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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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Analysis of IGF2 alterations. (A) Examples of the methylation status of CTCF6 analysed by a combined bisulphite restriction assay (COBRA). Bisulphite-modified PCR products were digested with MluI. Upper and lower lanes indicate unmethylated and methylated fragments, respectively. Numbers above lanes indicate the tumour number. Numbers below lanes show the percentage of methylated DNA fragments containing CTCF6. The mean value of the DNA methylation percentages calculated from three COBRA experiments is shown in Table 1. Methyl., control methylated DNA. The IGF2 status is shown above the tumour numbers. LOI, loss of IGF2 imprinting; LOH, loss of heterozygosity in the IGF2 region; ROI, retention of IGF2 imprinting. (B) Electrophoretic pattern of genomic DNA PCR products or RT–PCR products after AvaII digestion. Reverse transcriptase–PCR analysis shows LOI in two tumours and ROI in three tumours.
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fig1: Analysis of IGF2 alterations. (A) Examples of the methylation status of CTCF6 analysed by a combined bisulphite restriction assay (COBRA). Bisulphite-modified PCR products were digested with MluI. Upper and lower lanes indicate unmethylated and methylated fragments, respectively. Numbers above lanes indicate the tumour number. Numbers below lanes show the percentage of methylated DNA fragments containing CTCF6. The mean value of the DNA methylation percentages calculated from three COBRA experiments is shown in Table 1. Methyl., control methylated DNA. The IGF2 status is shown above the tumour numbers. LOI, loss of IGF2 imprinting; LOH, loss of heterozygosity in the IGF2 region; ROI, retention of IGF2 imprinting. (B) Electrophoretic pattern of genomic DNA PCR products or RT–PCR products after AvaII digestion. Reverse transcriptase–PCR analysis shows LOI in two tumours and ROI in three tumours.

Mentions: Combined bisulphite restriction assay showed that 21 and 33 tumours had hypermethylation and normal methylation at CTCF6, indicating LOH or LOI and retention of IGF2 imprinting (ROI), respectively (Table 1 and Figure 1). Single nucleotide polymorphism array analysis was performed in 43 of 54 tumours; all 21 tumours with hypermethylated CTCF6 and 22 of 33 tumours with normally methylated CTCF6. Combined results of both analyses indicated that 12 tumours had LOH (10 hypermethylated CTCF6 and UPD 2 hypermethylated CTCF6 and hemizygous 11p15 deletion), 9 had LOI (hypermethylated CTCF6 and retention of heterozygosity (ROH)) and 22 had ROI (normally methylated CTCF6 and ROH). Of 21 tumours whose RNA was available, 9 and 12 tumours had heterozygous and homozygous ApaI/AvaII sites in exon 9 of IGF2, respectively. Of the nine heterozygous tumours, seven showed monoallelic expression of IGF2, indicating ROI, and two showed biallelic expression of IGF2, indicating LOI, and the results were consistent with those examined by COBRA and SNP array analyses (Table 1). From these findings, 11 tumours with normally methylated CTCF6, in which SNP array analysis was not performed, were classified as those with ROI. Thus, combined results of COBRA, SNP array and allelic expression analyses showed 12 tumours with LOH, 9 tumours with LOI and 33 tumours with ROI. In addition, one cell line (HuH6) had LOI, and the other (HepG2) had LOH (UPD) of IGF2.


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)

Analysis of IGF2 alterations. (A) Examples of the methylation status of CTCF6 analysed by a combined bisulphite restriction assay (COBRA). Bisulphite-modified PCR products were digested with MluI. Upper and lower lanes indicate unmethylated and methylated fragments, respectively. Numbers above lanes indicate the tumour number. Numbers below lanes show the percentage of methylated DNA fragments containing CTCF6. The mean value of the DNA methylation percentages calculated from three COBRA experiments is shown in Table 1. Methyl., control methylated DNA. The IGF2 status is shown above the tumour numbers. LOI, loss of IGF2 imprinting; LOH, loss of heterozygosity in the IGF2 region; ROI, retention of IGF2 imprinting. (B) Electrophoretic pattern of genomic DNA PCR products or RT–PCR products after AvaII digestion. Reverse transcriptase–PCR analysis shows LOI in two tumours and ROI in three tumours.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2600691&req=5

fig1: Analysis of IGF2 alterations. (A) Examples of the methylation status of CTCF6 analysed by a combined bisulphite restriction assay (COBRA). Bisulphite-modified PCR products were digested with MluI. Upper and lower lanes indicate unmethylated and methylated fragments, respectively. Numbers above lanes indicate the tumour number. Numbers below lanes show the percentage of methylated DNA fragments containing CTCF6. The mean value of the DNA methylation percentages calculated from three COBRA experiments is shown in Table 1. Methyl., control methylated DNA. The IGF2 status is shown above the tumour numbers. LOI, loss of IGF2 imprinting; LOH, loss of heterozygosity in the IGF2 region; ROI, retention of IGF2 imprinting. (B) Electrophoretic pattern of genomic DNA PCR products or RT–PCR products after AvaII digestion. Reverse transcriptase–PCR analysis shows LOI in two tumours and ROI in three tumours.
Mentions: Combined bisulphite restriction assay showed that 21 and 33 tumours had hypermethylation and normal methylation at CTCF6, indicating LOH or LOI and retention of IGF2 imprinting (ROI), respectively (Table 1 and Figure 1). Single nucleotide polymorphism array analysis was performed in 43 of 54 tumours; all 21 tumours with hypermethylated CTCF6 and 22 of 33 tumours with normally methylated CTCF6. Combined results of both analyses indicated that 12 tumours had LOH (10 hypermethylated CTCF6 and UPD 2 hypermethylated CTCF6 and hemizygous 11p15 deletion), 9 had LOI (hypermethylated CTCF6 and retention of heterozygosity (ROH)) and 22 had ROI (normally methylated CTCF6 and ROH). Of 21 tumours whose RNA was available, 9 and 12 tumours had heterozygous and homozygous ApaI/AvaII sites in exon 9 of IGF2, respectively. Of the nine heterozygous tumours, seven showed monoallelic expression of IGF2, indicating ROI, and two showed biallelic expression of IGF2, indicating LOI, and the results were consistent with those examined by COBRA and SNP array analyses (Table 1). From these findings, 11 tumours with normally methylated CTCF6, in which SNP array analysis was not performed, were classified as those with ROI. Thus, combined results of COBRA, SNP array and allelic expression analyses showed 12 tumours with LOH, 9 tumours with LOI and 33 tumours with ROI. In addition, one cell line (HuH6) had LOI, and the other (HepG2) had LOH (UPD) of IGF2.

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