No evidence for involvement of SDHD in neuroblastoma pathogenesis

Abstract

Background: Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma.

Methods: SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria.

Results: Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype.

Conclusions: Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis.

Figure 1

Details of sequencing profiles: (A) Deletion of GGCA in cell line N206 causing skip of exon 3 and (B) Y93C mutation in cell line NMB; (C) RT-PCR (reverse transcriptase PCR) on cell lines grown with or without puromycine (T = treated, U = untreated) revealed a transcript variant in cell line N206, caused by the GGCA deletion (lane 1 and 2).

Figure 2

SDHD mRNA levels in NB cell lines (light gray), neuroblast cells (gray) and human normal control samples (white). Significantly reduced SDHD mRNA expression levels in NB cell lines with 11q23 allelic loss compared to 11q23 intact NB cell lines (P = 5.31E-06) and normal tissue samples (P = 1.49E-05).

Figure 3

Mitochondrial ultrastructure shows heterogeneity between cell lines (same final magnification for the 4 images, marker = 0.5 μm): (A) NB cell line N206: dilated crista spaces in small mitochondria with a dense matrix; (B) NB cell line NMB: small mitochondria with narrow cristae and light matrix, so-called orthodox configuration, (C) NB cell line SJNB-8: unusually large mitochondria in orthodox configuration (narrow cristae), some areas in the matrix are cleared and lack cristae; (D) NB cell line LA-N-2: very large mitochondria with dilated cristae and dense matrix.