Genetic regulatory effects in response to a high-cholesterol, high-fat diet in baboons.

Steady-state expression quantitative trait loci (eQTLs) explain only a fraction of disease-associated loci identified through genome-wide association studies (GWASs), while eQTLs involved in gene-by-environment (GxE) interactions have rarely been characterized in humans due to experimental challenges. Using a baboon model, we found hundreds of eQTLs that emerge in adipose, liver, and muscle after prolonged exposure to high dietary fat and cholesterol. Diet-responsive eQTLs exhibit genomic localization and genic features that are distinct from steady-state eQTLs. Furthermore, the human orthologs associated with diet-responsive eQTLs are enriched for GWAS genes associated with human metabolic traits, suggesting that context-responsive eQTLs with more complex regulatory effects are likely to explain GWAS hits that do not seem to overlap with standard eQTLs. Our results highlight the complexity of genetic regulatory effects and the potential of eQTLs with disease-relevant GxE interactions in enhancing the understanding of GWAS signals for human complex disease using non-human primate models.

ADELLE: A global testing method for Trans-eQTL mapping.

Understanding the genetic regulatory mechanisms of gene expression is a challenging and ongoing problem. Genetic variants that are associated with expression levels are readily identified when they are proximal to the gene (i.e., cis-eQTLs), but SNPs distant from the gene whose expression levels they are associated with (i.e., trans-eQTLs) have been much more difficult to discover, even though they account for a majority of the heritability in gene expression levels. A major impediment to the identification of more trans-eQTLs is the lack of statistical methods that are powerful enough to overcome the obstacles of small effect sizes and large multiple testing burden of trans-eQTL mapping. Here, we propose ADELLE, a powerful statistical testing framework that requires only summary statistics and is designed to be most sensitive to SNPs that are associated with multiple gene expression levels, a characteristic of many trans-eQTLs. In simulations, we show that ADELLE is more powerful than other methods at detecting SNPs that are associated with 0.2-2% of the traits. We apply ADELLE to a mouse advanced intercross line data set and show its ability to find trans-eQTLs that were not significant under a standard analysis. This demonstrates that ADELLE is a powerful tool at uncovering trans regulators of genetic expression.

BRASS: Permutation methods for binary traits in genetic association studies with structured samples.

In genetic association analysis of complex traits, permutation testing can be a valuable tool for assessing significance when the distribution of the test statistic is unknown or not well-approximated. This commonly arises, e.g, in tests of gene-set, pathway or genome-wide significance, or when the statistic is formed by machine learning or data adaptive methods. Existing applications include eQTL mapping, association testing with rare variants, inclusion of admixed individuals in genetic association analysis, and epistasis detection among many others. For genetic association testing in samples with population structure and/or relatedness, use of naive permutation can lead to inflated type 1 error. To address this in quantitative traits, the MVNpermute method was developed. However, for association mapping of a binary trait, the relationship between the mean and variance makes both naive permutation and the MVNpermute method invalid. We propose BRASS, a permutation method for binary traits, for use in association mapping in structured samples. In addition to modeling structure in the sample, BRASS allows for covariates, ascertainment and simultaneous testing of multiple markers, and it accommodates a wide range of test statistics. In simulation studies, we compare BRASS to other permutation and resampling-based methods in a range of scenarios that include population structure, familial relatedness, ascertainment and phenotype model misspecification. In these settings, we demonstrate the superior control of type 1 error by BRASS compared to the other 6 methods considered. We apply BRASS to assess genome-wide significance for association analyses in domestic dog for elbow dysplasia (ED) and idiopathic epilepsy (IE). For both traits we detect previously identified associations, and in addition, for ED, we detect significant association with a SNP on chromosome 35 that was not detected by previous analyses, demonstrating the potential of the method.

Characterization of gene expression patterns in response to an orthotospovirus infection between two diploid peanut species and their hybrid.

Tomato spotted wilt orthotospovirus (TSWV) transmitted by thrips causes significant yield loss in peanut (Arachis hypogaea L.) production. Use of peanut cultivars with moderate field resistance has been critical for TSWV management. However, current TSWV resistance is often not adequate, and the availability of sources of tetraploid resistance to TSWV is very limited. Allotetraploids derived by crossing wild diploid species could help introgress alleles that confer TSWV resistance into cultivated peanut. Thrips-mediated TSWV screening identified two diploids and their allotetraploid possessing the AA, BB, and AABB genomes Arachis stenosperma V10309, Arachis valida GK30011, and [A. stenosperma × A. valida]4x (ValSten1), respectively. These genotypes had reduced TSWV infection and accumulation in comparison with peanut of pure cultivated pedigree. Transcriptomes from TSWV-infected and non-infected samples from A. stenosperma, A. valida, and ValSten1 were assembled, and differentially expressed genes (DEGs) following TSWV infection were assessed. There were 3,196, 8,380, and 1,312 significant DEGs in A. stenosperma, A. valida, and ValSten1, respectively. A higher proportion of genes decreased in expression following TSWV infection for A. stenosperma and ValSten1, whereas a higher proportion of genes increased in expression following infection in A. valida. The number of DEGs previously annotated as defense-related in relation to abiotic and biotic stress was highest in A. valida followed by ValSten1 and A. stenosperma. Plant phytohormone and photosynthesis genes also were differentially expressed in greater numbers in A. valida followed by ValSten1 and A. stenosperma, with over half of those exhibiting decreases in expression.

Evaluation of Wild Peanut Species and Their Allotetraploids for Resistance against Thrips and Thrips-Transmitted Tomato Spotted Wilt Orthotospovirus (TSWV).

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) causes spotted wilt disease in peanut (Arachis hypogaea L.) and limits yield. Breeding programs have been developing TSWV-resistant cultivars, but availability of sources of resistance against TSWV in cultivated germplasm is extremely limited. Diploid wild Arachis species can serve as important sources of resistance, and despite ploidy barriers (cultivated peanut is tetraploid), their usage in breeding programs is now possible because of the knowledge and development of induced interspecific allotetraploid hybrids. This study screened 10 wild diploid Arachis and six induced allotetraploid genotypes via thrips-mediated TSWV transmission assays and thrips' feeding assays in the greenhouse. Three parameters were evaluated: percent TSWV infection, virus accumulation, and temporal severity of thrips feeding injury. Results indicated that the diploid A. stenosperma accession V10309 and its derivative-induced allotetraploid ValSten1 had the lowest TSWV infection incidences among the evaluated genotypes. Allotetraploid BatDur1 had the lowest thrips-inflicted damage at each week post thrips release, while diploid A. batizocoi accession K9484 and A. duranensis accession V14167 had reduced feeding damage one week post thrips release, and diploids A. valida accession GK30011 and A. batizocoi had reduced feeding damage three weeks post thrips releasethan the others. Overall, plausible TSWV resistance in diploid species and their allotetraploid hybrids was characterized by reduced percent TSWV infection, virus accumulation, and feeding severity. Furthermore, a few diploids and tetraploid hybrids displayed antibiosis against thrips. These results document evidence for resistance against TSWV and thrips in wild diploid Arachis species and peanut-compatible-induced allotetraploids.

Genetic regulatory effects in response to a high cholesterol, high fat diet in baboons.

Steady-state expression quantitative trait loci (eQTLs) explain only a fraction of disease-associated loci identified through genome-wide association studies (GWAS), while eQTLs involved in gene-by-environment (GxE) interactions have rarely been characterized in humans due to experimental challenges. Using a baboon model, we found hundreds of eQTLs that emerge in adipose, liver, and muscle after prolonged exposure to high dietary fat and cholesterol. Diet-responsive eQTLs exhibit genomic localization and genic features that are distinct from steady-state eQTLs. Furthermore, the human orthologs associated with diet-responsive eQTLs are enriched for GWAS genes associated with human metabolic traits, suggesting that context-responsive eQTLs with more complex regulatory effects are likely to explain GWAS hits that do not seem to overlap with standard eQTLs. Our results highlight the complexity of genetic regulatory effects and the potential of eQTLs with disease-relevant GxE interactions in enhancing the understanding of GWAS signals for human complex disease using nonhuman primate models.

Application of Equal Local Levels to Improve Q-Q Plot Testing Bands with R Package qqconf.

Quantile-Quantile (Q-Q) plots are often difficult to interpret because it is unclear how large the deviation from the theoretical distribution must be to indicate a lack of fit. Most Q-Q plots could benefit from the addition of meaningful global testing bands, but the use of such bands unfortunately remains rare because of the drawbacks of current approaches and packages. These drawbacks include incorrect global Type I error rate, lack of power to detect deviations in the tails of the distribution, relatively slow computation for large data sets, and limited applicability. To solve these problems, we apply the equal local levels global testing method, which we have implemented in the R Package qqconf, a versatile tool to create Q-Q plots and probability-probability (P-P) plots in a wide variety of settings, with simultaneous testing bands rapidly created using recently-developed algorithms. qqconf can easily be used to add global testing bands to Q-Q plots made by other packages. In addition to being quick to compute, these bands have a variety of desirable properties, including accurate global levels, equal sensitivity to deviations in all parts of the null distribution (including the tails), and applicability to a range of null distributions. We illustrate the use of qqconf in several applications: assessing normality of residuals from regression, assessing accuracy of p values, and use of Q-Q plots in genome-wide association studies.

Impact of Host Resistance to Tomato Spotted Wilt Orthotospovirus in Peanut Cultivars on Virus Population Genetics and Thrips Fitness.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) is a major constraint to peanut production in the southeastern United States. Peanut cultivars with resistance to TSWV have been widely used for over twenty years. Intensive usage of resistant cultivars has raised concerns about possible selection pressure against TSWV and a likelihood of resistance breakdown. Population genetics of TSWV isolates collected from cultivars with varying levels of TSWV resistance was investigated using five TSWV genes. Phylogenetic trees of genes did not indicate host resistance-based clustering of TSWV isolates. Genetic variation in TSWV isolates and neutrality tests suggested recent population expansion. Mutation and purifying selection seem to be the major forces driving TSWV evolution. Positive selection was found in N and RdRp genes but was not influenced by TSWV resistance. Population differentiation occurred between isolates collected from 1998 and 2010 and from 2016 to 2019 but not between isolates from susceptible and resistant cultivars. Evaluated TSWV-resistant cultivars differed, albeit not substantially, in their susceptibility to thrips. Thrips oviposition was reduced, and development was delayed in some cultivars. Overall, no evidence was found to support exertion of selection pressure on TSWV by host resistance in peanut cultivars, and some cultivars differentially affected thrips fitness than others.

Discrepancies in Serology-Based and Nucleic Acid-Based Detection and Quantitation of Tomato Spotted Wilt Orthotospovirus in Leaf and Root Tissues from Symptomatic and Asymptomatic Peanut Plants.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) causes spotted wilt disease in peanuts. A serological test (DAS-ELISA) is often used to detect TSWV in peanut leaf samples. However, in a few studies, DAS-ELISA detected more TSWV infection in root than leaf samples. It was not clear if the increased detection was due to increased TSWV accumulation in root tissue or merely an overestimation. Additionally, it was unclear if TSWV detection in asymptomatic plants would be affected by the detection technique. TSWV infection in leaf and root tissue from symptomatic and asymptomatic plants was compared via DAS-ELISA, RT-PCR, and RT-qPCR. TSWV incidence did not vary by DAS-ELISA, RT-PCR, and RT-qPCR in leaf and root samples of symptomatic plants or in leaf samples of asymptomatic plants. In contrast, significantly more TSWV infection and virus load were detected in root samples of asymptomatic plants via DAS-ELISA than other techniques suggesting that DAS-ELISA overestimated TSWV incidence and load. TSWV loads from symptomatic plants via RT-qPCR were higher in leaf than root samples, while TSWV loads in leaf and root samples from asymptomatic plants were not different but were lower than those in symptomatic plants. These findings suggested that peanut tissue type and detection technique could affect accurate TSWV detection and/or quantitation.

Defense-Related Gene Expression Following an Orthotospovirus Infection Is Influenced by Host Resistance in Arachis hypogaea.

Planting resistant cultivars is the most effective tactic to manage the thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) in peanut plants. However, molecular mechanisms conferring resistance to TSWV in resistant cultivars are unknown. In this study, transcriptomes of TSWV-susceptible (SunOleic 97R) and field-resistant (Tifguard) peanut cultivars with and without TSWV infection were assembled and differentially expressed genes (DEGs) were compared. There were 4605 and 2579 significant DEGs in SunOleic 97R and Tifguard, respectively. Despite the lower number of DEGs in Tifguard, an increased proportion of defense-related genes were upregulated in Tifguard than in the susceptible cultivar. Examples included disease resistance (R) proteins, leucine-rich repeats, stilbene synthase, dicer, and calmodulin. Pathway analysis revealed the increased downregulation of genes associated with defense and photosynthesis in the susceptible cultivar rather than in the resistant cultivar. These results suggest that essential physiological functions were less perturbed in the resistant cultivar than in the susceptible cultivar and that the defense response following TSWV infection was more robust in the resistant cultivar than in the susceptible cultivar.

Species Composition, Temporal Abundance and Distribution of Insect Captures Inside and Outside Commercial Peanut Shelling Facilities.

Insect populations were studied within two commercial peanut shelling facilities located in the southeastern United States. Commercially available pheromone/kairomone-baited dome traps and pheromone-baited flight traps were deployed throughout processing and shipping portions of the shelling plants and serviced weekly over one year. Lasioderma serricorne, Tribolium castaneum, Typhaea stercorea, Carpophilus spp., Plodia interpunctella and Cadra cautella were the most common captures across locations. Lasioderma serricorne made up 87% and 88% of all captures in dome traps in plants one and two, respectively. While L. serricorne was not captured during the winter months in flight traps, it was captured with near 100% frequency in dome traps, suggesting that populations persisted throughout the year inside the facilities. Tribolium castaneum populations were active year round. Across insect species and trap type, temperature was a significant covariate for explaining variation in insect counts. After accounting for the effect of temperature, there were always more insects captured in the processing portions of the facilities compared to the shipping areas. A negative linear relationship was observed between captures of L. serricorne and T. castaneum and trap distance from in-shell peanuts entering the shelling facilities. Conversely, fungivores were more evenly distributed throughout all parts of the shelling plants. These data suggest that management efforts should be focused where in-shell peanuts enter to reduce breeding and harborage sites for grain feeding insects.

Genome-wide Associations Reveal Human-Mouse Genetic Convergence and Modifiers of Myogenesis, CPNE1 and STC2.

Muscle bulk in adult healthy humans is highly variable even after height, age, and sex are accounted for. Low muscle mass, due to fewer and/or smaller constituent muscle fibers, would exacerbate the impact of muscle loss occurring in aging or disease. Genetic variability substantially influences muscle mass differences, but causative genes remain largely unknown. In a genome-wide association study (GWAS) on appendicular lean mass (ALM) in a population of 85,750 middle-aged (aged 38-49 years) individuals from the UK Biobank (UKB), we found 182 loci associated with ALM (p < 5 × 10-8). We replicated associations for 78% of these loci (p < 5 × 10-8) with ALM in a population of 181,862 elderly (aged 60-74 years) individuals from UKB. We also conducted a GWAS on hindlimb skeletal muscle mass of 1,867 mice from an advanced intercross between two inbred strains (LG/J and SM/J); this GWAS identified 23 quantitative trait loci. Thirty-eight positional candidates distributed across five loci overlapped between the two species. In vitro studies of positional candidates confirmed CPNE1 and STC2 as modifiers of myogenesis. Collectively, these findings shed light on the genetics of muscle mass variability in humans and identify targets for the development of interventions for treatment of muscle loss. The overlapping results between humans and the mouse model GWAS point to shared genetic mechanisms across species.

Comparison of transcriptomes of an orthotospovirus vector and non-vector thrips species.

Thrips transmit one of the most devastating plant viruses worldwide-tomato spotted wilt tospovirus (TSWV). Tomato spotted wilt tospovirus is a type species in the genus Orthotospovirus and family Tospoviridae. Although there are more than 7,000 thrips species, only nine thrips species are known to transmit TSWV. In this study, we investigated the molecular factors that could affect thrips ability to transmit TSWV. We assembled transcriptomes of a vector, Frankliniella fusca [Hinds], and a non-vector, Frankliniella tritici [Fitch], and performed qualitative comparisons of contigs associated with virus reception, virus infection, and innate immunity. Annotations of F. fusca and F. tritici contigs revealed slight differences across biological process and molecular functional groups. Comparison of virus cell surface receptors revealed that homologs of integrin were present in both species. However, homologs of another receptor, heperan sulfate, were present in F. fusca alone. Contigs associated with virus replication were identified in both species, but a contig involved in inhibition of virus replication (radical s-adenosylmethionine) was only present in the non-vector, F. tritici. Additionally, some differences in immune signaling pathways were identified between vector and non-vector thrips. Detailed investigations are necessary to functionally characterize these differences between vector and non-vector thrips and assess their relevance in orthotospovirus transmission.

Kinpute: using identity by descent to improve genotype imputation.

MOTIVATION: Genotype imputation, though generally accurate, often results in many genotypes being poorly imputed, particularly in studies where the individuals are not well represented by standard reference panels. When individuals in the study share regions of the genome identical by descent (IBD), it is possible to use this information in combination with a study-specific reference panel (SSRP) to improve the imputation results. Kinpute uses IBD information-due to recent, familial relatedness or distant, unknown ancestors-in conjunction with the output from linkage disequilibrium (LD) based imputation methods to compute more accurate genotype probabilities. Kinpute uses a novel method for IBD imputation, which works even in the absence of a pedigree, and results in substantially improved imputation quality. RESULTS: Given initial estimates of average IBD between subjects in the study sample, Kinpute uses a novel algorithm to select an optimal set of individuals to sequence and use as an SSRP. Kinpute is designed to use as input both this SSRP and the genotype probabilities output from other LD-based imputation software, and uses a new method to combine the LD imputed genotype probabilities with IBD configurations to substantially improve imputation. We tested Kinpute on a human population isolate where 98 individuals have been sequenced. In half of this sample, whose sequence data was masked, we used Impute2 to perform LD-based imputation and Kinpute was used to obtain higher accuracy genotype probabilities. Measures of imputation accuracy improved significantly, particularly for those genotypes that Impute2 imputed with low certainty. AVAILABILITY AND IMPLEMENTATION: Kinpute is an open-source and freely available C++ software package that can be downloaded from. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome wide association analysis in a mouse advanced intercross line.

The LG/J x SM/J advanced intercross line of mice (LG x SM AIL) is a multigenerational outbred population. High minor allele frequencies, a simple genetic background, and the fully sequenced LG and SM genomes make it a powerful population for genome-wide association studies. Here we use 1,063 AIL mice to identify 126 significant associations for 50 traits relevant to human health and disease. We also identify thousands of cis- and trans-eQTLs in the hippocampus, striatum, and prefrontal cortex of ~200 mice. We replicate an association between locomotor activity and Csmd1, which we identified in an earlier generation of this AIL, and show that Csmd1 mutant mice recapitulate the locomotor phenotype. Our results demonstrate the utility of the LG x SM AIL as a mapping population, identify numerous novel associations, and shed light on the genetic architecture of mammalian behavior.

Resistance to Thrips in Peanut and Implications for Management of Thrips and Thrips-Transmitted Orthotospoviruses in Peanut.

Thrips are major pests of peanut (Arachis hypogaea L.) worldwide, and they serve as vectors of devastating orthotospoviruses such as Tomato spotted wilt virus (TSWV) and Groundnut bud necrosis virus (GBNV). A tremendous effort has been devoted to developing peanut cultivars with resistance to orthotospoviruses. Consequently, cultivars with moderate field resistance to viruses exist, but not much is known about host resistance to thrips. Integrating host plant resistance to thrips in peanut could suppress thrips feeding damage and reduce virus transmission, will decrease insecticide usage, and enhance sustainability in the production system. This review focuses on details of thrips resistance in peanut and identifies future directions for incorporating thrips resistance in peanut cultivars. Research on thrips-host interactions in peanut is predominantly limited to field evaluations of feeding damage, though, laboratory studies have revealed that peanut cultivars could differentially affect thrips feeding and thrips biology. Many runner type cultivars, field resistant to TSWV, representing diverse pedigrees evaluated against thrips in the greenhouse revealed that thrips preferred some cultivars over others, suggesting that antixenosis "non-preference" could contribute to thrips resistance in peanut. In other crops, morphological traits such as leaf architecture and waxiness and spectral reflectance have been associated with thrips non-preference. It is not clear if foliar morphological traits in peanut are associated with reduced preference or non-preference of thrips and need to be evaluated. Besides thrips non-preference, thrips larval survival to adulthood and median developmental time were negatively affected in some peanut cultivars and in a diploid peanut species Arachis diogoi (Hoehne) and its hybrids with a Virginia type cultivar, indicating that antibiosis (negative effects on biology) could also be a factor influencing thrips resistance in peanut. Available field resistance to orthotospoviruses in peanut is not complete, and cultivars can suffer substantial yield loss under high thrips and virus pressure. Integrating thrips resistance with available virus resistance would be ideal to limit losses. A discussion of modern technologies such as transgenic resistance, marker assisted selection and RNA interference, and future directions that could be undertaken to integrate resistance to thrips and to orthotospoviruses in peanut cultivars is included in this article.

Observing the Effect of Soil-Applied Insecticides on Wireworm (Coleoptera: Elateridae) Behavior and Mortality Using Radiographic Imaging.

Radiographic imaging was used to study corn wireworm, Melanotus communis (Gyllenhal) (Coleoptera: Elateridae), behavior in the soil relative to Irish potato, Solanum tuberosum L. (Solanales: Solanaceae), placed above a barrier of insecticide-treated soil. Behavioral response to three insecticides was evaluated by 1) calculating the percentage of wireworms that breached the zone of insecticide-treated soil, 2) comparing the variability in the mean distance between each wireworm and the center of the tuber in each arena compartment, 3) comparing the cumulative distance of wireworm travel, and 4) comparing incidence and severity of wireworm-inflicted tuber injury. The percentage of wireworms that breached the insecticide zone was significantly lower in the bifenthrin treatment than the untreated check, but not different from clothianidin or fipronil. Limited difference between treatments in relation to the cumulative distance traveled was observed where bifenthrin inhibited travel distance more than other insecticides tested. No difference in variability of movement in relation to the food source was observed between treatments. Bifenthrin and fipronil treatments resulted in the greatest reduction in wireworm injury incidence and severity compared to the untreated check. While bifenthrin suppressed wireworm movement and feeding more than other insecticides tested, exposure to fipronil resulted in higher wireworm mortality at all sample dates.

Investigating multiple sclerosis genetic susceptibility on the founder population of east-central Sardinia via association and linkage analysis of immune-related loci.

BACKGROUND: A wealth of single-nucleotide polymorphisms (SNPs) responsible for multiple sclerosis (MS) susceptibility have been identified; however, they explain only a fraction of MS heritability. OBJECTIVES: We contributed to discovery of new MS susceptibility SNPs by studying a founder population with high MS prevalence. METHODS: We analyzed ImmunoChip data from 15 multiplex families and 94 unrelated controls from the Nuoro Province, Sardinia, Italy. We tested each SNP for both association and linkage with MS, the linkage being explored in terms of identity-by-descent (IBD) sharing excess and using gene dropping to compute a corresponding empirical p-value. By targeting regions that are both associated and in linkage with MS, we increase chances of identifying interesting genomic regions. RESULTS: We identified 486 MS-associated (p < 1 × 10-4) and 18,426 MS-linked (p < 0.05) SNPs. A total of 111 loci were both linked and associated with MS, 18 of them pointing to 14 non-major histocompatibility complex (MHC) genes, and 93 of them located in the MHC region. CONCLUSION: We discovered new suggestive signals and confirmed some previously identified ones. We believe this to represent a significant step toward an understanding of the genetic basis of MS.

A LASSO penalized regression approach for genome-wide association analyses using related individuals: application to the Genetic Analysis Workshop 19 simulated data.

We propose a novel LASSO (least absolute shrinkage and selection operator) penalized regression method used to analyze samples consisting of (potentially) related individuals. Developed in the context of linear mixed models, our method models the relatedness of individuals in the sample through a random effect whose covariance structure is a linear function of known matrices with elements combinations of the condensed coefficients of identity between the individuals in the sample. We implement our method to analyze the simulated family data provided by the 19th Genetic Analysis Workshop in an effort to identify loci regulating the simulated trait of systolic blood pressure. The analyses were performed with full knowledge of the simulation model. Our findings demonstrate that we can significantly reduce the rate of false positive signals by incorporating the relatedness of the study participants.