Detection of FMR1 Trinucleotide Repeat Expansion Mutations Using Southern Blot and PCR Methodologies

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 ). These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.