Molecular analysis of fragile X syndrome

The gene responsible for fragile X syndrome, fragile X mental retardation-1 (FMR1), contains an unstable repeat sequence of (CGG)(n). Additionally, an upstream promoter region of the gene--a CpG island--is abnormally methylated in most affected individuals. Amplification of CGG repeats and abnormal methylation show a correlation with affected status. The first basic and alternate protocols in this unit outline the use of the polymerase chain reaction (PCR) to rapidly detect the size of the repeat amplification. The second basic protocol describes the assessment of fragile X syndrome by direct Southern blot hybridization of genomic DNA digested with a pair of restriction enzymes, one of which is methylation-sensitive. Extent of amplification and level of methylation can be simultaneously detected. A combination of the two techniques can be used to characterize the genotypes of individual members in identified fragile X families.