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Numerous studies demonstrated the lack of transferability of polygenic score (PGS) models across populations and the problem arising from unequal presentation of ancestries across genetic studies. However, even within European ancestry there are ethnic groups that are rarely presented in genetic studies. For instance, Russians, being one of the largest, diverse, and yet understudied group in Europe. In this study, we evaluated the reliability of genotype imputation for the Russian cohort by testing several commonly used imputation reference panels (e.g. HRC, 1000G, HGDP). HRC, in comparison with two other panels, showed the most accurate results based on both imputation accuracy and allele frequency concordance between masked and imputed genotypes. We built polygenic score models based on GWAS results from the UK biobank, measured the explained phenotypic variance in the Russian cohort attributed to polygenic scores for 11 phenotypes, collected in the clinic for each participant, and finally explored the role of allele frequency discordance between the UK biobank and the study cohort in the resulting PGS performance.

Protein-truncating variants (PTVs) affecting dyslipidemia risk may point to therapeutic targets for cardiometabolic disease. Our objective was to identify PTVs that were associated with both lipid levels and the risk of coronary artery disease (CAD) or type 2 diabetes (T2D) and assess their possible associations with risks of other diseases. To achieve this aim, we leveraged the enrichment of PTVs in the Finnish population and tested the association of low-frequency PTVs in 1,209 genes with serum lipid levels in the Finrisk Study (n = 23,435). We then tested which of the lipid-associated PTVs were also associated with the risks of T2D or CAD, as well as 2,683 disease endpoints curated in the FinnGen Study (n = 218,792). Two PTVs were associated with both lipid levels and the risk of CAD or T2D: triglyceride-lowering variants in ANGPTL8 (-24.0[-30.4 to -16.9] mg/dL per rs760351239-T allele, P = 3.4 × 10-9) and ANGPTL4 (-14.4[-18.6 to -9.8] mg/dL per rs746226153-G allele, P = 4.3 × 10-9). The risk of T2D was lower in carriers of the ANGPTL4 PTV (OR = 0.70[0.60-0.81], P = 2.2 × 10-6) than noncarriers. The odds of CAD were 47% lower in carriers of a PTV in ANGPTL8 (OR = 0.53[0.37-0.76], P = 4.5 × 10-4) than noncarriers. Finally, the phenome-wide scan of the ANGPTL8 PTV showed that the ANGPTL8 PTV carriers were less likely to use statin therapy (68,782 cases, OR = 0.52[0.40-0.68], P = 1.7 × 10-6) compared to noncarriers. Our findings provide genetic evidence of potential long-term efficacy and safety of therapeutic targeting of dyslipidemias.

Information about individual-level genetic ancestry is central to population genetics, forensics and genomic medicine. So far, studies have typically considered genetic ancestry on a broad continental level, and there is much less understanding of how more detailed genetic ancestry profiles can be generated and how accurate and reliable they are. Here, we assess these questions by developing a framework for individual-level ancestry estimation within a single European country, Finland, and we apply the framework to track changes in the fine-scale genetic structure throughout the 20th century. We estimate the genetic ancestry for 18,463 individuals from the National FINRISK Study with respect to up to 10 genetically and geographically motivated Finnish reference groups and illustrate the annual changes in the fine-scale genetic structure over the decades from 1920s to 1980s for 12 geographic regions of Finland. We detected major changes after a sudden, internal migration related to World War II from the region of ceded Karelia to the other parts of the country as well as the effect of urbanization starting from the 1950s. We also show that while the level of genetic heterogeneity in general increases towards the present day, its rate of change has considerable differences between the regions. To our knowledge, this is the first study that estimates annual changes in the fine-scale ancestry profiles within a relatively homogeneous European country and demonstrates how such information captures a detailed spatial and temporal history of a population. We provide an interactive website for the general public to examine our results.

An integrated augmented reality (AR) surgical navigation system that potentially improves intra-operative visualization of concealed anatomical structures. Integration of real-time tracking technology with a laser pico-projector allows the surgical surface to be augmented by projecting virtual images of lesions and critical structures created by multimodality imaging. We aim to quantitatively and qualitatively evaluate the performance of a prototype interactive AR surgical navigation system through a series of pre-clinical studies. Four pre-clinical animal studies using xenograft mouse models were conducted to investigate system performance. A combination of CT, PET, SPECT, and MRI images were used to augment the mouse body during image-guided procedures to assess feasibility. A phantom with machined features was employed to quantitatively estimate the system accuracy. All the image-guided procedures were successfully performed. The tracked pico-projector correctly and reliably depicted virtual images on the animal body, highlighting the location of tumour and anatomical structures. The phantom study demonstrates the system was accurate to 0.55 ± 0.33mm. This paper presents a prototype real-time tracking AR surgical navigation system that improves visualization of underlying critical structures by overlaying virtual images onto the surgical site. This proof-of-concept pre-clinical study demonstrated both the clinical applicability and high precision of the system which was noted to be accurate to

ObjectiveIn stroke survivors, a treatment-resistant problem is inability to volitionally differentiate upper limb wrist extension versus flexion. When one intends to extend the wrist, the opposite occurs, wrist flexion, rendering the limb non-functional. Conventional therapeutic approaches have had limited success in achieving functional recovery of patients with chronic and severe upper extremity impairments. Functional magnetic resonance imaging (fMRI) neurofeedback is an emerging strategy that has shown potential for stroke rehabilitation. There is a lack of information regarding unique blood-oxygenation-level dependent (BOLD) cortical activations uniquely controlling execution of wrist extension versus uniquely controlling wrist flexion. Therefore, a first step in providing accurate neural feedback and training to the stroke survivor is to determine the feasibility of classifying (or differentiating) brain activity uniquely associated with wrist extension from that of wrist flexion, first in healthy adults.ApproachWe studied brain signal of 10 healthy adults, who performed wrist extension and wrist flexion during fMRI data acquisition. We selected four types of analyses to study the feasibility of differentiating brain signal driving wrist extension versus wrist flexion, as follows: 1) general linear model (GLM) analysis; 2) support vector machine (SVM) classification; 3) 'Winner Take All'; and 4) Relative Dominance.ResultsWith these four methods and our data, we found that few voxels were uniquely active during either wrist extension or wrist flexion. SVM resulted in only minimal classification accuracies. There was no significant difference in activation magnitude between wrist extension versus flexion; however, clusters of voxels showed extension signal > flexion signal and other clusters vice versa. Spatial patterns of activation differed among subjects.SignificanceWe encountered a number of obstacles to obtaining clear group results in healthy adults. These obstacles included the following: high variability across healthy adults in all measures studied; close proximity of uniquely active voxels to voxels that were common to both the extension and flexion movements; in general, higher magnitude of signal for the voxels common to both movements versus the magnitude of any given uniquely active voxel for one type of movement. Our results indicate that greater precision in imaging will be required to develop a truly effective method for differentiating wrist extension versus wrist flexion from fMRI data.

Using Twitter to implement public health awareness campaigns is on the rise, but campaign monitoring and evaluation are largely dependent on basic Twitter Analytics. To establish the potential of social network theory-based metrics in better understanding public health campaigns, we analyzed real-time user interactions on Twitter during the 2020 World Breastfeeding Week (WBW) as an exemplar case. Social network analysis (SNA), including community and influencer identification, as well as topic modeling were used to compare the activity of n = 29,958 campaign participants and n = 10,694 reference users from the six-months pre-campaign period. Users formed more inter-connected relationships during the campaign, retweeting and mentioning each other 46,161 times compared to 10,662 times in the prior six months. Campaign participants formed identifiable communities that were not only based on their geolocation, but also based on interests and professional background. While influencers who dominated the WBW conversations were disproportionally members of the scientific community, the campaign did mobilize influencers from the general public who seemed to play a "bridging" role between the public and the scientific community. Users communicated about the campaign beyond its original themes to also discuss breastfeeding within the context of social and racial inequities. Applying SNA allowed understanding of the breastfeeding campaign's messaging and engagement dynamics across communities and influencers. Moving forward, WBW could benefit from improving targeting to enhance geographic coverage and user interactions. As this exemplar case indicates, social network theory and analysis can be used to inform other public health campaigns with data on user interactions that go beyond traditional metrics.

ObjectivePilot testing of real time functional magnetic resonance imaging (rt-fMRI) and real time functional near infrared spectroscopy (rt-fNIRS) as brain computer interface (BCI) neural feedback systems combined with motor learning for motor recovery in chronic severely impaired stroke survivors.ApproachWe enrolled a four-case series and administered three sequential rt-fMRI and ten rt-fNIRS neural feedback sessions interleaved with motor learning sessions. Measures were: Arm Motor Assessment Tool, functional domain (AMAT-F; 13 complex functional tasks), Fugl-Meyer arm coordination scale (FM); active wrist extension range of motion (ROM); volume of activation (fMRI); and fNIRS HbO concentration. Performance during neural feedback was assessed, in part, using percent successful brain modulations during rt-fNIRS.Main resultsPre-/post-treatment mean clinically significant improvement in AMAT-F (.49 ± 0.22) and FM (10.0 ± 3.3); active wrist ROM improvement ranged from 20° to 50°. Baseline to follow-up change in brain signal was as follows: fMRI volume of activation was reduced in almost all ROIs for three subjects, and for one subject there was an increase or no change; fNIRS HbO was within normal range, except for one subject who increased beyond normal at post-treatment. During rt-fNIRS neural feedback training, there was successful brain signal modulation (42%-78%).SignificanceSeverely impaired stroke survivors successfully engaged in spatially focused BCI systems, rt-fMRI and rt-fNIRS, to clinically significantly improve motor function. At the least, equivalency in motor recovery was demonstrated with prior long-duration motor learning studies (without neural feedback), indicating that no loss of motor improvement resulted from substituting neural feedback sessions for motor learning sessions. Given that the current neural feedback protocol did not prevent the motor improvements observed in other long duration studies, even in the presence of fewer sessions of motor learning in the current work, the results support further study of neural feedback and its potential for recovery of motor function in stroke survivors. In future work, expanding the sophistication of either or both rt-fMRI and rt-fNIRS could hold the potential for further reducing the number of hours of training needed and/or the degree of recovery. ClinicalTrials.gov ID: NCT02856035.

Protein-altering variants that are protective against human disease provide in vivo validation of therapeutic targets. Here we use genotyping data from UK Biobank (n = 337,151 unrelated White British individuals) and FinnGen (n = 176,899) to conduct a search for protein-altering variants conferring lower intraocular pressure (IOP) and protection against glaucoma. Through rare protein-altering variant association analysis, we find a missense variant in ANGPTL7 in UK Biobank (rs28991009, p.Gln175His, MAF = 0.8%, genotyped in 82,253 individuals with measured IOP and an independent set of 4,238 glaucoma patients and 250,660 controls) that significantly lowers IOP (β = -0.53 and -0.67 mmHg for heterozygotes, -3.40 and -2.37 mmHg for homozygotes, P = 5.96 x 10-9 and 1.07 x 10-13 for corneal compensated and Goldman-correlated IOP, respectively) and is associated with 34% reduced risk of glaucoma (P = 0.0062). In FinnGen, we identify an ANGPTL7 missense variant at a greater than 50-fold increased frequency in Finland compared with other populations (rs147660927, p.Arg220Cys, MAF Finland = 4.3%), which was genotyped in 6,537 glaucoma patients and 170,362 controls and is associated with a 29% lower glaucoma risk (P = 1.9 x 10-12 for all glaucoma types and also protection against its subtypes including exfoliation, primary open-angle, and primary angle-closure). We further find three rarer variants in UK Biobank, including a protein-truncating variant, which confer a strong composite lowering of IOP (P = 0.0012 and 0.24 for Goldman-correlated and corneal compensated IOP, respectively), suggesting the protective mechanism likely resides in the loss of interaction or function. Our results support inhibition or down-regulation of ANGPTL7 as a therapeutic strategy for glaucoma.

A concerted action on the part of international agencies and national governments has resulted in the near-eradication of poliomyelitis. However, both the oral polio vaccine (OPV) and the inactivated polio vaccine (IPV) have deficiencies which make them suboptimal for use after global eradication. OPV is composed of attenuated Sabin strains and stimulates robust immunity, but may revert to neurovirulent forms in the intestine which can be shed and infect susceptible contacts. The majority of IPV products are manufactured using pathogenic strains inactivated with formalin. Upon eradication, the production of large quantities of pathogenic virus will present an increased biosecurity hazard. A logical ideal endgame vaccine would be an inactivated form of an attenuated strain that could afford protective immunity while safely producing larger numbers of doses per unit of virus stock than current vaccines. We report here the development of an ionizing radiation (IR)-inactivated Sabin-based vaccine using a reconstituted Mn-decapeptide (MDP) antioxidant complex derived from the radioresistant bacterium Deinococcus radiodurans. In bacteria, Mn2+-peptide antioxidants protect proteins from oxidative damage caused by extreme radiation exposure. Here we show for the first time, that MDP can protect immunogenic neutralizing epitopes in picornaviruses. MDP protects epitopes in Polio Virus 1 and 2 Sabin strains (PV1-S and PV2-S, respectively), but viral genomic RNA is not protected during supralethal irradiation. IR-inactivated Sabin viruses stimulated equivalent or improved neutralizing antibody responses in Wistar rats compared to the commercially used IPV products. Our approach reduces the biosecurity risk of the current PV vaccine production method by utilizing the Sabin strains instead of the wild type neurovirulent strains. Additionally, the IR-inactivation approach could provide a simpler, faster and less costly process for producing a more immunogenic IPV. Gamma-irradiation is a well-known method of virus inactivation and this vaccine approach could be adapted to any pathogen of interest.

Breastfeeding is one of many health practices known to support the survival and health of mother and infant, yet low breastfeeding rates persist globally. These rates may be influenced by limited diffusion of evidence-based research and guidelines from the scientific community (SC). As recently highlighted by the National Academy of Sciences, there is a need for the SC to diffuse its findings to the public more effectively online, as means to counteract the spread of misinformation. In response to this call, we gathered data from Twitter for one month from major breastfeeding hashtags resulting in an interconnected social network (n = 3,798 users). We then identified 59 influencers who disproportionately influenced information flow using social network analysis. These influencers were from the SC (e.g. academics, researchers, health care practitioners), as well as interested citizens (IC) and companies. We then conducted an ego-network analysis of influencer networks, developed ego maps, and compared diffusion metrics across the SC, IC and company influencers. We also qualitatively analyzed their tweets (n = 711) to understand the type of information being diffused. SC influencers were the least efficient communicators. Although having the highest tweeting activity (80% of tweets), they did not reach more individuals compared to IC and companies (two-step ego size: 220± 99, 188 ± 124, 169 ± 97 respectively, P = 0.28). Content analysis of tweets suggest IC are more active than the SC in diffusing evidence-based breastfeeding knowledge, with 35% of their tweets around recent research findings compared to only 12% by the SC. Nonetheless, in terms of outreach to the general public, the two-step networks of SC influences were more heterogenous than ICs (55.7 ± 5.07, 50.9 ± 12.0, respectively, P

[This corrects the article DOI: 10.1371/journal.pgen.1007329.].

BackgroundGene-microbiome interactions are important in aetiology and pathogenesis of inflammatory bowel disease, a chronic inflammatory disorder of the gastrointestinal tract consisting of Crohn's disease and ulcerative colitis. Scarce studies on gene-microbiome interactions show very little overlap in their results. Therefore, it is of utmost importance that gene-microbiome studies are repeated. We aimed to replicate the association between the SLC39A8 [Thr]391 risk allele and gut microbiome composition in patients with inflammatory bowel disease and healthy controls.MethodsWe collected faecal samples, peripheral blood and extensive phenotype data from 291 patients with inflammatory bowel disease and 476 healthy controls. Carrier status information was obtained from whole exome sequencing data, generated using the Illumina HiSeq. The gut microbiome composition was determined by tag-sequencing the 16S rRNA gene. Associations between carrier status and disease were tested using the Wilcoxon-Mann-Whitney test. Associations between carriers and gut microbiome composition were determined using principal coordinate analyses, variance explained, alpha diversity and additive general linear models in inflammatory bowel disease, healthy controls and all groups combined.ResultsCrohn's disease patients were more often carriers of the missense variant (21/171, 12.3%) than controls (30/476, 6.3%) (OR = 2.1, P = 0.01). We could not identify associations between carrier status and overall gut microbiome composition and microbial richness in all tested groups after correcting for potential confounding factors. We did identify 37 different operational taxonomical units to be associated with carrier status among the tested groups. Two of these 37 were identified before in the discovery study.ConclusionsWe could confirm the genetic association of the SLC39A8 [Thr]391 risk allele with Crohn's disease but we could only limited replicate the association in gut microbiome composition. Independent replication of gene-microbiome studies is warranted to identify true biological mechanisms.

Post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs) are important product quality attributes (PQAs) that can potentially impact drug stability, safety, and efficacy. The PTMs of a mAb may change remarkably in the bloodstream after drug administration compared to in vitro conditions. Thus, monitoring in vivo PTM changes of mAbs helps evaluate the criticality of PQAs during the product risk assessment. In addition, quantitation of the subject exposures to PTM variants helps assess the impact of PTMs on the safety and efficacy of therapeutic mAbs. Here, we developed an immunocapture-liquid chromatography/mass spectrometry (LC/MS) method to quantify in vivo PTM changes a therapeutic mAb overtime in single- and multiple-dose monkey pharmacokinetic (PK) studies. We also built mathematical models to predict the in vivo serum concentrations of PQAs, the subject exposures to PQAs, and the relative abundance of PQAs in single- and multiple-dose regimens. The model predictions are in good agreement with the experimental results. The immunocapture-LC/MS method and mathematical models enable bioanalytical chemists to quantitatively assess the criticality of PQAs during drug development.

As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at https://ibd.broadinstitute.org, also available in gnomAD at http://gnomad.broadinstitute.org). We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10-100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease (GBA, p.Asn409Ser, 8-fold enrichment); Canavan disease (ASPA, p.Glu285Ala, 12-fold enrichment); and Tay-Sachs disease (HEXA, c.1421+1G>C, 27-fold enrichment; p.Tyr427IlefsTer5, 12-fold enrichment). We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples (0.97 s.d. higher, p

Mesalamine is commonly used to treat ulcerative colitis (UC). Although mesalamine acts topically, in vitro data suggest that intracellular transport is required for its beneficial effect. Genetic variants in mucosal transport proteins may affect this uptake, but the clinical relevance of these variants has not been studied. The aim of this study was to determine whether variants in genes involved in cellular transport affect the response to mesalamine in UC.Subjects with UC from a 6-week clinical trial using multiple doses of mesalamine were genotyped using a genome-wide array that included common exome variants. Analysis focused on cellular transport gene variants with a minor allele frequency >5%. Mesalamine response was defined as improvement in Week 6 Physician's Global Assessment (PGA) and non-response as a lack of improvement in Week 6 PGA. Quality control thresholds included an individual genotyping rate of >90%, SNP genotyping rate of >98%, and exclusion for subjects with cryptic relatedness. All included variants met Hardy-Weinberg equilibrium (p>0.001).457 adults with UC were included with 280 responders and 177 non-responders. There were no common variants in transporter genes that were associated with response to mesalamine. The genetic risk score of responders was similar to that of non-responders (p = 0.18). Genome-wide variants demonstrating a trend towards mesalamine response included ST8SIA5 (p = 1x10-5).Common transporter gene variants did not affect response to mesalamine in adult UC. The response to mesalamine may be due to rare genetic events or environmental factors such as the intestinal microbiome.

Understanding chronic ionizing radiation (CIR) effects is of utmost importance to protecting human health and the environment. Diverse bacteria and fungi inhabiting extremely radioactive waste and disaster sites (e.g. Hanford, Chernobyl, Fukushima) represent new targets of CIR research. We show that many microorganisms can grow under intense gamma-CIR dose rates of 13-126 Gy/h, with fungi identified as a particularly CIR-resistant group of eukaryotes: among 145 phylogenetically diverse strains tested, 78 grew under 36 Gy/h. Importantly, we demonstrate that CIR resistance can depend on cell concentration and that certain resistant microbial cells protect their neighbors (not only conspecifics, but even radiosensitive species from a different phylum), from high-level CIR. We apply a mechanistically-motivated mathematical model of CIR effects, based on accumulation/removal kinetics of reactive oxygen species (ROS) and antioxidants, in bacteria (3 Escherichia coli strains and Deinococcus radiodurans) and in fungi (Candida parapsilosis, Kazachstania exigua, Pichia kudriavzevii, Rhodotorula lysinophila, Saccharomyces cerevisiae, and Trichosporon mucoides). We also show that correlations between responses to CIR and acute ionizing radiation (AIR) among studied microorganisms are weak. For example, in D. radiodurans, the best molecular correlate for CIR resistance is the antioxidant enzyme catalase, which is dispensable for AIR resistance; and numerous CIR-resistant fungi are not AIR-resistant. Our experimental findings and quantitative modeling thus demonstrate the importance of investigating CIR responses directly, rather than extrapolating from AIR. Protection of radiosensitive cell-types by radioresistant ones under high-level CIR is a potentially important new tool for bioremediation of radioactive sites and development of CIR-resistant microbiota as radioprotectors.

BACKGROUND:We set out to determine the accuracy of 3D-navigated mandibular and maxillary osteotomies with the ultimate aim to integrate virtual cutting guides and 3D-navigation into ablative and reconstructive head and neck surgery. METHODS:Four surgeons (two attending, two clinical fellows) completed 224 unnavigated and 224 3D-navigated osteotomies on anatomical models according to preoperative 3D plans. The osteotomized bones were scanned and analyzed. RESULTS:Median distance from the virtual plan was 2.1 mm unnavigated (IQR 2.6 mm, ≥3 mm in 33%) and 1.2 mm 3D-navigated (IQR 1.1 mm, ≥3 mm in 6%) (P

Using robust, integrated analysis of multiple genomic datasets, we show that genes depleted for non-synonymous de novo mutations form a subnetwork of 72 members under strong selective constraint. We further show this subnetwork is preferentially expressed in the early development of the human hippocampus and is enriched for genes mutated in neurological Mendelian disorders. We thus conclude that carefully orchestrated developmental processes are under strong constraint in early brain development, and perturbations caused by mutation have adverse outcomes subject to strong purifying selection. Our findings demonstrate that selective forces can act on groups of genes involved in the same process, supporting the notion that purifying selection can act coordinately on multiple genes. Our approach provides a statistically robust, interpretable way to identify the tissues and developmental times where groups of disease genes are active.

The radioprotective capacity of a rationally-designed Mn2+-decapeptide complex (MDP), based on Mn antioxidants in the bacterium Deinococcus radiodurans, was investigated in a mouse model of radiation injury. MDP was previously reported to be extraordinarily radioprotective of proteins in the setting of vaccine development. The peptide-component (DEHGTAVMLK) of MDP applied here was selected from a group of synthetic peptides screened in vitro for their ability to protect cultured human cells and purified enzymes from extreme damage caused by ionizing radiation (IR). We show that the peptides accumulated in Jurkat T-cells and protected them from 100 Gy. MDP preserved the activity of T4 DNA ligase exposed to 60,000 Gy. In vivo, MDP was nontoxic and protected B6D2F1/J (female) mice from acute radiation syndrome. All irradiated mice treated with MDP survived exposure to 9.5 Gy (LD70/30) in comparison to the untreated mice, which displayed 63% lethality after 30 days. Our results show that MDP provides early protection of white blood cells, and attenuates IR-induced damage to bone marrow and hematopoietic stem cells via G-CSF and GM-CSF modulation. Moreover, MDP mediated the immunomodulation of several cytokine concentrations in serum including G-CSF, GM-CSF, IL-3 and IL-10 during early recovery. Our results present the necessary prelude for future efforts towards clinical application of MDP as a promising IR countermeasure. Further investigation of MDP as a pre-exposure prophylactic and post-exposure therapeutic in radiotherapy and radiation emergencies is warranted.

The aim of this study was to demonstrate the role of advanced fabrication technology across a broad spectrum of head and neck surgical procedures, including applications in endoscopic sinus surgery, skull base surgery, and maxillofacial reconstruction. The initial case studies demonstrated three applications of rapid prototyping technology are in head and neck surgery: i) a mono-material paranasal sinus phantom for endoscopy training ii) a multi-material skull base simulator and iii) 3D patient-specific mandible templates. Digital processing of these phantoms is based on real patient or cadaveric 3D images such as CT or MRI data. Three endoscopic sinus surgeons examined the realism of the endoscopist training phantom. One experienced endoscopic skull base surgeon conducted advanced sinus procedures on the high-fidelity multi-material skull base simulator. Ten patients participated in a prospective clinical study examining patient-specific modeling for mandibular reconstructive surgery. Qualitative feedback to assess the realism of the endoscopy training phantom and high-fidelity multi-material phantom was acquired. Conformance comparisons using assessments from the blinded reconstructive surgeons measured the geometric performance between intra-operative and pre-operative reconstruction mandible plates. Both the endoscopy training phantom and the high-fidelity multi-material phantom received positive feedback on the realistic structure of the phantom models. Results suggested further improvement on the soft tissue structure of the phantom models is necessary. In the patient-specific mandible template study, the pre-operative plates were judged by two blinded surgeons as providing optimal conformance in 7 out of 10 cases. No statistical differences were found in plate fabrication time and conformance, with pre-operative plating providing the advantage of reducing time spent in the operation room. The applicability of common model design and fabrication techniques across a variety of otolaryngological sub-specialties suggests an emerging role for rapid prototyping technology in surgical education, procedure simulation, and clinical practice.

Contactins and Contactin-Associated Proteins, and Contactin-Associated Protein-Like 2 (CNTNAP2) in particular, have been widely cited as autism risk genes based on findings from homozygosity mapping, molecular cytogenetics, copy number variation analyses, and both common and rare single nucleotide association studies. However, data specifically with regard to the contribution of heterozygous single nucleotide variants (SNVs) have been inconsistent. In an effort to clarify the role of rare point mutations in CNTNAP2 and related gene families, we have conducted targeted next-generation sequencing and evaluated existing sequence data in cohorts totaling 2704 cases and 2747 controls. We find no evidence for statistically significant association of rare heterozygous mutations in any of the CNTN or CNTNAP genes, including CNTNAP2, placing marked limits on the scale of their plausible contribution to risk.

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5-5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.

De novo mutations affect risk for many diseases and disorders, especially those with early-onset. An example is autism spectrum disorders (ASD). Four recent whole-exome sequencing (WES) studies of ASD families revealed a handful of novel risk genes, based on independent de novo loss-of-function (LoF) mutations falling in the same gene, and found that de novo LoF mutations occurred at a twofold higher rate than expected by chance. However successful these studies were, they used only a small fraction of the data, excluding other types of de novo mutations and inherited rare variants. Moreover, such analyses cannot readily incorporate data from case-control studies. An important research challenge in gene discovery, therefore, is to develop statistical methods that accommodate a broader class of rare variation. We develop methods that can incorporate WES data regarding de novo mutations, inherited variants present, and variants identified within cases and controls. TADA, for Transmission And De novo Association, integrates these data by a gene-based likelihood model involving parameters for allele frequencies and gene-specific penetrances. Inference is based on a Hierarchical Bayes strategy that borrows information across all genes to infer parameters that would be difficult to estimate for individual genes. In addition to theoretical development we validated TADA using realistic simulations mimicking rare, large-effect mutations affecting risk for ASD and show it has dramatically better power than other common methods of analysis. Thus TADA's integration of various kinds of WES data can be a highly effective means of identifying novel risk genes. Indeed, application of TADA to WES data from subjects with ASD and their families, as well as from a study of ASD subjects and controls, revealed several novel and promising ASD candidate genes with strong statistical support.

The nucleoid-associated protein FIS is a global regulator of gene expression and chromosome structure in Escherichia coli and Salmonella enterica. Despite the importance of FIS for infection and intracellular invasion, very little is known about the regulation of S. enterica fis expression. Under standard laboratory growth conditions, fis is highly expressed during rapid growth but is then silenced as growth slows. However, if cells are cultured in non-aerated conditions, fis expression is sustained during stationary phase. This led us to test whether the redox-sensing transcription factors ArcA and FNR regulate S. enterica fis. Deletion of FNR had no detectable effect, whereas deletion of ArcA had the unexpected effect of further elevating fis expression in stationary phase. ArcA required RpoS for induction of fis expression, suggesting that ArcA indirectly affects fis expression. Other putative regulators were found to play diverse roles: FIS acted directly as an auto-repressor (as expected), whereas CRP had little direct effect on fis expression. Deleting regions of the fis promoter led to the discovery of a novel anaerobically-induced transcription start site (Pfis-2) upstream of the primary transcription start site (Pfis-1). Promoter truncation also revealed that the shortest functional fis promoter was incapable of sustained expression. Moreover, fis expression was observed to correlate directly with DNA supercoiling in non-aerated conditions. Thus, the full-length S. enterica fis promoter region may act as a topological switch that is sensitive to stress-induced duplex destabilisation and up-regulates expression in non-aerated conditions.

Genome-wide association studies and follow-up meta-analyses in Crohn's disease (CD) and ulcerative colitis (UC) have recently identified 163 disease-associated loci that meet genome-wide significance for these two inflammatory bowel diseases (IBD). These discoveries have already had a tremendous impact on our understanding of the genetic architecture of these diseases and have directed functional studies that have revealed some of the biological functions that are important to IBD (e.g. autophagy). Nonetheless, these loci can only explain a small proportion of disease variance (~14% in CD and 7.5% in UC), suggesting that not only are additional loci to be found but that the known loci may contain high effect rare risk variants that have gone undetected by GWAS. To test this, we have used a targeted sequencing approach in 200 UC cases and 150 healthy controls (HC), all of French Canadian descent, to study 55 genes in regions associated with UC. We performed follow-up genotyping of 42 rare non-synonymous variants in independent case-control cohorts (totaling 14,435 UC cases and 20,204 HC). Our results confirmed significant association to rare non-synonymous coding variants in both IL23R and CARD9, previously identified from sequencing of CD loci, as well as identified a novel association in RNF186. With the exception of CARD9 (OR = 0.39), the rare non-synonymous variants identified were of moderate effect (OR = 1.49 for RNF186 and OR = 0.79 for IL23R). RNF186 encodes a protein with a RING domain having predicted E3 ubiquitin-protein ligase activity and two transmembrane domains. Importantly, the disease-coding variant is located in the ubiquitin ligase domain. Finally, our results suggest that rare variants in genes identified by genome-wide association in UC are unlikely to contribute significantly to the overall variance for the disease. Rather, these are expected to help focus functional studies of the corresponding disease loci.

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10(-8)-1.2×10(-43)). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p

Genome-wide association (GWA) studies have identified numerous, replicable, genetic associations between common single nucleotide polymorphisms (SNPs) and risk of common autoimmune and inflammatory (immune-mediated) diseases, some of which are shared between two diseases. Along with epidemiological and clinical evidence, this suggests that some genetic risk factors may be shared across diseases-as is the case with alleles in the Major Histocompatibility Locus. In this work we evaluate the extent of this sharing for 107 immune disease-risk SNPs in seven diseases: celiac disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. We have developed a novel statistic for Cross Phenotype Meta-Analysis (CPMA) which detects association of a SNP to multiple, but not necessarily all, phenotypes. With it, we find evidence that 47/107 (44%) immune-mediated disease risk SNPs are associated to multiple-but not all-immune-mediated diseases (SNP-wise P(CPMA)

Technological advances make it possible to use high-throughput sequencing as a primary discovery tool of medical genetics, specifically for assaying rare variation. Still this approach faces the analytic challenge that the influence of very rare variants can only be evaluated effectively as a group. A further complication is that any given rare variant could have no effect, could increase risk, or could be protective. We propose here the C-alpha test statistic as a novel approach for testing for the presence of this mixture of effects across a set of rare variants. Unlike existing burden tests, C-alpha, by testing the variance rather than the mean, maintains consistent power when the target set contains both risk and protective variants. Through simulations and analysis of case/control data, we demonstrate good power relative to existing methods that assess the burden of rare variants in individuals.

Lifelong, many somatic tissues are replenished by specialized adult stem cells. These stem cells are generally rare, infrequently dividing, occupy a unique niche, and can rapidly respond to injury to maintain a steady tissue size. Despite these commonalities, few shared regulatory mechanisms have been identified. Here, we scrutinized data comparing genes expressed in murine long-term hematopoietic stem cells with their differentiated counterparts and observed that a disproportionate number were members of the developmentally-important, monoallelically expressed imprinted genes. Studying a subset, which are members of a purported imprinted gene network (IGN), we found their expression in HSCs rapidly altered upon hematopoietic perturbations. These imprinted genes were also predominantly expressed in stem/progenitor cells of the adult epidermis and skeletal muscle in mice, relative to their differentiated counterparts. The parallel down-regulation of these genes postnatally in response to proliferation and differentiation suggests that the IGN could play a mechanistic role in both cell growth and tissue homeostasis.

Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in line with observations in Mendelian disease.

Crohn's disease (CD) and celiac disease (CelD) are chronic intestinal inflammatory diseases, involving genetic and environmental factors in their pathogenesis. The two diseases can co-occur within families, and studies suggest that CelD patients have a higher risk to develop CD than the general population. These observations suggest that CD and CelD may share common genetic risk loci. Two such shared loci, IL18RAP and PTPN2, have already been identified independently in these two diseases. The aim of our study was to explicitly identify shared risk loci for these diseases by combining results from genome-wide association study (GWAS) datasets of CD and CelD. Specifically, GWAS results from CelD (768 cases, 1,422 controls) and CD (3,230 cases, 4,829 controls) were combined in a meta-analysis. Nine independent regions had nominal association p-value

The considerable uncertainty regarding cancer risks associated with inherited mutations of BRCA2 is due to unknown factors. To investigate whether common genetic variants modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study in BRCA2 mutation carriers. In stage 1 using the Affymetrix 6.0 platform, 592,163 filtered SNPs genotyped were available on 899 young (

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn(2+) and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn(2+)-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.

Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied "pathway" analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package.

Mitochondrial dysfunction has been observed in skeletal muscle of people with diabetes and insulin-resistant individuals. Furthermore, inherited mutations in mitochondrial DNA can cause a rare form of diabetes. However, it is unclear whether mitochondrial dysfunction is a primary cause of the common form of diabetes. To date, common genetic variants robustly associated with type 2 diabetes (T2D) are not known to affect mitochondrial function. One possibility is that multiple mitochondrial genes contain modest genetic effects that collectively influence T2D risk. To test this hypothesis we developed a method named Meta-Analysis Gene-set Enrichment of variaNT Associations (MAGENTA; http://www.broadinstitute.org/mpg/magenta). MAGENTA, in analogy to Gene Set Enrichment Analysis, tests whether sets of functionally related genes are enriched for associations with a polygenic disease or trait. MAGENTA was specifically designed to exploit the statistical power of large genome-wide association (GWA) study meta-analyses whose individual genotypes are not available. This is achieved by combining variant association p-values into gene scores and then correcting for confounders, such as gene size, variant number, and linkage disequilibrium properties. Using simulations, we determined the range of parameters for which MAGENTA can detect associations likely missed by single-marker analysis. We verified MAGENTA's performance on empirical data by identifying known relevant pathways in lipid and lipoprotein GWA meta-analyses. We then tested our mitochondrial hypothesis by applying MAGENTA to three gene sets: nuclear regulators of mitochondrial genes, oxidative phosphorylation genes, and approximately 1,000 nuclear-encoded mitochondrial genes. The analysis was performed using the most recent T2D GWA meta-analysis of 47,117 people and meta-analyses of seven diabetes-related glycemic traits (up to 46,186 non-diabetic individuals). This well-powered analysis found no significant enrichment of associations to T2D or any of the glycemic traits in any of the gene sets tested. These results suggest that common variants affecting nuclear-encoded mitochondrial genes have at most a small genetic contribution to T2D susceptibility.

Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn's disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions--that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/).

It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease. A strong founder effect, severe isolation, and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae, Federated States of Micronesia, who exhibit a high prevalence of obesity and other metabolic disorders. We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects, making these alleles readily detectable in genome-wide association analysis. However, mapping in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based. We performed genome-wide association analysis for 15 quantitative traits in 2,906 members of the Kosrae population, using novel approaches to manage the extreme relatedness in the sample. As positive controls, we observe association to known loci for plasma cholesterol, triglycerides, and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose. We show that our study is well powered to detect common alleles explaining >/=5% phenotypic variance. However, no such large effects were observed with genome-wide significance, arguing that even in such a severely inbred population, common alleles typically have modest effects. Finally, we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae, despite the major differences in population genetics and environment.

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype-phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance--i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.