FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of fema le carriers of a mutated FMR1 allele. In view of the tight relationship bet ween replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-c hromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 he terozygous for a mutated FMR1 allele with a trinucleotide repeat number var ying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with F MR1 and X-chromosome a-satellite probes to interphase cells of the various genotypes: the a-satellite probe was used to distinguish between early repl icating (active) and late replicating (inactive) X-chromosomes, and the FMR 1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the ina ctive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, rega rdless of repeat size. We conclude therefore that: (1) the FMR1 locus is su bjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat s ize, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication a ssociated with X-inactivation.