The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias

Additional file 1:

RASSF methylation profile. The data provided represent analysis of RASSF members and additional data relating to RASSF6 and RASSF10 in childhood leukaemia. Figure S1: RASSF methylation profile of leukaemia cell lines. The methylation status of RASSF1A and RASSF2 was determined using MSP whereas the methylation status of RASSF3-6 were determined using COBRA. RASSF1A, RASSF5A and RASSF6 were hypermethylated in leukaemia cell lines whereas RASSF2 and RASSF3 showed no evidence of methylation. RASSF4 was methylated in both leukaemia cell lines and normal blood. For MSP assays M = methylated specific PCR; U = unmethylated specific PCR. For COBRA assays U = undigested PCR product; T = TaqI digested PCR product. Figure S2: RASSF methylation profile of primary leukaemias. RASSF genes showing evidence of hypermethylation in leukaemia cell lines were investigated for methylation in B-ALL and T-ALL leukaemias. RASSF1A and RASSF5A were infrequently methylated in T-ALL (2/12 and 2/24 respectively). RASSF4 was methylated in 25/25 T-ALL but in only 7/50 B-ALL leukaemias, however given the low level methylation of RASSF4 observed in normal blood this gene was not investigated further. RASSF6 was methylated in 48/51 (94%) B-ALL and 12/29 (41%) T-ALL but not in normal bone marrow (BM) or blood control samples. For MSP assays M = methylated specific PCR; U = unmethylated specific PCR. For COBRA assays U = undigested PCR product; T = TaqI digested PCR product. Figure S3: Bisulphite sequencing of the RASSF6 CpG island. As in figure 1C up to 12 alleles from B-ALL, T-ALL leukaemia cell lines, normal blood and normal bone marrow were cloned and sequenced. The methylation index (MI) is given for each. Figure S4: RASSF6 expression in normal tissues and leukaemia cell lines. A, RASSF6 mRNA was detected in all tissues analysed. B, Restoration of RASSF6 mRNA expression in methylated leukaemia cell lines following treatment with 5azaDC and TSA showing the additional cell lines JKT and REH. GAPDH was used as a control for equal loading. Figure S5: Methylation of the RASSF8 CpG island infrequently inactivates RASSF8 in leukaemias. A, Direct bisulphite sequencing of RASSF8 CpG island PCR products. Filled black circle represents a completely methylated CpG dinucleotide, open circle represents a completely unmethylated CpG dinucleotide and filled gray circle represents a partially methylated CpG dinucleotide. B, Methylation of the RASSF8 CpG island correlates with loss or downregulation of RASSF8 expression which is restored following 5azaDC treatment.

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Additional file 2:

RASSF6 methylation indexes and PCR primers used within this study. The data provided summarise RASSF6 methylation index data in cell lines, primary tumour and control samples (Table S1). PCR primers used in this study are also listed (Table S2). Table S1: A summary of the methylation index of the RASSF6 CpG island in leukaemia cell lines, B-ALL, T-ALL and normal blood and bone marrow control samples. Table S2: MSP and COBRA primer sequences used within this study.

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