Investigation of the role of SDHB inactivation in sporadic phaeochromocytoma and neuroblastoma

Abstract

Germline mutations in the succinate dehydrogenase (SDH) (mitochondrial respiratory chain complex II) subunit B gene, SDHB, cause susceptibility to head and neck paraganglioma and phaeochromocytoma. Previously, we did not identify somatic SDHB mutations in sporadic phaeochromocytoma, but SDHB maps to 1p36, a region of frequent loss of heterozygosity (LOH) in neuroblastoma as well. Hence, to evaluate SDHB as a candidate neuroblastoma tumour suppressor gene (TSG) we performed mutation analysis in 46 primary neuroblastomas by direct sequencing, but did not identify germline or somatic SDHB mutations. As TSGs such as RASSF1A are frequently inactivated by promoter region hypermethylation, we designed a methylation-sensitive PCR-based assay to detect SDHB promoter region methylation. In 21% of primary neuroblastomas and 32% of phaeochromocytomas (32%) methylated (and unmethylated) alleles were detected. Although promoter region methylation was also detected in two neuroblastoma cell lines, this was not associated with silencing of SDHB expression, and treatment with a demethylating agent (5-azacytidine) did not increase SDH activity. These findings suggest that although germline SDHB mutations are an important cause of phaeochromocytoma susceptibility, somatic inactivation of SDHB does not have a major role in sporadic neural crest tumours and SDHB is not the target of 1p36 allele loss in neuroblastoma and phaeochromocytoma.

Figure 1

SDHB promoter sequence. In boxes shaded in light grey are the MSP primer sequences.CpG islands are in bold and numbered from 1 to 23. The numbers in brackets are nucleotide position in relation to the ATG start codon (highlighted in light grey).

Figure 2

(A) Partial chromatogram of cloned MSP product from a methylated neuroblastoma tumour (St158T). Methylated cytosine appear as a G signal in the complementary strand. The number indicates the position of CpG sites. (B) Similar chromatogram obtained from an unmethylated neuroblastoma tumour (St111T).

Figure 3

Summary of chromosome 1p loss of heterozygosity analysis in phaeochromocytomas. Filled circles indicate LOH; shaded circles indicate retention of heterozygosity and open circles indicate noninformative cases. Microsatellite markers are ordered from telomere to centromere (Genome Browser-Human assembly, July 2003; http://genome.ucsc.edu).

Figure 4

MSP analysis of SDHB methylation in sporadic neuroblastoma (St111T, St158T and St119T) tumours and in sporadic phaeochromocytoma (T29, T33, T21 and T20) tumours. Bisulphite-modified DNA was amplified with primer pair specific for unmethylated (U) and methylated (M) alleles as described in the text. In vitro methylated DNA was used as a positive control (+) for amplification with methylated DNA-specific primers.

Figure 5

Expression analysis of SDHB gene in neuroblastoma cell line SK-N-AS and SK-N-SH, before (−) and after (+) 5 aza-2-deoxycytidine treatment. Western blotting was performed essentially as described in the Material and Methods. Indian ink staining was included as a loading control.