The use of high-resolution melting techniques for mutation screening of diseases caused by trinucleotide repeats expansion, with emphasis on the AR gene
BACKGROUND Trinucleotide repeat expansion (TRE) diseases are genetic diseases caused by an increase in the number of CAG, CGG, and CTG codons. CAG repeat expansion in exon 1 of the androgen receptor (AR) gene is known to be associated with disorders of sex development (DSD) and spinal and bulbar muscular atrophy (SBMA). Because the traditional Southern blot for CAG repeat expansion is laborious and time-consuming, this study was aimed to use high-resolution melting (HRM) analysis to screen the CAG repeat length of the AR gene in Indonesian patients with DSD.
METHODS In total, 30 male patients with DSD (46, XY), one male patient with SBMA, and 30 healthy males (control) were included in the study. The CAG repeat length was determined using HRM analysis, and Sanger sequencing was used to confirm the CAG repeat length.
RESULTS For the DSD cases and controls, the melting temperature (Tm) was within the normal range of 89–91.05°C; however, Tm was 92.65°C for the SBMA case. Sanger sequencing confirmed that DSD cases had 13–27 CAG repeats, and the SBMA case had 54 CAG repeats.
CONCLUSIONS HRM analysis using polymerase chain reaction is a sensitive, effective, and rapid technique for screening CAG repeat expansion in exon 1 of the AR gene. This is the first technique for AR gene screening that may be applicable to other TRE diseases.
Sanders DW, Brangwynne CP. RNA repeats put a freeze on cells. Nature. 2017;546(7657):215-6. https://doi.org/10.1038/nature22503
La Spada AR, Taylor JP. Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat Rev Genet. 2010;11(4):247−58. https://doi.org/10.1038/nrg2748
Ryan CP, Crespi BJ. Androgen receptor polyglutamine repeat number: Models of selection and disease susceptibility. Evol Appl. 2013;6(2):180−96. https://doi.org/10.1111/j.1752-4571.2012.00275.x
Pan B, Li R, Chen Y, Tang Q, Wu W, Chen L, et al. Genetic association between androgen receptor gene cag repeat length polymorphism and male infertility: a meta-analysis. Medicine (Baltimore). 2016;95(10):e2878. https://doi.org/10.1097/MD.0000000000002878
Tirabassi G, Cignarelli A, Perrini S, Delli Muti N, Furlani G, Gallo M, et al. Influence of CAG repeat polymorphism on the targets of testosterone action. Int J Endocrinol. 2015;2015:298107. https://doi.org/10.1155/2015/298107
La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular athrophy. Nature. 1991;352(6330):77-9. https://doi.org/10.1038/352077a0
Adamovic T, Nordenskjöld A. The CAG repeat polymorphism in the androgen receptor gene modifies the risk for hypospadias in Caucasians. BMC Med Genet. 2012;13:109. https://doi.org/10.1186/1471-2350-13-109
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003;49(6):853−60. https://doi.org/10.1373/49.6.853
Sharma N, Nautiyal SC, Kumari S, Kumari K, Singh V, Kumar K, et al. High resolution melt curve analysis- an innovative approach for molecular diagnosis. Webmed Cent. 2013;4(3):WMC003998.
Lim GX, Yeo M, Koh YY, Winarni TI, Rajan-Babu IS, Chong SS, et al. Validation of a commercially available test that enables the quantification of the numbers of CGG trinucleotide repeat expansion in FMR1 gene. PLoS One. 2017;12(3):e0173279. https://doi.org/10.1371/journal.pone.0173279
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215. https://doi.org/10.1093/nar/16.3.1215
Greenland KJ, Beilin J, Castro J, Varghese PN, Zajac JD. Polymorphic CAG repeat length in the androgen receptor gene and association with neurodegeneration in a heterozygous female carrier of Kennedy's disease. J Neurol. 2004;251(1):35−41. https://doi.org/10.1007/s00415-004-0266-x
Tahmasbpour E, Balasubramanian D, Agarwal A. A multi-faceted approach to understanding male infertility: gene mutations, molecular defects and assisted reproductive techniques (ART). J Assist Reprod Genet. 2014;31(9):1115−37. https://doi.org/10.1007/s10815-014-0280-6
Muroya K, Sasagawa I, Suzuki Y, Nakada T, Ishii T, Ogata T. Hypospadias and the androgen receptor gene: mutation screening and CAG repeat length analysis. Mol Hum Reprod. 2001;7(5):409−13. https://doi.org/10.1093/molehr/7.5.409
Fietz D, Geyer J, Kliesch S, Gromoll J, Bergmann M. Evaluation of CAG repeat length of androgen receptor expressing cells in human testes showing different pictures of spermatogenic impairment. Histochem Cell Biol. 2011;136(6):689−97. https://doi.org/10.1007/s00418-011-0871-6
Juniarto AZ, van der Zwan YG, Santosa A, Hersmus R, de Jong FH, Olmer R, et al. Application of the new classification on patients with a disorder of sex development in Indonesia. Int J Endocrinol. 2012;2012:237084. https://doi.org/10.1155/2012/237084
Jancik S, Drabek J, Berkovcova J, Xu YZ, Stankova M, Klein J, et al. A comparison of direct sequencing, pyrosequencing, high resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas. J Exp Clin Cancer Res. 2012;31(1):79. https://doi.org/10.1186/1756-9966-31-79
Marino M, Monzani ML, Brigante G, Cioni K, Madeo B, Santi D, et al. High-resolution melting is a sensitive, cost-effective, time-saving technique for BRAF V600E detection in thyroid fnab washing liquid: a prospective cohort study. Eur Thyroid J. 2015;4(2):73−81. https://doi.org/10.1159/000430092
Ghasemi Z, Hashemi M, Ejabati M, Ebrahimi SM, Manjili HK, Sharafi A, et al. Development of a high-resolution melting analysis method for CYP2C19*17 genotyping in healthy volunteers. Avicenna J Med Biotechnol. 2016;8(4):193−9.
Gottlieb B, Beitel LK, Nadarajah A, Paliouras M, Trifiro M. The androgen receptor gene mutations database: 2012 update. Hum Mutat. 2012;33(5):887−94. https://doi.org/10.1002/humu.22046
Davey RA, Grossmann M. Androgen receptor structure, function and biology: from bench to bedside. Clin Biochem Rev. 2016;37(1):3−15.
Grunseich C, Rinaldi C, Fischbeck KH. Spinal and bulbar muscular atrophy: pathogenesis and clinical management. Oral Dis. 2014;20(1):6−9. https://doi.org/10.1111/odi.12121
Copyright (c) 2019 Mahayu Dewi Ariani, Stefani Harumsari, Nurin Aisyiyah Listyasari, Agustini Utari, Achmad Zulfa Juniarto, Sultana MH Faradz
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Authors who publish with Medical Journal of Indonesia agree to the following terms:
- Authors retain copyright and grant Medical Journal of Indonesia right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial License that allows others to remix, adapt, build upon the work non-commercially with an acknowledgment of the work’s authorship and initial publication in Medical Journal of Indonesia.
- Authors are permitted to copy and redistribute the journal's published version of the work non-commercially (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Medical Journal of Indonesia.