Genome-wide approach to identify second gene targets for malignant rhabdoid tumors using high-density oligonucleotide microarrays.
Bottom Line: High-resolution analysis also disclosed the recurrent hemizygous/homozygous deletions of 7q35-q36.1, involving the CNTNAP2 locus in three specimens.Mutations analysis of CNTNAP2 showed a novel R157C missense mutation in a primary case, and methylation analysis showed recurrent hypermethylation of CNTNAP2 in three of nine cell lines.These results demonstrated that CNTNAP2 is one of the additional gene targets, other than SMARCB1, in MRT.
Affiliation: Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.Show MeSH
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Mentions: The SNP-chip analysis was carried out for 21 MRT specimens, including 12 fresh tumors and 9 cell lines, using Affymetrix GeneChip 50K XbaI/HindIII and/or 250K NspI/StyI mapping arrays (Table S1). Although many specimens had no matched control DNA and suffered from varying degrees of normal cell contamination, the allelic compositions were accurately determined in most specimens using our CNAG/AsCNAR programs (Fig.1a). In our SNP-chip analysis, the most frequent copy number change detected in MRT was deletion of chromosome 22q11.2 (Fig.1a,b). In total, 20 of 21 specimens (95.2%) had LOH or homozygous deletions at 22q11.2 involving the SMARCB1 locus (Fig.1a,b). In eight samples, uniparental disomy (UPD) of 22q segments caused homozygous mutations/deletions of SMARCB1 (Fig.1b). Ten samples had homozygous focal deletions commonly involving a 175-kb region (ch22:22,353,181-22,528,353), which exclusively included SMARCB1. Subsequent mutation analysis revealed that five samples with heterozygous deletion or UPD at the 22q11.2 locus had mutations in SMARCB1 (Table1). An MRT-derived cell line with 22qUPD (YAMRT) harbored a small deletion involving exons 1–3, which was not detectable by SNP-chip analysis. In our cohort, two specimens showed hemizygous deletion at the SMARCB1 locus, and one case showed no genetic changes within this locus. Immunohistochemical analyses of these three cases showed positive results for vimentin but negative findings for muscle lineage markers and SMARCB1, supporting the diagnosis of MRT or AT/RT. Thus, to investigate whether abnormal methylation is associated with inactivation of SMARCB1, bisulfate sequencing for the promoter region of SMARCB1 was carried out in these three cases. As shown in Figure1(c), two cases having hemizygous deletions at the SMARCB1 locus displayed complete methylation of the CpG island. However, one case without any genetic abnormality of the SMARCB1 locus lacked PCR products (both methylated and unmethylated) for the promoter region (Fig.1c), suggesting that this case may harbor a small deletion involving the promoter region of SMARCB1, which escaped SNP array detection. In total, 20 of the 21 MRT samples had biallelic aberrations of SMARCB1, indicating genetic homogeneity of MRT. Molecular allelokaryotyping profiles were essentially similar between cell lines and primary tumors, providing some rationale for the combined analysis of both specimens in this study (Fig.1b).
Affiliation: Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.