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Evidence for Epigenetic Alterations in Turner Syndrome Opens up Feasibility of New Pharmaceutical Interventions Shriram N. Rajpathaka and Deepti D. Deobagkara,b,* a
Department of Zoology, Centre for Advanced Studies, University of Pune, Pune 411007, India; bBioinformatics Center, University of Pune, Pune 411007, India Abstract: DNA methylation is an important regulatory component which influences phenotypes by modulating gene expression. Changes in DNA methylation may lead to altered phenotypes and ability of an organism to respond to stress leading to subsequent manifestation of life style diseases, cancer, etc. The human X chromosome represents a classical model for epigenetic processes governing differential regulation of homologous chromosomes. X monosomy (45, XO) leads to Turner's syndrome in human with mild to severe phenotypes. Using a novel cDNA based high throughput approach of assessing genome wide methylation; we have examined the methylation landscape in human fibroblasts in 45, XO and 46, XX individuals. We report here that as expected methylation of X linked genes is different in these two situations. It was observed that methylation of several autosomal genes is also affected in this X monosomy state. Genes involved in bone remodeling, glucose sensitivity and ovarian function appear to be altered in addition to genes involved in epigenetic regulatory processes. This opens up interesting possibility of misregulation of DNA methylation in the X monosomy state resulting in altered gene expression and altered phenotypes. This may be one of the reasons for the variance, differential severity and penetrance in case of Turner’s syndrome. We propose that a systematic analysis of the molecular genetic mechanisms governing this epigenetic regulation will open up new therapeutic interventions which will certainly help in reducing severity of the disease and help in better management of X monosomy (Turner’s syndrome).
Keywords: DNA methylation ,Turner’s syndrome, epigenetics, altered penetrance and variability, pharmacological interventions, Epigenetic therapeutics. INTRODUCTION Epigenetics is the study of heritable changes in gene expression that occur without any change in DNA sequence [1]. Epigenetic information is added on the DNA by specific enzymatic mechanisms such as DNA methylation, histone modifications, mechanisms involving siRNAs, miRNAs, etc. DNA methylation is a postreplicative epigenetic modification which can influence DNA conformation; DNA- protein interactions and can lead to change in the gene expression. It is present across biological systems from micro organisms to insects [2] through plants till mammals [3]. Mammalian DNA contains 1 – 2 mol % of 5-methylcytosine; with 2 – 10 % of all the cytosine residues being modified [3]. DNA methylation is involved in diverse biological functions like normal embryonic development in mice and humans [4], allele specific imprinting [5], regulation of gene expression, maintenance of chromosome structure and stability [6, 7, 8]. DNA methylation changes with the stage of development and is tissue specific. It has been shown that diseases such as cancer [9], metabolic and lifestyle disorders (e.g diabetes), imprinting disorders (e.g. Prader–Willi syndrome) [10], pediatric syndromes (e.g. Immunodeficiency, Centromere instability and Facial anomalies syndrome, Rett syndrome) [11] are associated with altered DNA methylation, chromosomal abnormalities and instability. Cells with altered DNA methylation appear to be more susceptible to undergoing chromosomal loss, gain, or rearrangement [12]. Chromosome instability, main cause of aneuploidy, is also shown to be governed by epigenetic factors [13]. Turner’s syndrome (TS) or X chromosome monosomy, is a chromosomal disorder caused by loss of whole X chromosome (XO) or loss of some part of the X chromosome (structural abnormalities, iso Xq, ring X etc.). It affects approximately 1 in *Address correspondence to this author at the Department of Zoology, Centre for Advanced Studies, University of Pune, Pune 411007, India; Tel: 09921184871; Fax: +91 25690087; E-mails:
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1,800–2,500 live female births [14]. Approximately 50% of TS individuals exhibit complete loss of one X chromosome while other females shows cryptic mosaicism [15]. Turner mosaics usually have a less severe phenotype than XO individuals [16]. The X chromosome, with the exception of the pseudoautosomal region, is subjected to X inactivation. Current evidences suggest characteristic features associated with TS presumably arise due to haploinsufficiency of one (or more) X-linked genes that normally escape X-inactivation [17, 18]. TS display characteristic neuropsychological and physiological features like short stature, ovarian dysfunction, osteoporosis, cardiovascular abnormalities, renal disorders, diabetes mellitus type II etc. Other features include defects in attention, visuospatial skills, memory based functions etc. Many X linked genes are proposed as candidate genes for such clinical phenotypes for example SHOX, PHOG are considered as strong candidate genes for short stature and bone deformities in TS individuals [19, 20].Genes like ZFX [21], SHOX [22], DIPAH2 [23] are shown to be associated with premature ovarian failure in Turner individuals. It has been proposed and shown that mutations in many of the autosomal genes are also responsible for diseased conditions. Mutations in the autosomal genes like FSH, ER alpha, LH receptor, are shown to be associated with ovarian dysfunction or premature ovarian failure [24, 25]. Several autosomal genes have been identified as candidate genes for osteoporosis [26]. Thus X linked as well as autosomal genes are linked to diseased phenotypes which are similar to Turner’s syndrome. Interestingly studies reported that autosomal abnormalities like Trisomy of 13 and 18 are also associated with ovarian dysfunction [27] while Trisomy of 21 i.e. down’s syndrome, is associated with congenital heart defects and global DNA hypomethylation [28]. It is well known that autosomy of other human chromosomes causes early embryonic lethality. Reports suggest that chromosomal abnormalities like aneuploidy have a link with epigenetic processes. In our laboratory by using a novel cDNA based microarray approach, we have shown differential methylation in human X chromosome in 45XO and 47 XXX conditions [29, 30].
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In this work we have investigated the genome wide methylation status of the autosomes and X chromosome in 45, XO and 46, XX individuals and provide evidence for altered DNA methylation. MATERIALS AND METHODS Genomic DNA Samples DNA used was from fibroblast cell lines of Caucasian population with karyotype 45, XO (Cat. no.NA00857) and 46, XX (Cat. no. ND 29194) supplied by Coriell Cell Repositories, USA. The lymphocyte DNA for 45, XO and 46, XX of Indian population was obtained from, CCMB, Hyderabad, India. Human 19K cDNA Arrays The Human 19KcDNA arrays were purchased from the University Health Network, Toronto, Canada. (www.microarrays.ca), these contain duly annotated Human 19, 200 cDNA spots. DNA Hybridization and Methylation Detection by Methylation Microarray Methylation microarray was done using standard protocol as described earlier [29]. Genomic DNA was sonicated (2μg) into approximately 1.5- 2 kb fragments, in Hybridization buffer (Perkin Elmer Ltd.). Sonicated DNA was denatured by heating at 900C, followed by immediate cooling. This denatured DNA and 1X Hybridization buffer (total volume 300 l) was added on slide and hybridization was carried out at 42oC, 15-16 hrs on Quantifoil Hybridization station. The Slides were washed with medium stringency buffers, and blocked with blocking reagent, TNB (Tris-Cl 50mM, pH 7.5; EDTA 1mM; NaCl 150 mM, BSA 2%) for 30 min at room temperature. Then slide was incubated with monoclonal 5mC antibody (1:1,000 in TNT buffer Tris-Cl 100mM, pH 7.5; NaCl 150 mM, Tween 20, 0.01%) for 60 min at 25oC. Slides were washed with TNT buffer 3 times for 15' with agitation at 25oC; and then incubated with the anti mouse Cy3 labeled IgG (1:1,000 in TNT buffer) for 60' at 25°C. Washing was repeated as described above and then slides were dried by centrifugation at 1,500 G for 2min at 37oC. The slides were scanned using a Perkin Elmer ScanArray Express at 10 μm resolution at a wavelength of 543 nm. For each DNA (XO-XX fibroblast and XO-XX lymphocytes) three technical repeats were carried out. Microarray Data Analysis a) Data Normalization LOWESS normalization was used as the default parameter for the array scanning apparatus. The microarray slides were provided with spots containing 3XSSC and Arabidopsis thaliana DNA which provided a background signal. The fluorescence values from these spots were considered to be representatives of non specific binding of the DNA to the slides and these values were used to determine the true positives in the data. These values were together termed as control values. The control values from each individual array were used to normalize the fluorescence for all the genes of that array in order to provide a uniform scale for comparison of genes between arrays. Assuming an error of 20 % in the ability to detect true positives, signals which were higher than 20% of the control value were considered a true positive. The normalization was performed by dividing the fluorescence values with the cutoff value of the respective arrays. For comparison of the genes between arrays the normalized values of fluorescence were used. The generally accepted criterion of signal to noise ratio above 3.0 might be a little too stringent in this case in determining the true positives. Thus the signal to noise ratio cut off used was 1.0 and not the usual standard value of 3.0. b) Microarray Data Comparison within Slides The fold intensity values were compared between replicates. Any genes showing a positive signal in at least 2 out of 3 replicates
Rajpathak and Deobagkar
was considered as a potential true positive. Potential true positives were calculated for each condition analyzed. This provided the number of total methylated genes for all the samples. Genes that are common between XO fibroblast and XO lymphocytes and between XX fibroblast – XX lymphocytes were identified. A comparative analysis using t-test was carried out between signal intensities (methylation level) of these common genes in order to generate genes list showing significantly different or similar methylation in each genotype. In Silico Analysis for True Positive Methylated Genes a) DAVID(The Database for Annotation, Visualization and Integrated Discovery, http://david.abcc.ncifcrf.gov/):DAVID provides a comprehensive set of functional annotation tools for investigators to understand biological meaning behind large list of genes. It can identify enriched biological themes, particularly GO term; Discover enriched functional-related gene groups, Cluster redundant annotation terms, Visualize genes on BioCarta & KEGG pathway maps, Link gene-disease associations [31]. Methylated genes, specific for each tissue for each genotype were analyzed by Functional tools from DAVID database. Pathway analysis was carried out with P value cutoff 0.1. For annotation clustering medium classification stringency was used. b) String (Protein- Protein Interaction Database) (http://stringdb.org/) STRING is a database of known and predicted protein interacti ons. The interactions include direct (physical) and indirect (functional) associations [32]. String database was used to identify other proteins that can interact with target proteins (corresponding to the gene found to be methylated in our analysis). This string analysis can possibly predict the functional interactions among proteins in the cell identify the biological process associated with particular protein. RESULTS The current study was aimed at identification of DNA methylation patterns in Turner’s and in normal individuals. We have employed a novel whole genome methylome detection approach using immunochemical technique and cDNA microarray. Here genomic DNA is sheared to an average size of 1-2 Kb and hybridised with the cDNA microarray slide. The hybridised genomic DNA is retained on the slide after washing. The cytosine methylation in the genomic DNA is detected using monoclonal anti5mC antibody and Cy3 labelled secondary antibody. Since the probe size is 1-2 Kb, methylation in the region surrounding the gene including promoter region will also be detected. In our analysis many of the X linked as well as autosomal genes showed differential DNA methylation in 45, XO and 46, XX conditions. METHYLATED GENES FROM FIBROBLAST OF NORMAL (46, XX) AND TURNER’S (45, XO) INDIVIDUAL In normal (46, XX) individual, a total 1663 genes were found to be methylated, with 51 X linked genes and 1326 autosomal genes (remaining 286 genes were not annotated). In 45, XO condition, a total of 1950 genes were methylated, with 53 X linked genes and 1560 autosomal genes (remaining 337 genes were not annotated). Annotation clustering analysis of fibroblast genes from 46, XX individual, showed that genes involved in insulin pathway (enrichment score 0.60), estrogen stimulation pathway (enrichment score 1.04), Gonadotropin-releasing hormone (GnRH) signaling pathways (enrichment score 0.64), female sex differentiation and ovulation cycle (enrichment score 1.04) are found to be methylated. Genes involved in epigenetic regulation such as PHD finger domain, PWWP domain, bromodomain were identified as methylated (enrichment score 0.70). Pathway analysis identified methylated
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genes involved in oocyte meiosis pathway (1.4%), GnRH signaling pathway (1.2%). etc. (Supplementary Tables 1, 2). DAVID clustering analysis of methylated genes of 45,XO revealed genes associated with nuclear chromosome condensation, chromatin remodeling complex involving histone modification (enrichment score 1.5); transcriptional repression (enrichment score 1.29), gamete generation and germ cell development (enrichment score 1.0) and in kidney and urinogenital system development (enrichment score 0.9 and 0.83). Interestingly some methylated genes are found to be involved Type II diabetes (P< 0.015), pancreatic cancer (P Drosophila melanogaster diaphanous Gene Is Disrupted in a Patient with Premature Ovarian Failure: Evidence for Conserved Function in Oogenesis and Implications for Human Sterility. Amer J Hum Gen 1998; 62: 533-541. Persani L, Rossetti R, Cacciatore C. Genes involved in human premature ovarian failure. J Mol Endo 2010; 45: 257-279. Simpson JL, Rajkovic A. Ovarian differentiation and gonadal failure. Amer J Med Gen 1999; 89: 186-200. Liu YZ, Liu YJ, Recker RR, Deng HW. Molecular studies of identification of genes for osteoporosis: the 2002 update. J Endo 2003; 177: 147-196. Cunniff C, Jones KL, Benirschke K. Ovarian dysgenesis in individuals with chromosomal abnormalities. Hum Gen 1991; 86: 552-556. ObermannBorst SA, van Driel LMJW, Helbing WA,et.al. Congenital heart defects and biomarkers of methylation in children: a case–control study. Euro J Clin Inv 2011; 41: 143-150. Kelkar A, Deobagkar D. A novel method to assess the full genome methylation profile using monoclonal antibody combined with the high throughput based microarray approach. Epigenetics, 2009; 4: 415-420. Kelkar A, Deobagkar D. Methylation profile of genes on the human X chromosome. Epigenetics, 2010; 5: 612-618. Huang DW, Sherman BT, Richard A, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Proto 2009; 4: 44 – 57. Szklarczyk D, Franceschini A, Kuhn M,et.al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucl Acid Res 2011; 39: D561D568. Castrillon DH, Wasserman SA. Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene. Development, 1994; 120: 3367-3377. Blaschke RJ, Rappold G. The pseudoautosomal regions,< i> SHOX and disease. Curr. opin. Gen. Dev. 2006; 16: 233-239 BURTON KA, VAN EE CC, Purcell K, WINSHIP I, SHELLING AN. Autosomal translocation associated with premature ovarian failure. J. med. Gen., 2000; 37: e2-e2. Bashamboo A & McElreavey K. NR5A1/SF-1 and development and function of the ovary. Annales d’Endocrinologie, 2010; 71: 177–182. Liu YJ, Shen H, Xiao P,et.al. Molecular genetic studies of gene identification for osteoporosis: a 2004 update. J. Bone Min. Res. 2006; 21: 1511-1535. Liu YZ, Liu YJ, Recker RR, Deng HW. Molecular studies of identification of genes for osteoporosis: the 2002 update. J. Endocrino. 2003; 177: 147-196. Tenf P, Yamashita H, Sampath TK,et.al. Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein4. J. Bio. Chem. 1994; 269: 16985-16988. Kawaguchi H. Hormones and osteoporosis update [Insulin/IGF-I and bone]. Clinical calcium, 2009; 19: 1015.
Rajpathak and Deobagkar [41]
[42] [43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52] [53]
[54] [55]
[56] [57]
[58]
[59] [60] [61]
[62]
Ogata N, Kawaguchi H. Involvement of insulin and IGF-1 signalling molecules in bone metabolism . Clinical calcium, 2008; 18: 614. Akune T, Ogata N, Hoshi K,et.al. Insulin receptor substrate-2 maintains predominance of anabolic function over catabolic function of osteoblasts. J. cell Bio. 2002; 159: 147-156. Bakalov VK, Cheng C, Zhou J, Bondy CA. X-chromosome gene dosage and the risk of diabetes in Turner syndrome. J. Clin. Endocrino. Met. 2009; 94: 3289-3296. Tanaka M, Murakami K, Ozaki S,et.al. DIP2 discointeracting protein 2 homolog A (Drosophila) is a candidate receptor for follistatinrelated protein/follistatinlike 1–analysis of their binding with TGF superfamily proteins. FEBS Journal, 2010; 277: 42784289. Scheidegger KJ, Cenni B, Picard D, Delafontaine P. Estradiol decreases IGF-1 and IGF-1 receptor expression in rat aortic smooth muscle cells. J. Bio. Chem. 2000; 275: 38921-38928. Eertmans F, Dhooge W, De Wever O, Bracke M, Comhaire F, Kaufman JM. Estrogen receptor alpha (ERalpha) and insulin-like growth factor I receptor (IGF-IR) cross-talk in the gonadotropic alphaT3-1 cell line. J. Cell Physio. 2007;212:583-590. Chang SY, Tsai MY, Huang FJ, Kung FT. Expression of insulinlike growth factor (IGF), IGF receptor, and IGF-binding protein messenger ribonucleic acids in luteinized granulosa cells from different size follicles after controlled ovarian hyperstimulation. J. Assist. Rep. Gen. 2002; 19: 121-126. Takahashi C, Fujito A, Kazuka M, Sugiyama R, Ito H, Isaka K. Anti-Müllerian hormone substance from follicular fluid is positively associated with success in oocyte fertilization during in vitro fertilization. Fertility and sterility, 2008; 89: 586-591. Kevenaar ME, Themmen APN, Van Kerkwijk AJ,et.al. Variants in the ACVR1 gene are associated with AMH levels in women with polycystic ovary syndrome. Hum. Repro. 2009; 24: 241-249. Townson SM, Kang K, Lee AV, Oesterreich S. Structure-function analysis of the estrogen receptor alpha corepressor scaffold attachment factor-B1: identification of a potent transcriptional repression domain. J. Bio. Chem. 2004 ;279: 26074-26081. Jiang S, Meyer R, Kang K, Osborne CK, Wong J, Oesterreich S. Scaffold attachment factor SAFB1 suppresses estrogen receptor mediated transcription in part via interaction with nuclear receptor corepressor. Mol. Endocrino. 2006; 20: 311-320. Zulian E, Sartorato P, Schiavi F,et.al. The M235T polymorphism of the angiotensinogen gene in women with polycystic ovary syndrome. Fertility and sterility, 2005; 84: 1520-1521. Ellsworth BS, Egashira N, Haller JL,et.al. FOXL2 in the pituitary: molecular, genetic, and developmental analysis. Mol. Endocrino., 2006; 20: 2796-2805. Bakalov VK, Cooley MM, Quon MJ,et.al. Impaired insulin secretion in the Turner metabolic syndrome. J. Clin. Endocrino. Meta. 2004; 89: 3516-3520. Bierne H, Tham TN, Batsche E,et.al. Human BAHD1 promotes heterochromatic gene silencing. Proc. Nat. Acad. Sci. 2009; 106: 13826-13831. Frojdo S, Durand C, Molin L,et.al. Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1. Mol. Cell. Endocrino. 2011. Wu MY, Tsai TF, Beaudet AL. Deficiency of Rbbp1/Arid4a and Rbbp1l1/Arid4b alters epigenetic modifications and suppresses an imprinting defect in the PWS/AS domain. Genes & development, 2006; 20: 2859-2870. Peng L, Yuan Z, Ling H,et.al. SIRT1 Deacetylates the DNA methyltransferase 1 (DNMT1) protein and alters its activities. Mol. Cell. Bio. 2011; 31: 4720-4734. Bjornsson HT, Daniele Fallin M, Feinberg AP. An integrated epigenetic and genetic approach to common human disease. TRENDS in Gen. 2004; 20: 350-358. Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation. Cell, 1980; 20: 85. Issa JPJ, Garcia-Manero G, Giles FJ,et.al. Phase 1 study of lowdose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies. Blood, 2004; 103: 1635-1640. Jha AK, Nikbakht M, Kaur J. Cervical cancer: Promoter hypermethylation and its reversal. J. Pharm. Res. 2011; 4.
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Korkmaz A, Manchester LC, Topal T, Ma S, Tan DX, Reiter RJ. Epigenetic mechanisms in human physiology and diseases. J. Exp.
Received: April 20, 2013
Accepted: July 18, 2013
Inte. Medi. 2011; 1: 139-147.
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