Germline copy number variation of genes involved in chromatin remodelling in families suggestive of Li-Fraumeni syndrome with brain tumours

Abstract

Germline alterations of the tumour suppressor TP53 gene are detected approximately in 25% of the families suggestive of Li-Fraumeni syndrome (LFS), characterised by a genetic predisposition to a wide tumour spectrum, including soft-tissue sarcomas, osteosarcomas, premenopausal breast cancers, brain tumours, adrenocortical tumours, plexus choroid tumours, leukaemia and lung cancer. The aim of this study was to determine the contribution of germline copy number variations (CNVs) to LFS in families without detectable TP53 mutation. Using a custom-designed high-resolution array CGH, we evaluated the presence of rare germline CNVs in 64 patients fulfilling the Chompret criteria for LFS, but without any detectable TP53 alteration. In 15 unrelated patients, we detected 20 new CNVs absent in 600 controls. Remarkably, in four patients who had developed each brain tumour, the detected CNV overlap the KDM1A, MTA3, TRRAP or SIRT3 genes encoding p53 partners involved in histone methylation or acetylation. Focused analysis of SIRT3 showed that the CNV encompassing SIRT3 leads to SIRT3 overexpression, and that in vitro SIRT3 overexpression prevents apoptosis, increases G2/M and results in a hypermethylation of numerous genes. This study supports the causal role of germline alterations of genes involved in chromatin remodelling in genetic predisposition to cancer and, in particular, to brain tumours.

Figure 1

Detection in LFS patients of CNVs targeting the TP53 locus, using a custom-designed 180K array CGH. Detection in three LFS patients, using custom-designed 180K array CGH of a complete deletion (a), a partial deletion removing the promoter region and exon 1 (b) and a duplication of exons 2–4 (c). (d) Probe coverage of the TP53 locus in the human catalogue 180K array CGH. CNVs detected by the ADM-2 algorithm are indicated by shaded area (green for deletion; red for duplication). Sizes of rearrangements are represented by double-headed arrows. Mapping of the corresponding genomic regions with the name of the genes (ATP1B2, TP53, WRAP53) are indicated on left.

Figure 2

Germline CNVs encompassing the KDM1A, MTA3, TRRAP or SIRT3 genes involved in chromatin remodelling and detected in four unrelated patients suggestive of LFS. Each panel (a–d) presents the detection of CNV by array CGH; the validation of the detected CNV by QMPSF (fluorescence profile obtained from the patient (red) was superimposed to that obtained from a control (blue) and adjusted using the height of a control amplicon); the partial pedigree of the family in whom the CNV was detected (analysed genotypes are indicated). (a) A 318-kb deletion partially removing the KDM1A gene (exons 3–19) in patient ID 53. (b) A 172-kb duplication of the MTA3 gene (promoter and exons 1–9) in patient ID 33. (c) A 169-kb duplication covering the TRRAP gene in patient ID 63. (d) A 133-kb duplication including the SIRT3 gene in patient ID 17.

Figure 3

SIRT3 overexpression prevents apoptosis and increases G2/M phase in the 8MG glioma-derived cell line. (a) Apoptosis analysis using TMRM analysis. (b) Cell cycle analysis by flow cytometer. The arrows indicate the increase of apoptotic cell number and of G2/M phase, resulting from SIRT3 knockdown or overexpression, respectively. Abbreviations: 8MG, parental 8MG cell line; SIRT3-8MG, SIRT3-overexpressing 8MG cells; 8MG+SIRT3 siRNA, parental 8MG cell line transfected with siRNA-targeting SIRT3; SIRT3-8MG+SIRT3 siRNA, SIRT3-overexpressing 8MG cells transfected with siRNA-targeting SIRT3.

Figure 4

SIRT3 overexpression induces hypermethylation in the 8MG glioma-derived cell line. Methylation profiles of bisulphite-treated and -untreated samples from SIRT3-overexpressing cells (SIRT3) and GFP-overexpressing cells (Control) were analysed using the 450K Infinium methylation bead arrays (see Subjects and methods). The figure presents the heatmap of significant probes using beta values; the scale indicates the level of methylation, from 0 (no methylation, green) to 1 (100% methylation, red). (a) CpG sites hypermethylated in SIRT3-overexpressing cells. (b) CpG sites hypomethylated in SIRT3-overexpressing cells. (c) Distribution of CpG sites hypermethylated in SIRT3-overexpressing cells. (d) Distribution of CpG sites hypomethylated in SIRT3-overexpressing cells.