Abstract
Fragile X mental retardation 1 (FMR1) CGG repeat expansions cause fragile X syndrome—the leading monogenic form of intellectual disability—and increase the risk for fragile X-associated tremor ataxia syndrome and fragile X-associated primary ovarian insufficiency. Southern blot (SB) analysis is the current gold standard test for FMR1 molecular diagnosis. Several polymerase chain reaction (PCR)-based methods are now available for sizing FMR1 CGG repeat expansions. These methods offer higher diagnostic sensitivity and specificity compared to SB analysis, significantly reduce the turnaround time and increase throughput. In this chapter, we describe a triplet-repeat primed PCR protocol that employs capillary electrophoresis to resolve the derived amplicon products, enabling precise determination of the FMR1 genotypes in both males and females and characterization of the CGG repeat structure.
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Yu TW, Berry-Kravis E (2014) Autism and fragile X syndrome. Semin Neurol 34:258–265
Hill MK, Archibald AD, Cohen J, Metcalfe SA (2010) A systematic review of population screening for fragile X syndrome. Genet Med 12:396–410
Monaghan KG, Lyon E, Spector EB, erican College of Medical G, Genomics (2013) ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics. Genet Med 15:575–586
Saul RA, Tarleton JC (1993) FMR1-related disorders. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, LJH B, Stephens K, Amemiya A (eds) GeneReviews®. University of Washington, Seattle, WA
Usdin K, Hayward BE, Kumari D, Lokanga RA, Sciascia N, Zhao XN (2014) Repeat-mediated genetic and epigenetic changes at the FMR1 locus in the Fragile X-related disorders. Front Genet 5:226
Willemsen R, Levenga J, Oostra BA (2011) CGG repeat in the FMR1 gene: size matters. Clin Genet 80:214–225
Tassone F et al (2012) FMR1 CGG allele size and prevalence ascertained through newborn screening in the United States. Genome Med 4:100
Maenner MJ et al (2013) FMR1 CGG expansions: prevalence and sex ratios. Am J Med Genet B Neuropsychiatr Genet 162B:466–473
Seltzer MM, Baker MW, Hong J, Maenner M, Greenberg J, Mandel D (2012) Prevalence of CGG expansions of the FMR1 gene in a US population-based sample. Am J Med Genet B Neuropsychiatr Genet 159B:589–597
Jin P, Warren ST (2000) Understanding the molecular basis of fragile X syndrome. Hum Mol Genet 9:901–908
Nolin SL et al (2003) Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles. Am J Hum Genet 72:454–464
Yrigollen CM, Durbin-Johnson B, Gane L, Nelson DL, Hagerman R, Hagerman PJ, Tassone F (2012) AGG interruptions within the maternal FMR1 gene reduce the risk of offspring with fragile X syndrome. Genet Med 14:729–736
Fernandez-Carvajal I, Lopez Posadas B, Pan R, Raske C, Hagerman PJ, Tassone F (2009) Expansion of an FMR1 grey-zone allele to a full mutation in two generations. J Mol Diagn 11:306–310
Tassone F (2015) Advanced technologies for the molecular diagnosis of fragile X syndrome. Expert Rev Mol Diagn 15:1465–1473
Nolin SL, Glicksman A, Ding X, Ersalesi N, Brown WT, Sherman SL, Dobkin C (2011) Fragile X analysis of 1112 prenatal samples from 1991 to 2010. Prenat Diagn 31:925–931
Nolin SL et al (2013) Fragile X AGG analysis provides new risk predictions for 45-69 repeat alleles. Am J Med Genet A 161A:771–778
Chen L et al (2010) An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis. J Mol Diagn 12:589–600
Chen L et al (2011) High-resolution methylation polymerase chain reaction for fragile X analysis: evidence for novel FMR1 methylation patterns undetected in Southern blot analyses. Genet Med 13:528–538
Filipovic-Sadic S et al (2010) A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome. Clin Chem 56:399–408
Hantash FM et al (2010) Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening. Genet Med 12:162–173
Rajan-Babu IS, Law HY, Yoon CS, Lee CG, Chong SS (2015) Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis. Expert Rev Mol Med 17:e7
Rajan-Babu IS, Teo CR, Lian M, Lee CG, Law HY, Chong SS (2015) Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis of Fragile X Mental Retardation 1-related disorders. Expert Rev Mol Diagn 15:431–441
Zhou Y, Law HY, Boehm CD, Yoon CS, Cutting GR, Ng IS, Chong SS (2004) Robust fragile X (CGG)n genotype classification using a methylation specific triple PCR assay. J Med Genet 41:e45
Zhou Y, Lum JM, Yeo GH, Kiing J, Tay SK, Chong SS (2006) Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis. Clin Chem 52:1492–1500
Rajan-Babu IS, Chong SS (2016) Molecular correlates and recent advancements in the diagnosis and screening of FMR1-related disorders. Genes (Basel) 7(10):87
Warner JP, Barron LH, Goudie D, Kelly K, Dow D, Fitzpatrick DR, Brock DJ (1996) A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet 33:1022–1026
Lyon E, Laver T, Yu P, Jama M, Young K, Zoccoli M, Marlowe N (2010) A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis. J Mol Diagn 12:505–511
Fu YH et al (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67:1047–1058
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Rajan-Babu, IS., Chong, S.S. (2019). Triplet-Repeat Primed PCR and Capillary Electrophoresis for Characterizing the Fragile X Mental Retardation 1 CGG Repeat Hyperexpansions. In: Phillips, T.M. (eds) Clinical Applications of Capillary Electrophoresis. Methods in Molecular Biology, vol 1972. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9213-3_14
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DOI: https://doi.org/10.1007/978-1-4939-9213-3_14
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