REPLICATION TIMING OF THE VARIOUS FMR1 ALLELES DETECTED BY FISH - INFERENCES REGARDING THEIR TRANSCRIPTIONAL STATUS

Following tile application of two-color fluorescence in-situ hybridiza tion (FISH) tu human interphase cells, we examined the replication tim ing of the fragile-X locus relative to the non-transcribed late replic ating alpha-satellite region of chromosome-X, a built-in intracellular reference locus. In this assay,: an unreplicated locus is identified by a single hybridization signal (singlet; S), whereas a replicated lo cus is identified by a duplicated signal (doublet; D), Hence, followin g, simultaneous hybridization with the FMR1 and alpha-satellite probes , male cells with one singlet and one doublet signal per cell (SD cell s) indicate S-phase cells where only one of the two loci has replicate d, The studied cell samples (lymphocytes and amniocytes) were derived from normal males. fragile-X male patients, and premutation male carri ers., Three distinct populations of SD cells were identified among the various samples. Tile first population had a high !frequency of cells showing a doublet FMR1; this pattern, indicating early replication of FMR1, characterized the SD cell population of normal males. The secon d population had a high frequency of cells showing a singlet FMR1; thi s pattern, indicating very late replication of FMR1, characterized til e SD population of ii agile-X patients. The third population had about one half of the cells showing a singlet FMR1 and the other half with a doublet FMR1, indicating somatic variation in the replication timing of FMR1; this pattern was seen in the SD cell population of premutati on carriers. The replication status of the FMR1 locus in the cells of patients was altered from late to early in the presence of 5-azadeoxyc ytidine, an activator of various silent genes. Based on the vast amoun t of information showing that expressed loci replicate early, whereas unexpressed loci replicate late, we inferred from the replication stat us of the FMR1 locus that: (I) the normal FMR1 allele is transcription ally active in lymphocytes and amniocytes; (2) the fully mutated FMR1 allele is transcriptionally silent; (3) the transcriptional activity o f the premutated allele is somewhat disturbed; (4) 5-azadeoxycytidine activates the fully mutated FMR1 allele.