A. A diagram mapping all copy number abnormalities on chromosome 1. Copy number status is schematically represented along chromosome 1 (1pter on the left to 1qter on the right) by colour-coded bars as indicated. Chromosomal band 1p36 is indicated at the top. Previously reported minimum regions of deletion by LOH analysis in various gliomas are schematically shown by grey bars (references, Hashimoto (Hashimoto et al., 2003), Iuchi (Iuchi et al., 2002), Felsberg (Felsberg et al., 2004), Barbashina (Barbashina et al., 2005), Nigro (Nigro et al., 2001), Smith (Smith et al., 1999), Dong (Dong et al., 2004)). The 1p36 region shown in more detail in Fig. 2B is boxed. A region homologous to chromosome 7 is indicated by a grey arrow all tumours with chromosome 7 gain (as judged by the 5Mb clone results) showed copy number gain in this region, most likely because of sequence homology. Codes for the 1994 tumours are in blue. B. A detailed view of copy number abnormalities at 1p36.21-33 for cases that had deletions within the region. Only deletions are shown. The boundaries of the largest homozygous deletion (GB84), telomeric break point at RP3-438L4 and centromeric at RP11-807G9, are indicated by dotted lines. For more precise borders of homozygous deletions see Fig. 3. Chromosomal bands are indicated below.

Analysis of TNFRSF9 alterations. A. Multiplex PCR at TNFRSF9 exon 5. The densitometrically calculated copy numbers are shown below the gel photographs (normal = 2, homozygous deletions (< 0.6) are indicated by bold text and underlining). c= control locus (WI-3306, an STS at 2q21). An arrow indicates the bands representing the target locus. B. MSP at the CpG island of TNFRSF9. The primer pair used for TNFRSF9 specifically amplifies bisulfite-modified methylated sequences. PCR products for ACTB using the primer pair that amplifies bisulfite-modified sequences regardless of methylation status are shown to indicate successful bisulfite modification. G-DNA, unmodified genomic DNA; Blood, modified DNA from WBC; SssI, SssI methylase treated DNA as positive control; Neg, no template. C. Bisulfite sequencing of TNFRSF9 in GB105. Upper panel is a diagram of the CpG island of TNFRSF9 (abscissa, nucleotide position; left ordinate, GC%; shaded area, CpG island). The positions of CpG dinucleotides in the island are indicated by vertical red bars. Frequency of methylation at each CpG dinucleotides in the 10 sequenced clones, each of which is presented in diagram form in the lower panel (closed circle, methylated; open circle, unmethylated), is superimposed in the upper panel (left ordinate, methylation%). The position of MSP primer pair (PC4317/PC4318) is indicated on the bottom of upper panel and the CpG dinucleotides recognized by the primers are indicated in boxes in the lower panel. D. mRNA expression levels of TNFRSF9 and RERE in glioma cell line U251 and breast carcinoma cell line MCF7 after treatment with 5′-Aza-2′-deoxycytidine for either 48 hours or 96 hours. Expression levels relative to that of the mock experiment are shown.

Log2 ratio plots of chromosome 1 in two glioblastomas, GB219 and GB52. Insets show microsatellite data displaying allelic balance (D1S247 in GB219 and D1S2655 in GB52) and imbalance (D1S2667 in GB219 and GB52). Positions of clones that harbour the corresponding markers are indicated by grey spots. Hemizygous and homozygous deletions are indicated by arrows (see also Fig. 2). A grey arrow in GB52 indicates the region that appeared as copy number gain due to homology to a part of chromosome 7 which showed trisomy in this tumour (see Fig. 2 legend).

A. Top; A schematic diagram of homozygous deletions (indicated by closed boxes). Middle; A schematic diagram of physical locations of the clones on the array, the genes in the region, the corresponding chromosomal bands and nucleotide position ruler (modified after Ensembl 33.35e, September 2005). Orientation of transcription is indicated by an arrow above corresponding genes. Bottom; A schematic diagram of exon positions of RERE and SLC45A1/DNB5 as well as the BAC clones. B-D. Multiplex PCR at an STS in RP11-185B14, RERE exon 23 and SLC45A1/DNB5 exon 8 (B), RERE exon 13 and 14 (C) and SLC45A1/DNB5 exon 1 (D). Xenografts are indicated by suffix X after the code corresponding to their parental primary tumours. The densitometrically calculated copy numbers are shown below the gel photographs (normal = 2, homozygous deletions (< 0.6) are indicated by bold text and underlining). c, control locus (WI-3306, an STS at 2q21). Arrows indicate the bands representing the target locus.

A. A log2 ratio plot of chromosome 1 in GB263. Homozygous deletion at 1p36.22-p36.23 (see also Fig. 3) and amplification at 1q44 are indicated by arrows. B. Superimposed log2 ratio plots of GB291, GB225, GB153 and GB263 around the amplicons at 1q44. A schematic diagram of physical locations of clones and genes in the minimal region of overlap, corresponding chromosomal bands and nucleotide position ruler are shown below the plot (modified after Ensembl 33.35e, September 2005).