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Pharmacists' tasks are multifaceted and include, for example, vital counseling and communication skills. Objective Structured Clinical Examinations (OSCEs) could be used to train pharmacy students in these skills. Our study sought to determine the efficacy of our OSCE training approach for training pharmacy students' counseling and communication skills on diabetes mellitus compared to a control group. This randomized controlled study was conducted with pharmacy students using a pre-post-design. The intervention group completed diabetes OSCE training, while the control group solved diabetes patient cases using subjective, objective, assessment, and plan notes. Before and after the respective training, both groups completed OSCEs evaluating counseling and communication skills. Before each OSCE encounter, the participants completed a self-assessment questionnaire and, upon completion of the seminar, filled out a satisfaction survey. The OSCE-trained group demonstrated a significantly greater increase in counseling and communication skills and self-confidence than the control group. Both groups were generally satisfied with the seminar. These results demonstrate that our OSCE training approach allows for the effective training of pharmacy students' diabetes counseling and communication skills and suggests the inclusion of such a skill-based approach more widely in pharmacy students' education.

A modern approach to clinical skill education is the use of educational videos, yet there is a shortage of literature investigating the effect of self-instruction videos (SIVs) in pharmacy students. Therefore, our objective was to investigate whether an SIV is non-inferior compared to face-to-face instruction (FTFI) in acquiring blood pressure measurement skills. The participants in this randomized controlled study were pharmacy students. The control group was taught by FTFI, while the intervention group watched an SIV. Before and after the instruction, the participants' performance was assessed by an objective structured clinical examination (OSCE). The participants completed a self-assessment survey before each OSCE session. Moreover, the participants' perception and satisfaction were assessed using another survey. The OSCE score and self-assessment score increased significantly from pre- to post-instruction in both groups. The SIV was non-inferior compared to FTFI in terms of the OSCE score, considering a predefined non-inferiority margin of -10%. The participants' self-assessment yielded inconclusive results for non-inferiority. Both instructional approaches were well received. Considering our findings, SIVs might be a valuable option for teaching pharmacy students' blood pressure measurement skills. However, depending on the skill intended to be taught, a combination of an instructional video with instructor-led teaching may be necessary.

Alcohol is the most widely used recreational drug in Western countries. It affects the psychophysical performance in different ways, e.g. by reducing cognitive functions, causing coordination disturbances or impairing vision. Visual impairments both concern oculomotor and visual sensory functions, such as decreased mesopic vision, decreased field of vision and an increase of saccadic eye movements. During cycling trials with alcoholised test persons, repeated measurements of (1.) the time needed to read a 50-word text, (2.) the time to perform a swing test by tenfold touching the moving fingertip of the examiner, and (3.) the amplitude of fusion were carried out. The results of these tests were further evaluated to test the hypothesis that impaired vision is significantly correlated to reduced cycling performances of alcoholised persons. In a second step, it was examined which test is most useful to identify alcohol intoxicated cyclists. The ophthalmologic examination results of the groups of best and worst cycling-performing test persons at blood alcohol levels between 0.10% and 0.15% were set into relation to the documented allocated demerits. Additionally, the individual results of these persons were compared to the state of soberness. The time needed to read a 50-word text significantly correlated with the cycling performance. As this is an easy and objective test, it might contribute to a synoptic evaluation of the psychophysical performance of a drunken cyclist., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Pharmacists have the responsibility to conduct medication therapy management and appropriately counsel patients and physicians on medicines. Blended learning and objective structured clinical examinations (OSCEs) may have the potential to address these skills. This study aimed to assess whether a blended learning program consisting of e-learning and OSCEs could enhance pharmacy students' abilities to conduct pharmaceutical consultations with type 2 diabetes mellitus patients., Methods: The blended learning program consisted of e-learning, formative OSCEs, and summative OSCEs. The e-learning aimed to bring the students to the same level of diabetes knowledge. The students' baseline consultation skills were measured by a summative pre-training OSCE. After the formative OSCEs, a summative post-training OSCE quantified the change in performance. During the pre- and post-training OSCEs, analytical checklists were used to assess consultation skills and a global rating scale assessed communication skills. Surveys were used to measure students' self-assessment and satisfaction., Results: The study demonstrated significant improvement in students' pharmaceutical knowledge of diabetes mellitus, consultation performances on diabetes mellitus, and self-assessment scores. Most students responded positively on the satisfaction survey., Conclusions: This study demonstrated that a blended learning approach with e-learning and OSCEs can improve students' diabetes consultations and communication skills. These results support the use of this instructional technique for teaching patient counseling. Further studies should consider a control group to assess whether the improvement of the consultation performance is a result of the training or solely a learning effect due to the repetition of summative OSCEs., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Biomedical research studies have generated large multi-omic datasets to study complex diseases like Alzheimer's disease (AD). An important aim of these studies is the identification of candidate genes that demonstrate congruent disease-related alterations across the different data types measured by the study. We developed a new method to detect such candidate genes in large multi-omic case-control studies that measure multiple data types in the same set of samples. The method is based on a gene-centric integrative coefficient quantifying to what degree consistent differences are observed in the different data types. For statistical inference, a Bayesian hierarchical model is used to study the distribution of the integrative coefficient. The model employs a conditional autoregressive prior to integrate a functional gene network and to share information between genes known to be functionally related. We applied the method to an AD dataset consisting of histone acetylation, DNA methylation, and RNA transcription data from human cortical tissue samples of 233 subjects, and we detected 816 genes with consistent differences between persons with AD and controls. The findings were validated in protein data and in RNA transcription data from two independent AD studies. Finally, we found three subnetworks of jointly dysregulated genes within the functional gene network which capture three distinct biological processes: myeloid cell differentiation, protein phosphorylation and synaptic signaling. Further investigation of the myeloid network indicated an upregulation of this network in early stages of AD prior to accumulation of hyperphosphorylated tau and suggested that increased CSF1 transcription in astrocytes may contribute to microglial activation in AD. Thus, we developed a method that integrates multiple data types and external knowledge of gene function to detect candidate genes, applied the method to an AD dataset, and identified several disease-related genes and processes demonstrating the usefulness of the integrative approach., Competing Interests: The authors have declared that no competing interests exist.

Introduction: In a case of child pornography, only the dorsum of the offender's hand was clearly visible. After identification of a suspect, the question arose of whether and how it is possible to identify or exclude the suspect as perpetrator according to the morphology of the hand vein pattern., Material and Methods: A simple approach to use the hand vein pattern in crime suspects as a tool for identification was tested. In this study, the hand vein patterns of 30 study participants were analysed from conventional frames on videography. A standardised grid system consisting of six lines and four sectors was applied on the dorsum of the hands. Vein branchings within the sectors and line crossings of the veins were counted, leading to a total of 11 variables for each hand., Results: A positive identification of each of the 30 test participants was possible for each hand when taking only the first five variables into account. A random overlapping prediction was obtained by statistically simulating hand vein patterns of different numbers of persons using this sample. Considering the hand vein frequencies in this sample, the results indicate that the chance for two persons having the same pattern is smaller than 1:1000., Conclusions: It can be concluded that the introduced grid system approach can be an appropriate simple and non-costly tool for the analysis of the pattern of hand veins for identification purposes., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology. One strategy for studying the genetic risk factors of NSCL/P is to consider gene-gene interaction (G × G) among gene pathways having a role in craniofacial development. The present study aimed to investigate the G × G among cell adhesion gene pathway., Methods: We carried out an interaction analysis of eight genes involved in cell adherens junctions among 806 NSCL/P Chinese case-parent trios originally recruited for a genome-wide association study (GWAS). Regression-based approach was used to test for two-way G × G interaction, while machine learning algorithm was run for exploring both two-way and multi-way interaction that may affect the risk of NSCL/P., Results: A two-way ACTN1 × CTNNB1 interaction reached the adjusted significance level. The single nucleotide polymorphisms pair composed of rs17252114 (CTNNB1) and rs1274944 (ACTN1) yielded a p value of .0002, and this interaction was also supported by the logic regression algorithm. Higher order interactions involving ACTN1, CTNNB1, and CDH1 were picked out by logic regression, suggesting a potential role in NSCL/P risk., Conclusion: This study suggests for the first time evidence of both two-way and multi-way G × G interactions among cell adhesion genes contributing to the NSCL/P risk., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

To contribute to the ongoing discussion about threshold limits of Δ9-tetrahydrocannabinol (THC) in road traffic, a driving simulator study with 15 habitually cannabis consuming test persons was conducted. Probands were tested on different routes after consumption of a maximum of three cannabis joints, each containing 300 μg THC/kg body weight (sober testing as well as testing directly, 3 and 6 h after cannabis consumption). Accompanying the drives, medical examinations including a blood sampling were performed. Driving faults and distinctive features in the medical examinations were allocated certain penalty points, which were then summed up and evaluated using the ANOVA model. The results showed that very high CIF values > 30 as well as serum THC concentrations > 15 ng/ml significantly increased the number of penalty points, but no direct correlation to the THC concentrations in serum and/or CIF values was detected. Instead, the point in time after cannabis consumption seems to play an important role concerning driving safety: significantly more driving faults were committed directly after consumption. Three hours after consumption, no significant increase of driving faults was seen. Six hours after consumption (during the so-called subacute phase), an increase of driving faults could be noted although not significant. Considering the limitation of our study (e.g. small test group, no placebo test persons, long lasting test situation with possible tiredness), further studies focusing on the time dependant impact of cannabis consumption on road traffic are required.

Background: Little evidence exists to support pharmacotherapeutic strategies for heart failure management in paediatrics. A recent Europe-wide survey suggests that this translates into substantial variability in clinical practice., Objective: To conduct a formal discussion among an expert group of paediatric cardiology physicians on controversial aspects regarding the pharmacotherapy of children heart failure, facilitate consensus, and highlight areas of agreement and disagreement., Methods: A two-round modified Delphi process was conducted between July and August 2015. Topics addressed were predominantly selected from the results of a previous Europe-wide survey. Fourteen statements were presented for discussion grouped under three categories; Angiotensin-converting-enzyme-inhibitors: Considerations for optimal dosage; Angiotensin-converting-enzyme-inhibitors for the management of CHDs; Neurohumoral antagonists for the management of dilated cardiomyopathy-related heart failure., Results: A total of 13 paediatricians dedicated to cardiology from across Europe and the United States of America completed the study; of them, 92% had a working experience in the field of more than 10 years and were working in a specific paediatric cardiology unit. Agreement on the acceptance/rejection of 11 statements was achieved. Results show agreement on the importance of a set of topics relevant to the standardisation of the therapy as well as consensus upon specific therapeutic attitudes., Conclusions: We have found areas of common thinking and motivation, which can provide a means of triggering scientific collaboration. Our results might also contribute to disseminate available paediatric evidence and promote reducing unjustified variability in everyday practice. Until solid evidence is available, other research methods can contribute to advancing the goal of safe and effective paediatric heart failure pharmacotherapy.

Motivation: Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) is a biochemical method for detecting interaction sites of proteins with mRNA. This method introduces T-to-C substitutions at sequenced cDNA that help to detect binding sites on mRNA. However, T-to-C substitutions can also occur due to other reasons such as mismatches or SNPs. Only few statistical procedures exist for detecting binding sites in PAR-CLIP data. Most of these methods do not account for other types of substitutions than those induced by PAR-CLIP, and therefore, also report positions with high T-to-C substitution rates, e.g. SNPs, as binding sites. Moreover, none of these procedures allow to include additional information, e.g. the type of mRNA region, relevant for the biology of microRNA-binding sites., Results: We have developed BayMAP, a procedure based on a fully Bayesian hierarchical model that takes other sources of substitutions into account. Furthermore, this model enables the incorporation of additional information into the analysis of PAR-CLIP data. This incorporation does not only permit a better detection of binding sites, but also a better understanding of the data and the biology of binding sites. In applications to simulated PAR-CLIP data, BayMAP distinguishes binding sites from noise better than existing methods. Additionally, it yields good estimates of the influence of the additional information. We here demonstrate BayMAP's usability for real datasets even when noisy data is present., Availability and Implementation: BayMAP is freely available as an R package at http://stat.math.uni-duesseldorf.de/baymap., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: The beneficial effect of improving air quality on lung function in the elderly remains unclear. We examined associations between decline in air pollutants and lung function, and effect modifications by genetics and body mass index (BMI), in elderly German women., Methods: Data were analysed from the prospective SALIA (Study on the influence of Air pollution on Lung function, Inflammation and Aging) study (n=601). Spirometry was conducted at baseline (1985-1994; age 55 years), in 2007-2010 and in 2012-2013. Air pollution concentrations at home addresses were determined for each time-point using land-use regression models. Global Lung Initiative 2012 z-scores were calculated. Weighted genetic risk scores (GRSs) were determined from lung function-related risk alleles and used to investigate interactions with improved air quality. Multiple linear mixed models were fitted., Results: Air pollution levels decreased substantially during the study period. Reduction of air pollution was associated with an increase in z-scores for forced expiratory volume in 1 s (FEV 1 ) and the FEV 1 /forced vital capacity ratio. For a decrease of 10 µg·m -3 in nitrogen dioxide (NO 2 ), the z-score for FEV 1 increased by 0.14 (95% CI 0.01-0.26). However, with an increasing number of lung function-related risk alleles, the benefit from improved air quality decreased (GRS×NO 2 interaction: p=0.029). Interactions with BMI were not significant., Conclusions: Reduction of air pollution is associated with a relative improvement of lung function in elderly women, but also depends on their genetic make-up., Competing Interests: Conflict of interest: D. Hüls has nothing to disclose. Conflict of interest: D. Sugiri has nothing to disclose. Conflict of interest: M.J. Abramson reports grants from Pfizer, grants from Boehringer Ingelheim, assistance with conference attendance and honoraria for consultancy from Sanofi, outside the submitted work. Conflict of interest: B. Hoffmann has nothing to disclose. Conflict of interest: H. Schwender has nothing to disclose. Conflict of interest: K. Ickstadt has nothing to disclose. Conflict of interest: U. Krämer has nothing to disclose. Conflict of interest: T. Schikowski has nothing to disclose., (Copyright ©ERS 2019.)

Many medical studies aim to identify factors associated with a time to an event such as survival time or time to relapse. Often, in particular, when binary variables are considered in such studies, interactions of these variables might be the actual relevant factors for predicting, e.g., the time to recurrence of a disease. Testing all possible interactions is often not possible, so that procedures such as logic regression are required that avoid such an exhaustive search. In this article, we present an ensemble method based on logic regression that can cope with the instability of the regression models generated by logic regression. This procedure called survivalFS also provides measures for quantifying the importance of the interactions forming the logic regression models on the time to an event and for the assessment of the individual variables that take the multivariate data structure into account. In this context, we introduce a new performance measure, which is an adaptation of Harrel's concordance index. The performance of survivalFS and the proposed importance measures is evaluated in a simulation study as well as in an application to genotype data from a urinary bladder cancer study. Furthermore, we compare the performance of survivalFS and its importance measures for the individual variables with the variable importance measure used in random survival forests, a popular procedure for the analysis of survival data. These applications show that survivalFS is able to identify interactions associated with time to an event and to outperform random survival forests.

Objective: To characterise heart failure (HF) maintenance pharmacotherapy for children across Europe and investigate how angiotensin-converting enzyme inhibitors (ACE-I) are used in this setting., Methods: A Europe-wide web-based survey was conducted between January and May 2015 among European paediatricians dedicated to cardiology., Results: Out of 200-eligible, 100 physicians representing 100 hospitals in 27 European countries participated. All participants reported prescribing ACE-I to treat dilated cardiomyopathy-related HF and 97% in the context of congenital heart defects; 87% for single ventricle physiology. Twenty-six per cent avoid ACE-I in newborns. Captopril was most frequently selected as first-choice for newborns (73%) and infants and toddlers (66%) and enalapril for children (56%) and adolescents (58%). Reported starting and maintenance doses varied widely. Up to 72% of participants follow formal creatinine increase limits for decision-making when up-titrating; however, heterogeneity in the cut-off points selected existed. ACE-I formulations prescribed by 47% of participants are obtained from more than a single source. Regarding symptomatic HF maintenance therapy, 25 different initial drug combinations were reported, although 79% select a regimen that includes ACE-I and diuretic (thiazide and/or loop), 61% ACE-I and aldosterone antagonist; 44% start with beta-blocker, 52% use beta-blockers as an add-on drug. Of the 89 participants that prescribe pharmacotherapy to asymptomatic patients, 40% do not use ACE-I monotherapy or ACE-I-beta-blocker two-drug only combination., Conclusions: Despite some reluctance to use them in newborns, ACE-I seem key in paediatric HF treatment strategies. Use in single ventricle patients seems frequent, in apparent contradiction with current paediatric evidence. Disparate dosage criteria and potential formulation-induced variability suggest significant differences may exist in the risk-benefit profile children are exposed to. No uniformity seems to exist in the drug regimens in use. The information collected provides relevant insight into real-life clinical practice and may facilitate research to identify the best therapeutic options for HF children., Competing Interests: Competing interests: All authors report grants from European Union Seventh Framework Programme during the conduct of the study.

We apply hierarchical clustering (HC) of DNA k-mer counts on multiple Fastq files. The tree structures produced by HC may reflect experimental groups and thereby indicate experimental effects, but clustering of preparation groups indicates the presence of batch effects. Hence, HC of DNA k-mer counts may serve as a diagnostic device. In order to provide a simple applicable tool we implemented sequential analysis of Fastq reads with low memory usage in an R package (seqTools) available on Bioconductor. The approach is validated by analysis of Fastq file batches containing RNAseq data. Analysis of three Fastq batches downloaded from ArrayExpress indicated experimental effects. Analysis of RNAseq data from two cell types (dermal fibroblasts and Jurkat cells) sequenced in our facility indicate presence of batch effects. The observed batch effects were also present in reads mapped to the human genome and also in reads filtered for high quality (Phred > 30). We propose, that hierarchical clustering of DNA k-mer counts provides an unspecific diagnostic tool for RNAseq experiments. Further exploration is required once samples are identified as outliers in HC derived trees.

Background Pharmacists who engage in Pharmaceutical Care need skills to optimise responsible medication use and increase medication adherence. Objectives We developed and evaluated a blended-learning programme for German community pharmacists that focused on conducting consultations in chronic diseases. Setting Community pharmacists in Germany. Method Interventional study with pre-post design. We combined e-learning with Objective Standardised Clinical Examinations (OSCEs) for emergency situations, initiation/implementation of medication therapy and detection of symptoms of four chronic diseases. Specific procedures were defined in the Pharmaceutical Action Plan. Skills were measured with a global analytical marking sheet derived from the Medication Related Consultation Framework and scored with the Canadian criticality/relevancy matrix. Time limits matched real practice scenarios. Main outcome measures Changes in knowledge (difference of test results before and after e-learning) and changes in skills (difference in scores of the OSCEs before and after training). Results 22 out of the 26 pharmacists enrolled, completed the study. The number of correctly answered questions increased significantly after the e-learning for all four indications with a mean number of additional correct answers between 3.86 and 4.9 points out of 15 (p < 0.001). The sums of the analytical checklist points in percentages increased significantly in all topics from the baseline summative OSCE to the final summative OSCE between 6.14 and 31.85% (p < 0.001). The maximum duration of consultation per patient was less than 10 min in all OSCEs. Conclusion The use of e-learning and OSCEs was well received by participants and is a successful method to deliver practical Pharmaceutical Care training.

Objective: Multiple studies have suggested nonsyndromic cleft lip with or without cleft palate (NSCL/P), and lung cancer may have common genetic etiology. Previous studies have showed genetic variants in nicotinic cholinergic receptor genes (CHRNs) may influence risk of lung cancer. We aimed to explore the effect of CHRNs on risk of NSCL/P considering gene-gene (GxG) interaction for these genes., Subjects and Methods: We selected 120 markers in 14 CHRNs to test for GxG interaction using 806 Chinese case-parent trios recruited from an international consortium established for a GWAS of oral clefts., Results: Totally, two pairs of SNPs yielded significant GxG interactions after Bonferroni correction (rs935865 and rs2337980 with p = 4.04 × 10 -5 , rs2741335 and rs3743077 with p = 4.80 × 10 -4 ), and these pairwise interactions were confirmed in permutation tests. In addition, the relative risk (RR) of the putative interaction between rs935865 and rs2337980 was 1.10 (95% CI: 0.92~1.31)., Conclusions: While the single SNP association and the gene-environment interaction analysis of 14 CHRN genes yielded no signal, this study did demonstrate the importance of considering potential GxG interaction for exploring etiology of NSCL/P. This study suggests an important role for particular combinations of SNPs in CHRN genes in influencing risk to NSCL/P, which needs further study., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

The relationship between grey matter volume (GMV) patterns and age can be captured by multivariate pattern analysis, allowing prediction of individuals' age based on structural imaging. Raw data, voxel-wise GMV and non-sparse factorization (with Principal Component Analysis, PCA) show good performance but do not promote relatively localized brain components for post-hoc examinations. Here we evaluated a non-negative matrix factorization (NNMF) approach to provide a reduced, but also interpretable representation of GMV data in age prediction frameworks in healthy and clinical populations. This examination was performed using three datasets: a multi-site cohort of life-span healthy adults, a single site cohort of older adults and clinical samples from the ADNI dataset with healthy subjects, participants with Mild Cognitive Impairment and patients with Alzheimer's disease (AD) subsamples. T1-weighted images were preprocessed with VBM8 standard settings to compute GMV values after normalization, segmentation and modulation for non-linear transformations only. Non-negative matrix factorization was computed on the GM voxel-wise values for a range of granularities (50-690 components) and LASSO (Least Absolute Shrinkage and Selection Operator) regression were used for age prediction. First, we compared the performance of our data compression procedure (i.e., NNMF) to various other approaches (i.e., uncompressed VBM data, PCA-based factorization and parcellation-based compression). We then investigated the impact of the granularity on the accuracy of age prediction, as well as the transferability of the factorization and model generalization across datasets. We finally validated our framework by examining age prediction in ADNI samples. Our results showed that our framework favorably compares with other approaches. They also demonstrated that the NNMF based factorization derived from one dataset could be efficiently applied to compress VBM data of another dataset and that granularities between 300 and 500 components give an optimal representation for age prediction. In addition to the good performance in healthy subjects our framework provided relatively localized brain regions as the features contributing to the prediction, thereby offering further insights into structural changes due to brain aging. Finally, our validation in clinical populations showed that our framework is sensitive to deviance from normal structural variations in pathological aging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Small ubiquitin-like modification, also known as sumoylation, is a crucial post-translational regulatory mechanisms involved in development of the lip and palate. Recent studies reported two sumoylation target genes, MSX1 and TP63, to have achieved genome-wide level significance in tests of association with nonsyndromic clefts. Here, we performed a candidate gene analysis considering gene-gene and gene-environment interaction for SUMO1, MSX1, and TP63 to further explore the etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P)., Methods: A total of 130 single-nucleotide polymorphisms (SNPs) in or near SUMO1, MSX1, and TP63 was analyzed among 1,038 Asian NSCL/P trios ascertained through an international consortium. Conditional logistic regression models were used to explore gene-gene (G × G) and gene-environment (G × E) interaction involving maternal environmental tobacco smoke and multivitamin supplementation. Bonferroni correction was used for G × E analysis and permutation tests were used for G × G analysis., Results: While transmission disequilibrium tests and gene-environment interaction analysis showed no significant results, we did find signals of gene-gene interaction between SNPs near MSX1 and TP63. Three pairwise interactions yielded significant p values in permutation tests (rs884690 and rs9290890 with p = 9.34 × 10 -5 and empirical p = 1.00 × 10 -4 , rs1022136 and rs4687098 with p = 2.41 × 10 -4 and empirical p = 2.95 × 10 -4 , rs6819546 and rs9681004 with p = 5.15 × 10 -4 and empirical p = 3.02 × 10 -4 )., Conclusion: Gene-gene interaction between MSX1 and TP63 may influence the risk of NSCL/P in Asian populations. Our study provided additional understanding of the genetic etiology of NSCL/P and underlined the importance of considering gene-gene interaction in the etiology of this common craniofacial malformation., (© 2018 Wiley Periodicals, Inc.)

Secretome analysis faces several challenges including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it to the analysis of secreted proteins of C2C12 skeletal muscle cells since the skeletal muscle has been identified as an important endocrine organ secreting myokines as signaling molecules. First, we compared culture supernatants with corresponding cell lysates by mass spectrometry-based proteomics and label-free quantification. We identified 672 protein groups as candidate secreted proteins due to their higher abundance in the secretome. On the basis of Brefeldin A mediated blocking of classical secretory processes, we estimated a sensitivity of >80% for the detection of classical secreted proteins for our experimental approach. In the second step, the peptide level information was integrated with UniProt based protein information employing the newly developed bioinformatics tool "Lysate and Secretome Peptide Feature Plotter" (LSPFP) to detect proteolytic protein processing events that might occur during secretion. Concerning the proof of concept, we identified truncations of the cytoplasmic part of the protein Plexin-B2. Our workflow provides an efficient combination of experimental workflow and data analysis to identify putative secreted and proteolytic processed proteins.

Objective: To evaluate randomized controlled trials (RCTs) that included interventions provided by community pharmacists for patients with type 1 and 2 diabetes, the analysis of each component of the intervention(s), and the description of the training that the pharmacists received., Data Sources: The literature research was conducted in PubMed and in the Cochrane Central Register of Controlled Trials (January 2000 to April 2016) for RCTs with interventions provided by community pharmacists for patients with diabetes. Corresponding authors were contacted about missing data and intervention and training design., Study Selection and Data Extraction: RCTs published in English or German were included if pharmaceutical care or medication therapy management was conducted by community pharmacists with diabetes patients. Basic information, intervention and training design data were extracted., Data Synthesis: The literature research resulted in 11 eligible studies for further analysis. The corresponding authors of 6 studies responded to our request and sent their raw data. The calculated meta-analytical effect of 640 analyzed patients was a hemoglobin A 1C (A1C) difference of -0.66%, with a 95% CI of -0.86% to -0.45%. The analysis revealed that most intervention elements had a significant positive meta-analytical effect on the A1C values., Conclusions: Our meta-analysis suggests that community pharmacist-led interventions can improve glycemic control in patients with type 1 and 2 diabetes. The most effective intervention components were patient centered and interdisciplinary. Pharmaceutical care interventions should, therefore, include the following components: sending feedback to the physician, setting individual goals, reviewing medication, and assessing patients' health beliefs and medication knowledge.

Background: Weighted genetic risk scores (GRS), defined as weighted sums of risk alleles of single nucleotide polymorphisms (SNPs), are statistically powerful for detection gene-environment (GxE) interactions. To assign weights, the gold standard is to use external weights from an independent study. However, appropriate external weights are not always available. In such situations and in the presence of predominant marginal genetic effects, we have shown in a previous study that GRS with internal weights from marginal genetic effects ("GRS-marginal-internal") are a powerful and reliable alternative to single SNP approaches or the use of unweighted GRS. However, this approach might not be appropriate for detecting predominant interactions, i.e. interactions showing an effect stronger than the marginal genetic effect., Methods: In this paper, we present a weighting approach for such predominant interactions ("GRS-interaction-training") in which parts of the data are used to estimate the weights from the interaction terms and the remaining data are used to determine the GRS. We conducted a simulation study for the detection of GxE interactions in which we evaluated power, type I error and sign-misspecification. We compared this new weighting approach to the GRS-marginal-internal approach and to GRS with external weights., Results: Our simulation study showed that in the absence of external weights and with predominant interaction effects, the highest power was reached with the GRS-interaction-training approach. If marginal genetic effects were predominant, the GRS-marginal-internal approach was more appropriate. Furthermore, the power to detect interactions reached by the GRS-interaction-training approach was only slightly lower than the power achieved by GRS with external weights. The power of the GRS-interaction-training approach was confirmed in a real data application to the Traffic, Asthma and Genetics (TAG) Study (N = 4465 observations)., Conclusion: When appropriate external weights are unavailable, we recommend to use internal weights from the study population itself to construct weighted GRS for GxE interaction studies. If the SNPs were chosen because a strong marginal genetic effect was hypothesized, GRS-marginal-internal should be used. If the SNPs were chosen because of their collective impact on the biological mechanisms mediating the environmental effect (hypothesis of predominant interactions) GRS-interaction-training should be applied.

Motivation: Genes showing congruent differences in several genomic variables between two biological conditions are crucial to unravel causalities behind phenotypes of interest. Detecting such genes is important in biomedical research, e.g. when identifying genes responsible for cancer development. Small sample sizes common in next-generation sequencing studies are a key challenge, and there are still only very few statistical methods to analyze more than two genomic variables in an integrative, model-based way. Here, we present a novel bioinformatics approach to detect congruent differences between two biological conditions in a larger number of different measurements such as various epigenetic marks or mRNA transcript levels., Results: We propose a coefficient quantifying the degree to which genes present consistent alterations in multiple (more than two) genomic variables when comparing samples presenting a condition of interest (e.g. cancer) to a reference group. A hierarchical Bayesian model is employed to assess uncertainty on a gene level, incorporating information on functional relationships between genes. We demonstrate the approach on different data sets containing RNA-seq gene transcripton and up to four ChIP-seq histone modification measurements. Both the coefficient-based ranking and the inference based on the model lead to a plausible prioritizing of candidate genes when analyzing multiple genomic variables., Availability and Implementation: BUGS code in the Supplement., Contact: m.schaefer@uni-duesseldorf.de., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Merging data from multiple samples is required to detect low expressed transcripts or splicing events that might be present only in a subset of samples. However, the exact number of required replicates enabling the detection of such rare events often remains a mystery but can be approached through probability theory. Here, we describe a probabilistic model, relating the number of observed events in a batch of samples with observation probabilities. Therein, samples appear as a heterogeneous collection of events, which are observed with some probability. The model is evaluated in a batch of 54 transcriptomes of human dermal fibroblast samples. The majority of putative splice-sites (alignment gap-sites) are detected in (almost) all samples or only sporadically, resulting in an U-shaped pattern for observation probabilities. The probabilistic model systematically underestimates event numbers due to a bias resulting from finite sampling. However, using an additional assumption, the probabilistic model can predict observed event numbers within a <10% deviation from the median. Single samples contain a considerable amount of uniquely observed putative splicing events (mean 7122 in alignments from TopHat alignments and 86,215 in alignments from STAR). We conclude that the probabilistic model provides an adequate description for observation of gap-sites in transcriptome data. Thus, the calculation of required sample sizes can be done by application of a simple binomial model to sporadically observed random events. Due to the large number of uniquely observed putative splice-sites and the known stochastic noise in the splicing machinery, it appears advisable to include observation of rare splicing events into analysis objectives. Therefore, it is beneficial to take scores for the validation of gap-sites into account., Competing Interests: The authors declare no conflict of interest.

Anosognosia is common in patients with Alzheimer disease (AD) even in early stages. Although neural correlates and the impact of cognitive dysfunctions have been described, possible psychodynamic processes such as a repressive coping style as described in other illnesses, have not been examined. Our study aimed to examine possible psychological influence factors on illness perception embracing a repressive coping style and cognitive functions in AD patients in the diagnostic process. Fifty-four subjects with mild AD diagnosed in our memory clinic were enrolled. Anosognosia was evaluated using a patient-caregiver discrepancy rating. All patients underwent comprehensive neuropsychological testing. In addition, characteristics of a repressive coping style were assessed. In total, 79.6% of our patients showed a lack of awareness at least to some degree. 33.3% of the patients were classified as repressors. Repressors and nonrepressors did not differ in cognition, or the unawareness score. Multivariate regression analysis showed that repressive coping style did not significantly contribute to anosognosia, but that verbal memory and naming ability had a strong influence. Although our data indicate that a high proportion of patients with mild AD show characteristics of repressive coping, this possible defense mechanism had no influence on the awareness of illness-related deficits measured by caregiver patient discrepancy.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with a complex and heterogeneous etiology. A recent genome-wide association study (GWAS) among Chinese populations has identified a new region at 16p13.3 as being associated with NSCL/P, which requires further replication. Here, we attempted to replicate and further clarify the genetic association between this region and NSCL/P, as well as testing for potential gene-gene (G × G) and gene-environment (G × E) interactions. We conducted transmission disequilibrium tests on 69 single nucleotide polymorphisms (SNPs) mapping to 16p13.3 among 806 Chinese case-parent trios ascertained through an international consortium where a GWAS of oral clefts was conducted. G × G, as well as G × E interactions involving maternal environmental tobacco smoke (ETS) and multivitamin supplementation, were explored using conditional logistic regression model. We applied Cordell's method as implemented in the R package TRIO to test for possible interactions. While no SNPs showed evidence of linkage and association with NSCL/P after Bonferroni correction, we found signals of G × G interactions between SNPs in 16p13.3. Nine pairs of SNP-SNP interactions attained significance after Bonferroni correction, among which the most significant interaction was found between rs2072346 (ADCY9) and rs11646137 (intergenic region, P = 7.2 × 10 -5 ). Linkage disequilibrium (LD) analysis revealed only low level of LD between these SNPs. This study failed to confirm the significant association between SNPs within 16p13.3 and the risk of NSCL/P, but underlined the importance of taking into account potential G × G interactions for the genetic association analysis of NSCL/P., (© 2017 Wiley Periodicals, Inc.)

Genomic alignments of sequenced cellular messenger RNA contain gapped alignments which are interpreted as consequence of intron removal. The resulting gap-sites, genomic locations of alignment gaps, are landmarks representing potential splice-sites. As alignment algorithms report gap-sites with a considerable false discovery rate, validations are required. We describe two quality scores, gap quality score ( gqs ) and weighted gap information score ( wgis ), developed for validation of putative splicing events: While gqs solely relies on alignment data wgis additionally considers information from the genomic sequence. FASTQ files obtained from 54 human dermal fibroblast samples were aligned against the human genome (GRCh38) using TopHat and STAR aligner. Statistical properties of gap-sites validated by gqs and wgis were evaluated by their sequence similarity to known exon-intron borders. Within the 54 samples, TopHat identifies 1,000,380 and STAR reports 6,487,577 gap-sites. Due to the lack of strand information, however, the percentage of identified GT-AG gap-sites is rather low. While gap-sites from TopHat contain ≈89% GT-AG, gap-sites from STAR only contain ≈42% GT-AG dinucleotide pairs in merged data from 54 fibroblast samples. Validation with gqs yields 156,251 gap-sites from TopHat alignments and 166,294 from STAR alignments. Validation with wgis yields 770,327 gap-sites from TopHat alignments and 1,065,596 from STAR alignments. Both alignment algorithms, TopHat and STAR, report gap-sites with considerable false discovery rate, which can drastically be reduced by validation with gqs and wgis ., Competing Interests: The authors declare no conflict of interest.

Ageing, the progressive functional decline of virtually all tissues, affects numerous living organisms. Main phenotypic alterations of human skin during the ageing process include reduced skin thickness and elasticity which are related to extracellular matrix proteins. Dermal fibroblasts, the main source of extracellular fibrillar proteins, exhibit complex alterations during in vivo ageing and any of these are likely to be accompanied or caused by changes in gene expression. We investigated gene expression of short term cultivated in vivo aged human dermal fibroblasts using RNA-seq. Therefore, fibroblast samples derived from unaffected skin were obtained from 30 human donors. The donors were grouped by gender and age (Young: 19 to 25 years, Middle: 36 to 45 years, Old: 60 to 66 years). Two samples were taken from each donor, one from a sun-exposed and one from a sun-unexposed site. In our data, no consistently changed gene expression associated with donor age can be asserted. Instead, highly correlated expression of a small number of genes associated with transforming growth factor beta signalling was observed. Also, known gene expression alterations of in vivo aged dermal fibroblasts seem to be non-detectable in cultured fibroblasts.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans, affecting 1 in 700 live births. This malformation has a complex etiology where multiple genes and several environmental factors influence risk. At least a dozen different genes have been confirmed to be associated with risk of NSCL/P in previous studies. However, all the known genetic risk factors cannot fully explain the observed heritability of NSCL/P, and several authors have suggested gene-gene (G × G) interaction may be important in the etiology of this complex and heterogeneous malformation. We tested for G × G interactions using common single nucleotide polymorphic (SNP) markers from targeted sequencing in 13 regions identified by previous studies spanning 6.3 Mb of the genome in a study of 1,498 NSCL/P case-parent trios. We used the R-package trio to assess interactions between polymorphic markers in different genes, using a 1 degree of freedom (1df) test for screening, and a 4 degree of freedom (4df) test to assess statistical significance of epistatic interactions. To adjust for multiple comparisons, we performed permutation tests. The most significant interaction was observed between rs6029315 in MAFB and rs6681355 in IRF6 (4df P = 3.8 × 10 -8 ) in case-parent trios of European ancestry, which remained significant after correcting for multiple comparisons. However, no significant interaction was detected in trios of Asian ancestry., (© 2016 WILEY PERIODICALS, INC.)

Resting-state functional connectivity analysis has become a widely used method for the investigation of human brain connectivity and pathology. The measurement of neuronal activity by functional MRI, however, is impeded by various nuisance signals that reduce the stability of functional connectivity. Several methods exist to address this predicament, but little consensus has yet been reached on the most appropriate approach. Given the crucial importance of reliability for the development of clinical applications, we here investigated the effect of various confound removal approaches on the test-retest reliability of functional-connectivity estimates in two previously defined functional brain networks. Our results showed that gray matter masking improved the reliability of connectivity estimates, whereas denoising based on principal components analysis reduced it. We additionally observed that refraining from using any correction for global signals provided the best test-retest reliability, but failed to reproduce anti-correlations between what have been previously described as antagonistic networks. This suggests that improved reliability can come at the expense of potentially poorer biological validity. Consistent with this, we observed that reliability was proportional to the retained variance, which presumably included structured noise, such as reliable nuisance signals (for instance, noise induced by cardiac processes). We conclude that compromises are necessary between maximizing test-retest reliability and removing variance that may be attributable to non-neuronal sources.

To assess the effects of cannabis on the ability required to ride a bicycle, repetitive practical cycling tests and medical examinations were carried out before and after inhalative consumption of cannabis. A maximum of three joints with body weight-adapted THC content (300 μg THC per kg body weight) could be consumed by each test subject. Fourteen regular cannabis-consuming test subjects were studied (12 males, 2 females). In summary, only a few driving faults were observed even under the influence of very high THC concentrations. A defined THC concentration that leads to an inability to ride a bicycle cannot be presented. The test subjects showed only slight distinctive features that can be documented using a medical test routinely run for persons under suspicion of driving under the influence of alcohol or drugs.

Most comparisons of blood alcohol concentrations (BAC) and breath alcohol concentrations (BrAC) are either derived from drinking trials with rigid drinking protocols or from investigative authorities' data with considerable time differences between the determination of BAC and BrAC. In general, only comparisons of relatively low BAC-BrAC pairs are available. Therefore, the relationship between BAC and BrAC was examined even for high BAC above 2g/kg. The results of a large-scale drinking test under realistic conditions with 78 test persons and short time intervals between BAC and BrAC measurements are presented. It was shown that the conversion factor Q varies greatly (between 1571:1 and 2394:1) and increases with increasing BAC. A constant conversion factor that is suitable for variable forensic purposes could not be presented., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The forkhead box F2 gene (FOXF2) located in chromosome 6p25.3 has been shown to play a crucial role in palatal development in mouse and rat models. To date, no evidence of linkage or association has been reported for this gene in humans with oral clefts., Methods: Allelic transmission disequilibrium tests were used to robustly assess evidence of linkage and association with nonsyndromic cleft lip with or without cleft palate for nine single nucleotide polymorphisms (SNPs) in and around FOXF2 in both Asian and European trios using PLINK., Results: Statistically significant evidence of linkage and association was shown for two SNPs (rs1711968 and rs732835) in 216 Asian trios where the empiric P values with permutation tests were 0.0016 and 0.005, respectively. The corresponding estimated odds ratios for carrying the minor allele at these SNPs were 2.05 (95% confidence interval = 1.41, 2.98) and 1.77 (95% confidence interval = 1.26, 2.49), respectively., Conclusion: Our results provided statistical evidence of linkage and association between FOXF2 and nonsyndromic cleft lip with or without cleft palate., Competing Interests: The authors declare no conflict of interest., (© 2015 Wiley Periodicals, Inc.)

Background: Physiological and psychological changes during puberty and a low adherence to complex treatment regimens often result in poor glycemic control in adolescents with type 1 diabetes mellitus (T1DM). The benefit of pharmaceutical care in adults with diabetes mellitus type 2 has been explored; however, evidence in adolescents with T1DM is scarce., Objective: To evaluate the impact of pharmaceutical care in adolescents with T1DM provided by pharmacists, in collaboration with physicians and diabetes educators on important clinical outcomes (e.g., HbA1c and severe hypoglycemia), Setting: At the outpatient Helios Paediatric Clinic and at the 12 regular community pharmacies of the study patients with 14 pharmacists in the Krefeld area, Germany, and at the University Pediatric Clinic with one clinical pharmacist on-site in Sarajevo, Bosnia-Herzegovina., Methods: A randomized, controlled, prospective, multicenter study in 68 adolescents with T1DM. The intervention group received monthly structured pharmaceutical care visits delivered by pharmacists plus supplementary visits and phone calls on an as needed basis, for 6 months. The control group received usual diabetic care. Data were collected at baseline and after 3 and 6 months., Main Outcome Measures: The between-group difference in the change from baseline in glycosylated hemoglobin (HbA1c) and the number of severe hypoglycemic events in both groups., Results: The improvement from baseline in HbA1c was significantly greater in the intervention group than in the control group after 6 months (change from baseline -0.54 vs. +0.32%, p = 0.0075), even after adjustment for country-specific variables (p = 0.0078). However, the effect was more pronounced after only 3 months (-1.09 vs. +0.23%, p = 0.00002). There was no significant between-group difference in the number of severe hypoglycemia events. (p = 0.1276)., Conclusion: This study suggests that multidisciplinary PhC may add value in the management of T1DM in adolescents with inadequate glycemic control. However, the optimal methods on how to achieve sustained, long-term improvements in this challenging population require further study.

To examine the effects of alcohol regarding the fitness required to ride a bicycle, practical cycling tests, accompanied by medical examinations, were carried out at different blood alcohol concentrations. Seventy-eight persons were included in the trials (41 males, 37 females). Eighty-three evaluable trials were obtained. Men committed less coordinative driving faults with comparable blood alcohol concentrations. Single highly alcoholized men were able to safely ride their bicycle; however, each of the female test persons had at least one severe driving fault at blood alcohol levels above 1.43 g/kg. Women tended to exhibit signs of alcoholization in the medical examination reports earlier than men.

To investigate the effects of alcohol on the ability to ride a bicycle, practical cycling tests were carried out at different blood alcohol concentrations (BAC). For this purpose, various alcoholic beverages could be consumed from around 2 p.m. until 11 p.m. Afterwards, the test persons spent the night on the trial site and were provided with dormitory sleeping accommodation. On the following morning, beginning at around 8 a.m., a final cycling test was performed. The performances of those test persons who had returned to state of soberness and of those with residual blood alcohol levels were compared to the performances on the day before. The practical ability to ride a bicycle was significantly reduced in the postalcoholic state compared to the rides of the day before. The relative cycling performance in the postalcoholic state was comparable to the rides under the influence of BAC of around 0.30 g/kg. There were no remarkable differences between the groups with and without residual blood alcohol levels regarding the rides on the next morning. Therefore, it can be assumed that the direct influence of residual blood alcohol levels plays a minor role for the ability to ride a bicycle in the postalcoholic state. Instead, the side effects of the high amounts of alcohol that were consumed the night before are crucial.

The open source environment R isf the most widely used software to statistically explore biological data sets including sequence alignments. BAM is the de facto standard file format for sequence alignment. With rbamtools, we provide now a full spectrum of accessibility to BAM for R users such as reading, writing, extraction of subsets and plotting of alignment depth where the script syntax closely follows the SAM/BAM format. Additionally, rbamtools enables fast accumulative tabulation of splicing events over multiple BAM files., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

To determine the threshold for the absolute inability to ride a bicycle, practical cycling tests and medical examinations at different blood alcohol concentrations were performed. Special attention was given to additional medical examinations, reaction tests and alcohol consumption under real-life conditions. Seventy-eight test subjects were included in the trials (37 females, 41 males). Five test subjects participated twice; thus, there were a total of 83 evaluable trials. Alcohol-related deficits were already identifiable at very low BACs. A significant increase in gross motoric disturbances compared to the soberness state did not regularly occur until a BAC of at least 0.8 g/kg was reached. At the BAC of 1.4 g/kg and above, no test subjects were able to achieve or surpass their sober driving results. Isolated highly alcoholised test subjects rode the bike in a manner that was not conspicuously different than the other sober test persons. Contrary to the assumptions of current German legal practise, it cannot be stated that all people are 'absolutely impaired' to the point of being incapable of riding bicycle at BACs of at least 1.6 g/kg.

Background: DNA copy number variants play an important part in the development of common birth defects such as oral clefts. Individual patients with multiple birth defects (including oral clefts) have been shown to carry small and large chromosomal deletions., Methods: We investigated the role of polymorphic copy number deletions by comparing transmission rates of deletions from parents to offspring in case-parent trios of European ancestry ascertained through a cleft proband with trios ascertained through a normal offspring. DNA copy numbers in trios were called using the joint hidden Markov model in the freely available PennCNV software. All statistical analyses were performed using Bioconductor tools in the open source environment R., Results: We identified a 67 kb region in the gene MGAM on chromosome 7q34, and a 206 kb region overlapping genes ADAM3A and ADAM5 on chromosome 8p11, where deletions are more frequently transmitted to cleft offspring than control offspring., Conclusions: These genes or nearby regulatory elements may be involved in the etiology of oral clefts., (© 2015 Wiley Periodicals, Inc.)

The porphyrias are a group of inherited metabolic diseases resulting from enzymatic deficiencies of specific haem biosynthetic enzymes. They can be classified as primarily acute and non-acute types. Clinically, the acute hepatic porphyrias (AHPs) are characterised by acute neurovisceral attacks. Patients with AHP may be at increased risk for development of hepatocellular carcinoma (HCC). However, systematic studies on the occurrence of other malignancies in patients with the AHPs have not been performed to date. Here, we studied the development of HCC and distinct malignant tumours in patients with the AHPs registered in a single European porphyria specialist centre. A questionnaire was designed and sent to all individuals (n = 122) diagnosed between 1970 and 2012 of whom a valid address was available (n = 82), requesting information on their personal and family history of cancer. Statistical analysis was performed to calculate incidence, prevalence and relative risk of HCC. To calculate confidence intervals, a Poisson distribution was assumed. Forty-nine patients (59.8%) returned a completed questionnaire. Overall, HCC was diagnosed in one female (2.1%), and the remaining patients reported on six distinct malignancies. We were able to confirm that HCC is an important complication in AHP. The patients in our cohort had an approximately 35-fold increased risk of developing HCC, similar to observations in other European countries. In addition, we detected colon, breast, uterine and thyroid cancer as well as lymphoma and a liver metastasis in patients with AHP. However, considering the small number of tumours and patients studied here, the data should be interpreted with caution, and further studies on cancer occurrence in AHP patients will require a multicentre setting.

Case-parent trio studies considering genotype data from children affected by a disease and their parents are frequently used to detect single nucleotide polymorphisms (SNPs) associated with disease. The most popular statistical tests for this study design are transmission/disequilibrium tests (TDTs). Several types of these tests have been developed, for example, procedures based on alleles or genotypes. Therefore, it is of great interest to examine which of these tests have the highest statistical power to detect SNPs associated with disease. Comparisons of the allelic and the genotypic TDT for individual SNPs have so far been conducted based on simulation studies, since the test statistic of the genotypic TDT was determined numerically. Recently, however, it has been shown that this test statistic can be presented in closed form. In this article, we employ this analytic solution to derive equations for calculating the statistical power and the required sample size for different types of the genotypic TDT. The power of this test is then compared with the one of the corresponding score test assuming the same mode of inheritance as well as the allelic TDT based on a multiplicative mode of inheritance, which is equivalent to the score test assuming an additive mode of inheritance. This is, thus, the first time the power of these tests are compared based on equations, yielding instant results and omitting the need for time-consuming simulation studies. This comparison reveals that these tests have almost the same power, with the score test being slightly more powerful., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common disorder with complex etiology. The Bone Morphogenetic Protein 4 gene (BMP4) has been considered a prime candidate gene with evidence accumulated from animal experimental studies, human linkage studies, as well as candidate gene association studies. The aim of the current study is to test for linkage and association between BMP4 and NSCL/P that could be missed in genome-wide association studies (GWAS) when genotypic (G) main effects alone were considered., Methodology/principal Findings: We performed the analysis considering G and interactions with multiple maternal environmental exposures using additive conditional logistic regression models in 895 Asian and 681 European complete NSCL/P trios. Single nucleotide polymorphisms (SNPs) that passed the quality control criteria among 122 genotyped and 25 imputed single nucleotide variants in and around the gene were used in analysis. Selected maternal environmental exposures during 3 months prior to and through the first trimester of pregnancy included any personal tobacco smoking, any environmental tobacco smoke in home, work place or any nearby places, any alcohol consumption and any use of multivitamin supplements. A novel significant association held for rs7156227 among Asian NSCL/P and non-syndromic cleft lip and palate (NSCLP) trios after Bonferroni correction which was not seen when G main effects alone were considered in either allelic or genotypic transmission disequilibrium tests. Odds ratios for carrying one copy of the minor allele without maternal exposure to any of the four environmental exposures were 0.58 (95%CI = 0.44, 0.75) and 0.54 (95%CI = 0.40, 0.73) for Asian NSCL/P and NSCLP trios, respectively. The Bonferroni P values corrected for the total number of 117 tested SNPs were 0.0051 (asymptotic P = 4.39*10(-5)) and 0.0065 (asymptotic P = 5.54*10(-5)), accordingly. In European trios, no significant association was seen for any SNPs after Bonferroni corrections for the total number of 120 tested SNPs., Conclusions/significance: Our findings add evidence from GWAS to support the role of BMP4 in susceptibility to NSCL/P originally identified in linkage and candidate gene association studies.

Case-parent trio studies are commonly employed in genetics to detect variants underlying common complex disease risk. Both commercial and freely available software suites for genetic data analysis usually contain methods for case-parent trio designs. A user might, however, experience limitations with these packages, which can include missing functionality to extend the software if a desired analysis has not been implemented, and the inability to programmatically capture all the software versions used for low-level processing and high-level inference of genomic data, a critical consideration in particular for high-throughput experiments. Here, we present a software vignette (i.e., a manual with step by step instructions and examples to demonstrate software functionality) for reproducible genome-wide analyses of case-parent trio data using the open source Bioconductor package trio. The workflow for the practitioner uses data from previous genetic trio studies to illustrate functions for marginal association tests, assessment of parent-of-origin effects, power and sample size calculations, and functions to detect gene-gene and gene-environment interactions associated with disease., (© 2014 WILEY PERIODICALS, INC.)

Background: Urothelial carcinoma (UC) is characterized by multiple recurrent chromosomal changes on a background of increasing genomic instability. To define target genes of recurrent deletions and amplifications, we explored which gene alterations are common in UC, in two recently established cell lines, BC44 and BC61., Materials and Methods: Genes located in regions of gain or deletion in the cell lines were identified by array comparative genomic hybridization (aCGH). Six published microarray datasets were analyzed for genes differentially expressed between urothelial tumor vs. normal tissues. Gene expression and chromosomal changes were compared in BC61 cells., Results: The cell lines share homozygous deletions at 9p21 around CDKN2A and amplifications at 11q13.2 around CCND1. In both cell lines 11 genes were commonly lost and 115 gained. Across UC in general, 230 genes were expressed stronger and 349 weaker; a subset displaying corresponding genetic changes in the cell lines. The commonly affected subset contains well-investigated genes like E2F1 and CCNE1, but also several genes not yet sufficiently investigated in UC., Discussion: Our approach highlights genes involved in cell cycle regulation, apoptosis and signal transduction as commonly deregulated across UC. Nevertheless, many chromosomal regions undergoing recurrent changes harbor several commonly deregulated genes that may act jointly in UC development and progression., (Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.)

Background: Copy number variants (CNVs) may play an important part in the development of common birth defects such as oral clefts, and individual patients with multiple birth defects (including clefts) have been shown to carry small and large chromosomal deletions. In this paper we investigate de novo deletions defined as DNA segments missing in an oral cleft proband but present in both unaffected parents. We compare de novo deletion frequencies in children of European ancestry with an isolated, non-syndromic oral cleft to frequencies in children of European ancestry from randomly sampled trios., Results: We identified a genome-wide significant 62 kilo base (kb) non-coding region on chromosome 7p14.1 where de novo deletions occur more frequently among oral cleft cases than controls. We also observed wider de novo deletions among cleft lip and palate (CLP) cases than seen among cleft palate (CP) and cleft lip (CL) cases., Conclusions: This study presents a region where de novo deletions appear to be involved in the etiology of oral clefts, although the underlying biological mechanisms are still unknown. Larger de novo deletions are more likely to interfere with normal craniofacial development and may result in more severe clefts. Study protocol and sample DNA source can severely affect estimates of de novo deletion frequencies. Follow-up studies are needed to further validate these findings and to potentially identify additional structural variants underlying oral clefts.

Nonsyndromic cleft palate (CP) is one of the most common human birth defects and both genetic and environmental risk factors contribute to its etiology. We conducted a genome-wide association study (GWAS) using 550 CP case-parent trios ascertained in an international consortium. Stratified analysis among trios with different ancestries was performed to test for GxE interactions with common maternal exposures using conditional logistic regression models. While no single nucleotide polymorphism (SNP) achieved genome-wide significance when considered alone, markers in SLC2A9 and the neighboring WDR1 on chromosome 4p16.1 gave suggestive evidence of gene-environment interaction with environmental tobacco smoke (ETS) among 259 Asian trios when the models included a term for GxE interaction. Multiple SNPs in these two genes were associated with increased risk of nonsyndromic CP if the mother was exposed to ETS during the peri-conceptual period (3 months prior to conception through the first trimester). When maternal ETS was considered, fifteen of 135 SNPs mapping to SLC2A9 and 9 of 59 SNPs in WDR1 gave P values approaching genome-wide significance (10(-6)

To locate multiple interacting quantitative trait loci (QTL) influencing a trait of interest within experimental populations, usually methods as the Cockerham's model are applied. Within this framework, interactions are understood as the part of the joined effect of several genes which cannot be explained as the sum of their additive effects. However, if a change in the phenotype (as disease) is caused by Boolean combinations of genotypes of several QTLs, this Cockerham's approach is often not capable to identify them properly. To detect such interactions more efficiently, we propose a logic regression framework. Even though with the logic regression approach a larger number of models has to be considered (requiring more stringent multiple testing correction) the efficient representation of higher order logic interactions in logic regression models leads to a significant increase of power to detect such interactions as compared to a Cockerham's approach. The increase in power is demonstrated analytically for a simple two-way interaction model and illustrated in more complex settings with simulation study and real data analysis.

Statistical approaches to evaluate interactions between single nucleotide polymorphisms (SNPs) and SNP-environment interactions are of great importance in genetic association studies, as susceptibility to complex disease might be related to the interaction of multiple SNPs and/or environmental factors. With these methods under active development, algorithms to simulate genomic data sets are needed to ensure proper type I error control of newly proposed methods and to compare power with existing methods. In this paper we propose an efficient method for a haplotype-based simulation of case-parent trios when the disease risk is thought to depend on possibly higher-order epistatic interactions or gene-environment interactions with binary exposures., (Copyright © 2013 S. Karger AG, Basel.)

Background : Isolated, nonsyndromic cleft lip with or without cleft palate is a common human congenital malformation with a complex and heterogeneous etiology. Genes coding for fibroblast growth factors and their receptors (FGF/FGFR genes) are excellent candidate genes. Methods : We tested single-nucleotide polymorphic markers in 10 FGF/FGFR genes (including FGFBP1, FGF2, FGF10, FGF18, FGFR1, FGFR2, FGF19, FGF4, FGF3, and FGF9) for genotypic effects, interactions with one another, and with common maternal environmental exposures in 221 Asian and 76 Maryland case-parent trios ascertained through a child with isolated, nonsyndromic cleft lip with or without cleft palate. Results : Both FGFR1 and FGF19 yielded evidence of linkage and association in the transmission disequilibrium test, confirming previous evidence. Haplotypes of three single-nucleotide polymorphisms in FGFR1 were nominally significant among Asian trios. Estimated odds ratios for individual single-nucleotide polymorphic markers and haplotypes of multiple markers in FGF19 ranged from 1.31 to 1.87. We also found suggestive evidence of maternal genotypic effects for markers in FGF2 and FGF10 among Asian trios. Tests for gene-environment (G × E) interaction between markers in FGFR2 and maternal smoking or multivitamin supplementation yielded significant evidence of G × E interaction separately. Tests of gene-gene (G × G) interaction using Cordell's method yielded significant evidence between single-nucleotide polymorphisms in FGF9 and FGF18, which was confirmed in an independent sample of trios from an international consortium. Conclusion : Our results suggest several genes in the FGF/FGFR family may influence risk for isolated, nonsyndromic cleft lip with or without cleft palate through distinct biological mechanisms.

Background: In studies of case-parent trios, we define copy number variants (CNVs) in the offspring that differ from the parental copy numbers as de novo and of interest for their potential functional role in disease. Among the leading array-based methods for discovery of de novo CNVs in case-parent trios is the joint hidden Markov model (HMM) implemented in the PennCNV software. However, the computational demands of the joint HMM are substantial and the extent to which false positive identifications occur in case-parent trios has not been well described. We evaluate these issues in a study of oral cleft case-parent trios., Results: Our analysis of the oral cleft trios reveals that genomic waves represent a substantial source of false positive identifications in the joint HMM, despite a wave-correction implementation in PennCNV. In addition, the noise of low-level summaries of relative copy number (log R ratios) is strongly associated with batch and correlated with the frequency of de novo CNV calls. Exploiting the trio design, we propose a univariate statistic for relative copy number referred to as the minimum distance that can reduce technical variation from probe effects and genomic waves. We use circular binary segmentation to segment the minimum distance and maximum a posteriori estimation to infer de novo CNVs from the segmented genome. Compared to PennCNV on simulated data, MinimumDistance identifies fewer false positives on average and is comparable to PennCNV with respect to false negatives. Genomic waves contribute to discordance of PennCNV and MinimumDistance for high coverage de novo calls, while highly concordant calls on chromosome 22 were validated by quantitative PCR. Computationally, MinimumDistance provides a nearly 8-fold increase in speed relative to the joint HMM in a study of oral cleft trios., Conclusions: Our results indicate that batch effects and genomic waves are important considerations for case-parent studies of de novo CNV, and that the minimum distance is an effective statistic for reducing technical variation contributing to false de novo discoveries. Coupled with segmentation and maximum a posteriori estimation, our algorithm compares favorably to the joint HMM with MinimumDistance being much faster.