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 ).