ANALYSIS OF A CGG SEQUENCE AT THE FMR-I LOCUS IN FRAGILE-X FAMILIES AND IN THE GENERAL-POPULATION

In this study, we have characterized a CGG repeat at the FMR-1 locus i n more than 100 families (more than 500 individuals) presenting for fr agile X testing and in 247 individuals from the general population. Bo th Southern blot and PCR-based assays were evaluated for their ability to detect premutations, full mutations, and variability in normal all ele sizes. Among the Southern blot assays, the probes Ox1.9 or StB12.3 with a double restriction-enzyme digest were the most sensitive in de tecting both small and large amplifications and, in addition, provided information on methylation of an adjacent CpG island. In the PCR-base d assays, analysis of PCR products on denaturing DNA sequencing gels a llowed the most accurate determination of CGG repeat number up to appr oximately 130 repeats. A combination of a Southern blot assay with a d ouble digest and the PCR-sequencing-gel assay detected the spectrum of amplification-type mutations at the FMR-1 locus. In the patient popul ation, a CGG repeat of 51 was the largest to be stably inherited, and a repeat of 57 was the smallest size of premutation to be unstably inh erited. When premutations were transmitted by females, the size of rep eat correlated with risk of expansion to a full mutation in the next g eneration. Full mutations (large repeats typically associated with an abnormal methylation pattern and mitotic instability) were associated with clinical and cytogenetic manifestations in males but not necessar ily in females. In the control population, the CGG repeat ranged from 13 to 61, but 94% of alleles had fewer than 40 repeats. The most frequ ent allele (34%) was a repeat of 30. One female had an allele (61 repe ats) within a range consistent with fragile X premutations, while two other individuals each had a repeat of 52. This suggests that the freq uency of unstable alleles in the general population may be approximate ly 1%.