To date, most genetic association studies of tobacco use have been conducted in European American subjects using the phenotype of smoking quantity (cigarettes per day). However, smoking quantity is a very imprecise measure of exposure to tobacco smoke constituents. Analyses of alternate phenotypes and populations may improve our understanding of tobacco addiction genetics. Cotinine is the major metabolite of nicotine, and measuring serum cotinine levels in smokers provides a more objective measure of nicotine dose than smoking quantity. Previous genetic association studies of serum cotinine have focused on individual genes. We conducted a genetic association study of the biomarker in African American (N=365) and European American (N=315) subjects from the Coronary Artery Risk Development in Young Adults study using a chip containing densely-spaced tag SNPs in ∼2100 genes. We found that rs11187065, located in the non-coding region (intron 1) of insulin-degrading enzyme (IDE), was the most strongly associated SNP (p=8.91 × 10−6) in the African American cohort, whereas rs11763963, located on chromosome 7 outside of a gene transcript, was the most strongly associated SNP in European Americans (p=1.53 × 10−6). We then evaluated how the top variant association in each population performed in the other group. We found that the association of rs11187065 in IDE was also associated with the phenotype in European Americans (p=0.044). Our top SNP association in European Americans, rs11763963 was non-polymorphic in our African American sample. It has been previously shown that psychostimulant self-administration is reduced in animals with lower insulin because of interference with dopamine transmission in the brain reward centers. Our finding provides a platform for further investigation of this, or additional mechanisms, involving the relationship between insulin and self-administered nicotine dose.
cotinine; nicotine; IDE; CARDIA; MORF4L1; IMAT-Broad-CARe; behavioral science; neurogenetics; addiction & substance abuse; pharmacogenetics/pharmacogenomics; cotinine; nicotine; IDE; CARDIA; MORF4L1
Based on interactions among transcription factors, oncogenes, tumor suppressors and microRNAs, a Boolean model of cancer network regulated by miR-17-92 cluster is constructed, and the network is associated with the control of G1/S transition in the mammalian cell cycle. The robustness properties of this regulatory network are investigated by virtue of the Boolean network theory. It is found that, during G1/S transition in the cell cycle process, the regulatory networks are robustly constructed, and the robustness property is largely preserved with respect to small perturbations to the network. By using the unique process-based approach, the structure of this network is analyzed. It is shown that the network can be decomposed into a backbone motif which provides the main biological functions, and a remaining motif which makes the regulatory system more stable. The critical role of miR-17-92 in suppressing the G1/S cell cycle checkpoint and increasing the uncontrolled proliferation of the cancer cells by targeting a genetic network of interacting proteins is displayed with our model.
Serum urate concentrations are highly heritable and elevated serum urate is a key risk factor for gout. Genome-wide association studies (GWAS) of serum urate in African American (AA) populations are lacking. We conducted a meta-analysis of GWAS of serum urate levels and gout among 5820 AA and a large candidate gene study among 6890 AA and 21 708 participants of European ancestry (EA) within the Candidate Gene Association Resource Consortium. Findings were tested for replication among 1996 independent AA individuals, and evaluated for their association among 28 283 EA participants of the CHARGE Consortium. Functional studies were conducted using 14C-urate transport assays in mammalian Chinese hamster ovary cells. In the discovery GWAS of serum urate, three loci achieved genome-wide significance (P< 5.0 × 10−8): a novel locus near SGK1/SLC2A12 on chromosome 6 (rs9321453, P= 1.0 × 10−9), and two loci previously identified in EA participants, SLC2A9 (P= 3.8 × 10−32) and SLC22A12 (P= 2.1 × 10−10). A novel rare non-synonymous variant of large effect size in SLC22A12, rs12800450 (minor allele frequency 0.01, G65W), was identified and replicated (beta −1.19 mg/dl, P= 2.7 × 10−16). 14C-urate transport assays showed reduced urate transport for the G65W URAT1 mutant. Finally, in analyses of 11 loci previously associated with serum urate in EA individuals, 10 of 11 lead single-nucleotide polymorphisms showed direction-consistent association with urate among AA. In summary, we identified and replicated one novel locus in association with serum urate levels and experimentally characterize the novel G65W variant in URAT1 as a functional allele. Our data support the importance of multi-ethnic GWAS in the identification of novel risk loci as well as functional variants.
Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it's target genes p21 and BIM via MYC.
Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell’s plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.
Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the μ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed.
Forty-eight hours after treating SH-SY5Y cells with morphine (10 μM), naloxone (10 μM) induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513) at 1–10 μM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718) did not. Interestingly, CCK-8 (0.1-1 μM), a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1–10 μM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor.
This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.
Cholecystokinin octapeptide; CCK1 receptor; CCK2 receptor; Morphine; Cellular dependence; cAMP overshoot
Urea transporter UT-B is the major urea transporter in erythrocytes and the descending vasa recta in the kidney. In this study, we investigated the effects of long-term UT-B deficiency on functional and structural defect in the kidney of 16-and 52-week-old UT-B-null mice.
UT-B-knockout mice were generated by targeted gene disruption and lacked UT-B protein expression in all organs. The urinary concentrating ability of mice was studied in terms of daily urine output, urine osmolality, and urine and plasma chemistries. Changes in renal morphology were evaluated by hematoxylin and eosin staining.
The UT-B-null mice showed defective urine concentrating ability. The daily urine output in UT-B-null mice (2.5 ± 0.1 ml) was 60% higher and urine osmolality (985 ± 151 mosm) was significantly lower than that in wild-type mice (1463 ± 227 mosm). The 52-week-old UT-B-null mice exhibited polyuria after water deprivation, although urine osmolality was increased. At 52 weeks of age, over 31% of UT-B-null mice exhibited renal medullary atrophy because of severe polyuria and hydronephrosis.
Long-term UT-B deficiency causes severe renal dysfunction and structural damage. These results demonstrate the important role of UT-B in countercurrent exchange and urine concentration.
urea transporter; renal function; kidney; UT-B; knock-out
One of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. Characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. The completion of the bovine genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of the genetic variations present in cattle. Here we describe the whole-genome resequencing of two Bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle.
The genomes of a Black Angus bull and a Holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the ABI SOLiD system. Comparisons of the sequences with the Btau4.0 reference assembly yielded 7 million single nucleotide polymorphisms (SNPs), 24% of which were identified in both animals. Of the total SNPs found in Holstein, Black Angus, and in both animals, 81%, 81%, and 75% respectively are novel. In-depth annotations of the data identified more than 16 thousand distinct non-synonymous SNPs (85% novel) between the two datasets. Alignments between the SNP-altered proteins and orthologues from numerous species indicate that many of the SNPs alter well-conserved amino acids. Several SNPs predicted to create or remove stop codons were also found. A comparison between the sequencing SNPs and genotyping results from the BovineHD high-density genotyping chip indicates a detection rate of 91% for homozygous SNPs and 81% for heterozygous SNPs. The false positive rate is estimated to be about 2% for both the Black Angus and Holstein SNP sets, based on follow-up genotyping of 422 and 427 SNPs, respectively. Comparisons of read depth between the two bulls along the reference assembly identified 790 putative copy-number variations (CNVs). Ten randomly selected CNVs, five genic and five non-genic, were successfully validated using quantitative real-time PCR. The CNVs are enriched for immune system genes and include genes that may contribute to lactation capacity. The majority of the CNVs (69%) were detected as regions with higher abundance in the Holstein bull.
Substantial genetic differences exist between the Black Angus and Holstein animals sequenced in this work and the Hereford reference sequence, and some of this variation is predicted to affect evolutionarily conserved amino acids or gene copy number. The deeply annotated SNPs and CNVs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering DNA differences.
We present a case of a patient with rare anatomy of a maxillary second molar with three mesiobuccal root canals and a maxillary third molar with four separate roots, identified using multi-slice computed topography (CT) and three-dimensional reconstruction techniques. The described case enriched/might enrich our knowledge about possible anatomical aberrations of maxillary molars. In addition, we demonstrate the role of multi-slice CT as an objective tool for confirmatory diagnosis and successful endodontic management.
maxillary second molar; mesiobuccal root; maxillary third molar; multi-slice computed topography; three-dimensional reconstruction
AIM: To describe survival trends in patients in Northeast China diagnosed as gastric cancer.
METHODS: A review of all inpatient and outpatient records of gastric cancer patients was conducted in the First Affiliated Hospital of China Medical University. All the gastric cancer patients who satisfied the inclusion criteria from January 1, 1980 through December 31, 2003 were included in the study. The main outcomes were based on median survival and 3-year and 5-year survival rates, by decade of diagnosis.
RESULTS: From 1980 through 2003, the median survival for patients with gastric cancer (n = 1604) increased from 33 mo to 49 mo. The decade of diagnosis was not significantly associated with patient survival for gastric cancer (P = 0.084 for overall survival, and P = 0.150 for 5-year survival); however, the survival rate of the 2000s was remarkably higher than that of the 1980s (P = 0.019 for overall survival, and P = 0.027 for 5-year survival).
CONCLUSION: There was no significant difference of survival among each period; however, the survival rate of the 2000s was remarkably higher than that of the 1980s.
Survival trends; Gastric cancer; Northeast China
Total white blood cell (WBC) and neutrophil counts are lower among individuals of African descent due to the common African-derived “null” variant of the Duffy Antigen Receptor for Chemokines (DARC) gene. Additional common genetic polymorphisms were recently associated with total WBC and WBC sub-type levels in European and Japanese populations. No additional loci that account for WBC variability have been identified in African Americans. In order to address this, we performed a large genome-wide association study (GWAS) of total WBC and cell subtype counts in 16,388 African-American participants from 7 population-based cohorts available in the Continental Origins and Genetic Epidemiology Network. In addition to the DARC locus on chromosome 1q23, we identified two other regions (chromosomes 4q13 and 16q22) associated with WBC in African Americans (P<2.5×10−8). The lead SNP (rs9131) on chromosome 4q13 is located in the CXCL2 gene, which encodes a chemotactic cytokine for polymorphonuclear leukocytes. Independent evidence of the novel CXCL2 association with WBC was present in 3,551 Hispanic Americans, 14,767 Japanese, and 19,509 European Americans. The index SNP (rs12149261) on chromosome 16q22 associated with WBC count is located in a large inter-chromosomal segmental duplication encompassing part of the hydrocephalus inducing homolog (HYDIN) gene. We demonstrate that the chromosome 16q22 association finding is most likely due to a genotyping artifact as a consequence of sequence similarity between duplicated regions on chromosomes 16q22 and 1q21. Among the WBC loci recently identified in European or Japanese populations, replication was observed in our African-American meta-analysis for rs445 of CDK6 on chromosome 7q21 and rs4065321 of PSMD3-CSF3 region on chromosome 17q21. In summary, the CXCL2, CDK6, and PSMD3-CSF3 regions are associated with WBC count in African American and other populations. We also demonstrate that large inter-chromosomal duplications can result in false positive associations in GWAS.
Although recent genome-wide association studies have identified common genetic variants associated with total white blood cell (WBC) and WBC sub-type counts in European and Japanese ancestry populations, whether these or other loci account for differences in WBC count among African Americans is unknown. By examining >16,000 African Americans, we show that, in addition to the previously identified Duffy Antigen Receptor for Chemokines (DARC) locus on chromosome 1, another variant, rs9131, and other nearby variants on human chromosome 4 are associated with total WBC count in African Americans. The variants span the CXCL2 gene, which encodes an inflammatory mediator involved in WBC production and migration. We show that the association is not restricted to African Americans but is also present in independent samples of European Americans, Hispanic Americans, and Japanese. This finding is potentially important because WBC mediate or have altered counts in a variety of acute and chronic disorders.
The asymmetric unit of the title compound, [Cs2(C2N10)]n, comprises a Cs+ cation, and one-half of a 5,5′-diazenediylditetrazolide anion. The Cs+ cation is six-coordinated by N atoms from six 5,5′-diazenediylditetrazolide ligands. Each 5,5′-diazenediylditetrazolide ligand is surrounded by 12 Cs+ cations, coordinating through ten N atoms. The Cs+ cations are arranged in a chain along the a-axis direction with a Cs⋯Cs separation of 4.4393 (10) Å. Such coordination leads to the formation of the three-dimensional framework.
The asymmetric unit of the title compound, [Rb(C7H3N2O7)]n, comprises an Rb+ cation and a 3,5-dinitrosalicylate ligand. The Rb+ cation is 10-coordinated by O atoms from eight 3,5-dinitrosalicylate anions and is linked by three μ2-O atoms, forming a zigzag chain along the b-axis direction, which is further linked by the phenyl groups, giving the three-dimensional framework. The crystal structure involves intra-anionic O—H⋯O hydrogen bonds and strong π–π stacking interactions [centroid-centroid distance = 3.6755 (7) Å].
The PR interval on the electrocardiogram reflects atrial and atrioventricular nodal conduction time. The PR interval is heritable, provides important information about arrhythmia risk, and has been suggested to differ among human races. Genome-wide association (GWA) studies have identified common genetic determinants of the PR interval in individuals of European and Asian ancestry, but there is a general paucity of GWA studies in individuals of African ancestry. We performed GWA studies in African American individuals from four cohorts (n = 6,247) to identify genetic variants associated with PR interval duration. Genotyping was performed using the Affymetrix 6.0 microarray. Imputation was performed for 2.8 million single nucleotide polymorphisms (SNPs) using combined YRI and CEU HapMap phase II panels. We observed a strong signal (rs3922844) within the gene encoding the cardiac sodium channel (SCN5A) with genome-wide significant association (p<2.5×10−8) in two of the four cohorts and in the meta-analysis. The signal explained 2% of PR interval variability in African Americans (beta = 5.1 msec per minor allele, 95% CI = 4.1–6.1, p = 3×10−23). This SNP was also associated with PR interval (beta = 2.4 msec per minor allele, 95% CI = 1.8–3.0, p = 3×10−16) in individuals of European ancestry (n = 14,042), but with a smaller effect size (p for heterogeneity <0.001) and variability explained (0.5%). Further meta-analysis of the four cohorts identified genome-wide significant associations with SNPs in SCN10A (rs6798015), MEIS1 (rs10865355), and TBX5 (rs7312625) that were highly correlated with SNPs identified in European and Asian GWA studies. African ancestry was associated with increased PR duration (13.3 msec, p = 0.009) in one but not the other three cohorts. Our findings demonstrate the relevance of common variants to African Americans at four loci previously associated with PR interval in European and Asian samples and identify an association signal at one of these loci that is more strongly associated with PR interval in African Americans than in Europeans.
We performed genome-wide association studies in African American participants from four population-based cohorts to identify genetic variation that correlates with variation in PR interval duration, an electrocardiographic measure of conduction through the atria and atrioventricular node. We observed a strong signal within the gene encoding the cardiac sodium channel, SCN5A, with genome-wide significant association (p<2.5×10−8) in two cohorts and in a meta-analysis of four cohorts with African Americans. We replicated this association in two additional cohorts of African Americans and in Europeans (p = 3×10−16). The signal explains 2% of PR duration variability in African Americans and 0.5% in Europeans. In further meta-analysis, we observed genome-wide significant associations for single nucleotide polymorphisms in SCN10A, MEIS1, TBX5, corresponding to signals observed in people of European and Asian descent. We found an association of genetic ancestry and PR interval in one but not the other three cohorts. Our findings provide the first demonstration of the relevance of these loci to individuals of African ancestry and identify an association signal from SCN5A that is more strongly associated with PR interval in African Americans.
The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models.
The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies.
This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical “stress proteins”, such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of “stress” genes for studies understanding marine ectotherms' capacities to cope with environmental change.
The title compound, C10H9NO3, was synthesized by the condensation reaction of isatin (systematic name 1H-indole-2,3-dione) with glycol in presence of p-toluenesulfonic acid. The indol-2-one ring system is essentially planar [N—C—C—C torsion angle = 3.1 (2)°], and the 1,3-dioxolane ring is slightly distorted. The crystal structure exhibits intermolecular N—H⋯O hydrogen bonds.
In the title compound, C14H10N2O2, the dihedral angle between the indole and benzene rings is 61.63 (4)°. In the crystal structure, centrosymmetrically related molecules are linked into dimers by N—H⋯O hydrogen bonds, generating rings of graph-set motif R
2(8). The dimers are further connected into a three-dimensional network by O—H⋯O and C—H⋯O hydrogen bonds.
The receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) pathway is frequently altered in cancer, but the underlying mechanism leading to tumorigenesis by activated mTOR remains less clear. Here we show that mTOR is a positive regulator of Notch signaling in mouse and human cells, acting through induction of the STAT3/p63/Jagged signaling cascade. Furthermore, in response to differential cues from mTOR, we found that Notch served as a molecular switch to shift the balance between cell proliferation and differentiation. We determined that hyperactive mTOR signaling impaired cell differentiation of murine embryonic fibroblasts via potentiation of Notch signaling. Elevated mTOR signaling strongly correlated with enhanced Notch signaling in poorly differentiated but not in well-differentiated human breast cancers. Both human lung lymphangioleiomyomatosis (LAM) and mouse kidney tumors with hyperactive mTOR due to tumor suppressor TSC1 or TSC2 deficiency exhibited enhanced STAT3/p63/Notch signaling. Furthermore, tumorigenic potential of cells with uncontrolled mTOR signaling was suppressed by Notch inhibition. Our data therefore suggest that perturbation of cell differentiation by augmented Notch signaling might be responsible for the underdifferentiated phenotype displayed by certain tumors with an aberrantly activated RTK/PI3K/AKT/mTOR pathway. Additionally, the STAT3/p63/Notch axis may be a useful target for the treatment of cancers exhibiting hyperactive mTOR signaling.
To identify susceptibility loci for bipolar disorder, we tested 1.8 million variants in 4,387 cases and 6,209 controls and identified a region of strong association (rs10994336, P = 9.1 × 10-9) in ANK3 (ankyrin G). We also found further support for the previously reported CACNA1C (alpha 1C subunit of the L-type voltage-gated calcium channel; combined P = 7.0 × 10-8, rs1006737). Our results suggest that ion channelopathies may be involved in the pathogenesis of bipolar disorder.
Despite the widespread application of RNA interference (RNAi) as a research tool for diverse purposes, the key step of strand selection of siRNAs during the formation of RNA-induced silencing complex (RISC) remains poorly understood. Here, using siRNAs targeted to the complementary region of Survivin and the effector protease receptor 1 (EPR-1), we show that both strands of the siRNA duplex can find their target mRNA and are equally eligible for assembly into Argonaute 2 (Ago2) of RISC in HEK293 cells. Transfection of the synthetic siRNA duplexes with different thermodynamic profiles or short hairpin RNA (shRNA) vectors that generate double-stranded RNAs (dsRNAs), permitting processing specifically from either the 5′ or 3′ end of the incipient siRNA, results in the degradation of the respective target mRNAs of either strand of the siRNA duplex with comparable efficiencies. Thus, while most RNAi reactions may follow the thermodynamic asymmetry rule in strand selection, our study suggests an exceptional mode for certain siRNAs in which both strands of the duplex are competent in sponsoring RNAi, and implies additional factors that might dictate the RNAi targets.
The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid β-peptide (Aβ) in Alzheimer’s Disease (AD). We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset AD. These variants, which occur in at least two different clusters of intronic sequences may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways, and that when SORL1 is under-expressed, APP is sorted into Aβ-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing AD.
Single nucleotide polymorphisms (SNPs) may be correlated due to linkage disequilibrium (LD). Association studies look for both direct and indirect associations with disease loci. In a Random Forest (RF) analysis, correlation between a true risk SNP and SNPs in LD may lead to diminished variable importance for the true risk SNP. One approach to address this problem is to select SNPs in linkage equilibrium (LE) for analysis. Here, we explore alternative methods for dealing with SNPs in LD: change the tree-building algorithm by building each tree in an RF only with SNPs in LE, modify the importance measure (IM), and use haplotypes instead of SNPs to build a RF.
We evaluated the performance of our alternative methods by simulation of a spectrum of complex genetics models. When a haplotype rather than an individual SNP is the risk factor, we find that the original Random Forest method performed on SNPs provides good performance. When individual, genotyped SNPs are the risk factors, we find that the stronger the genetic effect, the stronger the effect LD has on the performance of the original RF. A revised importance measure used with the original RF is relatively robust to LD among SNPs; this revised importance measure used with the revised RF is sometimes inflated. Overall, we find that the revised importance measure used with the original RF is the best choice when the genetic model and the number of SNPs in LD with risk SNPs are unknown. For the haplotype-based method, under a multiplicative heterogeneity model, we observed a decrease in the performance of RF with increasing LD among the SNPs in the haplotype.
Our results suggest that by strategically revising the Random Forest method tree-building or importance measure calculation, power can increase when LD exists between SNPs. We conclude that the revised Random Forest method performed on SNPs offers an advantage of not requiring genotype phase, making it a viable tool for use in the context of thousands of SNPs, such as candidate gene studies and follow-up of top candidates from genome wide association studies.
The plasmin system is involved in the degradation of Aβ peptides, the accumulation of which in brain is a hallmark of Alzheimer’s disease (AD). In a North European case-control AD dataset we studied 14 common variations in the PLG, PAI-1, PLAT and PLI genes encoding components of the plasmin system. Among the four polymorphisms in the PLAT, PAI-1 and PLI genes showing nominally significant evidence for an association with AD (allele p-value = 0.01–0.00003) the strongest association was detected for the deletion allele in the Alu-repeat region of the PLAT gene. However, none of these positive results were confirmed in follow-up studies using an independent Canadian case-control cohort and two familial AD datasets of North European and Caribbean Hispanic origin. Thus, the current survey does not support the notion that common polymorphisms in the plasmin genes influence the development of AD.
Alzheimer’s disease; plasmin; polymorphism; risk factor
A broad region on chromosome 12p13 has been intensely investigated for novel genetic variants associated with Alzheimer disease (AD). We examined this region with 23 microsatellite markers using 124 North European (NE) families and 209 Caribbean Hispanic families with late-onset AD (FAD). Significant evidence for linkage was present in a 5 cM interval near 20 cM in both the NE FAD (LOD=3.5) and the Caribbean Hispanic FAD (LOD=2.2) datasets. We further investigated these families and an independent NE case-control dataset using 14 single nucleotide polymorphisms (SNPs). The initial screening of the region at ~20 cM in the NE case-control dataset revealed significant association between AD and seven SNPs in several genes, with the strongest result for rs2532500 in TAPBPL (p=0.006). For rs3741916 in GAPDH, the C allele, rather than the G allele as was observed by Li and colleagues (2004), was the risk allele. When the two family datasets were examined, none of the SNPs were significant in NE families, but two SNPs were associated with AD in Caribbean Hispanics: rs740850 in NCAPD2 (p=0.0097) and rs1060620 in GAPDH (p=0.042). In a separate analysis combining the Caribbean Hispanic families and NE cases and controls, rs740850 was significant after correcting for multiple testing (empirical p=0.0048). Subsequent haplotype analyses revealed that two haplotype sets -- haplotype C-A at SNPs 6-7 within NCAPD2 in Caribbean Hispanics, and haplotypes containing C-A-T at SNPs 8-10 within GAPDH in Caribbean Hispanic family and NE case-control datasets -- were associated with AD. Taken together, these SNPs may be in linkage disequilibrium with a pathogenic variant(s) on or near NCAPD2 and GAPDH.
Alzheimer disease; GAPDH; NCAPD2; linkage; association
Several studies [1–3] have reported an association of Alzheimer disease (AD) with polymorphic markers in SORL1. Data from a recently published genome wide association study in AD  have been made publically available. We tested the association of AD with SORL1 in this dataset (TGEN), which included 31 SORL1 SNPs, 8 of which overlapped the original study . Six SNPs, near the 3′ region of SORL1 containing SNPs which were strongly associated with AD in previous studies, showed significant association in the TGEN dataset. These results provide an independent replication of the association between AD and SORL1.
Alzheimer disease; SORL1; association; genome-wide association study