Admixture-matched case-control study: a practical approach for genetic association studies in admixed populations

Case-control genetic association studies in admixed populations are known to be susceptible to genetic confounding due to population stratification. The transmission/disequilibrium test (TDT) approach can avoid this problem. However, the TDT is expensive and impractical for late-onset diseases. Case-control study designs, in which, cases and controls are matched by admixture, can be an appealing and a suitable alternative for genetic association studies in admixed populations. In this study, we applied this matching strategy when recruiting our African American participants in the Study of African American, Asthma, Genes and Environments. Group admixture in this cohort consists of 83% African ancestry and 17% European ancestry, which was consistent with reports from other studies. By carrying out several complementary analyses, our results show that there is a substructure in the cohort, but that the admixture distributions are almost identical in cases and controls, and also in cases only. We performed association tests for asthma-related traits with ancestry, and only found that FEV(1), a measure for baseline pulmonary function, was associated with ancestry after adjusting for socio-economic and environmental risk factors (P=0.01). We did not observe an excess of type I error rate in our association tests for ancestry informative markers and asthma-related phenotypes when ancestry was not adjusted in the analyses. Furthermore, using the association tests between genetic variants in a known asthma candidate gene, beta(2) adrenergic receptor (beta(2)AR) and DeltaFEF(25-75), an asthma-related phenotype, as an example, we demonstrated population stratification was not a confounder in our genetic association. Our present work demonstrates that admixture-matched case-control strategies can efficiently control population stratification confounding in admixed populations.     

Multi-parent populations in crops: a toolbox integrating genomics and genetic mapping with breeding

Crop populations derived from experimental crosses enable the genetic dissection of complex traits and support modern plant breeding. Among these, multi-parent populations now play a central role. By mixing and recombining the genomes of multiple founders, multi-parent populations combine many commonly sought beneficial properties of genetic mapping populations. For example, they have high power and resolution for mapping quantitative trait loci, high genetic diversity and minimal population structure. Many multi-parent populations have been constructed in crop species, and their inbred germplasm and associated phenotypic and genotypic data serve as enduring resources. Their utility has grown from being a tool for mapping quantitative trait loci to a means of providing germplasm for breeding programmes. Genomics approaches, including de novo genome assemblies and gene annotations for the population founders, have allowed the imputation of rich sequence information into the descendent population, expanding the breadth of research and breeding applications of multi-parent populations. Here, we report recent successes from crop multi-parent populations in crops. We also propose an ideal genotypic, phenotypic and germplasm ‘package’ that multi-parent populations should feature to optimise their use as powerful community resources for crop research, development and breeding.Subject terms: Plant genetics, Plant breeding, Agricultural genetics, Quantitative trait

Over recent years, numerous multi-parent populations (MPPs) have been successfully developed in crops (Huang et al. 2015; Cockram and Mackay 2018). MPPs bring together key genomic, phenotypic and germplasm resources to form a platform for research and development. In this review, we examine three themes covering new developments in crop MPP research: (1) we survey the rapidly expanding variety of crop MPPs, explaining how differences in their design and construction affect their power and precision in mapping quantitative trait loci (QTL), on which we provide a brief primer. (2) We review the use of genomic technologies in MPPs, which have proven particularly suitable for gathering dense genomic information across large populations. We make general recommendations for collecting genotypic resources in MPPs. (3) We discuss successful applications of MPPs, particularly where they have been used for breeding and pre-breeding. This includes the identification of QTL, the application of genomic prediction to MPPs, and the direct use of MPP lines as germplasm for varietal release or pre-breeding. These recent developments have shown the potential of MPPs for crop improvement.     

High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.     

Multiplexed mass spectrometric immunoassay in biomarker research: a novel approach to the determination of a myocardial infarct

Reported here is the development of a multiplexed mass spectrometric immunoassay (MSIA) for the detection of myocardial infarction (MI). The assay is the product of a study that systematically progresses from biomarker discovery--to identification and verification--to assay design, data analysis, and statistical challenge. During targeted population proteomics investigations, two novel biomarkers, serum amyloid A1alpha and S-sulfated transthyretin, were found to be responsive to MI. These putative markers were subsequently screened in larger cohorts of individuals to verify their responsiveness toward MI. Upon verification, a multiplexed assay was designed that was capable of simultaneously monitoring the new markers plus a previously established MI-marker (myoglobin). The multiplexed MSIA was applied to two 96-sample sets comprised of 48-MI/48-healthy and 19-MI/77-healthy, which served as training and case cohorts, respectively. Data evaluation using either preset reference levels or multivariate analysis exhibited sensitivities and specificities of >97%. These findings illustrate the importance of using systematic approaches in clinical proteomics to discover biomarkers and produce high-performance assays relevant to disease.     

Management of subdivided populations in conservation programs: development of a novel dynamic system

Within the context of a conservation program the management of subdivided populations implies a compromise between the control of the global genetic diversity, the avoidance of high inbreeding levels, and, sometimes, the maintenance of a certain degree of differentiation between subpopulations. We present a dynamic and flexible methodology, based on genealogical information, for the maximization of the genetic diversity (measured through the global population coancestry) in captive subdivided populations while controlling/restricting the levels of inbreeding. The method is able to implement specific restrictions on the desired relative levels of coancestry between and within subpopulations. By accounting for the particular genetic population structure, the method determines the optimal contributions (i.e., number of offspring) of each individual, the number of migrants, and the particular subpopulations involved in the exchange of individuals. Computer simulations are used to illustrate the procedure and its performance in a range of reasonable scenarios. The method performs well in most situations and is shown to be more efficient than the commonly accepted one-migrant-per-generation strategy.     

Heterogeneous capture-recapture models with covariates: a partial likelihood approach for closed populations

In practice, when analyzing data from a capture-recapture experiment it is tempting to apply modern advanced statistical methods to the observed capture histories. However, unless the analysis takes into account that the data have only been collected from individuals who have been captured at least once, the results may be biased. Without the development of new software packages, methods such as generalized additive models, generalized linear mixed models, and simulation-extrapolation cannot be readily implemented. In contrast, the partial likelihood approach allows the analysis of a capture-recapture experiment to be conducted using commonly available software. Here we examine the efficiency of this approach and apply it to several data sets.     

Challenges for Routine Health System Data Management in a Large Public Programme to Prevent Mother-to-Child HIV Transmission in South Africa

Background

Recent changes to South Africa''s prevention of mother-to-child transmission of HIV (PMTCT) guidelines have raised hope that the national goal of reducing perinatal HIV transmission rates to less than 5% can be attained. While programmatic efforts to reach this target are underway, obtaining complete and accurate data from clinical sites to track progress presents a major challenge. We assessed the completeness and accuracy of routine PMTCT data submitted to the district health information system (DHIS) in three districts of Kwazulu-Natal province, South Africa.

Methodology/Principal Findings

We surveyed the completeness and accuracy of data reported for six key PMTCT data elements between January and December 2007 from all 316 clinics and hospitals in three districts. Through visits to randomly selected sites, we reconstructed reports for the same six PMTCT data elements from clinic registers and assessed accuracy of the monthly reports previously submitted to the DHIS. Data elements were reported only 50.3% of the time and were “accurate” (i.e. within 10% of reconstructed values) 12.8% of the time. The data element “Antenatal Clients Tested for HIV” was the most accurate data element (i.e. consistent with the reconstructed value) 19.8% of the time, while “HIV PCR testing of baby born to HIV positive mother” was the least accurate with only 5.3% of clinics meeting the definition of accuracy.

Conclusions/Significance

Data collected and reported in the public health system across three large, high HIV-prevalence Districts was neither complete nor accurate enough to track process performance or outcomes for PMTCT care. Systematic data evaluation can determine the magnitude of the data reporting failure and guide site-specific improvements in data management. Solutions are currently being developed and tested to improve data quality.     

The ProteinChip Biomarker System from Ciphergen Biosystems: a novel proteomics platform for rapid biomarker discovery and validation

Protein biochips are emerging in two distinct formats. The first involves high-density immobilized arrays of recognition molecules (e.g. antibodies), where target binding is monitored indirectly (e.g. via fluorescence). This technology is in its infancy, being limited by the availability of suitable binding molecules that can cope effectively with protein diversity. The second format involves the capture of proteins by biochemical or intermolecular interaction, coupled with direct detection by MS. This technology is available as the ProteinChip Biomarker System. ProteinChip technology uses surface-enhanced laser desorption/ionization processes to analyse proteins directly from biological samples. Chromatographic surfaces are placed on to ProteinChip Arrays and used to capture subclasses of proteins, dependent on their physical properties. Time-of-flight MS then assigns native molecular masses to the captured proteins. Reproducible protein profiles can be generated from crude biological fluids (e.g. cell lysates, urine or serum). The technology is being applied to a wide range of disciplines, from plant sciences to cancer research, and will be reviewed here.     

A novel population health approach: Using fish retinoblastoma gene profiles as a surrogate for humans

Retinoblastoma, a tumor suppressor gene, is frequently mutated in diverse types of human tumors. We have previously shown that two types of fish tumor, eye and liver, also possess mutant Rb genes. Our aim is to determine if the Rb allele status is linked to environmentally-induced cancer and whether this information in fish can be used to predict future phenotype. This is a proof-of-concept investigation to elucidate if fish may act as surrogates in assessing pollution-induced tumor incidence and inform regulatory authorities of potential long-term population health consequences. Marine flatfish, Limanda limanda, that display either normal liver histopathology, liver adenoma or liver hepatocellular carcinoma were analysed for the presence of Rb gene alterations. Several Rb alterations were detected in the fish displaying adenoma and carcinoma, and not in the surrounding normal tissue from the same individuals. The profile is similar to that reported in humans in that they spread across the gene, particularly exons 8-23, and a functionally important region of the protein. This Rb allele data was then used to build statistical classifier sets, linking Rb status with tumor pathology. Further flatfish caught from coastal-water areas of differing contaminant burden around the UK were subsequently analysed for the presence of Rb alterations. Using novel pattern matching statistics of the classifier sets compared with the coastal samples, the coastal fish were considered more similar to the characterised disease phenotype than the normal phenotype. Preliminary data suggests that using a statistical approach, based on classifying sets of histopathologically-defined tumor states, makes it possible to predict the phenotype of wild fish based on the status of the Rb allele. Since the Rb gene is orthologous, fish populations could act as surrogates for human populations in an eco-epidemiological investigation of the combined roles of genetics and environmental exposures in the tumorigenesis process.     

A comprehensive approach uncovers hidden diversity in freshwater mussels (Bivalvia: Unionidae) with the description of a novel species

Major geological processes have shaped biogeographical patterns of riverine biota. The Edwards Plateau of central Texas, USA, exhibits unique aquatic communities and endemism, including several species of freshwater mussels. Lampsilis bracteata (Gould, 1855) is endemic to the Edwards Plateau region; however, its phylogenetic relationship with other species in the Gulf coastal rivers and Mississippi River basin is unknown. We evaluated phylogenetic relationships, shell morphologies and soft anatomy characters of L. bracteata and a closely related congener, Lampsilis hydiana (Lea, 1838) throughout their ranges. Our results showed the presence of an undescribed species: Lampsilis bergmanni sp.n. Lampsilis bracteata and L. bergmanni sp.n. share similar shell morphologies and soft anatomy characters; however, they are genetically distinct. Geological processes, such as faulting and sea-level changes during the Miocene to Pliocene, are likely to have facilitated diversification of Lampsilis species, resulting in isolation of L. bracteata on the Edwards Plateau and diversification between L. bergmanni sp.n. and L. hydiana. We conclude that L. bracteata range is restricted to the Colorado River basin, whereas L. bergmanni sp.n. occurs only in upstream reaches of the Guadalupe River basin. Conservation actions are warranted for both species due to their restricted distributions and potential anthropogenic threats.     

Serum sulfatides as a novel biomarker for cardiovascular disease in patients with end-stage renal failure

Sulfatides, normal components of serum lipoproteins, may play an important role in cardiovascular disease due to their various modulatory functions in haemostasis. The incidence of cardiovascular disease in patients with end-stage renal failure undergoing maintenance hemodialysis has been reported to be approximately 10 to 30 times higher than that in the general population. To elucidate the possible roles of serum sulfatides in this high incidence, we measured the level of sulfatides in 59 such patients, by converting them to lysosulfatides according to a recently developed quantitative, qualitative, high-throughput technique using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mean level of sulfatides in patients 3.58 ± 1.18 nmol/ml was significantly lower than that in age-matched normal subjects (8.21 ± 1.50 nmol/ml; P < 0.001). Patients receiving maintenance hemodialysis over a longer period had lower levels of sulfatides. When the mean levels of sulfatides were compared between patients with cardiovascular disease (N = 22) and those without the disease (N = 37), the level in the former group 2.85 ± 0.67 nmol/ml was found to be significantly lower than that in the latter group 4.01 ± 1.22 nmol/ml (P < 0.001). These findings reveal a close correlation between low levels of serum sulfatides and a high risk of cardiovascular disease in these patients. Determination of the level of serum sulfatides can contribute to predictions of the incidence of cardiovascular disease in patients with end-stage renal failure undergoing maintenance hemodialysis.     

Highly specific localization of promoter regions in large genomic sequences by PromoterInspector: a novel context analysis approach

We present a new algorithm called PromoterInspector to locate eukaryotic polymase II promoter regions in large genomic sequences with a high degree of specificity. PromoterInspector focuses on the genetic context of promoters, rather than their exact location. Application of PromoterInspector can serve as a crucial pre-processing step for other methods to locate exactly, or to analyze promoters.PromoterInspector does not depend on heuristics, because it is purely based on libraries of IUPAC words extracted from training sequences by an unsupervised learning approach. We compared PromoterInspector to in silico promoter prediction tools using the sequences from the review by J.W. Fickett. PromoterInspector compared favourably on Fickett's evaluation scheme. A true positive to false positive ratio of 2.3 was obtained, surpassing the best ratio of 0.6, reported for TSSG. The application of our method to several large genomic sequences of over 1.3 million base-pairs in total resulted in even more specific predictions. The coverage of annotated promoters was comparable to other in silico promoter prediction methods, while the true positive predictions increased by up to 100% of total matches.PromoterInspector scans 100 kb in less than one minute on a workstation, and thus is especially applicable for large genome analysis. The method is available at http://genomatix.gsf. de/cgi-bin/promoterinspector/promoterinspector.pl.     

Peptide differential display: a novel approach for phase transition in locusts

Today, the question of the physiological cause of phase transition, the transition from the solitary to the gregarious phase, in locusts remains unanswered. We hereby present a novel approach by which we have attempted to determine whether different phases express or release different peptides in similar physiological conditions. For this purpose, a peptidomic analysis of the corpora cardiaca and hemolymph of crowded and isolated locusts of Schistocerca gregaria was performed using high performance liquid chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry. A comparison between the two conditions reveals differences in the number and amount of peptides present in the corpora cardiaca and the hemolymph. Further research will have to identify these phase specific differences and their role in locust phase polymorphism.     

Terminal branch haplotype analysis: a novel approach to investigate newly arisen variants of mitochondrial DNA in natural populations.

The discrimination of recent mutational derivatives from ancestral variation is a critical antecedent to any effort which aims to identify the factors modulating the rates of origin and persistence of new mutants. We propose that newly arisen mtDNA variants, which we designate as terminal branch haplotypes (TBHs), can be recognized by joint sequencing and phylogenetic analysis. This study examined mtDNA diversity in natural populations of the brown bullhead (Ameiurus nebulosus) from four heavily contaminated sites and three relatively pristine locations. While sequence analysis of the mtDNA D-loop region revealed that TBHs were prevalent in these populations, contaminant exposure appeared to play a minor role in their generation. Instead, most TBHs likely arose due to spontaneous mutations with variation in their incidence among sites reflecting the impact of demographic factors.     

FlyCounter: a simple software for counting large populations of small clumped objects in the laboratory

While several software programs exist to count bacterial colonies on a Petri plate, no suitable solution is available for quick and reliable enumeration of small, live insects. We have written a program called FlyCounter that can obtain counts from images, even if insects are highly clumped in space. We also describe a simple and inexpensive system for anesthetizing and capturing high-quality images of the small insects. Taken together, our process is fast, fully automatic, and has a low percentage of error (~1%-4% on average). Although we have tested our software on fruit flies, it should be simple to extend to other organisms of similar size.