FISH analysis of replication and transcription of chromosome X loci: new approach for genetic analysis of Rett syndrome

Differential replication staining using the 5-bromo-2′-deoxyuridine+Hoechst 33258 technique has been carried out on a series of 28 girls with Rett syndrome (RTT). The results indicated that regions Xq23 and Xq28 of inactive chromosome X could contain early replicating and, therefore, transcriptionally active loci in RTT. Interphase fluorescence in situ hybridization studies of replication timing, using chromosome X-specific genomic DNA probes, was applied to determine the loci with altered replication and transcription in RTT. Randomly selected P1 artificial chromosome (PAC) clones for Xp, Xcen and Xq were used. Two PAC clones from Xq28 (anonymous clone 24.23.0 and 671D9, containing MeCP2 locus) probably escape inactivation in late replicating chromosome X in some RTT patients. Therefore, region Xq28 could contain the genes escaping X inactivation and with expression from the human active and inactive X chromosomes. These results support the hypothesis proposing the disturbances in dosage compensation effect due to aberrant activation of genes in inactive chromosome X in RTT (bi-allelic expression instead of mono-allelic). Our results indicate that the normal allele of the MeCP2 gene could escape X inactivation and reduce the pathogenic effect of mutated allele in RTT.