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Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma.

Honda S, Arai Y, Haruta M, Sasaki F, Ohira M, Yamaoka H, Horie H, Nakagawara A, Hiyama E, Todo S, Kaneko Y - Br. J. Cancer (2008)

Bottom Line: Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers.In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB.Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Diagnosis, Saitama Cancer Center, Research Institute for Clinical Oncology, 818 Komuro, Ina, Saitama 362-0806, Japan.

ABSTRACT
IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT-PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.

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(A) Diagram of the IGF2 P3 promoter region. Individual CpG dinucleotides located upstream of exon 6 (from −1068 to −702 bp) are represented by circles. Horizontal arrows indicate locations of PCR primers used for MSP and bisulphite sequencing. (B) Examples of the promoter methylation status using methylation-specific PCR. Polymerase chain reaction products of methylated or unmethylated P3 promoters from HB tumours are shown. Numbers above horizontal bars indicate the tumour number. M, methylated promoter; U, unmethylated promoter. (C) Bisulphite sequencing analysis of the methylation status of P3 promoter in HuH6 and one tumour (no. 1), which displayed complete methylation and complete unmethylation, respectively. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. (D) Levels of P3 transcripts in tumours with partially methylated P3 promoter and tumours with unmethylated P3 promoter.
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fig4: (A) Diagram of the IGF2 P3 promoter region. Individual CpG dinucleotides located upstream of exon 6 (from −1068 to −702 bp) are represented by circles. Horizontal arrows indicate locations of PCR primers used for MSP and bisulphite sequencing. (B) Examples of the promoter methylation status using methylation-specific PCR. Polymerase chain reaction products of methylated or unmethylated P3 promoters from HB tumours are shown. Numbers above horizontal bars indicate the tumour number. M, methylated promoter; U, unmethylated promoter. (C) Bisulphite sequencing analysis of the methylation status of P3 promoter in HuH6 and one tumour (no. 1), which displayed complete methylation and complete unmethylation, respectively. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. (D) Levels of P3 transcripts in tumours with partially methylated P3 promoter and tumours with unmethylated P3 promoter.

Mentions: In the MSP analysis of each promoter, the P2 promoter region was partially methylated in 19 tumours and normal liver tissues and the P4 promoter region was unmethylated in all 20 tumours and normal liver tissues. Therefore, the methylation status of P2 or P4 promoter region was not correlated with the expression level of P2- or P4-specific transcripts. The P3 promoter region was partially methylated in 11 tumours, HuH6 and normal liver tissues and unmethylated in 9 tumours and HepG2 (Table 1, Figure 4A and B). The results of MSP analysis in one tumour (no. 1) and HuH6 were confirmed by bisulphite sequencing (Figure 4C). Nine tumours with the unmethylated P3 promoter had higher levels of P3 transcripts than 11 tumours with the partially methylated P3 promoter (P=0.005) (Figure 4D). The P3 promoter was unmethylated in 5 of 7 tumours with IGF2 alterations; UPD, 11p15 loss or LOI, but in 4 of 13 tumours with ROI. Thus, the incidence of tumours with unmethylated P3 promoter tended to be higher in tumours with hypermethylated H19 DMR than in tumours with normally methylated H19 DMR (P=0.1).


Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma.

Honda S, Arai Y, Haruta M, Sasaki F, Ohira M, Yamaoka H, Horie H, Nakagawara A, Hiyama E, Todo S, Kaneko Y - Br. J. Cancer (2008)

(A) Diagram of the IGF2 P3 promoter region. Individual CpG dinucleotides located upstream of exon 6 (from −1068 to −702 bp) are represented by circles. Horizontal arrows indicate locations of PCR primers used for MSP and bisulphite sequencing. (B) Examples of the promoter methylation status using methylation-specific PCR. Polymerase chain reaction products of methylated or unmethylated P3 promoters from HB tumours are shown. Numbers above horizontal bars indicate the tumour number. M, methylated promoter; U, unmethylated promoter. (C) Bisulphite sequencing analysis of the methylation status of P3 promoter in HuH6 and one tumour (no. 1), which displayed complete methylation and complete unmethylation, respectively. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. (D) Levels of P3 transcripts in tumours with partially methylated P3 promoter and tumours with unmethylated P3 promoter.
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getmorefigures.php?uid=PMC2600691&req=5

fig4: (A) Diagram of the IGF2 P3 promoter region. Individual CpG dinucleotides located upstream of exon 6 (from −1068 to −702 bp) are represented by circles. Horizontal arrows indicate locations of PCR primers used for MSP and bisulphite sequencing. (B) Examples of the promoter methylation status using methylation-specific PCR. Polymerase chain reaction products of methylated or unmethylated P3 promoters from HB tumours are shown. Numbers above horizontal bars indicate the tumour number. M, methylated promoter; U, unmethylated promoter. (C) Bisulphite sequencing analysis of the methylation status of P3 promoter in HuH6 and one tumour (no. 1), which displayed complete methylation and complete unmethylation, respectively. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. (D) Levels of P3 transcripts in tumours with partially methylated P3 promoter and tumours with unmethylated P3 promoter.
Mentions: In the MSP analysis of each promoter, the P2 promoter region was partially methylated in 19 tumours and normal liver tissues and the P4 promoter region was unmethylated in all 20 tumours and normal liver tissues. Therefore, the methylation status of P2 or P4 promoter region was not correlated with the expression level of P2- or P4-specific transcripts. The P3 promoter region was partially methylated in 11 tumours, HuH6 and normal liver tissues and unmethylated in 9 tumours and HepG2 (Table 1, Figure 4A and B). The results of MSP analysis in one tumour (no. 1) and HuH6 were confirmed by bisulphite sequencing (Figure 4C). Nine tumours with the unmethylated P3 promoter had higher levels of P3 transcripts than 11 tumours with the partially methylated P3 promoter (P=0.005) (Figure 4D). The P3 promoter was unmethylated in 5 of 7 tumours with IGF2 alterations; UPD, 11p15 loss or LOI, but in 4 of 13 tumours with ROI. Thus, the incidence of tumours with unmethylated P3 promoter tended to be higher in tumours with hypermethylated H19 DMR than in tumours with normally methylated H19 DMR (P=0.1).

Bottom Line: Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers.In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB.Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Diagnosis, Saitama Cancer Center, Research Institute for Clinical Oncology, 818 Komuro, Ina, Saitama 362-0806, Japan.

ABSTRACT
IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT-PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.

Show MeSH
Related in: MedlinePlus