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

Figures & data

Table 1. Information on the CpG loci interrogated for each gene

Gene name	Chromo-somal location	Bona fide CpG island	# of Infinium probes and location relative to CpG island	Amplification and Sequencing Primers / Infinium Probes	CpGs read out of total number of CpGs in amplicon
PBRM1	3p21	Yes	1; outside	F – TGGTGGTTGTAGTAATTTTTAGA R - GAGGGGTAAGGGAGGTGAG	23/29
KDM6A	Xp11	Yes	1; outside	F – GATAAAGTTGGTGTGTTGGTTT R – TAGTTTGATAGTRGAGGAGAG	21/28
SETD2	3p21	Yes	2; outside	F – GGTTTATTGTTTYGGAGAGTTAT R - TGAGGGTGAGAGGGAGAGA
BAP1	3p21	Yes	1; outside	assay #ADS1756FS1, EpigenDX	7/7
			1; within	F- GATGAATAAGGGTTGGTTGGAGT R – GTTTGTTTGATTATTATTTTTTTTTTTTG PSQ - TGGTTGGAGTTGGAGA	3/6
KDM5C	Xp11	Yes	1; within	cg04927982_CGTGCACCGCCGGTCCATCCGGAAAGACGATCCGGCAAACTAATTACAAT	6
ARID1A	1p36	Yes	2; within	cg11856093_CAGGCCAGGGCTTTGTTGTCCGCCATGTTGTTGGTGGAAGACGGCGGCCG	4
				cg17385674_ACCCTCTTTGCAAGCCCGAAAGAATGACTGATCATTGTTCAGACGATTCG	3
MLL2	12q13	No	1	cg13007988_CGGGGAGACCTGTTGGTGCCAAGAAAGAGATCTATATGCCTACTAAGTCT	1

Gene name according to NCBI; chromosomal location according to NCBI; CpG island according to Takai and Jones criteria by CpG Island Searcher; number of probes in Infinium humanmethylation27 beadchip and position of probe relative to Takai and Jones CpG island; sequence of primers used and Infinium probes examined in our study, Y and R indicate degenerate T or C in forward and reverse primer respectively, primer sequences for the most 5′ area of BAP1 are proprietary and available as a commercial kit (Epigen DX, Hopkinton, MA, USA); number of CpG loci read from total number of CpG loci in amplicon due to loss of sequence read at the 5′ end of the bisulfite sequencing amplicon or the 3′ end of the pyrosequencing amplicon.

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.

Table 2. Clinicopathological data for the 50 ccRCC

ccRCC	Stage I	Stage II	Stage III	Stage IV
Grade I	3
Grade II	20		2
Grade III	4	1	3	8
Grade IV				9