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Genome-wide approach to identify second gene targets for malignant rhabdoid tumors using high-density oligonucleotide microarrays.

Takita J, Chen Y, Kato M, Ohki K, Sato Y, Ohta S, Sugita K, Nishimura R, Hoshino N, Seki M, Sanada M, Oka A, Hayashi Y, Ogawa S - Cancer Sci. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

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Recurrent deletions of chromosome 7q35–q36 and CNTNAP2 alterations in malignant rhabdoid tumor (MRT). (a) Deletions of chromosome 7q35–q36 in three specimens detected by single nucleotide polymorphism (SNP) array. For each panel, total copy numbers (tCNs; red dots), moving averages of tCNs for five consecutive SNPs (blue line), an ideogram of the relevant chromosome, location of heterozygous SNP calls (green bars), and allele-specific copy numbers (AsCNs) averaged for five consecutive SNPs (red and green lines for larger and smaller alleles, respectively) are plotted. (b) Expression and mutation analyses of MRT. Upper panel shows RT-PCR analysis of CNTNAP2 in nine cell lines. Sequence chromatogram of R157C missense mutation detected in a fresh tumor, MRT-8, is shown in the lower panel. (c) Bisulfate modification- and methylation-specific PCR for CNTNAP2 in cell lines. Hypermethylation of CpG islands in KYM-1, TTC-549, and RTK(J)-4N cell lines is shown in the upper panel. The lower panel shows control. CpG islands are marked by asterisks. (d) Representative results of re-expression of transcriptionally silenced CNTNAP2 after treatment with 5-aza-deoxycytidine in MRT cell lines. Reverse transcription-PCR analysis of KYM-1 cell line harvested following 72 h of incubation with control media (−) and 5 μM 5-aza-2-deoxycytidine (+). SJNB-1 neuroblastoma cell line, which expressed abundant CNTNAP2, was used as a positive control.
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fig02: Recurrent deletions of chromosome 7q35–q36 and CNTNAP2 alterations in malignant rhabdoid tumor (MRT). (a) Deletions of chromosome 7q35–q36 in three specimens detected by single nucleotide polymorphism (SNP) array. For each panel, total copy numbers (tCNs; red dots), moving averages of tCNs for five consecutive SNPs (blue line), an ideogram of the relevant chromosome, location of heterozygous SNP calls (green bars), and allele-specific copy numbers (AsCNs) averaged for five consecutive SNPs (red and green lines for larger and smaller alleles, respectively) are plotted. (b) Expression and mutation analyses of MRT. Upper panel shows RT-PCR analysis of CNTNAP2 in nine cell lines. Sequence chromatogram of R157C missense mutation detected in a fresh tumor, MRT-8, is shown in the lower panel. (c) Bisulfate modification- and methylation-specific PCR for CNTNAP2 in cell lines. Hypermethylation of CpG islands in KYM-1, TTC-549, and RTK(J)-4N cell lines is shown in the upper panel. The lower panel shows control. CpG islands are marked by asterisks. (d) Representative results of re-expression of transcriptionally silenced CNTNAP2 after treatment with 5-aza-deoxycytidine in MRT cell lines. Reverse transcription-PCR analysis of KYM-1 cell line harvested following 72 h of incubation with control media (−) and 5 μM 5-aza-2-deoxycytidine (+). SJNB-1 neuroblastoma cell line, which expressed abundant CNTNAP2, was used as a positive control.

Mentions: Although recurrent copy number changes other than 22q11.2 deletions were less frequent in MRT (Fig.1a), seven loci of gains and eight loci of losses were commonly detected in multiple samples (Table2). Importantly, some of the regions contained potential gene targets that were known to be associated with tumorigenesis of other cancers, such as CCNL1, POT1, CNTNAP2, and PRPTD (Table2).18,21,22 Detection of homozygous deletions was also of interest because they provide an important clue to pinpoint tumor suppressor loci. In fact, SMARCB1 was identified from homozygously deleted regions at 22q11.2.4 Of note, we found a homozygous deletion of the CNTNAP2 locus at 7q35–q36 in one specimen (STM-91-01), and another two cases (TTC-549 and MRT-7) showed a hemizygous deletion in this region (Fig.2a). The commonly deleted region of the CNTNAP2 locus was expanded in a 534-kb region (ch7:146,183,963-146,718-030) at 7q35–q36, and a homozygous deletion was involved in only exons 9–10 of CNTNAP2. Unfortunately, we were not able to completely exclude the possibility that this may represent copy number variations (CNV) rather than real homozygous/hemizygous deletions, because these homozygous deletions were found in established MRT cell lines. However, in our SNP array database, we did not find any CNV at the CNTNAP2 locus in 100 normal samples, but hemizygous/homozygous deletions were observed in two neuroblastoma cell lines (NB-16 and NB-19) (data not shown), suggesting that this deletions would be somatic events rather than CNV. Complete or incomplete losses of genetic materials at seven loci were confirmed by quantitative genomic PCR (Fig. S1).


Genome-wide approach to identify second gene targets for malignant rhabdoid tumors using high-density oligonucleotide microarrays.

Takita J, Chen Y, Kato M, Ohki K, Sato Y, Ohta S, Sugita K, Nishimura R, Hoshino N, Seki M, Sanada M, Oka A, Hayashi Y, Ogawa S - Cancer Sci. (2014)

Recurrent deletions of chromosome 7q35–q36 and CNTNAP2 alterations in malignant rhabdoid tumor (MRT). (a) Deletions of chromosome 7q35–q36 in three specimens detected by single nucleotide polymorphism (SNP) array. For each panel, total copy numbers (tCNs; red dots), moving averages of tCNs for five consecutive SNPs (blue line), an ideogram of the relevant chromosome, location of heterozygous SNP calls (green bars), and allele-specific copy numbers (AsCNs) averaged for five consecutive SNPs (red and green lines for larger and smaller alleles, respectively) are plotted. (b) Expression and mutation analyses of MRT. Upper panel shows RT-PCR analysis of CNTNAP2 in nine cell lines. Sequence chromatogram of R157C missense mutation detected in a fresh tumor, MRT-8, is shown in the lower panel. (c) Bisulfate modification- and methylation-specific PCR for CNTNAP2 in cell lines. Hypermethylation of CpG islands in KYM-1, TTC-549, and RTK(J)-4N cell lines is shown in the upper panel. The lower panel shows control. CpG islands are marked by asterisks. (d) Representative results of re-expression of transcriptionally silenced CNTNAP2 after treatment with 5-aza-deoxycytidine in MRT cell lines. Reverse transcription-PCR analysis of KYM-1 cell line harvested following 72 h of incubation with control media (−) and 5 μM 5-aza-2-deoxycytidine (+). SJNB-1 neuroblastoma cell line, which expressed abundant CNTNAP2, was used as a positive control.
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Related In: Results  -  Collection

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fig02: Recurrent deletions of chromosome 7q35–q36 and CNTNAP2 alterations in malignant rhabdoid tumor (MRT). (a) Deletions of chromosome 7q35–q36 in three specimens detected by single nucleotide polymorphism (SNP) array. For each panel, total copy numbers (tCNs; red dots), moving averages of tCNs for five consecutive SNPs (blue line), an ideogram of the relevant chromosome, location of heterozygous SNP calls (green bars), and allele-specific copy numbers (AsCNs) averaged for five consecutive SNPs (red and green lines for larger and smaller alleles, respectively) are plotted. (b) Expression and mutation analyses of MRT. Upper panel shows RT-PCR analysis of CNTNAP2 in nine cell lines. Sequence chromatogram of R157C missense mutation detected in a fresh tumor, MRT-8, is shown in the lower panel. (c) Bisulfate modification- and methylation-specific PCR for CNTNAP2 in cell lines. Hypermethylation of CpG islands in KYM-1, TTC-549, and RTK(J)-4N cell lines is shown in the upper panel. The lower panel shows control. CpG islands are marked by asterisks. (d) Representative results of re-expression of transcriptionally silenced CNTNAP2 after treatment with 5-aza-deoxycytidine in MRT cell lines. Reverse transcription-PCR analysis of KYM-1 cell line harvested following 72 h of incubation with control media (−) and 5 μM 5-aza-2-deoxycytidine (+). SJNB-1 neuroblastoma cell line, which expressed abundant CNTNAP2, was used as a positive control.
Mentions: Although recurrent copy number changes other than 22q11.2 deletions were less frequent in MRT (Fig.1a), seven loci of gains and eight loci of losses were commonly detected in multiple samples (Table2). Importantly, some of the regions contained potential gene targets that were known to be associated with tumorigenesis of other cancers, such as CCNL1, POT1, CNTNAP2, and PRPTD (Table2).18,21,22 Detection of homozygous deletions was also of interest because they provide an important clue to pinpoint tumor suppressor loci. In fact, SMARCB1 was identified from homozygously deleted regions at 22q11.2.4 Of note, we found a homozygous deletion of the CNTNAP2 locus at 7q35–q36 in one specimen (STM-91-01), and another two cases (TTC-549 and MRT-7) showed a hemizygous deletion in this region (Fig.2a). The commonly deleted region of the CNTNAP2 locus was expanded in a 534-kb region (ch7:146,183,963-146,718-030) at 7q35–q36, and a homozygous deletion was involved in only exons 9–10 of CNTNAP2. Unfortunately, we were not able to completely exclude the possibility that this may represent copy number variations (CNV) rather than real homozygous/hemizygous deletions, because these homozygous deletions were found in established MRT cell lines. However, in our SNP array database, we did not find any CNV at the CNTNAP2 locus in 100 normal samples, but hemizygous/homozygous deletions were observed in two neuroblastoma cell lines (NB-16 and NB-19) (data not shown), suggesting that this deletions would be somatic events rather than CNV. Complete or incomplete losses of genetic materials at seven loci were confirmed by quantitative genomic PCR (Fig. S1).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus