate host defenses leading to severe cellular damage. We also identified adaptive metabolic responses triggered by nutrient signals and hypoxic/acidic conditions in the host, likely facilitating pathogen persistence and proliferation in soft tissues. Key discoveries included that oxidative stress genes, virulence genes, genes related to amino acid and maltodextrin utilization, and several two-component transcriptional regulators were highly expressed in vivo. This study is the first global analysis of the GAS transcriptome during invasive infection. Coupled with parallel analysis of the covR mutant strain, novel insights have been made into the regulation of GAS virulence in vivo, resulting in new avenues for targeted therapeutic and vaccine research.

Objective: This paper utilises a Poisson frailty regression model with clustering to report on the failure rates of sealants placed and repaired during a three-year follow-up school-based comprehensive preventive programme., Basic Research Design: The programme included annual application/reapplication of light-cured fissure sealants in 210 12-year-old subjects over three years. Sealants placed at the commencement of each year of the programme were assessed for retention 12 months later and repair/reapplication was undertaken at that time for those surfaces judged to require it. An application of a Poisson regression model with random effects ("frailties") and clustering was used in this paper to allow for heterogeneity between subjects. Whilst it appears that the direct utilization of frailty models in determining fissure sealant failure rates has not been reported, it is clearly an appropriate use of this statistical method. A total of 1,544 sealants were placed during the study period. Of these, 1,038 (67.2%) were first placements at baseline and 506 (32.8%) were repairs/reapplications. Of these repairs/reapplications, 206 (40.7%) were performed at first year follow-up and 300 (59.3%) at second year follow-up., Results: The Failure Rate Ratios were statistically significantly greater for second molars cf. first molars (FRR=1.33, p=0.008) and for lower molars cf upper molars (FRR=1.32, p<0.001). Failure rate ratios for sealants placed in the distal occlusal pit and fissures cf. mesial occlusal pit and fissures were significantly higher (FRR=1.33, p<0.001). A significant interaction was found for molar type versus pit and fissure site (p=0.009). There was no significant interaction between molar type and arch., Conclusions: Sealants placed on second molars, those placed on lower molars and those placed on the distal fissure sites have a higher failure rate. As individual tooth sites in the mouth are not independent, any statistical analysis should allow for these associations when assessing sealant retention.

Background: The Oral Health Assessment Tool (OHAT) was a component of the Best Practice Oral Health Model for Australian Residential Care study. The OHAT provided institutional carers with a simple, eight category screening tool to assess residents' oral health, including those with dementia. This analysis presents OHAT reliability and validity results., Methods: A convenience sample of 21 residential care facilities (RCFs) in urban and rural Victoria, NSW and South Australia used the OHAT at baseline, three-months and six-months to assess intra- and inter-carer reliability and concurrent validity., Results: Four hundred and fifty five residents completed all study phases. Intra-carer reliability for OHAT categories: percent agreement ranged from 74.4 per cent for oral cleanliness, to 93.9 per cent for dental pain; Kappa statistics were in moderate range (0.51-0.60) for lips, saliva, oral cleanliness, and for all other categories in range of 0.61-0.80 (substantial agreement) (p < 0.05). Inter-carer reliability for OHAT categories: percent agreement ranged from 72.6 per cent for oral cleanliness to 92.6 per cent for dental pain; Kappa statistics were in moderate range (0.48-0.60) for lips, tongue, gums, saliva, oral cleanliness, and for all other categories in range of 0.61-0.80 (substantial agreement) (p < 0.05). Intraclass correlation coefficients for OHAT total scores were 0.78 for intra-carer and 0.74 for inter-carer reliability. Validity analyses of the OHAT categories and examination findings showed complete agreement for the lips category, with the natural teeth, dentures, and tongue categories having high significant correlations and percent agreements. The gums category had significant moderate correlation and percent agreement. Non-significant and low correlations and percent agreements were evident for the saliva, oral cleanliness and dental pain categories., Conclusion: The Oral Health Assessment Tool was evaluated as being a reliable and valid screening assessment tool for use in residential care facilities, including those with cognitively impaired residents.

Objective: To identify similarities and differences in oral health attitudes, behaviour and values among freshman dental students., Design: Cross-cultural survey of dental students., Setting: 18 cultural areas., Participants and Methods: 904 first-year dental students completed the Hiroshima University-Dental Behavioural Inventory (HU-DBI) translated into their own languages. Individual areas were clustered by similarity in responses to the questions., Results: The first group displayed an 'occidental-culture orientation' with the exception of Brazil (Cluster 1 comprised: Australia, United Kingdom, Ireland, Belgium and Brazil, Cluster 2: Germany, Italy, Finland and France). The second group displayed an 'oriental-cultural orientation' with the exception of Greece and Israel (Cluster 3 comprised: China and Indonesia, and Cluster 4: Japan, Korea, Israel, Hong Kong, Malaysia, Thailand and Greece). Australia and United Kingdom were the countries that were most alike. Ireland was the 'neighbour' to these countries. Greece and Malaysia had similar patterns of oral health behaviour although geographic conditions are very different. Although it was considered that in Hong Kong, occidental nations have affected the development of education, it remained in the oriental-culture group. Comparison with the data from the occidentals indicates that a higher percentage of the orientals put off going to the dentist until they have toothache (p < 0.001). Only a small proportion of the occidentals (8%) reported a perception of inevitability in having false teeth, whereas 33% of the orientals held this fatalistic belief (p = 0.001)., Conclusions: Grouping the countries into key cultural orientations and international clusters yielded plausible results, using the HU-DBI.

Motivation: Owing to the experimental cost and difficulty in obtaining biological materials, it is essential to consider appropriate sample sizes in microarray studies. With the growing use of the False Discovery Rate (FDR) in microarray analysis, an FDR-based sample size calculation is essential., Method: We describe an approach to explicitly connect the sample size to the FDR and the number of differentially expressed genes to be detected. The method fits parametric models for degree of differential expression using the Expectation-Maximization algorithm., Results: The applicability of the method is illustrated with simulations and studies of a lung microarray dataset. We propose to use a small training set or published data from relevant biological settings to calculate the sample size of an experiment., Availability: Code to implement the method in the statistical package R is available from the authors.

Motivation: In high-throughput genomic and proteomic experiments, investigators monitor expression across a set of experimental conditions. To gain an understanding of broader biological phenomena, researchers have until recently been limited to post hoc analyses of significant gene lists., Method: We describe a general framework, significance analysis of function and expression (SAFE), for conducting valid tests of gene categories ab initio. SAFE is a two-stage, permutation-based method that can be applied to various experimental designs, accounts for the unknown correlation among genes and enables permutation-based estimation of error rates., Results: The utility and flexibility of SAFE is illustrated with a microarray dataset of human lung carcinomas and gene categories based on Gene Ontology and the Protein Family database. Significant gene categories were observed in comparisons of (1) tumor versus normal tissue, (2) multiple tumor subtypes and (3) survival times., Availability: Code to implement SAFE in the statistical package R is available from the authors., Supplementary Information: http://www.bios.unc.edu/~fwright/SAFE.

The molecular basis for bacterial responses to host signals during natural infections is poorly understood. The gram-positive bacterial pathogen group A Streptococcus (GAS) causes human mucosal, skin, and life-threatening systemic infections. During the transition from a throat or skin infection to an invasive infection, GAS must adapt to changing environments and host factors. To better understand how GAS adapts, we used transcript profiling and functional analysis to investigate the transcriptome of a wild-type serotype M1 GAS strain in human blood. Global changes in GAS gene expression occur rapidly in response to human blood exposure. Increased transcription was observed for many genes that likely enhance bacterial survival, including those encoding superantigens and host-evasion proteins regulated by a multiple gene activator called Mga. GAS also coordinately expressed genes involved in proteolysis, transport, and catabolism of oligopeptides to obtain amino acids in this protein-rich host environment. Comparison of the transcriptome of the wild-type strain to that of an isogenic deletion mutant (DeltacovR) mutated in the two-component regulatory system designated CovR-CovS reinforced the hypothesis that CovR-CovS has an important role linking key biosynthetic, catabolic, and virulence functions during transcriptome restructuring. Taken together, the data provide crucial insights into strategies used by pathogenic bacteria for thwarting host defenses and surviving in human blood.

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.

Somatic loss of heterozygosity (LOH) has been widely reported in breast cancer as a means of identifying putative tumor-suppressor genes. However, individual studies have rarely spanned more than a single chromosome, and the varying criteria used to declare LOH complicate efforts to formally differentiate regions of consistent versus sporadic (random) loss. We report here the compilation of an extensive database from 151 published LOH studies of breast cancer, with summary data from >15,000 tumors and primary allelotypes from >4,300 tumors. Allelic loss was evaluated at 1,168 marker loci, with large variation in the density of informative observations across the genome. Using studies in which primary allelotype information was available, we employed a likelihood-based approach with a formal chromosomal instability and selection model. The approach seeks direct evidence for preferential loss at each locus compared with nearby loci, accounts for heterogeneity across studies, and enables the direct comparison of candidate regions across the genome. Striking preferential loss was observed (in descending order of significance) in specific regions of chromosomes 7q, 16q, 13q, 17p, 8p, 21q, 3p, 18q, 2q, and 19p, as well as other regions, in many cases coinciding with previously identified candidate genes or known fragile sites. Many of these observations were not possible from any single LOH study, and our results suggest that many previously reported LOH results are not systematic or reproducible. Our approach provides a comparative framework for further investigation of regions exhibiting LOH and identifies broad genomic regions for which there exist few data.