An integrative systems genetic analysis of mammalian lipid metabolism.

Dysregulation of lipid homeostasis is a precipitating event in the pathogenesis and progression of hepatosteatosis and metabolic syndrome. These conditions are highly prevalent in developed societies and currently have limited options for diagnostic and therapeutic intervention. Here, using a proteomic and lipidomic-wide systems genetic approach, we interrogated lipid regulatory networks in 107 genetically distinct mouse strains to reveal key insights into the control and network structure of mammalian lipid metabolism. These include the identification of plasma lipid signatures that predict pathological lipid abundance in the liver of mice and humans, defining subcellular localization and functionality of lipid-related proteins, and revealing functional protein and genetic variants that are predicted to modulate lipid abundance. Trans-omic analyses using these datasets facilitated the identification and validation of PSMD9 as a previously unknown lipid regulatory protein. Collectively, our study serves as a rich resource for probing mammalian lipid metabolism and provides opportunities for the discovery of therapeutic agents and biomarkers in the setting of hepatic lipotoxicity.

Development of novel activin-targeted therapeutics.

Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-ß proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, "fastener" residues (Lys(45), Tyr(96), His(97), and Ala(98); activin A numbering) that confer latency to other TGF-ß proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC(50) 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC(50) ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass.

Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq.

BACKGROUND: Massively parallel cDNA sequencing (RNA-seq) experiments are gradually superseding microarrays in quantitative gene expression profiling. However, many biologists are uncertain about the choice of differentially expressed gene (DEG) analysis methods and the validity of cost-saving sample pooling strategies for their RNA-seq experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2, edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving mice amygdalae micro-punches, using high-throughput qPCR on independent biological replicate samples. Moreover, we sequenced RNA-pools and compared their results with sequencing corresponding individual RNA samples. RESULTS: False-positivity rate of Cuffdiff2 and false-negativity rates of DESeq2 and TSPM were high. Among the four investigated DEG analysis methods, sensitivity and specificity of edgeR was relatively high. We documented the pooling bias and that the DEGs identified in pooled samples suffered low positive predictive values. CONCLUSIONS: Our results highlighted the need for combined use of more sensitive DEG analysis methods and high-throughput validation of identified DEGs in future RNA-seq experiments. They indicated limited utility of sample pooling strategies for RNA-seq in similar setups and supported increasing the number of biological replicate samples.

Genomewide association between GLCCI1 and response to glucocorticoid therapy in asthma.

BACKGROUND: The response to treatment for asthma is characterized by wide interindividual variability, with a significant number of patients who have no response. We hypothesized that a genomewide association study would reveal novel pharmacogenetic determinants of the response to inhaled glucocorticoids. METHODS: We analyzed a small number of statistically powerful variants selected on the basis of a family-based screening algorithm from among 534,290 single-nucleotide polymorphisms (SNPs) to determine changes in lung function in response to inhaled glucocorticoids. A significant, replicated association was found, and we characterized its functional effects. RESULTS: We identified a significant pharmacogenetic association at SNP rs37972, replicated in four independent populations totaling 935 persons (P=0.0007), which maps to the glucocorticoid-induced transcript 1 gene (GLCCI1) and is in complete linkage disequilibrium (i.e., perfectly correlated) with rs37973. Both rs37972 and rs37973 are associated with decrements in GLCCI1 expression. In isolated cell systems, the rs37973 variant is associated with significantly decreased luciferase reporter activity. Pooled data from treatment trials indicate reduced lung function in response to inhaled glucocorticoids in subjects with the variant allele (P=0.0007 for pooled data). Overall, the mean (±SE) increase in forced expiratory volume in 1 second in the treated subjects who were homozygous for the mutant rs37973 allele was only about one third of that seen in similarly treated subjects who were homozygous for the wild-type allele (3.2±1.6% vs. 9.4±1.1%), and their risk of a poor response was significantly higher (odds ratio, 2.36; 95% confidence interval, 1.27 to 4.41), with genotype accounting for about 6.6% of overall inhaled glucocorticoid response variability. CONCLUSIONS: A functional GLCCI1 variant is associated with substantial decrements in the response to inhaled glucocorticoids in patients with asthma.

Maternal overnutrition programs changes in the expression of skeletal muscle genes that are associated with insulin resistance and defects of oxidative phosphorylation in adult male rat offspring.

Children of obese mothers have increased risk of metabolic syndrome as adults. Here we report the effects of a high-fat diet in the absence of maternal obesity at conception on skeletal muscle metabolic and transcriptional profiles of adult male offspring. Female Sprague Dawley rats were fed a diet rich in saturated fat and sucrose [high-fat diet (HFD): 23.5% total fat, 9.83% saturated fat, 20% sucrose wt:wt] or a normal control diet [(CD) 7% total fat, 0.5% saturated fat, 10% sucrose wt:wt] for the 3 wk prior to mating and throughout pregnancy and lactation. Maternal weights were not different at conception; however, HFD-fed dams were 22% heavier than controls during pregnancy. On a normal diet, the male offspring of HFD-fed dams were not heavier than controls but demonstrated features of insulin resistance, including elevated plasma insulin concentration [40.1 ± 2.5 (CD) vs 56.2 ± 6.1 (HFD) mU/L; P = 0.023]. Next-generation mRNA sequencing was used to identify differentially expressed genes in the offspring soleus muscle, and gene set enrichment analysis (GSEA) was used to detect coordinated changes that are characteristic of a biological function. GSEA identified 15 upregulated pathways, including cytokine signaling (P < 0.005), starch and sucrose metabolism (P < 0.017), inflammatory response (P < 0.024), and cytokine-cytokine receptor interaction (P < 0.037). A further 8 pathways were downregulated, including oxidative phosphorylation (P < 0.004), mitochondrial matrix (P < 0.006), and electron transport/uncoupling (P < 0.022). Phosphorylation of the insulin signaling protein kinase B was reduced [2.86 ± 0.63 (CD) vs 1.02 ± 0.27 (HFD); P = 0.027] and mitochondrial complexes I, II, and V protein were downregulated by 50-68% (P < 0.005). On a normal diet, the male offspring of HFD-fed dams did not become obese adults but developed insulin resistance, with transcriptional evidence of muscle cytokine activation, inflammation, and mitochondrial dysfunction. These data indicate that maternal overnutrition, even in the absence of prepregnancy obesity, can promote metabolic dysregulation and predispose offspring to type 2 diabetes.

Creating reusable tools from scripts: the Galaxy Tool Factory.

MOTIVATION: Galaxy is a software application supporting high-throughput biology analyses and work flows, available as a free on-line service or as source code for local deployment. New tools can be written to extend Galaxy, and these can be shared using public Galaxy Tool Shed (GTS) repositories, but converting even simple scripts into tools requires effort from a skilled developer. RESULTS: The Tool Factory is a novel Galaxy tool that automates the generation of all code needed to execute user-supplied scripts, and wraps them into new Galaxy tools for upload to a GTS, ready for review and installation through the Galaxy administrative interface. AVAILABILITY AND IMPLEMENTATION: The Galaxy administrative interface supports automated installation from the main GTS. Source code and support are available at the project website, https://bitbucket.org/fubar/galaxytoolfactory. The Tool Factory is implemented as an installable Galaxy tool. CONTACT: ross.lazarus@channing.harvard.edu.

Mapping of numerous disease-associated expression polymorphisms in primary peripheral blood CD4+ lymphocytes.

Genome-wide association studies of human gene expression promise to identify functional regulatory genetic variation that contributes to phenotypic diversity. However, it is unclear how useful this approach will be for the identification of disease-susceptibility variants. We generated gene expression profiles for 22 184 mRNA transcripts using RNA derived from peripheral blood CD4+ lymphocytes, and genome-wide genotype data for 516 512 autosomal markers in 200 subjects. We screened for cis-acting variants by testing variants mapping within 50 kb of expressed transcripts for association with transcript abundance using generalized linear models. Significant associations were identified for 1585 genes at a false discovery rate of 0.05 (corresponding to P-values ranging from 1 × 10(-91) to 7 × 10(-4)). Importantly, we identified evidence of regulatory variation for 119 previously mapped disease genes, including 24 examples where the variant with the strongest evidence of disease-association demonstrates strong association with specific transcript abundance. The prevalence of cis-acting variants among disease-associated genes was 63% higher than the genome-wide rate in our data set (P = 6.41 × 10(-6)), and although many of the implicated loci were associated with immune-related diseases (including asthma, connective tissue disorders and inflammatory bowel disease), associations with genes implicated in non-immune-related diseases including lipid profiles, anthropomorphic measurements, cancer and neurologic disease were also observed. Genetic variants that confer inter-individual differences in gene expression represent an important subset of variants that contribute to disease susceptibility. Population-based integrative genetic approaches can help identify such variation and enhance our understanding of the genetic basis of complex traits.

Genome-wide association analysis identifies PDE4D as an asthma-susceptibility gene.

Asthma, a chronic airway disease with known heritability, affects more than 300 million people around the world. A genome-wide association (GWA) study of asthma with 359 cases from the Childhood Asthma Management Program (CAMP) and 846 genetically matched controls from the Illumina ICONdb public resource was performed. The strongest region of association seen was on chromosome 5q12 in PDE4D. The phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog, Drosophila) gene (PDE4D) is a regulator of airway smooth-muscle contractility, and PDE4 inhibitors have been developed as medications for asthma. Allelic p values for top SNPs in this region were 4.3 x 10(-07) for rs1588265 and 9.7 x 10(-07) for rs1544791. Replications were investigated in ten independent populations with different ethnicities, study designs, and definitions of asthma. In seven white and Hispanic replication populations, two PDE4D SNPs had significant results with p values less than 0.05, and five had results in the same direction as the original population but had p values greater than 0.05. Combined p values for 18,891 white and Hispanic individuals (4,342 cases) in our replication populations were 4.1 x 10(-04) for rs1588265 and 9.2 x 10(-04) for rs1544791. In three black replication populations, which had different linkage disequilibrium patterns than the other populations, original findings were not replicated. Further study of PDE4D variants might lead to improved understanding of the role of PDE4D in asthma pathophysiology and the efficacy of PDE4 inhibitor medications.

Instrumenting the health care enterprise for discovery research in the genomic era.

Tens of thousands of subjects may be required to obtain reliable evidence relating disease characteristics to the weak effects typically reported from common genetic variants. The costs of assembling, phenotyping, and studying these large populations are substantial, recently estimated at three billion dollars for 500,000 individuals. They are also decade-long efforts. We hypothesized that automation and analytic tools can repurpose the informational byproducts of routine clinical care, bringing sample acquisition and phenotyping to the same high-throughput pace and commodity price-point as is currently true of genome-wide genotyping. Described here is a demonstration of the capability to acquire samples and data from densely phenotyped and genotyped individuals in the tens of thousands for common diseases (e.g., in a 1-yr period: N = 15,798 for rheumatoid arthritis; N = 42,238 for asthma; N = 34,535 for major depressive disorder) in one academic health center at an order of magnitude lower cost. Even for rare diseases caused by rare, highly penetrant mutations such as Huntington disease (N = 102) and autism (N = 756), these capabilities are also of interest.

Integrating clinical practice and public health surveillance using electronic medical record systems.

Electronic medical record (EMR) systems have rich potential to improve integration between primary care and the public health system at the point of care. EMRs make it possible for clinicians to contribute timely, clinically detailed surveillance data to public health practitioners without changing their existing workflows or incurring extra work. New surveillance systems can extract raw data from providers' EMRs, analyze them for conditions of public health interest, and automatically communicate results to health departments. We describe a model EMR-based public health surveillance platform called Electronic Medical Record Support for Public Health (ESP). The ESP platform provides live, automated surveillance for notifiable diseases, influenza-like illness, and diabetes prevalence, care, and complications. Results are automatically transmitted to state health departments.

Genetic adaptation of the antibacterial human innate immunity network.

BACKGROUND: Pathogens have represented an important selective force during the adaptation of modern human populations to changing social and other environmental conditions. The evolution of the immune system has therefore been influenced by these pressures. Genomic scans have revealed that immune system is one of the functions enriched with genes under adaptive selection. RESULTS: Here, we describe how the innate immune system has responded to these challenges, through the analysis of resequencing data for 132 innate immunity genes in two human populations. Results are interpreted in the context of the functional and interaction networks defined by these genes. Nucleotide diversity is lower in the adaptors and modulators functional classes, and is negatively correlated with the centrality of the proteins within the interaction network. We also produced a list of candidate genes under positive or balancing selection in each population detected by neutrality tests and showed that some functional classes are preferential targets for selection. CONCLUSIONS: We found evidence that the role of each gene in the network conditions the capacity to evolve or their evolvability: genes at the core of the network are more constrained, while adaptation mostly occurred at particular positions at the network edges. Interestingly, the functional classes containing most of the genes with signatures of balancing selection are involved in autoinflammatory and autoimmune diseases, suggesting a counterbalance between the beneficial and deleterious effects of the immune response.

Genome-wide significant predictors of metabolites in the one-carbon metabolism pathway.

Low plasma B-vitamin levels and elevated homocysteine have been associated with cancer, cardiovascular disease and neurodegenerative disorders. Common variants in FUT2 on chromosome 19q13 were associated with plasma vitamin B12 levels among women in a genome-wide association study in the Nurses' Health Study (NHS) NCI-Cancer Genetic Markers of Susceptibility (CGEMS) project. To identify additional loci associated with plasma vitamin B12, homocysteine, folate and vitamin B6 (active form pyridoxal 5'-phosphate, PLP), we conducted a meta-analysis of three GWA scans (total n = 4763, consisting of 1658 women in NHS-CGEMS, 1647 women in Framingham-SNP-Health Association Resource (SHARe) and 1458 men in SHARe). On chromosome 19q13, we confirm the association of plasma vitamin B12 with rs602662 and rs492602 (P-value = 1.83 x 10(-15) and 1.30 x 10(-14), respectively) in strong linkage disequilibrium (LD) with rs601338 (P = 6.92 x 10(-15)), the FUT2 W143X nonsense mutation. We identified additional genome-wide significant loci for plasma vitamin B12 on chromosomes 6p21 (P = 4.05 x 10(-08)), 10p12 (P-value=2.87 x 10(-9)) and 11q11 (P-value=2.25 x 10(-10)) in genes with biological relevance. We confirm the association of the well-studied functional candidate SNP 5,10-methylene tetrahydrofolate reductase (MTHFR) Ala222Val (dbSNP ID: rs1801133; P-value=1.27 x 10(-8)), on chromosome 1p36 with plasma homocysteine and identify an additional genome-wide significant locus on chromosome 9q22 (P-value=2.06 x 10(-8)) associated with plasma homocysteine. We also identified genome-wide associations with variants on chromosome 1p36 with plasma PLP (P-value=1.40 x 10(-15)). Genome-wide significant loci were not identified for plasma folate. These data reveal new biological candidates and confirm prior candidate genes for plasma homocysteine, plasma vitamin B12 and plasma PLP.

On the replication of genetic associations: timing can be everything!

The failure of researchers to replicate genetic-association findings is most commonly attributed to insufficient statistical power, population stratification, or various forms of between-study heterogeneity or environmental influences.(1) Here, we illustrate another potential cause for nonreplications that has so far not received much attention in the literature. We illustrate that the strength of a genetic effect can vary by age, causing "age-varying associations." If not taken into account during the design and the analysis of a study, age-varying genetic associations can cause nonreplication. By using the 100K SNP scan of the Framingham Heart Study, we identified an age-varying association between a SNP in ROBO1 and obesity and hypothesized an age-gene interaction. This finding was followed up in eight independent samples comprising 13,584 individuals. The association was replicated in five of the eight studies, showing an age-dependent relationship (one-sided combined p = 3.92 x 10(-9), combined p value from pediatric cohorts = 2.21 x 10(-8), combined p value from adult cohorts = 0.00422). Furthermore, this study illustrates that it is difficult for cross-sectional study designs to detect age-varying associations. If the specifics of age- or time-varying genetic effects are not considered in the selection of both the follow-up samples and in the statistical analysis, important genetic associations may be missed.

Comprehensive genetic assessment of a functional TLR9 promoter polymorphism: no replicable association with asthma or asthma-related phenotypes.

BACKGROUND: Prior studies suggest a role for a variant (rs5743836) in the promoter of toll-like receptor 9 (TLR9) in asthma and other inflammatory diseases. We performed detailed genetic association studies of the functional variant rs5743836 with asthma susceptibility and asthma-related phenotypes in three independent cohorts. METHODS: rs5743836 was genotyped in two family-based cohorts of children with asthma and a case-control study of adult asthmatics. Association analyses were performed using chi square, family-based and population-based testing. A luciferase assay was performed to investigate whether rs5743836 genotype influences TLR9 promoter activity. RESULTS: Contrary to prior reports, rs5743836 was not associated with asthma in any of the three cohorts. Marginally significant associations were found with FEV1 and FVC (p = 0.003 and p = 0.008, respectively) in one of the family-based cohorts, but these associations were not significant after correcting for multiple comparisons. Higher promoter activity of the CC genotype was demonstrated by luciferase assay, confirming the functional importance of this variant. CONCLUSION: Although rs5743836 confers regulatory effects on TLR9 transcription, this variant does not appear to be an important asthma-susceptibility locus.

A role for Wnt signaling genes in the pathogenesis of impaired lung function in asthma.

RATIONALE: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes. OBJECTIVES: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma. METHODS: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV(1), FVC, and FEV(1)/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study. MEASUREMENTS AND MAIN RESULTS: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7-16 wk) and the canalicular (gestational age: 17-26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV(1) (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV(1)/FVC (combined P = 0.003). CONCLUSIONS: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.

Multistudy fine mapping of chromosome 2q identifies XRCC5 as a chronic obstructive pulmonary disease susceptibility gene.

RATIONALE: Several family-based studies have identified genetic linkage for lung function and airflow obstruction to chromosome 2q. OBJECTIVES: We hypothesized that merging results of high-resolution single nucleotide polymorphism (SNP) mapping in four separate populations would lead to the identification of chronic obstructive pulmonary disease (COPD) susceptibility genes on chromosome 2q. METHODS: Within the chromosome 2q linkage region, 2,843 SNPs were genotyped in 806 COPD cases and 779 control subjects from Norway, and 2,484 SNPs were genotyped in 309 patients with severe COPD from the National Emphysema Treatment Trial and 330 community control subjects. Significant associations from the combined results across the two case-control studies were followed up in 1,839 individuals from 603 families from the International COPD Genetics Network (ICGN) and in 949 individuals from 127 families in the Boston Early-Onset COPD Study. MEASUREMENTS AND MAIN RESULTS: Merging the results of the two case-control analyses, 14 of the 790 overlapping SNPs had a combined P < 0.01. Two of these 14 SNPs were consistently associated with COPD in the ICGN families. The association with one SNP, located in the gene XRCC5, was replicated in the Boston Early-Onset COPD Study, with a combined P = 2.51 x 10(-5) across the four studies, which remains significant when adjusted for multiple testing (P = 0.02). Genotype imputation confirmed the association with SNPs in XRCC5. CONCLUSIONS: By combining data from COPD genetic association studies conducted in four independent patient samples, we have identified XRCC5, an ATP-dependent DNA helicase, as a potential COPD susceptibility gene.

A risk allele for focal segmental glomerulosclerosis in African Americans is located within a region containing APOL1 and MYH9.

Genetic variation at the MYH9 locus is linked to the high incidence of focal segmental glomerulosclerosis (FSGS) and non-diabetic end-stage renal disease among African Americans. To further define risk alleles with FSGS we performed a genome-wide association analysis using more than one million single-nucleotide polymorphisms in 56 African-American and 61 European-American patients with biopsy-confirmed FSGS. Results were compared to 1641 European Americans and 1800 African Americans as unselected controls. While no association was observed in the cohort of European Americans, the case-control comparison of African Americans found variants within a 60 kb region of chromosome 22 containing part of the APOL1 and MYH9 genes associated with increased risk of FSGS. This region spans different linkage disequilibrium blocks, and variants associating with disease within this region are in linkage disequilibrium with variants which have shown signals of natural selection. APOL1 is a strong candidate for a gene that has undergone recent natural selection and is known to be involved in the infection by Trypanosoma brucei, a parasite common in Africa that has recently adapted to infect human hosts. Further studies will be required to establish which variants are causally related to kidney disease, what mutations caused the selective sweep, and to ultimately determine if these are the same.

Asthma-susceptibility variants identified using probands in case-control and family-based analyses.

BACKGROUND: Asthma is a chronic respiratory disease whose genetic basis has been explored for over two decades, most recently via genome-wide association studies. We sought to find asthma-susceptibility variants by using probands from a single population in both family-based and case-control association designs. METHODS: We used probands from the Childhood Asthma Management Program (CAMP) in two primary genome-wide association study designs: (1) probands were combined with publicly available population controls in a case-control design, and (2) probands and their parents were used in a family-based design. We followed a two-stage replication process utilizing three independent populations to validate our primary findings. RESULTS: We found that single nucleotide polymorphisms with similar case-control and family-based association results were more likely to replicate in the independent populations, than those with the smallest p-values in either the case-control or family-based design alone. The single nucleotide polymorphism that showed the strongest evidence for association to asthma was rs17572584, which replicated in 2/3 independent populations with an overall p-value among replication populations of 3.5E-05. This variant is near a gene that encodes an enzyme that has been implicated to act coordinately with modulators of Th2 cell differentiation and is expressed in human lung. CONCLUSIONS: Our results suggest that using probands from family-based studies in case-control designs, and combining results of both family-based and case-control approaches, may be a way to augment our ability to find SNPs associated with asthma and other complex diseases.

Distributed health data networks: a practical and preferred approach to multi-institutional evaluations of comparative effectiveness, safety, and quality of care.

BACKGROUND: Comparative effectiveness research, medical product safety evaluation, and quality measurement will require the ability to use electronic health data held by multiple organizations. There is no consensus about whether to create regional or national combined (eg, "all payer") databases for these purposes, or distributed data networks that leave most Protected Health Information and proprietary data in the possession of the original data holders. OBJECTIVES: Demonstrate functions of a distributed research network that supports research needs and also address data holders concerns about participation. Key design functions included strong local control of data uses and a centralized web-based querying interface. RESEARCH DESIGN: We implemented a pilot distributed research network and evaluated the design considerations, utility for research, and the acceptability to data holders of methods for menu-driven querying. We developed and tested a central, web-based interface with supporting network software. Specific functions assessed include query formation and distribution, query execution and review, and aggregation of results. RESULTS: This pilot successfully evaluated temporal trends in medication use and diagnoses at 5 separate sites, demonstrating some of the possibilities of using a distributed research network. The pilot demonstrated the potential utility of the design, which addressed the major concerns of both users and data holders. No serious obstacles were identified that would prevent development of a fully functional, scalable network. CONCLUSIONS: Distributed networks are capable of addressing nearly all anticipated uses of routinely collected electronic healthcare data. Distributed networks would obviate the need for centralized databases, thus avoiding numerous obstacles.

Balancing selection is the main force shaping the evolution of innate immunity genes.

The evolutionarily recent geographic expansion of humans, and the even more recent development of large, relatively dense human settlements, has exposed our species to new pathogenic environments. Potentially lethal pathogens are likely to have exerted important selective pressures on our genome, so immunity genes can be expected to show molecular signatures of the adaptation of human populations to these recent conditions. While genes related to the acquired immunity system have indeed been reported to show traces of local adaptation, little is known about the response of the innate immunity system. In this study, we analyze the variability patterns in different human populations of fifteen genes related to innate immunity. We have used both single nucleotide polymorphism and sequence data, and through the analysis of interpopulation differentiation, the linkage disequilibrium pattern, and intrapopulation diversity, we have discovered some signatures of positive and especially balancing selection in these genes, thus confirming the importance of the immune system genetic plasticity in the evolutionary adaptive process. Interestingly, the strongest evidence is found in three TLR genes and CD14. These innate immunity genes play a pivotal role, being involved in the primary recognition of pathogens. In general, more evidences of selection appear in the European populations, in some case possibly related to severe population specific pressures. However, we also describe evidence from African populations, which may reflect parallel or long-term selective forces acting in different geographic areas.