3D-FISH analysis reveals chromatid cohesion defect during interphase in Roberts syndrome

Abstract

Background: Roberts syndrome (RBS) is a rare autosomal recessive disorder mainly characterized by growth retardation, limb defects and craniofacial anomalies. Characteristic cytogenetic findings are "railroad track" appearance of chromatids and premature centromere separation in metaphase spreads. Mutations in the ESCO2 (establishment of cohesion 1 homolog 2) gene located in 8p21.1 have been found in several families. ESCO2, a member of the cohesion establishing complex, has a role in the effective cohesion between sister chromatids. In order to analyze sister chromatids topography during interphase, we performed 3D-FISH using pericentromeric heterochromatin probes of chromosomes 1, 4, 9 and 16, on preserved nuclei from a fetus with RBS carrying compound heterozygous null mutations in the ESCO2 gene.

Results: Along with the first observation of an abnormal separation between sister chromatids in heterochromatic regions, we observed a statistically significant change in the intranuclear localization of pericentromeric heterochromatin of chromosome 1 in cells of the fetus compared to normal cells, demonstrating for the first time a modification in the spatial arrangement of chromosome domains during interphase.

Conclusion: We hypothesize that the disorganization of nuclear architecture may result in multiple gene deregulations, either through disruption of DNA cis interaction -such as modification of chromatin loop formation and gene insulation - mediated by cohesin complex, or by relocation of chromosome territories. These changes may modify interactions between the chromatin and the proteins associated with the inner nuclear membrane or the pore complexes. This model offers a link between the molecular defect in cohesion and the complex phenotypic anomalies observed in RBS.

Keywords: Cohesinopathy; ESCO2; Heterochromatin; Limb development.

Figure 1

Clinical and cytogenetical description of the fetus. (A): Fetus of 18 weeks gestation with multiple congenital anomalies: hypertelorism, micrognathia, tetraphocomelia and oligodactyly. Scale bar = 2 cm (B): C-banded metaphase chromosomes from the affected fetus showing the pathognomonic cytogenetic anomaly in Roberts Syndrome: chromosomes with premature centromere separation: PCS (black arrows) and heterochromatin puffing. Scale bar = 5 μm.

Figure 2

3D FISH of Roberts sub-chromosomal domain territories. Views of Imaris® reconstructions of trophoblasts and fibroblasts nuclei after three-dimensional FISH with probes for PH1 (Chr1), centromeric régions of chromosome 4 (Chr4), 9 (Chr9) and 16 (Chr16). Cells were counterstained with DAPI (blue). (A) Control cells hybridized with PH1 probe (trophoblasts: red probe and a fibroblast: green probe). (B) RBS cells with split signal of one or two territories (chr 1, chr 4, chr 9 and chr 16). (C) Focus on split spots of PH1 showing the bridge between the two signals (arrows).

Figure 3

Statistical representation of CB2 radial and mutual positions. (A): Distribution of each PH1 (=CB2 probe) radial position of normal cells (black rings) and Roberts cells (blue triangles). The median was calculated both for normal cells (M^rn, black line) and Roberts cells (M^rR, blue line). Radial distances are expressed as a proportion of the radius. M^rn = 0.5285 and M^rR = 0.6700: Wilcoxon test was significant (alpha = 0.05) with a p value of 0.0018 showing a significant relocation of PH1 territory towards the edge of the nucleus in Roberts cells. (B): Distribution of CB2 mutual position of normal and Roberts cells. The median was calculated both for normal cells (M^mn,black line) and Roberts cells (M^mR, blue line). Mutual distances are expressed as a proportion of the diameter. M^mn = 0.4527 and M^mR = 0.3813: Wilcoxon test was significant (alpha = 0.05) with a p value of 0.0829 showing a significant rapprochement of homologous PH1 territories in Roberts cells.