The Study of X Chromosome Inactivation Mechanism in Klinefelter's Syndrome by cDNA Microarray Experiment

  • Jeong, Yu-Mi (Functional Genomics Lab) ;
  • Chung, In-Hyuk (Functional Genomics Lab) ;
  • Park, Jung Hoon (Functional Genomics Lab) ;
  • Lee, Sook-Hwan (Genome Research Center for Reproductive Medicine and Infertility) ;
  • Chung, Tae-Gyu (Department of Urology, CHA General Hospital, College of Medicine, Pochon CHA University) ;
  • Kim, Yong Sung (The Center for Functional analysis of Human Genome, KRIBB) ;
  • Kim, Nam-Soon (The Center for Functional analysis of Human Genome, KRIBB) ;
  • Yoo, Hyang-Sook (The Center for Functional analysis of Human Genome, KRIBB) ;
  • Lee, Suman (Functional Genomics Lab, Genome Research Center for Reproductive Medicine and Infertility)
  • Published : 2004.03.01

Abstract

To investigate the XIST gene expression and its effect in a Klinefelter's patient, we used Klinefelter's syndrome (XXY) patient with azoospermia and also used a normal male (XY) and a normal female (XX) as the control, We were performed cytogenetic analysis, Y chromosomal microdeletion assay (Yq), semi-quantitative RT-PCR, and the Northern blot for Klinefelter's syndrome (KS) patient, a female and a male control, We extracted total RNA from the KS patient, and from the normal cells of the female and male control subjects using the RNA prep kit (Qiagen), cDNA microarray contained 218 human X chromosome-specific genes was fabricated. Each total RNA was reverse transcribed to the first strand cDNA and was labeled with Cy-3 and Cy-5 fluorescein, The microarray was scanned by ScanArray 4000XL system. XIST transcripts were detected from the Klinefelters patient and the female by RT-PCR and Northern blot analysis, but not from the normal male, In the cDNA microarray experiment, we found 24 genes and 14 genes are highly expressed in KS more than the normal male and females, respectively. We concluded that highly expressed genes in KS may be a resulted of the abnormal X inactivation mechanism.

Keywords

References

  1. Brockdorff, N., Ashworth, A., Kay, G. F., McCabe, V. M., Norris, D. P., Cooper, P. J., Swift, S., and Rastan, S. (1992). The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell 71, 515-526 https://doi.org/10.1016/0092-8674(92)90519-I
  2. Brown, C. J., Ballabio, A., Rupert, J. L., Lafreniere, R. G., Grompe, M., Tonlorenzi, R., and Willard, H. F. (1991). A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349, 38-44 https://doi.org/10.1038/349038a0
  3. Brown, C. J., Hendrich, B. D., Rupert, J. L., Lafreniere, R. G., Xing, Y., Lawrence, J., and Willard, H. F. (1992). The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71, 527-542 https://doi.org/10.1016/0092-8674(92)90520-M
  4. Eichhorn, E. J., Bedollo, J. B., Malloy, C. R., Hatfield, B. A., Deitchman, D., Brown, M., Willard, J. E., and Grayburn, p. A. (1990). Effect of beta-adrenergic blockade on myocardial function and energetics in congestive heart failure. Improvements in hemodynamic, contractile, and diastolic performance with bucindolol. Circulation 82, 473-483 https://doi.org/10.1161/01.CIR.82.2.473
  5. Fisher, E. M., Beer-Romero, P., Brown, L. G., Ridley, A., McNeil, J. A., Lawrence, J. B., Willard, H. F., Bieber, F. R., and Page, D. C. (1990). Homologous ribosomal protein genes on the human X and Y chromosomes: escape from X inactivation and possible implications for Turner syndrome. Cell 63, 1205-1218 https://doi.org/10.1016/0092-8674(90)90416-C
  6. Hasle, H., Mellemgaard, A., Nielsen, J., and Hansen, J. (1995). Cancer incidence in men with Klinefelter syndrome. Br. J. Gancer 71. 416-420 https://doi.org/10.1038/bjc.1995.85
  7. Hong, Y. K., Ontiveros, S. D., Chen, C., and Strauss, W. M. (1999). A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation. Proc. Natl. Acad. Sci. USA 96, 6829-6834 https://doi.org/10.1073/pnas.96.12.6829
  8. Hong, Y. K., Ontiveros, S. D., and Strauss, W. M. (2000). A revision of the human XIST gene organization and structural comparison with mouse Xist. Mamm. Genome 11, 220-224 https://doi.org/10.1007/s003350010040
  9. Kawakami, T., Okamoto, K., Sugihara, H., Hattori, T., Reeve, A. E., Ogawa, O., and Okada, Y. (2003). The roles of supernumerical X chromosomes and XIST expression in testicular germ cell tumors. J. Ural. 169, 1546-1552. https://doi.org/10.1097/01.ju.0000044927.23323.5a
  10. Lafreniere, R. G., Brown, C. J., Rider, S., Chelly, J., Taillon-Miller, p., Chinault, A. C., Monaco, A. p., and Willard, H. F. (1993). 2.6 Mb YAC contig of the human X inactivation center region in Xq13: physical linkage of the RPS4X, PHKA1, XlST and DXS128E genes. Hum. Mol. Genet. 2, 1105-1115 https://doi.org/10.1093/hmg/2.8.1105
  11. Memili, E., Hong, Y. K., Kim, D. H., Ontiveros, S. D., and Strauss, W. M. (2001). Murine Xist RNA isoforms are different at their 3' ends: a role for differential polyadenylation. Gene 266, 131-137 https://doi.org/10.1016/S0378-1119(01)00353-5
  12. Rapley, E. A, Crockford, G. P., Teare, D., Biggs, P., Seal, S., Barfoot, R, Edwards, S., Hamoudi, R, Heimdal, K., Fossa, S. D., Tucker, K., Donald, J., Collins, F., Friedlander, M., Hogg, D., Goss, P., Heidenreich, A., Ormiston, W., Daly, P. A., Forman, D., Oliver, T. D., Leahy, M., Huddart, R., Cooper, C. S., Bodmer, J. G., Easton, D. F., Stratton, M. R., and Bishop, D. T. (2000). Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours. Nat. Genet. 24, 197-200 https://doi.org/10.1038/72877
  13. Salido, E. C., Yen, P. H., Mohandas, T. K., and Shapiro, L. J. (1992). Expression of the X-inactivation-associated gene XIST during spermatogenesis. Nat. Genet. 2, 196-199 https://doi.org/10.1038/ng1192-196
  14. Simoni, M. (2001). Molecular diagnosis of Y chromosome microdeletions in Europe: state-of-the-art and quality control. Hum. Reprod. 16, 402-409 https://doi.org/10.1093/humrep/16.3.402
  15. Simoni, M., Bakker, E., Eurlings, M. C., Matlhijs, G., Moro, E., Muller, C. R, and Vogt, P. H. (1999). Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int. J. Androl. 22, 292-299 https://doi.org/10.1046/j.1365-2605.1999.00193.x
  16. Sudbrak, R., Wieczorek, G., Nuber, U. A., Mann, W., Kirchner, R., Erdogan, F., Brown, C. J., Wohrle, D., Sterk, P., Kalscheuer, V. M., Berger, W., Lehrach, H., and Ropers, H. H. (2001). X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications. Hum. Mol. Genet. 10, 77-83 https://doi.org/10.1093/hmg/10.1.77