Microdeletion syndromes disclose replication timing alterations of genes unrelated to the missing DNA

Abstract

Background: The temporal order of allelic replication is interrelated to the epigenomic profile. A significant epigenetic marker is the asynchronous replication of monoallelically-expressed genes versus the synchronous replication of biallelically-expressed genes. The present study sought to determine whether a microdeletion in the genome affects epigenetic profiles of genes unrelated to the missing segment. In order to test this hypothesis, we checked the replication patterns of two genes - SNRPN, a normally monoallelically expressed gene (assigned to 15q11.13), and the RB1, an archetypic biallelically expressed gene (assigned to 13.q14) in the genomes of patients carrying the 22q11.2 deletion (DiGeorge/Velocardiofacial syndrome) and those carrying the 7q11.23 deletion (Williams syndrome).

Results: The allelic replication timing was determined by fluorescence in situ hybridization (FISH) technology performed on peripheral blood cells. As expected, in the cells of normal subjects the frequency of cells showing asynchronous replication for SNRPN was significantly (P < 10-12) higher than the corresponding value for RB1. In contrast, cells of the deletion-carrying patients exhibited a reversal in this replication pattern: there was a significantly lower frequency of cells engaging in asynchronous replication for SNRPN than for RB1 (P < 10-4 and P < 10-3 for DiGeorge/Velocardiofacial and Williams syndromes, respectively). Accordingly, the significantly lower frequency of cells showing asynchronous replication for SNRPN than for RB1 is a new epigenetic marker distinguishing these deletion syndrome genotypes from normal ones.

Conclusion: In cell samples of each deletion-carrying individual, an aberrant, reversed pattern of replication is delineated, namely, where a monoallelic gene replicates more synchronously than a biallelic gene. This inverted pattern, which appears to be non-deletion-specific, clearly distinguishes cells of deletion-carriers from normal ones. As such, it offers a potential epigenetic marker for suspecting a hidden microdeletion that is too small to be detected by conventional karyotyping methods.

Figure 1

Cells from a patient with DGS/VCFS, following two-color FISH with TUPLE1 (red) and ARSA (green). Right, a metaphase-spread with the normal chromosome 22, identified by double labeling (red and green signals) and the deletion-carrying one, distinguished by the absence of the red signal; left, an interphase cell exemplifying two ARSA (green) signals and a single TUPLE1 (red) signal. The green signal that is closer to the red was assumed to belong to the normal homologue, while the more distant one to the deletion-carrying homologue.

Figure 2

Fluorescent signals in PHA-stimulated lymphocytes at interphase, following FISH with RB1. (a) – (c) cells with two singlets (SS cells) in which neither allele has replicated; (d) – (f) cells with two doublets (DD cells) in which both alleles have replicated; and (g) – (i) cells with one singlet and one doublet (SD cells), which are S-phase cells in which one allele has replicated while its partner has not.

Figure 3

The frequency of SD cells for RB1 and SNRPN. (a) and (b), samples from control individuals; (c) and (d), samples from patients with DGS/VCFS; and (e) and (f), samples from patients with Williams syndrome. The P values in frames (a), (c) and (e) represent the level of significance of the differences in the frequency of SD cells between RB1 and SNRPN within a given group of samples. The mean frequency and standard deviation for each locus in each group of samples are also shown (last bar in each frame).

Figure 4

Mean frequency values for SD, SS and DD cells for RB1 and SNRPN in control and patient samples. Solid bars present the control sample (cases C1–C15 for RB1 and C16–C25 for SNRPN); striped bars present the sample of patients with DGS/VCFS (cases V1–V10 for RB1, and V1, V5–V7, V9 and V11–V15 for SNRPN); and dotted bars present the sample of patients with Williams syndrome (cases W1–W10).

Figure 5

Frequency of SD cells for RB1 in BrdU-labeled and unlabeled cell populations. Control individuals (cases C1–C8), a patient with DGS/VCFS (case V1) and a patient with Williams syndrome (case W1). Each sample is shown with two SD values for RB1, one in an unlabeled cell population (open bars) and the second in a BrdU labeled population (solid bars).

Figure 6

Frequency of SD cells for ARSA in samples from control individuals and from patients with DGS/VCFS. In frame (c) the frequency of SD cells in each individual sample was separated into two sub-categories: cells in which the ARSA signal of the deletion-carrying homologue shows a singlet (S-signal, open bar) and those in which the ARSA signal of the deletion-carrying chromosome reveals a doublet (D-signal, solid bar). The mean frequencies and standard deviations for each group of samples are also given (last bar(s) in each frame). The P value in frame (a) represents the significance of the difference between the values in frames (a) and (b); the P value in frame (c) shows the significance of the difference between the S and D values for the ARSA signal of the deletion-carrying homologue within the total ARSA SD cell population.