Recurrent RECQL4 imbalance and increased gene expression levels are associated with structural chromosomal instability in sporadic osteosarcoma

Abstract

Osteosarcoma (OS) is an aggressive bone tumor with complex abnormal karyotypes and a highly unstable genome, exhibiting both numerical- and structural-chromosomal instability (N- and S-CIN). Chromosomal rearrangements and genomic imbalances affecting 8q24 are frequent in OS. RECQL4 gene maps to this cytoband and encodes a putative helicase involved in the fidelity of DNA replication and repair. This protective genomic function of the protein is relevant because often patients with Rothmund-Thomson syndrome have constitutional mutations of RECQL4 and carry a very high risk of developing OS. To determine the relative level of expression of RECQL4 in OS, 18 sporadic tumors were studied by reverse transcription-polymerase chain reaction. All tumors overexpressed RECQL4 in comparison to control osteoblasts, and fluorescence in situ hybridization analysis of tumor DNA showed that expression levels were strongly copy number-dependent. Relative N- and S-CIN levels were determined by classifying copy number transitions within array comparative genomic hybridization profiles and by enumerating the frequency of break-apart fluorescence in situ hybridization within 8q24 using region-specific and control probes. Although there was no evidence that disruption of 8q24 in OS led to an elevated expression of RECQL4, there was a marked association between increased overall levels of S-CIN, determined by copy number transition frequency and higher levels of RECQL4.

Figure 1

RECQL4 is overexpressed in OS and is associated with S-CIN. The histogram is showing the fold change of RECQL4 mRNA compared with normal osteoblasts (NO) measured by semiquantitative RT-PCR. Rothmund-Thomson fibroblasts (RTS) and normal tissues (kidney and testis) were used as negative and positive controls, respectively. Each value is the result of three independent experiments. The SD is shown above each bar. The dashed line represents the mean value calculated from OS sample data. The lower panel part describes the chromosomal 8 instability (CIN) in the context of overall ploidy for 10 samples for which material was available for FISH studies. The absence of N-CIN or S-CIN is shown as (-). Identification of S-CIN or N-CIN is shown as (+). The overall ploidy status was established according to the FISH data using cen(3), cen(7), and cen(17). OS tumors that are showing an RECQL4 expression value close or more than the mean are showing S-CIN, whereas the ones less than the mean do not. The N-CIN and S-CIN are independent, and a high level of RECQL4 expression is associated with the appearance of S-CIN on the 8q24 region.

Figure 2

Association between RECQL4 expression levels and varying N-CIN and S-CIN frequencies. (A) Differing frequencies of N-CIN (y-axis) as determined by analyzing imbalance profiles of whole chromosomes or entire arms for the 10 OS tumors (x-axis) after aCGH analysis. (B) Differing frequencies of S-CIN (y-axis) as determined by analyzing CNT distributions across each genomic profile derived from the ten OS tumors (x-axis) after aCGH analysis. The OS samples are ordered from left to right according to their RECQL4 expression level (lowest to highest) and are represented as a triangle in which the adjacent side represents the highest RECQL4 expression level.

Figure 3

(A–F) RECQL4/cen(8) dual-color interphase FISH experiment on OS tumors samples' fixed sections. RECQL4 probe is shown in red, whereas the cen(8) is in green. OS samples exhibited a normal pattern [OS1 (A)], a loss [OS2 (B)], an abnormal pattern associated with a 1:1 ratio [OS7 (C) and OS9 (D)], or a gain of RECQL4 [OS15 (E) and OS13 (F)]. (G–J) Structural CIN of the 8q24 cytoband. Dual-color interphase FISH was done on fixed tissue sections using the MYC probe (red) located on 8q24.21 and the RP11-349C2 probe (green) located on 8q24.3. The signal pattern of contiguous red and green signals on OS7 (G) and OS1 (H) identifies these samples as no S-CIN for the 8q24 cytoband. In contrast, for OS13 (I) and OS15 (J), the red and green signals are scattered all around the nuclei, characterizing these two samples as S-CIN for the 8q24 region.