Unbiased and Locally Efficient Estimation of Genetic Effect on Quantitative Trait in the Presence of Population Admixture

Summary Population admixture can be a confounding factor in genetic association studies. Family‐based methods ( Rabinowitz and Larid, 2000 , Human Heredity 50, 211–223) have been proposed in both testing and estimation settings to adjust for this confounding, especially in case‐only association studies. The family‐based methods rely on conditioning on the observed parental genotypes or on the minimal sufficient statistic for the genetic model under the null hypothesis. In some cases, these methods do not capture all the available information due to the conditioning strategy being too stringent. General efficient methods to adjust for population admixture that use all the available information have been proposed ( Rabinowitz, 2002 , Journal of the American Statistical Association 92, 742–758). However these approaches may not be easy to implement in some situations. A previously developed easy‐to‐compute approach adjusts for admixture by adding supplemental covariates to linear models ( Yang et al., 2000 , Human Heredity 50, 227–233). Here is shown that this augmenting linear model with appropriate covariates strategy can be combined with the general efficient methods in Rabinowitz (2002) to provide computationally tractable and locally efficient adjustment. After deriving the optimal covariates, the adjusted analysis can be carried out using standard statistical software packages such as SAS or R . The proposed methods enjoy a local efficiency in a neighborhood of the true model. The simulation studies show that nontrivial efficiency gains can be obtained by using information not accessible to the methods that rely on conditioning on the minimal sufficient statistics. The approaches are illustrated through an analysis of the influence of apolipoprotein E (APOE) genotype on plasma low‐density lipoprotein (LDL) concentration in children.     

A Hierarchical Bayesian Design for Phase I Trials of Novel Combinations of Cancer Therapeutic Agents

Summary We propose a hierarchical model for the probability of dose‐limiting toxicity (DLT) for combinations of doses of two therapeutic agents. We apply this model to an adaptive Bayesian trial algorithm whose goal is to identify combinations with DLT rates close to a prespecified target rate. We describe methods for generating prior distributions for the parameters in our model from a basic set of information elicited from clinical investigators. We survey the performance of our algorithm in a series of simulations of a hypothetical trial that examines combinations of four doses of two agents. We also compare the performance of our approach to two existing methods and assess the sensitivity of our approach to the chosen prior distribution.     

Analysis of continuous covariables in epidemiological studies: Dose‐response modelling and confounder adjustment

The analysis of continuous covariables with regression models commonly used in epidemiology are reviewed and compared. While some methods have been in use for decades, other more recent methods are not yet common or have not yet been formally described. It is shown that recently developed methods such as fractional polynomials and others are very useful to obtain dose‐response curves or for confounder adjustment. Different methods have their specific merits making it difficult to give general recommendations. The application of some of the methods is demonstrated with real data examples from epidemiological studies. Some suggestions for practical strategies in analysing continuous covariables are given.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Confirmation and fine‐mapping of clinical mastitis and somatic cell score QTL in Nordic Holstein cattle

A genome‐wide association study of 2098 progeny‐tested Nordic Holstein bulls genotyped for 36 387 SNPs on 29 autosomes was conducted to confirm and fine‐map quantitative trait loci (QTL) for mastitis traits identified earlier using linkage analysis with sparse microsatellite markers in the same population. We used linear mixed model analysis where a polygenic genetic effect was fitted as a random effect and single SNPs were successively included as fixed effects in the model. We detected 143 SNP‐by‐trait significant associations (P < 0.0001) on 20 chromosomes affecting mastitis‐related traits. Among them, 21 SNP‐by‐trait combinations exceeded the genome‐wide significant threshold. For 12 chromosomes, both the present association study and the previous linkage study detected QTL, and of these, six were in the same chromosomal locations. Strong associations of SNPs with mastitis traits were observed on bovine autosomes 6, 13, 14 and 20. Possible candidate genes for these QTL were identified. Identification of SNPs in linkage disequilibrium with QTL will enable marker‐based selection for mastitis resistance. The candidate genes identified should be further studied to detect candidate polymorphisms underlying these QTL.     

Genetic mapping of recurrent exertional rhabdomyolysis in a population of North American Thoroughbreds

Recurrent exertional rhabdomyolysis is a heritable disorder that results in painful skeletal muscle cramping with exercise in up to 10% of all Thoroughbred racehorses. Here, we report a genome‐wide association study with 48 282 SNPs analyzed among 48 case and 37 control Thoroughbreds. The most significant SNPs spanned approximately 13 Mb on ECA16, and the P‐value of the most significant SNP after correcting for population structure was 8.0 × 10^?6. This region on ECA16 was further evaluated by genotyping 247 SNPs in both the initial population and a second population of 34 case and 98 control Thoroughbreds. Several SNPs across the 13‐Mb region on ECA16 showed significance when each population was analyzed separately; however, the exact positions of the most significant SNPs within this region on ECA16 varied between populations. This variability in location may be attributed to lack of power owing to insufficient sample sizes within each population individually, or to the relative distribution of long, conserved haplotypes, characteristic of the Thoroughbred breed. Future genome‐wide association studies with additional horses would likely improve the power to resolve casual loci located on ECA16 and increase the likelihood of detecting any additional loci on other chromosomes contributing to disease susceptibility.     

Mapping the four‐horned locus and testing the polled locus in three Chinese sheep breeds

Four‐horned sheep are an ideal animal model for illuminating the genetic basis of horn development. The objective of this study was to locate the genetic region responsible for the four‐horned phenotype and to verify a previously reported polled locus in three Chinese breeds. A genome‐wide association study (GWAS) was performed using 34 two‐horned and 32 four‐horned sheep from three Chinese indigenous breeds: Altay, Mongolian and Sishui Fur sheep. The top two significant single nucleotide polymorphisms (SNPs) associated with the four‐horned phenotype were both located in a region spanning positions 132.6 to 132.7 Mb on sheep chromosome 2. Similar locations for the four‐horned trait were previously identified in Jacob, Navajo‐Churro, Damara and Sishui Fur sheep, suggesting a common genetic component underlying the four‐horned phenotype. The two identified SNPs were both downstream of the metaxin 2 (MTX2) gene and the HOXD gene cluster. For the top SNP—OAR2:g.132619300G>A—the strong associations of the AA and AG genotypes with the four‐horned phenotype and the GG genotype with the two‐horned phenotype indicated the dominant inheritance of the four‐horned trait. No significant SNPs for the polled phenotype were identified in the GWAS analysis, and a PCR analysis for the detection of the 1.8‐kb insertion associated with polled sheep in other breeds failed to verify the association with polledness in the three Chinese breeds. This study supports the hypothesis that two different loci are responsible for horn existence and number. This study contributes to the understanding of the molecular regulation of horn development and enriches the knowledge of qualitative traits in domestic animals.     

Pilot study to assess the validity of the single assessment process as a screening tool for dental treatment needs in older people

Objective: To assess the feasibility of using a questionnaire‐based needs assessment tool (D‐E‐N‐T‐A‐L) to screen for dental treatment need as part of the single assessment process (SAP) for older people in Sheffield. Materials and methods: Test validation study comparing questionnaire‐assessed and normative need in two consecutive samples of older adults: 48 living at home in the transition phase of older age and 29 frail older adults living in care homes. Each answered the six D‐E‐N‐T‐A‐L questions as part of SAP and a dental examination was carried out within 2 weeks in participants’ homes. Question‐defined need was then compared to the normative need. Results: Questionnaire‐defined and normative need were high in both the transitional group (83% and 90% respectively) and the frail group (83% and 62%). These high levels of need meant that the sensitivity and positive predictive values of D‐E‐N‐T‐A‐L were high, but the specificity and negative predictive values were low. Conclusion: The high levels of need in these patient groups suggests that preliminary questionnaire‐based screening is an unnecessary step. A clinical examination of all older people undergoing SAP may be necessary. Further research may be warranted on the use of questionnaires to assess dental treatment needs among people with different attendance patterns.     

Powerful testing via hierarchical linkage disequilibrium in haplotype association studies

Marginal tests based on individual SNPs are routinely used in genetic association studies. Studies have shown that haplotype‐based methods may provide more power in disease mapping than methods based on single markers when, for example, multiple disease‐susceptibility variants occur within the same gene. A limitation of haplotype‐based methods is that the number of parameters increases exponentially with the number of SNPs, inducing a commensurate increase in the degrees of freedom and weakening the power to detect associations. To address this limitation, we introduce a hierarchical linkage disequilibrium model for disease mapping, based on a reparametrization of the multinomial haplotype distribution, where every parameter corresponds to the cumulant of each possible subset of a set of loci. This hierarchy present in the parameters enables us to employ flexible testing strategies over a range of parameter sets: from standard single SNP analyses through the full haplotype distribution tests, reducing degrees of freedom and increasing the power to detect associations. We show via extensive simulations that our approach maintains the type I error at nominal level and has increased power under many realistic scenarios, as compared to single SNP and standard haplotype‐based studies. To evaluate the performance of our proposed methodology in real data, we analyze genome‐wide data from the Wellcome Trust Case‐Control Consortium.     

Influences of extreme weather,climate and pesticide use on invertebrates in cereal fields over 42 years

Cereal fields are central to balancing food production and environmental health in the face of climate change. Within them, invertebrates provide key ecosystem services. Using 42 years of monitoring data collected in southern England, we investigated the sensitivity and resilience of invertebrates in cereal fields to extreme weather events and examined the effect of long‐term changes in temperature, rainfall and pesticide use on invertebrate abundance. Of the 26 invertebrate groups examined, eleven proved sensitive to extreme weather events. Average abundance increased in hot/dry years and decreased in cold/wet years for Araneae, Cicadellidae, adult Heteroptera, Thysanoptera, Braconidae, Enicmus and Lathridiidae. The average abundance of Delphacidae, Cryptophagidae and Mycetophilidae increased in both hot/dry and cold/wet years relative to other years. The abundance of all 10 groups usually returned to their long‐term trend within a year after the extreme event. For five of them, sensitivity to cold/wet events was lowest (translating into higher abundances) at locations with a westerly aspect. Some long‐term trends in invertebrate abundance correlated with temperature and rainfall, indicating that climate change may affect them. However, pesticide use was more important in explaining the trends, suggesting that reduced pesticide use would mitigate the effects of climate change.     

A genome‐wide association study suggests new candidate genes for milk production traits in Chinese Holstein cattle

A genome‐wide association study (GWAS) was conducted on 15 milk production traits in Chinese Holstein. The experimental population consisted of 445 cattle, each genotyped by the GGP (GeneSeek genomic profiling)‐BovineLD V3 SNP chip, which had 26 151 public SNPs in its manifest file. After data cleaning, 20 326 SNPs were retained for the GWAS. The phenotypes were estimated breeding values of traits, provided by a public dairy herd improvement program center that had been collected once a month for 3 years. Two statistical models, a fixed‐effect linear regression model and a mixed‐effect linear model, were used to estimate the association effects of SNPs on each of the phenotypes. Genome‐wide significant and suggestive thresholds were set at 2.46E‐06 and 4.95E‐05 respectively. The two statistical models concurrently identified two genome‐wide significant (P < 0.05) SNPs on milk production traits in this Chinese Holstein population. The positional candidate genes, which were the ones closest to these two identified SNPs, were EEF2K (eukaryotic elongation factor 2 kinase) and KLHL1 (kelch like family member 1). These two genes could serve as new candidate genes for milk yield and lactation persistence, yet their roles need to be verified in further function studies.     

Population-based case-control study of isolated congenital cataract

BACKGROUND: The aim of this study was to detect possible etiological factors in the origin of isolated congenital cataracts. METHODS: The data set of the Hungarian Case-Control Surveillance of Congenital Abnormalities, 1980-2002, contains 111 cases of isolated congenital cataract and 111 matched control pairs without the defect, 37,837 population controls without any defects, and 22,744 malformed controls with other nonocular abnormalities. Exposure data and family history are based on prospective medical records, retrospective maternal information, and information obtained by regional nurses during a home visit with nonrespondent mothers. RESULTS: A positive family history indicated an autosomal-dominant origin in 10% of cases. Rubella infections occurred more frequently in case mothers than in control mothers before vaccination against rubella virus was instituted. A higher prevalence of influenza or common cold during pregnancy was found in the case group (55.9%) than in the population control group (18.5%; adjusted odds ratios [ORs], 5.8; 95% confidence interval (CI), 4.0-8.4) or in the malformed control group (21.7%; adjusted OR, 4.7; 95% CI, 3.2-6.9). The prevalence of acute infectious diseases of the respiratory system during pregnancy was also higher in the case group (26.1%) than in the population control group (9.1%; adjusted OR, 3.8; 95% CI, 2.5-5.8), or the malformed control group (9.3%; adjusted OR, 3.4; 95% CI, 2.3-5.3). The higher risk for isolated congenital cataract in cases of mothers with influenza or common cold and acute infectious diseases of the respiratory system during pregnancy was not found after administration of antifever therapy. CONCLUSIONS: Some isolated congenital cataracts are preventable by rubella vaccination and probably by influenza vaccination in the epidemic period. In addition, our results suggest that using antifever therapy for fever-related respiratory diseases may restrict the teratogenic risk of hyperthermia.     

Genetic contributions to precocity traits in racing Thoroughbreds

Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high‐density SNP array data, additive SNP heritability () was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome‐wide association studies (GWAS) for age at first race and age at best race, a 1.98‐Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10^?12 and P = 1.89 × 10^?14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10^?5). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity.     

A Multiphase Design Strategy for Dealing with Participation Bias

Summary A recently funded study of the impact of oral contraceptive use on the risk of bone fracture employed the randomized recruitment scheme of Weinberg and Wacholder (1990, Biometrics 46, 963–975). One potential complication in the bone fracture study is the potential for differential response rates between cases and controls; participation rates in previous, related studies have been around 70%. Although data from randomized recruitment schemes may be analyzed within the two‐phase study framework, ignoring potential differential participation may lead to biased estimates of association. To overcome this, we build on the two‐phase framework and propose an extension by introducing an additional stage of data collection aimed specifically at addressing potential differential participation. Four estimators that correct for both sampling and participation bias are proposed; two are general purpose and two are for the special case where covariates underlying the participation mechanism are discrete. Because the fracture study is ongoing, we illustrate the methods using infant mortality data from North Carolina.     

Detecting and adjusting for small-study effects in meta-analysis

Publication bias and related types of small-study effects threaten the validity of systematic reviews. The existence of small-study effects has been demonstrated in empirical studies. Small-study effects are graphically diagnosed by inspection of the funnel plot. Though observed funnel plot asymmetry cannot be easily linked to a specific reason, tests based on funnel plot asymmetry have been proposed. Beyond a vast range of funnel plot tests, there exist several methods for adjusting treatment effect estimates for these biases. In this article, we consider the trim-and-fill method, the Copas selection model, and more recent regression-based approaches. The methods are exemplified using a meta-analysis from the literature and compared in a simulation study, based on binary response data. They are also applied to a large set of meta-analyses. Some fundamental differences between the approaches are discussed. An assumption common to the trim-and-fill method and the Copas selection model is that the small-study effect is caused by selection. The trim-and-fill method corresponds to an unknown implicit model generated by the symmetry assumption, whereas the Copas selection model is a parametric statistical model. However, it requires a sensitivity analysis. Regression-based approaches are easier to implement and not based on a specific selection model. Both simulations and applications suggest that in the presence of strong selection both the trim-and-fill method and the Copas selection model may not fully eliminate bias, while regression-based approaches seem to be a promising alternative.     

A Comparison of Formulae for Calculating Cost‐Efficient Sample Sizes of Case‐Control Studies with an Internal Validation Scheme

When a case‐control study is planned to include an internal validation study, the sample size of the study and the proportion of validated observations has to be calculated. There are a variety of alternative methods to accomplish this. In this article some possible procedures will be compared in order to clarify whether considerable differences in the suggested optimal designs occur, dependent on the used method.