Molecular karyotyping and gene expression analysis in childhood cancer patients

Abstract

The genetic etiology of sporadic childhood cancer cases remains unclear. We recruited a cohort of 20 patients who survived a childhood malignancy and then developed a second primary cancer (2N), and 20 carefully matched patients who survived a childhood cancer without developing a second malignancy (1N). Twenty matched cancer-free (0N) and additional 1000 (0N) GHS participants served as controls. Aiming to identify new candidate loci for cancer predisposition, we compared the genome-wide DNA copy number variations (CNV) with the RNA-expression data obtained after in vitro irradiation of primary fibroblasts. In 2N patients, we detected a total of 142 genes affected by CNV. A total of 53 genes of these were not altered in controls. Six genes (POLR3F, SEC23B, ZNF133, C16orf45, RRN3, and NTAN1) that we found to be overexpressed after irradiation were also duplicated in the genome of the 2N patients. For the 1N collective, 185 genes were affected by CNV and 38 of these genes were not altered in controls. Five genes (ZCWPW2, SYNCRIP, DHX30, DHRS4L2, and THSD1) were located in duplicated genomic regions and exhibited altered RNA expression after irradiation. One gene (ABCC6) was partially duplicated in one 1N and one 2N patient. Analysis of methylation levels of THSD1 and GSTT2 genes which were detected in duplicated regions and are frequently aberrantly methylated in cancer showed no changes in patient's fibroblasts. In summary, we describe rare and radiation-sensitive genes affected by CNV in childhood sporadic cancer cases, which may have an impact on cancer development. KEY MESSAGES: • Rare CNV's may have an impact on cancer development in sporadic, non-familial, non-syndromic childhood cancer cases. • In our cohort, each patient displayed a unique pattern of cancer-related gene CNVs, and only few cases shared similar CNV. • Genes that are transcriptionally regulated after radiation can be located in CNVs in cancer patients and controls. • THSD1 and GSTT2 methylation is not altered by CNV.

Keywords: Childhood cancer; Copy number variation; Gene expression; Primary secondary cancer; Radiation.

Fig. 1

FISH analysis of duplications in 16p13.11 (2N4) and 2q11.1 (2N7). Possible tandem duplication in one 2N cancer patient (with brighter signals than on corresponding chromosomes indicated with white arrow) (a). Copy number qPCR analysis may indicate duplications in a mosaic state (b)

Fig. 2

SNP array copy number analysis (a) and verification using a qPCR approach (b). Case 2N12 shows a deletion in chromosome 2q32.1 and case 2N9 shows a duplication in 19q13.41. qPCR analyses with specific primers show a heterozygous/homozygous deletion

Fig. 3

QPCR experiments in three independent 0N cell lines. The analysis of ten genes was performed with irradiated (2, 5, 8 Gy) and control (0 Gy) primary fibroblast cell lines at 15 min, 2 h, and 24 h post-treatment. Significant differences in expression are indicated by asterisk

Fig. 4

Analysis of the duplication and gene expression of the THSD1 gene. The scheme shows duplication in 13q14.3 including the THSD1 gene (a). qPCR copy number analysis of this region reveals a heterozygous duplication of the DNA fragment in 1N13 (b). RNA expression analysis using qPCR in index patient 1N13 and three independent 2N and 1N cell lines. The analysis was performed with irradiated (2, 5, 8 Gy) and control (0 Gy) cells at 2 h and 24 h post-treatment (c)

Fig. 5

Detail analysis of a deletion in Chr.15q14 of two matched patients. Genomic deletion in 15q14, including the GOLGAG8A gene (a). RNA expression analysis using qPCR in 1N8, 2N8, and corresponding 0N11 patients. The analysis was performed with irradiated (2, 5 Gy) and control (0 Gy) cells at 2 h post-treatment (b). qPCR copy number analysis of this region reveals heterozygous deletions of the DNA fragment in a heterozygous state (c)

Fig. 6

Methylation analysis of THSD1 and GSTT2 genes. Depicted are the mean methylation values of six CpGs for GSTT2 and ten CpGs for THSD1. The tumor cell lines ZR-75-1 and T47D display hypermethylation of THSD1 in contrast to 0N20, 2N19, 1N13, and other samples (a). The A549, MCF7, and EFO27 tumor cell lines show hypermethylation of GSTT2 compared with 0N7, 2N7, and the remaining samples (b)