Identifying Relationships among Genomic Disease Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions

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/).     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

Analysis of Trace Elements During Different Developmental Stages of Somatic Embryogenesis in Plantago ovata Forssk Using Energy Dispersive X-ray Fluorescence

Energy dispersive X-ray fluorescence (ED-XRF) technique has been used for the determination of trace element profile during different developmental stages of somatic embryogenic callus of an economically important medicinal plant, Plantago ovata Forssk. Somatic embryogenesis is a plant tissue culture-based technique, which is used for plant regeneration and crop improvement. In the present investigation, elemental content was analysed using ED-XRF technique during different developmental stages and also determine the effect of additives—casein hydrolysate and coconut water on the trace elemental profile of embryogenic callus tissue of P. ovata. Subsequent experiments showed significant alteration in the concentration of K, Ca, Mn, Fe, Zn, Cu, Br, and Sr in both the embryogenic and non-embryogenic callus. Higher K, Ca, Fe, Cu, and Zn accumulation was in embryogenic tissue stage compared to other stages, suggesting these elements are crucial for successful embryogenesis. The results suggest that this information could be useful for formulating a media for in vitro embryo induction of P. ovata.     

DDB2, an Essential Mediator of Premature Senescence

Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.DDB2 is encoded by the nucleotide excision repair (NER) XPE gene (17, 24, 33). Unlike other NER gene-deficient cells or xeroderma pigmentosum (XP) cells, the XPE cells exhibit only a mild deficiency in NER (55). However, because of its high affinity for cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, several studies implicated DDB2 in the early damaged-DNA recognition step of NER (61). However, a direct role of DDB2 in NER is a point of controversy (28, 41, 57). Lower organisms (yeasts), in which other XP genes are conserved, apparently do not encode a DDB2 homolog (55, 64). We showed that DDB2 associates with Cul4, a component of an E3 ubiquitin ligase complex that is now known to involve the DDB2 binding protein DDB1 as its adapter (48). The Cul4-DDB1 E3 ligase associates with a number of substrate-specific adapter proteins to target substrates for ubiquitination (30, 35). DDB2 is believed to be one of those substrate adapters, which allows Cul4-DDB1 to target specific proteins. Two studies suggested that the Cul4A-DDB1-DDB2 complex could participate in the ubiquitination of histones, indicating a role of DDB2 in chromatin remodeling (23, 59). Other investigators suggested a role of Cul4A-DDB1-DDB2 in the ubiquitination of XPC (15, 52). We recently found that DDB2 is involved also in targeting p21 for proteolysis and demonstrated that DDB2 stimulated NER by regulating the level of p21 (51).It was shown elsewhere that DDB2^−/− mouse embryonic fibroblasts (MEFs) are resistant to UV-induced apoptosis (20, 21). Recently, we extended those observations by demonstrating that DDB2^−/− MEFs or DDB2-deficient human cells are resistant to apoptosis induced by a variety of DNA-damaging agents (50). Moreover, DDB2^−/− MEFs are deficient in E2F1-induced apoptosis. The resistance to apoptosis is linked also to high-level accumulation of p21 because deletion of p21 restored apoptosis. The polyubiquitination of p21 is significantly reduced in DDB2-deficient cells (50), suggesting that after DNA damage DDB2 plays a key role in polyubiquitinating p21. Also, we observed evidence for a physical association between DDB2 and p21, which was increased in UV-irradiated cells (50), indicating that DDB2 plays a direct role in targeting p21 for proteolysis after DNA damage. These observations provided evidence that DDB2, in addition to stimulating NER, plays a significant role in terminating DNA damage checkpoint, allowing cells with extensive DNA damage to undergo apoptosis.In addition to its role in the inhibition of cell cycle and apoptosis, p21 has been implicated also in cellular senescence, as its level increases in senescent cells (7). Cellular senescence is defined as a proliferative arrest of a cell after a limited number of cell divisions while the cell remains metabolically and synthetically active (6, 63). Senescence can be triggered by both extrinsic factors such as oncogenic stress, DNA damage, oxidative stress, and culture shock and intrinsic factors such as telomere regression in human cells (19). When grown in cell culture medium, human diploid fibroblasts undergo 60 to 80 population doublings, after which they cease proliferation as a result of telomere erosion and enter into the stage of replicative senescence characterized by enlarged and flattened morphology, increased granularity, and enhanced senescence-associated β-galactosidase (SA-β-Gal) activity (13). In contrast, telomere length does not limit the ability of the murine fibroblasts to proliferate in culture. It was shown that the supraphysiological level of oxygen or reactive oxygen species (ROS) under which the cells are grown led murine fibroblasts to senesce (39). ROS accumulation or oxidative stress induces the senescent phenotype in response to oncogenic stress as well as in response to DNA-damaging agents (56). These pathways have been termed premature senescence, which recapitulates molecular features of replicative senescence. Premature senescence induced by oncogene expression is a significant mechanism of tumor suppression involving the Ink4a/Arf locus (47). Moreover, DNA damage-induced premature senescence is significant, as many anticancer drugs have been shown to induce premature senescence of tumor cells (12, 44).Because DDB2^−/− MEFs express p21 at a high level, we expected those cells to undergo premature senescence at an earlier passage than the wild-type (WT) MEFs. Surprisingly, we found that DDB2^−/− MEFs escape senescence at a very high frequency. Moreover, DDB2^−/− MEFs or DDB2-deficient human cells are resistant to premature senescence induced by a variety of agents, including oncogenic stress, exogenous oxidative stress, and DNA damage. The lack of premature senescence in the presence of high-level p21, especially after DNA damage, suggests that DDB2 functions in the senescence program through a mechanism that is downstream of the p21 pathway senescence. Here we show that DDB2 participates in the senescence program by inducing persistent accumulation of ROS.     

Absence of adipose differentiation related protein upregulates hepatic VLDL secretion,relieves hepatosteatosis,and improves whole body insulin resistance in leptin-deficient mice

We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep^ob/ob mice. We bred Adfp^−/− mice with Lep^ob/ob mice to create Lep^ob/ob/Adfp^−/− and Lep^ob/ob/Adfp^+/+ mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep^ob/ob/Adfp^+/+ mice, Lep^ob/ob/Adfp^−/− mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep^ob/ob/Adfp^−/− mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep^ob/ob/Adfp^−/− mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.     

PGE2 induces IL-6 in orbital fibroblasts through EP2 receptors and increased gene promoter activity: implications to thyroid-associated ophthalmopathy

Background

IL-6 plays an important role in the pathogenesis of Graves'' disease and its orbital component, thyroid-associated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE₂, underlying the inflammatory phenotype of these cells.

Methodology/Principal Findings

Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE₂ on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP₂ receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE₂-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE₂ provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE₂ promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts.

Conclusion/Significance

These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO.     

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.     

Role of an ABC Importer in Mycobacterial Drug Resistance

Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIP^r) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIP^r.