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

Abstract

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 female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying 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 FMR1 and X-chromosome alpha-satellite probes to interphase cells of the various genotypes: the alpha-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 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 inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, 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 associated with X-inactivation.

Modification in the inherent mode of allelic replication in lymphocytes of patients suffering from renal cell carcinoma: A novel genetic alteration associated with malignancy

“…Recently, a new method based on fluorescence in situ hybridization (FISH) applied to interphase cells was developed to follow the temporal order of DNA replication. The FISH replication assay is relatively simple and fast, and in contrast to the classical replication timing methods allows identification of individual alleles within a single cell with no need to rely on allelic polymorphism or methylation level differences between 2 allelic counterparts (Selig et al, 1992;Boggs and Chinault, 1997;Yeshaya et al, 1999).…”

Recombinant Human DNA (Cytosine-5) Methyltransferase

“…The timing of DNA replication for the FMR1 locus and its flanking sequences (ϳ1 megabase pair total) is lengthened in fragile X males (50,51), in concert with transcriptional silencing (52). A delay in replication is also observed in chromosomes of carrier individuals (53)(54)(55) where the CGG⅐CCG tract is only moderately expanded and methylation is not observed (22). This suggests that repeat expansion leads to delay in replication irrespective of its methylation status.…”

On the Classification of a Small Imbalanced Cytogenetic Image Database

“…When applying FISH to interphase human cells, the conformation of a fluorescent signal changes during the S-phase of the cell cycle in an ordered manner [10], [11], [12]. In the beginning of the cell cycle, the fluorescent signal appears as a single dot ("singlet"; S), representing a prereplication state.…”

“…This is the reason why the majority of work employing the FISH replication assay has dealt only with the S and D conformations by either ignoring the other two shapes or summing the S and R1 signals and the R2 and D signals into two "new" S and D entities. Nevertheless, the use of the intermediate types R1 and R2 enables raising the sensitivity (resolution) of the FISH replication method, allowing the detection of minor changes in the replication patterns of specific genes associated with genetic abnormalities [12]. Moreover, since the R1 and R2 types are detected in a significant proportion of S-phase cells, especially when exploring -satellite sequences [11], ignoring these types in the analysis may lead to reduction of the sample size and distortion of the frequencies of occurrence of signal types.…”

“…However, since the conformation of the inspected sequence and, thus, of the fluorescent signal, changes during DNA replication along the cell cycle [10], non-dot-like signals are frequently found in many FISH applications and especially in clinical routine [11], [12].…”