Abstract
Fragile X syndrome, caused by the loss or diminution of the FMR1 (FRAXA - chromosomal locus Xq27.3) encoded protein, FMRP, results in mild to moderate mental retardation as its hallmark. Patients with the syndrome often vary dramatically in presentation with a range of intellectual and behavioral deficits, and provide a diagnostic challenge for clinicians due to the subtle nature of the physical phenotype (1,2). Insta bility of a CGG repeat segment contained within FMR1 exon 1 is the molecular basis for nearly all mutations (>99%) in the gene and leads to reduced or complete loss of FMRP (3–8). The variable phenotype occurs related to variation in FMR1 expression mediated by the extent of CGG repeat expansion and a secondary epigenetic feature: the aberrant hypermethylation of CpG dinucleotides contained in the CGG repeat segment and surrounding regions of the gene (9). Thus, molecular genetic studies of FMR1 are utilized to confirm a clinical diagnosis of fragile X syndrome, and perhaps just as importantly, to exclude an alteration in FMR1 as an explanation for nonspecific mental retardation in a patient. For clinical molecular diagnosis, the variety of FMR1 alleles and the myriad of possible alterations in the gene present a diagnostic challenge for which no one detection method has proven fully satisfactory. Here, a dual approach to FMR1 repeat expansion mutation detection utilizing Southern blot and polymerase chain reaction (PCR) methodologies is presented (10,11). The reader is referred to published technical standards for fragile X analysis to supplement the interpretation of molecular genetic results for patients (12).
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References
Tarleton, J. C. and Saul, R. A. (1993) Molecular genetic advances in fragile X syndrome. J. Pediatr. 122, 169–185.
Tarleton, J. and Saul, R. A. (updated June, 2000) Fragile X Syndrome, in: GeneClinics: Medical Genetics Knowledge Base [database online]. Copyright, University of Washington, Seattle, USA. Available online at http://www.geneclinics.org/profiles/fragileX.Accessed1/8/01.
Oberle, I., Rousseau, F., Heitz, D., Kretz, C., Devys, D., Hanauer, A., et al. (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252, 1097–1102.
Kremer, E. J., Pritchard, M., Lynch, M., Yu, S., Holman, K., Baker, E., et al. (1991) Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252, 1711–1714.
Verkerk, A. J. M. H., Pieretti, M., Sutcliffe, J. S., Fu, Y-H., Kuhl, D. P. A., Pizzuti, A., et al. (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905–914.
Yu, S., Pritchard, M., Kremer, E., Lynch, M., Nancarrow, J., Baker, E., et al. (1991) Fragile X genotype characterized by an unstable region of DNA. Science 252, 1179–1181.
Sutcliffe, J. S., Nelson, D. L., Zhang, F., Pieretti, M., Caskey, C. T., Saxe, D., and Warren, S. T. (1992) DNA methylation represses FMR-1 transcription in fragile X syndrome. Hum. Mol. Genet. 1, 397–400.
Pieretti, M., Zhang, F., Fu, Y-H., Warren, S. T., Oostra, B. A., Caskey, C. T., and Nelson, D. L. (1991) Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 66, 817–822.
McConkie-Rosell, A., Lachiewicz, A. M., Spiridigliozzi, G. A., Tarleton, J., Schoenwald, S., Phelan, M. C., et al. (1993) Evidence that methylation of the FMR-1 locus is responsible for variable phenotypic expression of the fragile X syndrome. Am. J. Hum. Genet. 53, 800–809.
Rousseau, F., Heitz, D., Biancalana, V., Blumenfeld, S., Kretz, C., Boue, J., et al. (1991) Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. N. Engl. J. Med. 325, 1673–1681.
Fu, Y-H., Kuhl, D. P. A., Pizzuti, A., Pieretti, M., Sutcliffe, J. S., Richards, S., 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.
Maddalena, A., Richards, C.S., McGinniss, M.J., Brothman, A., Desnick, R.J., Grier, R.E., et al. (2001) Technical standards and guidelines for fragile X: the first of a series of disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics. Genet. Med. 3, 200–205.
Nolin, S. L., Lewis, F.A., III, Ye, L. L., Houck, G.E., Glicksman, A. E., Limprasert, P., et al. (1996) Familial transmission of the FMR1 repeat. Am. J. Hum. Genet. 59, 1252–1261.
Warren, S. T. and Nelson, D. L. (1994) Advances in molecular analysis of fragile X Syndrome. JAMA 271, 536–542.
Eichler, E. E., Holden, J. J. A., Popovich, B. W., Reiss, A. L., Snow, K., Thibodeau, S. N., et al. (1994) Length of uninterrupted CGG repeats determines instability in the FMR1 gene. Nat. Genet. 8, 88–94.
Kunst, C. B. and Warren, S. T. (1994) Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles. Cell 77, 853–861.
Orrico, A., Galli, L., Dotti, M. T., Plewnia, K., Censini, S., and Federico, A. (1998) Mosa-icism for full mutation and normal-sized allele of the FMR1 gene: a new case. Am. J. Med. Genet. 78, 341–344.
Schmucker, B. and Seidel, J. (1999) Mosaicism for a full mutation and a normal size allele in two fragile X males. Am. J. Med. Genet. 84, 221–225.
Gedeon, A. K., Baker, E., Robinson, H., Partington, M. W., Gross, B., Manca, A., et al. (1992) Fragile X syndrome without CCG amplification has an FMR1 deletion. Nat. Genet. 1, 341–344.
Tarleton, J., Richie, R., Schwartz, C., Rao, K., Aylsworth, A. S., and Lachiewicz, A. (1993) An extensive de novo deletion removing FMR1 in a patient with mental retardation and the fragile X syndrome phenotype. Hum. Mol. Genet. 2, 1973–1974.
Wohrle, D., Kotzot, D., Hirst, M. C., Manca, A., Korn, B., Schmidt, A., et al. (1992) A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile X syndrome. Am. J. Hum. Genet. 51, 299–306.
Hammond, L. S, Macias, M. M, Tarleton, J. C., and Pai, G. S. (1997) Fragile X syndrome and deletions in FMR1: new case and review of the Lature. Am. J. Med. Genet. 72, 430–434.
De Boulle, K., Verkerk, A. J. M. H., Reyniers, E., Vits, L., Hendrickx, J., Van Roy, B., et al. (1993) A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nat. Genet. 3, 31–35.
Lugenbeel, K. A., Peier, A. M., Carson, N. L., Chudley, A. E., and Nelson, D. L. (1995) Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome. Nat. Genet. 10, 483–485.
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Tarleton, J. (2003). Detection of FMR1 Trinucleotide Repeat Expansion Mutations Using Southern Blot and PCR Methodologies. In: Potter, N.T. (eds) Neurogenetics. Methods in Molecular Biology™, vol 217. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-330-5:29
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DOI: https://doi.org/10.1385/1-59259-330-5:29
Publisher Name: Springer, Totowa, NJ
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