Bisulfite treatment of CG-rich track of trinucleotide repeat expansion disorder: Make the sequence less CG rich


Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Trinucleotide repeat (TNR) expansion is a kind of mutation with instability in the number of microsatellite repeats. This nature of mutation leads to the different kinds of neurological and neuromuscular disorders; among them, fragile-X syndrome is the main cause of intellectual disability in which the increasing number of CGG TNR in 5' untranslated region is the main reason for epigenetic silencing of Fragile X mental retardation 1 gene. The aim of this study is to decrease the CG content of the candidate region to facilitate amplification by conventional polymerase chain reaction (PCR). Bisulfite treatment of the genomic DNA results in conversion of unmethylated cytosine to uridine and may overcome the diagnostic pitfalls. Materials and Methods: The whole blood DNA was extracted and bisulfite treated. Then any simplification in PCR process of desire sequence were assayed through following conventional PCR using specifically designed primers for converted sequence. Bisulfite-treated PCR product of a nearby sequence confirmed our results as a conversion control. Results: Both the control and the candidate sequences undergoing bisulfite treatment were successfully amplified by PCR. Conclusions: Decreasing the GC content of the sequence by bisulfite treating could be a new approach to overcome difficulties in amplifying GC-rich sequences.


Budworth H, McMurray CT. A brief history of triplet repeat diseases. In: Trinucleotide Repeat Protocols. Methods Mol Biol Springer; 2013. p. 3-17.  Back to cited text no. 1
Orr HT, Zoghbi HY. Trinucleotide repeat disorders. Annu Rev Neurosci 2007;30:575-621.  Back to cited text no. 2
La Spada AR, Taylor JP. Repeat expansion disease: Progress and puzzles in disease pathogenesis. Nat Rev Genet 2010;11:247-58.  Back to cited text no. 3
Cummings CJ, Zoghbi HY. Fourteen and counting: Unraveling trinucleotide repeat diseases. Hum Mol Genet 2000;9:909-16.  Back to cited text no. 4
Albrecht A, Mundlos S. The other trinucleotide repeat: Polyalanine expansion disorders. Curr Opin Genet Dev 2005;15:285-93.  Back to cited text no. 5
Kanwal M, Alyas S, Afzal M, Mansoor A, Abbasi R, Tassone F, et al. Molecular diagnosis of Fragile X syndrome in subjects with intellectual disability of unknown origin: Implications of its prevalence in regional Pakistan. PLoS One 2015;10:e0122213.  Back to cited text no. 6
Garber KB, Visootsak J, Warren ST. Fragile X syndrome. Eur J Hum Genet 2008;16:666-72.  Back to cited text no. 7
Gabis LV, Gruber N, Berkenstadt M, Shefer S, Attia OL, Mula D, et al. Fragile X premutation carrier epidemiology and symptomatology in Israel-Results from a tertiary child developmental center. Cerebellum 2016;15:595-8.  Back to cited text no. 8
Grigsby J. The fragile X mental retardation 1 gene (FMR1): Historical perspective, phenotypes, mechanism, pathology, and epidemiology. Clin Neuropsychol 2016;30:815-33.  Back to cited text no. 9
Strien J, Sanft J, Mall G. Enhancement of PCR amplification of moderate GC-containing and highly GC-rich DNA sequences. Mol Biotechnol 2013;54:1048-54.  Back to cited text no. 10
Meguid NA, Ismail MF, El-Mahdy RS, Barakat MA, El-Awady MK. Simple molecular diagnostic method for fragile X syndrome in Egyptian patients: Pilot study. Acta Biochim Pol 2014;61:259-63.  Back to cited text no. 11
Bhagya CH, Wijesundera Sulochana WS, Hemamali NP. Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA. Indian J Hum Genet 2013;19:78-83.  Back to cited text no. 12
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Chakrabarti R, Schutt CE. The enhancement of PCR amplification by low molecular weight amides. Nucleic Acids Res 2001;29:2377-81.  Back to cited text no. 13
Rajan-Babu IS, Chong SS. Triplet-repeat primed PCR and capillary electrophoresis for characterizing the fragile X mental retardation 1 CGG repeat hyperexpansions. In: Clinical Applications of Capillary Electrophoresis. Methods in Molecular Biology Springer; 2019. p. 199-210.  Back to cited text no. 14
Chen L, Hadd A, Sah S, Houghton JF, Filipovic-Sadic S, Zhang W, et al. High resolution methylation PCR for fragile X analysis: Evidence for novel FMR1 methylation patterns undetected in southern blot analyses. Genet Med 2011;13:528.  Back to cited text no. 15
Grasso M, Boon EM, Filipovic-Sadic S, van Bunderen PA, Gennaro E, Cao R, et al. A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis. J Mol Diagn 2014;16:23-31.  Back to cited text no. 16
Sasaki M, Anast J, Bassett W, Kawakami T, Sakuragi N, Dahiya R. Bisulfite conversion-specific and methylation-specific PCR: A sensitive technique for accurate evaluation of CpG methylation. Biochem Biophys Res Commun 2003;309:305-9.  Back to cited text no. 17
Li LC, Dahiya R. MethPrimer: Designing primers for methylation PCRs. Bioinformatics 2002;18:1427-31.  Back to cited text no. 18
Smith ZD, Gu H, Bock C, Gnirke A, Meissner A. High-throughput bisulfite sequencing in mammalian genomes. Methods 2009;48:226-32.  Back to cited text no. 19
Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ. Identification and resolution of artifacts in bisulfite sequencing. Methods 2002;27:101-7.  Back to cited text no. 20
Woo HD, Kim J. Global DNA hypomethylation in peripheral blood leukocytes as a biomarker for cancer risk: A meta-analysis. PLoS One 2012;7:e34615.  Back to cited text no. 21
Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci U S A 1993;90:11995-9.  Back to cited text no. 22
Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415-28.  Back to cited text no. 23
Shen L, Guo Y, Chen X, Ahmed S, Issa JP. Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 2007;42:48, 50, 52 passim.  Back to cited text no. 24