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Computational and experimental approaches to regulatory genetic variation
KTH, School of Biotechnology (BIO), Gene Technology.
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations.

The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease.

An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available.

The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene.

Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect.

Place, publisher, year, edition, pages
Stockholm: Bioteknologi , 2007.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2007:12
Keyword [en]
Molecular biology, Genetics, single nucleotide polymprhism (SNP), regulatory SNP, transcription factor binding site, phylogenetic footprinting, allelic imbalance, EMSA, CD36, cardiovascular disease.
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4593ISBN: 978-91-7178-827-6 (print)OAI: oai:DiVA.org:kth-4593DiVA: diva2:13020
Public defence
2008-01-18, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Available from: 2007-12-19 Created: 2007-12-19 Last updated: 2012-03-20Bibliographically approved
List of papers
1. In silico detection of sequence variations modifying transcriptional regulation
Open this publication in new window or tab >>In silico detection of sequence variations modifying transcriptional regulation
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2008 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 4, no 1, e5- p.Article in journal (Refereed) Published
Abstract [en]

Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN ( regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7864 (URN)10.1371/journal.pcbi.0040005 (DOI)000255407500008 ()2-s2.0-38949195057 (Scopus ID)
Note
QC 20100621Available from: 2007-12-19 Created: 2007-12-19 Last updated: 2012-03-20Bibliographically approved
2. Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells
Open this publication in new window or tab >>Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells
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2007 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5, E34- p.Article in journal (Refereed) Published
Abstract [en]

Using the relative expression levels of two SNIP alleles of a gene in the same sample is an effective approach for identifying cis-acting regulatory SNPs (rSNPs). In the current study, we established a process for systematic screening for cis-acting rSNPs using experimental detection of Al as an initial approach. We selected 160 expressed candidate genes that are involved in cancer and anticancer drug resistance for analysis of All in a panel of cell lines that represent different types of cancers and have been well characterized for their response patterns against anticancer drugs. Of these genes, 60 contained heterozygous SNPs in their coding regions, and 41 of the genes displayed imbalanced expression of the two cSNP alleles. Genes that displayed Al were subjected to bioinformatics-assisted identification of rSNPs that alter the strength of transcription factor binding. rSNPs in 15 genes were subjected to electrophoretic mobility shift assay, and in eight of these genes (APC, BCL2, CCND2, MLH1, PARP1, SLIT2, YES1, XRCC1) we identified differential protein binding from a nuclear extract between the SNIP alleles. The screening process allowed us to zoom in from 160 candidate genes to eight genes that may contain functional rSNPs in their promoter regions.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7865 (URN)10.1093/nar/gkl1152 (DOI)000246371200037 ()2-s2.0-34247133388 (Scopus ID)
Note
QC 20100621Available from: 2007-12-19 Created: 2007-12-19 Last updated: 2012-03-20Bibliographically approved
3. Alternative promoter usage of the membrane glycoprotein CD36
Open this publication in new window or tab >>Alternative promoter usage of the membrane glycoprotein CD36
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2006 (English)In: BMC Molecular Biology, ISSN 1471-2199, Vol. 7, 8- p.Article in journal (Refereed) Published
Abstract [en]

Background: CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation.

Results: We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters.

Conclusion: Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7866 (URN)10.1186/1471-2199-7-8 (DOI)000238398300001 ()2-s2.0-33744946520 (Scopus ID)
Note
QC 20100621Available from: 2007-12-19 Created: 2007-12-19 Last updated: 2012-03-20Bibliographically approved
4. Hormonal and nutritional regulation of alternative CD36 transcripts in rat liver: a role for growth hormone in alternative exon usage
Open this publication in new window or tab >>Hormonal and nutritional regulation of alternative CD36 transcripts in rat liver: a role for growth hormone in alternative exon usage
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2007 (English)In: BMC Molecular Biology, ISSN 1471-2199, Vol. 8, no 60, 12- p.Article in journal (Refereed) Published
Abstract [en]

Background: CD36 is a multiligand receptor involved in various metabolic pathways, including cellular uptake of long-chain fatty acids. Defect function or expression of CD36 can result in dyslipidemia or insulin resistance. We have previously shown that CD36 expression is female-predominant in rat liver. In the present study, hormonal and nutritional regulation of hepatic CD36 expression was examined in male and female rats. Since alternative transcription start sites have been described in murine and human Cd36, we investigated whether alternative CD36 transcripts are differentially regulated in rat liver during these conditions.

Results: Sequence information of the rat Cd36 5'-UTR was extended, showing that the gene structure of Cd36 in rat is similar to that previously described in mouse with at least two alternative first exons. The rat Cd36 exon 1a promoter was sequenced and found to be highly similar to murine and human Cd36. We show that alternative first exon usage is involved in the female-predominant expression of CD36 in rat liver and during certain hormonal states that induce CD36 mRNA abundance. Estrogen treatment or continuous infusion of growth hormone (GH) in male rats induced CD36 expression preferentially through the exon 1a promoter. Old age was associated with increased CD36 expression in male rats, albeit without any preferential first exon usage. Intermittent GH treatment in old male rats reversed this effect. Mild starvation (12 hours without food) reduced CD36 expression in female liver, whereas its expression was increased in skeletal muscle.

Conclusion: The results obtained in this study confirm and extend our previous observation that GH is an important regulator of hepatic CD36, and depending on the mode of treatment (continuous or intermittent) the gene might be either induced or repressed. We suggest that the effects of continuous GH secretion in females (which is stimulatory) and intermittent GH secretion in males (which is inhibitory) explains the sex-different expression of this gene. Furthermore, a female-specific repression of hepatic CD36 in response to food deprivation was found, which was in contrast to a stimulatory effect in skeletal muscle. This demonstrates a tissue-specific regulation of Cd36.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7867 (URN)10.1186/1471-2199-8-60 (DOI)000248476300001 ()2-s2.0-34547685783 (Scopus ID)
Note
QC 20100621Available from: 2007-12-19 Created: 2007-12-19 Last updated: 2012-03-20Bibliographically approved
5. In silico prediction of regulatory SNPs in the CD36 gene and evaluation of their effect in a clinical study for coronary artery disease
Open this publication in new window or tab >>In silico prediction of regulatory SNPs in the CD36 gene and evaluation of their effect in a clinical study for coronary artery disease
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:kth:diva-7868 (URN)
Note
QC 20100621Available from: 2007-12-19 Created: 2007-12-19 Last updated: 2010-06-21Bibliographically approved

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