Aberrant promoter hypermethylation of PBRM1, BAP1, SETD2, KDM6A and other chromatin-modifying genes is absent or rare in clear cell RCC

Abstract

Recent sequencing studies of clear cell (conventional) renal cell carcinoma (ccRCC) have identified inactivating point mutations in the chromatin-modifying genes PBRM1, KDM6A/UTX, KDM5C/JARID1C, SETD2, MLL2 and BAP1. To investigate whether aberrant hypermethylation is a mechanism of inactivation of these tumor suppressor genes in ccRCC, we sequenced the promoter region within a bona fide CpG island of PBRM1, KDM6A, SETD2 and BAP1 in bisulfite-modified DNA of a representative series of 50 primary ccRCC, 4 normal renal parenchyma specimens and 5 RCC cell lines. We also interrogated the promoter methylation status of KDM5C and ARID1A in the Cancer Genome Atlas (TCGA) ccRCC Infinium data set. PBRM1, KDM6A, SETD2 and BAP1 were unmethylated in all tumor and normal specimens. KDM5C and ARID1A were unmethylated in the TCGA 219 ccRCC and 119 adjacent normal specimens. Aberrant promoter hypermethylation of PBRM1, BAP1 and the other chromatin-modifying genes examined here is therefore absent or rare in ccRCC.

Keywords: ARID1A; BAP1; KDM5C; KDM6A; MLL2; PBRM1; SETD2; clear cell RCC; promoter methylation; renal cell carcinoma.

Figure 1. CpG island schematic of the genes studied. Vertical red lines represent individual CpG loci in the island. The TSS is indicated by a vertical rectangle and the ATG by a hatched box. The horizontal black line indicates the area sequenced and the nucleotide position given is relative to the location of the TSS from Ensembl.

Figure 2. Representative examples of bisulfite sequencing and pyrosequencing. (A) Bisulfite direct sequencing of PBRM1 in 50:50 unmethylated:fully methylated DNA control, a ccRCC and normal renal parenchyma. Methylation is visible as a cytosine peak superimposed on a thymine peak at CpG loci indicated by black arrows in the 50:50 control. (B) Bisulfite direct sequencing of the reverse strand of KDM6A in ccRCC. (C) Bisulfite direct sequencing of SETD2 in a ccRCC. (D) Bisulfite pyrosequencing of two areas of the BAP1 promoter CpG island in the 50:50 control and a ccRCC.