Absence of SDHD mutations in primary nasopharyngeal carcinomas

Dear Sir, Nasopharyngeal carcinoma (NPC) is a prevalent cancer among the southern Chinese. It is the fifth most common cancer in Hong Kong. Our previous NPC studies have demonstrated frequent genetic deletions on chromosomes 3p, 9, 11q, 13q, 14q and 16q.1 This suggested the involvement of multiple tumour-suppressor genes in NPC tumorigenesis. Further study of chromosome 9p21 illustrated homozygous deletion and aberrant methylation of the gene p16 which implied that it is the critical target NPC-related tumour-suppressor gene residing in this region.2 RASSF1A gene was identified to be the target tumour-suppressor gene located on 3p21.3 Loss of chromosome 11q23 was another one of the most consistent aberrations, as described in our previous studies.2,4 In a loss of heterozygosity (LOH) study of 11q, a high incidence of deletions was identified at regions 11q13 and 11q22-24.4 Similar results were reported in a high-resolution allelotype analysis, which detected 74.1% of 11q LOH in microdissected primary NPC.2 The latter study confined the minimal region of deletion to 11q21-q23. Tumour-suppressor genes residing in these regions are suspected to be involved in NPC tumorigenesis. The importance of this chromosomal region was illustrated in a functional analysis using a monochromosome transfer approach.5 Hybrids of an NPC cell line (HONE-1) and normal chromosome 11q22-23 region suppressed tumorigenicity of NPC cells in nude mice. These results strongly indicated the presence of NPC-associated tumour-suppressor genes in this region. LOH on chromosomal regions bearing mutated tumoursuppressor genes is critical in the development of epithelial tumours.6 LOH of the long arm of chromosome 11 is 1 of the common genetic aberrations in human cancers. Chromosome 11q deletion has been reported in several tumours, e.g., lung cancer, ovarian cancer, breast cancer, cervical carcinoma and NPC.7–10 In these LOH studies, 11q23 was illustrated to be the common minimal deletion region. Tumour-suppressor genes residing in this region are thought to be involved in the development of many solid malignancies. Search of candidate tumour-suppressor genes in 11q23 is still in progress. Two tumour-suppressor genes were mapped to this region: the ATM gene (mutated in ataxia-telangiectasia) and the PPP2R1B gene (protein phosphatase 2, structural/regulatory subunit A, ). However, extensive mutation screenings illustrated that these 2 genes were infrequently mutated in various solid tumours.11,12 The gene SDHD (succinate dehydrogenase complex, subunit D) was mapped to chromosome 11q23 by fluorescence in situ hybridization.13 The SDHD gene encodes the small subunits (cybS) of cytochrome b in the succinate–ubiquinone oxidoreductase complex (complex II) of human mitochondria.13 The 2 genes for the large and small subunits of cytochrome b were SDHC (cybL) and SDHD, which were mapped to 1q21 and 11q23, respectively. SDHD is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms.13,14 It spans over 19 kb at the genomic level. The translated region consists of 4 exons of 52, 117, 145 and 163 bp.14 Germline mutation of SDHD was reported in hereditary paraganglioma families.15 Loss of chromosome 11q23 region was frequently detected in families with paraganglioms. No imprinting of the SDHD gene was found in these families. Hence, mutation of the wild-type together with loss of the other copy of this gene suggested a tumour-suppressor role for the SDHD gene. In another study, somatic mutation of the SDHD gene was found in 1 of 18 apparently sporadic pheochromocytomas.16 To further investigate the involvement of SDHD mutation in sporadic cancers, mutations of this gene were screened in NPC and Burkitt’s lymphoma. Both of these cancer types show frequent chromosome 11q aberrations. In the present study, single-strand conformation polymorphism (SSCP) analysis was applied at each of the 4 exons of the SDHD gene. Four NPC cell lines (C666-1, CNE-1, CNE-2, HK-1), 3 xenografts (xeno1915, xeno-2117, xeno-NPC8) as well as 4 Burkitt’s lymphoma cell lines (Raji, Daudi, Jijoye, Namalwa) were first investigated by SSCP analysis. Thereafter, we also examined 43 primary NPCs and 5 Burkitt’s lymphomas. These NPC samples were recruited from our previous LOH study of chromosome 11q and allelotyping. Mutation screening of the SDHD gene was performed on 43 primary NPC biopsies as described previously.3 All 4 exons of the SDHD gene were examined with the previously described primers.15 After PCRs, the products were analyzed by electrophoresis on nondenaturing polyacrylamide gels with various concentrations using different running conditions. Bands showing a mobility shift were cut out and soaked in water to elute DNA. Isolated DNA was purified and reamplified for direct