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BACKGROUND: Experimental studies and small clinical trials have suggested that treatment with intranasal oxytocin may reduce social impairment in persons with autism spectrum disorder. Oxytocin has been administered in clinical practice to many children with autism spectrum disorder. METHODS: We conducted a 24-week, placebo-controlled phase 2 trial of intranasal oxytocin therapy in children and adolescents 3 to 17 years of age with autism spectrum disorder. Participants were randomly assigned in a 1:1 ratio, with stratification according to age and verbal fluency, to receive oxytocin or placebo, administered intranasally, with a total target dose of 48 international units daily. The primary outcome was the least-squares mean change from baseline on the Aberrant Behavior Checklist modified Social Withdrawal subscale (ABC-mSW), which includes 13 items (scores range from 0 to 39, with higher scores indicating less social interaction). Secondary outcomes included two additional measures of social function and an abbreviated measure of IQ. RESULTS: Of the 355 children and adolescents who underwent screening, 290 were enrolled. A total of 146 participants were assigned to the oxytocin group and 144 to the placebo group; 139 and 138 participants, respectively, completed both the baseline and at least one postbaseline ABC-mSW assessments and were included in the modified intention-to-treat analyses. The least-squares mean change from baseline in the ABC-mSW score (primary outcome) was −3.7 in the oxytocin group and −3.5 in the placebo group (least-squares mean difference, −0.2; 95% confidence interval, −1.5 to 1.0; P = 0.61). Secondary outcomes generally did not differ between the trial groups. The incidence and severity of adverse events were similar in the two groups. CONCLUSIONS: This placebo-controlled trial of intranasal oxytocin therapy in children and adolescents with autism spectrum disorder showed no significant between-group differences in the least-squares mean change from baseline on measures of social or cognitive functioning over a period of 24 weeks. (Funded by the National Institute of Child Health and Human Development; SOARS-B ClinicalTrials.gov number, NCT01944046.)

Men with circulating tumor cell (CTC) AR-V7–positive metastatic castration-resistant prostate cancer (mCRPC) have worse outcomes when treated with enzalutamide/abiraterone. However, most men lack CTC AR-V7 detection, and additional predictive biomarkers are needed. We conducted a retrospective secondary analysis of the prospective PROPHECY trial (NCT02269982) of men with mCRPC undergoing treatment with enzalutamide/abiraterone, analyzing pooled CTC and germline DNA for whole-genome copy-number alterations (CNA) in 73 samples from 48 men over time along with pooled CTC and germline whole-exome sequencing on 22 paired samples before and following progression on androgen receptor (AR) inhibitor therapy to identify somatic genomic alterations associated with acquired resistance. We observed broad interpatient and longitudinal CTC genomic heterogeneity from AR-V7–negative men with mCRPC, including common gains of KDM6A, MYCN, and AR, and loss of ZFHX3, BRCA1, and PTEN. Men who had progression-free survival of ≤3 months despite enzalutamide/abiraterone treatment were more likely to have baseline CTC genomic loss of CHD1, PTEN, PHLPP1, and ZFHX3 and gains of BRCA2, KDM5D, MYCN, and SPARC. After progression on abiraterone/enzalutamide, we observed clonal evolution of CTCs harboring TP53 mutations and gain of ATM, KDM6A, and MYC, and loss of NCOR1, PTEN, RB1, and RUNX2. CTC genomic findings were independently confirmed in a separate cohort of mCRPC men who progressed despite prior treatment with abiraterone/enzalutamide (NCT02204943). Implications: We identified common and reproducible genomic alterations in CTCs from AR-V7–negative mCRPC men associated with poor outcomes during enzalutamide/abiraterone treatment, including CNAs in genes linked to lineage plasticity and epigenetic signaling, DNA repair, AR, TP53/RB1, PTEN, and WNT pathways.

Changes in platelet physiology are associated with simultaneous changes in microRNA concentrations, suggesting a role for microRNA in platelet regulation. Here we investigated potential associations between microRNA and platelet reactivity (PR), a marker of platelet function, in two cohorts following a non-ST elevation acute coronary syndrome (NSTE-ACS) event. First, non-targeted microRNA concentrations and PR were compared in a case (N = 77) control (N = 76) cohort within the larger TRILOGY-ACS trial. MicroRNA significant in this analysis plus CVD-associated microRNAs from the literature were then quantified by targeted rt-PCR in the complete TRILOGY-ACS cohort (N = 878) and compared with matched PR samples. Finally, microRNA significant in the non-targeted & targeted analyses were verified in an independent post NSTE-ACS cohort (N = 96). From the non-targeted analysis, 14 microRNAs were associated with PR (Fold Change: 0.91–1.27, p-value: 0.004–0.05). From the targeted analysis, five microRNAs were associated with PR (Beta: −0.09–0.22, p-value: 0.004–0.05). Of the 19 significant microRNAs, three, miR-15b-5p, miR-93 and miR-126, were consistently associated with PR in the TRILOGY-ACS and independent Singapore post-ACS cohorts, suggesting the measurement of circulating microRNA concentrations may report on dynamic changes in platelet biology following a cardiovascular ischemic event.

Objective To describe the rationale, design, and methods of the Autism Centers of Excellence (ACE) network Study of Oxytocin in Autism to improve Reciprocal Social Behaviors (SOARS-B). Method This phase 2 clinical trial was designed to evaluate the use of intranasal oxytocin treatment to improve social difficulties in individuals with autism spectrum disorder (ASD). In total, 290 participants ages 3 to 17 years with a DSM-5 diagnosis of ASD were enrolled to receive 24 weeks of treatment with either oxytocin or a matched placebo at one of seven collaborating sites. Participants were subsequently treated with open-label oxytocin for 24 additional weeks. Post-treatment assessments were done approximately 4 weeks after treatment discontinuation. Plasma oxytocin and oxytocin receptor gene (OXTR) methylation level were measured at baseline, and week 8, 24 and 36 to explore potential relationships between these biomarkers and treatment response. Results This report describes the rationale, design, and methods of the SOARS-B clinical trial. Conclusions There is a tremendous unmet need for safe and effective pharmacological treatment options that target the core symptoms of ASD. Several studies support the hypothesis that intranasal oxytocin could improve social orienting and the salience of social rewards in ASD, thereby enhancing reciprocal social behaviors. However, due to conflicting results from a number of pilot studies on the prosocial effects of exogenous oxytocin, this hypothesis remains controversial and inconclusive. SOARS-B is the best powered study to date to address this hypothesis and promises to improve our understanding of the safety and efficacy of intranasal oxytocin in the treatment of social deficits in children with ASD.

The immune system plays a multifunctional role throughout the regenerative process, regulating both pro-/anti-inflammatory phases and progenitor cell function. In the present study, we identify the myokine/cytokine Meteorin-like (Metrnl) as a critical regulator of muscle regeneration. Mice genetically lacking Metrnl have impaired muscle regeneration associated with a reduction in immune cell infiltration and an inability to transition towards an anti-inflammatory phenotype. Isochronic parabiosis, joining wild-type and whole-body Metrnl knock-out (KO) mice, returns Metrnl expression in the injured muscle and improves muscle repair, providing supportive evidence for Metrnl secretion from infiltrating immune cells. Macrophage-specific Metrnl KO mice are also deficient in muscle repair. During muscle regeneration, Metrnl works, in part, through Stat3 activation in macrophages, resulting in differentiation to an anti-inflammatory phenotype. With regard to myogenesis, Metrnl induces macrophage-dependent insulin-like growth factor 1 production, which has a direct effect on primary muscle satellite cell proliferation. Perturbations in this pathway inhibit efficacy of Metrnl in the regenerative process. Together, these studies identify Metrnl as an important regulator of muscle regeneration and a potential therapeutic target to enhance tissue repair.

We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was enriched for prefibro- and fibro-, regulatory, reparative and prehypertrophic chondrocytes. A total of 61 cytokines and growth factors were predicted to regulate the 7 chondrocyte cell phenotypes. Based on production by > 1% of cells, 55% of the cytokines were produced by synovial cells (39% exclusive to synoviocytes and not expressed by chondrocytes) and their presence in osteoarthritic synovial fluid confirmed. The synoviocytes producing IL-1beta (a classic pathogenic cytokine in osteoarthritis), mainly inflammatory macrophages and dendritic cells, were characterized by co-expression of surface proteins corresponding to HLA-DQA1, HLA-DQA2, OLR1 or TLR2. Strategies to deplete these pathogenic intra-articular cell subpopulations could be a therapeutic option for human osteoarthritis.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-seq analysis of zebrafish granulomas as well as analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell-autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

The balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell-mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.

The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P, Fuchs endothelial corneal dystrophy (FECD) is one of the most common reasons for corneal transplantation, and is known to cluster in families. Here, the authors discover new genetic loci associated with FECD with sex-specific effects and implications for disease mechanism.

Background Diagnostic delays are common for multiple sclerosis (MS) since diagnosis typically depends on the presentation of nonspecific clinical symptoms together with radiologically-determined central nervous system (CNS) lesions. It is important to reduce diagnostic delays as earlier initiation of disease modifying therapies mitigates long-term disability. Developing a metabolomic blood-based MS biomarker is attractive, but prior efforts have largely focused on specific subsets of metabolite classes or analytical platforms. Thus, there are opportunities to interrogate metabolite profiles using more expansive and comprehensive approaches for developing MS biomarkers and for advancing our understanding of MS pathogenesis. Methods To identify putative blood-based MS biomarkers, we comprehensively interrogated the metabolite profiles in 12 non-Hispanic white, non-smoking, male MS cases who were drug naive for 3 months prior to biospecimen collection and 13 non-Hispanic white, non-smoking male controls who were frequency matched to cases by age and body mass index. We performed untargeted two-dimensional gas chromatography and time-of-flight mass spectrometry (GCxGC-TOFMS) and targeted lipidomic and amino acid analysis on serum. 325 metabolites met quality control and supervised machine learning was used to identify metabolites most informative for MS status. The discrimination potential of these select metabolites were assessed using receiver operator characteristic curves based on logistic models; top candidate metabolites were defined as having area under the curves (AUC) >80%. The associations between whole-genome expression data and the top candidate metabolites were examined, followed by pathway enrichment analyses. Similar associations were examined for 175 putative MS risk variants and the top candidate metabolites. Results 12 metabolites were determined to be informative for MS status, of which 6 had AUCs >80%: pyroglutamate, laurate, acylcarnitine C14:1, N-methylmaleimide, and 2 phosphatidylcholines (PC ae 40:5, PC ae 42:5). These metabolites participate in glutathione metabolism, fatty acid metabolism/oxidation, cellular membrane composition, and transient receptor potential channel signaling. Pathway analyses based on the gene expression association for each metabolite suggested enrichment for pathways associated with apoptosis and mitochondrial dysfunction. Interestingly, the predominant MS genetic risk allele HLA-DRB1×15:01 was associated with one of the 6 top metabolites. Conclusion Our analysis represents the most comprehensive description of metabolic changes associated with MS in serum, to date, with the inclusion of genomic and genetic information. We identified atypical metabolic processes that differed between MS patients and controls, which may enable the development of biological targets for diagnosis and treatment.

Osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone. Here, we used lineage tracing studies—labelling cells expressing Cx3cr1, Csf1r or Flt3—to identify the precursors of osteoclasts in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow haematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodelling in both physiological and pathological settings. Our single-cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently of the haematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP and HSC lineages indicated the possibility of cell–cell fusion between these two lineages. Cx3cr1+ yolk-sac macrophage descendants resided in the adult spleen, and parabiosis experiments showed that these cells migrated through the bloodstream to the remodelled bone after injury. Yahara et al. show that yolk-sac macrophages, which are derived from erythromyeloid progenitors, produce neonatal osteoclasts that support bone homeostasis and repair and also provide long-lasting osteoclasts in adult bone.

Centromeres are defined by a specialized chromatin organization that includes nucleosomes that contain the centromeric histone variant centromere protein A (CENP-A) instead of canonical histone H3. Studies in various organisms have shown that centromeric chromatin (i.e., CENP-A chromatin or centrochromatin) exhibits plasticity, in that it can assemble on different types of DNA sequences. However, once established on a chromosome, the centromere is maintained at the same position. In humans, this location is the highly homogeneous repetitive DNA alpha satellite. Mislocalization of centromeric chromatin to atypical locations can lead to genome instability, indicating that restriction of centromeres to a distinct genomic position is important for cell and organism viability. Here, we describe a rearrangement of Homo sapiens chromosome 17 (HSA17) that has placed alpha satellite DNA next to euchromatin. We show that on this mutant chromosome, CENP-A chromatin has spread from the alpha satellite into the short arm of HSA17, establishing a ∼700 kb hybrid centromeric domain that spans both repetitive and unique sequences and changes the expression of at least one gene over which it spreads. Our results illustrate the plasticity of human centromeric chromatin and suggest that heterochromatin normally constrains CENP-A chromatin onto alpha satellite DNA. This work highlights that chromosome rearrangements, particularly those that remove the pericentromere, create opportunities for centromeric nucleosomes to move into non-traditional genomic locations, potentially changing the surrounding chromatin environment and altering gene expression.

Scope Fumonisin (FB) occurs in maize and is an inhibitor of ceramide synthase (CerS). We determined the urinary FB1 (UFB1) and sphingoid base 1-phosphate levels in blood from women consuming maize in high and low FB exposure communities in Guatemala. Methods and results FB1 intake was estimated using the UFB1. Sphinganine 1-phosphate (Sa 1-P), sphingosine 1-phosphate (So 1-P), and the Sa 1-P/So 1-P ratio were determined in blood spots collected on absorbent paper at the same time as urine collection. In the first study, blood spots and urine were collected every 3 months (March 2011 to February 2012) from women living in low (Chimaltenango and Escuintla) and high (Jutiapa) FB exposure communities (1240 total recruits). The UFB1, Sa 1-P/So 1-P ratio, and Sa 1-P/mL in blood spots were significantly higher in the high FB1 intake community compared to the low FB1 intake communities. The results were confirmed in a follow-up study (February 2013) involving 299 women living in low (Sacatepequez) and high (Santa Rosa and Chiquimula) FB exposure communities. Conclusions High levels of FB1 intake are correlated with changes in Sa 1-P and the Sa 1-P/So 1-P ratio in human blood in a manner consistent with FB1 inhibition of CerS.

Substantial advances in the molecular biology of prostate cancer have led to the approval of multiple new systemic agents to treat men with metastatic castration-resistant prostate cancer (mCRPC). These treatments encompass androgen receptor directed therapies, immunotherapies, bone targeting radiopharmaceuticals and cytotoxic chemotherapies. There is, however, great heterogeneity in the degree of patient benefit with these agents, thus fueling the need to develop predictive biomarkers that are able to rationally guide therapy. Circulating tumor cells (CTCs) have the potential to provide an assessment of tumor-specific biomarkers through a non-invasive, repeatable "liquid biopsy" of a patient's cancer at a given point in time. CTCs have been extensively studied in men with mCRPC, where CTC enumeration using the Cellsearch® method has been validated and FDA approved to be used in conjunction with other clinical parameters as a prognostic biomarker in metastatic prostate cancer. In addition to enumeration, more sophisticated molecular profiling of CTCs is now feasible and may provide more clinical utility as it may reflect tumor evolution within an individual particularly under the pressure of systemic therapies. Here, we review technologies used to detect and characterize CTCs, and the potential biological and clinical utility of CTC molecular profiling in men with metastatic prostate cancer.

Objective— Exposure to mobile source emissions is nearly ubiquitous in developed nations and is associated with multiple adverse health outcomes. There is an ongoing need to understand the specificity of traffic exposure associations with vascular outcomes, particularly in individuals with cardiovascular disease. Approach and Results— We performed a cross-sectional study using 2124 individuals residing in North Carolina, United States, who received a cardiac catheterization at the Duke University Medical Center. Traffic-related exposure was assessed via 2 metrics: (1) the distance between the primary residence and the nearest major roadway; and (2) location of the primary residence in regions defined based on local traffic patterns. We examined 4 cardiovascular disease outcomes: hypertension, peripheral arterial disease, the number of diseased coronary vessels, and recent myocardial infarction. Statistical models were adjusted for race, sex, smoking, type 2 diabetes mellitus, body mass index, hyperlipidemia, and home value. Results are expressed in terms of the odds ratio (OR). A 23% decrease in residential distance to major roadways was associated with higher prevalence of peripheral arterial disease (OR=1.29; 95% confidence interval, 1.08–1.55) and hypertension (OR=1.15; 95% confidence interval, 1.01–1.31). Associations with peripheral arterial disease were strongest in men (OR=1.42; 95% confidence interval, 1.17–1.74) while associations with hypertension were strongest in women (OR=1.21; 95% confidence interval, 0.99–1.49). Neither myocardial infarction nor the number of diseased coronary vessels were associated with traffic exposure. Conclusions— Traffic-related exposure is associated with peripheral arterial disease and hypertension while no associations are observed for 2 coronary-specific vascular outcomes.

Summary Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

We performed a gene–smoking interaction analysis using families from an early-onset coronary artery disease cohort (GENECARD). This analysis was focused on validating and expanding results from previous studies implicating single nucleotide polymorphisms (SNPs) on chromosome 3 in smoking-mediated coronary artery disease. We analyzed 430 SNPs on chromosome 3 and identified 16 SNPs that showed a gene–smoking interaction at P < 0.05 using association in the presence of linkage—ordered subset analysis, a method that uses permutations of the data to empirically estimate the strength of the association signal. Seven of the 16 SNPs were in the Rho-GTPase pathway indicating a 1.87-fold enrichment for this pathway. A meta-analysis of gene–smoking interactions in three independent studies revealed that rs9289231 in KALRN had a Fisher’s combined P value of 0.0017 for the interaction with smoking. In a gene-based meta-analysis KALRN had a P value of 0.026. Finally, a pathway-based analysis of the association results using WebGestalt revealed several enriched pathways including the regulation of the actin cytoskeleton pathway as defined by the Kyoto Encyclopedia of Genes and Genomes.

Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P

Glioblastoma is universally fatal because of its propensity for rapid recurrence due to highly migratory tumor cells. Unraveling the genomic complexity that underlies this migratory characteristic could provide therapeutic targets that would greatly complement current surgical therapy. Using multiple high-resolution genomic screening methods, we identified a single locus, adherens junctional associated protein 1 (AJAP1) on chromosome 1p36 that is lost or epigenetically silenced in many glioblastomas. We found AJAP1 expression absent or reduced in 86% and 100% of primary glioblastoma tumors and cell lines, respectively, and the loss of expression correlates with AJAP1 methylation. Restoration of AJAP1 gene expression by transfection or demethylation agents results in decreased tumor cell migration in glioblastoma cell lines. This work shows the significant loss of expression of AJAP1 in glioblastoma and provides evidence of its role in the highly migratory characteristic of these tumors. Mol Cancer Res; 10(2); 208–17. ©2012 AACR.

Importance One in 88 children in the United States is diagnosed as having autism spectrum disorder. Significant interest centers on understanding the environmental factors that may contribute to autism risk. Objective To examine whether induced (stimulating uterine contractions prior to the onset of spontaneous labor) and/or augmented (increasing the strength, duration, or frequency of uterine contractions with spontaneous onset of labor) births are associated with increased odds of autism. Design, Setting, and Participants We performed an epidemiological analysis using multivariable logistic regression modeling involving the North Carolina Detailed Birth Record and Education Research databases. The study featured 625 042 live births linked with school records, including more than 5500 children with a documented exceptionality designation for autism. Exposures Induced or augmented births. Main Outcomes and Measures Autism as assessed by exceptionality designations in child educational records. Results Compared with children born to mothers who received neither labor induction nor augmentation, children born to mothers who were induced and augmented, induced only, or augmented only experienced increased odds of autism after controlling for potential confounders related to socioeconomic status, maternal health, pregnancy-related events and conditions, and birth year. The observed associations between labor induction/augmentation were particularly pronounced in male children. Conclusions and Relevance Our work suggests that induction/augmentation during childbirth is associated with increased odds of autism diagnosis in childhood. While these results are interesting, further investigation is needed to differentiate among potential explanations of the association including underlying pregnancy conditions requiring the eventual need to induce/augment, the events of labor and delivery associated with induction/augmentation, and the specific treatments and dosing used to induce/augment labor (eg, exogenous oxytocin and prostaglandins).

Objective The genetic contributions to the multifactorial disorder osteoarthritis (OA) have been increasingly recognized. The goal of the current study was to use OA-related biomarkers of severity and disease burden as quantitative traits to identify genetic susceptibility loci for OA. Methods In a large multigenerational extended family (n = 350), we measured 5 OA-related biomarkers: hyaluronan (HA), cartilage oligomeric matrix protein (COMP), N-propeptide of type IIA collagen (PIIANP), C-propeptide of type II procollagen (CPII), and type II collagen neoepitope (C2C). Single-nucleotide polymorphism markers (n = 6,090) covering the whole genome were genotyped using the Illumina HumanLinkage-12 BeadChip. Variance components analysis, as implemented in the Sequential Oligogenic Linkage Analysis Routines, was used to estimate heritabilities of the quantitative traits and to calculate 2-point and multipoint logarithm of odds (LOD) scores using a polygenic model. Results After adjusting for age and sex, we found that 4 of the 5 biomarkers exhibited significant heritability (PIIANP 0.57, HA 0.49, COMP 0.43, C2C 0.30; P ≤ 0.01 for all). Fourteen of the 19 loci that had multipoint LOD scores of >1.5 were near to or overlapped with previously reported OA susceptibility loci. Four of these loci were identified by more than 1 biomarker. The maximum multipoint LOD scores for the heritable quantitative biomarker traits were 4.3 for PIIANP (chromosome 8p23.2), 3.2 for COMP (chromosome 8q11.1), 2.0 for HA (chromosome 6q16.3), and 2.0 for C2C (chromosome 5q31.2). Conclusion Herein, we report the first evidence of genetic susceptibility loci identified by OA-related biomarkers in an extended family. Our results demonstrate that serum concentrations of PIIANP, HA, COMP, and C2C have substantial heritable components, and using these biomarkers, several genetic loci potentially contributing to the genetic diversity of OA were identified.

Leukotrienes are arachidonic acid derivatives long known for their inflammatory properties and their involvement with a number of human diseases, most particularly asthma. Recently, leukotriene-based inflammation has also been shown to play an important role in atherosclerosis: ALOX5AP and LTA4H, both genes in the leukotriene biosynthesis pathway, have individually been shown to be associated with various cardiovascular disease (CVD) phenotypes. To assess the role of the leukotriene pathway in CVD pathogenesis, we performed genetic association studies of ALOX5AP and LTA4H in a family based study of early onset coronary artery disease (EOCAD) (GENECARD, 1,101 families) and in a non-familial dataset of EOCAD (CATHGEN, 656 cases and 405 controls). We found weak to moderate association between single nucleotide polymorphisms (SNPs) in ALOX5AP and LTA4H with EOCAD. The previously reported four-SNP haplotype (HapA) in ALOX5AP showed association with EOCAD in CATHGEN (P = 0.02), while controlling for age, race and CVD risk factors. HapK, the previously reported ten-SNP haplotype in LTA4H was associated with EOCAD in CATHGEN (P = 0.04). Another previously reported four-SNP haplotype in ALOX5AP (HapB) was not significant in our sample (P = 0.39). The overall lack of (or weak) association of single SNPs as compared with the haplotype results demonstrates the need for analyzing multiple SNPs within each gene in such studies. Interestingly, we detected an association of SNPs in ALOX5 (P < 0.05), the target of ALOX5AP, with CVD. Using a pathway-based approach, we also detected statistical evidence for interactions among ALOX5, ALOX5AP and LTA4H using RNA expression data from a collection of freshly harvested human aortas with varying degrees of atherosclerosis. The GENECARD families did not demonstrate evidence for linkage or association with ALOX5, ALOX5AP or LTA4H. Our results support a modest role for the leukotriene pathway in atherosclerosis pathogenesis, reveal important genomic interactions within the pathway, and suggest the importance of using pathway-based modeling for evaluating the genomics of atherosclerosis susceptibility.

Use of drug-eluting stents (DES) has reduced in-stent restenosis after percutaneous coronary intervention (PCI); however, DES are associated with late stent thrombosis. There is no accurate way to predict in-stent restenosis, although risk factors for atherosclerosis overlap those for in-stent restenosis. Therefore, we evaluated atherosclerosis candidate genes for association with in-stent restenosis.We identified 46 consecutive cases that had undergone PCI with bare-metal stents who subsequently developed symptomatic in-stent restenosis of the target lesion (/=75% luminal narrowing) within 6 months. Forty-six age-, race-, vessel-diameter- and sex-matched controls without in-stent restenosis after PCI with bare-metal stent were also identified. Single-nucleotide polymorphisms (SNPs, N=82) from 39 candidate atherosclerosis genes were genotyped. Multivariable logistic regression models were used to test for association.Five SNPs were associated with in-stent restenosis. Three ALOX5AP SNPs were most strongly associated, two with increased risk (OR 3.74, p=0.01; OR 3.46, p=0.02), and the third with decreased risk of in-stent restenosis (OR 0.09, p=0.004). Two ALOX5AP haplotypes were associated with in-stent restenosis (HapB: OR 3.13, p=0.03); and a haplotype similar to HapA: OR 0.14, p=0.0009).ALOX5AP, a gene within the inflammatory leukotriene pathway linked to and associated with coronary atherosclerosis, is also associated with in-stent restenosis. Genotyping these variants may help identify those at risk for in-stent restenosis who would benefit most from use of DES.

Background Neural tube defects (NTDs), including spina bifida and anencephaly, are the second most common birth defect with an incidence of 1/1000. Genetic factors are believed to contribute to NTD risk and family-based studies can be useful for identifying such risk factors. Methods We ascertained 1066 NTD families (1467 affected patients), including 307 multiplex NTD families. We performed pedigree analysis to describe the inheritance patterns, pregnancy outcomes, and recurrence risks to relatives of various types. Results Myelomeningocele or spina bifida (66.9%) and cranial defects (17.7%) were the most common NTD subtypes observed. The overall male:female ratio for affected individuals was 0.82, and there were even fewer males among individuals with an upper level NTD (0.62). Among twins, 2 of the 5 monozygotic twins and only 3 of 35 dizygotic twins were concordant, while 27% of the same sex twins were concordant, but none of the different sex twins. The estimated 6.3% recurrence risk to siblings (CI 0.04-0.08) is consistent with previous reports. Families with two or more affected individuals show a higher proportion of female transmitters (p = 0.0002). Additionally, the number of affected relatives in maternal compared to paternal lineages was more than double (p = 0.006). There were significantly more miscarriages, infant deaths, and stillborn pregnancies of the maternal aunts and uncles (p Conclusions Our data provide several lines of evidence consistent with a maternal effect, as well as a sex-influenced effect, in the etiology of NTDs.

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD–unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately two-fold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we demonstrate that engineered haplodeficiency of IL-2 gene expression not only reduces T cell IL-2 production by two-fold but also mimics the autoimmune dysregulatory effects of the naturally-occurring susceptibility alleles of IL-2. Reduced IL-2 production achieved by both genetic mechanisms correlates with fewer and less functional CD4+CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.

TMEM231, a functional component of the MKS complex at the ciliary transition zone, is mutated in orofaciodigital syndrome type 3 and Meckel syndrome., The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.

We compared the performance of gas chromatography time-of-flight mass spectrometry (GC-MS) and comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) for metabolite biomarker discovery. Metabolite extracts from 109 human serum samples were analyzed on both platforms with a pooled serum sample analyzed after every 9 biological samples for the purpose of quality control (QC). The experimental data derived from the pooled QC samples showed that the GC×GC-MS platform detected about three times as many peaks as the GC-MS platform at a signal-to-noise ratio SNR ≥ 50, and three times the number of metabolites were identified by mass spectrum matching with a spectral similarity score Rsim ≥ 600. Twenty-three metabolites had statistically significant abundance changes between the patient samples and the control samples in the GC-MS data set while 34 metabolites in the GC×GC-MS data set showed statistically significant differences. Among these two groups of metabolite biomarkers, nine metabolites were detected in both the GC-MS and GC×GC-MS data sets with the same direction and similar magnitude of abundance changes between the control and patient sample groups. Manual verification indicated that the difference in the number of the biomarkers discovered using these two platforms was mainly due to the limited resolution of chromatographic peaks by the GC-MS platform, which can result in severe peak overlap making subsequent spectrum deconvolution for metabolite identification and quantification difficult.

The proteins encoded by the classical HLA class I and class II genes in the major histocompatibility complex (MHC) are highly polymorphic and play an essential role in self/non-self immune recognition. HLA variation is a crucial determinant of transplant rejection and susceptibility to a large number of infectious and autoimmune disease1. Yet identification of causal variants is problematic due to linkage disequilibrium (LD) that extends across multiple HLA and non-HLA genes in the MHC2,3. We therefore set out to characterize the LD patterns between the highly polymorphic HLA genes and background variation by typing the classical HLA genes and >7,500 common single nucleotide polymorphisms (SNPs) and deletion/insertion polymorphisms (DIPs) across four population samples. The analysis provides informative tag SNPs that capture some of the variation in the MHC region and that could be used in initial disease association studies, and provides new insight into the evolutionary dynamics and ancestral origins of the HLA loci and their haplotypes.

Multiple sclerosis (MS) is a common disease of the central nervous system characterized by inflammation, myelin loss, gliosis, varying degrees of axonal pathology, and progressive neurological dysfunction. Multiple sclerosis exhibits many of the characteristics that distinguish complex genetic disorders including polygenic inheritance and environmental exposure risks. Here, we used a highly efficient multilocus genotyping assay representing variation in 34 genes associated with inflammatory pathways to explore gene–gene interactions and disease susceptibility in a well-characterized African-American case–control MS data set. We applied the multifactor dimensionality reduction (MDR) test to detect epistasis, and identified single-IL4R(Q576R)- and three-IL4R(Q576R), IL5RA(-80), CD14(-260)- locus association models that predict MS risk with 75–76% accuracy (P < 0.01). These results demonstrate the importance of exploring both main effects and gene–gene interactions in the study of complex diseases.

Summary: Single nucleotide polymorphisms (SNPs) are commonly used for association studies to find genes responsible for complex genetic diseases. With the recent advance of SNP technology, researchers are able to assay thousands of SNPs in a single experiment. But the process of manually choosing thousands of genotyping SNPs for tens or hundreds of genes is time consuming. We have developed a web-based program, SNPselector, to automate the process. SNPselector takes a list of gene names or a list of genomic regions as input and searches the Ensembl genes or genomic regions for available SNPs. It prioritizes these SNPs on their tagging for linkage disequilibrium, SNP allele frequencies and source, function, regulatory potential and repeat status. SNPselector outputs result in compressed Excel spreadsheet files for review by the user. Availability: SNPselector is freely available at http://primer.duhs.duke.edu/ Contact: mike.hauser@duke.edu

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

Neural tube defects (NTD) have a strong genetic component, with up to 70% of variance in human prevalence determined by heritable factors. Although the identification of causal DNA variants by sequencing candidate genes from functionally relevant pathways and model organisms has provided some success, alternative approaches are demanded.Next generation sequencing platforms are facilitating the production of massive amounts of sequencing data, primarily from the protein coding regions of the genome, at a faster rate and cheaper cost than has previously been possible. These platforms are permitting the identification of variants (de novo, rare, and common) that are drivers of NYTD etiology, and the cost of the approach allows for the screening of increased numbers of affected and unaffected individuals from NTD families and in simplex cases.The next generation sequencing platforms represent a powerful tool in the armory of the genetics researcher to identify the causal genetic basis of NTDs.

Chiari Type I Malformation (CMI) is characterized by herniation of the cerebellar tonsils through the base of the skull. Although cerebellar tonsillar herniation (CTH) is hypothesized to result from an underdeveloped posterior cranial fossa (PF), patients are frequently diagnosed by the extent of CTH without cranial morphometric assessment. We recently completed the largest CMI whole genome qualitative linkage screen to date. Despite an initial lack of statistical evidence, stratified analyses using clinical criteria to reduce heterogeneity resulted in a striking increase in evidence for linkage. The present study focused on the use of cranial base morphometrics to further dissect this heterogeneity and increase power to identify disease genes. We characterized the genetic contribution for a series of PF traits and evaluated the use of heritable, disease-relevant PF traits in ordered subset analysis (OSA). Consistent with a genetic hypothesis for CMI, much of the PF morphology was found to be heritable and multiple genomic regions were strongly implicated from OSA, including regions on Chromosomes 1 (LOD = 3.07, p = 3 × 10(-3) ) and 22 (LOD = 3.45, p = 6 × 10(-5) ) containing several candidates warranting further investigation. This study underscores the genetic heterogeneity of CMI and the utility of PF traits in CMI genetic studies.

Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 5′ splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 5′ splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.

Scope Fumonisins (FB) are mycotoxins found in maize. The purpose of this study was to (i) determine the relationship between FB(1) , FB(2) , and FB(3) intake and urinary excretion in humans, (ii) validate a method to isolate urinary FB on C(18) -SPE cartridges for international shipment, and (iii) test the method using samples from Guatemala. Methods and results Volunteers (n = 10) consumed 206 grams/day of tortillas and biscuits prepared from masa flour and a product containing maize flour. Volunteers estimated their daily urine output and samples were analyzed for FB(1) , FB(2) , and FB(3) and hydrolyzed FB(1) . Only FB(1) was detected in urine suggesting lower absorption of FB(2) and FB(3) . Excretion was highly variable peaking soon after consumption began and decreasing rapidly after consumption stopped. Within 5 days after consumption ended, FB(1) was not detected in urine. In a study with eight volunteers, the average total urinary FB(1) was 0.5% of the intake. FB(1) was detected in 61% (107/177) of the samples collected in Guatemala. Conclusion The results support the use of urinary FB(1) to assess ongoing exposure in population-based studies. However, relating the FB(1) concentration in urine to dietary intake of FB by individual subjects will be complicated due to interindividual variability and the rapidity of clearance.

Background Myelomeningocele (MMC) commonly causes impairments in body structure and functions as well as cognitive disabilities that can have an adverse effect on adult life. Improved medical care has resulted in increased numbers of individuals with MMC surviving to adulthood, however little is known about the impact of MMC on the lives of adults age 25 years or older. Objective To gain a better understanding of outcomes in education, employment, relationships, reproduction and life satisfaction of adults with MMC. Methods A primarily quantitative multiple-choice questionnaire designed to capture outcomes in education, employment, relationships and reproduction, along with a previously validated life satisfaction checklist (LiSat-11), was completed by adults with MMC. Relationships between demographic variables, outcomes and life satisfaction were determined using cross tabulation analysis, logistic regression and linear regression. Results Ninety adults with MMC, age 25–85 years (median age 32), reported a diverse range of outcomes in education, employment, relationships and reproduction. The most consistent variable associated with difficulty attaining adult milestones was hydrocephalus, the presence of which reduced the likelihood of living independently (p ≤ 0.001), having a partner (p = 0.003) and reproducing (p ≤ 0.001), but did not contribute to reduced life satisfaction. Conclusions Adults with MMC, especially those without hydrocephalus, can obtain gainful employment, live independently, form partner relationships and have children, and these achievements contribute to life satisfaction. While MMC does not affect overall reported life satisfaction for adults, attention should be paid to specific domains with less reported satisfaction.

Although Chiari Type I (CM-I) and Type 0 (CM-0) malformations have been previously characterized clinically and radiologically, there have been no studies focusing on the possible genetic link between these disorders. The goal of this study was to identify families in whom CM-0 and CM-I co-occurred and to further assess the similarities between these disorders.Families were ascertained through a proband with CM-I. Detailed family histories were obtained to identify first-degree relatives diagnosed with CM-0. Several criteria were used to exclude individuals with acquired forms of CM-I and/or syringomyelia. Individuals were excluded with syndromic, traumatic, infectious, or tumor-related syringomyelia, as well as CM-I due to a supratentorial mass, hydrocephalus, history of cervical or cranial surgery unrelated to CM-I, or development of symptoms following placement of a lumbar shunt. Medical records and MR images were used to characterize CM-I and CM-0 individuals clinically and radiologically.Five families were identified in which the CM-I proband had a first-degree relative with CM-0. Further assessment of affected individuals showed similar clinical and radiological features between CM-0 and CM-I individuals, although CM-I patients in general had more severe symptoms and skull base abnormalities than their CM-0 relatives. Overall, both groups showed improvement in symptoms and/or syrinx size following craniocervical decompression surgery.There is accumulating evidence suggesting that CM-0 and CM-I may be caused by a common underlying developmental mechanism. The data in this study are consistent with this hypothesis, showing similar clinical and radiological features between CM-0 and CM-I individuals, as well as the occurrence of both disorders within families. Familial clustering of CM-0 and CM-I suggests that these disorders may share an underlying genetic basis, although additional epigenetic and/or environmental factors are likely to play an important role in the development of CM-0 versus CM-I.

Neural tube defects (NTDs) are common human birth defects with a complex etiology. To develop a comprehensive knowledge of the genes expressed during normal neurulation, we established transcriptomes from human neural tube fragments during and after neurulation using long Serial Analysis of Gene Expression (long-SAGE).Rostral and caudal neural tubes were dissected from normal human embryos aged between 26 and 32 days of gestation. Tissues from the same region and Carnegie stage were pooled (n ≥ 4) and total RNA extracted to construct four long-SAGE libraries. Tags were mapped using the UniGene Homo sapiens 17 bp tag-to-gene best mapping set. Differentially expressed genes were identified by chi-square or Fisher's exact test, and validation was performed for a subset of those transcripts using in situ hybridization. In silico analyses were performed with BinGO and EXPANDER.We observed most genes to be similarly regulated in rostral and caudal regions, but expression profiles differed during and after closure. In silico analysis found similar enrichments in both regions for biologic process terms, transcription factor binding and miRNA target motifs. Twelve genes potentially expressing alternate isoforms by region or developmental stage, and the microRNAs miR-339-5p, miR-141/200a, miR-23ab, and miR-129/129-5p are among several potential candidates identified here for future research.Time appears to influence gene expression in the developing central nervous system more than location. These data provide a novel complement to traditional strategies of identifying genes associated with human NTDs and offer unique insight into the genes associated with normal human neurulation.

Background— Risk-stratifying heart failure patients for primary prevention implantable cardioverter-defibrillators (ICDs) remains a challenge, especially for blacks, who have an increased incidence of sudden cardiac death but have been underrepresented in clinical trials. We hypothesized that the S1103Y cardiac sodium channel SCN5A variant influences the propensity for ventricular arrhythmias in black patients with heart failure and reduced ejection fraction. Methods and Results— Blacks (n=112) with ejection fractions P =0.03). Controlling for baseline characteristics, the adjusted hazard ratio using a Cox proportional hazard model for ICD therapy in Y1103 allele carriers was 4.33 (95% confidence interval, 1.60 to 11.73, P = Conclusions— This is the first report that the S1103Y variant is associated with a higher incidence of ventricular arrhythmias in blacks with heart failure and reduced ejection fraction.

Tenascin-C (TNC) is an extracellular matrix protein implicated in biological processes important for atherosclerotic plaque development and progression, including smooth muscle cell migration and proliferation. Previously, we observed differential expression of TNC in atherosclerotic aortas compared with healthy aortas. The goal of this study was to investigate whether common genetic variation within TNC is associated with risk of atherosclerosis and coronary artery disease (CAD) in three independent datasets. We genotyped 35 single nucleotide polymorphisms (SNPs), including 21 haplotype tagging SNPs, in two of these datasets: human aorta tissue samples (n = 205) and the CATHGEN cardiovascular study (n = 1,325). Eleven of these 35 SNPs were then genotyped in a third dataset, the GENECARD family study of early-onset CAD (n = 879 families). Three SNPs representing a block of linkage disequilibrium, rs3789875, rs12347433, and rs4552883, were significantly associated with atherosclerosis in multiple datasets and demonstrated consistent, but suggestive, genetic effects in all analyses. In combined analysis rs3789875 and rs12347433 were statistically significant after Bonferroni correction for 35 comparisons, p = 2 × 10(-6) and 5 × 10(-6), respectively. The SNP rs12347433 is a synonymous coding SNP and may be biologically relevant to the mechanism by which tenascin-C influences the pathophysiology of CAD and atherosclerosis. This is the first report of genetic association between polymorphisms in TNC and atherosclerosis or CAD.

We have used the public sequencing and annotation of the mouse genome to delimit the previously resolved type 1 diabetes (T1D) insulin-dependent diabetes (Idd)18 interval to a region on chromosome 3 that includes the immunologically relevant candidate gene, Vav3. To test the candidacy of Vav3, we developed a novel congenic strain that enabled the resolution of Idd18 to a 604-kb interval, designated Idd18.1, which contains only two annotated genes: the complete sequence of Vav3 and the last exon of the gene encoding NETRIN G1, Ntng1. Targeted sequencing of Idd18.1 in the NOD mouse strain revealed that allelic variation between NOD and C57BL/6J (B6) occurs in noncoding regions with 138 single nucleotide polymorphisms concentrated in the introns between exons 20 and 27 and immediately after the 3′ untranslated region. We observed differential expression of VAV3 RNA transcripts in thymocytes when comparing congenic mouse strains with B6 or NOD alleles at Idd18.1. The T1D protection associated with B6 alleles of Idd18.1/Vav3 requires the presence of B6 protective alleles at Idd3, which are correlated with increased IL-2 production and regulatory T cell function. In the absence of B6 protective alleles at Idd3, we detected a second T1D protective B6 locus, Idd18.3, which is closely linked to, but distinct from, Idd18.1. Therefore, genetic mapping, sequencing, and gene expression evidence indicate that alteration of VAV3 expression is an etiological factor in the development of autoimmune β-cell destruction in NOD mice. This study also demonstrates that a congenic strain mapping approach can isolate closely linked susceptibility genes.

Multiple sclerosis (MS) is a debilitating neuroimmunological and neurodegenerative disease affecting >4,00,000 individuals in the United States. Population and family-based studies have suggested that there is a strong genetic component. Numerous genomic linkage screens have identified regions of interest for MS loci. Our own second-generation genome-wide linkage study identified a handful of non-major histocompatibility complex regions with suggestive linkage. Several of these regions were further examined using single-nucleotide polymorphisms (SNPs) with average spacing between SNPs of approximately 1.0 Mb in a dataset of 173 multiplex families. The results of that study provided further evidence for the involvement of the chromosome 1q43 region. This region is of particular interest given linkage evidence in studies of other autoimmune and inflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus. In this follow-up study, we saturated the region with approximately 700 SNPs (average spacing of 10 kb per SNP) in search of disease-associated variation within this region. We found preliminary evidence to suggest that common variation within the RGS7 locus may be involved in disease susceptibility.

Previous association mapping on chromosome 3q13-21 detected evidence for association at the limbic system-associated membrane protein (LSAMP) gene in individuals with late-onset coronary artery disease (CAD). LSAMP has never been implicated in the pathogenesis of CAD. We sought to thoroughly characterize the association and the gene. Non-redundant single nucleotide polymorphisms (SNPs) across the gene were examined in an initial dataset (168 cases with late-onset CAD, 149 controls). Stratification analysis on left main CAD (N = 102) revealed stronger association, which was further validated in a validation dataset (141 cases with left main CAD, 215 controls), a third control dataset (N = 255), and a family-based dataset (N = 2954). A haplotype residing in a novel alternative transcript of the LSAMP gene was significant in all independent case-control datasets (p = 0.0001 to 0.0205) and highly significant in the joint analysis (p = 0.00004). Lower expression of the novel alternative transcript was associated with the risk haplotype (p = 0.0002) and atherosclerosis burden in human aortas (p = 0.0001). Furthermore, silencing LSAMP expression in human aortic smooth muscle cells (SMCs) substantially augmented SMC proliferation (p

Astrocytic, oligodendroglial and mixed gliomas are the commonest gliomas in adults. They have distinct phenotypes and clinical courses, but as they exist as a continuous histological spectrum, differentiating them can be difficult. Co-deletions of total 1p and 19q are found in the majority of oligodendrogliomas and considered as a diagnostic marker and a prognostic indicator. The 1p status of astrocytomas has not yet been thoroughly examined. Using a chromosome 1 tile path array, we investigated 108 adult astrocytic tumours for copy number alterations. Total 1p deletions were rare (2%), however partial deletions involving 1p36 were frequently identified in anaplastic astrocytomas (22%) and glioblastomas (34%). Multivariate analysis showed that patients with total 1p deletions had significantly longer survival (P=0.005). In nine glioblastomas homozygous deletions at 1p36 were identified. No somatic mutations were found among the five genes located in the homozygously deleted region. However, the CpG island of TNFRSF9 was hypermethylated in 19% of astrocytic tumours and 87% of glioma cell lines. TNFRSF9 expression was upregulated after demethylation of glioma cell lines. Akt3 amplifications were found in four glioblastomas. Our results indicate that 1p deletions are common anaplastic astrocytomas and glioblastomas but are distinct from the 1p abnormalities in oligodendrogliomas.

Fuchs endothelial corneal dystrophy (FECD) is a common progressive disorder of the corneal endothelium that becomes symptomatic with aging.1 It appears as guttae progressively form on Descemet’s membrane along with corneal edema and stromal clouding leading to impaired vision. FECD has been one of the main indications for corneal transplantation in many countries, including the United States,2 with the main treatment modality being a penetrating keratoplasty, until the advent of various types of endothelial keratoplasty. FECD typically becomes symptomatic during the fifth or sixth decade of life and predominantly affects women, who comprise three of four affected individuals.3 The pathogenesis of FECD remains unknown, but the corneal endothelial cells are arrested in the G1 phase of the cell cycle and do not undergo mitosis.4 During a normal state, the cornea is kept in a state of deturgescence by endothelial cell Na+/K+ ATPases that remove fluid from the stroma and by tight junctions between endothelial cells that limit fluid entry.1 This maintains the stroma’s ordered arrangement of collagen and with it, the transparency of the cornea. In states of low endothelial cell density, the loss of the endothelium’s barrier function causes corneal edema. Damage to the endothelial cell layer therefore results in irreparable tissue damage that necessitates endothelial or penetrating keratoplasty.1 It is well established that FECD can affect multiple family members and that several genetic factors underlie the development of the disorder.5 However, the use of genetic approaches to identify the genes underlying the development of FECD is complicated by the late onset of the disease, so that parents and even siblings of the proband are often unavailable for study. In addition, younger individuals in a family may not clinically exhibit the disease because they may not be old enough to manifest the disorder. The first progress toward identifying gene mutations associated with familial FECD was provided by a rare subtype of FECD characterized by onset in early childhood. Early-onset FECD was found to be caused by a missense mutation, Q455K, in the COL8A2 gene (MIM: 120252), which encodes for the α2 subtype of collagen VIII, a major component of Descemet’s membrane. Three other missense mutations within COL8A2—R155Q, R304Q, and R434H—were found in patients with late-onset FECD, but their role remains uncertain, as R155Q has the same frequency in normal controls.6 Another mutation in COL8A2, L450W, that is believed to be pathogenic has been detected in a family with atypical FECD.7,8 Although mutations in COL8A2 have been linked to early-onset FECD, to date, mutations in the COL8A2 gene have not been associated with the much more common late-onset FECD.9–11 Heterozygous single nucleotide polymorphisms in the SLC4A11 gene on chromosome 20 (20p13) have recently been detected in four Chinese and Indian patients with the common phenotype of FECD,12 but they contribute to only ∼5% of the genetic burden of the disease. Family-based studies have mapped late-onset FECD susceptibility loci to 13ptel-13q12.1313 and 18q21.2-q21.32,14 in one and three large multigenerational FECD families, respectively. However, to date, there have been no FECD genetic linkage studies involving a large number of affected families. In this study, we performed the first whole-genome linkage scan for late-onset FECD in 22 families (linkage panel IV; Illumina, Inc., San Diego, CA).

Fuchs corneal dystrophy (FCD) is a degenerative genetic disorder of the corneal endothelium that represents one of the most common causes of corneal transplantation in the United States. Despite its high prevalence (4% over the age of 40), the underlying genetic basis of FCD is largely unknown. Here we report missense mutations in TCF8, a transcription factor whose haploinsufficiency causes posterior polymorphous corneal dystrophy (PPCD), in a cohort of late-onset FCD patients. In contrast to PPCD-causing mutations, all of which are null, FCD-associated mutations encode rare missense changes suggested to cause loss of function by an in vivo complementation assay. Importantly, segregation of a recurring p.Q840P mutation in a large, multigenerational FCD pedigree showed this allele to be sufficient but not necessary for pathogenesis. Execution of a genome-wide scan conditioned for the presence of the 840P allele identified an additional late-onset FCD locus on chromosome 9p, whereas haplotype analysis indicated that the presence of the TCF8 allele and the disease haplotype on 9p leads to a severe FCD manifestation with poor prognosis. Our data suggest that PPCD and FCD are allelic variants of the same disease continuum and that genetic interaction between genes that cause corneal dystrophies can modulate the expressivity of the phenotype.