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Sample records for functional promoter variant

  1. Genetic and Functional Sequence Variants of the SIRT3 Gene Promoter in Myocardial Infarction

    PubMed Central

    Yin, Xiaoyun; Pang, Shuchao; Huang, Jian; Cui, Yinghua; Yan, Bo

    2016-01-01

    Coronary artery disease (CAD), including myocardial infarction (MI), is a common complex disease that is caused by atherosclerosis. Although a large number of genetic variants have been associated with CAD, only 10% of CAD cases could be explained. It has been proposed that low frequent and rare genetic variants may be main causes for CAD. SIRT3, a mitochondrial deacetylase, plays important roles in mitochondrial function and metabolism. Lack of SIRT3 in experimental animal leads to several age-related diseases, including cardiovascular diseases. Therefore, SIRT3 gene variants may contribute to the MI development. In this study, SIRT3 gene promoter was genetically and functionally analyzed in large cohorts of MI patients (n = 319) and ethnic-matched controls (n = 322). Total twenty-three DNA sequence variants (DSVs) were identified, including 10 single-nucleotide polymorphisms (SNPs). Six novel heterozygous DSVs, g.237307A>G, g.237270G>A, g.237023_25del, g.236653C>A, g.236628G>C, g.236557T>C, and two SNPs g.237030C>T (rs12293349) and g.237022C>G (rs369344513), were identified in nine MI patients, but in none of controls. Three SNPs, g.236473C>T (rs11246029), g.236380_81ins (rs71019893) and g.236370C>G (rs185277566), were more significantly frequent in MI patients than controls (P<0.05). These DSVs and SNPs, except g.236557T>C, significantly decreased the transcriptional activity of the SIRT3 gene promoter in cultured HEK-293 cells and H9c2 cells. Therefore, these DSVs identified in MI patients may change SIRT3 level by affecting the transcriptional activity of SIRT3 gene promoter, contributing to the MI development as a risk factor. PMID:27078640

  2. Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element.

    PubMed

    Olds, Lynne C; Sibley, Eric

    2003-09-15

    Lactase persistence is a heritable, autosomal dominant, condition that results in a sustained ability to digest the milk sugar lactose throughout adulthood. The majority of the world's human population experiences a decline in production of the digestive enzyme lactase-phlorizin hydrolase during maturation. However, individuals with lactase persistence continue to express high levels of the lactase gene into adulthood. Lactase persistence has been strongly correlated with single nucleotide genetic variants, C/T_(13910) and G/A_(22018), located 13.9 and 22 kb upstream from the lactase structural gene. We aimed to characterize a functional role for the polymorphisms in regulating lactase gene transcription. DNA in the region of the C/T_(13910) or G/A_(22018) human lactase variants was cloned upstream of the 3.0 kb rat lactase gene promoter in a luciferase reporter construct. Human intestinal Caco-2 cells were transfected with the lactase variant/promoter-reporter constructs and assayed for promoter activity. A 200 bp region surrounding the C_(13910) variant, associated with lactase non-persistence, results in a 2.2-fold increase in lactase promoter activity. The T_(13910) variant, associated with lactase persistence, results in an even greater 2.8-fold increase. The DNA sequence of the C/T_(13910) variants differentially interacts with intestinal cell nuclear proteins on EMSAs. AP2 co-transfection results in a similar repression of the C/T_(13910) variant/promoter-reporter constructs. The DNA region of the C/T_(13910) lactase persistence/non-persistence variant functions in vitro as a cis element capable of enhancing differential transcriptional activation of the lactase promoter. Such differential regulation by the C and T variants is consistent with a causative role in the mechanism specifying the lactase persistence/non-persistence phenotypes in humans.

  3. Autism-Associated Promoter Variant in MET Impacts Functional and Structural Brain Networks

    PubMed Central

    Rudie, J. D.; Hernandez, L. M.; Brown, J. A.; Beck-Pancer, D.; Colich, N. L.; Gorrindo, P.; Thompson, P. M.; Geschwind, D. H.; Bookheimer, S. Y.; Levitt, P.; Dapretto, M.

    2012-01-01

    SUMMARY As genes that confer increased risk for autism spectrum disorder (ASD) are identified, a crucial next step is to determine how these risk factors impact brain structure and function and contribute to disorder heterogeneity. With three converging lines of evidence, we show that a common, functional ASD risk variant in the Met Receptor Tyrosine Kinase (MET) gene is a potent modulator of key social brain circuitry in children and adolescents with and without ASD. MET risk genotype predicted atypical fMRI activation and deactivation patterns to social stimuli (i.e., emotional faces), as well as reduced functional and structural connectivity in temporo-parietal regions known to have high MET expression, particularly within the default mode network. Notably, these effects were more pronounced in individuals with ASD. These findings highlight how genetic stratification may reduce heterogeneity and help elucidate the biological basis of complex neuropsychiatric disorders such as ASD. PMID:22958829

  4. A functional variant in the CD209 promoter is associated with DQ2-negative celiac disease in the Spanish population

    PubMed Central

    Núñez, C; Rueda, B; Martínez, A; Maluenda, C; Polanco, I; López-Nevot, MA; Ortega, E; Sierra, E; de la Concha, E Gómez; Urcelay, E; Martín, J

    2006-01-01

    AIM: To address the role of CD209 in celiac disease (CD) patients. Non-human leukocyte antigen (HLA) genetic factors in CD predisposition are poorly understood, and environmental factors like infectious pathogens may play a role. CD209 is a dendritic and macrophage surface molecule involved in pathogen recognition and immune activation. Recently, a functional variant in the promoter of the CD209 gene (-336A/G) has been shown to affect the transcriptional CD209 activity in vitro and it has been associated with a higher susceptibility to/or severity of infection. METHODS: The study population was composed of two case-control cohorts of 103 and 386 CD patients and 312 y 419 healthy controls as well as a panel of 257 celiac families. Genotyping for the -336A/G CD209 promoter polymorphism was performed using a TaqMan 5´ allelic discrimination assay. HLA-DQ was determined by hybridization with allele specific probes. RESULTS: Initially, the case-control and familial studies did not find any association of the -336 A/G CD209 genetic variant with CD susceptibility. However, the stratification by HLA-DQ2 did reveal a significant association of CD209 promoter polymorphism in the HLA-DQ2 (-) group (carrier A vs GG in DQ2 (-) vs DQ2 (+) patients (P = 0.026, OR = 3.71). CONCLUSION: The -336G CD209 allele seems to be involved in CD susceptibility in HLA-DQ2 (-) patients. Our results might suggest a possible role of pathogens in the onset of a minor group of CD patients. PMID:16865785

  5. Functional analysis of a promoter variant identified in the CFTR gene in cis of a frameshift mutation.

    PubMed

    Viart, Victoria; Des Georges, Marie; Claustres, Mireille; Taulan, Magali

    2012-02-01

    In monogenic diseases, the presence of several sequence variations in the same allele may complicate our understanding of genotype-phenotype relationships. We described new alterations identified in a cystic fibrosis (CF) patient harboring a 48C>G promoter sequence variation associated in cis of a 3532AC>GTA mutation and in trans with the F508del mutation. Functional analyses including in vitro experiments confirmed the deleterious effect of the 3532GTA frameshift mutation through the creation of a premature termination codon. The analyses also revealed that the 48G promoter variant has a negative effect on both transcription and mRNA level, thus demonstrating the importance of analyzing all mutations or sequence variations with potential impact on CF transmembrane conductance regulator processing, even when the two known disease-causing mutations have already been detected. Our results emphasize the need to perform, wherever possible, functional studies that may greatly assist the interpretation of the disease-causing potential of rare mutation-associated sequence variations.

  6. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo.

    PubMed

    Wang, Xiaowen; Li, Kai; Liu, Ling; Shi, Qiong; Song, Pu; Jian, Zhe; Guo, Sen; Wang, Gang; Li, Chunying; Gao, Tianwen

    2015-09-15

    Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR -129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that -129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with -129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than -129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.

  7. AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo

    PubMed Central

    Wang, Xiaowen; Li, Kai; Liu, Ling; Shi, Qiong; Song, Pu; Jian, Zhe; Guo, Sen; Wang, Gang; Li, Chunying; Gao, Tianwen

    2015-01-01

    Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR −129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that −129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with −129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than −129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo. PMID:26370050

  8. Role of the 5-HTTLPR and SNP Promoter Polymorphisms on Serotonin Transporter Gene Expression: a Closer Look at Genetic Architecture and In Vitro Functional Studies of Common and Uncommon Allelic Variants.

    PubMed

    Iurescia, Sandra; Seripa, Davide; Rinaldi, Monica

    2016-10-01

    The serotonin (5-hydroxytriptamine (5-HT)) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) is a variable number tandem repeats (VNTR) located in the promoter region of the human 5-HTT-encoding gene SLC6A4. This length polymorphism gives rise to different promoter variants, variously influencing SLC6A4 expression. Over the years, an extensive literature has investigated the relationships between these promoter variants and SLC6A4 gene expression, since these variants have been variously associated to complex neuropsychiatric conditions and traits. In this review, we detail the genetic architecture of the 5-HTTLPR allelic variants reported so far, with a closer look at the two single nucleotide polymorphisms (SNPs) rs25531 and rs25532 that lies in the VNTR and thus increase genetic variability of the SLC6A4 promoter. We summarize the hypothesized molecular mechanisms underlying this variation. We also provide an update on common and uncommon 5-HTTLPR allelic variants reviewing the available data on functional in vitro analysis of their regulatory effect on SLC6A4 gene transcription. Controversial findings are highlighted and critically discussed. A deeper knowledge of the "5-HTTLPR universe" will be useful to better understand the molecular basis of serotonin homeostasis and the pathological basis underlying serotonin-related neuropsychiatric conditions and traits.

  9. A splicing variant of Merlin promotes metastasis in hepatocellular carcinoma

    PubMed Central

    Luo, Zai-Li; Cheng, Shu-Qun; Shi, Jie; Zhang, Hui-Lu; Zhang, Cun-Zhen; Chen, Hai-Yang; Qiu, Bi-Jun; Tang, Liang; Hu, Cong-Li; Wang, Hong-Yang; Li, Zhong

    2015-01-01

    Merlin, which is encoded by the tumour suppressor gene Nf2, plays a crucial role in tumorigenesis and metastasis. However, little is known about the functional importance of Merlin splicing forms. In this study, we show that Merlin is present at low levels in human hepatocellular carcinoma (HCC), particularly in metastatic tumours, where it is associated with a poor prognosis. Surprisingly, a splicing variant of Merlin that lacks exons 2, 3 and 4 (Δ2–4Merlin) is amplified in HCC and portal vein tumour thrombus (PVTT) specimens and in the CSQT2 cell line derived from PVTT. Our studies show that Δ2–4Merlin interferes with the capacity of wild-type Merlin to bind β-catenin and ERM, and it is expressed in the cytoplasm rather than at the cell surface. Furthermore, Δ2–4Merlin overexpression increases the expression levels of β-catenin and stemness-related genes, induces the epithelium–mesenchymal-transition phenotype promoting cell migration in vitro and the formation of lung metastasis in vivo. Our results indicate that the Δ2–4Merlin variant disrupts the normal function of Merlin and promotes tumour metastasis. PMID:26443326

  10. MICA variant promotes allosensitization after kidney transplantation.

    PubMed

    Tonnerre, Pierre; Gérard, Nathalie; Chatelais, Mathias; Poli, Caroline; Allard, Stéphanie; Cury, Sylvie; Bressollette, Céline; Cesbron-Gautier, Anne; Charreau, Béatrice

    2013-05-01

    MHC class I-related chain A (MICA) antigens are surface glycoproteins strongly implicated in innate immunity, and the MICA gene is highly polymorphic. Clinical observations suggest a role for donor MICA antigens expressed on transplant endothelial cells in the alloimmune response, but the effect of MICA genotype is not well understood. Here, we investigated the immunologic effect of the A5.1 mutation, related to the common MICA*008 allele. Compared with wild-type endothelial cells (ECs), homozygosity for MICA A5.1 associated with an endothelial phenotype characterized by 7- to 10-fold higher levels of MICA mRNA and MICA proteins at the cell surface, as well as exclusive release in exosomes instead of enzymatic cleavage. Mechanistically, we did not detect quantitative changes in regulatory microRNAs. Functionally, A5.1 ECs enhanced NKG2D interaction and natural killer cell activation, promoting NKG2D-dependent lysis of ECs. In kidney transplant recipients, polyreactive anti-MICA sera bound preferentially to ECs from MICA A5.1 donors, suggesting that MICA*008(A5.1) molecules are the preferential antigenic determinants on ECs of grafts. Furthermore, the incidence of MICA A5.1 mismatch revealed a statistically significant association between donor MICA A5.1 and both anti-MICA sensitization and increased proteinuria in kidney recipients. Taken together, these results identify the A5.1 mutation as an immunodominant factor and a potential risk factor for transplant survival.

  11. Exome-based Variant Detection in Core Promoters.

    PubMed

    Kim, Yeong C; Cui, Jian; Luo, Jiangtao; Xiao, Fengxia; Downs, Bradley; Wang, San Ming

    2016-07-28

    Core promoter controls the initiation of transcription. Core promoter sequence change can disrupt transcriptional regulation, lead to impairment of gene expression and ultimately diseases. Therefore, comprehensive characterization of core promoters is essential to understand normal and abnormal gene expression in biomedical studies. Here we report the development of EVDC (Exome-based Variant Detection in Core promoters) method for genome-scale analysis of core-promoter sequence variation. This method is based on the fact that exome sequences contain the sequences not only from coding exons but also from non-coding region including core promoters generated by random fragmentation in exome sequencing process. Using exome data from three cell types of CD4+ T cells, CD19+ B cells and neutrophils of a single individual, we characterized the features of core promoter-mapped exome sequences, and analysed core-promoter variation in this individual genome. We also compared the core promoters between YRI (Yoruba in Ibadan, Nigeria) and the CEU (Utah residents of European decedent) populations using the exome data generated by the 1000 Genome project, and observed much higher variation in YRI population than in CEU population. Our study demonstrates that the EVDC method provides a simple but powerful means for genome-wile de novo characterization of core promoter sequence variation.

  12. Exome-based Variant Detection in Core Promoters

    PubMed Central

    Kim, Yeong C.; Cui, Jian; Luo, Jiangtao; Xiao, Fengxia; Downs, Bradley; Wang, San Ming

    2016-01-01

    Core promoter controls the initiation of transcription. Core promoter sequence change can disrupt transcriptional regulation, lead to impairment of gene expression and ultimately diseases. Therefore, comprehensive characterization of core promoters is essential to understand normal and abnormal gene expression in biomedical studies. Here we report the development of EVDC (Exome-based Variant Detection in Core promoters) method for genome-scale analysis of core-promoter sequence variation. This method is based on the fact that exome sequences contain the sequences not only from coding exons but also from non-coding region including core promoters generated by random fragmentation in exome sequencing process. Using exome data from three cell types of CD4+ T cells, CD19+ B cells and neutrophils of a single individual, we characterized the features of core promoter-mapped exome sequences, and analysed core-promoter variation in this individual genome. We also compared the core promoters between YRI (Yoruba in Ibadan, Nigeria) and the CEU (Utah residents of European decedent) populations using the exome data generated by the 1000 Genome project, and observed much higher variation in YRI population than in CEU population. Our study demonstrates that the EVDC method provides a simple but powerful means for genome-wile de novo characterization of core promoter sequence variation. PMID:27464681

  13. Exome-based Variant Detection in Core Promoters.

    PubMed

    Kim, Yeong C; Cui, Jian; Luo, Jiangtao; Xiao, Fengxia; Downs, Bradley; Wang, San Ming

    2016-01-01

    Core promoter controls the initiation of transcription. Core promoter sequence change can disrupt transcriptional regulation, lead to impairment of gene expression and ultimately diseases. Therefore, comprehensive characterization of core promoters is essential to understand normal and abnormal gene expression in biomedical studies. Here we report the development of EVDC (Exome-based Variant Detection in Core promoters) method for genome-scale analysis of core-promoter sequence variation. This method is based on the fact that exome sequences contain the sequences not only from coding exons but also from non-coding region including core promoters generated by random fragmentation in exome sequencing process. Using exome data from three cell types of CD4+ T cells, CD19+ B cells and neutrophils of a single individual, we characterized the features of core promoter-mapped exome sequences, and analysed core-promoter variation in this individual genome. We also compared the core promoters between YRI (Yoruba in Ibadan, Nigeria) and the CEU (Utah residents of European decedent) populations using the exome data generated by the 1000 Genome project, and observed much higher variation in YRI population than in CEU population. Our study demonstrates that the EVDC method provides a simple but powerful means for genome-wile de novo characterization of core promoter sequence variation. PMID:27464681

  14. COMT gene locus: new functional variants.

    PubMed

    Meloto, Carolina B; Segall, Samantha K; Smith, Shad; Parisien, Marc; Shabalina, Svetlana A; Rizzatti-Barbosa, Célia M; Gauthier, Josée; Tsao, Douglas; Convertino, Marino; Piltonen, Marjo H; Slade, Gary Dmitri; Fillingim, Roger B; Greenspan, Joel D; Ohrbach, Richard; Knott, Charles; Maixner, William; Zaykin, Dmitri; Dokholyan, Nikolay V; Reenilä, Ilkka; Männistö, Pekka T; Diatchenko, Luda

    2015-10-01

    Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3' untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes. PMID:26207649

  15. COMT gene locus: new functional variants

    PubMed Central

    Meloto, Carolina B.; Segall, Samantha K.; Smith, Shad; Parisien, Marc; Shabalina, Svetlana A.; Rizzatti-Barbosa, Célia M.; Gauthier, Josée; Tsao, Douglas; Convertino, Marino; Piltonen, Marjo H.; Slade, Gary Dmitri; Fillingim, Roger B.; Greenspan, Joel D.; Ohrbach, Richard; Knott, Charles; Maixner, William; Zaykin, Dmitri; Dokholyan, Nikolay V.; Reenilä, Ilkka; Männistö, Pekka T.; Diatchenko, Luda

    2015-01-01

    Abstract Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3′ untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes. PMID:26207649

  16. Functional Promoter Variant rs2868371 of HSPB1 Is Associated With Risk of Radiation Pneumonitis After Chemoradiation for Non-Small Cell Lung Cancer

    SciTech Connect

    Pang, Qingsong; Wei, Qingyi; Xu, Ting; Yuan, Xianglin; Lopez Guerra, Jose Luis; Levy, Lawrence B.; Liu, Zhensheng; Gomez, Daniel R.; Zhuang, Yan; Wang, Li-E.; Mohan, Radhe; Komaki, Ritsuko; Liao, Zhongxing

    2013-04-01

    Purpose: To date, no biomarkers have been found to predict, before treatment, which patients will develop radiation pneumonitis (RP), a potentially fatal toxicity, after chemoradiation for lung cancer. We investigated potential associations between single nucleotide polymorphisms (SNPs) in HSPB1 and risk of RP after chemoradiation for non-small cell lung cancer (NSCLC). Methods and Materials: Subjects were patients with NSCLC treated with chemoradiation at 1 institution. The training data set comprised 146 patients treated from 1999 to July 2004; the validation data set was 125 patients treated from August 2004 to March 2010. We genotyped 2 functional SNPs of HSPB1 (rs2868370 and rs2868371) from all patients. We used Kaplan-Meier analysis to assess the risk of grade ≥2 or ≥3 RP in both data sets and a parametric log-logistic survival model to evaluate the association of HSPB1 genotypes with that risk. Results: Grade ≥3 RP was experienced by 13% of those with CG/GG and 29% of those with CC genotype of HSPB1 rs2868371 in the training data set (P=.028); corresponding rates in the validation data set were 2% CG/GG and 14% CC (P=.02). Univariate and multivariate analysis confirmed the association of CC of HSPB1 rs2868371 with higher risk of grade ≥3 RP than CG/GG after adjustment for sex, age, performance status, and lung mean dose. This association was validated both in the validation data set and with Harrell's C statistic. Conclusions: The CC genotype of HSPB1 rs2868371 was associated with severe RP after chemoradiation for NSCLC.

  17. Functional annotation of non-coding sequence variants

    PubMed Central

    Ritchie, Graham R. S.; Dunham, Ian; Zeggini, Eleftheria; Flicek, Paul

    2016-01-01

    Identifying functionally relevant variants against the background of ubiquitous genetic variation is a major challenge in human genetics. For variants that fall in protein-coding regions our understanding of the genetic code and splicing allow us to identify likely candidates, but interpreting variants that fall outside of genic regions is more difficult. Here we present a new tool, GWAVA, which supports prioritisation of non-coding variants by integrating a range of annotations. PMID:24487584

  18. The power of multiplexed functional analysis of genetic variants.

    PubMed

    Gasperini, Molly; Starita, Lea; Shendure, Jay

    2016-10-01

    New technologies have recently enabled saturation mutagenesis and functional analysis of nearly all possible variants of regulatory elements or proteins of interest in single experiments. Here we discuss the past, present, and future of such multiplexed (functional) assays for variant effects (MAVEs). MAVEs provide detailed insight into sequence-function relationships, and they may prove critical for the prospective clinical interpretation of genetic variants. PMID:27583640

  19. Ghrelin and its promoter variant associated with cardiac hypertrophy.

    PubMed

    Ukkola, O; Pääkkö, T; Kesäniemi, Y A

    2012-07-01

    The roles of ghrelin, a peptide hormone that has a role in regulating food intake and energy homeostasis, in the cardiovascular system have not yet been unambiguously established. We evaluated the association between plasma ghrelin concentrations and -501A>C single-nucleotide polymorphism (SNP) in the ghrelin gene 5' flanking area and echocardiographic measurements in 1037 middle-aged subjects. Left ventricular mass index (LVMI) was calculated according to Devereux's method. The ambulatory blood pressure (BP) was recorded using the fully automatic SpaceLabs 90207 oscillometric unit. Results suggested that plasma ghrelin was not related to mean ambulatory BP values. However, the highest plasma ghrelin tertile was associated with increased intraventricular septum (P=0.043) and posterior ventricular wall (P=0.002) thicknesses as well as left ventricular mass (P=0.05). After adjustment for age, sex, body mass index and systolic BP, the association persisted between ghrelin tertiles and intraventricular septum (P=0.05) and posterior ventricular wall (P=0.001) thicknesses. The SNP -501A>C polymorphism was associated with LVMI after adjustments for age, sex and systolic BP. In conclusion, ghrelin and its promoter variant are associated with cardiac hypertrophy indexes independent of BP. Positive correlation between ghrelin levels and increased wall thickness parameters may reflect compensatory up-regulation of ghrelin concentrations or direct effects of ghrelin on myocardium. The effects of the SNP seem not to be mediated through its effects on ghrelin plasma levels. PMID:21614024

  20. The personal genome browser: visualizing functions of genetic variants.

    PubMed

    Juan, Liran; Teng, Mingxiang; Zang, Tianyi; Hao, Yafeng; Wang, Zhenxing; Yan, Chengwu; Liu, Yongzhuang; Li, Jie; Zhang, Tianjiao; Wang, Yadong

    2014-07-01

    Advances in high-throughput sequencing technologies have brought us into the individual genome era. Projects such as the 1000 Genomes Project have led the individual genome sequencing to become more and more popular. How to visualize, analyse and annotate individual genomes with knowledge bases to support genome studies and personalized healthcare is still a big challenge. The Personal Genome Browser (PGB) is developed to provide comprehensive functional annotation and visualization for individual genomes based on the genetic-molecular-phenotypic model. Investigators can easily view individual genetic variants, such as single nucleotide variants (SNVs), INDELs and structural variations (SVs), as well as genomic features and phenotypes associated to the individual genetic variants. The PGB especially highlights potential functional variants using the PGB built-in method or SIFT/PolyPhen2 scores. Moreover, the functional risks of genes could be evaluated by scanning individual genetic variants on the whole genome, a chromosome, or a cytoband based on functional implications of the variants. Investigators can then navigate to high risk genes on the scanned individual genome. The PGB accepts Variant Call Format (VCF) and Genetic Variation Format (GVF) files as the input. The functional annotation of input individual genome variants can be visualized in real time by well-defined symbols and shapes. The PGB is available at http://www.pgbrowser.org/. PMID:24799434

  1. Detection of rare functional variants using group ISIS.

    PubMed

    Niu, Yue S; Hao, Ning; An, Lingling

    2011-11-29

    Genome-wide association studies have been firmly established in investigations of the associations between common genetic variants and complex traits or diseases. However, a large portion of complex traits and diseases cannot be explained well by common variants. Detecting rare functional variants becomes a trend and a necessity. Because rare variants have such a small minor allele frequency (e.g., <0.05), detecting functional rare variants is challenging. Group iterative sure independence screening (ISIS), a fast group selection tool, was developed to select important genes and the single-nucleotide polymorphisms within. The performance of the group ISIS and group penalization methods is compared for detecting important genes in the Genetic Analysis Workshop 17 data. The results suggest that the group ISIS is an efficient tool to discover genes and single-nucleotide polymorphisms associated to phenotypes.

  2. Fine-Mapping of Common Genetic Variants Associated with Colorectal Tumor Risk Identified Potential Functional Variants.

    PubMed

    Du, Mengmeng; Jiao, Shuo; Bien, Stephanie A; Gala, Manish; Abecasis, Goncalo; Bezieau, Stephane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J; Carlson, Christopher S; Casey, Graham; Chang-Claude, Jenny; Conti, David V; Curtis, Keith R; Duggan, David; Gallinger, Steven; Haile, Robert W; Harrison, Tabitha A; Hayes, Richard B; Hoffmeister, Michael; Hopper, John L; Hudson, Thomas J; Jenkins, Mark A; Küry, Sébastien; Le Marchand, Loic; Leal, Suzanne M; Newcomb, Polly A; Nickerson, Deborah A; Potter, John D; Schoen, Robert E; Schumacher, Fredrick R; Seminara, Daniela; Slattery, Martha L; Hsu, Li; Chan, Andrew T; White, Emily; Berndt, Sonja I; Peters, Ulrike

    2016-01-01

    Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. These SNPs may tag correlated variants with biological importance. Fine-mapping around GWAS loci can facilitate detection of functional candidates and additional independent risk variants. We analyzed 11,900 cases and 14,311 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. To fine-map genomic regions containing all known common risk variants, we imputed high-density genetic data from the 1000 Genomes Project. We tested single-variant associations with colorectal tumor risk for all variants spanning genomic regions 250-kb upstream or downstream of 31 GWAS-identified SNPs (index SNPs). We queried the University of California, Santa Cruz Genome Browser to examine evidence for biological function. Index SNPs did not show the strongest association signals with colorectal tumor risk in their respective genomic regions. Bioinformatics analysis of SNPs showing smaller P-values in each region revealed 21 functional candidates in 12 loci (5q31.1, 8q24, 11q13.4, 11q23, 12p13.32, 12q24.21, 14q22.2, 15q13, 18q21, 19q13.1, 20p12.3, and 20q13.33). We did not observe evidence of additional independent association signals in GWAS-identified regions. Our results support the utility of integrating data from comprehensive fine-mapping with expanding publicly available genomic databases to help clarify GWAS associations and identify functional candidates that warrant more onerous laboratory follow-up. Such efforts may aid the eventual discovery of disease-causing variant(s).

  3. Fine-Mapping of Common Genetic Variants Associated with Colorectal Tumor Risk Identified Potential Functional Variants.

    PubMed

    Du, Mengmeng; Jiao, Shuo; Bien, Stephanie A; Gala, Manish; Abecasis, Goncalo; Bezieau, Stephane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J; Carlson, Christopher S; Casey, Graham; Chang-Claude, Jenny; Conti, David V; Curtis, Keith R; Duggan, David; Gallinger, Steven; Haile, Robert W; Harrison, Tabitha A; Hayes, Richard B; Hoffmeister, Michael; Hopper, John L; Hudson, Thomas J; Jenkins, Mark A; Küry, Sébastien; Le Marchand, Loic; Leal, Suzanne M; Newcomb, Polly A; Nickerson, Deborah A; Potter, John D; Schoen, Robert E; Schumacher, Fredrick R; Seminara, Daniela; Slattery, Martha L; Hsu, Li; Chan, Andrew T; White, Emily; Berndt, Sonja I; Peters, Ulrike

    2016-01-01

    Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. These SNPs may tag correlated variants with biological importance. Fine-mapping around GWAS loci can facilitate detection of functional candidates and additional independent risk variants. We analyzed 11,900 cases and 14,311 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. To fine-map genomic regions containing all known common risk variants, we imputed high-density genetic data from the 1000 Genomes Project. We tested single-variant associations with colorectal tumor risk for all variants spanning genomic regions 250-kb upstream or downstream of 31 GWAS-identified SNPs (index SNPs). We queried the University of California, Santa Cruz Genome Browser to examine evidence for biological function. Index SNPs did not show the strongest association signals with colorectal tumor risk in their respective genomic regions. Bioinformatics analysis of SNPs showing smaller P-values in each region revealed 21 functional candidates in 12 loci (5q31.1, 8q24, 11q13.4, 11q23, 12p13.32, 12q24.21, 14q22.2, 15q13, 18q21, 19q13.1, 20p12.3, and 20q13.33). We did not observe evidence of additional independent association signals in GWAS-identified regions. Our results support the utility of integrating data from comprehensive fine-mapping with expanding publicly available genomic databases to help clarify GWAS associations and identify functional candidates that warrant more onerous laboratory follow-up. Such efforts may aid the eventual discovery of disease-causing variant(s). PMID:27379672

  4. Fine-Mapping of Common Genetic Variants Associated with Colorectal Tumor Risk Identified Potential Functional Variants

    PubMed Central

    Gala, Manish; Abecasis, Goncalo; Bezieau, Stephane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J.; Carlson, Christopher S.; Casey, Graham; Chang-Claude, Jenny; Conti, David V.; Curtis, Keith R.; Duggan, David; Gallinger, Steven; Haile, Robert W.; Harrison, Tabitha A.; Hayes, Richard B.; Hoffmeister, Michael; Hopper, John L.; Hudson, Thomas J.; Jenkins, Mark A.; Küry, Sébastien; Le Marchand, Loic; Leal, Suzanne M.; Newcomb, Polly A.; Nickerson, Deborah A.; Potter, John D.; Schoen, Robert E.; Schumacher, Fredrick R.; Seminara, Daniela; Slattery, Martha L.; Hsu, Li; Chan, Andrew T.; White, Emily; Berndt, Sonja I.; Peters, Ulrike

    2016-01-01

    Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. These SNPs may tag correlated variants with biological importance. Fine-mapping around GWAS loci can facilitate detection of functional candidates and additional independent risk variants. We analyzed 11,900 cases and 14,311 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. To fine-map genomic regions containing all known common risk variants, we imputed high-density genetic data from the 1000 Genomes Project. We tested single-variant associations with colorectal tumor risk for all variants spanning genomic regions 250-kb upstream or downstream of 31 GWAS-identified SNPs (index SNPs). We queried the University of California, Santa Cruz Genome Browser to examine evidence for biological function. Index SNPs did not show the strongest association signals with colorectal tumor risk in their respective genomic regions. Bioinformatics analysis of SNPs showing smaller P-values in each region revealed 21 functional candidates in 12 loci (5q31.1, 8q24, 11q13.4, 11q23, 12p13.32, 12q24.21, 14q22.2, 15q13, 18q21, 19q13.1, 20p12.3, and 20q13.33). We did not observe evidence of additional independent association signals in GWAS-identified regions. Our results support the utility of integrating data from comprehensive fine-mapping with expanding publicly available genomic databases to help clarify GWAS associations and identify functional candidates that warrant more onerous laboratory follow-up. Such efforts may aid the eventual discovery of disease-causing variant(s). PMID:27379672

  5. Lithium and GSK3-β Promoter Gene Variants Influence White Matter Microstructure in Bipolar Disorder

    PubMed Central

    Benedetti, Francesco; Bollettini, Irene; Barberi, Ignazio; Radaelli, Daniele; Poletti, Sara; Locatelli, Clara; Pirovano, Adele; Lorenzi, Cristina; Falini, Andrea; Colombo, Cristina; Smeraldi, Enrico

    2013-01-01

    Lithium is the mainstay for the treatment of bipolar disorder (BD) and inhibits glycogen synthase kinase 3-β (GSK3-β). The less active GSK3-β promoter gene variants have been associated with less detrimental clinical features of BD. GSK3-β gene variants and lithium can influence brain gray matter structure in psychiatric conditions. Diffusion tensor imaging (DTI) measures of white matter (WM) integrity showed widespred disruption of WM structure in BD. In a sample of 70 patients affected by a major depressive episode in course of BD, we investigated the effect of ongoing long-term lithium treatment and GSK3-β promoter rs334558 polymorphism on WM microstructure, using DTI and tract-based spatial statistics with threshold-free cluster enhancement. We report that the less active GSK3-β rs334558*C gene-promoter variants, and the long-term administration of the GSK3-β inhibitor lithium, were associated with increases of DTI measures of axial diffusivity (AD) in several WM fiber tracts, including corpus callosum, forceps major, anterior and posterior cingulum bundle (bilaterally including its hippocampal part), left superior and inferior longitudinal fasciculus, left inferior fronto-occipital fasciculus, left posterior thalamic radiation, bilateral superior and posterior corona radiata, and bilateral corticospinal tract. AD reflects the integrity of axons and myelin sheaths. We suggest that GSK3-β inhibition and lithium could counteract the detrimental influences of BD on WM structure, with specific benefits resulting from effects on specific WM tracts contributing to the functional integrity of the brain and involving interhemispheric, limbic, and large frontal, parietal, and fronto-occipital connections. PMID:22990942

  6. Risk variants in BMP4 promoters for nonsyndromic cleft lip/palate in a Chilean population

    PubMed Central

    2011-01-01

    Background Bone morphogenetic protein 4 gene (BMP4) plays a key role during maxillofacial development, since orofacial clefts are observed in animals when this gene is conditionally inactivated. We recently reported the existence of association between nonsyndromic cleft lip/palate (NSCLP) and BMP4 polymorphisms by detecting transmission deviations for haplotypes that include a region containing a BMP4 promoter in case-parent trios. The aim of the present study was to search for possible causal mutations within BMP4 promoters (BMP4.1 and BMP4.2). Methods We analyzed the sequence of BMP4.1 and BMP4.2 in 167 Chilean NSCLP cases and 336 controls. Results We detected three novel variants in BMP4.1 (c.-5514G > A, c.-5365C > T and c.-5049C > T) which could be considered as cleft risk factors due to their absence in controls. Additionally, rs2855530 G allele (BMP4.2) carriers showed an increased risk for NSCLP restricted to males (OR = 1.52; 95% C.I. = 1.07-2.15; p = 0.019). For this same SNP the dominant genotype model showed a higher frequency of G/G+G/C and a lower frequency of C/C in cases than controls in the total sample (p = 0.03) and in the male sample (p = 0.003). Bioinformatic prediction analysis showed that all the risk variants detected in this study could create new transcription factor binding motifs. Conclusions The sex-dependent association between rs2855530 and NSCLP could indirectly be related to the differential gene expression observed between sexes in animal models. We concluded that risk variants detected herein could potentially alter BMP4 promoter activity in NSCLP. Further functional and developmental studies are necessary to support this hypothesis. PMID:22182590

  7. Lithium and GSK3-β promoter gene variants influence white matter microstructure in bipolar disorder.

    PubMed

    Benedetti, Francesco; Bollettini, Irene; Barberi, Ignazio; Radaelli, Daniele; Poletti, Sara; Locatelli, Clara; Pirovano, Adele; Lorenzi, Cristina; Falini, Andrea; Colombo, Cristina; Smeraldi, Enrico

    2013-01-01

    Lithium is the mainstay for the treatment of bipolar disorder (BD) and inhibits glycogen synthase kinase 3-β (GSK3-β). The less active GSK3-β promoter gene variants have been associated with less detrimental clinical features of BD. GSK3-β gene variants and lithium can influence brain gray matter structure in psychiatric conditions. Diffusion tensor imaging (DTI) measures of white matter (WM) integrity showed widespred disruption of WM structure in BD. In a sample of 70 patients affected by a major depressive episode in course of BD, we investigated the effect of ongoing long-term lithium treatment and GSK3-β promoter rs334558 polymorphism on WM microstructure, using DTI and tract-based spatial statistics with threshold-free cluster enhancement. We report that the less active GSK3-β rs334558*C gene-promoter variants, and the long-term administration of the GSK3-β inhibitor lithium, were associated with increases of DTI measures of axial diffusivity (AD) in several WM fiber tracts, including corpus callosum, forceps major, anterior and posterior cingulum bundle (bilaterally including its hippocampal part), left superior and inferior longitudinal fasciculus, left inferior fronto-occipital fasciculus, left posterior thalamic radiation, bilateral superior and posterior corona radiata, and bilateral corticospinal tract. AD reflects the integrity of axons and myelin sheaths. We suggest that GSK3-β inhibition and lithium could counteract the detrimental influences of BD on WM structure, with specific benefits resulting from effects on specific WM tracts contributing to the functional integrity of the brain and involving interhemispheric, limbic, and large frontal, parietal, and fronto-occipital connections.

  8. Assessment of functional effects of unclassified genetic variants.

    PubMed

    Couch, Fergus J; Rasmussen, Lene Juel; Hofstra, Robert; Monteiro, Alvaro N A; Greenblatt, Marc S; de Wind, Niels

    2008-11-01

    Inherited predisposition to disease is often linked to reduced activity of a disease associated gene product. Thus, quantitation of the influence of inherited variants on gene function can potentially be used to predict the disease relevance of these variants. While many disease genes have been extensively characterized at the functional level, few assays based on functional properties of the encoded proteins have been established for the purpose of predicting the contribution of rare inherited variants to disease. Much of the difficulty in establishing predictive functional assays stems from the technical complexity of the assays. However, perhaps the most challenging aspect of functional assay development for clinical testing purposes is the absolute requirement for validation of the sensitivity and specificity of the assays and the determination of positive predictive values (PPVs) and negative predictive values (NPVs) of the assays relative to a "gold standard" measure of disease predisposition. In this commentary, we provide examples of some of the functional assays under development for several cancer predisposition genes (BRCA1, BRCA2, CDKN2A, and mismatch repair [MMR] genes MLH1, MSH2, MSH6, and PMS2) and present a detailed review of the issues associated with functional assay development. We conclude that validation is paramount for all assays that will be used for clinical interpretation of inherited variants of any gene, but note that in certain circumstances information derived from incompletely validated assays may be valuable for classification of variants for clinical purposes when used to supplement data derived from other sources. PMID:18951449

  9. Impact of Interacting Functional Variants in COMT on Regional Gray Matter Volume in Human Brain

    PubMed Central

    Honea, Robyn; Verchinski, Beth A.; Pezawas, Lukas; Kolachana, Bhaskar S.; Callicott, Joseph H.; Mattay, Venkata S.; Weinberger, Daniel R.; Meyer-Lindenberg, Andreas

    2009-01-01

    Background Functional variants in the catechol-O-methyltransferase (COMT) gene have been shown to impact cognitive function, cortical physiology and risk for schizophrenia. A recent study showed that previously reported effects of the functional val158met SNP (rs4680) on brain function are modified by other functional SNPs and haplotypes in the gene, though it was unknown if these effects are also seen in brain structure. Methods We used voxel-based morphometry to investigate the impact of multiple functional variants in COMT on gray matter volume in a large group of 151 healthy volunteers from the CBDB/NIMH Genetic Study of Schizophrenia. Results We found that the previously described rs4680 val risk variant affects hippocampal and dorsolateral prefrontal (DLPFC) gray matter volume. In addition, we found that this SNP interacts with a variant in the P2 promoter region (rs2097603) in predicting changes in hippocampal gray matter volume consistent with a nonlinear effect of extracellular dopamine. Conclusions We report evidence that interacting functional variants in COMT affect gray matter regional volume in hippocampus and DLPFC, providing further in vivo validation of the biological impact of complex genetic variation in COMT on neural systems relevant for the pathophysiology of schizophrenia and extending observations of nonlinear dependence of prefrontal neurons on extracellular dopamine to the domain of human brain structure. PMID:19071221

  10. Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris.

    PubMed

    Ruth, C; Zuellig, T; Mellitzer, A; Weis, R; Looser, V; Kovar, K; Glieder, A

    2010-09-01

    Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.

  11. Promoter Variants of the ADAM10 Gene and Their Roles in Temporal Lobe Epilepsy

    PubMed Central

    Tao, Hua; Zhao, Jianghao; Zhou, Xu; Ma, Zhonghua; Chen, Ying; Sun, Fuhai; Cui, Lili; Zhou, Haihong; Cai, Yujie; Chen, Yanyan; Zhao, Shu; Yao, Lifen; Zhao, Bin; Li, Keshen

    2016-01-01

    Previous evidence has indicated that downregulated ADAM10 gives rise to epileptic seizures in Alzheimer’s disease, and this study investigated the association of ADAM10 with temporal lobe epilepsy (TLE) from a genetic perspective. A total of 496 TLE patients and 528 healthy individuals were enrolled and genotyped for ADAM10 promoter variants (rs653765 G > A and rs514049 A > C). The alleles, genotypes, and haplotypes were then compared with clarify the association of these variants with TLE and their impacts upon age at onset, initial seizure types before treatments, and responses to drug treatments. In cohorts I, II, and I + II, the frequencies of the A allele and AA genotype at rs514049 were consistently increased in the cases compared with the controls (p = 0.020 and p = 0.009; p = 0.008 and p = 0.009; p = 0.000 and p = 0.000; q = 0.003 and q = 0.002, respectively). In contrast, the frequency of the AC haplotype (rs653765–rs514049) decreased in cohorts I + II (p = 0.013). Further analyses of the TLE patients indicated that the AA genotype functioned as a predisposing factor to drug-resistant TLE and the AC haplotype as a protective factor against generalized tonic–clonic seizures (GTCS) and drug-resistant TLE. This study is the first to demonstrate an association of the ADAM10 promoter variants with TLE. In particular, the AA genotype and AC haplotype showed their effects upon GTCS and drug-resistant TLE. PMID:27445971

  12. Radon Exposure, IL-6 Promoter Variants, and Lung Squamous Cell Carcinoma in Former Uranium Miners

    PubMed Central

    Leng, Shuguang; Thomas, Cynthia L.; Snider, Amanda M.; Picchi, Maria A.; Chen, Wenshu; Willis, Derall G.; Carr, Teara G.; Krzeminski, Jacek; Desai, Dhimant; Shantu, Amin; Lin, Yong; Jacobson, Marty R.; Belinsky, Steven A.

    2015-01-01

    Background: High radon exposure is a risk factor for squamous cell carcinoma, a major lung cancer histology observed in former uranium miners. Radon exposure can cause oxidative stress, leading to pulmonary inflammation. Interleukin-6 (IL-6) is a pro-carcinogenic inflammatory cytokine that plays a pivotal role in lung cancer development. Objectives: We assessed whether single nucleotide polymorphisms (SNPs) in the IL6 promoter are associated with lung cancer in former uranium miners with high occupational exposure to radon gas. Methods: Genetic associations were assessed in a case–control study of former uranium miners (242 cases and 336 controls). A replication study was performed using data from the Gene Environment Association Studies (GENEVA) Genome Wide Association Study (GWAS) of Lung Cancer and Smoking. Functional relevance of the SNPs was characterized using in vitro approaches. Results: We found that rs1800797 was associated with squamous cell carcinoma in miners and with a shorter time between the midpoint of the period of substantial exposure and diagnosis among the cases. Furthermore, rs1800797 was also associated with lung cancer among never smokers in the GENEVA dataset. Functional studies identified that the risk allele was associated with increased basal IL-6 mRNA level and greater promoter activity. Furthermore, fibroblasts with the risk allele showed greater induction of IL-6 secretion by hydrogen peroxide or benzo[a]pyrene diolepoxide treatments. Conclusions: An IL6 promoter variant was associated with lung cancer in uranium miners and never smokers in two external study populations. The associations are strongly supported by the functional relevance that the IL6 promoter SNP affects basal expression and carcinogen-induced IL-6 secretion. Citation: Leng S, Thomas CL, Snider AM, Picchi MA, Chen W, Willis DG, Carr TG, Krzeminski J, Desai D, Shantu A, Lin Y, Jacobson MR, Belinsky SA. 2016. Radon exposure, IL-6 promoter variants, and lung squamous

  13. Massively Parallel Functional Analysis of BRCA1 RING Domain Variants

    PubMed Central

    Starita, Lea M.; Young, David L.; Islam, Muhtadi; Kitzman, Jacob O.; Gullingsrud, Justin; Hause, Ronald J.; Fowler, Douglas M.; Parvin, Jeffrey D.; Shendure, Jay; Fields, Stanley

    2015-01-01

    Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing. PMID:25823446

  14. Promoter variants determine γ-aminobutyric acid homeostasis-related gene transcription in human epileptic hippocampi.

    PubMed

    Pernhorst, Katharina; Raabe, Anna; Niehusmann, Pitt; van Loo, Karen M J; Grote, Alexander; Hoffmann, Per; Cichon, Sven; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2011-12-01

    The functional consequences of single nucleotide polymorphisms associated with episodic brain disorders such as epilepsy and depression are unclear. Allelic associations with generalized epilepsies have been reported for single nucleotide polymorphisms rs1883415 (ALDH5A1; succinic semialdehyde dehydrogenase) and rs4906902 (GABRB3; GABAA β3), both of which are present in the 5' regulatory region of genes involved in γ-aminobutyric acid (GABA) homeostasis. To address their allelic association with episodic brain disorders and allele-specific impact on the transcriptional regulation of these genes in human brain tissue, DNA and messenger RNA (mRNA) isolated from hippocampi were obtained at epilepsy surgery of 146 pharmacoresistant mesial temporal lobe epilepsy (mTLE) patients and from 651 healthy controls. We found that the C allele of rs1883415 is accumulated to a greater extentin mTLE versus controls. By real-time quantitative reverse transcription-polymerase chain reaction analyses, individuals homozygous for the C allele showed higher ALDH5A1 mRNA expression. The rs4906902 G allele of the GABRB3 gene was overrepresented in mTLE patients with depression; individuals homozygous for the G allele showed reduced GABRB3 mRNA expression. Bioinformatic analyses suggest that rs1883415 and rs4906902 alter the DNA binding affinity of the transcription factors Egr-3 in ALDH5A1 and MEF-2 in GABRB3 promoters, respectively. Using in vitro luciferase transfection assays, we observed that, in both cases, the transcription factors regulate gene expression depending on the allelic variant in the same direction as in the human hippocampi. Our data suggest that distinct promoter variants may sensitize individuals for differential, potentially stimulus-induced alterations of GABA homeostasis-relevant gene expression. This might contribute to the episodic onset of symptoms and point to new targets for pharmacotherapies.

  15. Predicting functional decline in behavioural variant frontotemporal dementia.

    PubMed

    Josephs, Keith A; Whitwell, Jennifer L; Weigand, Stephen D; Senjem, Matthew L; Boeve, Bradley F; Knopman, David S; Smith, Glenn E; Ivnik, Robert J; Jack, Clifford R; Petersen, Ronald C

    2011-02-01

    Behavioural variant frontotemporal dementia is characterized by a change in comportment. It is associated with considerable functional decline over the course of the illness albeit with sometimes dramatic variability among patients. It is unknown whether any baseline features, or combination of features, could predict rate of functional decline in behavioural variant frontotemporal dementia. The aim of this study was to investigate the effects of different baseline clinical, neuropsychological, neuropsychiatric, genetic and anatomic predictors on the rate of functional decline as measured by the Clinical Dementia Rating Sum of Boxes scale. We identified 86 subjects with behavioural variant frontotemporal dementia that had multiple serial Clinical Dementia Rating Sum of Boxes assessments (mean 4, range 2-18). Atlas-based parcellation was used to generate volumes for specific regions of interest at baseline. Volumes were utilized to classify subjects into different anatomical subtypes using the advanced statistical technique of cluster analysis and were assessed as predictor variables. Composite scores were generated for the neuropsychological domains of executive, language, memory and visuospatial function. Behaviours from the brief questionnaire form of the Neuropsychiatric Inventory were assessed. Linear mixed-effects regression modelling was used to determine which baseline features predict rate of future functional decline. Rates of functional decline differed across the anatomical subtypes of behavioural variant frontotemporal dementia, with faster rates observed in the frontal dominant and frontotemporal subtypes. In addition, subjects with poorer performance on neuropsychological tests of executive, language and visuospatial function, less disinhibition, agitation/aggression and night-time behaviours at presentation, and smaller medial, lateral and orbital frontal lobe volumes showed faster rates of decline. In many instances, the effect of the predictor

  16. Engineered antibody Fc variants with enhanced effector function

    PubMed Central

    Lazar, Greg A.; Dang, Wei; Karki, Sher; Vafa, Omid; Peng, Judy S.; Hyun, Linus; Chan, Cheryl; Chung, Helen S.; Eivazi, Araz; Yoder, Sean C.; Vielmetter, Jost; Carmichael, David F.; Hayes, Robert J.; Dahiyat, Bassil I.

    2006-01-01

    Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcγ receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcγ receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy. PMID:16537476

  17. Engineered antibody Fc variants with enhanced effector function

    NASA Astrophysics Data System (ADS)

    Lazar, Greg A.; Dang, Wei; Karki, Sher; Vafa, Omid; Peng, Judy S.; Hyun, Linus; Chan, Cheryl; Chung, Helen S.; Eivazi, Araz; Yoder, Sean C.; Vielmetter, Jost; Carmichael, David F.; Hayes, Robert J.; Dahiyat, Bassil I.

    2006-03-01

    Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fc receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fc receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy. antibody-dependent cell-mediated cytotoxicity | FcR | protein engineering | cancer

  18. Engineered antibody Fc variants with enhanced effector function.

    PubMed

    Lazar, Greg A; Dang, Wei; Karki, Sher; Vafa, Omid; Peng, Judy S; Hyun, Linus; Chan, Cheryl; Chung, Helen S; Eivazi, Araz; Yoder, Sean C; Vielmetter, Jost; Carmichael, David F; Hayes, Robert J; Dahiyat, Bassil I

    2006-03-14

    Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

  19. Histone variants--the structure behind the function.

    PubMed

    Ausió, Juan

    2006-09-01

    In recent years, the chromatin field has witnessed a renewed interest in histone variants as pertaining to their structural role, but mainly because of the functional specificity they impart to chromatin. In this review, I am going to discuss several of the most recent structural studies on core histone (H2A.Bbd, H2A.Z, H2A.X, macroH2A, H3.3, CENP-A) and linker histone variants (histone H1 microheterogeneity) focusing on their role in nucleosome stability and chromatin fibre dynamics with special emphasis on their possible functional implications. The data accumulated to date indicates that histone variability plays an important role in the histone-mediated regulation of chromatin metabolism. Understanding and deciphering the underlying structural amino acid code behind such variability remains one of the most exciting future challenges in chromatin research.

  20. Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia

    PubMed Central

    Fazio, Leonardo; Caforio, Grazia; Blasi, Giuseppe; Rampino, Antonio; Romano, Raffaella; Di Giorgio, Annabella; Taurisano, Paolo; Papp, Audrey; Pinsonneault, Julia; Wang, Danxin; Nardini, Marcello; Popolizio, Teresa; Sadee, Wolfgang

    2009-01-01

    Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case–control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density. PMID:18829695

  1. Association Analysis of GABRB3 Promoter Variants with Heroin Dependence

    PubMed Central

    Chen, Chia-Hsiang; Huang, Chia-Chun; Liao, Ding-Lieh

    2014-01-01

    GABRB3 encoding the β3 subunit of GABAA receptor has been implicated in multiple neuropsychiatric disorders, including substance abuse. Previous studies reported that SNPs at the 5′ regulatory region of GABRB3 could regulate GABRB3 gene expression and associated with childhood absence epilepsy (CAE). The study aimed to investigate whether SNPs at the 5′ regulatory region of GABRB3 were associated with heroin dependence in our population. We first re-sequenced 1.5 kb of the 5′regulatory region of GABRB3 gene to examine the SNP profile in the genomic DNA of 365 control subjects. Then, we conducted a case-control association analysis between 576 subjects with heroin dependence (549 males, 27 females) and 886 controls (472 males, 414 females) by genotyping the rs4906902 as a tag SNP. We also conducted a reporter gene assay to assess the promoter activity of two major haplotypes derived from SNPs at this region. We detected 3 common SNPs (rs4906902, rs8179184 and rs20317) at this region that had strong pair-wise linkage disequilibrium. The C allele of rs4906902 was found to be associated with increased risk of heroin dependence (odds ratio:1.27, p = 0.002). Two major haplotypes (C-A-G and T-G-C) derived from these 3 SNPs accounted for 99% of this sample, and reporter gene activity assay showed that haplotype C-A-G that contained the C allele of the tag SNP rs4906902 had higher activity than haplotype T-G-C. Our data suggest that GABRB3 might be associated with heroin dependence, and increased expression of GABRB3 might contribute to the pathogenesis of heroin dependence. PMID:25025424

  2. 15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers

    PubMed Central

    Leng, Shuguang; Liu, Yushi; Weissfeld, Joel L.; Thomas, Cynthia L.; Han, Younghun; Picchi, Maria A.; Edlund, Christopher K.; Willink, Randall P.; Gaither Davis, Autumn L.; Do, Kieu C.; Nukui, Tomoko; Zhang, Xiequn; Burki, Elizabeth A.; Van Den Berg, David; Romkes, Marjorie; Gauderman, W. James; Crowell, Richard E.; Tesfaigzi, Yohannes; Stidley, Christine A.; Amos, Christopher I.; Siegfried, Jill M.; Gilliland, Frank D.

    2015-01-01

    Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis. Methods: A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided. Results: Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10–8). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10–9). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin. Conclusions: A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing. PMID:25713168

  3. Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations.

    PubMed

    Dogruluk, Turgut; Tsang, Yiu Huen; Espitia, Maribel; Chen, Fengju; Chen, Tenghui; Chong, Zechen; Appadurai, Vivek; Dogruluk, Armel; Eterovic, Agna Karina; Bonnen, Penelope E; Creighton, Chad J; Chen, Ken; Mills, Gordon B; Scott, Kenneth L

    2015-12-15

    Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers. Here we describe a mutation assessment pipeline enabled by high-throughput engineering of molecularly barcoded gene variant expression clones identified by tumor sequencing. We first used this platform to functionally assess tail mutations observed in PIK3CA, which encodes the catalytic subunit alpha of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) frequently mutated in cancer. Orthogonal screening for PIK3CA variant activity using in vitro and in vivo cell growth and transformation assays differentiated driver from passenger mutations, revealing that PIK3CA variant activity correlates imperfectly with its mutation frequency across breast cancer populations. Although PIK3CA mutations with frequencies above 5% were significantly more oncogenic than wild-type in all assays, mutations occurring at 0.07% to 5.0% included those with and without oncogenic activities that ranged from weak to strong in at least one assay. Proteomic profiling coupled with therapeutic sensitivity assays on PIK3CA variant-expressing cell models revealed variant-specific activation of PI3K signaling as well as other pathways that include the MEK1/2 module of mitogen-activated protein kinase pathway. Our data indicate that cancer treatments will need to increasingly consider the functional relevance of specific mutations in driver genes rather than considering all mutations in drivers as equivalent. PMID:26627007

  4. Functional Status in Behavioral Variant Frontotemporal Dementia: A Systematic Review

    PubMed Central

    Lima-Silva, Thais Bento; Bahia, Valéria Santoro; Nitrini, Ricardo; Yassuda, Mônica Sanches

    2013-01-01

    The aim was to conduct a systematic review of studies that described the functional profile of patients with behavioral variant frontotemporal dementia (bvFTD), published between 2000 and 2013. The bibliographic search was conducted using the terms “frontotemporal dementia” and “frontotemporal lobar degeneration” in combination with “independence,” “functionality,” “basic activities of daily living,” “disability,” and scales that measure functional performance: “Disability Assessment for Dementia-DAD,” “Functional Activities Questionnaire (FAQ),” “Direct Assessment of Functional Status (DAFS).” To be included in the review, the study had to mention the characterization of the functional status of patients with bvFTD in the objectives of the study, using a previously validated instrument of functional assessment. Fourteen studies met this criterion. The reviewed studies suggested that individuals with bvFTD have greater functional impairment when compared to those with other subtypes of frontotemporal lobar degeneration or Alzheimer's disease. The studies documented a significant association between cognitive impairment and measures of functionality in these patients. The cognitive profile of patients may predict faster functional decline. PMID:24308008

  5. Mitochondrial DNA Polymerase POLG1 Disease Mutations and Germline Variants Promote Tumorigenic Properties.

    PubMed

    Singh, Bhupendra; Owens, Kjerstin M; Bajpai, Prachi; Desouki, Mohamed Mokhtar; Srinivasasainagendra, Vinodh; Tiwari, Hemant K; Singh, Keshav K

    2015-01-01

    Germline mutations in mitochondrial DNA polymerase gamma (POLG1) induce mitochondrial DNA (mtDNA) mutations, depletion, and decrease oxidative phosphorylation. Earlier, we identified somatic mutations in POLG1 and the contribution of these mutations in human cancer. However, a role for germline variations in POLG1 in human cancers is unknown. In this study, we examined a role for disease associated germline variants of POLG1, POLG1 gene expression, copy number variation and regulation in human cancers. We analyzed the mutations, expression and copy number variation in POLG1 in several cancer databases and validated the analyses in primary breast tumors and breast cancer cell lines. We discovered 5-aza-2'-deoxycytidine led epigenetic regulation of POLG1, mtDNA-encoded genes and increased mitochondrial respiration. We conducted comprehensive race based bioinformatics analyses of POLG1 gene in more than 33,000 European-Americans and 5,000 African-Americans. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 protein at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans (allele frequency 0.03777) when compared to African-American (allele frequency 0.00151) population. We identified T251I and P587L missense variations in exonuclease and linker region of POLG1 also to be more prevalent in European-Americans. Expression of these variants increased glucose consumption, decreased ATP production and increased matrigel invasion. Interestingly, conditional expression of these variants revealed that matrigel invasion properties conferred by these germline variants were reversible suggesting a role of epigenetic regulators. Indeed, we identified a set of miRNA whose expression was reversible after variant expression was turned off. Together, our studies demonstrate altered genetic and epigenetic regulation of POLG1 in human cancers and suggest a role for POLG1 germline variants in promoting tumorigenic

  6. Identification and characterization of human xylosyltransferase II promoter single nucleotide variants.

    PubMed

    Faust, Isabel; Böker, Kai Oliver; Eirich, Christina; Akkermann, Dagmar; Kuhn, Joachim; Knabbe, Cornelius; Hendig, Doris

    2015-03-20

    The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity. XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry. Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant. This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong

  7. Identification and characterization of human xylosyltransferase II promoter single nucleotide variants.

    PubMed

    Faust, Isabel; Böker, Kai Oliver; Eirich, Christina; Akkermann, Dagmar; Kuhn, Joachim; Knabbe, Cornelius; Hendig, Doris

    2015-03-20

    The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity. XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry. Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant. This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong

  8. A radial basis function network approach for the computation of inverse continuous time variant functions.

    PubMed

    Mayorga, René V; Carrera, Jonathan

    2007-06-01

    This Paper presents an efficient approach for the fast computation of inverse continuous time variant functions with the proper use of Radial Basis Function Networks (RBFNs). The approach is based on implementing RBFNs for computing inverse continuous time variant functions via an overall damped least squares solution that includes a novel null space vector for singularities prevention. The singularities avoidance null space vector is derived from developing a sufficiency condition for singularities prevention that conduces to establish some characterizing matrices and an associated performance index.

  9. Identification and Functional Characterization of Novel Genetic Variations in the OCTN1 Promoter

    PubMed Central

    Park, Hyo Jin

    2014-01-01

    Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter. PMID:24757380

  10. Functional Inducible Nitric Oxide Synthase Gene Variants Associate With Hypertension

    PubMed Central

    Nikkari, Seppo T.; Määttä, Kirsi M.; Kunnas, Tarja A.

    2015-01-01

    Abstract Increased inducible nitric oxide synthase (iNOS) activity and expression has been associated with hypertension, but less is known whether the 2 known functional polymorphic sites in the iNOS gene (g.–1026 C/A (rs2779249), g.2087 G/A (rs2297518)) affect susceptibility to hypertension. The objective of this study was to investigate the association between the genetic variants of iNOS and diagnosed hypertension in a Finnish cohort. This study included 320 hypertensive cases and 439 healthy controls. All participants were 50-year-old men and women and the data were collected from the Tampere adult population cardiovascular risk study (TAMRISK). DNA was extracted from buccal swabs and iNOS single nucleotide polymorphisms (SNPs) were analyzed using KASP genotyping PCR. Data analysis was done by logistic regression. At the age of 50 years, the SNP rs2779249 (C/A) associated significantly with hypertension (P = 0.009); specifically, subjects carrying the A-allele had higher risk of hypertension compared to those carrying the CC genotype (OR = 1.47; CI = 1.08–2.01; P = 0.015). In addition, a 15-year follow-up period (35, 40, and 45 years) of the same individuals showed that carriers of the A-allele had more often hypertension in all of the studied age-groups. The highest risk for developing hypertension was obtained among 35-year-old subjects (odds ratio [OR] 3.83; confidence interval [CI] = 1.20–12.27; P = 0.024). Those carrying variant A had also significantly higher readings of both systolic (P = 0.047) and diastolic (P = 0.048) blood pressure during the follow-up. No significant associations between rs2297518 (G/A) variants alone and hypertension were found. However, haplotype analysis of rs2779249 and rs2297518 revealed that individuals having haplotype H3 which combines both A alleles (CA–GA, 19.7% of individuals) was more commonly found in the hypertensive group than in the normotensive group (OR = 2.01; CI = 1

  11. Association Study of CHRNA7 Promoter Variants with Sensory and Sensorimotor Gating in Schizophrenia Patients and Healthy Controls: A Danish Case-Control Study.

    PubMed

    Bertelsen, Birgitte; Oranje, Bob; Melchior, Linea; Fagerlund, Birgitte; Werge, Thomas M; Mikkelsen, Jens D; Tümer, Zeynep; Glenthøj, Birte Y

    2015-12-01

    Schizophrenia is a severe psychiatric disorder with a core component of impaired cognitive function still remaining as one of the greatest challenges in the pharmacological treatment of the disorder. The CHRNA7 gene, encoding the subunit of the human α7 nicotinic acetylcholine receptor (α7nAChR), is suggested as a susceptibility factor for schizophrenia. CHRNA7 has also been genetically linked to the P50 auditory evoked potential deficit, a candidate endophenotype of schizophrenia, but not to prepulse inhibition of the startle reflex (PPI). In this study, 95 antipsychotic-naïve schizophrenic patients and 450 unaffected controls were screened for CHRNA7 promoter variants to investigate the association with schizophrenia, P50 suppression and PPI. We found that the promoter variant -194C (rs28531779) was significantly associated with schizophrenia, but did not find any association of this variant with P50 suppression or PPI. In addition, individuals with CHRNA7 promoter variants had elevated startle magnitude in pulse-alone trials compared to individuals without a variant. The present findings provide further support for a role of the α7nAChR in schizophrenia and show a genetic link between CHRNA7 and startle magnitude, indicating that cholinergic neurotransmission involving the α7nAChR could be involved in sensory registration processes.

  12. Filtering genetic variants and placing informative priors based on putative biological function.

    PubMed

    Friedrichs, Stefanie; Malzahn, Dörthe; Pugh, Elizabeth W; Almeida, Marcio; Liu, Xiao Qing; Bailey, Julia N

    2016-02-03

    High-density genetic marker data, especially sequence data, imply an immense multiple testing burden. This can be ameliorated by filtering genetic variants, exploiting or accounting for correlations between variants, jointly testing variants, and by incorporating informative priors. Priors can be based on biological knowledge or predicted variant function, or even be used to integrate gene expression or other omics data. Based on Genetic Analysis Workshop (GAW) 19 data, this article discusses diversity and usefulness of functional variant scores provided, for example, by PolyPhen2, SIFT, or RegulomeDB annotations. Incorporating functional scores into variant filters or weights and adjusting the significance level for correlations between variants yielded significant associations with blood pressure traits in a large family study of Mexican Americans (GAW19 data set). Marker rs218966 in gene PHF14 and rs9836027 in MAP4 significantly associated with hypertension; additionally, rare variants in SNUPN significantly associated with systolic blood pressure. Variant weights strongly influenced the power of kernel methods and burden tests. Apart from variant weights in test statistics, prior weights may also be used when combining test statistics or to informatively weight p values while controlling false discovery rate (FDR). Indeed, power improved when gene expression data for FDR-controlled informative weighting of association test p values of genes was used. Finally, approaches exploiting variant correlations included identity-by-descent mapping and the optimal strategy for joint testing rare and common variants, which was observed to depend on linkage disequilibrium structure.

  13. IN VIVO Function of Rare G6pd Variants from Natural Populations of DROSOPHILA MELANOGASTER

    PubMed Central

    Eanes, Walter F.; Hey, Jody

    1986-01-01

    From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function. PMID:17246336

  14. Hepatitis B virus-induced hepatocellular carcinoma: functional roles of MICA variants.

    PubMed

    Tong, H V; Toan, N L; Song, L H; Bock, C-T; Kremsner, P G; Velavan, T P

    2013-10-01

    Hepatitis B virus infection is a high-risk factor for hepatocellular carcinoma. The human major histocompatibility complex class I chain-related gene A (MICA) is a ligand of the NKG2D receptor that modulates the NK and T-cell-mediated immune responses and is associated with several diseases. This study determined the effects of MICA polymorphisms during HBV infection and HBV-induced HCC. We conducted a case-controlled study in a Vietnamese cohort and genotyped ten functional MICA polymorphisms including the microsatellite motif in 552 clinically classified hepatitis B virus patients and 418 healthy controls. The serum soluble MICA levels (sMICA) were correlated with MICA variants and liver enzyme levels. We demonstrated a significant contribution of MICA rs2596542G/A promoter variant and nonsynonymous substitutions MICA-129Met/Val, MICA-251Gln/Arg, MICA-175Gly/Ser, triplet repeat polymorphism and respective haplotypes with HBV-induced HCC and HBV persistence. The circulating sMICA levels in HBV patient groups were elevated significantly compared with healthy controls. A significant contribution of studied MICA variants to sMICA levels was also observed. The liver enzymes alanine amino transferase (ALT), aspartate transaminase (AST), total bilirubin and direct bilirubin were positively correlated with sMICA levels suggesting sMICA as a biomarker for liver injury. We conclude that MICA polymorphisms play a crucial role in modulating innate immune responses, tumour surveillance and regulate disease susceptibility during HBV infection.

  15. Promoter variants in the MSMB gene associated with prostate cancer regulate MSMB/NCOA4 fusion transcripts

    PubMed Central

    Yeager, Meredith; Im, Kate; Gold, Bert; Schneider, Thomas D.; Fraumeni, Joseph F.; Chanock, Stephen J.; Anderson, Stephen K.; Dean, Michael

    2012-01-01

    Beta-microseminoprotein (MSP)/MSMB is an immunoglobulin superfamily protein synthesized by prostate epithelial cells and secreted into seminal plasma. Variants in the promoter of the MSMB gene have been associated with the risk of prostate cancer (PCa) in several independent genome-wide association studies. Both MSMB and an adjacent gene, NCOA4, are subjected to transcriptional control via androgen response elements. The gene product of NCOA4 interacts directly with the androgen receptor as a co-activator to enhance AR transcriptional activity. Here, we provide evidence for the expression of full-length MSMB-NCOA4 fusion transcripts regulated by the MSMB promoter. The predominant MSMB-NCOA4 transcript arises by fusion of the 5′UTR and exons 1–2 of the MSMB pre-mRNA, with exons 2–10 of the NCOA4 premRNA, producing a stable fusion protein, comprising the essential domains of NCOA4. Analysis of the splice sites of this transcript shows an unusually strong splice acceptor at NCOA4 exon 2 and the presence of Alu repeats flanking the exons potentially involved in the splicing event. Transfection experiments using deletion clones of the promoter coupled with luciferase reporter assays define a core MSMB promoter element located between –27 and –236 of the gene, and a negative regulatory element immediately upstream of the start codon. Computational network analysis reveals that the MSMB gene is functionally connected to NCOA4 and the androgen receptor signaling pathway. The data provide an example of how GWAS-associated variants may have multiple genetic and epigenetic effects. PMID:22661295

  16. Gene-environment interaction between the MMP9 C-1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease.

    PubMed

    Stankovic, Marija; Kojic, Snezana; Djordjevic, Valentina; Tomovic, Andrija; Nagorni-Obradovic, Ljudmila; Petrovic-Stanojevic, Natasa; Mitic-Milikic, Marija; Radojkovic, Dragica

    2016-07-01

    The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well-established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene-environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C-1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C-1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C-1562T genetic variant and cigarette smoking was assessed using a case-control model. The response of the C-1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C-1562T variant demonstrated a dose-dependent and allele-specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9-1562T allele and cigarette smoke in COPD, emphasising gene-environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447-454, 2016. © 2016 Wiley Periodicals, Inc. PMID:27270564

  17. Interpreting functional effects of coding variants: challenges in proteome-scale prediction, annotation and assessment.

    PubMed

    Shameer, Khader; Tripathi, Lokesh P; Kalari, Krishna R; Dudley, Joel T; Sowdhamini, Ramanathan

    2016-09-01

    Accurate assessment of genetic variation in human DNA sequencing studies remains a nontrivial challenge in clinical genomics and genome informatics. Ascribing functional roles and/or clinical significances to single nucleotide variants identified from a next-generation sequencing study is an important step in genome interpretation. Experimental characterization of all the observed functional variants is yet impractical; thus, the prediction of functional and/or regulatory impacts of the various mutations using in silico approaches is an important step toward the identification of functionally significant or clinically actionable variants. The relationships between genotypes and the expressed phenotypes are multilayered and biologically complex; such relationships present numerous challenges and at the same time offer various opportunities for the design of in silico variant assessment strategies. Over the past decade, many bioinformatics algorithms have been developed to predict functional consequences of single nucleotide variants in the protein coding regions. In this review, we provide an overview of the bioinformatics resources for the prediction, annotation and visualization of coding single nucleotide variants. We discuss the currently available approaches and major challenges from the perspective of protein sequence, structure, function and interactions that require consideration when interpreting the impact of putatively functional variants. We also discuss the relevance of incorporating integrated workflows for predicting the biomedical impact of the functionally important variations encoded in a genome, exome or transcriptome. Finally, we propose a framework to classify variant assessment approaches and strategies for incorporation of variant assessment within electronic health records.

  18. Human keratin 8 variants promote mouse acetaminophen hepatotoxicity coupled with JNK activation and protein adduct formation

    PubMed Central

    Guldiken, Nurdan; Zhou, Qin; Kucukoglu, Ozlem; Rehm, Melanie; Levada, Kateryna; Gross, Annika; Kwan, Raymond; James, Laura P.; Trautwein, Christian; Omary, M. Bishr; Strnad, Pavel

    2015-01-01

    Background and aims Keratins 8 and 18 (K8/K18) are the intermediate filaments proteins of simple-type digestive epithelia, and provide important cytoprotective function. K8/K18 variants predispose humans to chronic liver disease progression and to poor outcomes in acute acetaminophen (APAP)-related liver failure. Given that K8 G62C and R341H/R341C are common K8 variants in European and North American populations, we studied their biological significance using transgenic mice. Methods Mice that overexpress the human K8 variants R341H or R341C were generated and used together with previously described mice that overexpress wild-type (WT) K8 or K8 G62C. Mice were injected with 600 mg/kg APAP, or underwent bile duct ligation (BDL). Livers were evaluated by microarray analysis, quantitative RT-PCR, immunoblotting, histological and immunological staining, and biochemical assays. Results Under basal conditions, the K8 G62C/R341H/R341C variant-expressing mice did not show an obvious liver phenotype or altered keratin filament distribution, while K8 G62C/R341C animals had aberrant disulphide-crosslinked keratins. Animals carrying the K8 variants displayed limited gene expression changes but had lower nicotinamide N-methyl transferase (NNMT) levels and were predisposed to APAP-induced hepatotoxicity. NNMT represents a novel K8/K18-associated protein that becomes upregulated after K8/K18 transfection. The more pronounced liver damage was accompanied by increased and prolonged JNK activation; elevated APAP protein adducts; K8 hyperphosphorylation at S74/S432 with enhanced K8 solubility; and prominent pericentral keratin network disruption. No differences in APAP serum levels, glutathione or ATP levels were noted. BDL resulted in similar liver injury and biliary fibrosis in all mouse genotypes. Conclusion Expression of human K8 variants G62C, R341H, or R341C in mice predisposes to acute acetaminophen hepatotoxicity, thereby providing direct evidence for the importance of these

  19. Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.

    PubMed

    Carvalho, Marcelo A; Marsillac, Sylvia M; Karchin, Rachel; Manoukian, Siranoush; Grist, Scott; Swaby, Ramona F; Urmenyi, Turan P; Rondinelli, Edson; Silva, Rosane; Gayol, Luis; Baumbach, Lisa; Sutphen, Rebecca; Pickard-Brzosowicz, Jennifer L; Nathanson, Katherine L; Sali, Andrej; Goldgar, David; Couch, Fergus J; Radice, Paolo; Monteiro, Alvaro N A

    2007-02-15

    Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability.

  20. BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

    PubMed

    Fonseca, Dora Janeth; Ortega-Recalde, Oscar; Esteban-Perez, Clara; Moreno-Ortiz, Harold; Patiño, Liliana Catherine; Bermúdez, Olga María; Ortiz, Angela María; Restrepo, Carlos M; Lucena, Elkin; Laissue, Paul

    2014-11-01

    BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF.

  1. Inheritance of rare functional GCKR variants and their contribution to triglyceride levels in families

    PubMed Central

    Rees, Matthew G.; Raimondo, Anne; Wang, Jian; Ban, Matthew R.; Davis, Mindy I.; Barrett, Amy; Ranft, Jessica; Jagdhuhn, David; Waterstradt, Rica; Baltrusch, Simone; Simeonov, Anton; Collins, Francis S.; Hegele, Robert A.; Gloyn, Anna L.

    2014-01-01

    Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458 individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues using GCKR, encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously. Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels. Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive value of rare GCKR variants in individuals and the complex heritability of lipid traits. PMID:24879641

  2. An Essential Role for CD44 Variant Isoforms in Epidermal Langerhans Cell and Blood Dendritic Cell Function

    PubMed Central

    Weiss, Johannes M.; Sleeman, Jonathan; Renkl, Andreas C.; Dittmar, Henning; Termeer, Christian C.; Taxis, Sabine; Howells, Norma; Hofmann, Martin; Köhler, Gabriele; Schöpf, Erwin; Ponta, Helmut; Herrlich, Peter; Simon, Jan C.

    1997-01-01

    Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC. PMID:9166413

  3. Targeted resequencing of regulatory regions at schizophrenia risk loci: Role of rare functional variants at chromatin repressive states.

    PubMed

    González-Peñas, Javier; Amigo, Jorge; Santomé, Luis; Sobrino, Beatriz; Brenlla, Julio; Agra, Santiago; Paz, Eduardo; Páramo, Mario; Carracedo, Ángel; Arrojo, Manuel; Costas, Javier

    2016-07-01

    There is mounting evidence that regulatory variation plays an important role in genetic risk for schizophrenia. Here, we specifically search for regulatory variants at risk by sequencing promoter regions of twenty-three genes implied in schizophrenia by copy number variant or genome-wide association studies. After strict quality control, a total of 55,206bp per sample were analyzed in 526 schizophrenia cases and 516 controls from Galicia, NW Spain, using the Applied Biosystems SOLiD System. Variants were filtered based on frequency from public databases, chromatin states from the RoadMap Epigenomics Consortium at tissues relevant for schizophrenia, such as fetal brain, mid-frontal lobe, and angular gyrus, and prediction of functionality from RegulomeDB. The proportion of rare variants at polycomb repressive chromatin state at relevant tissues was higher in cases than in controls. The proportion of rare variants with predicted regulatory role was significantly higher in cases than in controls (P=0.0028, OR=1.93, 95% C.I.=1.23-3.04). Combination of information from both sources led to the identification of an excess of carriers of rare variants with predicted regulatory role located at polycomb repressive chromatin state at relevant tissues in cases versus controls (P=0.0016, OR=19.34, 95% C.I.=2.45-2495.26). The variants are located at two genes affected by the 17q12 copy number variant, LHX1 and HNF1B. These data strongly suggest that a specific epigenetic mechanism, chromatin remodeling by histone modification during early development, may be impaired in a subset of schizophrenia patients, in agreement with previous data. PMID:27066855

  4. Better prediction of functional effects for sequence variants

    PubMed Central

    2015-01-01

    Elucidating the effects of naturally occurring genetic variation is one of the major challenges for personalized health and personalized medicine. Here, we introduce SNAP2, a novel neural network based classifier that improves over the state-of-the-art in distinguishing between effect and neutral variants. Our method's improved performance results from screening many potentially relevant protein features and from refining our development data sets. Cross-validated on >100k experimentally annotated variants, SNAP2 significantly outperformed other methods, attaining a two-state accuracy (effect/neutral) of 83%. SNAP2 also outperformed combinations of other methods. Performance increased for human variants but much more so for other organisms. Our method's carefully calibrated reliability index informs selection of variants for experimental follow up, with the most strongly predicted half of all effect variants predicted at over 96% accuracy. As expected, the evolutionary information from automatically generated multiple sequence alignments gave the strongest signal for the prediction. However, we also optimized our new method to perform surprisingly well even without alignments. This feature reduces prediction runtime by over two orders of magnitude, enables cross-genome comparisons, and renders our new method as the best solution for the 10-20% of sequence orphans. SNAP2 is available at: https://rostlab.org/services/snap2web Definitions used Delta, input feature that results from computing the difference feature scores for native amino acid and feature scores for variant amino acid; nsSNP, non-synoymous SNP; PMD, Protein Mutant Database; SNAP, Screening for non-acceptable polymorphisms; SNP, single nucleotide polymorphism; variant, any amino acid changing sequence variant. PMID:26110438

  5. Functional variants of sphingosine-1-phosphate receptor 1 gene associate with asthma susceptibility

    PubMed Central

    Sun, Xiaoguang; Ma, Shwu-Fan; Wade, Michael S.; Flores, Carlos; Pino-Yanes, Maria; Moitra, Jaideep; Ober, Carole; Kittles, Rick; Husain, Aliya N.; Ford, Jean G.; Garcia, Joe G. N.

    2012-01-01

    Background The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma and the sphingosine-1-phosphate receptor, S1PR1, is an essential participant regulating lung vascular integrity and responses to lung inflammation. Objective We explored the contribution of polymorphisms in the S1PR1 gene (S1PR1) to asthma susceptibility. Methods A combination of gene re-sequencing for SNP discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was utilized. Results Immunohistochemistry studies demonstrated significantly decreased S1PR1 protein expression in pulmonary vessels in asthmatic lungs compared to non-asthmatic individuals (p<0.05). Direct DNA sequencing of 27 multiethnic samples identified 39 S1PR1 variants (18 novel SNPs). Association studies were performed based on genotyping results from cosmopolitan tagging SNPs in three case-control cohorts from Chicago and New York totaling 1061 subjects (502 cases and 559 controls). Promoter SNP rs2038366 (−1557G/T) was found to be associated with asthma (p=0.03) in European Americans. In African Americans, an association was found for both asthma and severe asthma for intronic SNP rs3753194 (c.−164+170A/G) (p=0.006 and p=0.040, respectively) and for promoter SNP rs59317557 (−532C/G) with severe asthma (p=0.028). Consistent with predicted in silico functionality, alleles of promoter SNPs rs2038366 (−1557G/T) and rs59317557 (−532C/G) influenced the activity of a luciferase S1PR1 reporter vector in transfected endothelial cells exposed to growth factors (EGF, PDGF, VEGF) known to be increased in asthmatic airways. Conclusion These data provide strong support for a role for S1PR1 gene variants in asthma susceptibility and severity. Clinical Implications Our results indicate S1PR1 is a novel asthma candidate gene and an attractive target for future therapeutic strategies. Capsule summary This study

  6. Regulation of Mouse 4-1BB Expression: Multiple Promoter Usages and a Splice Variant

    PubMed Central

    Kim, Jung D.; Kim, Chang H.; Kwon, Byoung S.

    2011-01-01

    The expression of 4-1BB has been known to be dependent on T cell activation. Recent studies have, however, revealed that 4-1BB expression is not restricted to T cells. We sought to determine the molecular basis for the differential gene expression. Here we report the expression pattern of two mouse 4-1BB transcripts, type I and type II. Whereas the type I transcript was specifically expressed on immune organ as previously reported, the type II transcript was ubiquitously expressed in tissues and various cell lines. However, both type I and type II transcript were highly induced on activated T cells. Primer extension assay of the two 4-1BB transcripts suggested that mouse 4-1BB had more than two transcripts. Using luciferase assay we have identified three promoter regions (PI, PII and PIII), which located on upstream region of second exon 1, first exon 1, and exon 2, respectively. In particular, the type I transcript was preferentially induced when naïve T cells are stimulated by anti-CD3 monoclonal antibody (mAb) since NF-κB specifically binds to the putative NF-κB element of PI. We have also shown that a splice variant, in which the transmembrane domain was deleted, could inhibit 4-1BB signaling. The splicing variant was highly induced by TCR stimulation. Our results reveal 4-1BB also has a negative regulation system through soluble 4-1BB produced from a splice variant induced under activation conditions. PMID:21347708

  7. NOVEL SPLICED VARIANTS OF IONOTROPIC GLUTAMATE RECEPTOR GLUR6 IN NORMAL HUMAN FIBROBLAST AND BRAIN CELLS ARE TRANSCRIBED BY TISSUE SPECIFIC PROMOTERS

    PubMed Central

    Zhawar, Vikramjit K.; Kaur, Gurpreet; deRiel, Jon K.; Kaur, G. Pal; Kandpal, Raj P.; Athwal, Raghbir S.

    2010-01-01

    The members of the ionotropic glutamate receptor family, namely, a-amino-3-hydroxy-S-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors, are important mediators of the rapid synaptic transmission in the central nervous system. We have investigated the splicing pattern and expression of the kainate receptor subunit GluR6 in human fibroblast cell lines and brain tissue. We demonstrate the expression of GluR6A variant specifically in brain, and four variants, namely, GluR6B, GluR6C, GluR6D and GluR6E in fibroblast cell lines. The variants GluR6D and GluR6E have not been described before, and appear to be specific for non-neuronal cells. Genomic analysis and cloning of the sequence preceding the transcribed region led to the identification of two tissue specific promoters designated as neuronal promoter PN and non-neuronal promoter PNN. We have used RNA ligase mediated RACE and in silico analyses to locate two sets of transcription start sites, and confirmed specific transcripts initiated by PN and PNN in brain cells and fibroblasts, respectively. The domain structure of variants GluR6D and GluR6E revealed the absence of three transmembrane domains. The lack of these domains suggests that the mature receptors arising from these variant subunits may not function as active channels. Based on these structural features in GluR6D and GluR6E, and the observations that GluR6B, GluR6C, GluR6D and GluR6E are exclusively expressed in non-neuronal cells, it is likely that these receptor subunits function as non-channel signaling proteins. PMID:20230879

  8. The Genomic Distribution and Function of Histone Variant HTZ-1 during C. elegans Embryogenesis

    PubMed Central

    Whittle, Christina M.; McClinic, Karissa N.; Ercan, Sevinc; Zhang, Xinmin; Green, Roland D.; Kelly, William G.; Lieb, Jason D.

    2008-01-01

    In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for development and viability in all animals tested to date. However, the function of H2A.Z in development remains unclear. Here, we use ChIP-chip, genetic mutation, RNAi, and immunofluorescence microscopy to interrogate the function of H2A.Z (HTZ-1) during embryogenesis in Caenorhabditis elegans, a key model of metazoan development. We find that HTZ-1 is expressed in every cell of the developing embryo and is essential for normal development. The sites of HTZ-1 incorporation during embryogenesis reveal a genome wrought by developmental processes. HTZ-1 is incorporated upstream of 23% of C. elegans genes. While these genes tend to be required for development and occupied by RNA polymerase II, HTZ-1 incorporation does not specify a stereotypic transcription program. The data also provide evidence for unexpectedly widespread independent regulation of genes within operons during development; in 37% of operons, HTZ-1 is incorporated upstream of internally encoded genes. Fewer sites of HTZ-1 incorporation occur on the X chromosome relative to autosomes, which our data suggest is due to a paucity of developmentally important genes on X, rather than a direct function for HTZ-1 in dosage compensation. Our experiments indicate that HTZ-1 functions in establishing or maintaining an essential chromatin state at promoters regulated dynamically during C. elegans embryogenesis. PMID:18787694

  9. In Silico Functional Pathway Annotation of 86 Established Prostate Cancer Risk Variants

    PubMed Central

    Loo, Lenora W. M.; Fong, Aaron Y. W.; Cheng, Iona; Le Marchand, Loïc

    2015-01-01

    Heritability is one of the strongest risk factors of prostate cancer, emphasizing the importance of the genetic contribution towards prostate cancer risk. To date, 86 established prostate cancer risk variants have been identified by genome-wide association studies (GWAS). To determine if these risk variants are located near genes that interact together in biological networks or pathways contributing to prostate cancer initiation or progression, we generated gene sets based on proximity to the 86 prostate cancer risk variants. We took two approaches to generate gene lists. The first strategy included all immediate flanking genes, up- and downstream of the risk variant, regardless of distance from the index variant, and the second strategy included genes closest to the index GWAS marker and to variants in high LD (r2 ≥0.8 in Europeans) with the index variant, within a 100 kb window up- and downstream. Pathway mapping of the two gene sets supported the importance of the androgen receptor-mediated signaling in prostate cancer biology. In addition, the hedgehog and Wnt/β-catenin signaling pathways were identified in pathway mapping for the flanking gene set. We also used the HaploReg resource to examine the 86 risk loci and variants high LD (r2 ≥0.8) for functional elements. We found that there was a 12.8 fold (p = 2.9 x 10-4) enrichment for enhancer motifs in a stem cell line and a 4.4 fold (p = 1.1 x 10-3) enrichment of DNase hypersensitivity in a prostate adenocarcinoma cell line, indicating that the risk and correlated variants are enriched for transcriptional regulatory motifs. Our pathway-based functional annotation of the prostate cancer risk variants highlights the potential regulatory function that GWAS risk markers, and their highly correlated variants, exert on genes. Our study also shows that these genes may function cooperatively in key signaling pathways in prostate cancer biology. PMID:25658610

  10. VARIANT: Command Line, Web service and Web interface for fast and accurate functional characterization of variants found by Next-Generation Sequencing

    PubMed Central

    Medina, Ignacio; De Maria, Alejandro; Bleda, Marta; Salavert, Francisco; Alonso, Roberto; Gonzalez, Cristina Y.; Dopazo, Joaquin

    2012-01-01

    The massive use of Next-Generation Sequencing (NGS) technologies is uncovering an unexpected amount of variability. The functional characterization of such variability, particularly in the most common form of variation found, the Single Nucleotide Variants (SNVs), has become a priority that needs to be addressed in a systematic way. VARIANT (VARIant ANalyis Tool) reports information on the variants found that include consequence type and annotations taken from different databases and repositories (SNPs and variants from dbSNP and 1000 genomes, and disease-related variants from the Genome-Wide Association Study (GWAS) catalog, Online Mendelian Inheritance in Man (OMIM), Catalog of Somatic Mutations in Cancer (COSMIC) mutations, etc). VARIANT also produces a rich variety of annotations that include information on the regulatory (transcription factor or miRNA-binding sites, etc.) or structural roles, or on the selective pressures on the sites affected by the variation. This information allows extending the conventional reports beyond the coding regions and expands the knowledge on the contribution of non-coding or synonymous variants to the phenotype studied. Contrarily to other tools, VARIANT uses a remote database and operates through efficient RESTful Web Services that optimize search and transaction operations. In this way, local problems of installation, update or disk size limitations are overcome without the need of sacrifice speed (thousands of variants are processed per minute). VARIANT is available at: http://variant.bioinfo.cipf.es. PMID:22693211

  11. Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis

    PubMed Central

    2013-01-01

    Background Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation. Results Here we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01). Conclusion In the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation. PMID:23815438

  12. Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making

    PubMed Central

    Thouvenot, Pierre; Ben Yamin, Barbara; Fourrière, Lou; Lescure, Aurianne; Boudier, Thomas; Del Nery, Elaine; Chauchereau, Anne; Goldgar, David E.; Stoppa-Lyonnet, Dominique; Nicolas, Alain; Millot, Gaël A.

    2016-01-01

    Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening. PMID:27272900

  13. Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

    PubMed Central

    Becker, M A; Raivio, K O; Bakay, B; Adams, W B; Nyhan, W L

    1980-01-01

    An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PP-ribose-P synthetase from the cells of this child showed four- to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition of enzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) activation in incompletely dialyzed extracts. Excessive maximal velocity of the enzyme reaction catalyzed by the mutant enzyme was indicated by: enzyme activities twice those of normal at all concentrations of Pi in chromatographed fibroblast extracts; normal affinity constants for substrates and for the activator, Mg2+; and twofold greater than normal activity per immunoreactive enzyme molecule. The mutant enzyme thus possessed deficient regulatory and superactive catalytic properties, two mechanisms previously demonstrated individually to underlie the excessive PPRribose-P and uric acid synthesis of affected members of families with superactive PP-ribose-P synthetases. Increased PP-ribose-P concentration (4-fold) and generation (2.7-fold) and enhanced rates of PP-ribose-P dependent purine synthetic reactions, including purine synthesis de novo, in S.M. fibroblasts confirmed the functional significance of this patient's mutant enzyme. Diminished stability of the variant PP-ribose-P synthetase was manifested in vitro by increased thermal lability and in vivo by deficiency of enzyme activity at Pi concentrations greater than 0.3 mM in hemolysates and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PP-ribose-P concentration and increased rates

  14. Promoting motor function by exercising the brain.

    PubMed

    Perrey, Stephane

    2013-01-01

    Exercise represents a behavioral intervention that enhances brain health and motor function. The increase in cerebral blood volume in response to physical activity may be responsible for improving brain function. Among the various neuroimaging techniques used to monitor brain hemodynamic response during exercise, functional near-infrared spectroscopy could facilitate the measurement of task-related cortical responses noninvasively and is relatively robust with regard to the subjects' motion. Although the components of optimal exercise interventions have not been determined, evidence from animal and human studies suggests that aerobic exercise with sufficiently high intensity has neuroprotective properties and promotes motor function. This review provides an insight into the effect of physical activity (based on endurance and resistance exercises) on brain function for producing movement. Since most progress in the study of brain function has come from patients with neurological disorders (e.g., stroke and Parkinson's patients), this review presents some findings emphasizing training paradigms for restoring motor function. PMID:24961309

  15. Interaction between SLC6A4 promoter variants and childhood trauma on the age at onset of bipolar disorders

    PubMed Central

    Etain, B.; Lajnef, M.; Henrion, A.; Dargél, A.A.; Stertz, L.; Kapczinski, F.; Mathieu, F.; Henry, C.; Gard, S.; Kahn, J. P.; Leboyer, M.; Jamain, S.; Bellivier, F.

    2015-01-01

    Age at onset (AAO) of bipolar disorders (BD) could be influenced both by a repeat length polymorphism (5HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4) and exposure to childhood trauma. We assessed 308 euthymic patients with BD for the AAO of their first mood episode and childhood trauma. Patients were genotyped for the 5HTTLPR (long/short variant) and the rs25531. Genotypes were classified on functional significance (LL, LS, SS). A sample of 126 Brazilian euthymic patients with BD was used for replication. In the French sample, the correlation between AAO and trauma score was observed only among ‘SS’ homozygotes (p = 0.002) but not among ‘L’ allele carriers. A history of at least one trauma decreased the AAO only in ‘SS’ homozygotes (p = 0.001). These results remained significant after correction using FDR. Regression models suggested an interaction between emotional neglect and ‘SS’ genotype on the AAO (p = 0.009) and no further interaction with other trauma subtypes. Partial replication was obtained in the Brazilian sample, showing an interaction between emotional abuse and ‘LS’ genotype on the AAO (p = 0.02). In conclusion, an effect of childhood trauma on AAO of BD was observed only in patients who carry a specific stress responsiveness-related SLC6A4 promoter genotype. PMID:26542422

  16. Multiple sclerosis associated genetic variants of CD226 impair regulatory T cell function.

    PubMed

    Piédavent-Salomon, Melanie; Willing, Anne; Engler, Jan Broder; Steinbach, Karin; Bauer, Simone; Eggert, Britta; Ufer, Friederike; Kursawe, Nina; Wehrmann, Sabine; Jäger, Jan; Reinhardt, Stefanie; Friese, Manuel A

    2015-11-01

    Recent association studies have linked numerous genetic variants with an increased risk for multiple sclerosis, although their functional relevance remains largely unknown. Here we investigated phenotypical and functional consequences of a genetic variant in the CD226 gene that, among other autoimmune diseases, predisposes to multiple sclerosis. Phenotypically, effector and regulatory CD4(+) memory T cells of healthy individuals carrying the predisposing CD226 genetic variant showed, in comparison to carriers of the protective variant, reduced surface expression of CD226 and an impaired induction of CD226 after stimulation. This haplotype-dependent reduction in CD226 expression on memory T cells was abrogated in patients with multiple sclerosis, as CD226 expression was comparable to healthy risk haplotype carriers irrespective of genetic variant. Functionally, FOXP3-positive regulatory T cells from healthy carriers of the genetic protective variant showed superior suppressive capacity, which was again abrogated in multiple sclerosis patients. Mimicking the phenotype of human CD226 genetic risk variant carriers, regulatory T cells derived from Cd226-deficient mice showed similarly reduced inhibitory activity, eventually resulting in an exacerbated disease course of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Therefore, by combining human and mouse analyses we show that CD226 exhibits an important role in the activation of regulatory T cells, with its genetically imposed dysregulation impairing regulatory T cell function.

  17. Post-GWAS methodologies for localisation of functional non-coding variants: ANGPTL3

    PubMed Central

    Oldoni, Federico; Palmen, Jutta; Giambartolomei, Claudia; Howard, Philip; Drenos, Fotios; Plagnol, Vincent; Humphries, Steve E.; Talmud, Philippa J.; Smith, Andrew J.P.

    2016-01-01

    Genome-wide association studies have confirmed the involvement of non-coding angiopoietin-like 3 (ANGPTL3) gene variants with coronary artery disease, levels of low-density lipoprotein cholesterol (LDL-C), triglycerides and ANGPTL3 mRNA transcript. Extensive linkage disequilibrium at the locus, however, has hindered efforts to identify the potential functional variants. Using regulatory annotations from ENCODE, combined with functional in vivo assays such as allele-specific formaldehyde-assisted isolation of regulatory elements, statistical approaches including eQTL/lipid colocalisation, and traditional in vitro methodologies including electrophoretic mobility shift assay and luciferase reporter assays, variants affecting the ANGPTL3 regulome were examined. From 253 variants associated with ANGPTL3 mRNA expression, and/or lipid traits, 46 were located within liver regulatory elements and potentially functional. One variant, rs10889352, demonstrated allele-specific effects on DNA-protein interactions, reporter gene expression and chromatin accessibility, in line with effects on LDL-C levels and expression of ANGPTL3 mRNA. The ANGPTL3 gene lies within DOCK7, although the variant is within non-coding regions outside of ANGPTL3, within DOCK7, suggesting complex long-range regulatory effects on gene expression. This study illustrates the power of combining multiple genome-wide datasets with laboratory data to localise functional non-coding variation and provides a model for analysis of regulatory variants from GWAS. PMID:26800306

  18. Functional characterization of common protein variants in the efflux transporter ABCC11 and identification of T546M as functionally damaging variant.

    PubMed

    Arlanov, R; Lang, T; Jedlitschky, G; Schaeffeler, E; Ishikawa, T; Schwab, M; Nies, A T

    2016-04-01

    Multidrug resistance protein 8 (ABCC11) is an efflux transporter for anionic lipophilic compounds, conferring resistance to antiviral and anticancer agents like 5-fluorouracil (5-FU). ABCC11 missense variants may contribute to variability in drug response but functional consequences, except for the 'earwax variant' c.538G>A, are unknown. Using the 'Screen and Insert' technology, we generated human embryonic kidney 293 cells stably expressing ABCC11 missense variants frequently occurring in different ethnic populations: c.57G>A, c.538G>A, c.950C>A, c.1637C>T, c.1942G>A, c.4032A>G. A series of in silico prediction analyses and in vitro plasma membrane vesicle uptake, immunoblotting and immunolocalization experiments were undertaken to investigate functional consequences. We identified c.1637C>T (T546M), previously associated with 5-FU-related toxicity, as a novel functionally damaging ABCC11 variant exhibiting markedly reduced transport function of 5-FdUMP, the active cytotoxic metabolite of 5-FU. Detailed analysis of 14 subpopulations revealed highest allele frequencies of c.1637C>T in Europeans and Americans (up to 11%) compared with Africans and Asians (up to 3%).

  19. AMPD1 functional variants associated with autism in Han Chinese population.

    PubMed

    Zhang, Lusi; Ou, Jianjun; Xu, Xiaojuan; Peng, Yu; Guo, Hui; Pan, Yongcheng; Chen, Jingjing; Wang, Tianyun; Peng, Hao; Liu, Qiong; Tian, Di; Pan, Qian; Zou, Xiaobin; Zhao, Jingping; Hu, Zhengmao; Xia, Kun

    2015-09-01

    Autism is a childhood neurodevelopmental disorder with high heterogeneity. Following our genome-wide associated loci with autism, we performed sequencing analysis of the coding regions, UTR and flanking splice junctions of AMPD1 in 830 Chinese autism individuals as well as 514 unrelated normal controls. Fourteen novel variants in the coding sequence were identified, including 11 missense variants and 3 synonymous mutations. Among these missense variants, 10 variants were absent in 514 control subjects, and conservative and functional prediction was carried out. Mitochondria activity and lactate dehydrogenase assay were performed in 5 patients' lymphoblast cell lines; p.P572S and p.S626C showed decreased mitochondrial complex I activity, and p.S626C increased lactate dehydrogenase release in medium. Conclusively, our data suggested that mutational variants in AMPD1 contribute to autism risk in Han Chinese population, uncovering the contribution of mutant protein to disease development that operates via mitochondria dysfunction and cell necrosis.

  20. Genetic Load of Loss-of-Function Polymorphic Variants in Great Apes.

    PubMed

    de Valles-Ibáñez, Guillem; Hernandez-Rodriguez, Jessica; Prado-Martinez, Javier; Luisi, Pierre; Marquès-Bonet, Tomàs; Casals, Ferran

    2016-03-01

    Loss of function (LoF) genetic variants are predicted to disrupt gene function, and are therefore expected to substantially reduce individual's viability. Knowing the genetic burden of LoF variants in endangered species is of interest for a better understanding of the effects of declining population sizes on species viability. In this study, we have estimated the number of LoF polymorphic variants in six great ape populations, based on whole-genome sequencing data in 79 individuals. Our results show that although the number of functional variants per individual is conditioned by the effective population size, the number of variants with a drastic phenotypic effect is very similar across species. We hypothesize that for those variants with high selection coefficients, differences in effective population size are not important enough to affect the efficiency of natural selection to remove them. We also describe that mostly CpG LoF mutations are shared across species, and an accumulation of LoF variants at olfactory receptor genes in agreement with its pseudogenization in humans and other primate species. PMID:26912403

  1. Genetic Load of Loss-of-Function Polymorphic Variants in Great Apes

    PubMed Central

    de Valles-Ibáñez, Guillem; Hernandez-Rodriguez, Jessica; Prado-Martinez, Javier; Luisi, Pierre; Marquès-Bonet, Tomàs; Casals, Ferran

    2016-01-01

    Loss of function (LoF) genetic variants are predicted to disrupt gene function, and are therefore expected to substantially reduce individual’s viability. Knowing the genetic burden of LoF variants in endangered species is of interest for a better understanding of the effects of declining population sizes on species viability. In this study, we have estimated the number of LoF polymorphic variants in six great ape populations, based on whole-genome sequencing data in 79 individuals. Our results show that although the number of functional variants per individual is conditioned by the effective population size, the number of variants with a drastic phenotypic effect is very similar across species. We hypothesize that for those variants with high selection coefficients, differences in effective population size are not important enough to affect the efficiency of natural selection to remove them. We also describe that mostly CpG LoF mutations are shared across species, and an accumulation of LoF variants at olfactory receptor genes in agreement with its pseudogenization in humans and other primate species. PMID:26912403

  2. Onco-lncRNA HOTAIR and its functional genetic variants in papillary thyroid carcinoma

    PubMed Central

    Zhu, Hui; Lv, Zheng; An, Changming; Shi, Meng; Pan, Wenting; Zhou, Liqing; Yang, Wenjun; Yang, Ming

    2016-01-01

    The role of long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) and its functional single nucleotide polymorphisms (SNPs) in papillary thyroid carcinoma (PTC) is still largely unclear. Therefore, we investigated the involvement of lncRNA HOTAIR and its three haplotype-tagging SNPs (htSNPs) in PTC. There was higher expression of HOTAIR in PTC tissues compared to normal tissues. A series of gain-loss assays demonstrated that HOTAIR acts as a PTC oncogene via promoting tumorigenic properties of PTC cells. Additionally, the functional HOTAIR rs920778 genetic variant was a PTC susceptibility SNP. Subjects with the HOTAIR rs920778 TT genotype had an odds ratio (OR) of 1.88, 1.25 and 1.61 (P = 6.0 × 10−6, P = 0.028 and P = 3.2 × 10−5) for developing PTC in Shandong, Jiangsu and Jilin case-control sets compared with subjects with the CC genotype. This statistically significant associations were only found between the rs920778 genetic polymorphism and PTC risk in females but not in males. The allele-specific regulation on HOTAIR expression by the rs920778 SNP was confirmed both in vitro and in vivo. Our results demonstrate that functional SNPs influencing lncRNA regulation may explain a part of PTC genetic basis. PMID:27549736

  3. Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture.

    PubMed

    Xue, Katherine S; Hooper, Kathryn A; Ollodart, Anja R; Dingens, Adam S; Bloom, Jesse D

    2016-01-01

    RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant's proficiency at cell entry with the other's proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies. PMID:26978794

  4. Overlap between Parkinson disease and Alzheimer disease in ABCA7 functional variants

    PubMed Central

    Nuytemans, Karen; Maldonado, Lizmarie; Ali, Aleena; John-Williams, Krista; Beecham, Gary W.; Martin, Eden; Scott, William K.

    2016-01-01

    Objective: Given their reported function in phagocytosis and clearance of protein aggregates in Alzheimer disease (AD), we hypothesized that variants in ATP-binding cassette transporter A7 (ABCA7) might be involved in Parkinson disease (PD). Methods: ABCA7 variants were identified using whole-exome sequencing (WES) on 396 unrelated patients with PD and 222 healthy controls. In addition, we used the publicly available WES data from the Parkinson's Progression Markers Initiative (444 patients and 153 healthy controls) as a second, independent data set. Results: We observed a higher frequency of loss-of-function (LOF) variants and rare putative highly functional variants (Combined Annotation Dependent Depletion [CADD] >20) in clinically diagnosed patients with PD than in healthy controls in both data sets. Overall, we identified LOF variants in 11 patients and 1 healthy control (odds ratio [OR] 4.94, Fisher exact p = 0.07). Four of these variants have been previously implicated in AD risk (p.E709AfsX86, p.W1214X, p.L1403RfsX7, and rs113809142). In addition, rare variants with CADD >20 were observed in 19 patients vs 3 healthy controls (OR 2.85, Fisher exact p = 0.06). Conclusion: The presence of ABCA7 LOF variants in clinically defined PD suggests that they might be risk factors for neurodegeneration in general, especially those variants hallmarked by protein aggregation. More studies will be needed to evaluate the overall impact of this transporter in neurodegenerative disease. PMID:27066581

  5. T-13910 DNA variant associated with lactase persistence interacts with Oct-1 and stimulates lactase promoter activity in vitro.

    PubMed

    Lewinsky, Rikke H; Jensen, Tine G K; Møller, Jette; Stensballe, Allan; Olsen, Jørgen; Troelsen, Jesper T

    2005-12-15

    Two phenotypes exist in the human population with regard to expression of lactase in adults. Lactase non-persistence (adult-type hypolactasia and lactose intolerance) is characterized by a decline in the expression of lactase-phlorizin hydrolase (LPH) after weaning. In contrast, lactase-persistent individuals have a high LPH throughout their lifespan. Lactase persistence and non-persistence are associated with a T/C polymorphism at position -13,910 upstream the lactase gene. A nuclear factor binds more strongly to the T-13,910 variant associated with lactase persistence than the C-13,910 variant associated with lactase non-persistence. Oct-1 and glyceraldehyde-3-phosphate dehydrogenase were co-purified by DNA affinity purification using the sequence of the T-13,910 variant. Supershift analyses show that Oct-1 binds directly to the T-13,910 variant, and we suggest that GAPDH is co-purified due to interactions with Oct-1. Expression of Oct-1 stimulates reporter gene expression from the T and the C-13,910 variant/LPH promoter constructs only when it is co-expressed with HNF1alpha. Binding sites for other intestinal transcription factors (GATA-6, HNF4alpha, Fox and Cdx-2) were identified in the region of the -13,910 T/C polymorphism. Three of these sites are required for the enhancer activity of the -13,910 region. The data suggest that the binding of Oct-1 to the T-13,910 variant directs increased lactase promoter activity and this might provide an explanation for the lactase persistence phenotype in the human population.

  6. Distribution and medical impact of loss-of-function variants in the Finnish founder population.

    PubMed

    Lim, Elaine T; Würtz, Peter; Havulinna, Aki S; Palta, Priit; Tukiainen, Taru; Rehnström, Karola; Esko, Tõnu; Mägi, Reedik; Inouye, Michael; Lappalainen, Tuuli; Chan, Yingleong; Salem, Rany M; Lek, Monkol; Flannick, Jason; Sim, Xueling; Manning, Alisa; Ladenvall, Claes; Bumpstead, Suzannah; Hämäläinen, Eija; Aalto, Kristiina; Maksimow, Mikael; Salmi, Marko; Blankenberg, Stefan; Ardissino, Diego; Shah, Svati; Horne, Benjamin; McPherson, Ruth; Hovingh, Gerald K; Reilly, Muredach P; Watkins, Hugh; Goel, Anuj; Farrall, Martin; Girelli, Domenico; Reiner, Alex P; Stitziel, Nathan O; Kathiresan, Sekar; Gabriel, Stacey; Barrett, Jeffrey C; Lehtimäki, Terho; Laakso, Markku; Groop, Leif; Kaprio, Jaakko; Perola, Markus; McCarthy, Mark I; Boehnke, Michael; Altshuler, David M; Lindgren, Cecilia M; Hirschhorn, Joel N; Metspalu, Andres; Freimer, Nelson B; Zeller, Tanja; Jalkanen, Sirpa; Koskinen, Seppo; Raitakari, Olli; Durbin, Richard; MacArthur, Daniel G; Salomaa, Veikko; Ripatti, Samuli; Daly, Mark J; Palotie, Aarno

    2014-07-01

    Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5-5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10⁻⁸) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10⁻¹¹⁷). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10⁻⁴), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.

  7. Distribution and medical impact of loss-of-function variants in the Finnish founder population.

    PubMed

    Lim, Elaine T; Würtz, Peter; Havulinna, Aki S; Palta, Priit; Tukiainen, Taru; Rehnström, Karola; Esko, Tõnu; Mägi, Reedik; Inouye, Michael; Lappalainen, Tuuli; Chan, Yingleong; Salem, Rany M; Lek, Monkol; Flannick, Jason; Sim, Xueling; Manning, Alisa; Ladenvall, Claes; Bumpstead, Suzannah; Hämäläinen, Eija; Aalto, Kristiina; Maksimow, Mikael; Salmi, Marko; Blankenberg, Stefan; Ardissino, Diego; Shah, Svati; Horne, Benjamin; McPherson, Ruth; Hovingh, Gerald K; Reilly, Muredach P; Watkins, Hugh; Goel, Anuj; Farrall, Martin; Girelli, Domenico; Reiner, Alex P; Stitziel, Nathan O; Kathiresan, Sekar; Gabriel, Stacey; Barrett, Jeffrey C; Lehtimäki, Terho; Laakso, Markku; Groop, Leif; Kaprio, Jaakko; Perola, Markus; McCarthy, Mark I; Boehnke, Michael; Altshuler, David M; Lindgren, Cecilia M; Hirschhorn, Joel N; Metspalu, Andres; Freimer, Nelson B; Zeller, Tanja; Jalkanen, Sirpa; Koskinen, Seppo; Raitakari, Olli; Durbin, Richard; MacArthur, Daniel G; Salomaa, Veikko; Ripatti, Samuli; Daly, Mark J; Palotie, Aarno

    2014-07-01

    Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5-5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10⁻⁸) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10⁻¹¹⁷). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10⁻⁴), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers. PMID

  8. Functionalized block copolymers as adhesion promoters

    SciTech Connect

    Kent, M.S.; Saunders, R.

    1995-03-01

    The goal of this work is to develop novel functionalized block copolymers to promote adhesion at inorganic substrate/polymer interfaces. We envision several potential advantages of functionalized block copolymers over small molecule coupling agents. Greater control over the structure of the interphase region should result through careful design of the backbone of the copolymer. The number of chains per area, the degree of entanglement between the copolymer and the polymer matrix, the number of sites per chain able to attach to the substrate, and the hydrophobicity of the interphase region can all be strongly affected by the choice of block lengths and the monomer sequence. In addition, entanglement between the copolymer and the polymer matrix, if achieved, should contribute significantly to adhesive strength. Our program involves four key elements: the synthesis of suitable functionalized block copolymers, characterization of the conformation of the copolymers at the interface by neutron reflectivity and atomic force microscopy, characterization of the degree of bonding by spectroscopy, and measurement of the mechanical properties of the interface. In this paper we discuss block copolymers designed as adhesion promoters for the copper/epoxy interface. We have synthesized a diblock with one block containing imidazole groups to bond to copper and a second block containing secondary amines to react with the epoxy matrix. We have also prepared a triblock copolymer containing a hydrophobic middle block. Below we describe the synthesis of the block copolymers by living, ring-opening metathesis polymerization (ROMP) and the first characterization data obtained by neutron reflectivity.

  9. Loss of HLTF function promotes intestinal carcinogenesis

    PubMed Central

    2012-01-01

    Background HLTF (Helicase-like Transcription Factor) is a DNA helicase protein homologous to the SWI/SNF family involved in the maintenance of genomic stability and the regulation of gene expression. HLTF has also been found to be frequently inactivated by promoter hypermethylation in human colon cancers. Whether this epigenetic event is required for intestinal carcinogenesis is unknown. Results To address the role of loss of HLTF function in the development of intestinal cancer, we generated Hltf deficient mice. These mutant mice showed normal development, and did not develop intestinal tumors, indicating that loss of Hltf function by itself is insufficient to induce the formation of intestinal cancer. On the Apcmin/+ mutant background, Hltf- deficiency was found to significantly increase the formation of intestinal adenocarcinoma and colon cancers. Cytogenetic analysis of colon tumor cells from Hltf -/-/Apcmin/+ mice revealed a high incidence of gross chromosomal instabilities, including Robertsonian fusions, chromosomal fragments and aneuploidy. None of these genetic alterations were observed in the colon tumor cells derived from Apcmin/+ mice. Increased tumor growth and genomic instability was also demonstrated in HCT116 human colon cancer cells in which HLTF expression was significantly decreased. Conclusion Taken together, our results demonstrate that loss of HLTF function promotes the malignant transformation of intestinal or colonic adenomas to carcinomas by inducing genomic instability. Our findings highly suggest that epigenetic inactivation of HLTF, as found in most human colon cancers, could play an important role in the progression of colon tumors to malignant cancer. PMID:22452792

  10. Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

    PubMed

    Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

    2014-12-01

    Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis.

  11. Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

    PubMed

    Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

    2014-12-01

    Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis. PMID:25193465

  12. Novel and functional ABCB1 gene variant in sporadic Parkinson's disease.

    PubMed

    Li, Yuequn; Li, Yonghua; Pang, Shuchao; Huang, Wenhui; Zhang, Aimei; Hawley, Robert G; Yan, Bo

    2014-04-30

    Parkinson's disease (PD) is a common progressive neurodegenerative disease. Most cases of PD are sporadic, which is caused by interaction of genetic and environmental factors. To date, genetic causes for sporadic PD remain largely unknown. ATP-binding cassette sub-family B member 1 (ABCB1) is a membrane-associated protein that acts as an efflux transporter for many substrates, including chemotherapeutic agents, anti-epilepsy medicine, antibiotics and drugs for PD. ABCB1 gene is widely expressed in human tissues, including endothelial cells of capillary blood vessels at blood-brain barrier sites. In PD patients, decreased ABCB1 levels have been reported. We speculated that misregulation of ABCB1 gene expression, caused by DNA sequence variants (DSVs) within its regulatory regions, may be involved in PD development. In this study, we genetically and functionally analyzed the proximal promoter of the human ABCB1 gene, which is required for constitutive expression, in sporadic PD patients and healthy controls. The results showed that a novel and heterozygous DSV g.117077G>A was identified in one PD patient, but in none of the controls. This DSV significantly altered the transcriptional activity of the ABCB1 gene promoter in transiently transfected HEK-293 cells. A heterozygous DSV g.116347T>C was only found in one control. Four single-nucleotide polymorphisms, g.116154T>C (rs28746504), g.117130A>G (rs2188524), g.117356C>G (rs34976462) and g.117372T>C (rs3213619), and one heterozygous deletion DSV g.116039del were found in PD patients and controls with similar frequencies. Therefore, our findings suggest that ABCB1 gene promoter DSVs may contribute to PD development as a rare risk factor. PMID:24572589

  13. Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major

    PubMed Central

    Andreani, Marco; Radio, Francesca Clementina; Testi, Manuela; De Bernardo, Carmelilia; Troiano, Maria; Majore, Silvia; Bertucci, Pierfrancesco; Polchi, Paola; Rosati, Renata; Grammatico, Paola

    2009-01-01

    Hepcidin is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide produced predominantly by hepatocytes. This molecule, encoded by the hepcidin antimicrobial peptide (HAMP) gene shows structural and functional properties consistent with a role in innate immunity. Moreover, as demonstrated in mice and humans, hepcidin is a major regulator of iron metabolism, and acts by binding to ferroportin and controlling its concentration and trafficking. In this study we investigated the influence that mutations in HAMP and/or hemocromatosis (HFE) genes might exert on iron metabolism in a group of poly-transfused thalassemic patients in preparation for bone marrow transplantation. Our results showed that the presence of the c.-582 A>G polymorphism (rs10421768) placed in HAMP promoter (HAMP-P) might play a role in iron metabolism, perhaps varying the transcriptional activation that occurs through E-boxes located within the promoter. PMID:19734422

  14. Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major.

    PubMed

    Andreani, Marco; Radio, Francesca Clementina; Testi, Manuela; De Bernardo, Carmelilia; Troiano, Maria; Majore, Silvia; Bertucci, Pierfrancesco; Polchi, Paola; Rosati, Renata; Grammatico, Paola

    2009-09-01

    Hepcidin is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide produced predominantly by hepatocytes. This molecule, encoded by the hepcidin antimicrobial peptide (HAMP) gene shows structural and functional properties consistent with a role in innate immunity. Moreover, as demonstrated in mice and humans, hepcidin is a major regulator of iron metabolism, and acts by binding to ferroportin and controlling its concentration and trafficking. In this study we investigated the influence that mutations in HAMP and/or hemocromatosis (HFE) genes might exert on iron metabolism in a group of poly-transfused thalassemic patients in preparation for bone marrow transplantation. Our results showed that the presence of the c.-582 A>G polymorphism (rs10421768) placed in HAMP promoter (HAMP-P) might play a role in iron metabolism, perhaps varying the transcriptional activation that occurs through E-boxes located within the promoter.

  15. Expression and functionality of histone H2A variants in cancer

    PubMed Central

    Monteiro, Fátima Liliana; Baptista, Tiago; Amado, Francisco; Vitorino, Rui; Jerónimo, Carmen; Helguero, Luisa A.

    2014-01-01

    Regulation of gene expression includes the replacement of canonical histones for non-allelic histone variants, as well as their multiple targeting by postranslational modifications. H2A variants are highly conserved between species suggesting they execute important functions that cannot be accomplished by canonical histones. Altered expression of many H2A variants is associated to cancer. MacroH2A variants are enriched in heterocromatic foci and are necessary for chromatin condensation. MacroH2A1.1 and macroH2A1.2 are two mutually exclusive isoforms. MacroH2A1.1 and macroH2A2 inhibit proliferation and are associated with better cancer prognosis; while macroH2A1.2 is associated to cancer progression. H2AX variant functions as a sensor of DNA damage and defines the cellular response towards DNA repair or apoptosis; therefore, screening approaches and therapeutic options targeting H2AX have been proposed. H2A.Z is enriched in euchromatin, acting as a proto-oncogene with established roles in hormone responsive cancers and overexpressed in endocrine-resistant disease. Other H2A family members have also been found altered in cancer, but their function remains unknown. Substantial progress has been made to understand histone H2A variants, their contribution to normal cellular function and to cancer development and progression. Yet, implementation of high resolution mass spectrometry is needed to further our knowledge on highly homologous H2A variants expression and function. PMID:25003966

  16. Misregulation effect of a novel allelic variant in the Z promoter region found in cis with the CYP21A2 p.P482S mutation: implications for 21-hydroxylase deficiency.

    PubMed

    Fernández, Cecilia S; Bruque, Carlos D; Taboas, Melisa; Buzzalino, Noemí D; Espeche, Lucia D; Pasqualini, Titania; Charreau, Eduardo H; Alba, Liliana G; Ghiringhelli, Pablo D; Dain, Liliana

    2015-09-01

    The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant-g.15626A>G-within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.

  17. The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5

    PubMed Central

    Lessel, Davor; Muhammad, Tariq; Casar Tena, Teresa; Moepps, Barbara; Burkhalter, Martin D.; Hitz, Marc-Phillip; Toka, Okan; Rentzsch, Axel; Schubert, Stephan; Schalinski, Adelheid; Bauer, Ulrike M. M.; Kubisch, Christian; Ware, Stephanie M.; Philipp, Melanie

    2016-01-01

    G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5. PMID:27618959

  18. The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5.

    PubMed

    Lessel, Davor; Muhammad, Tariq; Casar Tena, Teresa; Moepps, Barbara; Burkhalter, Martin D; Hitz, Marc-Phillip; Toka, Okan; Rentzsch, Axel; Schubert, Stephan; Schalinski, Adelheid; Bauer, Ulrike M M; Kubisch, Christian; Ware, Stephanie M; Philipp, Melanie

    2016-01-01

    G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5. PMID:27618959

  19. Homozygous loss-of-function variants in European cosmopolitan and isolate populations.

    PubMed

    Kaiser, Vera B; Svinti, Victoria; Prendergast, James G; Chau, You-Ying; Campbell, Archie; Patarcic, Inga; Barroso, Inês; Joshi, Peter K; Hastie, Nicholas D; Miljkovic, Ana; Taylor, Martin S; Enroth, Stefan; Memari, Yasin; Kolb-Kokocinski, Anja; Wright, Alan F; Gyllensten, Ulf; Durbin, Richard; Rudan, Igor; Campbell, Harry; Polašek, Ozren; Johansson, Åsa; Sauer, Sascha; Porteous, David J; Fraser, Ross M; Drake, Camilla; Vitart, Veronique; Hayward, Caroline; Semple, Colin A; Wilson, James F

    2015-10-01

    Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown—as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation.

  20. Homozygous loss-of-function variants in European cosmopolitan and isolate populations

    PubMed Central

    Kaiser, Vera B.; Svinti, Victoria; Prendergast, James G.; Chau, You-Ying; Campbell, Archie; Patarcic, Inga; Barroso, Inês; Joshi, Peter K.; Hastie, Nicholas D.; Miljkovic, Ana; Taylor, Martin S.; Enroth, Stefan; Memari, Yasin; Kolb-Kokocinski, Anja; Wright, Alan F.; Gyllensten, Ulf; Durbin, Richard; Rudan, Igor; Campbell, Harry; Polašek, Ozren; Johansson, Åsa; Sauer, Sascha; Porteous, David J.; Fraser, Ross M.; Drake, Camilla; Vitart, Veronique; Hayward, Caroline; Semple, Colin A.; Wilson, James F.

    2015-01-01

    Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown—as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation. PMID:26173456

  1. Genetic and functional analysis of the TBX3 gene promoter in indirect inguinal hernia.

    PubMed

    Zhao, Zhongqing; Tian, Wenjun; Wang, Lin; Wang, Haihua; Qin, Xianyun; Xing, Qining; Pang, Shuchao; Yan, Bo

    2015-01-01

    Inguinal hernia is a common developmental disease in children and most cases are indirect inguinal hernia (IIH). Genetic factors have been suggested to play important roles in IIH. Although IIH has been observed in several human syndromes, genetic causes and molecular mechanisms for IIH remain unknown. TBX3 is a member of the T-box family of transcription factors that are essential to the embryonic development. Human studies and animal experiments have demonstrated that TBX3 is required for the development of the heart, limbs, mammary glands and other tissues and organs. TBX3 gene expression has been detected in human fibroblast and tissues of abdominal wall. We speculated that TBX3 may be involved in the IIH formation. Since TBX3 activity is highly dosage-sensitive, a TBX3 gene promoter was genetically and functionally analyzed in IIH patients and ethnic-matched controls in this study. One heterozygous deletion variant (g.4820_4821del) was identified in one IIH patient, but in none of controls. The variant significantly decreased TBX3 gene promoter activities, likely by creating a binding site for sex-determining region Y (SRY), mobility group transcription factor. One heterozygous insertion variant (g.3913_3914ins) was only found in one control, which did not affect TBX3 gene promoter activities. Taken together, TBX3 gene variants may contribute to IIH as a rare risk factor by reducing TBX3 levels. PMID:25455105

  2. STRONG GRAVITATIONAL LENS MODELING WITH SPATIALLY VARIANT POINT-SPREAD FUNCTIONS

    SciTech Connect

    Rogers, Adam; Fiege, Jason D.

    2011-12-10

    Astronomical instruments generally possess spatially variant point-spread functions, which determine the amount by which an image pixel is blurred as a function of position. Several techniques have been devised to handle this variability in the context of the standard image deconvolution problem. We have developed an iterative gravitational lens modeling code called Mirage that determines the parameters of pixelated source intensity distributions for a given lens model. We are able to include the effects of spatially variant point-spread functions using the iterative procedures in this lensing code. In this paper, we discuss the methods to include spatially variant blurring effects and test the results of the algorithm in the context of gravitational lens modeling problems.

  3. Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

    PubMed

    Dang, Xiao-yong; Chu, Wei-wei; Shi, Heng-chuan; Yu, Shi-gang; Han, Hai-yin; Gu, Shu-Hua; Chen, Jie

    2015-01-25

    Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.

  4. Disease associations and altered immune function in CD45 138G variant carriers.

    PubMed

    Boxall, Sally; Stanton, Tara; Hirai, Kouzo; Ward, Victoria; Yasui, Tomoyo; Tahara, Hideki; Tamori, Akihiro; Nishiguchi, Shuhei; Shiomi, Susumu; Ishiko, Osamu; Inaba, Masaaki; Nishizawa, Yoshiki; Dawes, Ritu; Bodmer, Walter; Beverley, Peter C L; Tchilian, Elma Z

    2004-10-15

    The CD45 antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes. Expression of different patterns of alternatively spliced CD45 isoforms is associated with distinct functions. We recently identified a polymorphism in exon 6 (A138G) of the gene encoding CD45 (PTPRC) that results in altered CD45 splicing. The 138G allele is present at a high frequency among Japanese (23.7%), with 5.1% individuals homozygous for the G allele. In this study we show that the A138G polymorphism is the cause of altered CD45 isoform expression, promoting splicing towards low molecular weight CD45 isoforms. We further report that the frequency of A138G heterozygotes is significantly reduced in number in cohorts of patients with autoimmune Graves' disease or hepatitis B infection, whereas G138G homozygotes are absent from a cohort of Hashimoto's thyroiditis patients. We also show that 138G individuals exhibit altered cytokine production in vitro and an increased proportion of memory T cells. These data suggest that the 138G variant allele strongly influences these diseases by modulation of immune mechanisms and may have achieved its high frequency as a result of a natural selection probably related to pathogen resistance. PMID:15333587

  5. Genetic and functional analyses of ZIC3 variants in congenital heart disease

    PubMed Central

    Cowan, Jason; Tariq, Muhammad; Ware, Stephanie M.

    2013-01-01

    Mutations in zinc-finger in cerebellum 3 (ZIC3) result in heterotaxy or isolated congenital heart disease (CHD). The majority of reported mutations cluster in zinc-finger domains. We previously demonstrated that many of these lead to aberrant ZIC3 subcellular trafficking. A relative paucity of N- and C-terminal mutations has, however, prevented similar analyses in these regions. Notably, an N-terminal polyalanine expansion was recently identified in a patient with VACTERL, suggesting a potentially distinct function for this domain. Here, we report ZIC3 sequencing results from 440 unrelated patients with heterotaxy and CHD, the largest cohort yet examined. Variants were identified in 5.2% of sporadic male cases. This rate exceeds previous estimates of 1% and has important clinical implications for genetic testing and risk-based counseling. Eight of 11 were novel, including 5 N-terminal variants. Subsequent functional analyses included 4 additional reported but untested variants. Aberrant cytoplasmic localization and decreased luciferase transactivation were observed for all zinc-finger variants, but not for downstream or in-frame upstream variants, including both analyzed polyalanine expansions. Collectively, these results expand the ZIC3 mutational spectrum, support a higher than expected prevalence in sporadic cases, and suggest alternative functions for terminal mutations, highlighting a need for further study of these domains. PMID:24123890

  6. Cloning and characterization of functional subtype A HIV-1 envelope variants transmitted through breastfeeding.

    PubMed

    Rainwater, Stephanie M J; Wu, Xueling; Nduati, Ruth; Nedellec, Rebecca; Mosier, Donald; John-Stewart, Grace; Mbori-Ngacha, Dorothy; Overbaugh, Julie

    2007-03-01

    Previous studies of HIV-1 variants transmitted from mother-to-infant have focused primarily on computational analyses of partial envelope gene sequences, rather than analyses of functional envelope variants. There are very few examples of well-characterized functional envelope clones from mother-infant pairs, especially from envelope variants representing the most prevalent subtypes worldwide. To address this, we amplified the envelope variants present in 4 mother-infant transmission pairs, all of whom were infected with subtype A and three of whom presumably transmitted HIV-1 during the breastfeeding period. Functional envelope clones were constructed, either encoding full-length envelope sequences from the mother and baby or by making chimeric envelope clones in a common backbone sequence. The infant envelope sequences were genetically homogeneous compared to the maternal viruses, and pseudoviruses bearing these envelopes all used CCR5 as a coreceptor. The infant viruses were generally resistant to neutralization by maternal antibodies present near the time of transmission. There were no notable differences in sensitivity of the mother and infant envelope variants to neutralization by heterologous plasma or monoclonal antibodies 2G12 and b12, or to inhibition by sCD4, PSC-RANTES or TAK779. This collection of viral envelopes, which can be used for making pseudotyped viruses, may be useful for examining the efficacy of interventions to block mother-infant transmission, including sera from vaccine candidates, purified antibodies under consideration for passive immunization and viral entry inhibitors.

  7. Structural and Functional Analysis of the ApolipoproteinA-I A164S Variant

    PubMed Central

    Dalla-Riva, Jonathan; Lagerstedt, Jens O.; Petrlova, Jitka

    2015-01-01

    Apolipoprotein A-I (apoA-I) is the main protein involved in the formation of high-density lipoprotein (HDL), it is the principal mediator of the reverse cholesterol transfer (RCT) pathway and provides cardio-protection. In addition to functional wild-type apoA-I, several variants have been shown to associate with hereditary amyloidosis. In this study we have performed biophysical and biochemical analyses of the structure and functional properties of the A164S variant of apoA-I (1:500 in the Danish general population), which is the first known mutation of apoA-I that leads to an increased risk of ischaemic heart disease (IHD), myocardial infarction and mortality without associated low HDL cholesterol levels. Despite the fact that epidemiologically IHD is associated with low plasma levels of HDL, the A164S mutation is linked to normal plasma levels of lipids, HDL and apoA-I, suggesting impaired functionality of this variant. Using biophysical techniques (e.g., circular dichroism spectroscopy and electron microscopy) to determine secondary structure, stability and pro-amyloidogenic property of the lipid free A164S apoA-I variant, our observations suggest similarity in structural properties between apoA-I WT and apoA-I A164S. However, the A164S apoA-I variant exhibits lower binding affinity to lipids but forms similar sized HDL particles to those produced by WT. PMID:26605794

  8. Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis.

    PubMed

    Börner, Kenneth; Becker, Peter B

    2016-09-01

    SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange. PMID:27578180

  9. Promotion of waterpipe tobacco use, its variants and accessories in young adult newspapers: a content analysis of message portrayal

    PubMed Central

    Sterling, Kymberle L.; Fryer, Craig S.; Majeed, Ban; Duong, Melissa M.

    2015-01-01

    The objective of our study was to identify waterpipe tobacco smoking advertisements and those that promoted a range of products and accessories used to smoke waterpipe tobacco. The content of these advertisements was analyzed to understand the messages portrayed about waterpipe tobacco smoking in young adult (aged 18–30) newspapers. The study methods include monitoring of six newspapers targeting young adults from four major cities in the Southeastern United States over a 6-month period. A total of 87 advertisements were found; 73.5% (64) were distinct and content analyzed. The study results showed that of the advertisements analyzed, 25% advertised waterpipe tobacco smoking, 54.7% featured waterpipe tobacco smoking and other tobacco use, 14.1% featured non-tobacco waterpipe variants (i.e. vaporizers), and 6.3% featured waterpipe apparatus accessories (e.g. charcoal, hoses). The sociability (34%) and sensuality (29.7%) of waterpipe smoking were promoted themes. Alternative to cigarette use messages (3.1%), and harm-reduction messages (17.1%) emphasized that smoking waterpipe tobacco using the featured accessory or waterpipe variant was a healthier experience than cigarette smoking. The study concluded that the messages that promoted waterpipe tobacco smoking to young adults are parallel to those used to promote cigarette use. Tobacco control professionals should continue to monitor young adult newspapers as a source of waterpipe-related advertising. PMID:24957675

  10. A promoter variant of SHANK1 affects auditory working memory in schizophrenia patients and in subjects clinically at risk for psychosis.

    PubMed

    Lennertz, Leonhard; Wagner, Michael; Wölwer, Wolfgang; Schuhmacher, Anna; Frommann, Ingo; Berning, Julia; Schulze-Rauschenbach, Svenja; Landsberg, Martin W; Steinbrecher, Anja; Alexander, Michael; Franke, Petra E; Pukrop, Ralf; Ruhrmann, Stephan; Bechdolf, Andreas; Gaebel, Wolfgang; Klosterkötter, Joachim; Häfner, Heinz; Maier, Wolfgang; Mössner, Rainald

    2012-03-01

    Mutations in postsynaptic scaffolding genes contribute to autism, thus suggesting a role in pathological processes in neurodevelopment. Recently, two de novo mutations in SHANK3 were described in schizophrenia patients. In most cases, abnormal SHANK3 genotype was also accompanied by cognitive disruptions. The present study queries whether common SHANK variants may also contribute to neuropsychological dysfunctions in schizophrenia. We genotyped five common coding or promoter variants located in SHANK1, SHANK2 and SHANK3. A comprehensive test battery was used to assess neuropsychological functions in 199 schizophrenia patients and 206 healthy control subjects. In addition, an independent sample of 77 subjects at risk for psychosis was analyzed for replication of significant findings. We found the T allele of the SHANK1 promoter variant rs3810280 to lead to significantly impaired auditory working memory as assessed with digit span (12.5 ± 3.6 vs. 14.8 ± 4.1, P < .001) in schizophrenia cases, applying strict Bonferroni correction for multiple testing. This finding was replicated for forward digit span in the at-risk sample (7.1 ± 2.0 vs. 8.3 ± 2.0, P = .044). Previously, altered memory functions and reduced dendritic spines and postsynaptic density of excitatory synapses were reported in SHANK1 knock-out mice. Moreover, the atypical neuroleptic clozapine was found to increase SHANK1 density in rats. Our findings suggest a role of SHANK1 in working memory deficits in schizophrenia, which may arise from neurodevelopmental changes to prefrontal cortical areas.

  11. Loss-of-function variants influence the human serum metabolome.

    PubMed

    Yu, Bing; Li, Alexander H; Metcalf, Ginger A; Muzny, Donna M; Morrison, Alanna C; White, Simon; Mosley, Thomas H; Gibbs, Richard A; Boerwinkle, Eric

    2016-08-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study. PMID:27602404

  12. Loss-of-function variants influence the human serum metabolome

    PubMed Central

    Yu, Bing; Li, Alexander H.; Metcalf, Ginger A.; Muzny, Donna M.; Morrison, Alanna C.; White, Simon; Mosley, Thomas H.; Gibbs, Richard A.; Boerwinkle, Eric

    2016-01-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study. PMID:27602404

  13. Loss-of-function variants influence the human serum metabolome

    PubMed Central

    Yu, Bing; Li, Alexander H.; Metcalf, Ginger A.; Muzny, Donna M.; Morrison, Alanna C.; White, Simon; Mosley, Thomas H.; Gibbs, Richard A.; Boerwinkle, Eric

    2016-01-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study.

  14. Identification and functional characterization of four TRPA1 variants in Apolygus lucorum (Meyer-Dür)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As signal integrators that respond to various physical and chemical stimuli, transient receptor potential (TRP) channels fulfil critical functional roles in the sensory systems of both vertebrate and invertebrate organisms. Here, four variants of TRP ankyrin 1 (TRPA1) were identified and cloned from...

  15. Transactivation of the p21 promoter by BRCA1 splice variants in mammary epithelial cells: evidence for both common and distinct activities of wildtype and mutant forms.

    PubMed

    Lu, M; Arrick, B A

    2000-12-14

    We have evaluated the transcriptional activation of a human p21 promoter reporter construct by transfection of BRCA1 expression constructs into tumorigenic and nontumorigenic human breast cell lines. Two cell lines with wildtype p53 (MCF-7 and MCF10A) demonstrated transcriptional activation of the p21 promoter by full-length BRCA1 (BRCA1L) as well as by two splice variants that lack most of exon 11 (BRCA1S and BRCA1S-9,10). In contrast, two cell lines with mutant p53 (MDA-231 and HCC1937) were inactive. Co-transfection of BRCA1L with BRCA1S or BRCA1S-9,10 exhibited synergistic p21 promoter activation, due to augmented expression of the cytomegalovirus promoter-based BRCA1 expression constructs. We examined the transcriptional activity of two known sequence alterations in BRCA1, one that results in a carboxy-terminal truncation of BRCA1 and is clearly pathogenic, and the other a missense mutation that is suspected of predisposing to cancer. Although both mutations have been shown to be defective in some assays of transactivation, we observed both mutations to be fully active in activation of the p21 promoter when incorporated in the full-length BRCA1L. In contrast, these mutations rendered BRCA1S inactive. These observations indicate that such transcriptional assays cannot serve as the basis for a functional appraisal of BRCA1 sequence alterations encountered in the course of genetic testing.

  16. Prioritization of Genetic Variants in the microRNA Regulome as Functional Candidates in Genome-Wide Association Studies

    PubMed Central

    Bulik-Sullivan, Brendan; Selitsky, Sara; Sethupathy, Praveen

    2013-01-01

    Comprehensive analyses of results from genome-wide association studies (GWAS) have demonstrated that complex disease/trait-associated loci are enriched in gene regulatory regions of the genome. The search for causal regulatory variation has focused primarily on transcriptional elements, such as promoters and enhancers. microRNAs (miRNAs) are now widely appreciated as critical posttranscriptional regulators of gene expression and are thought to impart stability to biological systems. Naturally occurring genetic variation in the miRNA regulome is likely an important contributor to phenotypic variation in the human population. However, the extent to which polymorphic miRNA-mediated gene regulation underlies GWAS signals remains unclear. In this study, we have developed the most comprehensive bioinformatic analysis pipeline to date for cataloging and prioritizing variants in the miRNA regulome as functional candidates in GWAS. We highlight specific findings, including a variant in the promoter of the miRNA let-7 that may contribute to human height variation. We also provide a discussion of how our approach can be expanded in the future. Overall, we believe that the results of this study will be valuable for researchers interested in determining whether GWAS signals implicate the miRNA regulome in their disease/trait of interest. PMID:23595788

  17. Updated findings of the association and functional studies of DRD2/ANKK1 variants with addictions.

    PubMed

    Ma, Yunlong; Yuan, Wenji; Jiang, Xianzhong; Cui, Wen-Yan; Li, Ming D

    2015-02-01

    Both nicotine and alcohol addictions are severe public health hazards worldwide. Various twin and family studies have demonstrated that genetic factors contribute to vulnerability to these addictions; however, the susceptibility genes and the variants underlying them remain largely unknown. Of susceptibility genes investigated for addictions, DRD2 has received much attention. Considering new evidence supporting the association of DRD2 and its adjacent gene ankyrin repeat and kinase domain containing 1 (ANKK1) with various addictions, in this paper, we provide an updated view of the involvement of variants in DRD2 and ANKK1 in the etiology of nicotine dependence (ND) and alcohol dependence (AD) based on linkage, association, and molecular studies. This evidence shows that both genes are significantly associated with addictions; however the association with ANKK1 appears to be stronger. Thus, both more replication studies in independent samples and functional studies of some of these variants are warranted.

  18. Unbiased Functional Clustering of Gene Variants with a Phenotypic-Linkage Network

    PubMed Central

    Honti, Frantisek; Meader, Stephen; Webber, Caleb

    2014-01-01

    Groupwise functional analysis of gene variants is becoming standard in next-generation sequencing studies. As the function of many genes is unknown and their classification to pathways is scant, functional associations between genes are often inferred from large-scale omics data. Such data types—including protein–protein interactions and gene co-expression networks—are used to examine the interrelations of the implicated genes. Statistical significance is assessed by comparing the interconnectedness of the mutated genes with that of random gene sets. However, interconnectedness can be affected by confounding bias, potentially resulting in false positive findings. We show that genes implicated through de novo sequence variants are biased in their coding-sequence length and longer genes tend to cluster together, which leads to exaggerated p-values in functional studies; we present here an integrative method that addresses these bias. To discern molecular pathways relevant to complex disease, we have inferred functional associations between human genes from diverse data types and assessed them with a novel phenotype-based method. Examining the functional association between de novo gene variants, we control for the heretofore unexplored confounding bias in coding-sequence length. We test different data types and networks and find that the disease-associated genes cluster more significantly in an integrated phenotypic-linkage network than in other gene networks. We present a tool of superior power to identify functional associations among genes mutated in the same disease even after accounting for significant sequencing study bias and demonstrate the suitability of this method to functionally cluster variant genes underlying polygenic disorders. PMID:25166029

  19. The SmgGDS splice variant SmgGDS-558 is a key promoter of tumor growth and RhoA signaling in breast cancer

    PubMed Central

    Hauser, Andrew D.; Bergom, Carmen; Schuld, Nathan J.; Chen, Xiuxu; Lorimer, Ellen L.; Huang, Jian; Mackinnon, Alexander C.; Williams, Carol L.

    2014-01-01

    Breast cancer malignancy is promoted by the small GTPases RhoA and RhoC. SmgGDS is a guanine nucleotide exchange factor that activates RhoA and RhoC in vitro. We previously reported that two splice variants of SmgGDS, SmgGDS-607 and SmgGDS-558, have different characteristics in binding and transport of small GTPases. To define the role of SmgGDS in breast cancer, we tested the expression of SmgGDS in breast tumors, and the role of each splice variant in proliferation, tumor growth, Rho activation, and NF-κB transcriptional activity in breast cancer cells. We show upregulated SmgGDS protein expression in breast cancer samples compared to normal breast tissue. Additionally, Kaplan-Meier survival curves indicated that patients with high SmgGDS expression in their tumors had worse clinical outcomes. Knockdown of SmgGDS-558, but not SmgGDS-607, in breast cancer cells decreased proliferation, in vivo tumor growth, and RhoA activity. Futhermore, we found that SmgGDS promoted a Rho-dependent activation of the transcription factor NF-κB, which provides a potential mechanism to define how SmgGDS-mediated activation of RhoA promotes breast cancer. This study demonstrates that elevated SmgGDS expression in breast tumors correlates with poor survival, and that SmgGDS-558 plays a functional role in breast cancer malignancy. Taken together, these findings define SmgGDS-558 as a unique promoter of RhoA and NF-κB activity and a novel therapeutic target in breast cancer. PMID:24197117

  20. Hypomorphic MGAT5 polymorphisms promote multiple sclerosis cooperatively with MGAT1 and interleukin-2 and 7 receptor variants

    PubMed Central

    Li, Carey F.; Zhou, Raymond W.; Mkhikian, Haik; Newton, Barbara L.; Yu, Zhaoxia; Demetriou, Michael

    2014-01-01

    Deficiency of the Golgi N-glycan branching enzyme Mgat5 in mice promotes T cell hyperactivity, endocytosis of CTLA-4 and autoimmunity, including a spontaneous multiple sclerosis (MS)-like disease. Multiple genetic and environmental MS risk factors lower N-glycan branching in T cells. These include variants in interleukin-2 receptor-α (IL2RA), interleukin-7 receptor-α (IL7RA), and MGAT1, a Golgi branching enzyme upstream of MGAT5, as well as vitamin D3 deficiency and Golgi substrate metabolism. Here we describe linked intronic variants of MGAT5 that are associated with reduced N-glycan branching, CTLA-4 surface expression and MS (p = 5.79 × 10−9, n = 7,741), the latter additive with the MGAT1, IL2RA and IL7RA MS risk variants (p = 1.76 × 10−9, OR = 0.67−1.83, n = 3,518). PMID:23351704

  1. Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

    PubMed Central

    Shotton, David M.; Attaran, Amir

    1998-01-01

    Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity. PMID:9861010

  2. Multiple loss-of-function variants of taste receptors in modern humans.

    PubMed

    Fujikura, Kohei

    2015-01-01

    Despite recent advances in the knowledge of interindividual taste differences, the underlying genetic backgrounds have remained to be fully elucidated. Much of the taste variation among different mammalian species can be explained by pseudogenization of taste receptors. Here I investigated whether the most recent disruptions of taste receptor genes segregate with their intact forms in modern humans by analyzing 14 ethnically diverse populations. The results revealed an unprecedented prevalence of 25 segregating loss-of-function (LoF) taste receptor variants, identifying one of the most pronounced cases of functional population diversity in the human genome. LoF variant frequency in taste receptors (2.10%) was considerably higher than the overall LoF frequency in human genome (0.16%). In particular, molecular evolutionary rates of candidate sour (14.7%) and bitter (1.8%) receptors were far higher in humans than those of sweet (0.02%), salty (0.05%), and umami (0.17%) receptors compared with other carnivorous mammals, although not all of the taste receptors were identified. Many LoF variants are population-specific, some of which arose even after population differentiation, not before divergence of the modern and archaic human. I conclude that modern humans might have been losing some sour and bitter receptor genes because of high-frequency LoF variants. PMID:26307445

  3. Multiple loss-of-function variants of taste receptors in modern humans

    PubMed Central

    Fujikura, K.

    2015-01-01

    Despite recent advances in the knowledge of interindividual taste differences, the underlying genetic backgrounds have remained to be fully elucidated. Much of the taste variation among different mammalian species can be explained by pseudogenization of taste receptors. Here I investigated whether the most recent disruptions of taste receptor genes segregate with their intact forms in modern humans by analyzing 14 ethnically diverse populations. The results revealed an unprecedented prevalence of 25 segregating loss-of-function (LoF) taste receptor variants, identifying one of the most pronounced cases of functional population diversity in the human genome. LoF variant frequency in taste receptors (2.10%) was considerably higher than the overall LoF frequency in human genome (0.16%). In particular, molecular evolutionary rates of candidate sour (14.7%) and bitter (1.8%) receptors were far higher in humans than those of sweet (0.02%), salty (0.05%), and umami (0.17%) receptors compared with other carnivorous mammals, although not all of the taste receptors were identified. Many LoF variants are population-specific, some of which arose even after population differentiation, not before divergence of the modern and archaic human. I conclude that modern humans might have been losing some sour and bitter receptor genes because of high-frequency LoF variants. PMID:26307445

  4. A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src.

    PubMed Central

    Nori, M; Shawver, L K; Weber, M J

    1990-01-01

    To study the relationship between oncogenesis by v-src and normal cellular signalling pathways, we determined the effects of v-src on 3T3-TNR9 cells, a Swiss 3T3 variant which does not respond mitogenically to tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We found that src was unable to transform these variant cells, whether the oncogene was introduced by infection with a murine retrovirus vector or by transfection with plasmid DNA. 3T3-TNR9 cells were not inherently resistant to transformation, since infection with similar recombinant retroviruses containing either v-ras or v-abl did induce transformation. Further analysis of Swiss 3T3 and 3T3-TNR9 cell populations infected with the v-src-containing retrovirus revealed that although the amount of v-src DNA in each was approximately the same, the level of the v-src message and protein and the overall level of phosphotyrosine expressed in the infected variants was much less than in infected parental cells. Cotransfection experiments using separate v-src and neo plasmids revealed a decrease in the number of G418-resistant colonies when transfections of TNR9 cells occurred in the presence of the src-containing plasmid, suggesting a growth inhibitory effect of v-src on 3T3-TNR9 cells, as has also been found for TPA itself. Since v-src cannot transform this variant cell line, which does not respond mitogenically to the protein kinase C agonist TPA, we suggest that src makes use of the protein kinase C pathway as part of its signalling activities. Images PMID:2115120

  5. A SPECTRAL APPROACH INTEGRATING FUNCTIONAL GENOMIC ANNOTATIONS FOR CODING AND NONCODING VARIANTS

    PubMed Central

    IONITA-LAZA, IULIANA; MCCALLUM, KENNETH; XU, BIN; BUXBAUM, JOSEPH

    2015-01-01

    Over the past few years, substantial effort has been put into the functional annotation of variation in human genome sequence. Such annotations can play a critical role in identifying putatively causal variants among the abundant natural variation that occurs at a locus of interest. The main challenges in using these various annotations include their large numbers, and their diversity. Here we develop an unsupervised approach to integrate these different annotations into one measure of functional importance (Eigen), that, unlike most existing methods, is not based on any labeled training data. We show that the resulting meta-score has better discriminatory ability using disease associated and putatively benign variants from published studies (in both coding and noncoding regions) compared with the recently proposed CADD score. Across varied scenarios, the Eigen score performs generally better than any single individual annotation, representing a powerful single functional score that can be incorporated in fine-mapping studies. PMID:26727659

  6. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  7. Association between promoter region genetic variants of PTH SNPs and serum 25(OH)-vitamin D level

    PubMed Central

    Al-Daghri, Nasser M; Al-Attas, Omar S; Krishnaswamy, Soundararajan; Yakout, Sobhy M; Mohammed, Abdul Khader; Alenad, Amal M; Chrousos, George P; Alokail, Majed S

    2015-01-01

    Parathyroid hormone (PTH) plays a crucial role in calcium metabolism and skeletal development via altering vitamin D level. Besides, hypersecretion of PTH is implicated in the etiology of osteoporosis. In this study, we analyzed association between promoter region sequence variants of PTH gene and circulating 25-hydroxy-vitamin D (25(OH)D) level. Genotypes of PTH SNPs rs1459015, rs10500783 and rs10500784 and circulating serum 25(OH)D level of healthy adults (N=386) of different nationalities living in Riyadh were determined and relation between the different PTH allelic variants and corresponding mean 25(OH)D values were obtained using Analysis of Variance (ANOVA) and Bonferroni post-hoc test for multiple comparisons. We observed a high prevalence of vitamin D deficiency (<50 nmol/l) among all nationals which ranged from 59% among Indians to 82% among Yemeni. Comparison of the means of 25(OH)D levels corresponding to different genotypes of PTH SNPs indicated that the T allele of SNP rs1459015 was associated with higher 25(OH)D level in the Sudanese (P=0.03), while the T allele of SNP rs10500783 was associated with higher 25(OH)D level in Saudis (P=0.03). Analysis of results also indicated that the Sudanese carriers of the CC genotype of SNP rs1459015 had a higher risk of suffering from vitamin D deficiency (P=0.02). In conclusion, our study indicated significant association between specific PTH gene promoter region variants and altered levels of 25(OH)D and vitamin D deficiency among specific nationals. PMID:26339419

  8. A truncated splice variant of human lysyl oxidase-like 2 promotes migration and invasion in esophageal squamous cell carcinoma.

    PubMed

    Zou, Hai-Ying; Lv, Guo-Qing; Dai, Li-Hua; Zhan, Xiu-Hui; Jiao, Ji-Wei; Liao, Lian-Di; Zhou, Tai-Mei; Li, Chun-Quan; Wu, Bing-Li; Xu, Li-Yan; Li, En-Min

    2016-06-01

    Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family, which plays an important role in extracellular matrix protein biosynthesis and tumor progression. In the present study, we identified a novel splice variant, LOXL2Δ72, which encodes a peptide having the same N- and C-termini as wild-type LOXL2 (LOXL2WT), but lacks 72 nucleotides encoding 24 amino acids. LOXL2Δ72 had dramatically reduced enzymatic activity, and was no longer secreted. However, LOXL2Δ72 promoted greater cell migration and invasion than LOXL2WT. Furthermore, a dual luciferase reporter assay indicated that LOXL2Δ72 activates distinct signal transduction pathways compared to LOXL2WT, consistent with cDNA microarray data showing different expression levels of cell migration- and invasion-related genes induced following over-expression of each LOXL2 isoform. In particular, LOXL2Δ72 distinctly promoted esophageal squamous cell carcinoma (ESCC) cell migration via up-regulating the C-C motif chemokine ligand 28 (CCL28). Our results suggest that the new LOXL2 splice variant contributes to tumor progression by novel molecular mechanisms different from LOXL2WT.

  9. Candidate genes and functional noncoding variants identified in a canine model of obsessive-compulsive disorder

    PubMed Central

    2014-01-01

    Background Obsessive-compulsive disorder (OCD), a severe mental disease manifested in time-consuming repetition of behaviors, affects 1 to 3% of the human population. While highly heritable, complex genetics has hampered attempts to elucidate OCD etiology. Dogs suffer from naturally occurring compulsive disorders that closely model human OCD, manifested as an excessive repetition of normal canine behaviors that only partially responds to drug therapy. The limited diversity within dog breeds makes identifying underlying genetic factors easier. Results We use genome-wide association of 87 Doberman Pinscher cases and 63 controls to identify genomic loci associated with OCD and sequence these regions in 8 affected dogs from high-risk breeds and 8 breed-matched controls. We find 119 variants in evolutionarily conserved sites that are specific to dogs with OCD. These case-only variants are significantly more common in high OCD risk breeds compared to breeds with no known psychiatric problems. Four genes, all with synaptic function, have the most case-only variation: neuronal cadherin (CDH2), catenin alpha2 (CTNNA2), ataxin-1 (ATXN1), and plasma glutamate carboxypeptidase (PGCP). In the 2 Mb gene desert between the cadherin genes CDH2 and DSC3, we find two different variants found only in dogs with OCD that disrupt the same highly conserved regulatory element. These variants cause significant changes in gene expression in a human neuroblastoma cell line, likely due to disrupted transcription factor binding. Conclusions The limited genetic diversity of dog breeds facilitates identification of genes, functional variants and regulatory pathways underlying complex psychiatric disorders that are mechanistically similar in dogs and humans. PMID:24995881

  10. In Silico Characterization of Functional Divergence of Two Cathelicidin Variants in Indian Sheep

    PubMed Central

    Dhaliwal, Kamaljeet K; Arora, Jaspreet S; Mukhopadhyay, Chandra S; Dubey, Prem P

    2015-01-01

    The present work focuses on the in silico characterization of functional divergence of two ovine cathelicidin coding sequence (cds) variants (ie, Cath1 and Cath2) of Indian sheep. Overlapping partial cds of both the cathelicidin variants were cloned in pJet1.2/blunt vector and sequenced. Evolutionary analysis of the Cath2 and Cath1 indicated that the mammalian cathelicidins clustered separately from avian fowlicidins. The avian fowlicidins, which are very different from mammalian cathelicidins (Caths), clearly displayed signatures of purifying selection. The pairwise sequence alignments of translated amino acid sequences of these two sheep cathelicidins showed gaps in the antimicrobial domain of Cath1 variant; however, the amino terminal cathelin regions of both the Caths were conserved. Amino acid sequence analysis of full-length cathelicidins available at public database revealed that Cath1, Cath2, and Cath7 of different ruminant species (including our Cath1 and Cath2 variants) formed individual clads, suggesting that these types have evolved to target specific types of microbes. In silico analysis of Cath1 and Cath2 peptide sequences indicated that the C-terminal antimicrobial peptide domain of Cath2 is more immunogenic than that of the ovine Cath1 due to its higher positive antigenic index, making Cath1 a promising antigen for production of monoclonal antibodies. PMID:26380546

  11. Functional characteristics of the Staphylococcus aureus δ-toxin allelic variant G10S.

    PubMed

    Cheung, Gordon Y C; Yeh, Anthony J; Kretschmer, Dorothee; Duong, Anthony C; Tuffuor, Kwame; Fu, Chih-Lung; Joo, Hwang-Soo; Diep, Binh A; Li, Min; Nakamura, Yuumi; Nunez, Gabriel; Peschel, Andreas; Otto, Michael

    2015-12-10

    Staphylococcus aureus δ-toxin is a member of the phenol-soluble modulin (PSM) peptide family. PSMs have multiple functions in staphylococcal pathogenesis; for example, they lyse red and white blood cells and trigger inflammatory responses. Compared to other PSMs, δ-toxin is usually more strongly expressed but has only moderate cytolytic capacities. The amino acid sequences of S. aureus PSMs are well conserved with two exceptions, one of which is the δ-toxin allelic variant G10S. This variant is a characteristic of the subspecies S. argenteus and S. aureus sequence types ST1 and ST59, the latter representing the most frequent cause of community-associated infections in Asia. δ-toxin G10S and strains expressing that variant from plasmids or the genome had significantly reduced cytolytic and pro-inflammatory capacities, including in a strain background with pronounced production of other PSMs. However, in murine infection models, isogenic strains expressing the two δ-toxin variants did not cause measurable differences in disease severity. Our findings indicate that the widespread G10S allelic variation of the δ-toxin locus has a significant impact on key pathogenesis mechanisms, but more potent members of the PSM peptide family may overshadow that impact in vivo.

  12. Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

    PubMed Central

    Vodo, Dan; Sarig, Ofer; Ben-Asher, Edna; Olender, Tsviya; Bochner, Ron; Goldberg, Ilan; Nosgorodsky, Judith; Alkelai, Anna; Tatarskyy, Pavel; Peled, Alon; Baum, Sharon; Barzilai, Aviv; Ibrahim, Saleh M.; Zillikens, Detlef; Lancet, Doron; Sprecher, Eli

    2016-01-01

    Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease caused by disruption of intercellular adhesion due to auto-antibodies directed against epithelial components. Treatment is limited to immunosuppressive agents, which are associated with serious adverse effects. The propensity to develop the disease is in part genetically determined. We therefore reasoned that the delineation of PV genetic basis may point to novel therapeutic strategies. Using a genome-wide association approach, we recently found that genetic variants in the vicinity of the ST18 gene confer a significant risk for the disease. Here, using targeted deep sequencing, we identified a PV-associated variant residing within the ST18 promoter region (p<0.0002; odds ratio = 2.03). This variant was found to drive increased gene transcription in a p53/p63-dependent manner, which may explain the fact that ST18 is up-regulated in the skin of PV patients. We then discovered that when overexpressed, ST18 stimulates PV serum-induced secretion of key inflammatory molecules and contributes to PV serum-induced disruption of keratinocyte cell-cell adhesion, two processes previously implicated in the pathogenesis of PV. Thus, the present findings indicate that ST18 may play a direct role in PV and consequently represents a potential target for the treatment of this disease. PMID:27148741

  13. Kinetoplastid-specific histone variant functions are conserved in Leishmania major.

    PubMed

    Anderson, Britta A; Wong, Iris L K; Baugh, Loren; Ramasamy, Gowthaman; Myler, Peter J; Beverley, Stephen M

    2013-10-01

    Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.

  14. Admixture Mapping in Lupus Identifies Multiple Functional Variants within IFIH1 Associated with Apoptosis, Inflammation, and Autoantibody Production

    PubMed Central

    Looger, Loren L.; Han, Shizhong; Kim-Howard, Xana; Glenn, Stuart; Adler, Adam; Kelly, Jennifer A.; Niewold, Timothy B.; Gilkeson, Gary S.; Brown, Elizabeth E.; Alarcón, Graciela S.; Edberg, Jeffrey C.; Petri, Michelle; Ramsey-Goldman, Rosalind; Reveille, John D.; Vilá, Luis M.; Freedman, Barry I.; Tsao, Betty P.; Criswell, Lindsey A.; Jacob, Chaim O.; Moore, Jason H.; Vyse, Timothy J.; Langefeld, Carl L.; Guthridge, Joel M.; Gaffney, Patrick M.; Moser, Kathy L.; Scofield, R. Hal; Alarcón-Riquelme, Marta E.; Williams, Scott M.; Merrill, Joan T.; James, Judith A.; Kaufman, Kenneth M.; Kimberly, Robert P.; Harley, John B.; Nath, Swapan K.

    2013-01-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis. PMID

  15. Admixture mapping in lupus identifies multiple functional variants within IFIH1 associated with apoptosis, inflammation, and autoantibody production.

    PubMed

    Molineros, Julio E; Maiti, Amit K; Sun, Celi; Looger, Loren L; Han, Shizhong; Kim-Howard, Xana; Glenn, Stuart; Adler, Adam; Kelly, Jennifer A; Niewold, Timothy B; Gilkeson, Gary S; Brown, Elizabeth E; Alarcón, Graciela S; Edberg, Jeffrey C; Petri, Michelle; Ramsey-Goldman, Rosalind; Reveille, John D; Vilá, Luis M; Freedman, Barry I; Tsao, Betty P; Criswell, Lindsey A; Jacob, Chaim O; Moore, Jason H; Vyse, Timothy J; Langefeld, Carl L; Guthridge, Joel M; Gaffney, Patrick M; Moser, Kathy L; Scofield, R Hal; Alarcón-Riquelme, Marta E; Williams, Scott M; Merrill, Joan T; James, Judith A; Kaufman, Kenneth M; Kimberly, Robert P; Harley, John B; Nath, Swapan K

    2013-01-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22-24 (LOD=6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ~1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [P(meta) = 5.20×10(-14); odds ratio, 95% confidence interval = 0.82 (0.78-0.87)], and two missense variants, rs1990760 (Ala946Thr) [P(meta) = 3.08×10(-7); 0.88 (0.84-0.93)] and rs10930046 (Arg460His) [P(dom) = 1.16×10(-8); 0.70 (0.62-0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis. PMID:23441136

  16. Loss-of-Function Variants in Schizophrenia Risk and SETD1A as a Candidate Susceptibility Gene

    PubMed Central

    Takata, Atsushi; Xu, Bin; Ionita-Laza, Iuliana; Roos, J. Louw; Gogos, Joseph A.; Karayiorgou, Maria

    2015-01-01

    SUMMARY Loss-of-function (LOF) (i.e., nonsense, splice site, and frameshift) variants that lead to disruption of gene function are likely to contribute to the etiology of neuropsychiatric disorders. Here, we perform a systematic investigation of the role of both de novo and inherited LOF variants in schizophrenia using exome sequencing data from 231 case and 34 control trios. We identify two de novo LOF variants in the SETD1A gene, which encodes a subunit of his-tone methyltransferase, a finding unlikely to have occurred by chance, and provide evidence for a more general role of chromatin regulators in schizophrenia risk. Transmission pattern analyses reveal that LOF variants are more likely to be transmitted to affected individuals than controls. This is especially true for private LOF variants in genes intolerant to functional genetic variation. These findings highlight the contribution of LOF mutations to the genetic architecture of schizophrenia and provide important insights into disease pathogenesis. PMID:24853937

  17. Loss-of-function variants in schizophrenia risk and SETD1A as a candidate susceptibility gene.

    PubMed

    Takata, Atsushi; Xu, Bin; Ionita-Laza, Iuliana; Roos, J Louw; Gogos, Joseph A; Karayiorgou, Maria

    2014-05-21

    Loss-of-function (LOF) (i.e., nonsense, splice site, and frameshift) variants that lead to disruption of gene function are likely to contribute to the etiology of neuropsychiatric disorders. Here, we perform a systematic investigation of the role of both de novo and inherited LOF variants in schizophrenia using exome sequencing data from 231 case and 34 control trios. We identify two de novo LOF variants in the SETD1A gene, which encodes a subunit of histone methyltransferase, a finding unlikely to have occurred by chance, and provide evidence for a more general role of chromatin regulators in schizophrenia risk. Transmission pattern analyses reveal that LOF variants are more likely to be transmitted to affected individuals than controls. This is especially true for private LOF variants in genes intolerant to functional genetic variation. These findings highlight the contribution of LOF mutations to the genetic architecture of schizophrenia and provide important insights into disease pathogenesis. PMID:24853937

  18. Functional Genetic Variants of TNFSF15 and Their Association with Gastric Adenocarcinoma: A Case-Control Study

    PubMed Central

    Lu, Jie; Zhai, Kan; Cao, Lei; Rao, Juan; Liu, Yingwen; Zhang, Xuemei; Guo, Yongli

    2014-01-01

    The purpose of this study was to identify functional genetic variants in the promoter of tumor necrosis factor superfamily member 15 (TNFSF15) and evaluate their effects on the risk of developing gastric adenocarcinoma. Forty DNA samples from healthy volunteers were sequenced to identify single nucleotide polymorphisms (SNPs) in the TNFSF15 promoter. Two TNFSF15 SNPs (−358T>C and −638A>G) were identified by direct sequencing. Next, genotypes and haplotypes of 470 gastric adenocarcinoma patients and 470 cancer-free controls were analyzed. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Serologic tests for Helicobacter pylori infection were measured by enzyme-linked immuno-sorbent assay (ELISA). Subjects carrying the TNFSF15 −358CC genotype were at an elevated risk for developing gastric adenocarcinoma, compared with those with the −358TT genotype (OR 1.42, 95% CI, 1.10 to 2.03). H. pylori infection was a risk factor for developing gastric adenocarcinoma (OR 2.31, 95% CI, 1.76 to 3.04). In the H. pylori infected group, subjects with TNFSF15 −358CC genotype were at higher risks for gastric adenocarcinoma compared with those carrying −358TT genotype (OR: 2.01, 95%CI: 1.65 to 4.25), indicating that H. pylori infection further influenced gastric adenocarcinoma susceptibility. The −358 T>C polymorphism eliminates a nuclear factor Y (NF-Y) binding site and the −358C containing haplotypes showed significantly decreased luciferase expression compared with −358T containing haplotypes. Collectively these findings indicate that functional genetic variants in TNFSF15 may play a role in increasing susceptibility to gastric adenocarcinoma. PMID:25251497

  19. Functional testing strategy for coding genetic variants of unclear significance in MLH1 in Lynch syndrome diagnosis.

    PubMed

    Hinrichsen, Inga; Schäfer, Dieter; Langer, Deborah; Köger, Nicole; Wittmann, Margarethe; Aretz, Stefan; Steinke, Verena; Holzapfel, Stefanie; Trojan, Jörg; König, Rainer; Zeuzem, Stefan; Brieger, Angela; Plotz, Guido

    2015-02-01

    Lynch syndrome is caused by inactivating mutations in the MLH1 gene, but genetic variants of unclear significance frequently preclude diagnosis. Functional testing can reveal variant-conferred defects in gene or protein function. Based on functional defect frequencies and clinical applicability of test systems, we developed a functional testing strategy aimed at efficiently detecting pathogenic defects in coding MLH1 variants. In this strategy, tests of repair activity and expression are prioritized over analyses of subcellular protein localization and messenger RNA (mRNA) formation. This strategy was used for four unclear coding MLH1 variants (p.Asp41His, p.Leu507Phe, p.Gln689Arg, p.Glu605del + p.Val716Met). Expression was analyzed using a transfection system, mismatch repair (MMR) activity by complementation in vitro, mRNA formation by reverse transcriptase-PCR in carrier lymphocyte mRNA, and subcellular localization with dye-labeled fusion constructs. All tests included clinically meaningful controls. The strategy enabled efficient identification of defects in two unclear variants: the p.Asp41His variant showed loss of MMR activity, whereas the compound variant p.Glu605del + p.Val716Met had a defect of expression. This expression defect was significantly stronger than the pathogenic expression reference variant analyzed in parallel, therefore the defect of the compound variant is also pathogenic. Interestingly, the expression defect was caused additively by both of the compound variants, at least one of which is non-pathogenic when occurring by itself. Tests were neutral for p.Leu507Phe and p.Gln689Arg, and the results were consistent with available clinical data. We finally discuss the improved sensitivity and efficiency of the applied strategy and its limitations in analyzing unclear coding MLH1 variants.

  20. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  1. Associations of Filaggrin Gene Loss-of-Function Variants with Urinary Phthalate Metabolites and Testicular Function in Young Danish Men

    PubMed Central

    Jørgensen, Niels; Meldgaard, Michael; Frederiksen, Hanne; Andersson, Anna-Maria; Menné, Torkil; Johansen, Jeanne Duus; Carlsen, Berit Christina; Stender, Steen; Szecsi, Pal Bela; Skakkebæk, Niels Erik; De Meyts, Ewa Rajpert; Thyssen, Jacob P.

    2014-01-01

    Background: Filaggrin is an epidermal protein that is crucial for skin barrier function. Up to 10% of Europeans and 5% of Asians carry at least one null allele in the filaggrin gene (FLG). Reduced expression of filaggrin in carriers of the null allele is associated with facilitated transfer of allergens across the epidermis. We hypothesized that these individuals may have increased transdermal uptake of endocrine disruptors, including phthalates. Objectives: We investigated urinary excretion of phthalate metabolites and testicular function in young men with and without FLG loss-of-function variants in a cross-sectional study of 861 young men from the general Danish population. Methods: All men were genotyped for FLG R501X, 2282del4, and R2447X loss-of-function variants. We measured urinary concentrations of 14 phthalate metabolites and serum levels of reproductive hormones. We also evaluated semen quality. Results: Sixty-five men (7.5%) carried at least one FLG-null allele. FLG-null carriers had significantly higher urinary concentrations of several phthalate metabolites, including a 33% higher concentration of MnBP (mono-n-butyl phthalate; 95% CI: 16, 51%). FLG-null variants were not significantly associated with reproductive hormones or semen quality parameters. Conclusion: This study provides evidence that carriers of FLG loss-of-function alleles may have higher internal exposure to phthalates, possibly due to increased transepidermal absorption. FLG loss-of-function variants may indicate susceptible populations for which special attention to transepidermal absorption of chemicals and medication may be warranted. Citation: Joensen UN, Jørgensen N, Meldgaard M, Frederiksen H, Andersson AM, Menné T, Johansen JD, Carlsen BC, Stender S, Szecsi PB, Skakkebæk NE, Rajpert-De Meyts E, Thyssen JP. 2014. Associations of filaggrin gene loss-of-function variants with urinary phthalate metabolites and testicular function in young Danish men. Environ Health Perspect 122

  2. Common variants in Mendelian kidney disease genes and their association with renal function.

    PubMed

    Parsa, Afshin; Fuchsberger, Christian; Köttgen, Anna; O'Seaghdha, Conall M; Pattaro, Cristian; de Andrade, Mariza; Chasman, Daniel I; Teumer, Alexander; Endlich, Karlhans; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Kim, Young J; Taliun, Daniel; Li, Man; Feitosa, Mary; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C; Glazer, Nicole; Isaacs, Aaron; Rao, Madhumathi; Smith, Albert V; O'Connell, Jeffrey R; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Hwang, Shih-Jen; Atkinson, Elizabeth J; Lohman, Kurt; Cornelis, Marilyn C; Johansson, Asa; Tönjes, Anke; Dehghan, Abbas; Couraki, Vincent; Holliday, Elizabeth G; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B; Launer, Lenore J; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D; Boerwinkle, Eric; Schmidt, Helena; Hofer, Edith; Hu, Frank; Demirkan, Ayse; Oostra, Ben A; Turner, Stephen T; Ding, Jingzhong; Andrews, Jeanette S; Freedman, Barry I; Giulianini, Franco; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H-Erich; Zgaga, Lina; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H; Wright, Alan F; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G; Rivadeneira, Fernando; Aulchenko, Yurii S; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Stengel, Bénédicte; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Krämer, Bernhard K; Portas, Laura; Ford, Ian; Buckley, Brendan M; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Mitchell, Paul; Ciullo, Marina; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J Wouter; Probst-Hensch, Nicole M; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; van Duijn, Cornelia M; Borecki, Ingrid; Kardia, Sharon L R; Liu, Yongmei; Curhan, Gary C; Rudan, Igor; Gyllensten, Ulf; Wilson, James F; Franke, Andre; Pramstaller, Peter P; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline; Hayward, Caroline; Ridker, Paul M; Bochud, Murielle; Heid, Iris M; Siscovick, David S; Fox, Caroline S; Kao, W Linda; Böger, Carsten A

    2013-12-01

    Many common genetic variants identified by genome-wide association studies for complex traits map to genes previously linked to rare inherited Mendelian disorders. A systematic analysis of common single-nucleotide polymorphisms (SNPs) in genes responsible for Mendelian diseases with kidney phenotypes has not been performed. We thus developed a comprehensive database of genes for Mendelian kidney conditions and evaluated the association between common genetic variants within these genes and kidney function in the general population. Using the Online Mendelian Inheritance in Man database, we identified 731 unique disease entries related to specific renal search terms and confirmed a kidney phenotype in 218 of these entries, corresponding to mutations in 258 genes. We interrogated common SNPs (minor allele frequency >5%) within these genes for association with the estimated GFR in 74,354 European-ancestry participants from the CKDGen Consortium. However, the top four candidate SNPs (rs6433115 at LRP2, rs1050700 at TSC1, rs249942 at PALB2, and rs9827843 at ROBO2) did not achieve significance in a stage 2 meta-analysis performed in 56,246 additional independent individuals, indicating that these common SNPs are not associated with estimated GFR. The effect of less common or rare variants in these genes on kidney function in the general population and disease-specific cohorts requires further research.

  3. Common Variants in Mendelian Kidney Disease Genes and Their Association with Renal Function

    PubMed Central

    Fuchsberger, Christian; Köttgen, Anna; O’Seaghdha, Conall M.; Pattaro, Cristian; de Andrade, Mariza; Chasman, Daniel I.; Teumer, Alexander; Endlich, Karlhans; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Kim, Young J.; Taliun, Daniel; Li, Man; Feitosa, Mary; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C.; Glazer, Nicole; Isaacs, Aaron; Rao, Madhumathi; Smith, Albert V.; O’Connell, Jeffrey R.; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Hwang, Shih-Jen; Atkinson, Elizabeth J.; Lohman, Kurt; Cornelis, Marilyn C.; Johansson, Åsa; Tönjes, Anke; Dehghan, Abbas; Couraki, Vincent; Holliday, Elizabeth G.; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y.; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B.; Launer, Lenore J.; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D.; Boerwinkle, Eric; Schmidt, Helena; Hofer, Edith; Hu, Frank; Demirkan, Ayse; Oostra, Ben A.; Turner, Stephen T.; Ding, Jingzhong; Andrews, Jeanette S.; Freedman, Barry I.; Giulianini, Franco; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H.-Erich; Zgaga, Lina; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E.; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H.; Wright, Alan F.; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K.; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G.; Rivadeneira, Fernando; Aulchenko, Yurii S.; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Stengel, Bénédicte; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Krämer, Bernhard K.; Portas, Laura; Ford, Ian; Buckley, Brendan M.; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Mitchell, Paul; Ciullo, Marina; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J. Wouter; Probst-Hensch, Nicole M.; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R.; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; van Duijn, Cornelia M.; Borecki, Ingrid; Kardia, Sharon L.R.; Liu, Yongmei; Curhan, Gary C.; Rudan, Igor; Gyllensten, Ulf; Wilson, James F.; Franke, Andre; Pramstaller, Peter P.; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline; Hayward, Caroline; Ridker, Paul M.; Bochud, Murielle; Heid, Iris M.; Siscovick, David S.; Fox, Caroline S.; Kao, W. Linda; Böger, Carsten A.

    2013-01-01

    Many common genetic variants identified by genome-wide association studies for complex traits map to genes previously linked to rare inherited Mendelian disorders. A systematic analysis of common single-nucleotide polymorphisms (SNPs) in genes responsible for Mendelian diseases with kidney phenotypes has not been performed. We thus developed a comprehensive database of genes for Mendelian kidney conditions and evaluated the association between common genetic variants within these genes and kidney function in the general population. Using the Online Mendelian Inheritance in Man database, we identified 731 unique disease entries related to specific renal search terms and confirmed a kidney phenotype in 218 of these entries, corresponding to mutations in 258 genes. We interrogated common SNPs (minor allele frequency >5%) within these genes for association with the estimated GFR in 74,354 European-ancestry participants from the CKDGen Consortium. However, the top four candidate SNPs (rs6433115 at LRP2, rs1050700 at TSC1, rs249942 at PALB2, and rs9827843 at ROBO2) did not achieve significance in a stage 2 meta-analysis performed in 56,246 additional independent individuals, indicating that these common SNPs are not associated with estimated GFR. The effect of less common or rare variants in these genes on kidney function in the general population and disease-specific cohorts requires further research. PMID:24029420

  4. A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation

    PubMed Central

    Small, Donna M; Zani, Marie-Louise; Quinn, Derek J; Dallet-Choisy, Sandrine; Glasgow, Arlene MA; O'Kane, Cecilia; McAuley, Danny F; McNally, Paul; Weldon, Sinéad; Moreau, Thierry; Taggart, Clifford C

    2015-01-01

    Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden. PMID:25189740

  5. Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations.

    PubMed

    Chang, Suhwan; Biswas, Kajal; Martin, Betty K; Stauffer, Stacey; Sharan, Shyam K

    2009-10-01

    To date, inheritance of a mutant BRCA1 or BRCA2 gene is the best-established indicator of an increased risk of developing breast cancer. Sequence analysis of these genes is being used to identify BRCA1/2 mutation carriers, though these efforts are hampered by the high frequency of variants of unknown clinical significance (VUSs). Functional evaluation of such variants has been restricted due to lack of a physiologically relevant assay. In this study we developed a functional assay using mouse ES cells to study variants of BRCA1. We introduced BAC clones with human wild-type BRCA1 or variants into Brca1-null ES cells and confirmed that only wild-type and a known neutral variant rescued cell lethality. The same neutral variant was also able to rescue embryogenesis in Brca1-null mice. A test of several BRCT domain mutants revealed all to be deleterious, including a VUS. Furthermore, we used this assay to determine the effects of BRCA1 variants on cell cycle regulation, differentiation, and genomic stability. Importantly, we discovered that ES cells rescued by S1497A BRCA1 exhibited significant hypersensitivity after gamma-irradiation. Our results demonstrate that this ES cell-based assay is a powerful and reliable method for analyzing the functional impact of BRCA1 variants, which we believe could be used to determine which patients may require preventative treatments. PMID:19770520

  6. Genome-Wide Functional Annotation of Human Protein-Coding Splice Variants Using Multiple Instance Learning.

    PubMed

    Panwar, Bharat; Menon, Rajasree; Eksi, Ridvan; Li, Hong-Dong; Omenn, Gilbert S; Guan, Yuanfang

    2016-06-01

    The vast majority of human multiexon genes undergo alternative splicing and produce a variety of splice variant transcripts and proteins, which can perform different functions. These protein-coding splice variants (PCSVs) greatly increase the functional diversity of proteins. Most functional annotation algorithms have been developed at the gene level; the lack of isoform-level gold standards is an important intellectual limitation for currently available machine learning algorithms. The accumulation of a large amount of RNA-seq data in the public domain greatly increases our ability to examine the functional annotation of genes at isoform level. In the present study, we used a multiple instance learning (MIL)-based approach for predicting the function of PCSVs. We used transcript-level expression values and gene-level functional associations from the Gene Ontology database. A support vector machine (SVM)-based 5-fold cross-validation technique was applied. Comparatively, genes with multiple PCSVs performed better than single PCSV genes, and performance also improved when more examples were available to train the models. We demonstrated our predictions using literature evidence of ADAM15, LMNA/C, and DMXL2 genes. All predictions have been implemented in a web resource called "IsoFunc", which is freely available for the global scientific community through http://guanlab.ccmb.med.umich.edu/isofunc . PMID:27142340

  7. Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles | Office of Cancer Genomics

    Cancer.gov

    Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic.

  8. Effects of genetic variants in the promoter region of the bovine adiponectin (ADIPOQ) gene on marbling of Hanwoo beef cattle.

    PubMed

    Choi, Yoonjeong; Davis, Michael E; Chung, Hoyoung

    2015-07-01

    This study aimed to verify genetic effects of the bovine adiponectin (ADIPOQ) gene on carcass traits of Hanwoo cattle. The measured carcass traits were marbling score (MAR), backfat thickness (BFT), loineye area (LEA), and carcass weight (CAW). Selection of primers was based on the bovine ADIPOQ sequence, and the analysis amplified approximately 267 and 333 bp genomic segments, including 67 bp of insertions in the promoter region. Sequencing analysis confirmed genetic variants (g.81966235C>T, g.81966377T>C, and g.81966364D>I) that showed significant effects on MAR. The present results suggest that the identified SNPs are useful genetic markers for the improvement of carcass traits in Hanwoo cattle.

  9. Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts

    PubMed Central

    Bell, Daphne W.; Kim, Sang H.; Godwin, Andrew K.; Schiripo, Taryn A.; Harris, Patricia L.; Haserlat, Sara M.; Wahrer, Doke C.R.; Haiman, Christopher A.; Daly, Mary B.; Niendorf, Kristin B.; Smith, Matthew R.; Sgroi, Dennis C.; Garber, Judy E.; Olopade, Olufunmilayo I.; Marchand, Loic Le; Henderson, Brian E.; Altshuler, David; Haber, Daniel A.; Freedman, Matthew L.

    2011-01-01

    The CHEK2-1100delC mutation is recurrent in the population and is a moderate risk factor for breast cancer. To identify additional CHEK2 mutations potentially contributing to breast cancer susceptibility, we sequenced 248 cases with early-onset disease; functionally characterized new variants and conducted a population-based case–control analysis to evaluate their contribution to breast cancer risk. We identified 1 additional null mutation and 5 missense variants in the germline of cancer patients. In vitro, the CHEK2-H143Y variant resulted in gross protein destabilization, while others had variable suppression of in vitro kinase activity using BRCA1 as a substrate. The germline CHEK2-1100delC mutation was present among 8/1,646 (0.5%) sporadic, 2/400 (0.5%) early-onset and 3/302 (1%) familial breast cancer cases, but undetectable amongst 2,105 multiethnic controls, including 633 from the US. CHEK2-positive breast cancer families also carried a deleterious BRCA1 mutation. 1100delC appears to be the only recurrent CHEK2 mutation associated with a potentially significant contribution to breast cancer risk in the general population. Another recurrent mutation with attenuated in vitro function, CHEK2-P85L, is not associated with increased breast cancer susceptibility, but exhibits a striking difference in frequency across populations with different ancestral histories. These observations illustrate the importance of genotyping ethnically diverse groups when assessing the impact of low-penetrance susceptibility alleles on population risk. Our findings highlight the notion that clinical testing for rare missense mutations within CHEK2 may have limited value in predicting breast cancer risk, but that testing for the 1100delC variant may be valuable in phenotypically- and geographically-selected populations. PMID:17721994

  10. A genetic variant in the LDLR promoter is responsible for part of the LDL-cholesterol variability in primary hypercholesterolemia

    PubMed Central

    2014-01-01

    Background GWAS have consistently revealed that LDLR locus variability influences LDL-cholesterol in general population. Severe LDLR mutations are responsible for familial hypercholesterolemia (FH). However, most primary hypercholesterolemias are polygenic diseases. Although Cis-regulatory regions might be the cause of LDL-cholesterol variability; an extensive analysis of the LDLR distal promoter has not yet been performed. We hypothesized that genetic variants in this region are responsible for the LDLR association with LDL-cholesterol found in GWAS. Methods Four-hundred seventy-seven unrelated subjects with polygenic hypercholesterolemia (PH) and without causative FH-mutations and 525 normolipemic subjects were selected. A 3103 pb from LDLR (-625 to +2468) was sequenced in 125 subjects with PH. All subjects were genotyped for 4 SNPs (rs17242346, rs17242739, rs17248720 and rs17249120) predicted to be potentially involved in transcription regulation by in silico analysis. EMSA and luciferase assays were carried out for the rs17248720 variant. Multivariable linear regression analysis using LDL-cholesterol levels as the dependent variable were done in order to find out the variables that were independently associated with LDL-cholesterol. Results The sequencing of the 125 PH subjects did not show variants with minor allele frequency ≥ 10%. The T-allele from g.3131C > T (rs17248720) had frequencies of 9% (PH) and 16.4% (normolipemic), p < 0.00001. Studies of this variant with EMSA and luciferase assays showed a higher affinity for transcription factors and an increase of 2.5 times in LDLR transcriptional activity (T-allele vs C-allele). At multivariate analysis, this polymorphism with the lipoprotein(a) and age explained ≈ 10% of LDL-cholesterol variability. Conclusion Our results suggest that the T-allele at the g.3131 T > C SNP is associated with LDL-cholesterol levels, and explains part of the LDL-cholesterol variability. As a plausible

  11. Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

    PubMed

    Wobst, Jana; von Ameln, Simon; Wolf, Bernhard; Wierer, Michael; Dang, Tan An; Sager, Hendrik B; Tennstedt, Stephanie; Hengstenberg, Christian; Koesling, Doris; Friebe, Andreas; Braun, Siegmund L; Erdmann, Jeanette; Schunkert, Heribert; Kessler, Thorsten

    2016-07-01

    Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the β1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the β1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants. PMID:27342234

  12. Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene.

    PubMed

    Mercimek-Mahmutoglu, Saadet; Ndika, Joseph; Kanhai, Warsha; de Villemeur, Thierry Billette; Cheillan, David; Christensen, Ernst; Dorison, Nathalie; Hannig, Vickie; Hendriks, Yvonne; Hofstede, Floris C; Lion-Francois, Laurence; Lund, Allan M; Mundy, Helen; Pitelet, Gaele; Raspall-Chaure, Miquel; Scott-Schwoerer, Jessica A; Szakszon, Katalin; Valayannopoulos, Vassili; Williams, Monique; Salomons, Gajja S

    2014-04-01

    Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients, 50 different mutations in the GAMT gene have been identified with missense variants being the most common. Clinical and biochemical features of the patients with missense variants were obtained from their physicians using a questionnaire. In 20 patients, 17 missense variants, 25% had a severe, 55% a moderate, and 20% a mild phenotype. The effect of these variants on GAMT enzyme activity was overexpressed using primary GAMT-D fibroblasts: 17 variants retained no significant activity and are therefore considered pathogenic. Two additional variants, c.22C>A (p.Pro8Thr) and c.79T>C (p.Tyr27His) (the latter detected in control cohorts) are in fact not pathogenic as these alleles restored GAMT enzyme activity, although both were predicted to be possibly damaging by in silico analysis. We report 13 new patients with GAMT-D, six novel mutations and functional analysis of 19 missense variants, all being included in our public LOVD database. Our functional assay is important for the confirmation of the pathogenicity of identified missense variants in the GAMT gene. PMID:24415674

  13. Incorporating predicted functions of nonsynonymous variants into gene-based analysis of exome sequencing data: a comparative study

    PubMed Central

    2011-01-01

    Next-generation sequencing has opened up new avenues for the genetic study of complex traits. However, because of the small number of observations for any given rare allele and high sequencing error, it is a challenge to identify functional rare variants associated with the phenotype of interest. Recent research shows that grouping variants by gene and incorporating computationally predicted functions of variants may provide higher statistical power. On the other hand, many algorithms are available for predicting the damaging effects of nonsynonymous variants. Here, we use the simulated mini-exome data of Genetic Analysis Workshop 17 to study and compare the effects of incorporating the functional predictions of single-nucleotide polymorphisms using two popular algorithms, SIFT and PolyPhen-2, into a gene-based association test. We also propose a simple mixture model that can effectively combine test results based on different functional prediction algorithms. PMID:22373178

  14. PTPN22 R620W functional variant in type 1 diabetes and autoimmunity related traits.

    PubMed

    Chelala, Claude; Duchatelet, Sabine; Joffret, Marie-Line; Bergholdt, Regine; Dubois-Laforgue, Danièle; Ghandil, Pegah; Pociot, Flemming; Caillat-Zucman, Sophie; Timsit, José; Julier, Cécile

    2007-02-01

    The PTPN22 gene, encoding the lymphoid-specific protein tyrosine phosphatase, a negative regulator in the T-cell activation and development, has been associated with the susceptibility to several autoimmune diseases, including type 1 diabetes. Based on combined case-control and family-based association studies, we replicated the finding of an association of the PTPN22 C1858T (R620W) functional variant with type 1 diabetes, which was independent from the susceptibility status at the insulin gene and at HLA-DR (DR3/4 compared with others). The risk contributed by the 1858T allele was increased in patients with a family history of other autoimmune diseases, further supporting a general role for this variant on autoimmunity. In addition, we found evidence for an association of 1858T allele with the presence of GAD autoantibodies (GADA), which was restricted to patients with long disease duration (>10 years, P < 0.001). This may help define a subgroup of patients with long-term persistence of GADA. The risk conferred by 1858T allele on GAD positivity was additive, and our meta-analysis also supported an additive rather than dominant effect of this variant on type 1 diabetes, similar to previous reports on rheumatoid arthritis and systemic lupus erythematosus. PMID:17259401

  15. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks.

    PubMed

    Rozacky, Jenna; Nemec, Antoni A; Sweasy, Joann B; Kidane, Dawit

    2015-09-15

    DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  16. CAUSEL: An epigenome and genome editing pipeline for establishing function of non-coding GWAS variants

    PubMed Central

    Spisak, Sandor; Lawrenson, Kate; Fu, Yanfang; Csabai, Istvan; Cottman, Rebecca T.; Haiman, Christopher; Han, Ying; Seo, Ji-Heui; Lenci, Romina; Li, Qiyuan; Tisza, Viktoria; Szallasi, Zoltan; Herbert, Zachery T.; Chabot, Matthew; Pomerantz, Mark; Solymosi, Norbert; Gayther, Simon; Joung, J. Keith; Freedman, Matthew L.

    2016-01-01

    The vast majority of disease-associated single nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWAS) are located in the non-protein coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here, we describe a general pipeline in which candidate functional SNPs are first evaluated by fine-mapping, epigenomic profiling, and epigenome editing and then interrogated for causal function by using genome editing to create isogenic cell lines. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top scoring SNP. Epigenome editing confirmed that rs339331 possessed regulatory potential. Using transcription activator-like effector nuclease (TALEN)-mediated genome-editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the “T” risk allele increased transcription of the RFX6 gene, increased HOXB13 binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach to establish functional causality for non-coding sequence variants identified by GWAS. PMID:26398868

  17. Promoting Executive Function in the Classroom

    ERIC Educational Resources Information Center

    Meltzer, Lynn

    2010-01-01

    Accessible and practical, this book helps teachers incorporate executive function processes--such as planning, organizing, prioritizing, and self-checking--into the classroom curriculum. Chapters provide effective strategies for optimizing what K-12 students learn by improving how they learn. Noted authority Lynn Meltzer and her research…

  18. Promoting Efficacy Research on Functional Analytic Psychotherapy

    ERIC Educational Resources Information Center

    Maitland, Daniel W. M.; Gaynor, Scott T.

    2012-01-01

    Functional Analytic Psychotherapy (FAP) is a form of therapy grounded in behavioral principles that utilizes therapist reactions to shape target behavior. Despite a growing literature base, there is a paucity of research to establish the efficacy of FAP. As a general approach to psychotherapy, and how the therapeutic relationship produces change,…

  19. Rs6295 promoter variants of the serotonin type 1A receptor are differentially activated by c-Jun in vitro and correlate to transcript levels in human epileptic brain tissue.

    PubMed

    Pernhorst, Katharina; van Loo, Karen M J; von Lehe, Marec; Priebe, Lutz; Cichon, Sven; Herms, Stefan; Hoffmann, Per; Helmstaedter, Christoph; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2013-03-01

    Many brain disorders, including epilepsy, migraine and depression, manifest with episodic symptoms that may last for various time intervals. Transient alterations of neuronal function such as related to serotonin homeostasis generally underlie this phenomenon. Several nucleotide polymorphisms (SNPs) in gene promoters associated with these diseases have been described. For obvious reasons, their regulatory roles on gene expression particularly in human brain tissue remain largely enigmatic. The rs6295 G-/C-allelic variant is located in the promoter region of the human HTR1a gene, encoding the G-protein-coupled receptor for 5-hydroxytryptamine (5HT1AR). In addition to reported transcriptional repressor binding, our bioinformatic analyses predicted a reduced binding affinity of the transcription factor (TF) c-Jun for the G-allele. In vitro luciferase transfection assays revealed c-Jun to (a) activate the rs6295 C- significantly stronger than the G-allelic variant and (b) antagonize efficiently the repressive effect of Hes5 on the promoter. The G-allele of rs6295 is known to be associated with aspects of major depression and migraine. In order to address a potential role of rs6295 variants in human brain tissue, we have isolated DNA and mRNA from fresh frozen hippocampal tissue of pharmacoresistant temporal lobe epilepsy (TLE) patients (n=140) after epilepsy surgery for seizure control. We carried out SNP genotyping studies and mRNA analyses in order to determine HTR1a mRNA expression in human hippocampal samples stratified according to the rs6295 allelic variant. The mRNA expression of HTR1a was significantly more abundant in hippocampal mRNA of TLE patients homozygous for the rs6295 C-allele as compared to those with the GG-genotype. These data may point to a novel, i.e., rs6295 allelic variant and c-Jun dependent transcriptional 5HT1AR 'receptoropathy'. PMID:23333373

  20. Conceptualizing Parental Autonomy Support: Adolescent Perceptions of Promotion of Independence versus Promotion of Volitional Functioning

    ERIC Educational Resources Information Center

    Soenens, Bart; Vansteenkiste, Maarten; Lens, Willy; Luyckx, Koen; Goossens, Luc; Beyers, Wim; Ryan, Richard M.

    2007-01-01

    In current research on parenting, 2 ways of conceptualizing perceived parental autonomy support can be distinguished. Parental autonomy support can be defined in terms of promotion of independence (PI) or in terms of promotion of volitional functioning (PVF). This study aimed to establish the empirical distinctiveness of both conceptualizations…

  1. SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.

    PubMed

    Bowton, E; Saunders, C; Reddy, I A; Campbell, N G; Hamilton, P J; Henry, L K; Coon, H; Sakrikar, D; Veenstra-VanderWeele, J M; Blakely, R D; Sutcliffe, J; Matthies, H J G; Erreger, K; Galli, A

    2014-10-14

    Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission

  2. Influence of loss of function MC1R variants in genetic susceptibility of familial melanoma in Spain.

    PubMed

    de Torre, Carlos; Garcia-Casado, Zaida; Martínez-Escribano, Jorge A; Botella-Estrada, Rafael; Bañuls, Jose; Oliver, Vicente; Mercader, Pedro; Azaña, Jose M; Frias, Javier; Nagore, Eduardo

    2010-08-01

    We explored the presence of germline alterations in CDK4 exon 2, CDKN2A and MC1R in a hospital-based study of 89 melanoma cases from 89 families with at least two members affected by cutaneous melanoma. A total of 30% of the melanoma kindreds studied were carriers of CDKN2A variants, and three of these variants were known predominant alleles that have been identified earlier in Mediterranean populations (p.G101W, p.V59G and c.358delG). We observed a higher frequency of nonsynonymous MC1R variants in these Spanish melanoma kindreds (72%) with respect to the general population (60%). We observed a higher frequency of nonsynonymous MC1R variants in this Spanish melanoma kindred (72%) respect to general population (60%). A new classification of MC1R variants based on their functional effects over melanocortin-1 receptor, including the dominant-negative effect of some of them in heterozygotes, suggested an association of loss of function MC1R variants and multiple primary melanoma cases from melanoma kindred (odds ratio: 6.07, 95% confidence interval: 1.35-27.20). This study proposes the relevance of loss of function MC1R variants in the risk of melanoma in multiple primary melanoma cases with family history from areas with low melanoma incidence rate. PMID:20539244

  3. Strategies of Functional Foods Promote Sleep in Human Being

    PubMed Central

    Zeng, Yawen; Yang, Jiazhen; Du, Juan; Pu, Xiaoying; Yang, Xiaomen; Yang, Shuming; Yang, Tao

    2014-01-01

    Sleep is a vital segment of life, however, the mechanisms of diet promoting sleep are unclear and are the focus of research. Insomnia is a general sleep disorder and functional foods are known to play a key role in the prevention of insomnia. A number of studies have demonstrated that major insomnia risk factors in human being are less functional foods in dietary. There are higher functional components in functional foods promoting sleep, including tryptophan, GABA, calcium, potassium, melatonin, pyridoxine, L-ornithine and hexadecanoic acid; but wake-promoting neurochemical factors include serotonin, noradrenalin, acetylcholine, histamine, orexin and so on. The factors promoting sleep in human being are the functional foods include barley grass powder, whole grains, maca, panax, Lingzhi, asparagus powder, lettuce, cherry, kiwifruits, walnut, schisandra wine, and milk; Barley grass powder with higher GABA and calcium, as well as potassium is the most ideal functional food promoting sleep, however, the sleep duration for modern humans is associated with food structure of ancient humans. In this review, we put forward possible mechanisms of functional components in foods promoting sleep. Although there is clear relevance between sleep and diet, their molecular mechanisms need to be studied further. PMID:26005400

  4. Identification of a functional variant in the KIF5A-CYP27B1-METTL1-FAM119B locus associated with multiple sclerosis

    PubMed Central

    Alcina, Antonio; Fedetz, Maria; Fernández, Óscar; Saiz, Albert; Izquierdo, Guillermo; Lucas, Miguel; Leyva, Laura; García-León, Juan-Antonio; Abad-Grau, María del Mar; Alloza, Iraide; Antigüedad, Alfredo; Garcia-Barcina, María J; Vandenbroeck, Koen; Varadé, Jezabel; de la Hera, Belén; Arroyo, Rafael; Comabella, Manuel; Montalban, Xavier; Petit-Marty, Natalia; Navarro, Arcadi; Otaegui, David; Olascoaga, Javier; Blanco, Yolanda; Urcelay, Elena; Matesanz, Fuencisla

    2013-01-01

    Background and aim Several studies have highlighted the association of the 12q13.3–12q14.1 region with coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis (MS); however, the causal variants underlying diseases are still unclear. The authors sought to identify the functional variant of this region associated with MS. Methods Tag-single nucleotide polymorphism (SNP) analysis of the associated region encoding 15 genes was performed in 2876 MS patients and 2910 healthy Caucasian controls together with expression regulation analyses. Results rs6581155, which tagged 18 variants within a region where 9 genes map, was sufficient to model the association. This SNP was in total linkage disequilibrium (LD) with other polymorphisms that associated with the expression levels of FAM119B, AVIL, TSFM, TSPAN31 and CYP27B1 genes in different expression quantitative trait loci studies. Functional annotations from Encyclopedia of DNA Elements (ENCODE) showed that six out of these rs6581155-tagged-SNPs were located in regions with regulatory potential and only one of them, rs10877013, exhibited allele-dependent (ratio A/G=9.5-fold) and orientation-dependent (forward/reverse=2.7-fold) enhancer activity as determined by luciferase reporter assays. This enhancer is located in a region where a long-range chromatin interaction among the promoters and promoter-enhancer of several genes has been described, possibly affecting their expression simultaneously. Conclusions This study determines a functional variant which alters the enhancer activity of a regulatory element in the locus affecting the expression of several genes and explains the association of the 12q13.3–12q14.1 region with MS. PMID:23160276

  5. Functional MC1R-gene variants are associated with increased risk for severe photoaging of facial skin.

    PubMed

    Elfakir, Anissa; Ezzedine, Khaled; Latreille, Julie; Ambroisine, Laurence; Jdid, Randa; Galan, Pilar; Hercberg, Serge; Gruber, Florian; Malvy, Denis; Tschachler, Erwin; Guinot, Christiane

    2010-04-01

    The objective of this study was to assess the association between melanocortin-1 receptor (MC1R) variants and the severity of facial skin photoaging. The study population comprised 530 middle-aged French women. A trained dermatologist graded the severity of facial skin photoaging from photographs using a global scale. Logistic regressions were performed to assess the influence of MC1R polymorphisms on severe photoaging with adjustment for possible confounders (demographic and phenotypic data and sun exposure intensity). Among the fifteen MC1R variants identified, the nine most common were V60L, V92M, R151C, R160W, R163Q, R142H, D294H, D84E, and I155T. One hundred and eighty-five individuals (35%) were WT homozygotes, 261 (49%) had one common variant, 78 (15%) had two common variants, and six (1%) had at least one rare variant. After adjustment for possible confounders, the presence of two common variants was already a risk factor for severe photoaging (AOR (95% confidence interval): 2.33 (1.17-4.63)). This risk reached 5.61 (1.43-21.96) when two major diminished-function variants were present. Surprisingly, the minor variant, V92M, was associated with increased risk of photoaging (2.57 (1.23-5.35)). Our results suggest that genetic variations of MC1R are important determinants for severe photoaging.

  6. HTRA1 promoter variant differentiates polypoidal choroidal vasculopathy from exudative age-related macular degeneration.

    PubMed

    Ng, Tsz Kin; Liang, Xiao Ying; Lai, Timothy Y Y; Ma, Li; Tam, Pancy O S; Wang, Jian Xiong; Chen, Li Jia; Chen, Haoyu; Pang, Chi Pui

    2016-01-01

    Exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) share similar abnormal choroidal vasculature, but responses to treatments are different. In this study, we sequenced the whole HTRA1 gene and its promoter by direct sequencing in a Hong Kong Chinese PCV cohort. We identified rs11200638, c.34delCinsTCCT, c.59C>T, rs1049331 and rs2293870 significantly associated with PCV. Notably, rs2672598 was significantly associated with exudative AMD (p = 1.31 × 10(-4)) than PCV (p = 0.11). Logistic regression indicated that rs2672598 (p = 2.27 × 10(-3)) remained significant after adjusting for rs11200638 in exudative AMD. Moreover, the rs11200638-rs2672598 joint genotype AA-CC conferred higher risk to exudative AMD (43.11 folds) than PCV (3.68 folds). Promoter analysis showed that rs2672598 C-allele showed higher luciferase expression than wildtype T-allele (p = 0.026), independent of rs11200638 genotype (p = 0.621). Coherently, vitreous humor HTRA1 expression with rs2672598 CC genotype was significantly higher than that with TT genotype by 2.56 folds (p = 0.02). Furthermore, rs2672598 C-allele was predicted to alter the transcription factor binding sites, but not rs11200638 A-allele. Our results revealed that HTRA1 rs2672598 is more significantly associated with exudative AMD than PCV in ARMS2/HTRA1 region, and it is responsible for elevated HTRA1 transcriptional activity and HTRA1 protein expression. PMID:27338780

  7. HTRA1 promoter variant differentiates polypoidal choroidal vasculopathy from exudative age-related macular degeneration

    PubMed Central

    Ng, Tsz Kin; Liang, Xiao Ying; Lai, Timothy Y. Y.; Ma, Li; Tam, Pancy O. S.; Wang, Jian Xiong; Chen, Li Jia; Chen, Haoyu; Pang, Chi Pui

    2016-01-01

    Exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) share similar abnormal choroidal vasculature, but responses to treatments are different. In this study, we sequenced the whole HTRA1 gene and its promoter by direct sequencing in a Hong Kong Chinese PCV cohort. We identified rs11200638, c.34delCinsTCCT, c.59C>T, rs1049331 and rs2293870 significantly associated with PCV. Notably, rs2672598 was significantly associated with exudative AMD (p = 1.31 × 10−4) than PCV (p = 0.11). Logistic regression indicated that rs2672598 (p = 2.27 × 10−3) remained significant after adjusting for rs11200638 in exudative AMD. Moreover, the rs11200638-rs2672598 joint genotype AA-CC conferred higher risk to exudative AMD (43.11 folds) than PCV (3.68 folds). Promoter analysis showed that rs2672598 C-allele showed higher luciferase expression than wildtype T-allele (p = 0.026), independent of rs11200638 genotype (p = 0.621). Coherently, vitreous humor HTRA1 expression with rs2672598 CC genotype was significantly higher than that with TT genotype by 2.56 folds (p = 0.02). Furthermore, rs2672598 C-allele was predicted to alter the transcription factor binding sites, but not rs11200638 A-allele. Our results revealed that HTRA1 rs2672598 is more significantly associated with exudative AMD than PCV in ARMS2/HTRA1 region, and it is responsible for elevated HTRA1 transcriptional activity and HTRA1 protein expression. PMID:27338780

  8. A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells.

    PubMed

    Cigliola, Valentina; Populaire, Celine; Pierri, Ciro L; Deutsch, Samuel; Haefliger, Jacques-Antoine; Fadista, João; Lyssenko, Valeriya; Groop, Leif; Rueedi, Rico; Thorel, Fabrizio; Herrera, Pedro Luis; Meda, Paolo

    2016-01-01

    Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.

  9. Distribution of Gifsy-3 and of Variants of ST64B and Gifsy-1 Prophages amongst Salmonella enterica Serovar Typhimurium Isolates: Evidence that Combinations of Prophages Promote Clonality

    PubMed Central

    Hiley, Lester; Fang, Ning-Xia; Micalizzi, Gino R.; Bates, John

    2014-01-01

    Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems. PMID:24475087

  10. Promoter Variant-Dependent mRNA Expression of the MEF2A in Longissimus Dorsi Muscle in Cattle

    PubMed Central

    Starzyński, Rafał Radosław; Wicińska, Krystyna; Flisikowski, Krzysztof

    2012-01-01

    The myocyte enhancer factor 2A (MEF2A) gene encodes a member of the myocyte enhancer factor 2 (MEF2) protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to recent studies, MEF2 genes might be major regulators of postnatal skeletal muscle growth; thus, they are considered to be important, novel candidates for muscle development and body growth in farm animals. The aim of the present study was to search for polymorphisms in the bovine MEF2A gene and analyze their effect on the MEF2A mRNA expression level in the longissimus dorsi muscle of Polish Holstein-Fresian cattle. In total, 4094 bp of the whole coding sequence and the promoter region of MEF2A were re-sequenced in 30 animals, resulting in the detection of 6 novel variants as well as one previously reported SNP. Three linked mutations in the promoter region (-780T/G, g.-768T/G, and g.-222A/G) and only two genotypes were identified in two Polish breeds (TTA/TTA and TTA/GGG). Three SNPs in the coding region [g.1599G/A (421aa), g.1626G/A (429aa), and g.1641G/A (434aa)] appeared to be silent substitutions and segregated as two intragene haplotypes: GGG and AAA. Expression analysis showed that the mutations in the promoter region are highly associated with the MEF2A mRNA level in the longissimus dorsi muscle of bulls carrying two different genotypes. The higher MEF2A mRNA level was estimated in the muscle of bulls carrying the TTA/TTA (p<0.01) genotype as compared with those with TTA/GGG. The results obtained suggest that the nucleotide sequence mutation in MEF2A might be useful marker for body growth traits in cattle. PMID:22320864

  11. Adoptive T cell therapy promotes the emergence of genomically altered tumor escape variants.

    PubMed

    Kaluza, Karen M; Thompson, Jill M; Kottke, Timothy J; Flynn Gilmer, Heather C; Knutson, Darlene L; Vile, Richard G

    2012-08-15

    Adoptive T cell therapy has been proven effective against melanoma in mice and humans. However, because most responses are incomplete or transient, cures remain rare. To maximize the efficacy of this therapy, it will be essential to gain a better understanding of the processes which result in tumor relapse. We studied these processes using B16ova murine melanoma and adoptive transfer of OT-I T cells. Transfer of T cells as a single therapy provided a significant survival benefit for mice with established subcutaneous tumors. However, tumors which initially regressed often recurred. By analyzing tumors which emerged in the presence of a potent OT-I response, we identified a novel tumor escape mechanism in which tumor cells evaded T cell pressure by undergoing major genomic changes involving loss of the gene encoding the target tumor antigen. Furthermore, we show that these in vivo processes can be recapitulated in vitro using T cell/tumor cell co-cultures. A single round of in vitro co-culture led to significant loss of the ova gene and a tumor cell population with rapidly induced and diverse karyotypic changes. Although these current studies focus on the model OVA antigen, the finding that T cells can directly promote genomic instability has important implications for the development of adoptive T cell therapies.

  12. AR function in promoting metastatic prostate cancer

    PubMed Central

    Augello, Michael A.; Den, Robert B.

    2015-01-01

    Prostate cancer (PCa) remains a leading cause of cancer-related death in the USA. While localized lesions are effectively treated through radical prostatectomy and/or radiation therapy, treatment for metastatic disease leverages the addiction of these tumors on the androgen receptor (AR) signaling axis for growth and disease progression. Though initially effective, tumors resistant to AR-directed therapeutics ultimately arise (a stage of the disease known as castration-resistant prostate cancer) and are responsible for PCa-specific mortality. Importantly, an abundance of clinical and preclinical evidence strongly implicates AR signaling cascades in the development of metastatic disease in both early and late stages, and thus a concerted effort has been made to delineate the AR-specific programs that facilitate progression to metastatic PCa. A multitude of downstream AR targets as well as critical AR cofactors have been identified which impinge upon both the AR pathway as well as associated metastatic phenotypes. This review will highlight the functional significance of these pathways to disseminated disease and define the molecular underpinnings behind these unique, AR-driven, metastatic signatures. PMID:24425228

  13. Expression and function of a novel variant of estrogen receptor-α36 in murine airways.

    PubMed

    Jia, Shuping; Zhang, Xintian; He, David Z Z; Segal, Manav; Berro, Abdo; Gerson, Trevor; Wang, Zhaoyi; Casale, Thomas B

    2011-11-01

    Evidence suggests that estrogen signaling is involved in sex differences in the prevalence rates and control of asthma, but the expression patterns of estrogen receptor variants and estrogen function in the lung are not well established. We investigated the expression of major estrogen receptor variants occurring naturally and after the development of allergen-induced airway hyperreactivity in a murine model of allergic asthma, along with the role of estrogen signaling in small-airway ciliary motion and smooth muscle contraction. Female BALB/c mice were sensitized with ovalbumin, and estrogen receptor expression patterns were examined by immunofluorescence and Western blot analysis. Time-lapse video and photodiode-based displacement measurement systems were used to assess the effects of estrogen signaling on airway ciliary beat frequency and smooth muscle contraction. We found that a novel variant of estrogen receptor (ER)-α, ER-α36, is expressed in airway epithelial and smooth muscle cells. ER-α36 was predominately localized on the plasma membranes of airway cells. After sensitization to allergen, the expression levels of ER-α36 increased significantly (P < 0.01), whereas the expression of ER-β and ER-α66 did not significantly change. Estrogen treatment in vitro resulted in a rapid increase in airway cilia motion in a dose-dependent fashion, but did not exert any effect on airway smooth muscle contraction. We speculate that the up-regulation of estrogen receptor expression associated with allergen-induced airway hyperresponsiveness may constitute a protective mechanism to facilitate the clearance of mucus. The identification and localization of specific estrogen receptor subtypes in the lung could lead to newer therapeutic avenues aimed at addressing sex differences of asthma susceptibility. PMID:21642591

  14. Structural properties of prokaryotic promoter regions correlate with functional features.

    PubMed

    Meysman, Pieter; Collado-Vides, Julio; Morett, Enrique; Viola, Roberto; Engelen, Kristof; Laukens, Kris

    2014-01-01

    The structural properties of the DNA molecule are known to play a critical role in transcription. In this paper, the structural profiles of promoter regions were studied within the context of their diversity and their function for eleven prokaryotic species; Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, Pseudomonas auroginosa, Geobacter sulfurreducens Helicobacter pylori, Chlamydophila pneumoniae, Synechocystis sp., Synechoccocus elongates, Bacillus anthracis, and the archaea Sulfolobus solfataricus. The main anchor point for these promoter regions were transcription start sites identified through high-throughput experiments or collected within large curated databases. Prokaryotic promoter regions were found to be less stable and less flexible than the genomic mean across all studied species. However, direct comparison between species revealed differences in their structural profiles that can not solely be explained by the difference in genomic GC content. In addition, comparison with functional data revealed that there are patterns in the promoter structural profiles that can be linked to specific functional loci, such as sigma factor regulation or transcription factor binding. Interestingly, a novel structural element clearly visible near the transcription start site was found in genes associated with essential cellular functions and growth in several species. Our analyses reveals the great diversity in promoter structural profiles both between and within prokaryotic species. We observed relationships between structural diversity and functional features that are interesting prospects for further research to yet uncharacterized functional loci defined by DNA structural properties.

  15. Identification of a functional variant for colorectal cancer risk mapping to chromosome 5q31.1

    PubMed Central

    Chen, Xueqin; Li, Jiaoyuan; Liu, Cheng; Gong, Yajie; Yang, Yang; Zhu, Ying; Zhang, Yi; Tian, Jianbo; Chang, Jiang; Zhong, Rong; Gong, Jing; Miao, Xiaoping

    2016-01-01

    Genome-wide association studies (GWASs) have established chromosome 5q31.1 as a risk locus for colorectal cancer (CRC). We previously identified a potentially regulatory single nucleotide polymorphism (SNP) rs17716310 within 5q31.1. Now, we extended our study with another independent Chinese population, functional assays and analyses of TCGA (The Cancer Genome Atlas) data. Significant associations between rs17716310 and CRC risk were found in Present Study including 1075 CRC cases and 1999 controls (additive model: OR = 1.149, 95% CI = 1.027–1.286, P = 0.016), and in Combined Study including 1766 cases and 2708 controls (additive model: OR = 1.145, 95% CI = 1.045–1.254, P = 0.004). Dual luciferase reporter gene assays indicated that the variant C allele obviously increased transcriptional activity. Using TCGA datasets, we indicated rs17716310 as a cis expression quantitative trait locus (eQTL) for the gene SMAD5, whose expression was significantly higher in CRC tissues. These findings suggested that the functional polymorphism rs17716310 A > C might be a genetic modifier for CRC, promoting the expression of SMAD5 that belonged to the transforming growth factor beta (TGF-β) signaling pathway. PMID:27177089

  16. BDNF-Val66Met variant and adolescent stress interact to promote susceptibility to anorexic behavior in mice

    PubMed Central

    Madra, M; Zeltser, L M

    2016-01-01

    There is an urgent need to identify therapeutic targets for anorexia nervosa (AN) because current medications do not impact eating behaviors that drive AN's high mortality rate. A major obstacle to developing new treatments is the lack of animal models that recapitulate the pattern of disease onset typically observed in human populations. Here we describe a translational mouse model to study interactions between genetic, psychological and biological risk factors that promote anorexic behavior. We combined several factors that are consistently associated with increased risk of AN—adolescent females, genetic predisposition to anxiety imposed by the BDNF-Val66Met gene variant, social isolation stress and caloric restriction (CR). Approximately 40% of the mice with all of these risk factors will exhibit severe self-imposed dietary restriction, sometimes to the point of death. We systematically varied the risk factors outlined above to explore how they interact to influence anorexic behavior. We found that the Val66Met genotype markedly increases the likelihood and severity of abnormal feeding behavior triggered by CR, but only when CR is imposed in the peri-pubertal period. Incidence of anorexic behavior in our model is dependent on juvenile exposure to social stress and can be extinguished by adolescent handling, but is discordant from anxiety-like behavior. Thus, this study characterized gene × environment interactions during adolescence that could be the underlying driver of abnormal eating behavior in certain AN patients, and represents a promising system to identify possible targets for therapeutic intervention. PMID:27045846

  17. Functional antagonism of TMPRSS2-ERG splice variants in prostate cancer.

    PubMed

    Rastogi, Anshu; Tan, Shyh-Han; Mohamed, Ahmed A; Chen, Yongmei; Hu, Ying; Petrovics, Gyorgy; Sreenath, Taduru; Kagan, Jacob; Srivastava, Sudhir; McLeod, David G; Sesterhenn, Isabell A; Srivastava, Shiv; Dobi, Albert; Srinivasan, Alagarsamy

    2014-07-01

    The fusion between ERG coding sequences and the TMPRSS2 promoter is the most prevalent in prostate cancer (CaP). The presence of two main types of TMPRSS2-ERG fusion transcripts in CaP specimens, Type I and Type II, prompted us to hypothesize that the cumulative actions of different ERG variants may impact CaP development/progression. Using TMPRSS2-ERG3 (Type I) and TMPRSS2-ERG8 (Type II) expression vectors, we determined that the TMPRSS2- ERG8 encoded protein is deficient in transcriptional regulation compared to TMPRSS2-ERG3. Co-transfection of vectors resulted in decreased transcriptional regulation compared to TMPRSS2-ERG3 alone, suggesting transdominance of ERG8. Expression of exogenous ERG8 protein resulted in a decrease in endogenous ERG3 protein levels in TMPRSS2-ERG positive VCaP cells, with a concomitant decrease in C-MYC. Further, we showed a physical association between ERG3 and ERG8 in live cells by the bimolecular fluorescence complementation assay, providing a basis for the observed effects. Inhibitory effects of TMPRSS2-ERG8 on TMPRSS2- ERG3 were also corroborated by gene expression data from human prostate cancers, which showed a positive correlation between C-MYC expression and TMPRSS2-ERG3/TMPRSS2- ERG8 ratio. We propose that an elevated TMPRSS2-ERG3/TMPRSS2-ERG8 ratio results in elevated C-MYC in CaP, providing a strong rationale for the biomarker and therapeutic utility of ERG splice variants, along with C-MYC.

  18. Rare Functional Variant in TM2D3 is Associated with Late-Onset Alzheimer's Disease

    PubMed Central

    Grove, Megan L.; Naj, Adam; Vronskaya, Maria; DeStefano, Anita L.; Brody, Jennifer A.; Smith, Albert V.; Amin, Najaf; Sims, Rebecca; Ibrahim-Verbaas, Carla A.; Choi, Seung-Hoan; Lopez, Oscar L.; Beiser, Alexa; Ikram, M. Arfan; Garcia, Melissa E.; Hayward, Caroline; Ripatti, Samuli; Franks, Paul W.; Hallmans, Göran; Rolandsson, Olov; Jansson, Jan-Håkon; Porteous, David J.; Salomaa, Veikko; Eiriksdottir, Gudny; Rice, Kenneth M.; Bellen, Hugo J.; Levy, Daniel; Uitterlinden, Andre G.; Emilsson, Valur; Rotter, Jerome I.; Aspelund, Thor; O’Donnell, Christopher J.; Fitzpatrick, Annette L.; Launer, Lenore J.; Hofman, Albert; Wang, Li-San; Williams, Julie; Schellenberg, Gerard D.; Boerwinkle, Eric; Psaty, Bruce M.; Seshadri, Sudha; Shulman, Joshua M.; Gudnason, Vilmundur; van Duijn, Cornelia M.

    2016-01-01

    We performed an exome-wide association analysis in 1393 late-onset Alzheimer’s disease (LOAD) cases and 8141 controls from the CHARGE consortium. We found that a rare variant (P155L) in TM2D3 was enriched in Icelanders (~0.5% versus <0.05% in other European populations). In 433 LOAD cases and 3903 controls from the Icelandic AGES sub-study, P155L was associated with increased risk and earlier onset of LOAD [odds ratio (95% CI) = 7.5 (3.5–15.9), p = 6.6x10-9]. Mutation in the Drosophila TM2D3 homolog, almondex, causes a phenotype similar to loss of Notch/Presenilin signaling. Human TM2D3 is capable of rescuing these phenotypes, but this activity is abolished by P155L, establishing it as a functionally damaging allele. Our results establish a rare TM2D3 variant in association with LOAD susceptibility, and together with prior work suggests possible links to the β-amyloid cascade. PMID:27764101

  19. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function

    PubMed Central

    Margolis, Kara Gross; Li, Zhishan; Stevanovic, Korey; Saurman, Virginia; Anderson, George M.; Snyder, Isaac; Blakely, Randy D.; Gershon, Michael D.

    2016-01-01

    Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT4 agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT4–mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation. PMID:27111230

  20. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function.

    PubMed

    Margolis, Kara Gross; Li, Zhishan; Stevanovic, Korey; Saurman, Virginia; Israelyan, Narek; Anderson, George M; Snyder, Isaac; Veenstra-VanderWeele, Jeremy; Blakely, Randy D; Gershon, Michael D

    2016-06-01

    Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT4 agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT4-mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation. PMID:27111230

  1. Architectural and functional commonalities between enhancers and promoters

    PubMed Central

    Kim, Tae-Kyung; Shiekhattar, Ramin

    2015-01-01

    Summary With the explosion of genome-wide studies of regulated transcription, it has become clear that traditional definitions of enhancers and promoters need to be revisited. These control elements can now be characterized in terms of their local and regional architecture, their regulatory components including histone modifications and associated binding factors and their functional contribution to transcription. This review discusses unifying themes between promoters and enhancers in transcriptional regulatory mechanisms. PMID:26317464

  2. Functional Variants in DPYSL2 Sequence Increase Risk of Schizophrenia and Suggest a Link to mTOR Signaling

    PubMed Central

    Liu, Yaping; Pham, Xuan; Zhang, Lilei; Chen, Pei-lung; Burzynski, Grzegorz; McGaughey, David M.; He, Shan; McGrath, John A.; Wolyniec, Paula; Fallin, Margaret D.; Pierce, Megan S.; McCallion, Andrew S.; Pulver, Ann E.; Avramopoulos, Dimitrios; Valle, David

    2014-01-01

    Numerous linkage and association studies by our group and others have implicated DPYSL2 at 8p21.2 in schizophrenia. Here we explore DPYSL2 for functional variation that underlies these associations. We sequenced all 14 exons of DPYSL2 as well as 27 conserved noncoding regions at the locus in 137 cases and 151 controls. We identified 120 variants, eight of which we genotyped in an additional 729 cases and 1542 controls. Several were significantly associated with schizophrenia, including a three single-nucleotide polymorphism (SNP) haplotype in the proximal promoter, two SNPs in intron 1, and a polymorphic dinucleotide repeat in the 5′-untranslated region that alters sequences predicted to be involved in translational regulation by mammalian target of rapamycin signaling. The 3-SNP promoter haplotype and the sequence surrounding one of the intron 1 SNPs direct tissue-specific expression in the nervous systems of Zebrafish in a pattern consistent with the two endogenous dpysl2 paralogs. In addition, two SNP haplotypes over the coding exons and 3′ end of DPYSL2 showed association with opposing sex-specific risks. These data suggest that these polymorphic, schizophrenia-associated sequences function as regulatory elements for DPYSL2 expression. In transient transfection assays, the high risk allele of the polymorphic dinucleotide repeat diminished reporter expression by 3- to 4-fold. Both the high- and low-risk alleles respond to allosteric mTOR inhibition by rapamycin until, at high drug levels, allelic differences are eliminated. Our results suggest that reduced transcription and mTOR-regulated translation of certain DPYSL2 isoforms increase the risk for schizophrenia. PMID:25416705

  3. Isolation and Functional Characterization of Bidirectional Promoters in Rice

    PubMed Central

    Wang, Rui; Yan, Yan; Zhu, Menglin; Yang, Mei; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2016-01-01

    Bidirectional promoters, which show great application potential in genetic improvement of plants, have aroused great research interest recently. However, most bidirectional promoters were cloned individually in the studies of single genes. Here, we initiatively combined RNA-seq data and cDNA microarray data to discover the potential bidirectional promoters in rice genome. Based on the expression level and correlation of each adjacent and oppositely transcribed gene pair, we selected four candidate gene pairs. Then, the intergenic region between each pair was isolated and cloned into a dual reporter vector pDX2181 for functional identification. GUS and GFP assays of the transgenic plants indicated that all the intergenic regions showed bidirectional expression activity in various tissues. Through 5′ and 3′ deletion analysis on one of the above bidirectional promoters, we identified the enhancing region which sharply increased its bidirectional expression efficiency and the essential regions respectively responsible for its 5′ and 3′ basic expression activity. The bidirectional arrangement of the four gene pairs in six gramineous plants was also analyzed, showing the conserved characteristics of the four bidirectional promoters identified in our study. In addition, two novel cis-sequences conserved in the four bidirectional promoters were discovered by bioinformatic identification. Our study proposes a feasible method for selecting, cloning, and functionally identifying bidirectional promoters as well as for discovering their bidirectional regulatory regions and conserved sequences in rice. PMID:27303432

  4. Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant

    PubMed Central

    Almeida, Rodrigo; Ricaño-Ponce, Isis; Kumar, Vinod; Deelen, Patrick; Szperl, Agata; Trynka, Gosia; Gutierrez-Achury, Javier; Kanterakis, Alexandros; Westra, Harm-Jan; Franke, Lude; Swertz, Morris A.; Platteel, Mathieu; Bilbao, Jose Ramon; Barisani, Donatella; Greco, Luigi; Mearin, Luisa; Wolters, Victorien M.; Mulder, Chris; Mazzilli, Maria Cristina; Sood, Ajit; Cukrowska, Bozena; Núñez, Concepción; Pratesi, Riccardo; Withoff, Sebo; Wijmenga, Cisca

    2014-01-01

    Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of ∼1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 × 10−49), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 × 10−44). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 × 10−49), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD. PMID:24334606

  5. A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome

    PubMed Central

    Gauthier, Julie; Ouled Amar Bencheikh, Bouchra; Hamdan, Fadi F; Harrison, Steven M; Baker, Linda A; Couture, Françoise; Thiffault, Isabelle; Ouazzani, Reda; Samuels, Mark E; Mitchell, Grant A; Rouleau, Guy A; Michaud, Jacques L; Soucy, Jean- François

    2015-01-01

    Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by marked dilatation of the bladder and microcolon and decreased intestinal peristalsis. Recent studies indicate that heterozygous variants in ACTG2, which codes for a smooth muscle actin, cause MMIHS. However, such variants do not explain MMIHS cases that show an autosomal recessive mode of inheritance. We performed exome sequencing in a newborn with MMIHS and prune belly phenotype whose parents are consanguineous and identified a homozygous variant (c.3598A>T: p.Lys1200Ter) in MYH11, which codes for the smooth muscle myosin heavy chain. Previous studies showed that loss of Myh11 function in mice causes a bladder and intestinal phenotype that is highly reminiscent of MMIHS. All together, these observations strongly suggest that loss-of-function variants in MYH11 cause MMIHS. The documentation of variants in ACTG2 and MYH11 thus points to the involvement of the contractile apparatus of the smooth muscle in MMIHS. Interestingly, dominant-negative variants in MYH11 have previously been shown to cause thoracic aortic aneurism and dilatation. Different mechanisms of MYH11 disruption may thus lead to distinct patterns of smooth muscle dysfunction. PMID:25407000

  6. Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

    PubMed Central

    Bhatia, Shipra; Gordon, Christopher T.; Foster, Robert G.; Melin, Lucie; Abadie, Véronique; Baujat, Geneviève; Vazquez, Marie-Paule; Amiel, Jeanne; Lyonnet, Stanislas; van Heyningen, Veronica; Kleinjan, Dirk A.

    2015-01-01

    Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function. PMID:26030420

  7. Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung▿ †

    PubMed Central

    Starkey, Melissa; Hickman, Jason H.; Ma, Luyan; Zhang, Niu; De Long, Susan; Hinz, Aaron; Palacios, Sergio; Manoil, Colin; Kirisits, Mary Jo; Starner, Timothy D.; Wozniak, Daniel J.; Harwood, Caroline S.; Parsek, Matthew R.

    2009-01-01

    Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV

  8. Comparative functional characterization of novel non-syndromic GJB2 gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu

    PubMed Central

    Gordhandas, Jeenal A.; Pique, Lynn

    2016-01-01

    We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+ regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+ concentrations, suggesting that Ca2+ regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein. PMID:27761313

  9. Functional characterization of a GFAP variant of uncertain significance in an Alexander disease case within the setting of an individualized medicine clinic.

    PubMed

    Boczek, Nicole J; Sigafoos, Ashley N; Zimmermann, Michael T; Maus, Rachel L; Cousin, Margot A; Blackburn, Patrick R; Urrutia, Raul; Clark, Karl J; Patterson, Marc C; Wick, Myra J; Klee, Eric W

    2016-09-01

    A de novo GFAP variant, p.R376W, was identified in a child presenting with hypotonia, developmental delay, and abnormal brain MRI. Following the 2015 ACMG variant classification guidelines and the functional studies showing protein aggregate formation in vitro, p.R376W should be classified as a pathogenic variant, causative for Alexander disease.

  10. Functional characterization of a GFAP variant of uncertain significance in an Alexander disease case within the setting of an individualized medicine clinic.

    PubMed

    Boczek, Nicole J; Sigafoos, Ashley N; Zimmermann, Michael T; Maus, Rachel L; Cousin, Margot A; Blackburn, Patrick R; Urrutia, Raul; Clark, Karl J; Patterson, Marc C; Wick, Myra J; Klee, Eric W

    2016-09-01

    A de novo GFAP variant, p.R376W, was identified in a child presenting with hypotonia, developmental delay, and abnormal brain MRI. Following the 2015 ACMG variant classification guidelines and the functional studies showing protein aggregate formation in vitro, p.R376W should be classified as a pathogenic variant, causative for Alexander disease. PMID:27648269

  11. A rare functional noncoding variant at the GWAS-implicated MIR137/MIR2682 locus might confer risk to schizophrenia and bipolar disorder.

    PubMed

    Duan, Jubao; Shi, Jianxin; Fiorentino, Alessia; Leites, Catherine; Chen, Xiangning; Moy, Winton; Chen, Jingchun; Alexandrov, Boian S; Usheva, Anny; He, Deli; Freda, Jessica; O'Brien, Niamh L; McQuillin, Andrew; Sanders, Alan R; Gershon, Elliot S; DeLisi, Lynn E; Bishop, Alan R; Gurling, Hugh M D; Pato, Michele T; Levinson, Douglas F; Kendler, Kenneth S; Pato, Carlos N; Gejman, Pablo V

    2014-12-01

    Schizophrenia (SZ) genome-wide association studies (GWASs) have identified common risk variants in >100 susceptibility loci; however, the contribution of rare variants at these loci remains largely unexplored. One of the strongly associated loci spans MIR137 (miR137) and MIR2682 (miR2682), two microRNA genes important for neuronal function. We sequenced ∼6.9 kb MIR137/MIR2682 and upstream regulatory sequences in 2,610 SZ cases and 2,611 controls of European ancestry. We identified 133 rare variants with minor allele frequency (MAF) <0.5%. The rare variant burden in promoters and enhancers, but not insulators, was associated with SZ (p = 0.021 for MAF < 0.5%, p = 0.003 for MAF < 0.1%). A rare enhancer SNP, 1:g.98515539A>T, presented exclusively in 11 SZ cases (nominal p = 4.8 × 10(-4)). We further identified its risk allele T in 2 of 2,434 additional SZ cases, 11 of 4,339 bipolar (BP) cases, and 3 of 3,572 SZ/BP study controls and 1,688 population controls; yielding combined p values of 0.0007, 0.0013, and 0.0001 for SZ, BP, and SZ/BP, respectively. The risk allele T of 1:g.98515539A>T reduced enhancer activity of its flanking sequence by >50% in human neuroblastoma cells, predicting lower expression of MIR137/MIR2682. Both empirical and computational analyses showed weaker transcription factor (YY1) binding by the risk allele. Chromatin conformation capture (3C) assay further indicated that 1:g.98515539A>T influenced MIR137/MIR2682, but not the nearby DPYD or LOC729987. Our results suggest that rare noncoding risk variants are associated with SZ and BP at MIR137/MIR2682 locus, with risk alleles decreasing MIR137/MIR2682 expression.

  12. dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions

    PubMed Central

    Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

    2016-01-01

    The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. PMID:26989155

  13. dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions.

    PubMed

    Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

    2016-01-01

    The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. PMID:26989155

  14. Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant.

    PubMed

    Gupta, Saumya; Radhakrishnan, Aparna; Nitin, Rachana; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M; Gagneur, Julien; Sinha, Himanshu

    2016-01-01

    Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae During mitosis, the common TAO3 allele interacts with CBK1-a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2-a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait. PMID:27317780

  15. Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant

    PubMed Central

    Gupta, Saumya; Radhakrishnan, Aparna; Nitin, Rachana; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M.; Gagneur, Julien; Sinha, Himanshu

    2016-01-01

    Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae. During mitosis, the common TAO3 allele interacts with CBK1—a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2—a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait. PMID:27317780

  16. Pain modality- and sex-specific effects of COMT genetic functional variants.

    PubMed

    Belfer, Inna; Segall, Samantha K; Lariviere, William R; Smith, Shad B; Dai, Feng; Slade, Gary D; Rashid, Naim U; Mogil, Jeffrey S; Campbell, Claudia M; Edwards, Robert R; Liu, Qian; Bair, Eric; Maixner, William; Diatchenko, Luda

    2013-08-01

    The enzyme catechol-O-methyltransferase (COMT) metabolizes catecholamine neurotransmitters involved in a number of physiological functions, including pain perception. Both human and mouse COMT genes possess functional polymorphisms contributing to interindividual variability in pain phenotypes such as sensitivity to noxious stimuli, severity of clinical pain, and response to pain treatment. In this study, we found that the effects of Comt functional variation in mice are modality specific. Spontaneous inflammatory nociception and thermal nociception behaviors were correlated the most with the presence of the B2 SINE transposon insertion residing in the 3'UTR mRNA region. Similarly, in humans, COMT functional haplotypes were associated with thermal pain perception and with capsaicin-induced pain. Furthermore, COMT genetic variations contributed to pain behaviors in mice and pain ratings in humans in a sex-specific manner. The ancestral Comt variant, without a B2 SINE insertion, was more strongly associated with sensitivity to capsaicin in female vs male mice. In humans, the haplotype coding for low COMT activity increased capsaicin-induced pain perception in women, but not men. These findings reemphasize the fundamental contribution of COMT to pain processes, and provide a fine-grained resolution of this contribution at the genetic level that can be used to guide future studies in the area of pain genetics.

  17. SNPnexus: a web server for functional annotation of novel and publicly known genetic variants (2012 update).

    PubMed

    Dayem Ullah, Abu Z; Lemoine, Nicholas R; Chelala, Claude

    2012-07-01

    Broader functional annotation of single nucleotide variations is a valuable mean for prioritizing targets in further disease studies and large-scale genotyping projects. We originally developed SNPnexus to assess the potential significance of known and novel SNPs on the major transcriptome, proteome, regulatory and structural variation models in order to identify the phenotypically important variants. Being committed to providing continuous support to the scientific community, we have substantially improved SNPnexus over time by incorporating a broader range of variations such as insertions/deletions, block substitutions, IUPAC codes submission and region-based analysis, expanding the query size limit, and most importantly including additional categories for the assessment of functional impact. SNPnexus provides a comprehensive set of annotations for genomic variation data by characterizing related functional consequences at the transcriptome/proteome levels of seven major annotation systems with in-depth analysis of potential deleterious effects, inferring physical and cytogenetic mapping, reporting information on HapMap genotype/allele data, finding overlaps with potential regulatory elements, structural variations and conserved elements, and retrieving links with previously reported genetic disease studies. SNPnexus has a user-friendly web interface with an improved query structure, enhanced functional annotation categories and flexible output presentation making it practically useful for biologists. SNPnexus is freely available at http://www.snp-nexus.org.

  18. Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

    PubMed Central

    Yap, Damian B.; Lewis, M.E. Suzanne; Chijiwa, Chieko; Ramos‐Arroyo, Maria A.; Tkachenko, Natália; Milano, Valentina; Fradin, Mélanie; McKinnon, Margaret L.; Townsend, Katelin N.; Xu, Jieqing; Van Allen, M.I.; Ross, Colin J.D.; Dobyns, William B.; Weaver, David D.; Gibson, William T.

    2016-01-01

    ABSTRACT Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb‐repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, whereas WS‐associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS‐associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G>C (p.Asp185His) polymorphism in EZH2. PMID:26694085

  19. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  20. Association of a functional variant of the nitric oxide synthase 1 gene with personality, anxiety, and depressiveness.

    PubMed

    Kurrikoff, Triin; Lesch, Klaus-Peter; Kiive, Evelyn; Konstabel, Kenn; Herterich, Sabine; Veidebaum, Toomas; Reif, Andreas; Harro, Jaanus

    2012-11-01

    A functional promoter polymorphism of the nitric oxide synthase 1 gene first exon 1f variable number tandem repeat (NOS1 ex1f-VNTR) is associated with impulsivity and related psychopathology. Facets of impulsivity are strongly associated with personality traits; maladaptive impulsivity with neuroticism; and adaptive impulsivity with extraversion. Both high neuroticism and low extraversion predict anxiety and depressive symptoms. The aim of the present study was to evaluate the effect of the NOS1 ex1f-VNTR genotype and possible interaction with environmental factors on personality, anxiety, and depressiveness in a population-representative sample. Short allele carriers had higher neuroticism and anxiety than individuals with the long/long (l/l) genotype. Male short/short homozygotes also had higher extraversion. In the face of environmental adversity, females with a short allele had higher scores of neuroticism, anxiety, and depressiveness compared to the l/l genotype. Males were more sensitive to environmental conditions when they had the l/l genotype and low extraversion. In conclusion, the NOS1 ex1f-VNTR influences personality and emotional regulation dependent on gender and environment. Together with previous findings on the effect of the NOS1 genotype on impulse control, these data suggest that NOS1 should be considered another plasticity gene, because its variants are associated with different coping strategies.

  1. New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants.

    PubMed

    Rajagopalan, Rema; Salvi, Veena P; Jensenius, Jens Chr; Rawal, Nenoo

    2009-04-27

    Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (approximately 450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D. PMID:19428558

  2. Further evidence that the rs1858830 C variant in the promoter region of the MET gene is associated with autistic disorder.

    PubMed

    Jackson, Pamela B; Boccuto, Luigi; Skinner, Cindy; Collins, Julianne S; Neri, Giovanni; Gurrieri, Fiorella; Schwartz, Charles E

    2009-08-01

    Previous studies in three independent cohorts have shown that the rs1858830 C allele variant in the promoter region of the MET gene on chromosome 7q31 is associated with autism. Another study has found correlations between other alterations in the MET gene and autism in two unrelated cohorts. This study screened two cohorts, an Autistic Disorder cohort from South Carolina and a Pervasive Developmental Disorder (PDD) cohort from Italy, for the presence of the C allele variant in rs1858830. A significant increase in the C allele variant frequency was found in the South Carolina Autistic Disorder patients as compared to South Carolina Controls (chi(2)=5.8, df=1, P=0.02). In the South Carolina cohort, a significant association with Autistic Disorder was found when comparing the CC and CG genotypes to the GG genotype (odds ratio (OR)=1.64; 95% confidence interval (CI)=1.12-2.40; chi(2)=6.5, df=1, P=0.01) in cases and controls. In the Italian cohort, no significant association with PDD was found when comparing the CC or CG genotype to the GG genotype (OR=1.20; 95% CI=0.56-2.56; chi(2)=0.2, df=1, P=0.64). This study is the third independent study to find the rs1858830 C variant in the MET gene promoter to be associated with autism.

  3. Functional Differences of Very-Low-Density Lipoprotein Receptor Splice Variants in Regulating Wnt Signaling

    PubMed Central

    Chen, Qian; Takahashi, Yusuke; Oka, Kazuhiro

    2016-01-01

    The very-low-density lipoprotein receptor (VLDLR) negatively regulates Wnt signaling. VLDLR has two major alternative splice variants, VLDLRI and VLDLRII, but their biological significance and distinction are unknown. Here we found that most tissues expressed both VLDLRI and VLDLRII, while the retina expressed only VLDLRII. The shed soluble VLDLR extracellular domain (sVLDLR-N) was detected in the conditioned medium of retinal pigment epithelial cells, interphotoreceptor matrix, and mouse plasma, indicating that ectodomain shedding of VLDLR occurs endogenously. VLDLRII displayed a higher ectodomain shedding rate and a more potent inhibitory effect on Wnt signaling than VLDLRI in vitro and in vivo. O-glycosylation, which is present in VLDLRI but not VLDLRII, determined the differential ectodomain shedding rates. Moreover, the release of sVLDLR-N was inhibited by a metalloproteinase inhibitor, TAPI-1, while it was promoted by phorbol 12-myristate 13-acetate (PMA). In addition, sVLDLR-N shedding was suppressed under hypoxia. Further, plasma levels of sVLDLR-N were reduced in both type 1 and type 2 diabetic mouse models. We concluded that VLDLRI and VLDLRII had differential roles in regulating Wnt signaling and that decreased plasma levels of sVLDLR-N may contribute to Wnt signaling activation in diabetic complications. Our study reveals a novel mechanism for intercellular regulation of Wnt signaling through VLDLR ectodomain shedding. PMID:27528615

  4. Rare loss-of-function variants in SETD1A are associated with schizophrenia and developmental disorders.

    PubMed

    Singh, Tarjinder; Kurki, Mitja I; Curtis, David; Purcell, Shaun M; Crooks, Lucy; McRae, Jeremy; Suvisaari, Jaana; Chheda, Himanshu; Blackwood, Douglas; Breen, Gerome; Pietiläinen, Olli; Gerety, Sebastian S; Ayub, Muhammad; Blyth, Moira; Cole, Trevor; Collier, David; Coomber, Eve L; Craddock, Nick; Daly, Mark J; Danesh, John; DiForti, Marta; Foster, Alison; Freimer, Nelson B; Geschwind, Daniel; Johnstone, Mandy; Joss, Shelagh; Kirov, Georg; Körkkö, Jarmo; Kuismin, Outi; Holmans, Peter; Hultman, Christina M; Iyegbe, Conrad; Lönnqvist, Jouko; Männikkö, Minna; McCarroll, Steve A; McGuffin, Peter; McIntosh, Andrew M; McQuillin, Andrew; Moilanen, Jukka S; Moore, Carmel; Murray, Robin M; Newbury-Ecob, Ruth; Ouwehand, Willem; Paunio, Tiina; Prigmore, Elena; Rees, Elliott; Roberts, David; Sambrook, Jennifer; Sklar, Pamela; St Clair, David; Veijola, Juha; Walters, James T R; Williams, Hywel; Sullivan, Patrick F; Hurles, Matthew E; O'Donovan, Michael C; Palotie, Aarno; Owen, Michael J; Barrett, Jeffrey C

    2016-04-01

    By analyzing the whole-exome sequences of 4,264 schizophrenia cases, 9,343 controls and 1,077 trios, we identified a genome-wide significant association between rare loss-of-function (LoF) variants in SETD1A and risk for schizophrenia (P = 3.3 × 10(-9)). We found only two heterozygous LoF variants in 45,376 exomes from individuals without a neuropsychiatric diagnosis, indicating that SETD1A is substantially depleted of LoF variants in the general population. Seven of the ten individuals with schizophrenia carrying SETD1A LoF variants also had learning difficulties. We further identified four SETD1A LoF carriers among 4,281 children with severe developmental disorders and two more carriers in an independent sample of 5,720 Finnish exomes, both with notable neuropsychiatric phenotypes. Together, our observations indicate that LoF variants in SETD1A cause a range of neurodevelopmental disorders, including schizophrenia. Combining these data with previous common variant evidence, we suggest that epigenetic dysregulation, specifically in the histone H3K4 methylation pathway, is an important mechanism in the pathogenesis of schizophrenia.

  5. Rare loss-of-function variants in SETD1A are associated with schizophrenia and developmental disorders.

    PubMed

    Singh, Tarjinder; Kurki, Mitja I; Curtis, David; Purcell, Shaun M; Crooks, Lucy; McRae, Jeremy; Suvisaari, Jaana; Chheda, Himanshu; Blackwood, Douglas; Breen, Gerome; Pietiläinen, Olli; Gerety, Sebastian S; Ayub, Muhammad; Blyth, Moira; Cole, Trevor; Collier, David; Coomber, Eve L; Craddock, Nick; Daly, Mark J; Danesh, John; DiForti, Marta; Foster, Alison; Freimer, Nelson B; Geschwind, Daniel; Johnstone, Mandy; Joss, Shelagh; Kirov, Georg; Körkkö, Jarmo; Kuismin, Outi; Holmans, Peter; Hultman, Christina M; Iyegbe, Conrad; Lönnqvist, Jouko; Männikkö, Minna; McCarroll, Steve A; McGuffin, Peter; McIntosh, Andrew M; McQuillin, Andrew; Moilanen, Jukka S; Moore, Carmel; Murray, Robin M; Newbury-Ecob, Ruth; Ouwehand, Willem; Paunio, Tiina; Prigmore, Elena; Rees, Elliott; Roberts, David; Sambrook, Jennifer; Sklar, Pamela; St Clair, David; Veijola, Juha; Walters, James T R; Williams, Hywel; Sullivan, Patrick F; Hurles, Matthew E; O'Donovan, Michael C; Palotie, Aarno; Owen, Michael J; Barrett, Jeffrey C

    2016-04-01

    By analyzing the whole-exome sequences of 4,264 schizophrenia cases, 9,343 controls and 1,077 trios, we identified a genome-wide significant association between rare loss-of-function (LoF) variants in SETD1A and risk for schizophrenia (P = 3.3 × 10(-9)). We found only two heterozygous LoF variants in 45,376 exomes from individuals without a neuropsychiatric diagnosis, indicating that SETD1A is substantially depleted of LoF variants in the general population. Seven of the ten individuals with schizophrenia carrying SETD1A LoF variants also had learning difficulties. We further identified four SETD1A LoF carriers among 4,281 children with severe developmental disorders and two more carriers in an independent sample of 5,720 Finnish exomes, both with notable neuropsychiatric phenotypes. Together, our observations indicate that LoF variants in SETD1A cause a range of neurodevelopmental disorders, including schizophrenia. Combining these data with previous common variant evidence, we suggest that epigenetic dysregulation, specifically in the histone H3K4 methylation pathway, is an important mechanism in the pathogenesis of schizophrenia. PMID:26974950

  6. Population-Specific Risk of Type 2 Diabetes Conferred by HNF4A P2 Promoter Variants

    PubMed Central

    Barroso, Inês; Luan, Jian’an; Wheeler, Eleanor; Whittaker, Pamela; Wasson, Jon; Zeggini, Eleftheria; Weedon, Michael N.; Hunt, Sarah; Venkatesh, Ranganath; Frayling, Timothy M.; Delgado, Marcos; Neuman, Rosalind J.; Zhao, Jinghua; Sherva, Richard; Glaser, Benjamin; Walker, Mark; Hitman, Graham; McCarthy, Mark I.; Hattersley, Andrew T.; Permutt, M. Alan; Wareham, Nicholas J.; Deloukas, Panagiotis

    2008-01-01

    OBJECTIVE—Single nucleotide polymorphisms (SNPs) in the P2 promoter region of HNF4A were originally shown to be associated with predisposition for type 2 diabetes in Finnish, Ashkenazi, and, more recently, Scandinavian populations, but they generated conflicting results in additional populations. We aimed to investigate whether data from a large-scale mapping approach would replicate this association in novel Ashkenazi samples and in U.K. populations and whether these data would allow us to refine the association signal. RESEARCH DESIGN AND METHODS—Using a dense linkage disequilibrium map of 20q, we selected SNPs from a 10-Mb interval centered on HNF4A. In a staged approach, we first typed 4,608 SNPs in case-control populations from four U.K. populations and an Ashkenazi population (n = 2,516). In phase 2, a subset of 763 SNPs was genotyped in 2,513 additional samples from the same populations. RESULTS—Combined analysis of both phases demonstrated association between HNF4A P2 SNPs (rs1884613 and rs2144908) and type 2 diabetes in the Ashkenazim (n = 991; P < 1.6 × 10−6). Importantly, these associations are significant in a subset of Ashkenazi samples (n = 531) not previously tested for association with P2 SNPs (odds ratio [OR] ∼1.7; P < 0.002), thus providing replication within the Ashkenazim. In the U.K. populations, this association was not significant (n = 4,022; P > 0.5), and the estimate for the OR was much smaller (OR 1.04; [95%CI 0.91–1.19]). CONCLUSIONS—These data indicate that the risk conferred by HNF4A P2 is significantly different between U.K. and Ashkenazi populations (P < 0.00007), suggesting that the underlying causal variant remains unidentified. Interactions with other genetic or environmental factors may also contribute to this difference in risk between populations. PMID:18728231

  7. Association of adiponectin promoter variants with traits and clusters of metabolic syndrome in Arabs: family-based study.

    PubMed

    Zadjali, F; Al-Yahyaee, S; Hassan, M O; Albarwani, S; Bayoumi, R A

    2013-09-25

    Plasma levels of adiponectin are decreased in type 2 diabetes, obesity and hypertension. Our aim was to use a family-based analysis to identify the genetic variants of the adiponectin (ADIPOQ) gene that are associated with obesity, insulin resistance, dyslipidemia and hypertension, among Arabs. We screened 328 Arabs in one large extended family for single nucleotide polymorphisms (SNPs) in the promoter region of the ADIPOQ gene. Two common SNPs were detected: rs17300539 and rs266729. Evidences of association between traits related to the metabolic syndrome and the SNPs were studied by implementing quantitative genetic association analysis. Results showed that SNP rs266729 was significantly associated with body weight (p-value=0.001), waist circumference (p-value=0.037), BMI (p-value=0.015) and percentage of total body fat (p-value=0.003). Up to 4.1% of heritability of obesity traits was explained by the rs266729 locus. Further cross-sectional analysis showed that carriers of the G allele had significantly higher values of waist circumference, BMI and percentage of total body fat (p-values 0.014, 0.004 and 0.032, respectively). No association was detected between SNP rs266729 and other clusters of metabolic syndrome or their traits except for HOMA-IR and fasting plasma insulin levels, p-values 0.035 and 0.004, respectively. In contrast, both measured genotype and cross-sectional analysis failed to detect an association between the SNP rs17300539 with traits and clusters of metabolic syndrome. In conclusion, we showed family-based evidence of association of SNP rs266729 at ADIPOQ gene with traits defining obesity in Arab population. This is important for future prediction and prevention of obesity in population where obesity is in an increasing trend.

  8. MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants.

    PubMed

    Annibalini, Giosuè; Bielli, Pamela; De Santi, Mauro; Agostini, Deborah; Guescini, Michele; Sisti, Davide; Contarelli, Serena; Brandi, Giorgio; Villarini, Anna; Stocchi, Vilberto; Sette, Claudio; Barbieri, Elena

    2016-05-01

    Insulin-like growth factor (IGF) -1 is a pleiotropic hormone exerting mitogenic and anti-apoptotic effects. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. The biological significance of the IGF-1 splice variants and how the E-peptides affect the actions of mature IGF-1 are largely unknown. In this study we investigated the origin and conservation of the IGF-1 E-peptides and we compared the pattern of expression of the IGF-1 isoforms in vivo, in nine mammalian species, and in vitro using human and mouse IGF-1 minigenes. Our analysis showed that only IGF-1Ea is conserved among all vertebrates, whereas IGF-1Eb and IGF-1Ec are an evolutionary novelty originated from the exonization of a mammalian interspersed repetitive-b (MIR-b) element. Both IGF-1Eb and IGF-1Ec mRNAs were constitutively expressed in all mammalian species analyzed but their expression ratio varies greatly among species. Using IGF-1 minigenes we demonstrated that divergence in cis-acting regulatory elements between human and mouse conferred species-specific features to the exon 5 region. Finally, the protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains. In conclusion, exonization of a MIR-b element in the IGF-1 gene determined gain of exon 5 during mammalian evolution. Alternative splicing of this novel exon added new regulatory elements at the mRNA and protein level potentially able to regulate the mature IGF-1 across tissues and species. PMID:27048986

  9. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor

    PubMed Central

    Herraiz, Cecilia; Olivares, Conchi; Castejón-Griñán, Maria; Abrisqueta, Marta; Jiménez-Cervantes, Celia; García-Borrón, José Carlos

    2015-01-01

    The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters. PMID:26657157

  10. The G32E Functional Variant Reduces Activity of PPARD by Nuclear Export and Post-Translational Modification in Pigs

    PubMed Central

    Duan, Yanyu; Brenig, Bertram; Wu, Xiaohui; Ren, Jun; Huang, Lusheng

    2013-01-01

    Peroxisome proliferator-activated receptor beta/delta (PPARD) is a crucial and multifaceted determinant of diverse biological functions including lipid metabolism, embryonic development, inflammatory response, wound healing and cancer. Recently, we proposed a novel function of porcine PPARD (sPPARD) in external ear development. A missense mutation (G32E) in an evolutionary conservative domain of sPPARD remarkably increases external ear size in pigs. Here, we investigated the underlying molecular mechanism of the causal mutation at the cellular level. Using a luciferase reporter system, we showed that the G32E substitution reduced transcription activity of sPPARD in a ligand-dependent manner. By comparison of the subcellular localization of wild-type and mutated sPPARD in both PK-15 cells and pinna cartilage-derived primary chondrocytes, we found that the G32E substitution promoted CRM-1 mediated nuclear exportation of sPPARD. With the surface plasmon resonance technology, we further revealed that the G32E substitution had negligible effect on its ligand binding affinity. Finally, we used co-immunoprecipitation and luciferase reporter assays to show that the G32E substitution greatly reduced ubiquitination level by blocking ubiquitination of the crucial A/B domain and consequently decreased transcription activity of sPPARD. Taken together, our findings strongly support that G32E is a functional variant that plays a key role in biological activity of sPPARD, which advances our understanding of the underlying mechanism of sPPARD G32E for ear size in pigs. PMID:24058710

  11. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

    PubMed

    Herraiz, Cecilia; Olivares, Conchi; Castejón-Griñán, Maria; Abrisqueta, Marta; Jiménez-Cervantes, Celia; García-Borrón, José Carlos

    2015-01-01

    The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters. PMID:26657157

  12. Alternative Splice Variants Modulates Dominant-Negative Function of Helios in T-Cell Leukemia

    PubMed Central

    Zhao, Shaorong; Liu, Wei; Li, Yinghui; Liu, Pengjiang; Li, Shufang; Dou, Daolei; Wang, Yue; Yang, Rongcun; Xiang, Rong; Liu, Feifei

    2016-01-01

    The molecular defects which lead to multistep incidences of human T-cell leukemia have yet to be identified. The DNA-binding protein Helios (known as IKZF2), a member of the Ikaros family of Krüppel-like zinc-finger proteins, functions pivotally in T-cell differentiation and activation. In this study, we identify three novel short Helios splice variants which are T-cell leukemic specific, and demonstrate their dominant-negative function. We then test the cellular localization of distinct Helios isoforms, as well as their capability to form heterodimer with Ikaros, and the association with complexes comprising histone deacetylase (HDAC). In addition, the ectopic expression of T-cell leukemic Helios isoforms interferes with T-cell proliferation and apoptosis. The gene expression profiling and pathway analysis indicated the enrichment of signaling pathways essential for gene expression, translation, cell cycle checkpoint, and response to DNA damage stimulus. These data indicate the molecular function of Helios to be involved in the leukemogenesis and phenotype of T-cell leukemia, and also reveal Helios deregulation as a novel marker for T-cell leukemia. PMID:27681508

  13. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  14. Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

    PubMed

    Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Darlyuk-Saadon, Ilona; Bai, Chen; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-09-01

    MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect. PMID:27413009

  15. Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer.

    PubMed

    Bailey, Swneke D; Desai, Kinjal; Kron, Ken J; Mazrooei, Parisa; Sinnott-Armstrong, Nicholas A; Treloar, Aislinn E; Dowar, Mark; Thu, Kelsie L; Cescon, David W; Silvester, Jennifer; Yang, S Y Cindy; Wu, Xue; Pezo, Rossanna C; Haibe-Kains, Benjamin; Mak, Tak W; Bedard, Philippe L; Pugh, Trevor J; Sallari, Richard C; Lupien, Mathieu

    2016-10-01

    Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk-associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer. PMID:27571262

  16. Functional analysis of transcription factor binding sites in human promoters

    PubMed Central

    2012-01-01

    Background The binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. To characterize transcription factor binding site function on a large scale, we predicted and mutagenized 455 binding sites in human promoters. We carried out functional tests on these sites in four different immortalized human cell lines using transient transfections with a luciferase reporter assay, primarily for the transcription factors CTCF, GABP, GATA2, E2F, STAT, and YY1. Results In each cell line, between 36% and 49% of binding sites made a functional contribution to the promoter activity; the overall rate for observing function in any of the cell lines was 70%. Transcription factor binding resulted in transcriptional repression in more than a third of functional sites. When compared with predicted binding sites whose function was not experimentally verified, the functional binding sites had higher conservation and were located closer to transcriptional start sites (TSSs). Among functional sites, repressive sites tended to be located further from TSSs than were activating sites. Our data provide significant insight into the functional characteristics of YY1 binding sites, most notably the detection of distinct activating and repressing classes of YY1 binding sites. Repressing sites were located closer to, and often overlapped with, translational start sites and presented a distinctive variation on the canonical YY1 binding motif. Conclusions The genomic properties that we found to associate with functional TF binding sites on promoters -- conservation, TSS proximity, motifs and their variations -- point the way to improved accuracy in future TFBS predictions. PMID:22951020

  17. Identification and functional characterization of four transient receptor potential ankyrin 1 variants in Apolygus lucorum (Meyer-Dür).

    PubMed

    Fu, T; Hull, J J; Yang, T; Wang, G

    2016-08-01

    As signal integrators that respond to various physical and chemical stimuli, transient receptor potential (TRP) channels fulfil critical functional roles in the sensory systems of both vertebrate and invertebrate organisms. Here, four variants of TRP ankyrin 1 (TRPA1) were identified and cloned from the green plant bug, Apolygus lucorum. Spatiotemporal expression profiling across development and in different adult tissues revealed that the highest relative-transcript levels occurred in first-instar nymphs and antennae, respectively. In Xenopus laevis-based functional assays, Apo. lucorum TRPA1-A (AlucTRPA1-A), AlucTRPA1-B and AlucTRPA1-C were activated by increasing the temperature from 20 to 40 °C with no significant desensitization observed after repeated temperature stimuli. The activation temperature of AlucTRPA1-A and AlucTRPA1-B was < 25 °C, whereas the activation temperature of AlucTRPA1-C was between 25 and 30 °C. Amongst the variants, only AlucTRPA1-A and AlucTRPA1-C were directly activated by high concentrations of allyl isothiocyanate, cinnamaldehyde and citronellal. Taken together, these results suggest that AlucTRPA1 variants may function in vivo as both thermal and chemical sensors, with the four variants potentially mediating different physiological functions. This study not only enriches our understanding of TRPA1 function in Hemiptera (Miridae), but also offers a foundation for developing new pest control strategies. PMID:27038267

  18. Structural and functional analysis of the bovine Mx1 promoter.

    PubMed

    Yamada, Kohji; Nakatsu, Yuichiro; Onogi, Akio; Takasuga, Akiko; Sugimoto, Yoshikazu; Ueda, Junji; Watanabe, Tomomasa

    2009-04-01

    The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene. PMID:19203250

  19. Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

    SciTech Connect

    Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer; Hirsch, Hans H.; Rinaldo, Christine Hanssen

    2010-03-01

    High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.

  20. A Single Nucleotide Polymorphism in the Il17ra Promoter Is Associated with Functional Severity of Ankylosing Spondylitis

    PubMed Central

    Vidal-Castiñeira, Jose Ramón; López-Vázquez, Antonio; Diaz-Peña, Roberto; Diaz-Bulnes, Paula; Martinez-Camblor, Pablo; Coto, Eliecer; Coto-Segura, Pablo; Bruges-Armas, Jacome; Pinto, Jose Antonio; Blanco, Francisco Jose; Sánchez, Alejandra; Mulero, Juan; Queiro, Ruben; Lopez-Larrea, Carlos

    2016-01-01

    The aim of this study was to identify new genetic variants associated with the severity of ankylosing spondylitis (AS). We sequenced the exome of eight patients diagnosed with AS, selected on the basis of the severity of their clinical parameters. We identified 27 variants in exons and regulatory regions. The contribution of candidate variants found to AS severity was validated by genotyping two Spanish cohorts consisting of 180 cases/300 controls and 419 cases/656 controls. Relationships of SNPs and clinical variables with the Bath Ankylosing Spondylitis Disease Activity and Functional Indices BASDAI and BASFI were analyzed. BASFI was standardized by adjusting for the duration of the disease since the appearance of the first symptoms. Refining the analysis of SNPs in the two cohorts, we found that the rs4819554 minor allele G in the promoter of the IL17RA gene was associated with AS (p<0.005). This variant was also associated with the BASFI score. Classifying AS patients by the severity of their functional status with respect to BASFI/disease duration of the 60th, 65th, 70th and 75th percentiles, we found the association increased from p60 to p75 (cohort 1: p<0.05 to p<0.01; cohort 2: p<0.01 to p<0.005). Our findings indicate a genetic role for the IL17/ILRA axis in the development of severe forms of AS. PMID:27415816

  1. A Single Nucleotide Polymorphism in the Il17ra Promoter Is Associated with Functional Severity of Ankylosing Spondylitis.

    PubMed

    Vidal-Castiñeira, Jose Ramón; López-Vázquez, Antonio; Diaz-Peña, Roberto; Diaz-Bulnes, Paula; Martinez-Camblor, Pablo; Coto, Eliecer; Coto-Segura, Pablo; Bruges-Armas, Jacome; Pinto, Jose Antonio; Blanco, Francisco Jose; Sánchez, Alejandra; Mulero, Juan; Queiro, Ruben; Lopez-Larrea, Carlos

    2016-01-01

    The aim of this study was to identify new genetic variants associated with the severity of ankylosing spondylitis (AS). We sequenced the exome of eight patients diagnosed with AS, selected on the basis of the severity of their clinical parameters. We identified 27 variants in exons and regulatory regions. The contribution of candidate variants found to AS severity was validated by genotyping two Spanish cohorts consisting of 180 cases/300 controls and 419 cases/656 controls. Relationships of SNPs and clinical variables with the Bath Ankylosing Spondylitis Disease Activity and Functional Indices BASDAI and BASFI were analyzed. BASFI was standardized by adjusting for the duration of the disease since the appearance of the first symptoms. Refining the analysis of SNPs in the two cohorts, we found that the rs4819554 minor allele G in the promoter of the IL17RA gene was associated with AS (p<0.005). This variant was also associated with the BASFI score. Classifying AS patients by the severity of their functional status with respect to BASFI/disease duration of the 60th, 65th, 70th and 75th percentiles, we found the association increased from p60 to p75 (cohort 1: p<0.05 to p<0.01; cohort 2: p<0.01 to p<0.005). Our findings indicate a genetic role for the IL17/ILRA axis in the development of severe forms of AS. PMID:27415816

  2. Mechanisms of Loss of Functions of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

    PubMed Central

    Padhi, Aditya K.; Kumar, Hirdesh; Vasaikar, Suhas V.; Jayaram, Bhyravabhotla; Gomes, James

    2012-01-01

    Background Mutations in the coding region of angiogenin (ANG) gene have been found in patients suffering from Amyotrophic Lateral Sclerosis (ALS). Neurodegeneration results from the loss of angiogenic ability of ANG (protein coded by ANG). In this work, we performed extensive molecular dynamics (MD) simulations of wild-type ANG and disease associated ANG variants to elucidate the mechanism behind the loss of ribonucleolytic activity and nuclear translocation activity, functions needed for angiogenesis. Methodology/Principal Findings MD simulations were carried out to study the structural and dynamic differences in the catalytic site and nuclear localization signal residues between WT-ANG (Wild-type ANG) and six mutants. Variants K17I, S28N, P112L and V113I have confirmed association with ALS, while T195C and A238G single nucleotide polymorphisms (SNPs) encoding L35P and K60E mutants respectively, have not been associated with ALS. Our results show that loss of ribonucleolytic activity in K17I is caused by conformational switching of the catalytic residue His114 by 99°. The loss of nuclear translocation activity of S28N and P112L is caused by changes in the folding of the residues 31RRR33 that result in the reduction in solvent accessible surface area (SASA). Consequently, we predict that V113I will exhibit loss of angiogenic properties by loss of nuclear translocation activity and L35P by loss of both ribonucleolytic activity and nuclear translocation activity. No functional loss was inferred for K60E. The MD simulation results were supported by hydrogen bond interaction analyses and molecular docking studies. Conclusions/Significance Conformational switching of catalytic residue His114 seems to be the mechanism causing loss of ribonucleolytic activity and reduction in SASA of nuclear localization signal residues 31RRR33 results in loss of nuclear translocation activity in ANG mutants. Therefore, we predict that L35P mutant, would exhibit loss of angiogenic

  3. Promoters of Escherichia coli versus Promoter Islands: Function and Structure Comparison

    PubMed Central

    Panyukov, Valeriy V.; Ozoline, Olga N.

    2013-01-01

    Expression of bacterial genes takes place under the control of RNA polymerase with exchangeable σ-subunits and multiple transcription factors. A typical promoter region contains one or several overlapping promoters. In the latter case promoters have the same or different σ-specificity and are often subjected to different regulatory stimuli. Genes, transcribed from multiple promoters, have on average higher expression levels. However, recently in the genome of Escherichia coli we found 78 regions with an extremely large number of potential transcription start points (promoter islands, PIs). It was shown that all PIs interact with RNA polymerase in vivo and are able to form transcriptionally competent open complexes both in vitro and in vivo but their transcriptional activity measured by oligonucleotide microarrays was very low, if any. Here we confirmed transcriptional defectiveness of PIs by analyzing the 5′-end specific RNA-seq data, but showed their ability to produce short oligos (9–14 bases). This combination of functional properties indicated a deliberate suppression of transcriptional activity within PIs. According to our data this suppression may be due to a specific conformation of the DNA double helix, which provides an ideal platform for interaction with both RNA polymerase and the histone-like nucleoid protein H-NS. The genomic DNA of E.coli contains therefore several dozen sites optimized by evolution for staying in a heterochromatin-like state. Since almost all promoter islands are associated with horizontally acquired genes, we offer them as specific components of bacterial evolution involved in acquisition of foreign genetic material by turning off the expression of toxic or useless aliens or by providing optimal promoter for beneficial genes. The putative molecular mechanism underlying the appearance of promoter islands within recipient genomes is discussed. PMID:23717391

  4. Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

    PubMed

    Allum, Fiona; Shao, Xiaojian; Guénard, Frédéric; Simon, Marie-Michelle; Busche, Stephan; Caron, Maxime; Lambourne, John; Lessard, Julie; Tandre, Karolina; Hedman, Åsa K; Kwan, Tony; Ge, Bing; Rönnblom, Lars; McCarthy, Mark I; Deloukas, Panos; Richmond, Todd; Burgess, Daniel; Spector, Timothy D; Tchernof, André; Marceau, Simon; Lathrop, Mark; Vohl, Marie-Claude; Pastinen, Tomi; Grundberg, Elin

    2015-05-29

    Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.

  5. Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants

    PubMed Central

    Allum, Fiona; Shao, Xiaojian; Guénard, Frédéric; Simon, Marie-Michelle; Busche, Stephan; Caron, Maxime; Lambourne, John; Lessard, Julie; Tandre, Karolina; Hedman, Åsa K.; Kwan, Tony; Ge, Bing; Rönnblom, Lars; McCarthy, Mark I.; Deloukas, Panos; Richmond, Todd; Burgess, Daniel; Spector, Timothy D.; Tchernof, André; Marceau, Simon; Lathrop, Mark; Vohl, Marie-Claude; Pastinen, Tomi; Grundberg, Elin; Ahmadi, Kourosh R.; Ainali, Chrysanthi; Barrett, Amy; Bataille, Veronique; Bell, Jordana T.; Buil, Alfonso; Dermitzakis, Emmanouil T.; Dimas, Antigone S.; Durbin, Richard; Glass, Daniel; Hassanali, Neelam; Ingle, Catherine; Knowles, David; Krestyaninova, Maria; Lindgren, Cecilia M.; Lowe, Christopher E.; Meduri, Eshwar; di Meglio, Paola; Min, Josine L.; Montgomery, Stephen B.; Nestle, Frank O.; Nica, Alexandra C.; Nisbet, James; O'Rahilly, Stephen; Parts, Leopold; Potter, Simon; Sandling, Johanna; Sekowska, Magdalena; Shin, So-Youn; Small, Kerrin S.; Soranzo, Nicole; Surdulescu, Gabriela; Travers, Mary E.; Tsaprouni, Loukia; Tsoka, Sophia; Wilk, Alicja; Yang, Tsun-Po; Zondervan, Krina T.

    2015-01-01

    Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS. PMID:26021296

  6. Can common functional gene variants affect visual discrimination in metacontrast masking?

    PubMed

    Maksimov, Margus; Vaht, Mariliis; Harro, Jaanus; Bachmann, Talis

    2013-01-01

    Mechanisms of visual perception should be robustly fast and provide veridical information about environmental objects in order to facilitate survival and successful coping. Because species-specific brain mechanisms for fast vision must have evolved under heavy pressure for efficiency, it has been held that different human individuals see the physical world in the same way and produce psychophysical functions of visual discrimination that are qualitatively the same. For many years, this assumption has been implicitly accepted in vision research studying extremely fast, basic visual processes, including studies of visual masking. However, in recent studies of metacontrast masking surprisingly robust individual differences in the qualitative aspects of subjects' performance have been found. As the basic species-specific visual functions very likely are based on universal brain mechanisms of vision, these differences probably are the outcome of variability in ontogenetic development (i.e., formation of idiosyncrasic skills of perception). Such developmental differences can be brought about by variants of genes that are differentially expressed in the course of CNS development. The objective of this study was to assess whether visual discrimination in metacontrast masking is related to three widely studied genetic polymorphisms implicated in brain function and used here as independent variables. The findings suggest no main effects of BDNF Val66Met, NRG1/rs6994992, or 5-HTTLPR polymorphisms on metacontrast performance, but several notable interactions of genetic variables with gender, stage of the sequence of experimental trials, perceptual strategies, and target/mask shape congruence were found. Thus, basic behavioral functions of fast vision may be influenced by common genetic variability. Also, when left uncontrolled, genetic factors may seriously confound variables in vision research using masking, obscure clear theoretical interpretation, lead to unexplicable inter

  7. Deleterious Rare Variants Reveal Risk for Loss of GABAA Receptor Function in Patients with Genetic Epilepsy and in the General Population

    PubMed Central

    Hernandez, Ciria C.; Klassen, Tara L.; Jackson, Laurel G.; Gurba, Katharine; Hu, Ningning; Macdonald, Robert L.

    2016-01-01

    Genetic epilepsies (GEs) account for approximately 50% of all seizure disorders, and familial forms include mutations in single GABAA receptor subunit genes (GABRs). In 144 sporadic GE cases (GECs), exome sequencing of 237 ion channel genes identified 520 GABR variants. Among these variants, 33 rare variants in 11 GABR genes were present in 24 GECs. To assess functional risk of variants in GECs, we selected 8 variants found in GABRA, 3 in GABRB, and 3 in GABRG and compared them to 18 variants found in the general population for GABRA1 (n = 9), GABRB3 (n = 7), and GABRG2 (n = 2). To identify deleterious variants and gain insight into structure-function relationships, we studied the gating properties, surface expression and structural perturbations of the 32 variants. Significant reduction of GABAA receptor function was strongly associated with variants scored as deleterious and mapped within the N-terminal and transmembrane domains. In addition, 12 out of 17 variants mapped along the β+/α- GABA binding interface, were associated with reduction in channel gating and were predicted to cause structural rearrangements of the receptor by in silico simulations. Missense or nonsense mutations of GABRA1, GABRB3 and GABRG2 primarily impair subunit biogenesis. In contrast, GABR variants affected receptor function by impairing gating, suggesting that different mechanisms are operating in GABR epilepsy susceptibility variants and disease-causing mutations. The functional impact of single GABR variants found in individuals with sporadic GEs warrants the use of molecular diagnosis and will ultimately improve the treatment of genetic epilepsies by using a personalized approach. PMID:27622563

  8. Deleterious Rare Variants Reveal Risk for Loss of GABAA Receptor Function in Patients with Genetic Epilepsy and in the General Population.

    PubMed

    Hernandez, Ciria C; Klassen, Tara L; Jackson, Laurel G; Gurba, Katharine; Hu, Ningning; Noebels, Jeffrey L; Macdonald, Robert L

    2016-01-01

    Genetic epilepsies (GEs) account for approximately 50% of all seizure disorders, and familial forms include mutations in single GABAA receptor subunit genes (GABRs). In 144 sporadic GE cases (GECs), exome sequencing of 237 ion channel genes identified 520 GABR variants. Among these variants, 33 rare variants in 11 GABR genes were present in 24 GECs. To assess functional risk of variants in GECs, we selected 8 variants found in GABRA, 3 in GABRB, and 3 in GABRG and compared them to 18 variants found in the general population for GABRA1 (n = 9), GABRB3 (n = 7), and GABRG2 (n = 2). To identify deleterious variants and gain insight into structure-function relationships, we studied the gating properties, surface expression and structural perturbations of the 32 variants. Significant reduction of GABAA receptor function was strongly associated with variants scored as deleterious and mapped within the N-terminal and transmembrane domains. In addition, 12 out of 17 variants mapped along the β+/α- GABA binding interface, were associated with reduction in channel gating and were predicted to cause structural rearrangements of the receptor by in silico simulations. Missense or nonsense mutations of GABRA1, GABRB3 and GABRG2 primarily impair subunit biogenesis. In contrast, GABR variants affected receptor function by impairing gating, suggesting that different mechanisms are operating in GABR epilepsy susceptibility variants and disease-causing mutations. The functional impact of single GABR variants found in individuals with sporadic GEs warrants the use of molecular diagnosis and will ultimately improve the treatment of genetic epilepsies by using a personalized approach. PMID:27622563

  9. Modifying lipid rafts promotes regeneration and functional recovery.

    PubMed

    Tassew, Nardos G; Mothe, Andrea J; Shabanzadeh, Alireza P; Banerjee, Paromita; Koeberle, Paulo D; Bremner, Rod; Tator, Charles H; Monnier, Philippe P

    2014-08-21

    Ideal strategies to ameliorate CNS damage should promote both neuronal survival and axon regeneration. The receptor Neogenin promotes neuronal apoptosis. Its ligand prevents death, but the resulting repulsive guidance molecule a (RGMa)-Neogenin interaction also inhibits axonal growth, countering any prosurvival benefits. Here, we explore strategies to inhibit Neogenin, thus simultaneously enhancing survival and regeneration. We show that bone morphogenetic protein (BMP) and RGMa-dependent recruitment of Neogenin into lipid rafts requires an interaction between RGMa and Neogenin subdomains. RGMa or Neogenin peptides that prevent this interaction, BMP inhibition by Noggin, or reduction of membrane cholesterol all block Neogenin raft localization, promote axon outgrowth, and prevent neuronal apoptosis. Blocking Neogenin raft association influences axonal pathfinding, enhances survival in the developing CNS, and promotes survival and regeneration in the injured adult optic nerve and spinal cord. Moreover, lowering cholesterol disrupts rafts and restores locomotor function after spinal cord injury. These data reveal a unified strategy to promote both survival and regeneration in the CNS. PMID:25127134

  10. The -14010*C variant associated with lactase persistence is located between an Oct-1 and HNF1α binding site and increases lactase promoter activity.

    PubMed

    Jensen, Tine G K; Liebert, Anke; Lewinsky, Rikke; Swallow, Dallas M; Olsen, Jørgen; Troelsen, Jesper T

    2011-10-01

    In most people worldwide intestinal lactase expression declines in childhood. In many others, particularly in Europeans, lactase expression persists into adult life. The lactase persistence phenotype is in Europe associated with the -13910*T single nucleotide variant located 13,910 bp upstream the lactase gene in an enhancer region that affects lactase promoter activity. This variant falls in an Oct-1 binding site and shows greater Oct-1 binding than the ancestral variant and increases enhancer activity. Several other variants have been identified very close to the -13910 position, which are associated with lactase persistence in the Middle East and Africa. One of them, the -14010*C, is associated with lactase persistence in Africa. Here we show by deletion analysis that the -14010 position is located in a 144 bp region that reduces the enhancer activity. In transfections the -14010*C allele shows a stronger enhancer effect than the ancestral -4010*G allele. Binding sites for Oct-1 and HNF1α surrounding the -14010 position were identified by gel shift assays, which indicated that -14010*C has greater binding affinity to Oct-1 than -14010*G.

  11. Disentangling the Effects of Colocalizing Genomic Annotations to Functionally Prioritize Non-coding Variants within Complex-Trait Loci.

    PubMed

    Trynka, Gosia; Westra, Harm-Jan; Slowikowski, Kamil; Hu, Xinli; Xu, Han; Stranger, Barbara E; Klein, Robert J; Han, Buhm; Raychaudhuri, Soumya

    2015-07-01

    Identifying genomic annotations that differentiate causal from trait-associated variants is essential to fine mapping disease loci. Although many studies have identified non-coding functional annotations that overlap disease-associated variants, these annotations often colocalize, complicating the ability to use these annotations for fine mapping causal variation. We developed a statistical approach (Genomic Annotation Shifter [GoShifter]) to assess whether enriched annotations are able to prioritize causal variation. GoShifter defines the null distribution of an annotation overlapping an allele by locally shifting annotations; this approach is less sensitive to biases arising from local genomic structure than commonly used enrichment methods that depend on SNP matching. Local shifting also allows GoShifter to identify independent causal effects from colocalizing annotations. Using GoShifter, we confirmed that variants in expression quantitative trail loci drive gene-expression changes though DNase-I hypersensitive sites (DHSs) near transcription start sites and independently through 3' UTR regulation. We also showed that (1) 15%-36% of trait-associated loci map to DHSs independently of other annotations; (2) loci associated with breast cancer and rheumatoid arthritis harbor potentially causal variants near the summits of histone marks rather than full peak bodies; (3) variants associated with height are highly enriched in embryonic stem cell DHSs; and (4) we can effectively prioritize causal variation at specific loci.

  12. Expression pattern and function of alternative splice variants of glutamate-gated chloride channel in the housefly Musca domestica.

    PubMed

    Kita, Tomo; Ozoe, Fumiyo; Ozoe, Yoshihisa

    2014-02-01

    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. cDNAs encoding two alternative splice variants (MdGluClB and C) of the GluCl subunit were cloned from the housefly Musca domestica. The expression patterns of three variants, including the previously reported MdGluClA, differed among the body parts (head, thorax, abdomen, and leg) of the adult housefly and among developmental stages (embryo, larva, pupa, and adult). The MdGluClA and B transcripts were abundant in the central nervous system of the adult, whereas the MdGluClC transcript was expressed in the central nervous system and as the predominant variant in the peripheral tissues. The sensitivities to the agonist glutamate and the allosteric activator ivermectin B1a did not differ between channels containing MdGluCl variants when they were singly or co-expressed in Xenopus oocytes. By contrast, MdGluClA and B channels were more sensitive to the channel blockers fipronil and picrotoxinin than was MdGluClC channels. Heteromeric channels containing different subunit variants were more sensitive to picrotoxinin than were homomeric channels. Heteromeric channels were more sensitive to fipronil than were homomeric MdGluClC channels but not than homomeric MdGluClA and B channels. These results suggest that functionally indistinguishable but pharmacologically distinct GluCls are expressed in a spatially and temporally distinct manner in the housefly.

  13. MicroRNA Genetic Variation: From Population Analysis to Functional Implications of Three Allele Variants Associated with Cancer.

    PubMed

    Torruella-Loran, Ignasi; Laayouni, Hafid; Dobon, Begoña; Gallego, Alicia; Balcells, Ingrid; Garcia-Ramallo, Eva; Espinosa-Parrilla, Yolanda

    2016-10-01

    Nucleotide variants in microRNA regions have been associated with disease; nevertheless, few studies still have addressed the allele-dependent effect of these changes. We studied microRNA genetic variation in human populations and found that while low-frequency variants accumulate indistinctly in microRNA regions, the mature and seed regions tend to be depleted of high-frequency variants, probably as a result of purifying selection. Comparison of pairwise population fixation indexes among regions showed that the seed had higher population fixation indexes than the other regions, suggesting the existence of local adaptation in the seed region. We further performed functional studies of three microRNA variants associated with cancer (rs2910164:C > G in MIR146A, rs11614913:C > T in MIR196A2, and rs3746444:A > G in both MIR499A and MIR499B). We found differences in the expression between alleles and in the regulation of several genes involved in cancer, such as TP53, KIT, CDH1, CLH, and TERT, which may result in changes in regulatory networks related to tumorigenesis. Furthermore, luciferase-based assays showed that MIR499A could be regulating the cadherin CDH1 and the cell adhesion molecule CLH1 in an allele-dependent fashion. A better understanding of the effect of microRNA variants associated with disease could be key in our way to a more personalized medicine.

  14. Transcription Factor Erg Variants and Functional Diversification of Chondrocytes during Limb Long Bone Development

    PubMed Central

    Iwamoto, Masahiro; Higuchi, Yoshinobu; Koyama, Eiki; Enomoto-Iwamoto, Motomi; Kurisu, Kojiro; Yeh, Helena; Abrams, William R.; Rosenbloom, Joel; Pacifici, Maurizio

    2000-01-01

    During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27–amino acid segment located ∼80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes. PMID:10893254

  15. A functional map of the nopaline synthase promoter.

    PubMed Central

    Shaw, C H; Carter, G H; Watson, M D; Shaw, C H

    1984-01-01

    This paper describes the first functional map of a promoter expressed from the plant chromosome. We have constructed a series of overlapping deletion mutants within the region upstream of the Ti-plasmid encoded nopaline synthase (nos) gene. By monitoring nos expression in tumour tissue we have inferred a functional map of the nos promoter. The maximum length of sequence upstream of the transcription initiation point required to express wild type levels of nopaline synthase is 88 bp. Within this region, the "CAAT" box is essential for maximal activity; deletion of this sequence reduced apparent nos expression by over 80%. Presence of an intact or partial "TATA" box in the absence of the "CAAT" box supports a barely detectable level of nopaline synthase. Removal of all sequences upstream of the nos coding sequence results in no detectable activity. PMID:6493982

  16. Construction of cis-regulatory input functions of yeast promoters.

    PubMed

    Ratna, Prasuna; Becskei, Attila

    2011-01-01

    Promoters contain a large number of binding sites for transcriptional factors transmitting signals from a variety of cellular pathways. The promoter processes these input signals and sets the level of gene expression, the output of the gene. Here, we describe how to design genetic constructs and measure gene expression to deliver data suitable for quantitative analysis. Synthetic genetic constructs are well suited to precisely control and measure gene expression to construct cis-regulatory input functions. These functions can be used to predict gene expression based on signal intensities transmitted to activators and repressors in the gene regulatory region. Simple models of gene expression are presented for competitive and noncompetitive repressions. Complex phenomena, exemplified by synergistic silencing, are modeled by reaction-diffusion equations.

  17. Human Brown Fat Inducible Thioesterase Variant 2 (BFIT2) Cellular Localization and Catalytic Function#

    PubMed Central

    Chen, Danqi; Latham, John; Zhao, Hong; Bisoffi, Marco; Farelli, Jeremiah; Dunaway-Mariano, Debra

    2014-01-01

    The mammalian brown fat inducible thioesterase variant 2 (BFIT2), also known as ACOT11, is a multi-modular protein containing two consecutive hotdog-fold domains and a C-terminal steroidogenic acute regulatory protein related lipid transfer (START) domain (StarD14). In this study, we demonstrate that the N-terminal region of human BFIT2 (hBFIT2) constitutes a mitochondrial location signal sequence, which undergoes mitochondria-dependent posttranslational cleavage. The mature hBFIT2 is shown to be located in the mitochondrial matrix whereas the paralog “cytoplasmic acetyl-CoA hydrolase” (CACH, also known as ACOT12) was found in the cytoplam. In-vitro activity analysis of full-length hBFIT2 isolated from stably transfected HEK293 cells demonstrates selective thioesterase activity directed towards long chain fatty acyl-CoA thioesters, thus distinguishing BFIT2 catalytic function from that of CACH. The results from a protein-lipid overlay test indicate that the hBFIT2 StarD14 domain binds phosphatidylinositol 4-phosphate. PMID:22897136

  18. Dopamine Inactivation Efficacy Related to Functional DAT1 and COMT Variants Influences Motor Response Evaluation

    PubMed Central

    Bender, Stephan; Rellum, Thomas; Freitag, Christine; Resch, Franz; Rietschel, Marcella; Treutlein, Jens; Jennen-Steinmetz, Christine; Brandeis, Daniel; Banaschewski, Tobias; Laucht, Manfred

    2012-01-01

    Background Dopamine plays an important role in orienting, response anticipation and movement evaluation. Thus, we examined the influence of functional variants related to dopamine inactivation in the dopamine transporter (DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of motor processing in a contingent negative variation (CNV) task. Methods 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as motor postimperative negative variation were assessed. Adolescents were genotyped for the COMT Val158Met and two DAT1 polymorphisms (variable number tandem repeats in the 3′-untranslated region and in intron 8). Results The results revealed a significant interaction between COMT and DAT1, indicating that COMT exerted stronger effects on lateralized motor post-processing (centro-parietal motor postimperative negative variation) in homozygous carriers of a DAT1 haplotype increasing DAT1 expression. Source analysis showed that the time interval 500–1000 ms after the motor response was specifically affected in contrast to preceding movement anticipation and programming stages, which were not altered. Conclusions Motor slow negative waves allow the genomic imaging of dopamine inactivation effects on cortical motor post-processing during response evaluation. This is the first report to point towards epistatic effects in the motor system during response evaluation, i.e. during the post-processing of an already executed movement rather than during movement programming. PMID:22649558

  19. Associations of functional alanine-glyoxylate aminotransferase 2 gene variants with atrial fibrillation and ischemic stroke.

    PubMed

    Seppälä, Ilkka; Kleber, Marcus E; Bevan, Steve; Lyytikäinen, Leo-Pekka; Oksala, Niku; Hernesniemi, Jussi A; Mäkelä, Kari-Matti; Rothwell, Peter M; Sudlow, Cathie; Dichgans, Martin; Mononen, Nina; Vlachopoulou, Efthymia; Sinisalo, Juha; Delgado, Graciela E; Laaksonen, Reijo; Koskinen, Tuomas; Scharnagl, Hubert; Kähönen, Mika; Markus, Hugh S; März, Winfried; Lehtimäki, Terho

    2016-01-01

    Asymmetric and symmetric dimethylarginines (ADMA and SDMA) impair nitric oxide bioavailability and have been implicated in the pathogenesis of atrial fibrillation (AF). Alanine-glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of metabolizing both of the dimethylarginines. We hypothesized that two functional AGXT2 missense variants (rs37369, V140I; rs16899974, V498L) are associated with AF and its cardioembolic complications. Association analyses were conducted using 1,834 individulas with AF and 7,159 unaffected individuals from two coronary angiography cohorts and a cohort comprising patients undergoing clinical exercise testing. In coronary angiography patients without structural heart disease, the minor A allele of rs16899974 was associated with any AF (OR = 2.07, 95% CI 1.59-2.68), and with paroxysmal AF (OR = 1.98, 95% CI 1.44-2.74) and chronic AF (OR = 2.03, 95% CI 1.35-3.06) separately. We could not replicate the association with AF in the other two cohorts. However, the A allele of rs16899974 was nominally associated with ischemic stroke risk in the meta-analysis of WTCCC2 ischemic stroke cohorts (3,548 cases, 5,972 controls) and with earlier onset of first-ever ischemic stroke (360 cases) in the cohort of clinical exercise test patients. In conclusion, AGXT2 variations may be involved in the pathogenesis of AF and its age-related thromboembolic complications. PMID:26984639

  20. Expression and function of a variant T cell receptor complex lacking CD3-gamma

    PubMed Central

    1991-01-01

    A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface- radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3- epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma. PMID:1713248

  1. Frequent somatic CDH1 loss-of-function mutations in plasmacytoid variant bladder cancer.

    PubMed

    Al-Ahmadie, Hikmat A; Iyer, Gopa; Lee, Byron H; Scott, Sasinya N; Mehra, Rohit; Bagrodia, Aditya; Jordan, Emmet J; Gao, Sizhi Paul; Ramirez, Ricardo; Cha, Eugene K; Desai, Neil B; Zabor, Emily C; Ostrovnaya, Irina; Gopalan, Anuradha; Chen, Ying-Bei; Fine, Samson W; Tickoo, Satish K; Gandhi, Anupama; Hreiki, Joseph; Viale, Agnès; Arcila, Maria E; Dalbagni, Guido; Rosenberg, Jonathan E; Bochner, Bernard H; Bajorin, Dean F; Berger, Michael F; Reuter, Victor E; Taylor, Barry S; Solit, David B

    2016-04-01

    Plasmacytoid bladder cancer is an aggressive histologic variant with a high risk of disease-specific mortality. Using whole-exome and targeted sequencing, we find that truncating somatic alterations in the CDH1 gene occur in 84% of plasmacytoid carcinomas and are specific to this histologic variant. Consistent with the aggressive clinical behavior of plasmacytoid carcinomas, which frequently recur locally, CRISPR/Cas9-mediated knockout of CDH1 in bladder cancer cells enhanced cell migration.

  2. Cumulative role of rare and common putative functional genetic variants at NPAS3 in schizophrenia susceptibility.

    PubMed

    González-Peñas, Javier; Arrojo, Manuel; Paz, Eduardo; Brenlla, Julio; Páramo, Mario; Costas, Javier

    2015-10-01

    Schizophrenia may be considered a human-specific disorder arisen as a maladaptive by-product of human-specific brain evolution. Therefore, genetic variants involved in susceptibility to schizophrenia may be identified among those genes related to acquisition of human-specific traits. NPAS3, a transcription factor involved in central nervous system development and neurogenesis, seems to be implicated in the evolution of human brain, as it is the human gene with most human-specific accelerated elements (HAEs), i.e., .mammalian conserved regulatory sequences with accelerated evolution in the lineage leading to humans after human-chimpanzee split. We hypothesize that any nucleotide variant at the NPAS3 HAEs may lead to altered susceptibility to schizophrenia. Twenty-one variants at these HAEs detected by the 1000 genomes Project, as well as five additional variants taken from psychiatric genome-wide association studies, were genotyped in 538 schizophrenic patients and 539 controls from Galicia. Analyses at the haplotype level or based on the cumulative role of the variants assuming different susceptibility models did not find any significant association in spite of enough power under several plausible scenarios regarding direction of effect and the specific role of rare and common variants. These results suggest that, contrary to our hypothesis, the special evolution of the NPAS3 HAEs in Homo relaxed the strong constraint on sequence that characterized these regions during mammalian evolution, allowing some sequence changes without any effect on schizophrenia risk. PMID:25982957

  3. A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction.

    PubMed

    Shao, Qing; Lindstrom, Kristin; Shi, Ruoyang; Kelly, John; Schroeder, Audrey; Juusola, Jane; Levine, Kara L; Esseltine, Jessica L; Penuela, Silvia; Jackson, Michael F; Laird, Dale W

    2016-06-10

    Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650G→A) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this variant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 glycosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demonstrate a PANX1 missense change associated with human disease in the first report of a "PANX1-related disorder." PMID:27129271

  4. The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants.

    PubMed

    Mergy, Marc A; Gowrishankar, Raajaram; Gresch, Paul J; Gantz, Stephanie C; Williams, John; Davis, Gwynne L; Wheeler, C Austin; Stanwood, Gregg D; Hahn, Maureen K; Blakely, Randy D

    2014-11-01

    Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPH-evoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness.

  5. The catecholamine biosynthetic enzyme dopamine β-hydroxylase (DBH): first genome-wide search positions trait-determining variants acting additively in the proximal promoter

    PubMed Central

    Mustapic, Maja; Maihofer, Adam X.; Mahata, Manjula; Chen, Yuqing; Baker, Dewleen G.; O'Connor, Daniel T.; Nievergelt, Caroline M.

    2014-01-01

    Dopamine beta-hydroxylase (DBH) is the biosynthetic enzyme catalyzing formation of norepinephrine. Changes in DBH expression or activity have been implicated in the pathogenesis of cardiovascular and neuropsychiatric disorders. Genetic determination of DBH enzymatic activity and its secretion are only incompletely understood. We began with a genome-wide association search for loci contributing to DBH activity in human plasma. Initially, in a population sample of European ancestry, we identified the proximal DBH promoter as a region harboring three common trait-determining variants (top hit rs1611115, P = 7.2 × 10−51). We confirmed their effects on transcription and showed that the three variants each acted additively on gene expression. Results were replicated in a population sample of Native American descent (top hit rs1611115, P = 4.1 × 10−15). Jointly, DBH variants accounted for 57% of DBH trait variation. We further identified a genome-wide significant SNP at the LOC338797 locus on chromosome 12 as trans-quantitative trait locus (QTL) (rs4255618, P = 4.62 × 10−8). Conditional analyses on DBH identified a third genomic region contributing to DBH variation: a likely cis-QTL adjacent to DBH in SARDH (rs7040170, P = 1.31 × 10−14) on chromosome 9q. We conclude that three common SNPs in the DBH promoter act additively to control phenotypic variation in DBH levels, and that two additional novel loci (SARDH and LOC338797) may also contribute to the expression of this catecholamine biosynthetic trait. Identification of DBH variants with strong effects makes it possible to take advantage of Mendelian randomization approaches to test causal effects of this intermediate trait on disease. PMID:24986918

  6. Impact of beta2-adrenoreceptor gene variants on cardiac cavity size and systolic function in idiopathic dilated cardiomyopathy.

    PubMed

    Badenhorst, D; Norton, G R; Sliwa, K; Brooksbank, R; Essop, R; Sareli, P; Woodiwiss, A J

    2007-10-01

    In heart failure, the Arg16Gly and Gln27Glu polymorphisms of the beta2-adrenoreceptor (beta2-AR) gene are associated with exercise-capacity, clinical outcomes and response to beta-AR blocker therapy. Whether beta2-AR gene variants mediate these effects in-part through an impact on cardiac structural remodeling and pump function independent of the effects of beta-blockers is uncertain. We evaluated whether the Arg16Gly and Gln27Glu variants of the beta2-AR gene predict left ventricular ejection fraction (LVEF) and LV end diastolic diameter (LVEDD) in patients with idiopathic dilated cardiomyopathy (IDC) before and 6 months after receiving standard medical therapy other than beta-AR blockers. In all, 394 patients with IDC and 393 age and gender-matched controls were genotyped for the beta2-AR gene variants using restriction-fragment length polymorphism-based techniques. LVEF and dimensions were determined in 132 patients (of whom 71 were newly diagnosed) both at baseline and after 6 months. Genotype of neither variant was associated with the presence of IDC. Moreover, beta2-AR genotype did not determine LVEF or LV dimensions prior to initiating therapy. After 6 months of therapy, LVEF increased by 7.1+/-1.0 absolute units (P<0.0001) and LVEDD decreased by 0.27+/-0.06 cm (P<0.02). Adjusting for baseline values as well as gender, age, and type of angiotensin-converting enzyme inhibitor therapy received, genotype was associated with neither final LVEF and LVEDD, nor change in LVEF and LVEDD. In conclusion, these data suggest that in heart failure, the functional Arg16Gly and Gln27Glu variants of the beta2-AR gene have no independent effect on adverse structural remodeling and pump function.

  7. Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

    PubMed Central

    Lewis, Michele D.; Park, Hyun Woo; Brand, Randall E.; Gelrud, Andres; Anderson, Michelle A.; Banks, Peter A.; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B.; Amann, Stephen T.; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L.; Yadav, Dhiraj; Barmada, M. Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C.

    2014-01-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR

  8. Functional characterization and molecular mechanism exploration of three granulin epithelin precursor splice variants in biomineralization of the pearl oyster Pinctada fucata.

    PubMed

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi; Shi, Yu

    2016-02-01

    The granulin/epithelin precursor (GEP) encodes a glycoprotein precursor which exhibits pleiotropic tissue growth factor activity with multiple functions. Here, GEP was isolated and its role in the shell biomineralization process of the pearl oyster Pinctada fucata was investigated. Three forms of GEP mRNA were isolated from the pearl oyster (designated PfGEP-1, PfGEP-2 and PfGEP-3). Genomic DNA flanking the splicing region of the PfGEP variants was sequenced and it was found that PfGEP-2 splices out Exon 4, whereas PfGEP-3 splices out Exon 3 compared to PfGEP-1. PfGEP-1 (1505 amino acids) consists of 18 granulin domains, whereas PfGEP-2 (1459 amino acids) and PfGEP-3 (1471 amino acids) consist of 17.5 granulin domains, respectively. Analyses of PfGEP-1 and PfGEP-3 mRNA showed differential patterns in the tissues and developmental stages. Western blotting results showed that the three splice variants can translate to proteins in HEK293T cells. A knockdown experiment using PfGEP dsRNA showed decreased PfGEP-1/PfGEP-3 and PfMSX mRNA, and irregular crystallization of the nacreous layer using scanning electron microscopy. In luciferase assays, co-transfection of PfGEP-1 could activate as well as repress luciferase expression of the reporter plasmid driven by the PfMSX promoter, whereas PfGEP-3 stimulated the expression, elucidating the molecular mechanisms involved in the correlation between PfGEP and PfMSX. These results suggested that GEP variants might function differently during the biomineralization process, which provides new knowledge on the mechanism regulating nacre formation.

  9. A role for coding functional variants in HNF4A in Type 2 Diabetes susceptibility

    PubMed Central

    Jafar-Mohammadi, B; Groves, C J; Gjesing, A P; Herrera, B M; Winckler, W; Stringham, H M; Morris, A P; Lauritzen, T; Doney, A S F; Morris, A D; Weedon, M N; Swift, A J; Kuusisto, J; Laakso, M; Altshuler, D; Hattersley, A T; Collins, F S; Boehnke, M; Hansen, T; Pedersen, O; Palmer, C N A; Frayling, T M; Gloyn, A L; McCarthy, M I

    2011-01-01

    Aims/hypothesis Rare mutations in the gene (HNF4A) encoding the transcription factor HNF-4A account for ~5% of cases of maturity-onset diabetes of the young (MODY) and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%, T130I, MAF ~3.0%), known to influence downstream HNF-4A target gene expression, are of interest but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. Methods We genotyped both variants in at least 5745 cases and 14756 population controls from the UK and Denmark. We also undertook an expanded association-analysis including previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14279 cases and 26835 controls. Results We found no association between V255M and type 2 diabetes in either the initial (p=0.28) or expanded analysis (p=0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [1.08-1.28]; p=1.5×10−4), which was strengthened in the meta-analysis (OR 1.20 [1.10-1.30]; p=2.1×10−5). Conclusions/interpretation Our data are consistent with T130I as a low frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low frequency variants. PMID:20878384

  10. Associations of functional alanine-glyoxylate aminotransferase 2 gene variants with atrial fibrillation and ischemic stroke

    PubMed Central

    Seppälä, Ilkka; Kleber, Marcus E.; Bevan, Steve; Lyytikäinen, Leo-Pekka; Oksala, Niku; Hernesniemi, Jussi A.; Mäkelä, Kari-Matti; Rothwell, Peter M.; Sudlow, Cathie; Dichgans, Martin; Mononen, Nina; Vlachopoulou, Efthymia; Sinisalo, Juha; Delgado, Graciela E.; Laaksonen, Reijo; Koskinen, Tuomas; Scharnagl, Hubert; Kähönen, Mika; Markus, Hugh S.; März, Winfried; Lehtimäki, Terho

    2016-01-01

    Asymmetric and symmetric dimethylarginines (ADMA and SDMA) impair nitric oxide bioavailability and have been implicated in the pathogenesis of atrial fibrillation (AF). Alanine–glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of metabolizing both of the dimethylarginines. We hypothesized that two functional AGXT2 missense variants (rs37369, V140I; rs16899974, V498L) are associated with AF and its cardioembolic complications. Association analyses were conducted using 1,834 individulas with AF and 7,159 unaffected individuals from two coronary angiography cohorts and a cohort comprising patients undergoing clinical exercise testing. In coronary angiography patients without structural heart disease, the minor A allele of rs16899974 was associated with any AF (OR = 2.07, 95% CI 1.59-2.68), and with paroxysmal AF (OR = 1.98, 95% CI 1.44–2.74) and chronic AF (OR = 2.03, 95% CI 1.35–3.06) separately. We could not replicate the association with AF in the other two cohorts. However, the A allele of rs16899974 was nominally associated with ischemic stroke risk in the meta-analysis of WTCCC2 ischemic stroke cohorts (3,548 cases, 5,972 controls) and with earlier onset of first-ever ischemic stroke (360 cases) in the cohort of clinical exercise test patients. In conclusion, AGXT2 variations may be involved in the pathogenesis of AF and its age-related thromboembolic complications. PMID:26984639

  11. Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

    SciTech Connect

    Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny; Ko, Kyung Soo; Pak, Youngmi Kim . E-mail: ymkimpak@amc.seoul.kr

    2006-06-09

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivator activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.

  12. Characterization and functional analyses of a novel chicken CD8α variant X1 (CD8α1).

    PubMed

    Truong, A D; Ban, J; Park, B; Hong, Y H; Lillehoj, H S

    2016-07-01

    We provide the first description of cloning and of structural and functional analysis of a novel variant in the chicken cluster of differentiation 8 alpha (CD8a) family, termed the CD8α X1 (CD8α1) gene. Multiple alignments of CD8α1 with known CD8α and CD8β sequences of other species revealed relatively low conservation of AA residues involved in the specific and unique structural domains among CD8α genes. For example, cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, the O-linked glycosylation sites (XPXX motif) are not found in the chicken CD8α1 sequence, and the A β strand and complementarity-determining region 1 and 2 sequences are poorly conserved between chicken CD8α1 and avian CD8α. Furthermore, the alignment showed that the transmembrane regions show relatively high sequence similarity, whereas the cytoplasmic regions show relatively low similarity, indicating poor conservation. Moreover, the motif (CXCP) that is thought to be responsible for binding the p56 lymphocyte cell kinase subunit (p56) is missing in the CD8α1 sequence. The chicken CD8α1 genomic structure is similar to that of chicken CD8α, but their protein structures differ. Phylogenetic analysis showed that chicken CD8α1 grouped with known avian CD8α sequences but was somewhat distantly related to the CD8α molecules of other species. Moreover, we analyzed the signal transduction and cytokine response to CD8α1 treatment to determine the specific biological functions of chicken CD8α1 in immune cells. The results showed that chicken CD8α1 is a key regulator of the expression of genes that are associated and cooperate with transcription factors in the major histocompatibility complex class I and II promoter regions and activates Janus kinase (JAK) 1/2, signal transducer and activator of transcription (STAT), and suppressor of cytokine signaling (SOCS) 1 signaling-related genes. Immune cells that express functional CD8α1 induce

  13. The functional influences of common ABCB1 genetic variants on the inhibition of P-glycoprotein by Antrodia cinnamomea extracts.

    PubMed

    Sheu, Ming-Jyh; Teng, Yu-Ning; Chen, Ying-Yi; Hung, Chin-Chuan

    2014-01-01

    Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51 ± 0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56 ± 0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications.

  14. Functional variants of POC5 identified in patients with idiopathic scoliosis

    PubMed Central

    Patten, Shunmoogum A.; Margaritte-Jeannin, Patricia; Bernard, Jean-Claude; Alix, Eudeline; Labalme, Audrey; Besson, Alicia; Girard, Simon L.; Fendri, Khaled; Fraisse, Nicolas; Biot, Bernard; Poizat, Coline; Campan-Fournier, Amandine; Abelin-Genevois, Kariman; Cunin, Vincent; Zaouter, Charlotte; Liao, Meijiang; Lamy, Raphaelle; Lesca, Gaetan; Menassa, Rita; Marcaillou, Charles; Letexier, Melanie; Sanlaville, Damien; Berard, Jerome; Rouleau, Guy A.; Clerget-Darpoux, Françoise; Drapeau, Pierre; Moldovan, Florina; Edery, Patrick

    2015-01-01

    Idiopathic scoliosis (IS) is a spine deformity that affects approximately 3% of the population. The underlying causes of IS are not well understood, although there is clear evidence that there is a genetic component to the disease. Genetic mapping studies suggest high genetic heterogeneity, but no IS disease-causing gene has yet been identified. Here, genetic linkage analyses combined with exome sequencing identified a rare missense variant (p.A446T) in the centriolar protein gene POC5 that cosegregated with the disease in a large family with multiple members affected with IS. Subsequently, the p.A446T variant was found in an additional set of families with IS and in an additional 3 cases of IS. Moreover, POC5 variant p.A455P was present and linked to IS in one family and another rare POC5 variant (p.A429V) was identified in an additional 5 cases of IS. In a zebrafish model, expression of any of the 3 human IS-associated POC5 variant mRNAs resulted in spine deformity, without affecting other skeletal structures. Together, these findings indicate that mutations in the POC5 gene contribute to the occurrence of IS. PMID:25642776

  15. Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects

    PubMed Central

    Johnson, Ben; Lowe, Gillian C.; Futterer, Jane; Lordkipanidzé, Marie; MacDonald, David; Simpson, Michael A.; Sanchez-Guiú, Isabel; Drake, Sian; Bem, Danai; Leo, Vincenzo; Fletcher, Sarah J.; Dawood, Ban; Rivera, José; Allsup, David; Biss, Tina; Bolton-Maggs, Paula HB; Collins, Peter; Curry, Nicola; Grimley, Charlotte; James, Beki; Makris, Mike; Motwani, Jayashree; Pavord, Sue; Talks, Katherine; Thachil, Jecko; Wilde, Jonathan; Williams, Mike; Harrison, Paul; Gissen, Paul; Mundell, Stuart; Mumford, Andrew; Daly, Martina E.; Watson, Steve P.; Morgan, Neil V.

    2016-01-01

    Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified “pathogenic” or “likely pathogenic” variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia. PMID:27479822

  16. Plant growth-promoting rhizobacteria and root system functioning.

    PubMed

    Vacheron, Jordan; Desbrosses, Guilhem; Bouffaud, Marie-Lara; Touraine, Bruno; Moënne-Loccoz, Yvan; Muller, Daniel; Legendre, Laurent; Wisniewski-Dyé, Florence; Prigent-Combaret, Claire

    2013-01-01

    The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Among the latter, plant growth-promoting rhizobacteria (PGPR) colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by PGPR implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. PGPR also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Recent results provided first clues as to how PGPR signals could trigger these plant responses. Whether local and/or systemic, the plant molecular pathways involved remain often unknown. From an ecological point of view, it emerged that PGPR form coherent functional groups, whose rhizosphere ecology is influenced by a myriad of abiotic and biotic factors in natural and agricultural soils, and these factors can in turn modulate PGPR effects on roots. In this paper, we address novel knowledge and gaps on PGPR modes of action and signals, and highlight recent progress on the links between plant morphological and physiological effects induced by PGPR. We also show the importance of taking into account the size, diversity, and gene expression patterns of PGPR assemblages in the rhizosphere to better understand their impact on plant growth and functioning. Integrating mechanistic and ecological knowledge on PGPR populations in soil will be a prerequisite to develop novel management strategies for sustainable agriculture.

  17. Plant growth-promoting rhizobacteria and root system functioning

    PubMed Central

    Vacheron, Jordan; Desbrosses, Guilhem; Bouffaud, Marie-Lara; Touraine, Bruno; Moënne-Loccoz, Yvan; Muller, Daniel; Legendre, Laurent; Wisniewski-Dyé, Florence; Prigent-Combaret, Claire

    2013-01-01

    The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Among the latter, plant growth-promoting rhizobacteria (PGPR) colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by PGPR implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. PGPR also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Recent results provided first clues as to how PGPR signals could trigger these plant responses. Whether local and/or systemic, the plant molecular pathways involved remain often unknown. From an ecological point of view, it emerged that PGPR form coherent functional groups, whose rhizosphere ecology is influenced by a myriad of abiotic and biotic factors in natural and agricultural soils, and these factors can in turn modulate PGPR effects on roots. In this paper, we address novel knowledge and gaps on PGPR modes of action and signals, and highlight recent progress on the links between plant morphological and physiological effects induced by PGPR. We also show the importance of taking into account the size, diversity, and gene expression patterns of PGPR assemblages in the rhizosphere to better understand their impact on plant growth and functioning. Integrating mechanistic and ecological knowledge on PGPR populations in soil will be a prerequisite to develop novel management strategies for sustainable agriculture. PMID:24062756

  18. Smoking Gun or Circumstantial Evidence? Comparison of Statistical Learning Methods using Functional Annotations for Prioritizing Risk Variants.

    PubMed

    Gagliano, Sarah A; Ravji, Reena; Barnes, Michael R; Weale, Michael E; Knight, Jo

    2015-08-24

    Although technology has triumphed in facilitating routine genome sequencing, new challenges have been created for the data-analyst. Genome-scale surveys of human variation generate volumes of data that far exceed capabilities for laboratory characterization. By incorporating functional annotations as predictors, statistical learning has been widely investigated for prioritizing genetic variants likely to be associated with complex disease. We compared three published prioritization procedures, which use different statistical learning algorithms and different predictors with regard to the quantity, type and coding. We also explored different combinations of algorithm and annotation set. As an application, we tested which methodology performed best for prioritizing variants using data from a large schizophrenia meta-analysis by the Psychiatric Genomics Consortium. Results suggest that all methods have considerable (and similar) predictive accuracies (AUCs 0.64-0.71) in test set data, but there is more variability in the application to the schizophrenia GWAS. In conclusion, a variety of algorithms and annotations seem to have a similar potential to effectively enrich true risk variants in genome-scale datasets, however none offer more than incremental improvement in prediction. We discuss how methods might be evolved for risk variant prediction to address the impending bottleneck of the new generation of genome re-sequencing studies.

  19. Smoking Gun or Circumstantial Evidence? Comparison of Statistical Learning Methods using Functional Annotations for Prioritizing Risk Variants

    PubMed Central

    Gagliano, Sarah A.; Ravji, Reena; Barnes, Michael R.; Weale, Michael E.; Knight, Jo

    2015-01-01

    Although technology has triumphed in facilitating routine genome sequencing, new challenges have been created for the data-analyst. Genome-scale surveys of human variation generate volumes of data that far exceed capabilities for laboratory characterization. By incorporating functional annotations as predictors, statistical learning has been widely investigated for prioritizing genetic variants likely to be associated with complex disease. We compared three published prioritization procedures, which use different statistical learning algorithms and different predictors with regard to the quantity, type and coding. We also explored different combinations of algorithm and annotation set. As an application, we tested which methodology performed best for prioritizing variants using data from a large schizophrenia meta-analysis by the Psychiatric Genomics Consortium. Results suggest that all methods have considerable (and similar) predictive accuracies (AUCs 0.64–0.71) in test set data, but there is more variability in the application to the schizophrenia GWAS. In conclusion, a variety of algorithms and annotations seem to have a similar potential to effectively enrich true risk variants in genome-scale datasets, however none offer more than incremental improvement in prediction. We discuss how methods might be evolved for risk variant prediction to address the impending bottleneck of the new generation of genome re-sequencing studies. PMID:26300220

  20. Two variants of the Drosophila melanogaster retrotransposon gypsy (mdg4): structural and functional differences, and distribution in fly stocks.

    PubMed

    Lyubomirskaya, N V; Smirnova, J B; Razorenova, O V; Karpova, N N; Surkov, S A; Avedisov, S N; Kim, A I; Ilyin, Y V

    2001-04-01

    Two variants of the Drosophila melanogaster retrotransposon gypsy were subjected to detailed structural and functional analysis. A series of hybrid constructs containing various combinations of "active" and "inactive" gypsy copies were tested for their ability to produce new DNA copies in cultured cells by means of reverse transcription. It was shown that the previously demonstrated variations in retrotranspositional activity are associated with either one or both of two amino acid substitutions at the beginning of ORF2. The first substitution is located at the boundary between the putative protease and reverse transcriptase domains and, hence, may influence the processing of the polyprotein. The other substitution may alter reverse transcriptase activity since it is located in the second of the seven conserved domains of the RT gene. To address the question of the evolutionary relationship between the two gypsy variants, their distribution was analyzed in among various fly stocks. Southern analysis revealed that all D. melanogaster strains studied so far contain the "inactive" gypsy variant, while the "active" copies are present only in some strains; most of the latter were established from flies recently isolated from natural populations. Finally, in stocks carrying the flamenco mutation the "active" gypsy variant is much more abundant than the "inactive" form. Possible scenarios for the orgin of the "active" form of gypsy are discussed. PMID:11361349

  1. Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus.

    PubMed

    Dimarco, Eufrosina; Cascone, Eleonora; Bellavia, Daniele; Caradonna, Fabio

    2012-10-15

    We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a feature that had been considered a peculiarity of some plants. PMID:22967708

  2. Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation.

    PubMed

    Hammes, A; Guo, J K; Lutsch, G; Leheste, J R; Landrock, D; Ziegler, U; Gubler, M C; Schedl, A

    2001-08-10

    Alternative splicing of Wt1 results in the insertion or omission of the three amino acids KTS between zinc fingers 3 and 4. In vitro experiments suggest distinct molecular functions for + and -KTS isoforms. We have generated mouse strains in which specific isoforms have been removed. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. Homozygous mutants of both strains die after birth due to kidney defects. Strikingly, mice lacking +KTS isoforms show a complete XY sex reversal due to a dramatic reduction of Sry expression levels. Our data demonstrate distinct functions for the two splice variants and place the +KTS variants as important regulators for Sry in the sex determination pathway. PMID:11509181

  3. Functional variants of the interleukin-23 receptor gene in non-gastrointestinal autoimmune diseases.

    PubMed

    Safrany, E; Melegh, B

    2009-01-01

    Variants of the gene of the interleukin-23 receptor (IL23R) were first identified as susceptibility factors in association with inflammatory bowel diseases. Since then it became clear that different variants of the gene play also role in a number of other autoimmune diseases like psoriasis, rheumatoid arthritis, ankylosing spondylitis and multiple sclerosis while in others, like systemic sclerosis, systemic lupus erythematosus or Sjögren syndrome the same effect could not be seen. However, the results are very controversial both in terms of the various polymorphisms and also in population specificity. The aim of the current paper is to overview all available reports on IL23R gene polymorphisms in various autoimmune and inflammatory diseases and to try to give an explanation on the possible effect of the examined variants.

  4. In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population

    PubMed Central

    2012-01-01

    Background The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism. Methods We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections. Results We identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function. Conclusions The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised. PMID:23217119

  5. In Silico Structural and Functional Characterization of the RSUME Splice Variants

    PubMed Central

    Tedesco, Lucas; Silberstein, Susana; Sevlever, Gustavo; Paez-Pereda, Marcelo; Holsboer, Florian; Turjanski, Adrián G.; Arzt, Eduardo

    2013-01-01

    RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas. PMID:23469069

  6. Examining quality function deployment in safety promotion in Sweden.

    PubMed

    Kullberg, Agneta; Nordqvist, Cecilia; Lindqvist, Kent; Timpka, Toomas

    2014-09-01

    The first-hand needs and demands of laypersons are not always considered when safety promotion programmes are being developed. We compared focal areas for interventions identified from residents' statements of safety needs with focal areas for interventions identified by local government professionals in a Swedish urban community certified by the international Safe Community movement supported by the World Health Organization. Quantitative and qualitative data on self-expressed safety needs from 787 housing residents were transformed into an intervention design, using the quality function deployment (QFD) technique and compared with the safety intervention programme developed by professionals at the municipality administrative office. The outcome of the comparison was investigated with regard to implications for the Safe Community movement. The QFD analysis identified the initiation and maintenance of social integrative processes in housing areas as the most highly prioritized interventions among the residents, but failed to highlight the safety needs of several vulnerable groups (the elderly, infants and persons with disabilities). The intervention programme designed by the public health professionals did not address the social integrative processes, but it did highlight the vulnerable groups. This study indicates that the QFD technique is suitable for providing residential safety promotion efforts with a quality orientation from the layperson's perspective. Views of public health professionals have to be included to ascertain that the needs of socially deprived residents are adequately taken into account. QFD can augment the methodological toolbox for safety promotion programmes, including interventions in residential areas. PMID:23322486

  7. Examining quality function deployment in safety promotion in Sweden.

    PubMed

    Kullberg, Agneta; Nordqvist, Cecilia; Lindqvist, Kent; Timpka, Toomas

    2014-09-01

    The first-hand needs and demands of laypersons are not always considered when safety promotion programmes are being developed. We compared focal areas for interventions identified from residents' statements of safety needs with focal areas for interventions identified by local government professionals in a Swedish urban community certified by the international Safe Community movement supported by the World Health Organization. Quantitative and qualitative data on self-expressed safety needs from 787 housing residents were transformed into an intervention design, using the quality function deployment (QFD) technique and compared with the safety intervention programme developed by professionals at the municipality administrative office. The outcome of the comparison was investigated with regard to implications for the Safe Community movement. The QFD analysis identified the initiation and maintenance of social integrative processes in housing areas as the most highly prioritized interventions among the residents, but failed to highlight the safety needs of several vulnerable groups (the elderly, infants and persons with disabilities). The intervention programme designed by the public health professionals did not address the social integrative processes, but it did highlight the vulnerable groups. This study indicates that the QFD technique is suitable for providing residential safety promotion efforts with a quality orientation from the layperson's perspective. Views of public health professionals have to be included to ascertain that the needs of socially deprived residents are adequately taken into account. QFD can augment the methodological toolbox for safety promotion programmes, including interventions in residential areas.

  8. Neanderthal origin of the haplotypes carrying the functional variant Val92Met in the MC1R in modern humans.

    PubMed

    Ding, Qiliang; Hu, Ya; Xu, Shuhua; Wang, Chuan-Chao; Li, Hui; Zhang, Ruyue; Yan, Shi; Wang, Jiucun; Jin, Li

    2014-08-01

    Skin color is one of the most visible and important phenotypes of modern humans. Melanocyte-stimulating hormone and its receptor played an important role in regulating skin color. In this article, we present evidence of Neanderthal introgression encompassing the melanocyte-stimulating hormone receptor gene MC1R. The haplotypes from Neanderthal introgression diverged with the Altai Neanderthal 103.3 ka, which postdates the anatomically modern human-Neanderthal divergence. We further discovered that all of the putative Neanderthal introgressive haplotypes carry the Val92Met variant, a loss-of-function variant in MC1R that is associated with multiple dermatological traits including skin color and photoaging. Frequency of this Neanderthal introgression is low in Europeans (∼5%), moderate in continental East Asians (∼30%), and high in Taiwanese aborigines (60-70%). As the putative Neanderthal introgressive haplotypes carry a loss-of-function variant that could alter the function of MC1R and is associated with multiple traits related to skin color, we speculate that the Neanderthal introgression may have played an important role in the local adaptation of Eurasians to sunlight intensity.

  9. Fatal neonatal encephalopathy and lactic acidosis caused by a homozygous loss-of-function variant in COQ9.

    PubMed

    Danhauser, Katharina; Herebian, Diran; Haack, Tobias B; Rodenburg, Richard J; Strom, Tim M; Meitinger, Thomas; Klee, Dirk; Mayatepek, Ertan; Prokisch, Holger; Distelmaier, Felix

    2016-03-01

    Coenzyme Q10 (CoQ10) has an important role in mitochondrial energy metabolism by way of its functioning as an electron carrier in the respiratory chain. Genetic defects disrupting the endogenous biosynthesis pathway of CoQ10 may lead to severe metabolic disorders with onset in early childhood. Using exome sequencing in a child with fatal neonatal lactic acidosis and encephalopathy, we identified a homozygous loss-of-function variant in COQ9. Functional studies in patient fibroblasts showed that the absence of the COQ9 protein was concomitant with a strong reduction of COQ7, leading to a significant accumulation of the substrate of COQ7, 6-demethoxy ubiquinone10. At the same time, the total amount of CoQ10 was severely reduced, which was reflected in a significant decrease of mitochondrial respiratory chain succinate-cytochrome c oxidoreductase (complex II/III) activity. Lentiviral expression of COQ9 restored all these parameters, confirming the causal role of the variant. Our report on the second COQ9 patient expands the clinical spectrum associated with COQ9 variants, indicating the importance of COQ9 already during prenatal development. Moreover, the rescue of cellular CoQ10 levels and respiratory chain complex activities by CoQ10 supplementation points to the importance of an early diagnosis and immediate treatment. PMID:26081641

  10. Structure and functional regulation of the CD38 promoter

    SciTech Connect

    Sun Li; Iqbal, Jameel; Zaidi, Samir; Zhu Linglng; Zhang Xuefeng; Peng Yuanzheng; Moonga, Baljit S.; Zaidi, Mone . E-mail: mone.zaidi@mssm.edu

    2006-03-17

    CD38 has multiple roles in biology, including T lymphocyte signaling, neutrophil migration, neurotransmission, cell proliferation, apoptosis, and bone remodeling. To study the transcriptional control of the CD38 gene, we cloned a putative 1.8 kb promoter fragment from a rabbit genomic DNA library. Primer extension analysis indicated two transcription start sites consistent with the absence of a TATA box. Sequence analysis revealed several AP-1, AP-4, myo-D, GATA, and SP-1 sequences. MC3T3.E1 (osteoblast) or RAW-C3 (osteoclast precursor macrophage) cells were then transfected with the CD38 promoter or its deletion fragments ligated to the luciferase reporter gene. Except for the shortest 41 bp fragment, all fragments showed significant luciferase activity. There was a marked stimulation of basal activity in the 93 bp fragment that contained a GC box and SP-1 site. Furthermore, there were significant differences in the activity of the fragments in MC3T3.E1 and RAW-C3 cells. Intracellular Ca{sup 2+} elevations by ionomycin (10 {mu}M) in MC3T3.E1 cells inhibited promoter activity, except in the short 41 bp. In contrast, cAMP elevation by exposure to forskolin (100 {mu}M) inhibited activation of all fragments, except the 0.6 and 1.2 kb fragments. Finally, TNF-{alpha} stimulated promoter activity in RAW-C3 cells transfected with the 93 bp and 1.0 kb fragments, consistent with the stimulation of CD38 mRNA by TNF-{alpha}. Physiologically, therefore, modulation of the expression of the NAD{sup +}-sensing enzyme, CD38, by Ca{sup 2+}, cAMP, and cytokines, such as TNF-{alpha} may contribute to coupling the intense metabolic activity of osteoclasts and osteoblasts to their respective bone-resorbing and bone-forming functions.

  11. An interaction quantitative trait loci tool implicates epistatic functional variants in an apoptosis pathway in smallpox vaccine eQTL data.

    PubMed

    Lareau, C A; White, B C; Oberg, A L; Kennedy, R B; Poland, G A; McKinney, B A

    2016-06-01

    Expression quantitative trait loci (eQTL) studies have functionalized nucleic acid variants through the regulation of gene expression. Although most eQTL studies only examine the effects of single variants on transcription, a more complex process of variant-variant interaction (epistasis) may regulate transcription. Herein, we describe a tool called interaction QTL (iQTL) designed to efficiently detect epistatic interactions that regulate gene expression. To maximize biological relevance and minimize the computational and hypothesis testing burden, iQTL restricts interactions such that one variant is within a user-defined proximity of the transcript (cis-regulatory). We apply iQTL to a data set of 183 smallpox vaccine study participants with genome-wide association study and gene expression data from unstimulated samples and samples stimulated by inactivated vaccinia virus. While computing only 0.15% of possible interactions, we identify 11 probe sets whose expression is regulated through a variant-variant interaction. We highlight the functional epistatic interactions among apoptosis-related genes, DIABLO, TRAPPC4 and FADD, in the context of smallpox vaccination. We also use an integrative network approach to characterize these iQTL interactions in a posterior network of known prior functional interactions. iQTL is an efficient, open-source tool to analyze variant interactions in eQTL studies, providing better understanding of the function of epistasis in immune response and other complex phenotypes. PMID:27052692

  12. Nitrites derived from Foneiculum vulgare (fennel) seeds promotes vascular functions.

    PubMed

    Swaminathan, Akila; Sridhara, Sree Rama Chaitanya; Sinha, Swaraj; Nagarajan, Shunmugam; Balaguru, Uma Maheswari; Siamwala, Jamila H; Rajendran, Saranya; Saran, Uttara; Chatterjee, Suvro

    2012-12-01

    Recent evidence has demonstrated that nitrites play an important role in the cardiovascular system. Fennel (Foneiculum vulgare) seeds are often used as mouth fresheners after a meal in both the Indian sub-continent and around the world. The present study aims to quantify the nitrite and nitrates in fennel seeds as well as elucidating the effect of fennel derived-nitrites on vascular functions. Results from our study show that fennel seeds contain significantly higher amount of nitrites when compared to other commonly used post-meal seeds. Furthermore our study confirmed the functional effects of fennel derived-nitrites using in vitro and ex vivo models that describe the promotion of angiogenesis, cell migration, and vasorelaxation. We also showed that chewing fennel seeds enhanced nitrite content of saliva. Thus our study indicates the potential role of fennel derived-nitrites on the vascular system.

  13. Expression pattern and function of alternative splice variants of glutamate-gated chloride channel in the housefly Musca domestica.

    PubMed

    Kita, Tomo; Ozoe, Fumiyo; Ozoe, Yoshihisa

    2014-02-01

    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. cDNAs encoding two alternative splice variants (MdGluClB and C) of the GluCl subunit were cloned from the housefly Musca domestica. The expression patterns of three variants, including the previously reported MdGluClA, differed among the body parts (head, thorax, abdomen, and leg) of the adult housefly and among developmental stages (embryo, larva, pupa, and adult). The MdGluClA and B transcripts were abundant in the central nervous system of the adult, whereas the MdGluClC transcript was expressed in the central nervous system and as the predominant variant in the peripheral tissues. The sensitivities to the agonist glutamate and the allosteric activator ivermectin B1a did not differ between channels containing MdGluCl variants when they were singly or co-expressed in Xenopus oocytes. By contrast, MdGluClA and B channels were more sensitive to the channel blockers fipronil and picrotoxinin than was MdGluClC channels. Heteromeric channels containing different subunit variants were more sensitive to picrotoxinin than were homomeric channels. Heteromeric channels were more sensitive to fipronil than were homomeric MdGluClC channels but not than homomeric MdGluClA and B channels. These results suggest that functionally indistinguishable but pharmacologically distinct GluCls are expressed in a spatially and temporally distinct manner in the housefly. PMID:24291284

  14. Characterization of a cancer cell line that expresses a splicing variant form of 53BP1: Separation of checkpoint and repair functions in 53BP1

    SciTech Connect

    Iwabuchi, Kuniyoshi Matsui, Tadashi; Hashimoto, Mitsumasa; Matsumoto, Yoshihisa; Kurihara, Takayuki; Date, Takayasu

    2008-11-21

    53BP1 plays important roles in checkpoint signaling and repair for DNA double-strand breaks. We found that a colon cancer cell line, SW48, expressed a splicing variant form of 53BP1, which lacks the residues corresponding to exons 10 and 11. Activation of ATM and phosphorylation of ATM and ATR targets occurred in SW48 cells in response to X-irradiation, and these X-ray-induced responses were not enhanced by expression of full-length 53BP1 in SW48 cells, indicating that this splicing variant fully activates the major checkpoint signaling in SW48 cells. In contrast, the expression of full-length 53BP1 in SW48 cells promoted the repair of X-ray-induced DNA damage, evidenced by faster disappearance of X-ray-induced {gamma}-H2AX foci, a marker for DNA damage, and less residual chromosomal aberrations after X-irradiation. We conclude that the two major roles of 53BP1, the checkpoint signaling and repair for DNA damage, can be functionally separated.

  15. The functional "KL-VS" variant of KLOTHO is not associated with type 2 diabetes in 5028 UK Caucasians

    PubMed Central

    Freathy, Rachel M; Weedon, Michael N; Melzer, David; Shields, Beverley; Hitman, Graham A; Walker, Mark; McCarthy, Mark I; Hattersley, Andrew T; Frayling, Timothy M

    2006-01-01

    Background Klotho has an important role in insulin signalling and the development of ageing-like phenotypes in mice. The common functional "KL-VS" variant in the KLOTHO (KL) gene is associated with longevity in humans but its role in type 2 diabetes is not known. We performed a large case-control and family-based study to test the hypothesis that KL-VS is associated with type 2 diabetes in a UK Caucasian population. Methods We genotyped 1793 cases, 1619 controls and 1616 subjects from 509 families for the single nucleotide polymorphism (SNP) F352V (rs9536314) that defines the KL-VS variant. Allele and genotype frequencies were compared between cases and controls. Family-based analysis was used to test for over- or under-transmission of V352 to affected offspring. Results Despite good power to detect odds ratios of 1.2, there were no significant associations between alleles or genotypes and type 2 diabetes (V352 allele: odds ratio = 0.96 (0.84–1.09)). Additional analysis of quantitative trait data in 1177 healthy control subjects showed no association of the variant with fasting insulin, glucose, triglycerides, HDL- or LDL-cholesterol (all P > 0.05). However, the HDL-cholesterol levels observed across the genotype groups showed a similar, but non-significant, pattern to previously reported data. Conclusion This is the first large-scale study to examine the association between common functional variation in KL and type 2 diabetes risk. We have found no evidence that the functional KL-VS variant is a risk factor for type 2 diabetes in a large UK Caucasian case-control and family-based study. PMID:16753056

  16. The evolution and functional impact of human deletion variants shared with archaic hominin genomes.

    PubMed

    Lin, Yen-Lung; Pavlidis, Pavlos; Karakoc, Emre; Ajay, Jerry; Gokcumen, Omer

    2015-04-01

    Allele sharing between modern and archaic hominin genomes has been variously interpreted to have originated from ancestral genetic structure or through non-African introgression from archaic hominins. However, evolution of polymorphic human deletions that are shared with archaic hominin genomes has yet to be studied. We identified 427 polymorphic human deletions that are shared with archaic hominin genomes, approximately 87% of which originated before the Human-Neandertal divergence (ancient) and only approximately 9% of which have been introgressed from Neandertals (introgressed). Recurrence, incomplete lineage sorting between human and chimp lineages, and hominid-specific insertions constitute the remaining approximately 4% of allele sharing between humans and archaic hominins. We observed that ancient deletions correspond to more than 13% of all common (>5% allele frequency) deletion variation among modern humans. Our analyses indicate that the genomic landscapes of both ancient and introgressed deletion variants were primarily shaped by purifying selection, eliminating large and exonic variants. We found 17 exonic deletions that are shared with archaic hominin genomes, including those leading to three fusion transcripts. The affected genes are involved in metabolism of external and internal compounds, growth and sperm formation, as well as susceptibility to psoriasis and Crohn's disease. Our analyses suggest that these "exonic" deletion variants have evolved through different adaptive forces, including balancing and population-specific positive selection. Our findings reveal that genomic structural variants that are shared between humans and archaic hominin genomes are common among modern humans and can influence biomedically and evolutionarily important phenotypes.

  17. Patterns and functional implications of rare germline variants across 12 cancer types

    PubMed Central

    Lu, Charles; Xie, Mingchao; Wendl, Michael C.; Wang, Jiayin; McLellan, Michael D.; Leiserson, Mark D. M.; Huang, Kuan-lin; Wyczalkowski, Matthew A.; Jayasinghe, Reyka; Banerjee, Tapahsama; Ning, Jie; Tripathi, Piyush; Zhang, Qunyuan; Niu, Beifang; Ye, Kai; Schmidt, Heather K.; Fulton, Robert S.; McMichael, Joshua F.; Batra, Prag; Kandoth, Cyriac; Bharadwaj, Maheetha; Koboldt, Daniel C.; Miller, Christopher A.; Kanchi, Krishna L.; Eldred, James M.; Larson, David E.; Welch, John S.; You, Ming; Ozenberger, Bradley A.; Govindan, Ramaswamy; Walter, Matthew J.; Ellis, Matthew J.; Mardis, Elaine R.; Graubert, Timothy A.; Dipersio, John F.; Ley, Timothy J.; Wilson, Richard K.; Goodfellow, Paul J.; Raphael, Benjamin J.; Chen, Feng; Johnson, Kimberly J.; Parvin, Jeffrey D.; Ding, Li

    2015-01-01

    Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine. PMID:26689913

  18. Patterns and functional implications of rare germline variants across 12 cancer types.

    PubMed

    Lu, Charles; Xie, Mingchao; Wendl, Michael C; Wang, Jiayin; McLellan, Michael D; Leiserson, Mark D M; Huang, Kuan-Lin; Wyczalkowski, Matthew A; Jayasinghe, Reyka; Banerjee, Tapahsama; Ning, Jie; Tripathi, Piyush; Zhang, Qunyuan; Niu, Beifang; Ye, Kai; Schmidt, Heather K; Fulton, Robert S; McMichael, Joshua F; Batra, Prag; Kandoth, Cyriac; Bharadwaj, Maheetha; Koboldt, Daniel C; Miller, Christopher A; Kanchi, Krishna L; Eldred, James M; Larson, David E; Welch, John S; You, Ming; Ozenberger, Bradley A; Govindan, Ramaswamy; Walter, Matthew J; Ellis, Matthew J; Mardis, Elaine R; Graubert, Timothy A; Dipersio, John F; Ley, Timothy J; Wilson, Richard K; Goodfellow, Paul J; Raphael, Benjamin J; Chen, Feng; Johnson, Kimberly J; Parvin, Jeffrey D; Ding, Li

    2015-01-01

    Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine. PMID:26689913

  19. BANK1 functional variants are associated with susceptibility to diffuse systemic sclerosis in Caucasians

    PubMed Central

    Rueda, B; Gourh, P; Broen, J; Agarwal, S K; Simeon, C; Ortego-Centeno, N; Vonk, M C; Coenen, M; Riemekasten, G; Hunzelmann, N; Hesselstrand, R; Tan, F K; Reveille, J D; Assassi, S; Garcia-Hernandez, F J; Carreira, P; Camps, M; Fernandez-Nebro, A; de la Peña, P Garcia; Nearney, T; Hilda, D; Gónzalez-Gay, M A; Airo, P; Beretta, L; Scorza, R; Radstake, T R D J; Mayes, M D; Arnett, F C; Martin, J

    2010-01-01

    Objective To investigate the possible association of the BANK1 gene with genetic susceptibility to systemic sclerosis (SSc) and its subphenotypes. Methods A large multicentre case–control association study including 2380 patients with SSc and 3270 healthy controls from six independent case–control sets of Caucasian ancestry (American, Spanish, Dutch, German, Swedish and Italian) was conducted. Three putative functional BANK1 polymorphisms (rs17266594 T/C, rs10516487 G/A, rs3733197 G/A) were selected as genetic markers and genotyped by Taqman 5´ allelic discrimination assay. Results A significant association of the rs10516487 G and rs17266594 T alleles with SSc susceptibility was observed (pooled OR=1.12, 95% CI 1.03 to 1.22; p=0.01 and pooled OR=1.14, 95% CI 1.05 to 1.25; p=0.003, respectively), whereas the rs3733197 genetic variant showed no statistically significant deviation. Stratification for cutaneous SSc phenotype showed that the BANK1 rs10516487 G, rs17266594 T and rs3733197 G alleles were strongly associated with susceptibility to diffuse SSc (dcSSc) (pooled OR=1.20, 95% CI 1.05 to 1.37, p=0.005; pooled OR=1.23, 95% CI 1.08 to 1.41, p=0.001; pooled OR=1.15, 95% CI 1.02 to 1.31, p=0.02, respectively). Similarly, stratification for specific SSc autoantibodies showed that the association of BANK1 rs10516487, rs17266594 and rs3733197 polymorphisms was restricted to the subgroup of patients carrying anti-topoisomerase I antibodies (pooled OR=1.20, 95% CI 1.02 to 1.41, p=0.03; pooled OR=1.24, 95% CI 1.05 to 1.46, p=0.01; pooled OR=1.26, 95% CI 1.07 to 1.47, p=0.004, respectively). Conclusion The results suggest that the BANK1 gene confers susceptibility to SSc in general, and specifically to the dcSSc and anti-topoisomerase I antibody subsets. PMID:19815934

  20. Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma.

    PubMed

    Pan, Wenting; Yang, Jinyun; Wei, Jinyu; Chen, Hongwei; Ge, Yunxia; Zhang, Jingfeng; Wang, Zhiqiong; Zhou, Changchun; Yuan, Qipeng; Zhou, Liqing; Yang, Ming

    2015-01-01

    B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57-0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29-0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32-0.62, P =  .002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations. PMID:26132559

  1. RBP-Var: a database of functional variants involved in regulation mediated by RNA-binding proteins

    PubMed Central

    Mao, Fengbiao; Xiao, Luoyuan; Li, Xianfeng; Liang, Jialong; Teng, Huajing; Cai, Wanshi; Sun, Zhong Sheng

    2016-01-01

    Transcription factors bind to the genome by forming specific contacts with the primary DNA sequence; however, RNA-binding proteins (RBPs) have greater scope to achieve binding specificity through the RNA secondary structure. It has been revealed that single nucleotide variants (SNVs) that alter RNA structure, also known as RiboSNitches, exhibit 3-fold greater local structure changes than replicates of the same DNA sequence, demonstrated by the fact that depletion of RiboSNitches could result in the alteration of specific RNA shapes at thousands of sites, including 3′ UTRs, binding sites of microRNAs and RBPs. However, the network between SNVs and post-transcriptional regulation remains unclear. Here, we developed RBP-Var, a database freely available at http://www.rbp-var.biols.ac.cn/, which provides annotation of functional variants involved in post-transcriptional interaction and regulation. RBP-Var provides an easy-to-use web interface that allows users to rapidly find whether SNVs of interest can transform the secondary structure of RNA and identify RBPs whose binding may be subsequently disrupted. RBP-Var integrates DNA and RNA biology to understand how various genetic variants and post-transcriptional mechanisms cooperate to orchestrate gene expression. In summary, RBP-Var is useful in selecting candidate SNVs for further functional studies and exploring causal SNVs underlying human diseases. PMID:26635394

  2. Loss of functional connectivity is greater outside the default mode network in nonfamilial early-onset Alzheimer's disease variants.

    PubMed

    Lehmann, Manja; Madison, Cindee; Ghosh, Pia M; Miller, Zachary A; Greicius, Michael D; Kramer, Joel H; Coppola, Giovanni; Miller, Bruce L; Jagust, William J; Gorno-Tempini, Maria L; Seeley, William W; Rabinovici, Gil D

    2015-10-01

    The common and specific involvement of brain networks in clinical variants of Alzheimer's disease (AD) is not well understood. We performed task-free ("resting-state") functional imaging in 60 nonfamilial AD patients, including 20 early-onset AD (age at onset <65 years, amnestic/dysexecutive deficits), 24 logopenic aphasia (language deficits), and 16 posterior cortical atrophy patients (visual deficits), as well as 60 healthy controls. Seed-based connectivity analyses were conducted to assess differences between groups in 3 default mode network (DMN) components (anterior, posterior, and ventral) and 4 additional non-DMN networks: left and right executive-control, language, and higher visual networks. Significant decreases in connectivity were found across AD variants compared with controls in the non-DMN networks. Within the DMN components, patients showed higher connectivity in the anterior DMN, in particular in logopenic aphasia. No significant differences were found for the posterior and ventral DMN. Our findings suggest that loss of functional connectivity is greatest in networks outside the DMN in early-onset and nonamnestic AD variants and may thus be a better biomarker in these patients. PMID:26242705

  3. Key Functional Regions in the Histone Variant H2A.Z C-Terminal Docking Domain ▿

    PubMed Central

    Wang, Alice Y.; Aristizabal, Maria J.; Ryan, Colm; Krogan, Nevan J.; Kobor, Michael S.

    2011-01-01

    The incorporation of histone variants into nucleosomes represents one way of altering the chromatin structure to accommodate diverse functions. Histone variant H2A.Z has specific roles in gene regulation, heterochromatin boundary formation, and genomic integrity. The precise features required for H2A.Z to function and specify an identity different from canonical H2A remain to be fully explored. Analysis of the C-terminal docking domain of H2A.Z in Saccharomyces cerevisiae using epistatic miniarray profile (E-MAP) uncovered nuanced requirements of the H2A.Z C-terminal region for cell growth when additional genes were compromised. Moreover, the H2A.Z(1–114) truncation, lacking the last 20 amino acids of the protein, did not support regular H2A.Z functions, such as resistance to genotoxic stress, restriction of heterochromatin in its native context, GAL1 gene activation, and chromatin anchoring. The corresponding region of H2A could fully rescue the strong defects caused by loss of this functionally essential region in the C terminus of H2A.Z. Despite the dramatic reduction in function, the H2A.Z(1–114) truncation still bound the H2A.Z deposition complex SWR1-C, the histone chaperone Chz1, and histone H2B. These data are consistent with a model in which retaining the variant in chromatin after its deposition by SWR1-C is a crucial determinant of its function. PMID:21791612

  4. Two Functional Variants of IRF5 Influence the Development of Macular Edema in Patients with Non-Anterior Uveitis

    PubMed Central

    Cordero-Coma, Miguel; Ortego-Centeno, Norberto; Adán, Alfredo; Fonollosa, Alejandro; Díaz Valle, David; Pato, Esperanza; Blanco, Ricardo; Cañal, Joaquín; Díaz-Llopis, Manuel; de Ramón, Enrique; del Rio, María José; García Serrano, José Luis; Artaraz, Joseba; Martín-Villa, José Manuel; Llorenç, Víctor; Gorroño-Echebarría, Marina Begoña; Martín, Javier

    2013-01-01

    Objective Interferon (IFN) signaling plays a crucial role in autoimmunity. Genetic variation in interferon regulatory factor 5 (IRF5), a major regulator of the type I interferon induction, has been associated with risk of developing several autoimmune diseases. In the current study we aimed to evaluate whether three sets of correlated IRF5 genetic variants, independently associated with SLE and with different functional roles, are involved in uveitis susceptibility and its clinical subphenotypes. Methods Three IRF5 polymorphisms, rs2004640, rs2070197 and rs10954213, representative of each group, were genotyped using TaqMan® allelic discrimination assays in a total of 263 non-anterior uveitis patients and 724 healthy controls of Spanish origin. Results A clear association between two of the three analyzed genetic variants, rs2004640 and rs10954213, and the absence of macular edema was observed in the case/control analysis (PFDR=5.07E-03, OR=1.48, CI 95%=1.14-1.92 and PFDR=3.37E-03, OR=1.54, CI 95%=1.19-2.01, respectively). Consistently, the subphenotype analysis accordingly with the presence/absence of this clinical condition also reached statistical significance (rs2004640: P=0.037, OR=0.69, CI 95%=0.48-0.98; rs10954213: P=0.030, OR=0.67, CI 95%=0.47-0.96), thus suggesting that both IRF5 genetic variants are specifically associated with the lack of macular edema in uveitis patients. Conclusion Our results clearly showed for the first time that two functional genetic variants of IRF5 may play a role in the development of macular edema in non-anterior uveitis patients. Identifying genetic markers for macular edema could lead to the possibility of developing novel treatments or preventive therapies. PMID:24116155

  5. Transcriptome outlier analysis implicates schizophrenia susceptibility genes and enriches putatively functional rare genetic variants

    PubMed Central

    Duan, Jubao; Sanders, Alan R.; Moy, Winton; Drigalenko, Eugene I.; Brown, Eric C.; Freda, Jessica; Leites, Catherine; Göring, Harald H. H.; Gejman, Pablo V.

    2015-01-01

    We searched a gene expression dataset comprised of 634 schizophrenia (SZ) cases and 713 controls for expression outliers (i.e., extreme tails of the distribution of transcript expression values) with SZ cases overrepresented compared with controls. These outlier genes were enriched for brain expression and for genes known to be associated with neurodevelopmental disorders. SZ cases showed higher outlier burden (i.e., total outlier events per subject) than controls for genes within copy number variants (CNVs) associated with SZ or neurodevelopmental disorders. Outlier genes were enriched for CNVs and for rare putative regulatory variants, but this only explained a small proportion of the outlier subjects, highlighting the underlying presence of additional genetic and potentially, epigenetic mechanisms. PMID:26022996

  6. Transcriptome outlier analysis implicates schizophrenia susceptibility genes and enriches putatively functional rare genetic variants.

    PubMed

    Duan, Jubao; Sanders, Alan R; Moy, Winton; Drigalenko, Eugene I; Brown, Eric C; Freda, Jessica; Leites, Catherine; Göring, Harald H H; Gejman, Pablo V

    2015-08-15

    We searched a gene expression dataset comprised of 634 schizophrenia (SZ) cases and 713 controls for expression outliers (i.e., extreme tails of the distribution of transcript expression values) with SZ cases overrepresented compared with controls. These outlier genes were enriched for brain expression and for genes known to be associated with neurodevelopmental disorders. SZ cases showed higher outlier burden (i.e., total outlier events per subject) than controls for genes within copy number variants (CNVs) associated with SZ or neurodevelopmental disorders. Outlier genes were enriched for CNVs and for rare putative regulatory variants, but this only explained a small proportion of the outlier subjects, highlighting the underlying presence of additional genetic and potentially, epigenetic mechanisms.

  7. Meta-analysis of the serotonin transporter promoter variant (5-HTTLPR) in relation to adverse environment and antisocial behavior.

    PubMed

    Tielbeek, Jorim J; Karlsson Linnér, Richard; Beers, Koko; Posthuma, Danielle; Popma, Arne; Polderman, Tinca J C

    2016-07-01

    Several studies have suggested an association between antisocial, aggressive, and delinquent behavior and the short variant of the serotonin transporter gene polymorphism (5-HTTLPR). Yet, genome wide and candidate gene studies in humans have not convincingly shown an association between these behaviors and 5-HTTLPR. Moreover, individual studies examining the effect of 5-HTTLPR in the presence or absence of adverse environmental factors revealed inconsistent results. We therefore performed a meta-analysis to test for the robustness of the potential interaction effect of the "long-short" variant of the 5-HTTLPR genotype and environmental adversities, on antisocial behavior. Eight studies, comprising of 12 reasonably independent samples, totaling 7,680 subjects with an effective sample size of 6,724, were included in the meta-analysis. Although our extensive meta-analysis resulted in a significant interaction effect between the 5-HTTLPR genotype and environmental adversities on antisocial behavior, the methodological constraints of the included studies hampered a confident interpretation of our results, and firm conclusions regarding the direction of effect. Future studies that aim to examine biosocial mechanisms that influence the etiology of antisocial behavior should make use of larger samples, extend to genome-wide genetic risk scores and properly control for covariate interaction terms, ensuring valid and well-powered research designs. © 2016 Wiley Periodicals, Inc. PMID:26990155

  8. Meta-analysis of the serotonin transporter promoter variant (5-HTTLPR) in relation to adverse environment and antisocial behavior.

    PubMed

    Tielbeek, Jorim J; Karlsson Linnér, Richard; Beers, Koko; Posthuma, Danielle; Popma, Arne; Polderman, Tinca J C

    2016-07-01

    Several studies have suggested an association between antisocial, aggressive, and delinquent behavior and the short variant of the serotonin transporter gene polymorphism (5-HTTLPR). Yet, genome wide and candidate gene studies in humans have not convincingly shown an association between these behaviors and 5-HTTLPR. Moreover, individual studies examining the effect of 5-HTTLPR in the presence or absence of adverse environmental factors revealed inconsistent results. We therefore performed a meta-analysis to test for the robustness of the potential interaction effect of the "long-short" variant of the 5-HTTLPR genotype and environmental adversities, on antisocial behavior. Eight studies, comprising of 12 reasonably independent samples, totaling 7,680 subjects with an effective sample size of 6,724, were included in the meta-analysis. Although our extensive meta-analysis resulted in a significant interaction effect between the 5-HTTLPR genotype and environmental adversities on antisocial behavior, the methodological constraints of the included studies hampered a confident interpretation of our results, and firm conclusions regarding the direction of effect. Future studies that aim to examine biosocial mechanisms that influence the etiology of antisocial behavior should make use of larger samples, extend to genome-wide genetic risk scores and properly control for covariate interaction terms, ensuring valid and well-powered research designs. © 2016 Wiley Periodicals, Inc.

  9. The Evolution and Functional Impact of Human Deletion Variants Shared with Archaic Hominin Genomes

    PubMed Central

    Lin, Yen-Lung; Pavlidis, Pavlos; Karakoc, Emre; Ajay, Jerry; Gokcumen, Omer

    2015-01-01

    Allele sharing between modern and archaic hominin genomes has been variously interpreted to have originated from ancestral genetic structure or through non-African introgression from archaic hominins. However, evolution of polymorphic human deletions that are shared with archaic hominin genomes has yet to be studied. We identified 427 polymorphic human deletions that are shared with archaic hominin genomes, approximately 87% of which originated before the Human–Neandertal divergence (ancient) and only approximately 9% of which have been introgressed from Neandertals (introgressed). Recurrence, incomplete lineage sorting between human and chimp lineages, and hominid-specific insertions constitute the remaining approximately 4% of allele sharing between humans and archaic hominins. We observed that ancient deletions correspond to more than 13% of all common (>5% allele frequency) deletion variation among modern humans. Our analyses indicate that the genomic landscapes of both ancient and introgressed deletion variants were primarily shaped by purifying selection, eliminating large and exonic variants. We found 17 exonic deletions that are shared with archaic hominin genomes, including those leading to three fusion transcripts. The affected genes are involved in metabolism of external and internal compounds, growth and sperm formation, as well as susceptibility to psoriasis and Crohn’s disease. Our analyses suggest that these “exonic” deletion variants have evolved through different adaptive forces, including balancing and population-specific positive selection. Our findings reveal that genomic structural variants that are shared between humans and archaic hominin genomes are common among modern humans and can influence biomedically and evolutionarily important phenotypes. PMID:25556237

  10. Functional variant in methionine synthase reductase intron-1 is associated with pleiotropic congenital malformations.

    PubMed

    Cheng, Haiqin; Li, Huili; Bu, Zhaoli; Zhang, Qin; Bai, Baoling; Zhao, Hong; Li, Ren-Ke; Zhang, Ting; Xie, Jun

    2015-09-01

    Congenital malformations, such as neural tube defects (NTDs) and congenital heart disease (CHD), cause significant fetal mortality and childhood morbidity. NTDs are a common congenital anomaly, and are typically induced by higher maternal homocysteine (Hcy) levels and abnormal folate metabolism. The gene encoding methionine synthase reductase (MTRR) is essential for adequate remethylation of Hcy. Previous studies have focused on the coding region of genes involved in one-carbon metabolism, but recent research demonstrates that an allelic change in a non-coding region of MTRR (rs326119) increases the risk of CHD. We hypothesized that this variant might contribute to the etiology of NTDs as well, based on a common role during early embryogenesis. In the present study, 244 neural tube defect cases and 407 controls from northern China were analyzed to determine any association (by χ (2) test) between rs326119 and disease phenotypes. Significant increased risk of anencephaly was seen in MTRR variant rs326119 heterozygote (het) and homozygote (hom) individuals [odds ratios (OR)het = 1.81; ORhom = 2.05)]. Furthermore, this variant was also a risk factor for congenital malformations of the adrenal gland (OR = 1.85), likely due to multiple systemic malformations in the NTDs case population. Our present data indicate that the rs326119 non-coding variant of MTRR has a pleiotropic effect on the development of multiple tissues, especially during early stages in utero. This suggests the allelic state of MTRR is a significant clinical factor affecting Hcy levels and optimal folic supplementation. PMID:26045171

  11. Functional characterization of open chromatin in bidirectional promoters of rice.

    PubMed

    Fang, Yuan; Wang, Ximeng; Wang, Lei; Pan, Xiucai; Xiao, Jin; Wang, Xiu-E; Wu, Yufeng; Zhang, Wenli

    2016-01-01

    Bidirectional gene pairs tend to be highly coregulated and function in similar biological processes in eukaryotic genomes. Structural features and functional consequences of bidirectional promoters (BDPs) have received considerable attention among diverse species. However, the underlying mechanisms responsible for the bidirectional transcription and coexpression of BDPs remain poorly understood in plants. In this study, we integrated DNase-seq, RNA-seq, ChIP-seq and MNase-seq data and investigated the effect of physical DNase I hypersensitive site (DHS) positions on the transcription of rice BDPs. We found that the physical position of a DHS relative to the TSS of bidirectional gene pairs can affect the expression of the corresponding genes: the closer a DHS is to the TSS, the higher is the expression level of the genes. Most importantly, we observed that the distribution of DHSs plays a significant role in the regulation of transcription and the coexpression of gene pairs, which are possibly mediated by orchestrating the positioning of histone marks and canonical nucleosomes around BDPs. Our results demonstrate that the combined actions of chromatin structures with DHSs, which contain functional cis-elements for interaction with transcriptional machinery, may play an important role in the regulation of the bidirectional transcription or coexpression in rice BDPs. Our findings may help to enhance the understanding of DHSs in the regulation of bidirectional gene pairs. PMID:27558448

  12. Positive technology: using interactive technologies to promote positive functioning.

    PubMed

    Riva, Giuseppe; Baños, Rosa M; Botella, Cristina; Wiederhold, Brenda K; Gaggioli, Andrea

    2012-02-01

    It is generally assumed that technology assists individuals in improving the quality of their lives. However, the impact of new technologies and media on well-being and positive functioning is still somewhat controversial. In this paper, we contend that the quality of experience should become the guiding principle in the design and development of new technologies, as well as a primary metric for the evaluation of their applications. The emerging discipline of Positive Psychology provides a useful framework to address this challenge. Positive Psychology is the scientific study of optimal human functioning and flourishing. Instead of drawing on a "disease model" of human behavior, it focuses on factors that enable individuals and communities to thrive and build the best in life. In this paper, we propose the "Positive Technology" approach--the scientific and applied approach to the use of technology for improving the quality of our personal experience through its structuring, augmentation, and/or replacement--as a way of framing a suitable object of study in the field of cyberpsychology and human-computer interaction. Specifically, we suggest that it is possible to use technology to influence three specific features of our experience--affective quality, engagement/actualization, and connectedness--that serve to promote adaptive behaviors and positive functioning. In this framework, positive technologies are classified according to their effects on a specific feature of personal experience. Moreover, for each level, we have identified critical variables that can be manipulated to guide the design and development of positive technologies.

  13. Functional characterization of open chromatin in bidirectional promoters of rice

    PubMed Central

    Fang, Yuan; Wang, Ximeng; Wang, Lei; Pan, Xiucai; Xiao, Jin; Wang, Xiu-e; Wu, Yufeng; Zhang, Wenli

    2016-01-01

    Bidirectional gene pairs tend to be highly coregulated and function in similar biological processes in eukaryotic genomes. Structural features and functional consequences of bidirectional promoters (BDPs) have received considerable attention among diverse species. However, the underlying mechanisms responsible for the bidirectional transcription and coexpression of BDPs remain poorly understood in plants. In this study, we integrated DNase-seq, RNA-seq, ChIP-seq and MNase-seq data and investigated the effect of physical DNase I hypersensitive site (DHS) positions on the transcription of rice BDPs. We found that the physical position of a DHS relative to the TSS of bidirectional gene pairs can affect the expression of the corresponding genes: the closer a DHS is to the TSS, the higher is the expression level of the genes. Most importantly, we observed that the distribution of DHSs plays a significant role in the regulation of transcription and the coexpression of gene pairs, which are possibly mediated by orchestrating the positioning of histone marks and canonical nucleosomes around BDPs. Our results demonstrate that the combined actions of chromatin structures with DHSs, which contain functional cis-elements for interaction with transcriptional machinery, may play an important role in the regulation of the bidirectional transcription or coexpression in rice BDPs. Our findings may help to enhance the understanding of DHSs in the regulation of bidirectional gene pairs. PMID:27558448

  14. Multiple Independent Retroelement Insertions in the Promoter of a Stress Response Gene Have Variable Molecular and Functional Effects in Drosophila

    PubMed Central

    Merenciano, Miriam; Ullastres, Anna; González, Josefa

    2016-01-01

    Promoters are structurally and functionally diverse gene regulatory regions. The presence or absence of sequence motifs and the spacing between the motifs defines the properties of promoters. Recent alternative promoter usage analyses in Drosophila melanogaster revealed that transposable elements significantly contribute to promote diversity. In this work, we analyzed in detail one of the transposable element insertions, named FBti0019985, that has been co-opted to drive expression of CG18446, a candidate stress response gene. We analyzed strains from different natural populations and we found that besides FBti0019985, there are another eight independent transposable elements inserted in the proximal promoter region of CG18446. All nine insertions are solo-LTRs that belong to the roo family. We analyzed the sequence of the nine roo insertions and we investigated whether the different insertions were functionally equivalent by performing 5’-RACE, gene expression, and cold-stress survival experiments. We found that different insertions have different molecular and functional consequences. The exact position where the transposable elements are inserted matters, as they all showed highly conserved sequences but only two of the analyzed insertions provided alternative transcription start sites, and only the FBti0019985 insertion consistently affects CG18446 expression. The phenotypic consequences of the different insertions also vary: only FBti0019985 was associated with cold-stress tolerance. Interestingly, the only previous report of transposable elements inserting repeatedly and independently in a promoter region in D. melanogaster, were also located upstream of a stress response gene. Our results suggest that functional validation of individual structural variants is needed to resolve the complexity of insertion clusters. PMID:27517860

  15. Kinetic and Sequence-Structure-Function Analysis of Known LinA Variants with Different Hexachlorocyclohexane Isomers

    PubMed Central

    Kumari, Kirti; Pandey, Gunjan; Jackson, Colin J.; Russell, Robyn J.; Oakeshott, John G.; Lal, Rup

    2011-01-01

    Background Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (−)-α -HCH are also discussed. Methodology/Principal Findings Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2B90A A110T, A111C, A110T/A111C and LinA1B90A were constructed using the FoldX computer algorithm. Turnover rates (min−1) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA's, followed by the γ and then δ isomer. Conclusions/Significance The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins. PMID:21949868

  16. Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

    PubMed Central

    Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

    2016-01-01

    Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. PMID:25180293

  17. High-throughput functional comparison of promoter and enhancer activities.

    PubMed

    Nguyen, Thomas A; Jones, Richard D; Snavely, Andrew R; Pfenning, Andreas R; Kirchner, Rory; Hemberg, Martin; Gray, Jesse M

    2016-08-01

    Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated coactivator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, whereas others are not. Although the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators. PMID:27311442

  18. Genetic Variants in the STMN1 Transcriptional Regulatory Region Affect Promoter Activity and Fear Behavior in English Springer Spaniels

    PubMed Central

    Zhang, Hanying; Xu, Yinxue

    2016-01-01

    Stathmin 1 (STMN1) is a neuronal growth-associated protein that is involved in microtubule dynamics and plays an important role in synaptic outgrowth and plasticity. Given that STMN1 affects fear behavior, we hypothesized that genetic variations in the STMN1 transcriptional regulatory region affect gene transcription activity and control fear behavior. In this study, two single nucleotide polymorphisms (SNPs), g. -327 A>G and g. -125 C>T, were identified in 317 English Springer Spaniels. A bioinformatics analysis revealed that both were loci located in the canine STMN1 putative promoter region and affected transcription factor binding. A statistical analysis revealed that the TT genotype at g.-125 C>T produced a significantly greater fear level than that of the CC genotype (P < 0.05). Furthermore, the H4H4 (GTGT) haplotype combination was significantly associated with canine fear behavior (P < 0.01). Using serially truncated constructs of the STMN1 promoters and the luciferase reporter, we found that a 395 bp (−312 nt to +83 nt) fragment constituted the core promoter region. The luciferase assay also revealed that the H4 (GT) haplotype promoter had higher activity than that of other haplotypes. Overall, our results suggest that the two SNPs in the canine STMN1 promoter region could affect canine fear behavior by altering STMN1 transcriptional activity. PMID:27390866

  19. Functional conservation of the promoter regions of vertebrate tyrosinase genes.

    PubMed

    Sato, S; Tanaka, M; Miura, H; Ikeo, K; Gojobori, T; Takeuchi, T; Yamamoto, H

    2001-11-01

    Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5' flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5' flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5' flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed. PMID:11764277

  20. Functional characterization of the human organic cation transporter 2 variant p.270Ala>Ser.

    PubMed

    Zolk, Oliver; Solbach, Thomas F; König, Jörg; Fromm, Martin F

    2009-06-01

    The organic cation transporter 2 (OCT2, SLC22A2) plays an important role for renal drug elimination. Recent clinical studies indicate an impact of the frequent nonsynonymous c.808G>T (p.270Ala>Ser) polymorphism on renal clearance of metformin and the extent of the metformin-cimetidine interaction. The role of this polymorphism for renal disposition of endogenous compounds and drugs other than metformin has not been investigated. In addition, it is unclear whether the observed genotype dependence of an OCT2-mediated drug-drug interaction might occur also with other OCT inhibitors. To address these issues, we generated human embryonic kidney cells stably expressing wild-type OCT2 or the p.270Ala>Ser variant. No differences in protein expression levels and membrane incorporation pattern were observed between the two cell lines. The p.270Ala>Ser variant significantly impaired uptake kinetics of 1-methyl-4-phenylpyridinium, dopamine, norepinephrine, and propranolol. V(max) values were significantly reduced for uptake of all four compounds mediated by the p.270Ala>Ser variant compared with wild-type OCT2. In addition, a significant difference in the affinity to wild-type and mutant OCT2 was observed for dopamine (K(m) dopamine: 932 +/- 77 versus 1285 +/- 132 microM). Moreover, out of a set of 27 compounds p.270Ala>Ser OCT2 was significantly less sensitive to inhibition by cimetidine, flurazepam, metformin, mexiletine, propranolol, and verapamil than wild-type OCT2 (e.g., for propranolol: IC(50) wild type versus p.270Ala>Ser 189 versus 895 microM, P < 0.001). Our results indicate that the common OCT2 c.808G>T single nucleotide polymorphism significantly alters uptake of endogenous compounds and drugs. Moreover, for selected compounds the extent of OCT2-mediated drug interactions could depend on OCT2 c.808G>T genotype. PMID:19251820

  1. Increased dynamics in the 40–57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation

    PubMed Central

    Karsisiotis, Andreas Ioannis; Deacon, Oliver M.; Wilson, Michael T.; Macdonald, Colin; Blumenschein, Tharin M. A.; Moore, Geoffrey R.; Worrall, Jonathan A. R.

    2016-01-01

    Thrombocytopenia 4 is an inherited autosomal dominant thrombocytopenia, which occurs due to mutations in the human gene for cytochrome c that results in enhanced mitochondrial apoptotic activity. The Gly41Ser mutation was the first to be reported. Here we report stopped-flow kinetic studies of azide binding to human ferricytochrome c and its Gly41Ser variant, together with backbone amide H/D exchange and 15N-relaxation dynamics using NMR spectroscopy, to show that alternative conformations are kinetically and thermodynamically more readily accessible for the Gly41Ser variant than for the wild-type protein. Our work reveals a direct conformational link between the 40–57 Ω-loop in which residue 41 resides and the dynamical properties of the axial ligand to the heme iron, Met80, such that the replacement of glycine by serine promotes the dissociation of the Met80 ligand, thereby increasing the population of a peroxidase active state, which is a key non-native conformational state in apoptosis. PMID:27461282

  2. The Ser82 RAGE Variant Affects Lung Function and Serum RAGE in Smokers and sRAGE Production In Vitro

    PubMed Central

    Miller, Suzanne; Henry, Amanda P.; Hodge, Emily; Kheirallah, Alexander K.; Billington, Charlotte K.; Rimington, Tracy L.; Bhaker, Sangita K.; Obeidat, Ma’en; Melén, Erik; Merid, Simon K.; Swan, Caroline; Gowland, Catherine; Nelson, Carl P.; Stewart, Ceri E.; Bolton, Charlotte E.; Kilty, Iain; Malarstig, Anders; Parker, Stuart G.; Moffatt, Miriam F.; Wardlaw, Andrew J.; Hall, Ian P.; Sayers, Ian

    2016-01-01

    Introduction Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. Methods Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. Results Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. Conclusions This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying

  3. The Drosophila histone variant H2A.V works in concert with HP1 to promote kinetochore-driven microtubule formation

    PubMed Central

    Vernì, Fiammetta; Cenci, Giovanni

    2015-01-01

    Unlike other organisms that have evolved distinct H2A variants for different functions, Drosophila melanogaster has just one variant which is capable of filling many roles. This protein, H2A.V, combines the features of the conserved variants H2A.Z and H2A.X in transcriptional control/heterochromatin assembly and DNA damage response, respectively. Here we show that mutations in the gene encoding H2A.V affect chromatin compaction and perturb chromosome segregation in Drosophila mitotic cells. A microtubule (MT) regrowth assay after cold exposure revealed that loss of H2A.V impairs the formation of kinetochore-driven (k) fibers, which can account for defects in chromosome segregation. All defects are rescued by a transgene encoding H2A.V that lacks the H2A.X function in the DNA damage response, suggesting that the H2A.Z (but not H2A.X) functionality of H2A.V is required for chromosome segregation. We also found that loss of H2A.V weakens HP1 localization, specifically at the pericentric heterochromatin of metaphase chromosomes. Interestingly, loss of HP1 yielded not only telomeric fusions but also mitotic defects similar to those seen in H2A.V null mutants, suggesting a role for HP1 in chromosome segregation. We also show that H2A.V precipitates HP1 from larval brain extracts indicating that both proteins are part of the same complex. Moreover, we found that the overexpression of HP1 rescues chromosome missegregation and defects in the kinetochore-driven k-fiber regrowth of H2A.V mutants indicating that both phenotypes are influenced by unbalanced levels of HP1. Collectively, our results suggest that H2A.V and HP1 work in concert to ensure kinetochore-driven MT growth. PMID:25591068

  4. Positive technology: using interactive technologies to promote positive functioning.

    PubMed

    Riva, Giuseppe; Baños, Rosa M; Botella, Cristina; Wiederhold, Brenda K; Gaggioli, Andrea

    2012-02-01

    It is generally assumed that technology assists individuals in improving the quality of their lives. However, the impact of new technologies and media on well-being and positive functioning is still somewhat controversial. In this paper, we contend that the quality of experience should become the guiding principle in the design and development of new technologies, as well as a primary metric for the evaluation of their applications. The emerging discipline of Positive Psychology provides a useful framework to address this challenge. Positive Psychology is the scientific study of optimal human functioning and flourishing. Instead of drawing on a "disease model" of human behavior, it focuses on factors that enable individuals and communities to thrive and build the best in life. In this paper, we propose the "Positive Technology" approach--the scientific and applied approach to the use of technology for improving the quality of our personal experience through its structuring, augmentation, and/or replacement--as a way of framing a suitable object of study in the field of cyberpsychology and human-computer interaction. Specifically, we suggest that it is possible to use technology to influence three specific features of our experience--affective quality, engagement/actualization, and connectedness--that serve to promote adaptive behaviors and positive functioning. In this framework, positive technologies are classified according to their effects on a specific feature of personal experience. Moreover, for each level, we have identified critical variables that can be manipulated to guide the design and development of positive technologies. PMID:22149077

  5. Gain of Olig2 function in oligodendrocyte progenitors promotes remyelination.

    PubMed

    Wegener, Amélie; Deboux, Cyrille; Bachelin, Corinne; Frah, Magali; Kerninon, Christophe; Seilhean, Danielle; Weider, Matthias; Wegner, Michael; Nait-Oumesmar, Brahim

    2015-01-01

    The basic helix-loop-helix transcription factor Olig2 is a key determinant for the specification of neural precursor cells into oligodendrocyte progenitor cells. However, the functional role of Olig2 in oligodendrocyte migration and differentiation remains elusive both during developmental myelination and under demyelinating conditions of the adult central nervous system. To decipher Olig2 functions, we generated transgenic mice (TetOlig2:Sox10(rtTA/+)) overexpressing Olig2 in Sox10(+) oligodendroglial cells in a doxycycline inducible manner. We show that Olig2 overexpression increases the generation of differentiated oligodendrocytes, leading to precocious myelination of the central nervous system. Unexpectedly, we found that gain of Olig2 function in oligodendrocyte progenitor cells enhances their migration rate. To determine whether Olig2 overexpression in adult oligodendrocyte progenitor cells promotes oligodendrocyte regeneration for myelin repair, we induced lysophosphatidylcholine demyelination in the corpus callosum of TetOlig2:Sox10(rtTA/+) and control mice. We found that Olig2 overexpression enhanced oligodendrocyte progenitor cell differentiation and remyelination. To assess the relevance of these findings in demyelinating diseases, we also examined OLIG2 expression in multiple sclerosis lesions. We demonstrate that OLIG2 displays a differential expression pattern in multiple sclerosis lesions that correlates with lesion activity. Strikingly, OLIG2 was predominantly detected in NOGO-A(+) (now known as RTN4-A) maturing oligodendrocytes, which prevailed in active lesion borders, rather than chronic silent and shadow plaques. Taken together, our data provide proof of principle indicating that OLIG2 overexpression in oligodendrocyte progenitor cells might be a possible therapeutic mechanism for enhancing myelin repair.

  6. Gain of Olig2 function in oligodendrocyte progenitors promotes remyelination

    PubMed Central

    Wegener, Amélie; Deboux, Cyrille; Bachelin, Corinne; Frah, Magali; Kerninon, Christophe; Seilhean, Danielle; Weider, Matthias; Wegner, Michael

    2015-01-01

    The basic helix-loop-helix transcription factor Olig2 is a key determinant for the specification of neural precursor cells into oligodendrocyte progenitor cells. However, the functional role of Olig2 in oligodendrocyte migration and differentiation remains elusive both during developmental myelination and under demyelinating conditions of the adult central nervous system. To decipher Olig2 functions, we generated transgenic mice (TetOlig2:Sox10rtTA/+) overexpressing Olig2 in Sox10+ oligodendroglial cells in a doxycycline inducible manner. We show that Olig2 overexpression increases the generation of differentiated oligodendrocytes, leading to precocious myelination of the central nervous system. Unexpectedly, we found that gain of Olig2 function in oligodendrocyte progenitor cells enhances their migration rate. To determine whether Olig2 overexpression in adult oligodendrocyte progenitor cells promotes oligodendrocyte regeneration for myelin repair, we induced lysophosphatidylcholine demyelination in the corpus callosum of TetOlig2:Sox10rtTA/+ and control mice. We found that Olig2 overexpression enhanced oligodendrocyte progenitor cell differentiation and remyelination. To assess the relevance of these findings in demyelinating diseases, we also examined OLIG2 expression in multiple sclerosis lesions. We demonstrate that OLIG2 displays a differential expression pattern in multiple sclerosis lesions that correlates with lesion activity. Strikingly, OLIG2 was predominantly detected in NOGO-A+ (now known as RTN4-A) maturing oligodendrocytes, which prevailed in active lesion borders, rather than chronic silent and shadow plaques. Taken together, our data provide proof of principle indicating that OLIG2 overexpression in oligodendrocyte progenitor cells might be a possible therapeutic mechanism for enhancing myelin repair. PMID:25564492

  7. Fine mapping and functional analysis of a common variant in MSMB on chromosome 10q11.2 associated with prostate cancer susceptibility

    PubMed Central

    Lou, Hong; Yeager, Meredith; Li, Hongchuan; Bosquet, Jesus Gonzalez; Hayes, Richard B.; Orr, Nick; Yu, Kai; Hutchinson, Amy; Jacobs, Kevin B.; Kraft, Peter; Wacholder, Sholom; Chatterjee, Nilanjan; Feigelson, Heather Spencer; Thun, Michael J.; Diver, W. Ryan; Albanes, Demetrius; Virtamo, Jarmo; Weinstein, Stephanie; Ma, Jing; Gaziano, J. Michael; Stampfer, Meir; Schumacher, Fredrick R.; Giovannucci, Edward; Cancel-Tassin, Geraldine; Cussenot, Olivier; Valeri, Antoine; Andriole, Gerald L.; Crawford, E. David; Anderson, Stephen K.; Tucker, Margaret; Hoover, Robert N.; Fraumeni, Joseph F.; Thomas, Gilles; Hunter, David J.; Dean, Michael; Chanock, Stephen J.

    2009-01-01

    Two recent genome-wide association studies have independently identified a prostate cancer susceptibility locus on chromosome 10q11.2. The most significant single-nucleotide polymorphism (SNP) marker reported, rs10993994, is 57 bp centromeric of the first exon of the MSMB gene, which encodes β-microseminoprotein (prostatic secretory protein 94). In this study, a fine-mapping analysis using HapMap SNPs was conducted across a ≈65-kb region (chr10: 51168330–51234020) flanking rs10993994 with 13 tag SNPs in 6,118 prostate cancer cases and 6,105 controls of European origin from the Cancer Genetic Markers of Susceptibility (CGEMS) project. rs10993994 remained the most strongly associated marker with prostate cancer risk [P = 8.8 × 10−18; heterozygous odds ratio (OR) = 1.20, 95% confidence interval (CI): 1.11–1.30; homozygous OR = 1.64, 95% CI: 1.47–1.86 for the adjusted genotype test with 2 df]. In follow-up functional analyses, the T variant of rs10993994 significantly affected expression of in vitro luciferase reporter constructs. In electrophoretic mobility shift assays, the C allele of rs10993994 preferentially binds to the CREB transcription factor. Analysis of tumor cell lines with a CC or CT genotype revealed a high level of MSMB gene expression compared with cell lines with a TT genotype. These findings were specific to the alleles of rs10993994 and were not observed for other SNPs determined by sequence analysis of the proximal promoter. Together, our mapping study and functional analyses implicate regulation of expression of MSMB as a plausible mechanism accounting for the association identified at this locus. Further investigation is warranted to determine whether rs10993994 alone or in combination with additional variants contributes to prostate cancer susceptibility. PMID:19383797

  8. Promotion of Waterpipe Tobacco Use, Its Variants and Accessories in Young Adult Newspapers: A Content Analysis of Message Portrayal

    ERIC Educational Resources Information Center

    Sterling, Kymberle L.; Fryer, Craig S.; Majeed, Ban; Duong, Melissa M.

    2015-01-01

    The objective of our study was to identify waterpipe tobacco smoking advertisements and those that promoted a range of products and accessories used to smoke waterpipe tobacco. The content of these advertisements was analyzed to understand the messages portrayed about waterpipe tobacco smoking in young adult (aged 18-30) newspapers. The study…

  9. Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls.

    PubMed

    Guo, F; Yang, B; Ju, Z H; Wang, X G; Qi, C; Zhang, Y; Wang, C F; Liu, H D; Feng, M Y; Chen, Y; Xu, Y X; Zhong, J F; Huang, J M

    2014-02-01

    The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

  10. Novel Allelic Variants in the Canine Cyclooxgenase-2 (Cox-2) Promoter Are Associated with Renal Dysplasia in Dogs

    PubMed Central

    Whiteley, Mary H.; Bell, Jerold S.; Rothman, Debby A.

    2011-01-01

    Renal dysplasia (RD) in dogs is a complex disease with a highly variable phenotype and mode of inheritance that does not follow a simple Mendelian pattern. Cox-2 (Cyclooxgenase-2) deficient mice have renal abnormalities and a pathology that has striking similarities to RD in dogs suggesting to us that mutations in the Cox-2 gene could be the cause of RD in dogs. Our data supports this hypothesis. Sequencing of the canine Cox-2 gene was done from clinically affected and normal dogs. Although no changes were detected in the Cox-2 coding region, small insertions and deletions of GC boxes just upstream of the ATG translation start site were found. These sequences are putative SP1 transcription factor binding sites that may represent important cis-acting DNA regulatory elements that govern the expression of Cox-2. A pedigree study of a family of Lhasa apsos revealed an important statistical correlation of these mutant alleles with the disease. We examined an additional 22 clinical cases from various breeds. Regardless of the breed or severity of disease, all of these had one or two copies of the Cox-2 allelic variants. We suggest that the unusual inheritance pattern of RD is due to these alleles, either by changing the pattern of expression of Cox-2 or making Cox-2 levels susceptible to influences of other genes or environmental factors that play an unknown but important role in the development of RD in dogs. PMID:21346820

  11. Invasive Cortical Stimulation to Promote Recovery of Function After Stroke

    PubMed Central

    Plow, Ela B.; Carey, James R.; Nudo, Randolph J.; Pascual-Leone, Alvaro

    2011-01-01

    Background and Purpose Residual motor deficits frequently linger after stroke. Search for newer effective strategies to promote functional recovery is ongoing. Brain stimulation, as a means of directing adaptive plasticity, is appealing. Animal studies and Phase I and II trials in humans have indicated safety, feasibility, and efficacy of combining rehabilitation and concurrent invasive cortical stimulation. However, a recent Phase III trial showed no advantage of the combination. We critically review results of various trials and discuss the factors that contributed to the distinctive result. Summary of Review Regarding cortical stimulation, it is important to determine the (1) location of peri-infarct representations by integrating multiple neuroanatomical and physiological techniques; (2) role of other mechanisms of stroke recovery; (3) viability of peri-infarct tissue and descending pathways; (4) lesion geometry to ensure no alteration/displacement of current density; and (5) applicability of lessons generated from noninvasive brain stimulation studies in humans. In terms of combining stimulation with rehabilitation, we should understand (1) the principle of homeostatic plasticity; (2) the effect of ongoing cortical activity and phases of learning; and (3) that subject-specific intervention may be necessary. Conclusions Future cortical stimulation trials should consider the factors that may have contributed to the peculiar results of the Phase III trial and address those in future study designs. PMID:19359643

  12. Structural insights into anaphase-promoting complex function and mechanism

    PubMed Central

    Barford, David

    2011-01-01

    The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C. PMID:22084387

  13. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  14. An IL-4R alpha allelic variant, I50, acts as a gain-of-function variant relative to V50 for Stat6, but not Th2 differentiation.

    PubMed

    Stephenson, Linda; Johns, Mary H; Woodward, Emily; Mora, Ana L; Boothby, Mark

    2004-10-01

    Signaling through the IL-4R alpha-chain (IL-4Ralpha) is crucial for the development of Th2 cells, central effectors in atopic disease. Alleles of the IL-4Ralpha have been identified that have been variably associated with increased incidence of allergic disease, but there is little direct evidence that any variant is sufficient to alter a target that determines allergic pathophysiology or susceptibility. Variants of IL-4Ralpha encoding isoleucine instead of valine at position 50 (I50 vs V50, respectively) can signal increased Stat6-dependent transcriptional activity, whether in an I50, Q551 or I50, R551 haplotype. Strikingly, signaling through these receptors did not increase the efficiency of Th2 development or the IL-4 mediated repression of Th1 development or a target gene, IL-18Ralpha. Further, IL-4-induced proliferation was similar for Th2 cells independent of the variant expressed. Together these findings indicate that IL-4Ralpha variants that exhibit gain-of-function with respect to Stat6 do not act directly through alterations in Th2/Th1 induction after Ag exposure. The data further suggest that for such variants, any mechanistic involvement is based on a role in cellular targets of Th2 cytokines.

  15. Lactoferrin gene promoter variants and their association with clinical and subclinical mastitis in indigenous and crossbred cattle.

    PubMed

    Chopra, A; Gupta, I D; Verma, A; Chakravarty, A K; Vohra, V

    2015-01-01

    Lactoferrin (Lf) gene promoter was screened for the presence of single nucleotide polymphism in indigenous and crossbred cattle from North India and to evaluate its association with Mastitis. Study revealed the presence of genetic variation in regulatory region of bovine Lactoferrin gene using PCR-RFLP technique. Three genotypes namely GG, GH and HH were identified. A single nucleotide change, from guanine to adenine at 25th position was found to be significantly associated (p<0.05) with clinical mastitis in indigenous Sahiwal and crossbred Karan Fries cattle maintained at organised herd of National Dairy Research Institute, Karnal. A non-significant association was observed between subclinical mastitis, somatic cell score (SCS), and GG genotype in Karan Fries cattle, however, a lower SCS was observed in animals having GG genotype. Overall a lower incidence of clinical mastitis was recorded in those animals having GG genotype of Lf in Sahiwal and Karan Fries (KF) cattle. The SNP identified in the promoter region may effect expression lactoferrin protein, which may lead to different levels of antibacterial and anti-inflammatory activity of Lf gene. Results from this study indicated the probable role played by Lactoferrin promoter to serve as candidate gene for mastitis susceptibility among indigenous and crossbred milch cattle.

  16. Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci.

    PubMed

    Leslie, Elizabeth J; Taub, Margaret A; Liu, Huan; Steinberg, Karyn Meltz; Koboldt, Daniel C; Zhang, Qunyuan; Carlson, Jenna C; Hetmanski, Jacqueline B; Wang, Hang; Larson, David E; Fulton, Robert S; Kousa, Youssef A; Fakhouri, Walid D; Naji, Ali; Ruczinski, Ingo; Begum, Ferdouse; Parker, Margaret M; Busch, Tamara; Standley, Jennifer; Rigdon, Jennifer; Hecht, Jacqueline T; Scott, Alan F; Wehby, George L; Christensen, Kaare; Czeizel, Andrew E; Deleyiannis, Frederic W-B; Schutte, Brian C; Wilson, Richard K; Cornell, Robert A; Lidral, Andrew C; Weinstock, George M; Beaty, Terri H; Marazita, Mary L; Murray, Jeffrey C

    2015-03-01

    Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

  17. Identification of Functional Variants for Cleft Lip with or without Cleft Palate in or near PAX7, FGFR2, and NOG by Targeted Sequencing of GWAS Loci

    PubMed Central

    Leslie, Elizabeth J.; Taub, Margaret A.; Liu, Huan; Steinberg, Karyn Meltz; Koboldt, Daniel C.; Zhang, Qunyuan; Carlson, Jenna C.; Hetmanski, Jacqueline B.; Wang, Hang; Larson, David E.; Fulton, Robert S.; Kousa, Youssef A.; Fakhouri, Walid D.; Naji, Ali; Ruczinski, Ingo; Begum, Ferdouse; Parker, Margaret M.; Busch, Tamara; Standley, Jennifer; Rigdon, Jennifer; Hecht, Jacqueline T.; Scott, Alan F.; Wehby, George L.; Christensen, Kaare; Czeizel, Andrew E.; Deleyiannis, Frederic W.-B.; Schutte, Brian C.; Wilson, Richard K.; Cornell, Robert A.; Lidral, Andrew C.; Weinstock, George M.; Beaty, Terri H.; Marazita, Mary L.; Murray, Jeffrey C.

    2015-01-01

    Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans. PMID:25704602

  18. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race.

    PubMed

    Edelstein, Leonard C; Simon, Lukas M; Lindsay, Cory R; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E; Chen, Edward S; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A; Bray, Paul F

    2014-11-27

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists.

  19. Functional evaluation of TERT-CLPTM1L genetic variants associated with susceptibility of papillary thyroid carcinoma

    PubMed Central

    Ge, Minghua; Shi, Meng; An, Changming; Yang, Wenjun; Nie, Xilin; Zhang, Jian; Lv, Zheng; Li, Jinliang; Zhou, Liqing; Du, Zhongli; Yang, Ming

    2016-01-01

    TERT is the catalytic subunit of telomerase which plays an essential part in cellular immortality by maintaining telomere integrity. TERT is commonly over-expressed in human malignancies, indicating its key role in cell transformation. The chromosome 5p15.33 TERT-CLPTM1L region has been associated with susceptibility of multiple cancers via a genome-wide association approach. However, the involvement of this locus in papillary thyroid carcinoma (PTC) etiology is still largely unknown. We analyzed 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) of the TERT-CLPTM1L region in a two stage case-control design. After genotyping 2300 PTC patients and frequency-matched 2300 unaffected controls, we found that TERT rs2736100 genetic variant is significantly associated with elevated PTC risk. Ex vivo reporter gene assays indicated that the PTC susceptibility rs2736100 polymorphism locating in a potential TERT intronic enhancer has a genotype-specific effect on TERT expression. Correlations between rs2736100 genotypes and tissue-specific TERT expression supported the regulatory function of this genetic variant in vivo. Our data demonstrated that the functional TERT rs2736100 SNP as a novel genetic component of PTC etiology. This study, together with recent studies in other cancers, unequivocally establishes an essential role of TERT in cancers. PMID:27185198

  20. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    PubMed Central

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.

    2014-01-01

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. PMID:25293779

  1. A functional variant in the 5'-flanking region of the chicken serotonin transporter gene is associated with increased body weight and locomotor activity.

    PubMed

    Phi-van, Loc; Holtz, Marlis; Kjaer, Joergen B; van Phi, Valerie D; Zimmermann, Katrin

    2014-10-01

    In this study, we identified a polymorphism in the 5'-flanking region of the chicken serotonin transporter (5-HTT) gene. Sequencing analysis revealed that in comparison with the wild-type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5'-AATT-3') and a single nucleotide change (A→T). Using a polyacrylamide gel electrophoresis system, we found that the 360-bp DNA fragment containing the W variant with the wild-type sequence 5'-AATTAATT-3' shows intrinsic DNA curvature while the 356-bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real-time RT-PCR and ELISA demonstrated that the expression of 5-HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5-HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6 weeks and 16 weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.

  2. Racial Differences in DNA-Methylation of CpG Sites Within Preterm-Promoting Genes and Gene Variants.

    PubMed

    Salihu, H M; Das, R; Morton, L; Huang, H; Paothong, A; Wilson, R E; Aliyu, M H; Salemi, J L; Marty, P J

    2016-08-01

    Objective To evaluate the role DNA methylation may play in genes associated with preterm birth for higher rates of preterm births in African-American women. Methods Fetal cord blood samples from births collected at delivery and maternal demographic and medical information were used in a cross-sectional study to examine fetal DNA methylation of genes implicated in preterm birth among black and non-black infants. Allele-specific DNA methylation analysis was performed using a methylation bead array. Targeted maximum likelihood estimation was applied to examine the relationship between race and fetal DNA methylation of candidate preterm birth genes. Receiver-operating characteristic analyses were then conducted to validate the CpG site methylation marker within the two racial groups. Bootstrapping, a method of validation and replication, was employed. Results 42 CpG sites were screened within 20 candidate gene variants reported consistently in the literature as being associated with preterm birth. Of these, three CpG sites on TNFAIP8 and PON1 genes (corresponding to: cg23917399; cg07086380; and cg07404485, respectively) were significantly differentially methylated between black and non-black individuals. The three CpG sites showed lower methylation status among infants of black women. Bootstrapping validated and replicated results. Conclusion for Practice Our study identified significant differences in levels of methylation on specific genes between black and non-black individuals. Understanding the genetic/epigenetic mechanisms that lead to preterm birth may lead to enhanced prevention strategies to reduce morbidity and mortality by eventually providing a means to identify individuals with a genetic predisposition to preterm labor.

  3. Dissecting the role of the mitochondrial chaperone mortalin in Parkinson's disease: functional impact of disease-related variants on mitochondrial homeostasis

    PubMed Central

    Burbulla, Lena F.; Schelling, Carina; Kato, Hiroki; Rapaport, Doron; Woitalla, Dirk; Schiesling, Carola; Schulte, Claudia; Sharma, Manu; Illig, Thomas; Bauer, Peter; Jung, Stephan; Nordheim, Alfred; Schöls, Ludger; Riess, Olaf; Krüger, Rejko

    2010-01-01

    The mitochondrial chaperone mortalin has been linked to neurodegeneration in Parkinson's disease (PD) based on reduced protein levels in affected brain regions of PD patients and its interaction with the PD-associated protein DJ-1. Recently, two amino acid exchanges in the ATPase domain (R126W) and the substrate-binding domain (P509S) of mortalin were identified in Spanish PD patients. Here, we identified a separate and novel variant (A476T) in the substrate-binding domain of mortalin in German PD patients. To define a potential role as a susceptibility factor in PD, we characterized the functions of all three variants in different cellular models. In vitro import assays revealed normal targeting of all mortalin variants. In neuronal and non-neuronal human cell lines, the disease-associated variants caused a mitochondrial phenotype of increased reactive oxygen species and reduced mitochondrial membrane potential, which were exacerbated upon proteolytic stress. These functional impairments correspond with characteristic alterations of the mitochondrial network in cells overexpressing mutant mortalin compared with wild-type (wt), which were confirmed in fibroblasts from a carrier of the A476T variant. In line with a loss of function hypothesis, knockdown of mortalin in human cells caused impaired mitochondrial function that was rescued by wt mortalin, but not by the variants. Our genetic and functional studies of novel disease-associated variants in the mortalin gene define a loss of mortalin function, which causes impaired mitochondrial function and dynamics. Our results support the role of this mitochondrial chaperone in neurodegeneration and underscore the concept of impaired mitochondrial protein quality control in PD. PMID:20817635

  4. A functional variant that affects exon-skipping and protein expression of SP140 as genetic mechanism predisposing to multiple sclerosis.

    PubMed

    Matesanz, Fuencisla; Potenciano, Victor; Fedetz, Maria; Ramos-Mozo, Priscila; Abad-Grau, María del Mar; Karaky, Mohamad; Barrionuevo, Cristina; Izquierdo, Guillermo; Ruiz-Peña, Juan Luis; García-Sánchez, María Isabel; Lucas, Miguel; Fernández, Óscar; Leyva, Laura; Otaegui, David; Muñoz-Culla, Maider; Olascoaga, Javier; Vandenbroeck, Koen; Alloza, Iraide; Astobiza, Ianire; Antigüedad, Alfredo; Villar, Luisa María; Álvarez-Cermeño, José Carlos; Malhotra, Sunny; Comabella, Manuel; Montalban, Xavier; Saiz, Albert; Blanco, Yolanda; Arroyo, Rafael; Varadé, Jezabel; Urcelay, Elena; Alcina, Antonio

    2015-10-01

    Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohn's disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL. PMID:26152201

  5. A functional variant that affects exon-skipping and protein expression of SP140 as genetic mechanism predisposing to multiple sclerosis.

    PubMed

    Matesanz, Fuencisla; Potenciano, Victor; Fedetz, Maria; Ramos-Mozo, Priscila; Abad-Grau, María del Mar; Karaky, Mohamad; Barrionuevo, Cristina; Izquierdo, Guillermo; Ruiz-Peña, Juan Luis; García-Sánchez, María Isabel; Lucas, Miguel; Fernández, Óscar; Leyva, Laura; Otaegui, David; Muñoz-Culla, Maider; Olascoaga, Javier; Vandenbroeck, Koen; Alloza, Iraide; Astobiza, Ianire; Antigüedad, Alfredo; Villar, Luisa María; Álvarez-Cermeño, José Carlos; Malhotra, Sunny; Comabella, Manuel; Montalban, Xavier; Saiz, Albert; Blanco, Yolanda; Arroyo, Rafael; Varadé, Jezabel; Urcelay, Elena; Alcina, Antonio

    2015-10-01

    Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohn's disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL.

  6. Fine-Scale Mapping of the FGFR2 Breast Cancer Risk Locus: Putative Functional Variants Differentially Bind FOXA1 and E2F1

    PubMed Central

    Meyer, Kerstin B.; O’Reilly, Martin; Michailidou, Kyriaki; Carlebur, Saskia; Edwards, Stacey L.; French, Juliet D.; Prathalingham, Radhika; Dennis, Joe; Bolla, Manjeet K.; Wang, Qin; de Santiago, Ines; Hopper, John L.; Tsimiklis, Helen; Apicella, Carmel; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Van ’t Veer, Laura J.; Hogervorst, Frans B.; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Lux, Michael P.; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marme, Federick; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Milne, Roger L.; Zamora, M. Pilar; Arias, Jose I.; Benitez, Javier; Neuhausen, Susan; Anton-Culver, Hoda; Ziogas, Argyrios; Dur, Christina C.; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Schmutzler, Rita K.; Engel, Christoph; Ditsch, Nina; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Nevanlinna, Heli; Muranen, Taru A.; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Yatabe, Yasushi; Dörk, Thilo; Helbig, Sonja; Bogdanova, Natalia V.; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Chenevix-Trench, Georgia; Wu, Anna H.; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O.; Lambrechts, Diether; Thienpont, Bernard; Christiaens, Marie-Rose; Smeets, Ann; Chang-Claude, Jenny; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Bernard, Loris; Couch, Fergus J.; Olson, Janet E.; Wang, Xianshu; Purrington, Kristen; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; McLean, Catriona; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo-Hwang; Yip, Cheng-Har; Phuah, Sze-Yee; Kristensen, Vessela; Grenaker Alnæs, Grethe; Børresen-Dale, Anne-Lise; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A.E.M.; Seynaeve, Caroline M.; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J.; Lissowska, Jolanta; Czene, Kamila; Darabi, Hartef; Eriksson, Kimael; Hooning, Maartje J.; Martens, John W.M.; van den Ouweland, Ans M.W.; van Deurzen, Carolien H.M.; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Reed, Malcolm W.R.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Ghoussaini, Maya; Harrington, Patricia; Tyrer, Jonathan; Kang, Daehee; Choi, Ji-Yeob; Park, Sue K.; Noh, Dong-Young; Hartman, Mikael; Hui, Miao; Lim, Wei-Yen; Buhari, Shaik A.; Hamann, Ute; Försti, Asta; Rüdiger, Thomas; Ulmer, Hans-Ulrich; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Vachon, Celine; Slager, Susan; Fostira, Florentia; Pilarski, Robert; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Hou, Ming-Feng; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J.; Ponder, Bruce A.J.; Dunning, Alison M.; Easton, Douglas F.

    2013-01-01

    The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease. PMID:24290378

  7. Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5

    PubMed Central

    Dong, H-F; Wigmore, K; Carrington, MN; Dean, M; Turpin, JA; Howard, OMZ

    2006-01-01

    CCR5 is one of the primary coreceptors for Env-mediated fusion between cells and human immunodeficiency virus type 1 (HIV-1). Analyses of CCR5 variants in cohorts of HIV-1 high-risk individuals led to the identification of multiple single amino-acid substitutions, which may have functional consequences. This study focused on eight naturally occurring allelic variants located between amino-acid residues 60 and 334 of CCR5. All studied allelic variants were highly expressed on the cell surface of HEK-293 cells and permissive for HIV-1 infection. Variant G301V showed 3.5-fold increase in 50% effective concentration (EC50) for CCL4 (MIP 1beta) in a competitive binding assay. There was also a significant reduction in CCL5 (RANTES) EC50 for the R223Q, A335V and Y339F variants. The most unexpected functional abnormality was exhibited by the R60S variant that exhibited a loss of ligand-induced desensitization in chemotaxis assays, but showed normal CCL4 and CCL5 binding avidity. This mutation is located in the first intracellular loop, a domain that has not previously been shown to be involved in receptor desensitization. In conclusion, our results support earlier studies showing that these naturally occurring point mutations do not limit HIV-1 infection, and indicated that single amino-acid changes can have unexpected functional consequences. PMID:16015368

  8. Analysis of PGC-1{alpha} variants Gly482Ser and Thr612Met concerning their PPAR{gamma}2-coactivation function

    SciTech Connect

    Nitz, Inke . E-mail: initz@molnut.uni-kiel.de; Ewert, Agnes; Klapper, Maja; Doering, Frank

    2007-02-09

    Peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) is a cofactor involved in adaptive thermogenesis, fatty acid oxidation, and gluconeogenesis. Dysfunctions of this protein are likely to contribute to the development of obesity and the metabolic syndrome. This is in part but not definitely confirmed by results of population studies. The aim of this study was to investigate if common genetic variants rs8192678 (Gly482Ser) and rs3736265 (Thr612Met) in the PGC-1{alpha} gene lead to a functional consequence in cofactor activity using peroxisome proliferator-activated receptor-{gamma} 2 (PPAR{gamma}2) as interacting transcription factor. Reporter gene assays in HepG2 cells with wildtype and mutant proteins of both PGC1{alpha} and PPAR{gamma}2 (Pro12Ala, rs1801282) using the acyl-CoA-binding protein (ACBP) promoter showed no difference in coactivator activity. This is First study implicating that the Gly482Ser and Thr612Met polymorphisms in PGC-1{alpha} and Pro12Ala polymorphism in PPAR{gamma}2 do not affect the functional integrity of these proteins.

  9. The effects of omega-3 polyunsaturated fatty acids and genetic variants on methylation levels of the interleukin-6 gene promoter

    PubMed Central

    Ma, Yiyi; Smith, Caren E.; Lai, Chao-Qiang; Irvin, Marguerite R.; Parnell, Laurence D.; Lee, Yu-Chi; Pham, Lucia D.; Aslibekyan, Stella; Claas, Steven A.; Tsai, Michael Y.; Borecki, Ingrid B.; Kabagambe, Edmond K.; Ordovás, José M.; Absher, Devin M.; Arnett, Donna K.

    2016-01-01

    Scope Omega-3 PUFAs (n-3 PUFAs) reduce IL-6 gene expression, but their effects on transcription regulatory mechanisms are unknown. We aimed to conduct an integrated analysis with both population and in vitro studies to systematically explore the relationships among n-3 PUFA, DNA methylation, single nucleotide polymorphisms (SNPs), gene expression, and protein concentration of IL6. Methods and results Using data in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study and the Encyclopedia of DNA Elements (ENCODE) consortium, we found that higher methylation of IL6 promoter cg01770232 was associated with higher IL-6 plasma concentration (p = 0.03) and greater IL6 gene expression (p = 0.0005). Higher circulating total n-3 PUFA was associated with lower cg01770232 methylation (p = 0.007) and lower IL-6 concentration (p = 0.02). Moreover, an allele of IL6 rs2961298 was associated with higher cg01770232 methylation (p = 2.55 × 10−7). The association between n-3 PUFA and cg01770232 methylation was dependent on rs2961298 genotype (p = 0.02), but higher total n-3 PUFA was associated with lower cg01770232 methylation in the heterozygotes (p = 0.04) not in the homozygotes. Conclusion Higher n-3 PUFA is associated with lower methylation at IL6 promoter, which may be modified by IL6 SNPs. PMID:26518637

  10. The impact of genome-wide supported schizophrenia risk variants in the neurogranin gene on brain structure and function.

    PubMed

    Walton, Esther; Geisler, Daniel; Hass, Johanna; Liu, Jingyu; Turner, Jessica; Yendiki, Anastasia; Smolka, Michael N; Ho, Beng-Choon; Manoach, Dara S; Gollub, Randy L; Roessner, Veit; Calhoun, Vince D; Ehrlich, Stefan

    2013-01-01

    The neural mechanisms underlying genetic risk for schizophrenia, a highly heritable psychiatric condition, are still under investigation. New schizophrenia risk genes discovered through genome-wide association studies (GWAS), such as neurogranin (NRGN), can be used to identify these mechanisms. In this study we examined the association of two common NRGN risk single nucleotide polymorphisms (SNPs) with functional and structural brain-based intermediate phenotypes for schizophrenia. We obtained structural, functional MRI and genotype data of 92 schizophrenia patients and 114 healthy volunteers from the multisite Mind Clinical Imaging Consortium study. Two schizophrenia-associated NRGN SNPs (rs12807809 and rs12541) were tested for association with working memory-elicited dorsolateral prefrontal cortex (DLPFC) activity and surface-wide cortical thickness. NRGN rs12541 risk allele homozygotes (TT) displayed increased working memory-related activity in several brain regions, including the left DLPFC, left insula, left somatosensory cortex and the cingulate cortex, when compared to non-risk allele carriers. NRGN rs12807809 non-risk allele (C) carriers showed reduced cortical gray matter thickness compared to risk allele homozygotes (TT) in an area comprising the right pericalcarine gyrus, the right cuneus, and the right lingual gyrus. Our study highlights the effects of schizophrenia risk variants in the NRGN gene on functional and structural brain-based intermediate phenotypes for schizophrenia. These results support recent GWAS findings and further implicate NRGN in the pathophysiology of schizophrenia by suggesting that genetic NRGN risk variants contribute to subtle changes in neural functioning and anatomy that can be quantified with neuroimaging methods. PMID:24098564

  11. Sequencing PDX1 (insulin promoter factor 1) in 1788 UK individuals found 5% had a low frequency coding variant, but these variants are not associated with Type 2 diabetes

    PubMed Central

    Edghill, E L; Khamis, A; Weedon, M N; Walker, M; Hitman, G A; McCarthy, M I; Owen, K R; Ellard, S; T Hattersley, A; Frayling, T M

    2011-01-01

    Aim Genome-wide association studies have identified > 30 common variants associated with Type 2 diabetes (> 5% minor allele frequency). These variants have small effects on individual risk and do not account for a large proportion of the heritable component of the disease. Monogenic forms of diabetes are caused by mutations that occur in < 1:2000 individuals and follow strict patterns of inheritance. In contrast, the role of low frequency genetic variants (minor allele frequency 0.1–5%) in Type 2 diabetes is not known. The aim of this study was to assess the role of low frequency PDX1 (also called IPF1) variants in Type 2 diabetes. Methods We sequenced the coding and flanking intronic regions of PDX1 in 910 patients with Type 2 diabetes and 878 control subjects. Results We identified a total of 26 variants that occurred in 5.3% of individuals, 14 of which occurred once. Only D76N occurred in > 1%. We found no difference in carrier frequency between patients (5.7%) and control subjects (5.0%) (P = 0.46). There were also no differences between patients and control subjects when analyses were limited to subsets of variants. The strongest subset were those variants in the DNA binding domain where all five variants identified were only found in patients (P = 0.06). Conclusion Approximately 5% of UK individuals carry a PDX1 variant, but there is no evidence that these variants, either individually or cumulatively, predispose to Type 2 diabetes. Further studies will need to consider strategies to assess the role of multiple variants that occur in < 1 in 1000 individuals. PMID:21569088

  12. Functional genetic variation in the basal promoter of the organic cation/carnitine transporters OCTN1 (SLC22A4) and OCTN2 (SLC22A5).

    PubMed

    Tahara, Harunobu; Yee, Sook Wah; Urban, Thomas J; Hesselson, Stephanie; Castro, Richard A; Kawamoto, Michiko; Stryke, Doug; Johns, Susan J; Ferrin, Thomas E; Kwok, Pui-Yan; Giacomini, Kathleen M

    2009-04-01

    The organic cation/ergothioneine transporter OCTN1 (SLC22A4) and the high-affinity carnitine transporter OCTN2 (SLC22A5), play an important role in the disposition of xenobiotics and endogenous compounds. Here, we analyzed the sequence of the proximal promoter regions of OCTN1 and OCTN2 in four ethnic groups and determined the effects of the identified genetic variants on transcriptional activities and mRNA expression. Six variants were found in the proximal promoter of OCTN1, one of which showed high allele frequency ranging from 13 to 34% in samples from individuals with ancestries in Africa, Europe, China, and Mexico. OCTN1 haplotypes had similar activities as the reference in luciferase reporter assays. For OCTN2, three of the seven variants identified in the proximal promoter showed allele frequencies greater than 29.5% in all populations, with the exception of -207C>G (rs2631367) that was monomorphic in Asian Americans. OCTN2 haplotypes containing -207G, present in all populations, were associated with a gain of function in luciferase reporter assays. Consistent with reporter assays, OCTN2 mRNA expression levels in lymphoblastoid cell lines (LCLs) from gene expression analysis were greater in samples carrying a marker for -207G. This SNP seems to contribute to racial differences in OCTN2 mRNA expression levels in LCLs. Our study with healthy subjects (n = 16) homozygous for either -207C or -207G, showed no appreciable effect of this SNP on carnitine disposition. However, there were significant effects of gender on carnitine plasma levels (p < 0.01). Further in vivo studies of OCTN2 promoter variants on carnitine disposition and variation in drug response are warranted.

  13. Hereditary fructose intolerance: functional study of two novel ALDOB natural variants and characterization of a partial gene deletion.

    PubMed

    Esposito, Gabriella; Imperato, Maria Rosaria; Ieno, Luigi; Sorvillo, Rosa; Benigno, Vincenzo; Parenti, Giancarlo; Parini, Rossella; Vitagliano, Luigi; Zagari, Adriana; Salvatore, Francesco

    2010-12-01

    Hereditary fructose intolerance (HFI) is an autosomal recessive metabolic disease caused by impaired functioning of human liver aldolase (ALDOB). At least 54 subtle/point mutations and only two large intragenic deletions have been found in the ALDOB gene. Here we report two novel ALDOB variants (p.R46W and p.Y343H) and an intragenic deletion that we found in patients with suspected HFI. The residual catalytic activity of the recombinant p.R46W and p.Y343H variants toward F1P was particularly altered. We also characterized a large intragenic deletion that we found in six unrelated patients. This is the first report of six unrelated patients sharing the same ALDOB deletion, thus indicating a founder effect for this allele in our geographic area. Because this deletion involves ALDOB exon 5, it can mimic worldwide common pathogenic genotypes, that is, homozygous p.A150P and p.A175D. Finally, the identification of only one ALDOB mutation in symptomatic patients suggests that HFI symptoms can, albeit rarely, appear also in heterozygotes. Therefore, an excessive and continuous fructose dietary intake may have deleterious effects even in apparently asymptomatic HFI carriers.

  14. Use of density functional theory orbitals in the GVVPT2 variant of second-order multistate multireference perturbation theory.

    PubMed

    Hoffmann, Mark R; Helgaker, Trygve

    2015-03-01

    A new variation of the second-order generalized van Vleck perturbation theory (GVVPT2) for molecular electronic structure is suggested. In contrast to the established procedure, in which CASSCF or MCSCF orbitals are first obtained and subsequently used to define a many-electron model (or reference) space, the use of an orbital space obtained from the local density approximation (LDA) variant of density functional theory is considered. Through a final, noniterative diagonalization of an average Fock matrix within orbital subspaces, quasicanonical orbitals that are otherwise indistinguishable from quasicanonical orbitals obtained from a CASSCF or MCSCF calculation are obtained. Consequently, all advantages of the GVVPT2 method are retained, including use of macroconfigurations to define incomplete active spaces and rigorous avoidance of intruder states. The suggested variant is vetted on three well-known model problems: the symmetric stretching of the O-H bonds in water, the dissociation of N2, and the stretching of ground and excited states C2 to more than twice the equilibrium bond length of the ground state. It is observed that the LDA-based GVVPT2 calculations yield good results, of comparable quality to conventional CASSCF-based calculations. This is true even for the C2 model problem, in which the orbital space for each state was defined by the LDA orbitals. These results suggest that GVVPT2 can be applied to much larger problems than previously accessible.

  15. Functional variants in CYP1B1, KRAS and MTHFR genes are associated with shorter telomere length in postmenopausal women.

    PubMed

    Cerne, Jasmina Z; Pohar-Perme, Maja; Cerkovnik, Petra; Gersak, Ksenija; Novakovic, Srdjan

    2015-07-01

    Estrogens and antioxidants indirectly alleviate telomere attrition. However, available clinical data on the association between hormone exposure and telomere length are inconclusive. In the present study, we examined the effects of exogenous estrogen use and of some genetic factors implicated in estrogen metabolism and oxidative stress response on mean leukocyte telomere length. We studied 259 postmenopausal women. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695), MnSOD (rs4880), KRAS (rs61764370), and MTHFR (rs1801133 and rs1801131) polymorphisms. Mean leukocyte telomere length was measured using a quantitative real-time PCR assay. In multivariate analysis we found no association between oral contraceptives or hormone replacement therapy (HRT) and mean leukocyte telomere length. The presence of variant alleles in CYP1B1, KRAS and MTHFR genes was statistically significantly associated with shorter mean leukocyte telomere length. Further, the data provided evidence for the effect modification of the association between HRT and mean leukocyte telomere length by the CYP1B1, KRAS and MTHFR genotypes. Our findings suggest that functionally relevant genetic variants within estrogen and folate metabolic pathways may influence telomere length. We propose these genetic factors should be taken into consideration when interpreting associations between hormone exposure and telomere length.

  16. Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry

    PubMed Central

    Hawkins, Charles; Caruana, Julie; Schiksnis, Erin; Liu, Zhongchi

    2016-01-01

    Fragaria vesca is a species of diploid strawberry being developed as a model for the octoploid garden strawberry. This work sequenced and compared the genomes of three F. vesca accessions: ‘Hawaii 4′, ‘Rügen’, and ‘Yellow Wonder’. Genome-scale analyses of shared and distinct SNPs among these three accessions have revealed that ‘Rügen’ and ‘Yellow Wonder’ are more similar to each other than they are to ‘Hawaii 4’. Though all three accessions are inbred seven generations, each accession still possesses extensive heterozygosity, highlighting the inherent differences between individual plants even of the same accession. The identification of the impact of each SNP as well as the large number of Indel markers provides a foundation for locating candidate mutations underlying phenotypic variations among these F. vesca accessions and for mapping new mutations generated through forward genetics screens. Through systematic analysis of SNP variants affecting genes in anthocyanin biosynthesis and regulation, a candidate SNP in FveMYB10 was identified and then functionally confirmed to be responsible for the yellow color fruits made by many F. vesca accessions. As a whole, this study provides further resources for F. vesca and establishes a foundation for linking traits of economic importance to specific genes and variants. PMID:27377763

  17. Functional variants in CYP1B1, KRAS and MTHFR genes are associated with shorter telomere length in postmenopausal women.

    PubMed

    Cerne, Jasmina Z; Pohar-Perme, Maja; Cerkovnik, Petra; Gersak, Ksenija; Novakovic, Srdjan

    2015-07-01

    Estrogens and antioxidants indirectly alleviate telomere attrition. However, available clinical data on the association between hormone exposure and telomere length are inconclusive. In the present study, we examined the effects of exogenous estrogen use and of some genetic factors implicated in estrogen metabolism and oxidative stress response on mean leukocyte telomere length. We studied 259 postmenopausal women. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695), MnSOD (rs4880), KRAS (rs61764370), and MTHFR (rs1801133 and rs1801131) polymorphisms. Mean leukocyte telomere length was measured using a quantitative real-time PCR assay. In multivariate analysis we found no association between oral contraceptives or hormone replacement therapy (HRT) and mean leukocyte telomere length. The presence of variant alleles in CYP1B1, KRAS and MTHFR genes was statistically significantly associated with shorter mean leukocyte telomere length. Further, the data provided evidence for the effect modification of the association between HRT and mean leukocyte telomere length by the CYP1B1, KRAS and MTHFR genotypes. Our findings suggest that functionally relevant genetic variants within estrogen and folate metabolic pathways may influence telomere length. We propose these genetic factors should be taken into consideration when interpreting associations between hormone exposure and telomere length. PMID:25987236

  18. Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry.

    PubMed

    Hawkins, Charles; Caruana, Julie; Schiksnis, Erin; Liu, Zhongchi

    2016-01-01

    Fragaria vesca is a species of diploid strawberry being developed as a model for the octoploid garden strawberry. This work sequenced and compared the genomes of three F. vesca accessions: 'Hawaii 4', 'Rügen', and 'Yellow Wonder'. Genome-scale analyses of shared and distinct SNPs among these three accessions have revealed that 'Rügen' and 'Yellow Wonder' are more similar to each other than they are to 'Hawaii 4'. Though all three accessions are inbred seven generations, each accession still possesses extensive heterozygosity, highlighting the inherent differences between individual plants even of the same accession. The identification of the impact of each SNP as well as the large number of Indel markers provides a foundation for locating candidate mutations underlying phenotypic variations among these F. vesca accessions and for mapping new mutations generated through forward genetics screens. Through systematic analysis of SNP variants affecting genes in anthocyanin biosynthesis and regulation, a candidate SNP in FveMYB10 was identified and then functionally confirmed to be responsible for the yellow color fruits made by many F. vesca accessions. As a whole, this study provides further resources for F. vesca and establishes a foundation for linking traits of economic importance to specific genes and variants. PMID:27377763

  19. Gain-of-function missense variant in SLC12A2, encoding the bumetanide-sensitive NKCC1 cotransporter, identified in human schizophrenia.

    PubMed

    Merner, Nancy D; Mercado, Adriana; Khanna, Arjun R; Hodgkinson, Alan; Bruat, Vanessa; Awadalla, Philip; Gamba, Gerardo; Rouleau, Guy A; Kahle, Kristopher T

    2016-06-01

    Perturbations of γ-aminobutyric acid (GABA) neurotransmission in the human prefrontal cortex have been implicated in the pathogenesis of schizophrenia (SCZ), but the mechanisms are unclear. NKCC1 (SLC12A2) is a Cl(-)-importing cation-Cl(-) cotransporter that contributes to the maintenance of depolarizing GABA activity in immature neurons, and variation in SLC12A2 has been shown to increase the risk for schizophrenia via alterations of NKCC1 mRNA expression. However, no disease-causing mutations or functional variants in NKCC1 have been identified in human patients with SCZ. Here, by sequencing three large French-Canadian (FC) patient cohorts of SCZ, autism spectrum disorders (ASD), and intellectual disability (ID), we identified a novel heterozygous NKCC1 missense variant (p.Y199C) in SCZ. This variant is located in an evolutionarily conserved residue in the critical N-terminal regulatory domain and exhibits high predicted pathogenicity. No NKCC1 variants were detected in ASD or ID, and no KCC3 variants were identified in any of the three neurodevelopmental disorder cohorts. Functional experiments show Y199C is a gain-of-function variant, increasing Cl(-)-dependent and bumetanide-sensitive NKCC1 activity even in conditions in which the transporter is normally functionally silent (hypotonicity). These data are the first to describe a functional missense variant in SLC12A2 in human SCZ, and suggest that genetically encoded dysregulation of NKCC1 may be a risk factor for, or contribute to the pathogenesis of, human SCZ.

  20. Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations | Office of Cancer Genomics

    Cancer.gov

    Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers.

  1. Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus.

    PubMed

    Mahajan, Anubha; Sim, Xueling; Ng, Hui Jin; Manning, Alisa; Rivas, Manuel A; Highland, Heather M; Locke, Adam E; Grarup, Niels; Im, Hae Kyung; Cingolani, Pablo; Flannick, Jason; Fontanillas, Pierre; Fuchsberger, Christian; Gaulton, Kyle J; Teslovich, Tanya M; Rayner, N William; Robertson, Neil R; Beer, Nicola L; Rundle, Jana K; Bork-Jensen, Jette; Ladenvall, Claes; Blancher, Christine; Buck, David; Buck, Gemma; Burtt, Noël P; Gabriel, Stacey; Gjesing, Anette P; Groves, Christopher J; Hollensted, Mette; Huyghe, Jeroen R; Jackson, Anne U; Jun, Goo; Justesen, Johanne Marie; Mangino, Massimo; Murphy, Jacquelyn; Neville, Matt; Onofrio, Robert; Small, Kerrin S; Stringham, Heather M; Syvänen, Ann-Christine; Trakalo, Joseph; Abecasis, Goncalo; Bell, Graeme I; Blangero, John; Cox, Nancy J; Duggirala, Ravindranath; Hanis, Craig L; Seielstad, Mark; Wilson, James G; Christensen, Cramer; Brandslund, Ivan; Rauramaa, Rainer; Surdulescu, Gabriela L; Doney, Alex S F; Lannfelt, Lars; Linneberg, Allan; Isomaa, Bo; Tuomi, Tiinamaija; Jørgensen, Marit E; Jørgensen, Torben; Kuusisto, Johanna; Uusitupa, Matti; Salomaa, Veikko; Spector, Timothy D; Morris, Andrew D; Palmer, Colin N A; Collins, Francis S; Mohlke, Karen L; Bergman, Richard N; Ingelsson, Erik; Lind, Lars; Tuomilehto, Jaakko; Hansen, Torben; Watanabe, Richard M; Prokopenko, Inga; Dupuis, Josee; Karpe, Fredrik; Groop, Leif; Laakso, Markku; Pedersen, Oluf; Florez, Jose C; Morris, Andrew P; Altshuler, David; Meigs, James B; Boehnke, Michael; McCarthy, Mark I; Lindgren, Cecilia M; Gloyn, Anna L

    2015-01-01

    Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights. PMID:25625282

  2. Identification and Functional Characterization of G6PC2 Coding Variants Influencing Glycemic Traits Define an Effector Transcript at the G6PC2-ABCB11 Locus

    PubMed Central

    Mahajan, Anubha; Sim, Xueling; Ng, Hui Jin; Manning, Alisa; Rivas, Manuel A.; Highland, Heather M.; Locke, Adam E.; Grarup, Niels; Im, Hae Kyung; Cingolani, Pablo; Flannick, Jason; Fontanillas, Pierre; Fuchsberger, Christian; Gaulton, Kyle J.; Teslovich, Tanya M.; Rayner, N. William; Robertson, Neil R.; Beer, Nicola L.; Rundle, Jana K.; Bork-Jensen, Jette; Ladenvall, Claes; Blancher, Christine; Buck, David; Buck, Gemma; Burtt, Noël P.; Gabriel, Stacey; Gjesing, Anette P.; Groves, Christopher J.; Hollensted, Mette; Huyghe, Jeroen R.; Jackson, Anne U.; Jun, Goo; Justesen, Johanne Marie; Mangino, Massimo; Murphy, Jacquelyn; Neville, Matt; Onofrio, Robert; Small, Kerrin S.; Stringham, Heather M.; Syvänen, Ann-Christine; Trakalo, Joseph; Abecasis, Goncalo; Bell, Graeme I.; Blangero, John; Cox, Nancy J.; Duggirala, Ravindranath; Hanis, Craig L.; Seielstad, Mark; Wilson, James G.; Christensen, Cramer; Brandslund, Ivan; Rauramaa, Rainer; Surdulescu, Gabriela L.; Doney, Alex S. F.; Lannfelt, Lars; Linneberg, Allan; Isomaa, Bo; Tuomi, Tiinamaija; Jørgensen, Marit E.; Jørgensen, Torben; Kuusisto, Johanna; Uusitupa, Matti; Salomaa, Veikko; Spector, Timothy D.; Morris, Andrew D.; Palmer, Colin N. A.; Collins, Francis S.; Mohlke, Karen L.; Bergman, Richard N.; Ingelsson, Erik; Lind, Lars; Tuomilehto, Jaakko; Hansen, Torben; Watanabe, Richard M.; Prokopenko, Inga; Dupuis, Josee; Karpe, Fredrik; Groop, Leif; Laakso, Markku; Pedersen, Oluf; Florez, Jose C.; Morris, Andrew P.; Altshuler, David; Meigs, James B.; Boehnke, Michael; McCarthy, Mark I.; Lindgren, Cecilia M.; Gloyn, Anna L.

    2015-01-01

    Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights. PMID:25625282

  3. The origin, evolution, and functional impact of short insertion-deletion variants identified in 179 human genomes.

    PubMed

    Montgomery, Stephen B; Goode, David L; Kvikstad, Erika; Albers, Cornelis A; Zhang, Zhengdong D; Mu, Xinmeng Jasmine; Ananda, Guruprasad; Howie, Bryan; Karczewski, Konrad J; Smith, Kevin S; Anaya, Vanessa; Richardson, Rhea; Davis, Joe; MacArthur, Daniel G; Sidow, Arend; Duret, Laurent; Gerstein, Mark; Makova, Kateryna D; Marchini, Jonathan; McVean, Gil; Lunter, Gerton

    2013-05-01

    Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%-48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels.

  4. The origin, evolution, and functional impact of short insertion-deletion variants identified in 179 human genomes.

    PubMed

    Montgomery, Stephen B; Goode, David L; Kvikstad, Erika; Albers, Cornelis A; Zhang, Zhengdong D; Mu, Xinmeng Jasmine; Ananda, Guruprasad; Howie, Bryan; Karczewski, Konrad J; Smith, Kevin S; Anaya, Vanessa; Richardson, Rhea; Davis, Joe; MacArthur, Daniel G; Sidow, Arend; Duret, Laurent; Gerstein, Mark; Makova, Kateryna D; Marchini, Jonathan; McVean, Gil; Lunter, Gerton

    2013-05-01

    Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%-48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels. PMID

  5. Carbachol promotes gastrointestinal function during oral resuscitation of burn shock

    PubMed Central

    Hu, Sen; Che, Jin-Wei; Tian, Yi-Jun; Sheng, Zhi-Yong

    2011-01-01

    AIM: To investigate the effect of carbachol on gastrointestinal function in a dog model of oral resuscitation for burn shock. METHODS: Twenty Beagle dogs with intubation of the carotid artery, jugular vein and jejunum for 24 h were subjected to 35% total body surface area full-thickness burns, and were divided into three groups: no fluid resuscitation (NR, n = 10), in which animals did not receive fluid by any means in the first 24 h post-burn; oral fluid resuscitation (OR, n = 8), in which dogs were gavaged with glucose-electrolyte solution (GES) with volume and rate consistent with the Parkland formula; and oral fluid with carbachol group (OR/CAR, n = 8), in which dogs were gavaged with GES containing carbachol (20 μg/kg), with the same volume and rate as the OR group. Twenty-four hours after burns, all animals were given intravenous fluid replacement, and 72 h after injury, they received nutritional support. Hemodynamic and gastrointestinal parameters were measured serially with animals in conscious and cooperative state. RESULTS: The mean arterial pressure, cardiac output and plasma volume dropped markedly, and gastrointestinal tissue perfusion was reduced obviously after the burn injury in all the three groups. Hemodynamic parameters and gastrointestinal tissue perfusion in the OR and OR/CAR groups were promoted to pre-injury level at 48 and 72 h, respectively, while hemodynamic parameters in the NR group did not return to pre-injury level till 72 h, and gastrointestinal tissue perfusion remained lower than pre-injury level until 120 h post-burn. CO2 of the gastric mucosa and intestinal mucosa blood flow of OR/CAR groups were 56.4 ± 4.7 mmHg and157.7 ± 17.7 blood perfusion units (BPU) at 24 h post-burn, respectively, which were significantly superior to those in the OR group (65.8 ± 5.8 mmHg and 127.7 ± 11.9 BPU, respectively, all P < 0.05). Gastric emptying and intestinal absorption rates of GES were significantly reduced to the lowest level (52.8% and

  6. Loss-of-function variants of SETD5 cause intellectual disability and the core phenotype of microdeletion 3p25.3 syndrome

    PubMed Central

    Kuechler, Alma; Zink, Alexander M; Wieland, Thomas; Lüdecke, Hermann-Josef; Cremer, Kirsten; Salviati, Leonardo; Magini, Pamela; Najafi, Kimia; Zweier, Christiane; Czeschik, Johanna Christina; Aretz, Stefan; Endele, Sabine; Tamburrino, Federica; Pinato, Claudia; Clementi, Maurizio; Gundlach, Jasmin; Maylahn, Carina; Mazzanti, Laura; Wohlleber, Eva; Schwarzmayr, Thomas; Kariminejad, Roxana; Schlessinger, Avner; Wieczorek, Dagmar; Strom, Tim M; Novarino, Gaia; Engels, Hartmut

    2015-01-01

    Intellectual disability (ID) has an estimated prevalence of 2–3%. Due to its extreme heterogeneity, the genetic basis of ID remains elusive in many cases. Recently, whole exome sequencing (WES) studies revealed that a large proportion of sporadic cases are caused by de novo gene variants. To identify further genes involved in ID, we performed WES in 250 patients with unexplained ID and their unaffected parents and included exomes of 51 previously sequenced child–parents trios in the analysis. Exome analysis revealed de novo intragenic variants in SET domain-containing 5 (SETD5) in two patients. One patient carried a nonsense variant, and the other an 81 bp deletion located across a splice-donor site. Chromosomal microarray diagnostics further identified four de novo non-recurrent microdeletions encompassing SETD5. CRISPR/Cas9 mutation modelling of the two intragenic variants demonstrated nonsense-mediated decay of the resulting transcripts, pointing to a loss-of-function (LoF) and haploinsufficiency as the common disease-causing mechanism of intragenic SETD5 sequence variants and SETD5-containing microdeletions. In silico domain prediction of SETD5, a predicted SET domain-containing histone methyltransferase (HMT), substantiated the presence of a SET domain and identified a novel putative PHD domain, strengthening a functional link to well-known histone-modifying ID genes. All six patients presented with ID and certain facial dysmorphisms, suggesting that SETD5 sequence variants contribute substantially to the microdeletion 3p25.3 phenotype. The present report of two SETD5 LoF variants in 301 patients demonstrates a prevalence of 0.7% and thus SETD5 variants as a relatively frequent cause of ID. PMID:25138099

  7. Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.

    PubMed

    McClure-Begley, T D; Papke, R L; Stone, K L; Stokes, C; Levy, A D; Gelernter, J; Xie, P; Lindstrom, J; Picciotto, M R

    2014-03-01

    Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4β2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4β2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4β2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4β2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

  8. Individualized and time-variant model for the functional link between thermoregulation and sleep onset.

    PubMed

    Quanten, Stijn; de Valck, Elke; Cluydts, Raymond; Aerts, Jean-Marie; Berckmans, Daniel

    2006-06-01

    This study makes use of control system model identification techniques to examine the relationship between thermoregulation and sleep regulation. Specifically, data-based mechanistic (DBM) modelling is used to formulate and experimentally test the hypothesis, put forth by Gilbert et al., that there exists a connection between distal heat loss and sleepiness. Six healthy sleepers each spent three nights and the following day in the sleep laboratory: an adaptation, a cognitive arousal and a neutral testing day. In the cognitive arousal condition, a visit of a television camera crew took place and subjects were asked to be interviewed. During each of the three 25-min driving simulator tasks per day, the distal-to-proximal gradient and the electroencephalogram are recorded. It is observed from these experimental data that there exists a feedback connection between thermoregulation and sleep. In addition to providing experimental evidence in support of the Gilbert et al. (2004) hypothesis, the authors propose that the nature of the feedback connection is determined by the nature of sleep/wake state (i.e. NREM sleep versus unwanted sleepiness in active subjects). Besides this, an individualized and time-variant model for the linkage between thermoregulation and sleep onset is presented. This compact model feeds on real-time data regarding distal heat loss and sleepiness and contains a physically meaningful parameter that delivers an individual- and time-depending quantification of a well known biological features in the field of thermoregulation: the thermoregulatory error signal T(hypo)(t)-T(set)(t). A validation of these physical/biological features emphasizes the reliability and power of DBM in describing individual differences related to the sleep process.

  9. Effects of Common Genetic Variants Associated With Type 2 Diabetes and Glycemic Traits on α- and β-Cell Function and Insulin Action in Humans

    PubMed Central

    Jonsson, Anna; Ladenvall, Claes; Ahluwalia, Tarunveer Singh; Kravic, Jasmina; Krus, Ulrika; Taneera, Jalal; Isomaa, Bo; Tuomi, Tiinamaija; Renström, Erik; Groop, Leif; Lyssenko, Valeriya

    2013-01-01

    Although meta-analyses of genome-wide association studies have identified >60 single nucleotide polymorphisms (SNPs) associated with type 2 diabetes and/or glycemic traits, there is little information on whether these variants also affect α-cell function. The aim of the current study was to evaluate the effects of glycemia-associated genetic loci on islet function in vivo and in vitro. We studied 43 SNPs in 4,654 normoglycemic participants from the Finnish population-based Prevalence, Prediction, and Prevention of Diabetes-Botnia (PPP-Botnia) Study. Islet function was assessed, in vivo, by measuring insulin and glucagon concentrations during oral glucose tolerance test, and, in vitro, by measuring glucose-stimulated insulin and glucagon secretion from human pancreatic islets. Carriers of risk variants in BCL11A, HHEX, ZBED3, HNF1A, IGF1, and NOTCH2 showed elevated whereas those in CRY2, IGF2BP2, TSPAN8, and KCNJ11 showed decreased fasting and/or 2-h glucagon concentrations in vivo. Variants in BCL11A, TSPAN8, and NOTCH2 affected glucagon secretion both in vivo and in vitro. The MTNR1B variant was a clear outlier in the relationship analysis between insulin secretion and action, as well as between insulin, glucose, and glucagon. Many of the genetic variants shown to be associated with type 2 diabetes or glycemic traits also exert pleiotropic in vivo and in vitro effects on islet function. PMID:23557703

  10. Analysis of genetic variants in the IL4 promoter and VNTR loci in Indian patients with Visceral Leishmaniasis.

    PubMed

    Mishra, Anshuman; Jha, Aditya Nath; van Tong, Hoang; Singh, Vipin Kumar; Gomes, Carlos E M; Singh, Lalji; Velavan, Thirumalaisamy P; Thangaraj, Kumarasamy

    2014-12-01

    Visceral Leishmaniasis (VL) is the most severest form of Leishmaniasis and resistance to infection is mediated by cellular immune responses. Interleukin 4 (IL-4) orchestrates of Th2 and Th1 immune responses during infections. In this study, we aimed to investigate possible association between three functional IL-4 polymorphisms -590C/T (rs2243250), -34C/T (rs2070874) and 70bp VNTR (rs79071878 in intron3) with VL in an Indian cohort comprising of 197 VL patients and 193 healthy controls. The three investigated IL-4 polymorphisms were in strong linkage disequilibrium. The investigated IL-4 alleles, genotypes and the reconstructed haplotypes were not significantly distributed between the VL patients and healthy controls. Our study signifies no possible association of functional IL-4 polymorphisms with Indian VL and postulate other vital genes involved in the IL-4 pathway may provide genetic clues to elucidate of IL-4 regulation and immune-pathogenesis during VL.

  11. Functional analysis of bipartite begomovirus coat protein promoter sequences

    SciTech Connect

    Lacatus, Gabriela; Sunter, Garry

    2008-06-20

    We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters.

  12. Kinetic and Sequence-Structure-Function Analysis of LinB Enzyme Variants with β- and δ-Hexachlorocyclohexane

    PubMed Central

    Kumari, Kirti; Sharma, Pooja; Lal, Rup; Oakeshott, John G.; Pandey, Gunjan

    2014-01-01

    Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a ‘Persistent Organic pollutant’ by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity. PMID:25076214

  13. Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant.

    PubMed

    Yandell, C A; Francis, G L; Wheldrake, J F; Upton, Z

    1999-06-01

    Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

  14. AAV Vectors Expressing LDLR Gain-of-Function Variants Demonstrate Increased Efficacy in Mouse Models of Familial Hypercholesterolemia

    PubMed Central

    Somanathan, Suryanarayan; Jacobs, Frank; Wang, Qiang; Hanlon, Alexandra L; Wilson, James M; Rader, Daniel J

    2014-01-01

    Rationale Familial hypercholesterolemia (FH) is a genetic disorder that arises due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous FH (hoFH) is a candidate for gene therapy using adeno-associated viral (AAV) vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen AAV encoded LDLR expression. Objective We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction. Methods and Results Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and hIDOL. A panel of mutant hLDLRs was initially screened in vitro for escape from PCSK9. The variant hLDLR-L318D was further evaluated using a mouse model of hoFH lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme, APOBEC-1 (DKO). Administration of wild type hLDLR to DKO mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9 mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\\C818A construct avoided hIDOL regulation and achieved stable reductions in serum cholesterol. An AAV8.LDLR-L318D\\K809R\\C818A vector that carried all three amino acid substitutions conferred partial resistance to both hPCSK9 and hIDOL mediated degradation. Conclusion Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve upon a potential gene therapeutic approach to treat homozygous FH subjects. PMID:25023731

  15. A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis

    PubMed Central

    Eswarakumar, Veraragavan P.; Horowitz, Mark C.; Locklin, Rachel; Morriss-Kay, Gillian M.; Lonai, Peter

    2004-01-01

    The b and c variants of fibroblast growth factor receptor 2 (FGFR2) differ in sequence, binding specificity, and localization. Fgfr2b, expressed in epithelia, is required for limb outgrowth and branching morphogenesis, whereas the mesenchymal Fgfr2c variant is required by the osteocyte lineage for normal skeletogenesis. Gain-of-function mutations in human FGFR2c are associated with craniosynostosis syndromes. To confirm and extend this evidence, we introduced a Cys342Tyr replacement into Fgfr2c to create a gain-of-function mutation equivalent to a mutation in human Crouzon and Pfeiffer syndromes. Fgfr2cC342Y/+ heterozygote mice are viable and fertile with shortened face, protruding eyes, premature fusion of cranial sutures, and enhanced Spp1 expression in the calvaria. Homozygous mutants display multiple joint fusions, cleft palate, and trachea and lung defects, and die shortly after birth. They show enhanced Cbfa1/Runx2 expression without significant change in chondrocyte-specific Ihh, PTHrP, Sox9, Col2a, or Col10a gene expression. Histomorphometric analysis and bone marrow stromal cell culture showed a significant increase of osteoblast progenitors with no change in osteoclastogenic cells. Chondrocyte proliferation was decreased in the skull base at embryonic day 14.5 but not later. These results suggest that long-term aspects of the mutant phenotype, including craniosynostosis, are related to the Fgfr2c regulation of the osteoblast lineage. The effect on early chondrocyte proliferation but not gene expression suggests cooperation of Fgfr2c with Fgfr3 in the formation of the cartilage model for endochondral bone. PMID:15316116

  16. Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    PubMed Central

    Vallejos, Maricarmen; Carvajal, Felipe; Pino, Karla; Navarrete, Camilo; Ferres, Marcela; Huidobro-Toro, Juan Pablo; Sargueil, Bruno; López-Lastra, Marcelo

    2012-01-01

    The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed. PMID:22496887

  17. Venezuelan Equine Encephalitis Virus Variants Lacking Transcription Inhibitory Functions Demonstrate Highly Attenuated Phenotype

    PubMed Central

    Atasheva, Svetlana; Kim, Dal Young; Frolova, Elena I.

    2014-01-01

    ABSTRACT Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for the development of new rationally designed alphavirus vaccine candidates that combine efficient immunogenicity, high safety, and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study combine a variety of characteristics that independently contribute to a reduction in virulence. These constructs encode a noncytopathic VEEV capsid protein that is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications also affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses. IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging, and interaction with the host to design new VEEV vaccine candidates that demonstrate exceptionally high levels of safety due to a

  18. TNFalpha and IL-8 regulate the expression and function of CD44 variant proteins in human colon carcinoma cells.

    PubMed

    Barshishat, Michal; Ariel, Amiram; Cahalon, Liora; Chowers, Yehuda; Lider, Ofer; Schwartz, Betty

    2002-01-01

    The mechanisms underlying the inflammatory and metastatic processes share a number of similar pathways, such as those involving adhesion, migration and extravasation. In this article, the effects of pro-inflammatory cytokines on metastatic-related activities of colon cancer cells were tested. The expression and biological activity of the proteoglycan CD44 in low (LS174T) and high metastatic (HM7) cell lines following exposure to TNFalpha and IL-8 were assessed. Treated cells expressed more CD44 splice variants (CD44v), while CD44 standard protein (CD44s) expression remained unchanged. Treatment with TNFalpha induced IL-8 secretion and IL-8 gene transcription in a time-dependent manner. Both cytokines enhanced the ability of the cells to adhere to the CD44-specific ligand hyaluronic acid, an effect that was specifically blocked by an anti-IL-8 antibody. These results suggest that the effect of TNFalpha on IL-8 is responsible for the regulation of the expression of CD44 isoforms. Additional experiments showed that neither of the cytokines tested regulate the expression of CD44 gene regulation via activation of a well-characterized specific 22-bp epidermal growth factor regulatory element present in the CD44 promoter sequence, suggesting that this is not the mechanism of activation. We conclude that immuno-modulatory mediators can modify the expression of cell-to-cell or cell-to-matrix adhesion proteins, implicated in the determination of phenotypes associated with aggressiveness and metastasis of colon cancer cells. PMID:12090473

  19. Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

    PubMed

    Miller, Daniel E; Patel, Zubin H; Lu, Xiaoming; Lynch, Arthur T; Weirauch, Matthew T; Kottyan, Leah C

    2016-01-01

    Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype. PMID:27585267

  20. Human GRK4γ142V Variant Promotes Angiotensin II Type I Receptor-Mediated Hypertension via Renal Histone Deacetylase Type 1 Inhibition.

    PubMed

    Wang, Zheng; Zeng, Chunyu; Villar, Van Anthony M; Chen, Shi-You; Konkalmatt, Prasad; Wang, Xiaoyan; Asico, Laureano D; Jones, John E; Yang, Yu; Sanada, Hironobu; Felder, Robin A; Eisner, Gilbert M; Weir, Matthew R; Armando, Ines; Jose, Pedro A

    2016-02-01

    The influence of a single gene on the pathogenesis of essential hypertension may be difficult to ascertain, unless the gene interacts with other genes that are germane to blood pressure regulation. G-protein-coupled receptor kinase type 4 (GRK4) is one such gene. We have reported that the expression of its variant hGRK4γ(142V) in mice results in hypertension because of impaired dopamine D1 receptor. Signaling through dopamine D1 receptor and angiotensin II type I receptor (AT1R) reciprocally modulates renal sodium excretion and blood pressure. Here, we demonstrate the ability of the hGRK4γ(142V) to increase the expression and activity of the AT1R. We show that hGRK4γ(142V) phosphorylates histone deacetylase type 1 and promotes its nuclear export to the cytoplasm, resulting in increased AT1R expression and greater pressor response to angiotensin II. AT1R blockade and the deletion of the Agtr1a gene normalize the hypertension in hGRK4γ(142V) mice. These findings illustrate the unique role of GRK4 by targeting receptors with opposite physiological activity for the same goal of maintaining blood pressure homeostasis, and thus making the GRK4 a relevant therapeutic target to control blood pressure.

  1. Functional Relevance for Associations between Genetic Variants and Systemic Lupus Erythematosus

    PubMed Central

    Deng, Fei-Yan; Lei, Shu-Feng; Zhang, Yong-Hong; Zhang, Zeng-Li; Guo, Yu-Fan

    2013-01-01

    Systemic lupus erythematosus (SLE) is a serious prototype autoimmune disease characterized by chronic inflammation, auto-antibody production and multi-organ damage. Recent association studies have identified a long list of loci that were associated with SLE with relatively high statistical power. However, most of them only established the statistical associations of genetic markers and SLE at the DNA level without supporting evidence of functional relevance. Here, using publically available datasets, we performed integrative analyses (gene relationship across implicated loci analysis, differential gene expression analysis and functional annotation clustering analysis) and combined with expression quantitative trait loci (eQTLs) results to dissect functional mechanisms underlying the associations for SLE. We found that 14 SNPs, which were significantly associated with SLE in previous studies, have cis-regulation effects on four eQTL genes (HLA-DQA1, HLA-DQB1, HLA-DQB2, and IRF5) that were also differentially expressed in SLE-related cell groups. The functional evidence, taken together, suggested the functional mechanisms underlying the associations of 14 SNPs and SLE. The study may serve as an example of mining publically available datasets and results in validation of significant disease-association results. Utilization of public data resources for integrative analyses may provide novel insights into the molecular genetic mechanisms underlying human diseases. PMID:23341919

  2. Executive Function and the Promotion of Social-Emotional Competence

    ERIC Educational Resources Information Center

    Riggs, Nathaniel R.; Jahromi, Laudan B.; Razza, Rachel P.; Dillworth-Bart, Janean E.; Mueller, Ulrich

    2006-01-01

    Executive function is understood as an umbrella term encompassing a number of interrelated sub-skills necessary for purposeful, goal-directed activity. Research suggests a vital role for executive function in children's social-emotional development. However, executive function is rarely considered in models of intervention programs that attempt to…

  3. Algorithm for finding partitionings of hard variants of boolean satisfiability problem with application to inversion of some cryptographic functions.

    PubMed

    Semenov, Alexander; Zaikin, Oleg

    2016-01-01

    In this paper we propose an approach for constructing partitionings of hard variants of the Boolean satisfiability problem (SAT). Such partitionings can be used for solving corresponding SAT instances in parallel. For the same SAT instance one can construct different partitionings, each of them is a set of simplified versions of the original SAT instance. The effectiveness of an arbitrary partitioning is determined by the total time of solving of all SAT instances from it. We suggest the approach, based on the Monte Carlo method, for estimating time of processing of an arbitrary partitioning. With each partitioning we associate a point in the special finite search space. The estimation of effectiveness of the particular partitioning is the value of predictive function in the corresponding point of this space. The problem of search for an effective partitioning can be formulated as a problem of optimization of the predictive function. We use metaheuristic algorithms (simulated annealing and tabu search) to move from point to point in the search space. In our computational experiments we found partitionings for SAT instances encoding problems of inversion of some cryptographic functions. Several of these SAT instances with realistic predicted solving time were successfully solved on a computing cluster and in the volunteer computing project SAT@home. The solving time agrees well with estimations obtained by the proposed method. PMID:27190753

  4. Algorithm for finding partitionings of hard variants of boolean satisfiability problem with application to inversion of some cryptographic functions.

    PubMed

    Semenov, Alexander; Zaikin, Oleg

    2016-01-01

    In this paper we propose an approach for constructing partitionings of hard variants of the Boolean satisfiability problem (SAT). Such partitionings can be used for solving corresponding SAT instances in parallel. For the same SAT instance one can construct different partitionings, each of them is a set of simplified versions of the original SAT instance. The effectiveness of an arbitrary partitioning is determined by the total time of solving of all SAT instances from it. We suggest the approach, based on the Monte Carlo method, for estimating time of processing of an arbitrary partitioning. With each partitioning we associate a point in the special finite search space. The estimation of effectiveness of the particular partitioning is the value of predictive function in the corresponding point of this space. The problem of search for an effective partitioning can be formulated as a problem of optimization of the predictive function. We use metaheuristic algorithms (simulated annealing and tabu search) to move from point to point in the search space. In our computational experiments we found partitionings for SAT instances encoding problems of inversion of some cryptographic functions. Several of these SAT instances with realistic predicted solving time were successfully solved on a computing cluster and in the volunteer computing project SAT@home. The solving time agrees well with estimations obtained by the proposed method.

  5. The ETS Family Transcription Factors Etv5 and PU.1 Function in Parallel To Promote Th9 Cell Development.

    PubMed

    Koh, Byunghee; Hufford, Matthew M; Pham, Duy; Olson, Matthew R; Wu, Tong; Jabeen, Rukhsana; Sun, Xin; Kaplan, Mark H

    2016-09-15

    The IL-9-secreting Th9 subset of CD4 Th cells develop in response to an environment containing IL-4 and TGF-β, promoting allergic disease, autoimmunity, and resistance to pathogens. We previously identified a requirement for the ETS family transcription factor PU.1 in Th9 development. In this report, we demonstrate that the ETS transcription factor ETS variant 5 (ETV5) promotes IL-9 production in Th9 cells by binding and recruiting histone acetyltransferases to the Il9 locus at sites distinct from PU.1. In cells that are deficient in both PU.1 and ETV5 there is lower IL-9 production than in cells lacking either factor alone. In vivo loss of PU.1 and ETV5 in T cells results in distinct effects on allergic inflammation in the lung, suggesting that these factors function in parallel. Together, these data define a role for ETV5 in Th9 development and extend the paradigm of related transcription factors having complementary functions during differentiation.

  6. Predicting the functional consequences of non-synonymous DNA sequence variants--evaluation of bioinformatics tools and development of a consensus strategy.

    PubMed

    Frousios, Kimon; Iliopoulos, Costas S; Schlitt, Thomas; Simpson, Michael A

    2013-10-01

    The study of DNA sequence variation has been transformed by recent advances in DNA sequencing technologies. Determination of the functional consequences of sequence variant alleles offers potential insight as to how genotype may influence phenotype. Even within protein coding regions of the genome, establishing the consequences of variation on gene and protein function is challenging and requires substantial laboratory investigation. However, a series of bioinformatics tools have been developed to predict whether non-synonymous variants are neutral or disease-causing. In this study we evaluate the performance of nine such methods (SIFT, PolyPhen2, SNPs&GO, PhD-SNP, PANTHER, Mutation Assessor, MutPred, Condel and CAROL) and developed CoVEC (Consensus Variant Effect Classification), a tool that integrates the prediction results from four of these methods. We demonstrate that the CoVEC approach outperforms most individual methods and highlights the benefit of combining results from multiple tools. PMID:23831115

  7. Identification of a functional splice variant of 14-3-3E1 in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are a family of regulatory proteins involved in diverse cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. In this study, we report the identification and charact...

  8. Constitutively-active androgen receptor variants function independently of the HSP90 chaperone but do not confer resistance to HSP90 inhibitors.

    PubMed

    Gillis, Joanna L; Selth, Luke A; Centenera, Margaret M; Townley, Scott L; Sun, Shihua; Plymate, Stephen R; Tilley, Wayne D; Butler, Lisa M

    2013-05-01

    The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer.

  9. The Effect of Turmeric (Curcuma longa) Extract on the Functionality of the Solute Carrier Protein 22 A4 (SLC22A4) and Interleukin-10 (IL-10) Variants Associated with Inflammatory Bowel Disease

    PubMed Central

    McCann, Mark J.; Johnston, Sarah; Reilly, Kerri; Men, Xuejing; Burgess, Elaine J.; Perry, Nigel B.; Roy, Nicole C.

    2014-01-01

    Inflammatory bowel disease (IBD) is a chronic relapsing disease. Genetic predisposition to the disease reduces an individual’s capacity to respond appropriately to environmental challenges in the intestine leading to inappropriate inflammation. IBD patients often modify their diet to mitigate or reduce the severity of inflammation. Turmeric (Curcuma longa L., Zingiberaceae) has historically been used in Chinese, Hindu, and Ayurvedic medicine over several centuries to treat inflammatory disorders. To understand how turmeric may influence the consequences of a genetic predisposition to inappropriate inflammation, we used HEK293 cells to examine the in vitro capacity of turmeric extract and fractions to affect the functionality of two gene variants, solute carrier protein 22 A4 (SLC22A4, rs1050152) and interleukin-10 (IL-10, rs1800896) associated with IBD. We found that a turmeric extract and several chromatographically separated fractions beneficially affected the variants of SLC22A4 and IL-10 associated with IBD, by reducing inappropriate epithelial cell transport (SLC22A4, 503F) and increasing anti-inflammatory cytokine gene promoter activity (IL-10, −1082A). The effect of turmeric on the IL-10 variant was strongly associated with the curcumin content of the extract and its fractions. PMID:25314644

  10. The effect of turmeric (Curcuma longa) extract on the functionality of the solute carrier protein 22 A4 (SLC22A4) and interleukin-10 (IL-10) variants associated with inflammatory bowel disease.

    PubMed

    McCann, Mark J; Johnston, Sarah; Reilly, Kerri; Men, Xuejing; Burgess, Elaine J; Perry, Nigel B; Roy, Nicole C

    2014-10-13

    Inflammatory bowel disease (IBD) is a chronic relapsing disease. Genetic predisposition to the disease reduces an individual's capacity to respond appropriately to environmental challenges in the intestine leading to inappropriate inflammation. IBD patients often modify their diet to mitigate or reduce the severity of inflammation. Turmeric (Curcuma longa L., Zingiberaceae) has historically been used in Chinese, Hindu, and Ayurvedic medicine over several centuries to treat inflammatory disorders. To understand how turmeric may influence the consequences of a genetic predisposition to inappropriate inflammation, we used HEK293 cells to examine the in vitro capacity of turmeric extract and fractions to affect the functionality of two gene variants, solute carrier protein 22 A4 (SLC22A4, rs1050152) and interleukin-10 (IL-10, rs1800896) associated with IBD. We found that a turmeric extract and several chromatographically separated fractions beneficially affected the variants of SLC22A4 and IL-10 associated with IBD, by reducing inappropriate epithelial cell transport (SLC22A4, 503F) and increasing anti-inflammatory cytokine gene promoter activity (IL-10, -1082A). The effect of turmeric on the IL-10 variant was strongly associated with the curcumin content of the extract and its fractions.

  11. The effect of turmeric (Curcuma longa) extract on the functionality of the solute carrier protein 22 A4 (SLC22A4) and interleukin-10 (IL-10) variants associated with inflammatory bowel disease.

    PubMed

    McCann, Mark J; Johnston, Sarah; Reilly, Kerri; Men, Xuejing; Burgess, Elaine J; Perry, Nigel B; Roy, Nicole C

    2014-10-01

    Inflammatory bowel disease (IBD) is a chronic relapsing disease. Genetic predisposition to the disease reduces an individual's capacity to respond appropriately to environmental challenges in the intestine leading to inappropriate inflammation. IBD patients often modify their diet to mitigate or reduce the severity of inflammation. Turmeric (Curcuma longa L., Zingiberaceae) has historically been used in Chinese, Hindu, and Ayurvedic medicine over several centuries to treat inflammatory disorders. To understand how turmeric may influence the consequences of a genetic predisposition to inappropriate inflammation, we used HEK293 cells to examine the in vitro capacity of turmeric extract and fractions to affect the functionality of two gene variants, solute carrier protein 22 A4 (SLC22A4, rs1050152) and interleukin-10 (IL-10, rs1800896) associated with IBD. We found that a turmeric extract and several chromatographically separated fractions beneficially affected the variants of SLC22A4 and IL-10 associated with IBD, by reducing inappropriate epithelial cell transport (SLC22A4, 503F) and increasing anti-inflammatory cytokine gene promoter activity (IL-10, -1082A). The effect of turmeric on the IL-10 variant was strongly associated with the curcumin content of the extract and its fractions. PMID:25314644

  12. Functional architecture of two exclusively late stage pollen-specific promoters in rice (Oryza sativa L.).

    PubMed

    Yan, Shuo; Wang, Zhongni; Liu, Yuan; Li, Wei; Wu, Feng; Lin, Xuelei; Meng, Zheng

    2015-07-01

    Late stage pollen-specific promoters are important tools in crop molecular breeding. Several such promoters, and their functional motifs, have been well characterized in dicotyledonous plants such as tomato and tobacco. However, knowledge about the functional architecture of such promoters is limited in the monocotyledonous plant rice. Here, pollen-late-stage-promoter 1 (PLP1) and pollen-late-stage-promoter 2 (PLP2) were characterized using a stable transformation system in rice. Histochemical staining showed that the two promoters exclusively drive GUS expression in late-stage pollen grains in rice. 5' deletion analysis revealed that four regions, including the -1159 to -720 and the -352 to -156 regions of PLP1 and the -740 to -557 and the -557 to -339 regions of PLP2, are important in maintaining the activity and specificity of these promoters. Motif mutation analysis indicated that 'AGAAA' and 'CAAT' motifs in the -740 to -557 region of PLP2 act as enhancers in the promoter. Gain of function experiments indicated that the novel TA-rich motif 'TACATAA' and 'TATTCAT' in the core region of the PLP1 and PLP2 promoters is necessary, but not sufficient, for pollen-specific expression in rice. Our results provide evidence that the enhancer motif 'AGAAA' is conserved in the pollen-specific promoters of both monocots and eudicots, but that some functional architecture characteristics are different.

  13. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function.

    PubMed

    Tajnik, Mojca; Rogalska, Malgorzata Ewa; Bussani, Erica; Barbon, Elena; Balestra, Dario; Pinotti, Mirko; Pagani, Franco

    2016-05-01

    Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. PMID:27227676

  14. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

    PubMed Central

    Bussani, Erica; Barbon, Elena; Pinotti, Mirko; Pagani, Franco

    2016-01-01

    Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. PMID:27227676

  15. Functional Studies on the IBD Susceptibility Gene IL23R Implicate Reduced Receptor Function in the Protective Genetic Variant R381Q

    PubMed Central

    Pidasheva, Svetlana; Trifari, Sara; Phillips, Anne; Hackney, Jason A.; Ma, Yan; Smith, Ashley; Sohn, Sue J.; Spits, Hergen; Little, Randall D.; Behrens, Timothy W.; Honigberg, Lee; Ghilardi, Nico; Clark, Hilary F.

    2011-01-01

    Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the IL23R gene with IBD (Crohn's disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23RR381 and IL23RQ381 haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23RQ381 was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23RQ381 positive donors. Our study shows conclusively that IL23RQ381 is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD. PMID:22022372

  16. Functional domains of the Xenopus laevis 5S gene promoter.

    PubMed Central

    Pieler, T; Oei, S L; Hamm, J; Engelke, U; Erdmann, V A

    1985-01-01

    To study the fine structure of the Xenopus laevis somatic 5S gene internal control region, we have created 15 different transversions using mutagenic oligonucleotide primers. The effects of these mutations on 5S DNA transcription in vitro as well as on stable complex formation with transcription factor TF III A and TF III C in crude nuclear extracts were analyzed. Mutations in the common class III 5' promoter element (nucleotides 50-61 in the 5S gene) interfere with transcription activity and stable complex formation whenever they contradict the tDNA box A consensus sequence. The second promoter element is defined by a major sequence block (nucleotides 80-89, box C) and two additional internal residues (70 and 71) at a distance of roughly one helical turn from both the major 3' and 5' control sequences; these two 3' elements contain the primary TF III A binding domain. The remaining nucleotides (62-69 and 71-79) when mutated do not interfere with transcription activity or factor binding and thus they constitute two spacer elements within a symmetrically structured 5S gene promoter. An increase in the relative spacing of box A and box C by insertion of 3 bp between nucleotides 66 and 67 leads to a drastic reduction in transcription activity and the ability to form a stable complex with TF III A and/or TF III C. Thus, accurate spacing is essential for the proper orientation of TF III A on 5S DNA and/or TF III C binding. Images Fig. 1. Fig. 3. Fig. 4. PMID:3004969

  17. Growing and Growing: Promoting Functional Thinking with Geometric Growing Patterns

    ERIC Educational Resources Information Center

    Markworth, Kimberly A.

    2010-01-01

    Design research methodology is used in this study to develop an empirically-substantiated instruction theory about students' development of functional thinking in the context of geometric growing patterns. The two research questions are: (1) How does students' functional thinking develop in the context of geometric growing patterns? (2) What are…

  18. Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria.

    PubMed

    Pesarrodona, Mireia; Fernández, Yolanda; Foradada, Laia; Sánchez-Chardi, Alejandro; Conchillo-Solé, Oscar; Unzueta, Ugutz; Xu, Zhikun; Roldán, Mónica; Villegas, Sandra; Ferrer-Miralles, Neus; Schwartz, Simó; Rinas, Ursula; Daura, Xavier; Abasolo, Ibane; Vázquez, Esther; Villaverde, Antonio

    2016-06-01

    Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source. PMID:27078873

  19. Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria.

    PubMed

    Pesarrodona, Mireia; Fernández, Yolanda; Foradada, Laia; Sánchez-Chardi, Alejandro; Conchillo-Solé, Oscar; Unzueta, Ugutz; Xu, Zhikun; Roldán, Mónica; Villegas, Sandra; Ferrer-Miralles, Neus; Schwartz, Simó; Rinas, Ursula; Daura, Xavier; Abasolo, Ibane; Vázquez, Esther; Villaverde, Antonio

    2016-06-01

    Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source.

  20. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean

    PubMed Central

    Zhou, Yuan; Yang, Yan; Zhou, Xinjian; Chi, Yingjun; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution. PMID:27708406

  1. The Secret Agent Society Social Skills Program for Children with High-Functioning Autism Spectrum Disorders: A Comparison of Two School Variants

    ERIC Educational Resources Information Center

    Beaumont, Renae; Rotolone, Cassie; Sofronoff, Kate

    2015-01-01

    School is often considered an ideal setting for child social skills training due to the opportunities it provides for skills teaching, modeling, and practice. The current study evaluated the effectiveness of two variants of the Secret Agent Society social skills program for children with high-functioning autism spectrum disorders (HFASD) in a…

  2. Psychosocial Treatments to Promote Functional Recovery in Schizophrenia

    PubMed Central

    Kern, Robert S.; Glynn, Shirley M.; Horan, William P.; Marder, Stephen R.

    2009-01-01

    A number of psychosocial treatments are available for persons with schizophrenia that include social skills training, cognitive behavioral therapy, cognitive remediation, and social cognition training. These treatments are reviewed and discussed in terms of how they address key components of functional recovery such as symptom stability, independent living, work functioning, and social functioning. We also review findings on the interaction between pharmacological and psychosocial treatments and discuss future directions in pharmacological treatment of schizophrenia. Overall, these treatments provide a range of promising approaches to helping patients achieve better outcomes far beyond symptom stabilization. PMID:19176470

  3. Cellulase variants

    DOEpatents

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  4. Association Between Variants of PRDM1 and NDP52 and Crohn’s Disease, Based on Exome Sequencing and Functional Studies

    PubMed Central

    Ellinghaus, David; Zhang, Hu; Zeissig, Sebastian; Lipinski, Simone; Till, Andreas; Jiang, Tao; Stade, Björn; Bromberg, Yana; Ellinghaus, Eva; Keller, Andreas; Rivas, Manuel A; Skieceviciene, Jurgita; Doncheva, Nadezhda T; Liu, Xiao; Liu, Qing; Jiang, Fuman; Forster, Michael; Mayr, Gabriele; Albrecht, Mario; Häsler, Robert; Boehm, Bernhard O; Goodall, Jane; Berzuini, Carlo R; Lee, James; Andersen, Vibeke; Vogel, Ulla; Kupcinskas, Limas; Kayser, Manfred; Krawczak, Michael; Nikolaus, Susanna; Weersma, Rinse K; Ponsioen, Cyriel Y; Sans, Miquel; Wijmenga, Cisca; Strachan, David P; McArdle, Wendy L; Vermeire, Séverine; Rutgeerts, Paul; Sanderson, Jeremy D; Mathew, Christopher G; Vatn, Morten H; Wang, Jun; Nöthen, Markus M; Duerr, Richard H; Büning, Carsten; Brand, Stephan; Glas, Jürgen; Winkelmann, Juliane; Illig, Thomas; Latiano, Anna; Annese, Vito; Halfvarson, Jonas; D’Amato, Mauro; Daly, Mark J; Nothnagel, Michael; Karlsen, Tom H; Subramani, Suresh; Rosenstiel, Philip; Schreiber, Stefan; Parkes, Miles; Franke, Andre

    2013-01-01

    Background & Aims Genome-wide association studies (GWASs) have identified 140 Crohn’s disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies. Methods We sequenced whole exomes of 42 unrelated subjects with Crohn’s disease (CD) and 5 healthy individuals (controls), and then filtered single-nucleotide variants by incorporating association results from meta-analyses of CD GWASs and in silico mutation effect prediction algorithms. We then genotyped 9348 patients with CD, 2868 with ulcerative colitis, and 14,567 controls, and associated variants analyzed in functional studies using materials from patients and controls and in vitro model systems. Results We identified rare missense mutations in PR domain-containing1 (PRDM1) and associated these with CD. These increased proliferation of T cells and secretion of cytokines upon activation, and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWASs, correlated with reduced expression of PRDM1 in ileal biopsies and peripheral blood mononuclear cells (combined P=1.6×0−8). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P=4.83×10−9). We found that this variant impairs the regulatory functions of NDP52 to inhibit NFκB activation of genes that regulate inflammation and affect stability of proteins in toll-like receptor pathways. Conclusions We have extended GWAS results and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWASs and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and

  5. Identification and functional characterization in vivo of a novel splice variant of LDLR in rhesus macaques.

    PubMed

    Kassim, Sadik H; Vandenberghe, Luk H; Hovhannisyan, Ruben; Wilson, James M; Rader, Daniel J

    2011-08-16

    In the course of developing a low-density lipoprotein receptor (LDLR) gene therapy treatment for homozygous familial hypercholesterolemia (HoFH), we planned to examine the efficacy in a nonhuman primate model, the rhesus macaque heterozygous for an LDL receptor mutation fed a high-fat diet. Unexpectedly, our initial cDNA sequencing studies led to the identification of a heretofore unidentified splicing isoform of the rhesus LDLR gene. Compared with the publicly available GenBank reference sequence of rhesus LDLR, the novel isoform contains a 21 bp in frame insertion. This sequence coincides with part of exon 5 and creates a site for the restriction enzyme MscI. Using this site as a marker for the 21 bp in-frame insertion, we conducted a restriction enzyme screen to examine for the prevalence of the novel isoform in rhesus liver tissue cDNA and its homolog in human liver tissue cDNA. We found that the novel isoform is the predominant LDLR cDNA found in rhesus liver and the sole LDLR cDNA found in human liver. Finally, we compared the in vivo functionality of the novel and previously identified rhesus LDLR splicing isoforms in a mouse model of HoFH. PMID:21628398

  6. Identification and functional characterization in vivo of a novel splice variant of LDLR in rhesus macaques

    PubMed Central

    Kassim, Sadik H.; Vandenberghe, Luk H.; Hovhannisyan, Ruben; Rader, Daniel J.

    2011-01-01

    In the course of developing a low-density lipoprotein receptor (LDLR) gene therapy treatment for homozygous familial hypercholesterolemia (HoFH), we planned to examine the efficacy in a nonhuman primate model, the rhesus macaque heterozygous for an LDL receptor mutation fed a high-fat diet. Unexpectedly, our initial cDNA sequencing studies led to the identification of a heretofore unidentified splicing isoform of the rhesus LDLR gene. Compared with the publicly available GenBank reference sequence of rhesus LDLR, the novel isoform contains a 21 bp in frame insertion. This sequence coincides with part of exon 5 and creates a site for the restriction enzyme MscI. Using this site as a marker for the 21 bp in-frame insertion, we conducted a restriction enzyme screen to examine for the prevalence of the novel isoform in rhesus liver tissue cDNA and its homolog in human liver tissue cDNA. We found that the novel isoform is the predominant LDLR cDNA found in rhesus liver and the sole LDLR cDNA found in human liver. Finally, we compared the in vivo functionality of the novel and previously identified rhesus LDLR splicing isoforms in a mouse model of HoFH. PMID:21628398

  7. A functional NPSR1 gene variant and environment shape personality and impulsive action: a longitudinal study.

    PubMed

    Laas, Kariina; Reif, Andreas; Kiive, Evelyn; Domschke, Katharina; Lesch, Klaus-Peter; Veidebaum, Toomas; Harro, Jaanus

    2014-03-01

    Neuropeptide S and its receptor NPSR1 are involved in the regulation of arousal, attention and anxiety. We examined whether the NPSR1 gene functional polymorphism Asn¹⁰⁷Ile (rs324981, A>T) influences personality, impulsivity, and attention-deficit/hyperactivity disorder (ADHD)-related symptoms in a population-representative sample, and whether any eventual associations depend on age, sex, family relations and stressful life events (SLE). We used self-reports or teachers' ratings for both the younger (n=593) and older (n=583) cohort of the longitudinal Estonian Children Personality, Behaviour and Health Study. Males with the TT genotype displayed more ADHD-related symptoms. Adaptive impulsivity and Extraversion increased the most from age 18 to 25. While highest increases were observed in AA men, TT women exhibited the largest decreases. For participants with the AA genotype, Warmth in family was inversely associated with Neuroticism, and positively associated with Extraversion and Adaptive impulsivity. High exposure to SLE increased impulsivity and ADHD scores in TT genotype subjects. We conclude that the NPSR1 A/T polymorphism is associated with impulsivity, ADHD symptoms and personality, mirroring the activity- and anxiety-mediating role of NPSR1. Heterozygous individuals were the least sensitive to environmental factors, whereas subjects with the AA genotype and TT genotype reacted to different types of environmental adversities.

  8. Time-variant species pools shape competitive dynamics and biodiversity-ecosystem function relationships.

    PubMed

    Armitage, David W

    2016-09-14

    Biodiversity-ecosystem function (BEF) experiments routinely employ common garden designs, drawing samples from a local biota. The communities from which taxa are sampled may not, however, be at equilibrium. To test for temporal changes in BEF relationships, I assembled the pools of aquatic bacterial strains isolated at different time points from leaves on the pitcher plant Darlingtonia californica in order to evaluate the strength, direction and drivers of the BEF relationship across a natural host-associated successional gradient. I constructed experimental communities using bacterial isolates from each time point and measured their respiration rates and competitive interactions. Communities assembled from mid-successional species pools showed the strongest positive relationships between community richness and respiration rates, driven primarily by linear additivity among isolates. Diffuse competition was common among all communities but greatest within mid-successional isolates. These results demonstrate the dependence of the BEF relationship on the temporal dynamics of the local species pool, implying that ecosystems may respond differently to the addition or removal of taxa at different points in time during succession. PMID:27629035

  9. Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation

    PubMed Central

    Guo, Long; Yamashita, Hiroshi; Kou, Ikuyo; Takimoto, Aki; Meguro-Horike, Makiko; Horike, Shin-ichi; Sakuma, Tetsushi; Miura, Shigenori; Adachi, Taiji; Yamamoto, Takashi; Ikegawa, Shiro; Hiraki, Yuji; Shukunami, Chisa

    2016-01-01

    Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of

  10. Moderation of adult depression by the serotonin transporter promoter variant (5-HTTLPR), childhood abuse and adult traumatic events in a general population sample.

    PubMed

    Grabe, Hans Jörgen; Schwahn, Christian; Mahler, Jessie; Schulz, Andrea; Spitzer, Carsten; Fenske, Kristin; Appel, Katja; Barnow, Sven; Nauck, Matthias; Schomerus, Georg; Biffar, Reiner; Rosskopf, Dieter; John, Ulrich; Völzke, Henry; Freyberger, Harald Jürgen

    2012-04-01

    The impact of the promoter polymorphisms of the serotonin transporter (5-HTTLPR) on mood has been studied by two-way interaction models comprising one environmental factor and genotype variants. However, childhood abuse is assumed to be associated with different psychobiological long-term effects than adult traumatic events. Both types of trauma may interact on an individual basis throughout the lifespan moderating the impact of the 5-HTTLPR s allele on depressive disorders. Therefore, the hypothesis of a three-way interaction among the 5-HTTLPR, childhood abuse and adult traumatic experience was tested. Caucasian subjects (1,974) from the general population in Germany (Study of Health in Pomerania (SHIP)) were analyzed. Depressive symptoms were measured with the Beck Depression Inventory (BDI-II). Childhood abuse was assessed with the Childhood Trauma Questionnaire. Adult traumatic events were derived from the SCID interview (DSM-IV) on posttraumatic stress disorder (PTSD). Global three-way interactions among the 5-HTTLPR, adult traumatic experiences and childhood abuse (P = 0.0007) were found. Carriers of the ss or sl genotypes who had been exposed to childhood abuse and to more than two adult traumatic events had higher mean BDI-II scores (16.0 [95% CI 8.4-23.6]) compared to those carrying the ll genotype (7.6 [4.5-10.7]). These results were supported using a second, more severe definition of childhood abuse (P = 0.02). No two-way interactions were observed (P > 0.05). Childhood abuse and adult traumatic events may act synergistically in interaction with the s allele of the 5-HTTLPR to increase the risk for depressive symptoms independently from the lifetime diagnosis of PTSD.

  11. Analysis of Ancestral and Functionally Relevant CD5 Variants in Systemic Lupus Erythematosus Patients

    PubMed Central

    Consuegra, Marta; Bonet, Lizette; Carnero-Montoro, Elena; Armiger, Noelia; Caballero-Baños, Miguel; Arias, Maria Teresa; Benitez, Daniel; Ortego-Centeno, Norberto; de Ramón, Enrique; Sabio, José Mario; García–Hernández, Francisco J.; Tolosa, Carles; Suárez, Ana; González-Gay, Miguel A.; Bosch, Elena; Martín, Javier; Lozano, Francisco

    2014-01-01

    Objective CD5 plays a crucial role in autoimmunity and is a well-established genetic risk factor of developing RA. Recently, evidence of positive selection has been provided for the CD5 Pro224-Val471 haplotype in East Asian populations. The aim of the present work was to further analyze the functional relevance of non-synonymous CD5 polymorphisms conforming the ancestral and the newly derived haplotypes (Pro224-Ala471 and Pro224-Val471, respectively) as well as to investigate the potential role of CD5 on the development of SLE and/or SLE nephritis. Methods The CD5 SNPs rs2241002 (C/T; Pro224Leu) and rs2229177 (C/T; Ala471Val) were genotyped using TaqMan allelic discrimination assays in a total of 1,324 controls and 681 SLE patients of Spanish origin. In vitro analysis of CD3-mediated T cell proliferative and cytokine response profiles of healthy volunteers homozygous for the above mentioned CD5 haplotypes were also analyzed. Results T-cell proliferation and cytokine release were significantly increased showing a bias towards to a Th2 profile after CD3 cross-linking of peripheral mononuclear cells from healthy individuals homozygous for the ancestral Pro224-Ala471 (CC) haplotype, compared to the more recently derived Pro224-Val471 (CT). The same allelic combination was statistically associated with Lupus nephritis. Conclusion The ancestral Ala471 CD5 allele confers lymphocyte hyper-responsiveness to TCR/CD3 cross-linking and is associated with nephritis in SLE patients. PMID:25402503

  12. Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival.

    PubMed

    Potrony, Miriam; Carreras, Esther; Aranda, Fernando; Zimmer, Lisa; Puig-Butille, Joan-Anton; Tell-Martí, Gemma; Armiger, Noelia; Sucker, Antje; Giménez-Xavier, Pol; Martínez-Florensa, Mario; Carrera, Cristina; Malvehy, Josep; Schadendorf, Dirk; Puig, Susana; Lozano, Francisco

    2016-09-15

    Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma.

  13. Targeted Re-Sequencing Approach of Candidate Genes Implicates Rare Potentially Functional Variants in Tourette Syndrome Etiology

    PubMed Central

    Alexander, John; Potamianou, Hera; Xing, Jinchuan; Deng, Li; Karagiannidis, Iordanis; Tsetsos, Fotis; Drineas, Petros; Tarnok, Zsanett; Rizzo, Renata; Wolanczyk, Tomasz; Farkas, Luca; Nagy, Peter; Szymanska, Urszula; Androutsos, Christos; Tsironi, Vaia; Koumoula, Anastasia; Barta, Csaba; Sandor, Paul; Barr, Cathy L.; Tischfield, Jay; Paschou, Peristera; Heiman, Gary A.; Georgitsi, Marianthi

    2016-01-01

    Although the genetic basis of Tourette Syndrome (TS) remains unclear, several candidate genes have been implicated. Using a set of 382 TS individuals of European ancestry we investigated four candidate genes for TS (HDC, SLITRK1, BTBD9, and SLC6A4) in an effort to identify possibly causal variants using a targeted re-sequencing approach by next generation sequencing technology. Identification of possible disease causing variants under different modes of inheritance was performed using the algorithms implemented in VAAST. We prioritized variants using Variant ranker and validated five rare variants via Sanger sequencing in HDC and SLITRK1, all of which are predicted to be deleterious. Intriguingly, one of the identified variants is in linkage disequilibrium with a variant that is included among the top hits of a genome-wide association study for response to citalopram treatment, an antidepressant drug with off-label use also in obsessive compulsive disorder. Our findings provide additional evidence for the implication of these two genes in TS susceptibility and the possible role of these proteins in the pathobiology of TS should be revisited. PMID:27708560

  14. The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function.

    PubMed

    Díaz-Barreiro, A; Bernal-Quirós, M; Georg, I; Marañón, C; Alarcón-Riquelme, M E; Castillejo-López, C

    2016-03-01

    The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding. PMID:26821283

  15. Hurt, tired and queasy: Specific variants in the ATPase domain of the TRAP1 mitochondrial chaperone are associated with common, chronic "functional" symptomatology including pain, fatigue and gastrointestinal dysmotility.

    PubMed

    Boles, Richard G; Hornung, Holly A; Moody, Alastair E; Ortiz, Thomas B; Wong, Stacey A; Eggington, Julie M; Stanley, Christine M; Gao, Mu; Zhou, Hongyi; McLaughlin, Stephen; Zare, Amir S; Sheldon, Katherine M; Skolnick, Jeffrey; McKernan, Kevin J

    2015-07-01

    Functional disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. There are bidirectional associations between functional disorders and mitochondrial dysfunction. In this study, provided clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were retrospectively reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility. Very-highly conserved TRAP1 variants were identified in 73 of 930 unrelated patients. Functional symptomatology is strongly associated with specific variants in the ATPase binding pocket. In particular, the combined presence of all three functional categories is strongly associated with p.Ile253Val (OR 7.5, P = 0.0001) and with two other interacting variants (OR 18, P = 0.0005). Considering a 1-2% combined variant prevalence and high odds ratios, these variants may be an important factor in the etiology of functional symptomatology. PMID:26022780

  16. A functional variant in miR-155 regulation region contributes to lung cancer risk and survival.

    PubMed

    Xie, Kaipeng; Ma, Hongxia; Liang, Cheng; Wang, Cheng; Qin, Na; Shen, Wei; Gu, Yayun; Yan, Caiwang; Zhang, Kai; Dai, Ningbin; Zhu, Meng; Wu, Shuangshuang; Wang, Hui; Dai, Juncheng; Jin, Guangfu; Shen, Hongbing; Hu, Zhibin

    2015-12-15

    Emerging evidence suggested that upregulation of miR-155 could serve as a promising marker for the diagnosis and prognosis of non-small cell lung cancer (NSCLC). In the present study, we genotyped rs767649 (A > T) located in miR-155 regulation region in 1341 cases and 1982 controls, and analyzed the associations of rs767649 with NSCLC risk and survival. Consequently, rs767649 exhibited the significant associations with the risk (adjusted OR = 1.12, 95% CI = 1.01-1.24, P = 0.031) and prognosis of NSCLC (adjusted HR = 1.17, 95% CI = 1.03-1.32, P = 0.014). Meanwhile, rs767649 specifically interacted with radio-chemotherapy (P(int) = 0.013), and patients with both the rs767649-TT genotype and radio-chemotherapy had the highest hazard ratio (adjusted HR = 1.65, 95% CI = 1.26-2.16, P < 0.001). Furthermore, using functional assays and The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma (LUAD) dataset, we found that rs767649 variant allele could increase the transcriptional activity of miR-155, which in turn facilitated tumor growth and metastasis by inhibiting HBP1, TJP1, SMAD5 and PRKAR1A expression. Our findings suggested that rs767649 A > T might contribute to the increased risk and poor prognosis of NSCLC, highlighting the importance of rs767649 in the prevention and therapy of NSCLC. PMID:26543233

  17. A functional variant in miR-155 regulation region contributes to lung cancer risk and survival

    PubMed Central

    Wang, Cheng; Qin, Na; Shen, Wei; Gu, Yayun; Yan, Caiwang; Zhang, Kai; Dai, Ningbin; Zhu, Meng; Wu, Shuangshuang; Wang, Hui; Dai, Juncheng; Jin, Guangfu; Shen, Hongbing; Hu, Zhibin

    2015-01-01

    Emerging evidence suggested that upregulation of miR-155 could serve as a promising marker for the diagnosis and prognosis of non-small cell lung cancer (NSCLC). In the present study, we genotyped rs767649 (A > T) located in miR-155 regulation region in 1341 cases and 1982 controls, and analyzed the associations of rs767649 with NSCLC risk and survival. Consequently, rs767649 exhibited the significant associations with the risk (adjusted OR = 1.12, 95% CI = 1.01–1.24, P = 0.031) and prognosis of NSCLC (adjusted HR = 1.17, 95% CI = 1.03–1.32, P = 0.014). Meanwhile, rs767649 specifically interacted with radio-chemotherapy (Pint = 0.013), and patients with both the rs767649-TT genotype and radio-chemotherapy had the highest hazard ratio (adjusted HR = 1.65, 95% CI = 1.26–2.16, P < 0.001). Furthermore, using functional assays and The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma (LUAD) dataset, we found that rs767649 variant allele could increase the transcriptional activity of miR-155, which in turn facilitated tumor growth and metastasis by inhibiting HBP1, TJP1, SMAD5 and PRKAR1A expression. Our findings suggested that rs767649 A > T might contribute to the increased risk and poor prognosis of NSCLC, highlighting the importance of rs767649 in the prevention and therapy of NSCLC. PMID:26543233

  18. Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides.

    PubMed

    Troczka, Bartlomiej J; Williams, Alan J; Williamson, Martin S; Field, Linda M; Lüemmen, Peter; Davies, T G Emyr

    2015-10-01

    Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

  19. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays

    PubMed Central

    Ding, Qiulan; Yang, Likui; Dinarvand, Peyman

    2015-01-01

    Protein C (PC) is a vitamin K–dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband’s negative VTE pedigree. PMID:25651845

  20. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays.

    PubMed

    Ding, Qiulan; Yang, Likui; Dinarvand, Peyman; Wang, Xuefeng; Rezaie, Alireza R

    2015-04-01

    Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree. PMID:25651845

  1. Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Kanofsky, Konstantin; Hanko, Erik K R; Ahrendt, Sarah; Wehrs, Maren; Machens, Fabian; Hehl, Reinhard

    2016-01-01

    Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.

  2. Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Kanofsky, Konstantin; Hanko, Erik K R; Ahrendt, Sarah; Wehrs, Maren; Machens, Fabian; Hehl, Reinhard

    2016-01-01

    Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA. PMID:25819608

  3. Association of a Functional Variant in the Wnt Co-Receptor LRP6 with Early Onset Ileal Crohn's Disease

    PubMed Central

    Koslowski, Maureen J.; Teltschik, Zora; Beisner, Julia; Schaeffeler, Elke; Wang, Guoxing; Kübler, Irmgard; Gersemann, Michael; Cooney, Rachel; Jewell, Derek; Reinisch, Walter; Vermeire, Séverine; Rutgeerts, Paul; Schwab, Matthias; Stange, Eduard F.; Wehkamp, Jan

    2012-01-01

    Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD–associated genes including NOD2, ATG16L1, and recently the β-catenin–dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n = 1,893) and prospectively tested 2 additional European sample sets (Leuven: n = 688, Vienna: n = 1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p = 0.00034; for homozygous carriers: OR 4.1; p = 0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p = 0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent

  4. Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis.

    PubMed

    Arcipowski, Kelly M; Bulic, Marinka; Gurbuxani, Sandeep; Licht, Jonathan D

    2016-01-01

    Two of the most common myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are associated with exceedingly low survival rates despite recent therapeutic advances. While their etiology is not completely understood, evidence suggests that certain chromosomal abnormalities contribute to MDS and AML progression. Among the most frequent chromosomal abnormalities in these disorders are alterations of chromosome 7: either complete loss of one copy of chromosome 7 (-7) or partial deletion of 7q (del(7q)), both of which increase the risk of progression from MDS to AML and are associated with chemoresistance. Notably, 7q36.1, a critical minimally deleted region in 7q, includes the gene encoding the histone methyltransferase mixed-lineage leukemia 3 (MLL3), which is also mutated in a small percentage of AML patients. However, the mechanisms by which MLL3 loss contributes to malignancy are unknown. Using an engineered mouse model expressing a catalytically inactive form of Mll3, we found a significant shift in hematopoiesis toward the granulocyte/macrophage lineage, correlating with myeloid infiltration and enlargement of secondary lymphoid organs. Therefore, we propose that MLL3 loss in patients may contribute to the progression of MDS and AML by promoting myelopoiesis. PMID:27610619

  5. Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

    PubMed Central

    Arcipowski, Kelly M.; Bulic, Marinka; Gurbuxani, Sandeep

    2016-01-01

    Two of the most common myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are associated with exceedingly low survival rates despite recent therapeutic advances. While their etiology is not completely understood, evidence suggests that certain chromosomal abnormalities contribute to MDS and AML progression. Among the most frequent chromosomal abnormalities in these disorders are alterations of chromosome 7: either complete loss of one copy of chromosome 7 (-7) or partial deletion of 7q (del(7q)), both of which increase the risk of progression from MDS to AML and are associated with chemoresistance. Notably, 7q36.1, a critical minimally deleted region in 7q, includes the gene encoding the histone methyltransferase mixed-lineage leukemia 3 (MLL3), which is also mutated in a small percentage of AML patients. However, the mechanisms by which MLL3 loss contributes to malignancy are unknown. Using an engineered mouse model expressing a catalytically inactive form of Mll3, we found a significant shift in hematopoiesis toward the granulocyte/macrophage lineage, correlating with myeloid infiltration and enlargement of secondary lymphoid organs. Therefore, we propose that MLL3 loss in patients may contribute to the progression of MDS and AML by promoting myelopoiesis. PMID:27610619

  6. Functional analysis of pharmacogenetic variants of human organic cation/carnitine transporter 2 (hOCTN2) identified in Singaporean populations.

    PubMed

    Toh, Dorothy Su Lin; Murray, Michael; Pern Tan, Kuan; Mulay, Vishwaroop; Grewal, Thomas; Lee, Edmund Jon Deoon; Zhou, Fanfan

    2011-12-01

    The human organic cation/carnitine transporter-2 (hOCTN2; SLC22A5) mediates the cellular influx of organic cations such as carnitine, which is essential for fatty acid oxidation. Primary carnitine deficiency has been associated with a wide range of hOCTN2 gene mutations. Six novel nonsynonymous single nucleotide polymorphisms in the hOCTN2 gene were identified recently in Chinese and Indian populations of Singapore. The present study evaluated the impact of these polymorphisms on hOCTN2 function and expression in HEK-293 cells. Transport function was markedly impaired in variants that encoded amino acid substitutions D122Y (<20% of wild-type control) and K302E (∼45% of wild-type) in the large extracellular loop and large intracellular loop of hOCTN2, respectively. The function of the other four variants was unimpaired (E109K, V175M, K191N and A214V). From biotinylation and immunofluorescence experiments, the expression of the D122Y and K302E-hOCTN2 variants at the plasma membrane of HEK-293 cells was decreased relative to the wild-type hOCTN2 but total cellular expression was unchanged. Transporter kinetic studies indicated a decrease in the V(max) for l-carnitine influx by K302E-hOCTN2 to 49% of wild-type control, while K(m) remained unchanged; kinetic evaluation of D122Y-hOCTN2 was not possible due to its low transport function. The K302E-hOCTN2 variant was also more susceptible than the wild-type transporter to inhibition by the drugs cimetidine, pyrilamine and verapamil. These findings indicate that impaired plasma membrane targeting of the D122Y and K302E-hOCTN2 variants that occur in Singaporean populations contributes to decreased carnitine influx.

  7. Plant functional group diversity promotes soil protist diversity.

    PubMed

    Ledeganck, Pieter; Nijs, Ivan; Beyens, Louis

    2003-07-01

    We tested whether effects of plant diversity can propagate through food webs, down to heterotrophic protists not linked directly to plants. To this end we synthesised grassland ecosystems with varying numbers of plant functional groups (FGN) and assessed corresponding changes in testate amoebae communities. The number of plant species was kept constant. When FGN was increased from 1 to 3, species number and total community density of live testate amoebae were enhanced according to a linear and a saturating function, respectively. From FGN 1 to 2, the appearance of new testate amoebae species did not affect the presence of the resident species, whereas, from FGN 2 to 3 about one quarter of the resident testate amoebae species was replaced, without altering the total species number. Overall, density by species increased, while evenness of the testate amoebae community was not affected by FGN; although Trinema lineare, one of the most common species, became more abundant. The observed relationship between plant functional group diversity and testate amoebae diversity could shed new light on the biogeographical distribution patterns of protists.

  8. Plant functional group diversity promotes soil protist diversity.

    PubMed

    Ledeganck, Pieter; Nijs, Ivan; Beyens, Louis

    2003-07-01

    We tested whether effects of plant diversity can propagate through food webs, down to heterotrophic protists not linked directly to plants. To this end we synthesised grassland ecosystems with varying numbers of plant functional groups (FGN) and assessed corresponding changes in testate amoebae communities. The number of plant species was kept constant. When FGN was increased from 1 to 3, species number and total community density of live testate amoebae were enhanced according to a linear and a saturating function, respectively. From FGN 1 to 2, the appearance of new testate amoebae species did not affect the presence of the resident species, whereas, from FGN 2 to 3 about one quarter of the resident testate amoebae species was replaced, without altering the total species number. Overall, density by species increased, while evenness of the testate amoebae community was not affected by FGN; although Trinema lineare, one of the most common species, became more abundant. The observed relationship between plant functional group diversity and testate amoebae diversity could shed new light on the biogeographical distribution patterns of protists. PMID:13677451

  9. Cloning and Functional Characterization of Novel Variants and Tissue-Specific Expression of Alternative Amino and Carboxyl Termini of Products of Slc4a10

    PubMed Central

    Qin, Xue; Xie, Zhang-Dong; Wang, Qing K.; Liu, Mugen; Chen, Li-Ming

    2013-01-01

    Previous studies have shown that the electroneutral Na+/HCO3− cotransporter NBCn2 (SLC4A10) is predominantly expressed in the central nervous system (CNS). The physiological and pathological significances of NBCn2 have been well recognized. However, little is known about the tissue specificity of expression of different NBCn2 variants. Moreover, little is known about the expression of NBCn2 proteins in systems other than CNS. Here, we identified a set of novel Slc4a10 variants differing from the originally described ones by containing a distinct 5′ untranslated region encoding a new extreme amino-terminus (Nt). Electrophysiology measurements showed that both NBCn2 variants with alternative Nt contain typical electroneutral Na+-coupled HCO3− transport activity in Xenopus oocytes. Luciferase reporter assay showed that Slc4a10 contains two alternative promoters responsible for expression of the two types of NBCn2 with distinct extreme Nt. Western blotting showed that NBCn2 proteins with the original Nt are primarily expressed in CNS, whereas those with the novel Nt are predominantly expressed in the kidney and to a lesser extent in the small intestine. Due to alternative splicing, the known NBCn2 variants contain two types of carboxyl-termini (CT) differing in the optional inclusion of a PDZ-binding motif. cDNA cloning showed that virtually all NBCn2 variants expressed in epithelial tissues contain, but the vast majority of those from the neural tissues lack the PDZ-binding motif. We conclude that alternative transcription and splicing of Slc4a10 products are regulated in a tissue-specific manner. Our findings provide critical insights that will greatly influence the study of the physiology of NBCn2. PMID:23409100

  10. Interferon-gamma receptor 1 promoter polymorphisms: population distribution and functional implications.

    PubMed

    Rosenzweig, Sergio D; Schäffer, Alejandro A; Ding, Li; Sullivan, Rachel; Enyedi, Balasz; Yim, Jae-Joon; Cook, James L; Musser, James M; Holland, Steven M

    2004-07-01

    Different polymorphisms have been described in the minimal promoter region (MPR) of the interferon-gamma receptor 1 (IFNGR1), a molecule that plays a critical role in mycobacterial control. We sequenced the IFNGR1 MPR from African American, Caucasian and Korean controls, and from mycobacteria-infected patients. Six different single nucleotide polymorphisms (SNPs) were detected in the IFNGR1 MPR. The three ethnic groups showed different SNP distribution patterns, but no significant differences were detected between mycobacterial cases and controls. Two polymorphisms were found in all populations (G-611A, T-56C). We cloned the four allelic variants (var) of haplotype G-611A/T-56C into a luciferase reporter vector and determined their promoter activity. Polymorphisms at position -611 had a stronger effect on the promoter activity than those at position -56, and constructs carrying G-611 produced a stronger promoter activity than -611A constructs. The IFNGR1 MPR is a polymorphic region with at least two SNPs influencing its activity, but these are not associated with increased mycobacterial susceptibility.

  11. Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions

    PubMed Central

    Zinovyeva, Marina V.; Nikolaev, Lev G.; Azhikina, Tatyana L.

    2016-01-01

    Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression. PMID:27248499

  12. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

    PubMed Central

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients. PMID:26633385

  13. The Kiss2 receptor (Kiss2r) gene in Southern Bluefin Tuna, Thunnus maccoyii and in Yellowtail Kingfish, Seriola lalandi - functional analysis and isolation of transcript variants.

    PubMed

    Nocillado, J N; Biran, J; Lee, Y Y; Levavi-Sivan, B; Mechaly, A S; Zohar, Y; Elizur, A

    2012-10-15

    The kisspeptin system plays an essential role in reproductive function in vertebrates, particularly in the onset of puberty. We investigated the kisspeptin system in two Perciform teleosts, the Southern Bluefin Tuna (SBT; Thunnus maccoyii), and the Yellowtail Kingfish (YTK; Seriola lalandi), by characterising their kisspeptin 2 receptor (Kiss2r) genes. In addition to the full length Kiss2r cDNA sequences, we have isolated from SBT and YTK a transcript variant that retained an intron. We have further obtained three ytkKiss2r transcript variants that contained deletions. In vitro functional analysis of the full length SBT and YTK Kiss2r showed higher response to Kiss2-10 than to Kiss1-10, with stronger transduction via PKC than PKA. The full length ytkKiss2r and two deletion variants were differentially expressed in the brain of male, but not in female, juvenile YTK treated with increasing doses of Kiss2-10 peptide. In the gonads, the expression level of the ytkKiss2r transcripts did not vary significantly either in the male or female fish. This is the first time that transcript variants of the Kiss2r gene that contain deletions and show responsiveness to treatments with kisspeptin have been reported in any teleost. PMID:22824208

  14. Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein.

    PubMed

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2015-12-02

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.

  15. Promoting return of function in multiple sclerosis: An integrated approach

    PubMed Central

    Gacias, Mar; Casaccia, Patrizia

    2013-01-01

    Multiple sclerosis is a disease characterized by inflammatory demyelination, axonal degeneration and progressive brain atrophy. Most of the currently available disease modifying agents proved to be very effective in managing the relapse rate, however progressive neuronal damage continues to occur and leads to progressive accumulation of irreversible disability. For this reason, any therapeutic strategy aimed at restoration of function must take into account not only immunomodulation, but also axonal protection and new myelin formation. We further highlight the importance of an holistic approach, which considers the variability of therapeutic responsiveness as the result of the interplay between genetic differences and the epigenome, which is in turn affected by gender, age and differences in life style including diet, exercise, smoking and social interaction. PMID:24363985

  16. Promoting return of function in multiple sclerosis: An integrated approach.

    PubMed

    Gacias, Mar; Casaccia, Patrizia

    2013-10-01

    Multiple sclerosis is a disease characterized by inflammatory demyelination, axonal degeneration and progressive brain atrophy. Most of the currently available disease modifying agents proved to be very effective in managing the relapse rate, however progressive neuronal damage continues to occur and leads to progressive accumulation of irreversible disability. For this reason, any therapeutic strategy aimed at restoration of function must take into account not only immunomodulation, but also axonal protection and new myelin formation. We further highlight the importance of an holistic approach, which considers the variability of therapeutic responsiveness as the result of the interplay between genetic differences and the epigenome, which is in turn affected by gender, age and differences in life style including diet, exercise, smoking and social interaction. PMID:24363985

  17. The cenH3 histone variant defines centromeres in Giardia intestinalis.

    PubMed

    Dawson, S C; Sagolla, M S; Cande, W Z

    2007-04-01

    Histone H3 variants play critical roles in the functional specialization of chromatin by epigenetically marking centromeric chromatin and transcriptionally active or silent genes. Specifically, the cenH3 histone variant acts as the primary epigenetic determinant of the site of kinetochore assembly at centromeres. Although the function of histone variants is well studied in plants, animals, and fungi, there is little knowledge of the evolutionary conservation of histone variants and their function in most protists. We find that Giardia intestinalis--a diplomonad parasite with two equivalent nuclei--has two phylogenetically distinct histone H3 variants with N-terminal extensions and nonconserved promoters. To determine their role in chromatin dynamics, conventional H3 and the two H3 variants were GFP-tagged, and their subcellular location was monitored during interphase and mitosis. We demonstrate that one cenH3-like variant has a conserved function in epigenetically marking centromeres. The other H3 variant (H3B) has a punctate distribution on chromosomes, but does not colocalize with active transcriptional regions as indicated by H3K4 methylation. We suggest that H3B could instead mark noncentromeric heterochromatin. Giardia is a member of the Diplomonads and represents an ancient divergence from metazoans and fungi. We confirm the ancient role of histone H3 variants in modulating chromatin architecture, and suggest that monocentric chromosomes represent an ancestral chromosome morphology.

  18. Genetic Variants of APOC3 Promoter and HLA-B Genes in an HIV Infected Cohort in Northern South Africa: A Pilot Study

    PubMed Central

    Masebe, Tracy; Bessong, Pascal Obong; Ndip, Roland Ndip; Meyer, Debra

    2014-01-01

    Metabolic disorders and hypersensitivities affect tolerability and impact adherence to highly active antiretroviral therapy (HAART). The aim of this study was to determine the prevalence of C-482T/T-455C variants in the Apolipoprotein C3 (APOC3) promoter gene and Human leukocyte antigen (HLA)-B*57:01, known to impact lipid metabolic disorders and hypersensitivity respectively; and to correlate genotypes with gender, CD4+ cell count and viral load in an HIV infected cohort in northern South Africa. Frequencies of C-482 and T-455 polymorphisms in APOC3 were determined by restriction fragment length polymorphism analysis. Allele determination for HLA-B was performed with Assign SBT software in an HLA library. Analysis of APOC3 C-482 site revealed a prevalence of 196/199 (98.5%) for CC, 1/199 (0.5%) for CT and 2/199 (1.0%) for TT genotype (p = 0.000 with 1° of freedom; χ2 = 126.551). For the T-455 site, prevalences were: 69/199 (35%) for TT and 130/199 (65%) for the CC genotype (p = 0.000 with 1° of freedom; χ2 = 199). There was no association between gender and the presence of −482 (p = 1; χ2 = 0.00001) or −455 genotypes (p = 0.1628; χ2 = 1.9842). There was no significant difference in the increase in CD4+ cell count irrespective of genotypes. Significant increases in CD4+ cell count were observed in males and females considering the −455C genotype, but not in males for the −455T genotype. Viral load decreases were significant with the −455C and −482C genotypes irrespective of gender. HLA-B*57:01 was not identified in the study cohort. The apparently high prevalence of APOC3 T-455CC genotype needs confirmation with a larger samples size and triglyceride measurements to support screening of patients to pre-empt HAART associated lipid disorders. PMID:24972136

  19. Association of variants in genes encoding for macrophage-related functions with clinical outcome in patients with locoregional gastric cancer

    PubMed Central

    Sunakawa, Y.; Stremitzer, S.; Cao, S.; Zhang, W.; Yang, D.; Wakatsuki, T.; Ning, Y.; Yamauchi, S.; Stintzing, S.; Sebio, A.; El-khoueiry, R.; Matsusaka, S.; Parekh, A.; Barzi, A.; Azuma, M.; Watanabe, M.; Koizumi, W.; Lenz, H.-J.

    2015-01-01

    Background Nuclear factor-kappaB (NF-κB) and CCL2/CCR2 chemokine axis play a central role in tumor progression such as stimulation of angiogenesis, acceleration of tumor invasion and migration, and suppression of innate immunosurveillance in the macrophage-related functions. There have been few reports regarding association of the macrophage function-related genes with the clinical outcome in gastric cancer. We hypothesized that variants in genes encoding for NF-κB and CCL2/CCR2 axis may predict prognosis in gastric cancer and tested whether the functional single-nucleotide polymorphisms (SNPs) will be associated with clinical outcome in patients with gastric cancer across two independent groups. Patients and methods This study enrolled two cohorts which consisted of 160 Japanese patients and 104 US patients with locoregional gastric cancer. Genomic DNA was analyzed for association of 11 SNPs in NFKB1, RELA, CCL2, and CCR2 with clinical outcome using PCR-based direct DNA sequencing. Results The univariable analysis showed four SNPs had significant association with clinical outcome in the Japanese cohort, NFKB1 rs230510 remained significant upon multivariable analysis. The patients with the A allele of the NFKB1 rs230510 had significantly longer overall survival (OS) compared with those with the T/T genotype in both the Japanese and US cohort in the univariable analysis. In contrast, genotypes with the T allele of CCL2 rs4586 were significantly associated with shorter OS compared with the C/C genotype in the US cohort [hazard ratio (HR) 2.43; P = 0.015] but longer OS in the Japanese cohort (HR 0.58; P = 0.021), resulting in the statistically significant opposite impact on OS (P = 0.001). Conclusions Our study provides the first evidence that the NFKB1 rs230510 and CCL2 rs4586 are significantly associated with the clinical outcome in patients with locoregional gastric cancer. These results also suggest that the genetic predisposition of the host may dictate the

  20. Association between functional variants of the ICAM1 and CRP genes and metabolic syndrome in Taiwanese subjects.

    PubMed

    Hsu, Lung-An; Chang, Chi-Jen; Wu, Semon; Teng, Ming-Sheng; Chou, Hsin-Hua; Chang, Hsien-Hsun; Chang, Pi-Yueh; Ko, Yu-Lin

    2010-12-01

    Although inflammation has been shown to play an important role in metabolic syndrome (MetS), the association between inflammatory marker gene polymorphisms and the risk of MetS has not been fully elucidated. This study was initiated to investigate the association between functional variants of inflammatory marker genes and the risk of MetS in Taiwanese adults. The sample population comprised 615 unrelated subjects, of which 22% had MetS. The single nucleotide polymorphisms rs5491 on the intercellular adhesive molecule 1 (ICAM1) gene and rs3091244 on C-reactive protein (CRP) were genotyped. The ICAM1 rs5491 polymorphism was significantly associated with the level of soluble intercellular adhesive molecule 1 (P < .001). Both the ICAM1 rs5491 and the CRP rs3091244 were shown to have significant association with MetS after adjustment for age, sex, smoking, and body mass index, but not after adjustment for levels of the respective serum marker. Independent associations between the combined ICAM1-CRP (rs5491 and rs3091244) genotypes and MetS were found by multivariate analysis (P = .005). In subgroup analysis, association of combined genotypes with insulin resistance and MetS mainly occurred in subjects with central obesity. In conclusion, inflammatory marker gene polymorphisms play an important role in modulating the risk of insulin resistance and MetS for subjects with central obesity. These findings will contribute toward a better understanding of the mechanism of association between inflammatory markers and the risk of developing atherosclerotic disease.

  1. Time-Resolved Influences of Functional DAT1 and COMT Variants on Visual Perception and Post-Processing

    PubMed Central

    Bender, Stephan; Rellum, Thomas; Freitag, Christine; Resch, Franz; Rietschel, Marcella; Treutlein, Jens; Jennen-Steinmetz, Christine; Brandeis, Daniel; Banaschewski, Tobias; Laucht, Manfred

    2012-01-01

    Background Dopamine plays an important role in orienting and the regulation of selective attention to relevant stimulus characteristics. Thus, we examined the influences of functional variants related to dopamine inactivation in the dopamine transporter (DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of visual processing in a contingent negative variation (CNV) task. Methods 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as preceding visual evoked potential components were assessed. Results Significant additive main effects of DAT1 and COMT on the occipito-temporal early CNV were observed. In addition, there was a trend towards an interaction between the two polymorphisms. Source analysis showed early CNV generators in the ventral visual stream and in frontal regions. There was a strong negative correlation between occipito-temporal visual post-processing and the frontal early CNV component. The early CNV time interval 500–1000 ms after the visual cue was specifically affected while the preceding visual perception stages were not influenced. Conclusions Late visual potentials allow the genomic imaging of dopamine inactivation effects on visual post-processing. The same specific time-interval has been found to be affected by DAT1 and COMT during motor post-processing but not motor preparation. We propose the hypothesis that similar dopaminergic mechanisms modulate working memory encoding in both the visual and motor and perhaps other systems. PMID:22844499

  2. Novel loss-of-function variants in DIAPH1 associated with syndromic microcephaly, blindness, and early onset seizures.

    PubMed

    Al-Maawali, Almundher; Barry, Brenda J; Rajab, Anna; El-Quessny, Malak; Seman, Ann; Coury, Stephanie Newton; Barkovich, A James; Yang, Edward; Walsh, Christopher A; Mochida, Ganeshwaran H; Stoler, Joan M

    2016-02-01

    Exome sequencing identified homozygous loss-of-function variants in DIAPH1 (c.2769delT; p.F923fs and c.3145C>T; p.R1049X) in four affected individuals from two unrelated consanguineous families. The affected individuals in our report were diagnosed with postnatal microcephaly, early-onset epilepsy, severe vision impairment, and pulmonary symptoms including bronchiectasis and recurrent respiratory infections. A heterozygous DIAPH1 mutation was originally reported in one family with autosomal dominant deafness. Recently, however, a homozygous nonsense DIAPH1 mutation (c.2332C4T; p.Q778X) was reported in five siblings in a single family affected by microcephaly, blindness, early onset seizures, developmental delay, and bronchiectasis. The role of DIAPH1 was supported using parametric linkage analysis, RNA and protein studies in their patients' cell lines and further studies in human neural progenitors cells and a diap1 knockout mouse. In this report, the proband was initially brought to medical attention for profound metopic synostosis. Additional concerns arose when his head circumference did not increase after surgical release at 5 months of age and he was diagnosed with microcephaly and epilepsy at 6 months of age. Clinical exome analysis identified a homozygous DIAPH1 mutation. Another homozygous DIAPH1 mutation was identified in the research exome analysis of a second family with three siblings presenting with a similar phenotype. Importantly, no hearing impairment is reported in the homozygous affected individuals or in the heterozygous carrier parents in any of the families demonstrating the autosomal recessive microcephaly phenotype. These additional families provide further evidence of the likely causal relationship between DIAPH1 mutations and a neurodevelopmental disorder.

  3. Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

    PubMed

    Giniatullina, A; Maroteaux, G; Geerts, C J; Koopmans, B; Loos, M; Klaassen, R; Chen, N; van der Schors, R C; van Nierop, P; Li, K W; de Jong, J; Altrock, W D; Cornelisse, L N; Toonen, R F; van der Sluis, S; Sullivan, P F; Stiedl, O; Posthuma, D; Smit, A B; Groffen, A J; Verhage, M

    2015-08-01

    Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies. PMID:26045179

  4. Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

    PubMed

    Giniatullina, A; Maroteaux, G; Geerts, C J; Koopmans, B; Loos, M; Klaassen, R; Chen, N; van der Schors, R C; van Nierop, P; Li, K W; de Jong, J; Altrock, W D; Cornelisse, L N; Toonen, R F; van der Sluis, S; Sullivan, P F; Stiedl, O; Posthuma, D; Smit, A B; Groffen, A J; Verhage, M

    2015-08-01

    Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.