Chromosomal instability at common fragile sites in Seckel syndrome

Abstract

Seckel syndrome (SCKL) is a rare, genetically heterogeneous disorder, with dysmorphic facial appearance, growth retardation, microcephaly, mental retardation, variable chromosomal instability, and hematological disorders. To date, three loci have been linked to this syndrome, and recently, the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) was identified as the gene mutated at the SCKL1 locus. The ATR mutation affects splicing efficiency, resulting in low levels of ATR in affected individuals. Elsewhere, we reported increased instability at common chromosomal fragile sites in cells lacking the replication checkpoint gene ATR. Here, we tested whether cells from patients carrying the SCKL1 mutation would show increased chromosome breakage following replication stress. We found that, compared with controls, there is greater chromosomal instability, particularly at fragile sites, in SCKL1-affected patient cells after treatment with aphidicolin, an inhibitor of DNA polymerase alpha and other polymerases. The difference in chromosomal instability between control and patient cells increases at higher levels of aphidicolin treatment, suggesting that the low level of ATR present in these patients is not sufficient to respond appropriately to replication stress. This is the first human genetic syndrome associated with increased chromosome instability at fragile sites following replication stress, and these findings may be related to the phenotypic findings in patients with SCKL1.

Figure 1

Pedigrees of family with SCKL and western blot showing reduced ATR protein levels in affected patients. A and B, Pedigrees of the two Pakistani families with SCKL (SCKL1) included in this study. Identification numbers of the lymphoblast cell lines derived from these family members are noted below their respective symbols. C, Western blot of lymphoblast cell lines from these family members and two unrelated control lymphoblast lines (LB-5 and LB-12) probed with α-ATR. The blot was stripped and reprobed with α-tubulin to indicate relative protein-loading levels.

Figure 2

Metaphase showing an example of FISH for identification of fragile sites. This representative metaphase is from lymphoblast cell line DK0061, after treatment with 0.4 μM aphidicolin. FRA3B is indicated by hybridization with YAC probe 850A6 (green); both homologs are broken. FRA16D is indicated by hybridization with BAC probe 264L1 (red); both homologs are broken.

Figure 3

Cells from patients with SCKL1 have increased fragile-site expression. A, Average total chromosome gaps and breaks. n=50 metaphases from each of two replicates for each condition. Fragile-site induction was achieved by addition of 0.4 μM aphidicolin 48 h before harvest. Error bars indicate the 95% CI. B and C, Frequency of FRA3B (B) and FRA16D (C) expression in cell lines from patients with SCKL1 and control individual; n⩾100 hybridizations from each of two replicates for each condition. Fragile-site induction was achieved by addition of 0.4 μM aphidicolin 48 h before harvest. Error bars indicate the SE.

Figure 4

The difference in fragile site breaks between patient and control cells increases at higher levels of aphidicolin treatment. Cells were treated with 0.1, 0.3, 0.5, 0.7, or 0.9 μM aphidicolin 48 h before harvest. Of DK0066 metaphases analyzed at high aphidicolin concentrations, 16%–38% had a “shattered” appearance and were not included in these calculations; thus, the figures reported are underestimates (indicated by a double asterisk [**]). A, Average total chromosome gaps and breaks. n=25 metaphases from each of two replicates for each condition. Error bars indicate the 95% CI. B and C, Frequency of FRA3B (B) and FRA16D (C) expression in cell lines from a patient with SCKL and a control individual; n⩾50 hybridizations from each of two replicates for each condition. Error bars indicate the SE. D, Example of a “shattered” metaphase in the affected cell line DK0066 after treatment with 0.5 μM aphidicolin for 48 h before harvest.