Dewi Astuti

Gene Mutations in the Succinate Dehydrogenase Subunit SDHB Cause Susceptibility to Familial Pheochromocytoma and to Familial Paraganglioma

The pheochromocytomas are an important cause of secondary hypertension. Although pheochromocytoma susceptibility may be associated with germline mutations in the tumor-suppressor genes VHL and NF1 and in the proto-oncogene RET, the genetic basis for most cases of nonsyndromic familial pheochromocytoma is unknown. Recently, pheochromocytoma susceptibility has been associated with germline SDHD mutations. Germline SDHD mutations were originally described in hereditary paraganglioma, a dominantly inherited disorder characterized by vascular tumors in the head and the neck, most frequently at the carotid bifurcation. The gene products of two components of succinate dehydrogenase, SDHC and SDHD, anchor the gene products of two other components, SDHA and SDHB, which form the catalytic core, to the inner-mitochondrial membrane. Although mutations in SDHC and in SDHD may cause hereditary paraganglioma, germline SDHA mutations are associated with juvenile encephalopathy, and the phenotypic consequences of SDHB mutations have not been defined. To investigate the genetic causes of pheochromocytoma, we analyzed SDHB and SDHC, in familial and in sporadic cases. Inactivating SDHB mutations were detected in two of the five kindreds with familial pheochromocytoma, two of the three kindreds with pheochromocytoma and paraganglioma susceptibility, and 1 of the 24 cases of sporadic pheochromocytoma. These findings extend the link between mitochondrial dysfunction and tumorigenesis and suggest that germline SDHB mutations are an important cause of pheochromocytoma susceptibility.

Germline SDHB mutations and familial renal cell carcinoma.

Familial renal cell carcinoma (RCC) is a heterogeneous disorder that is most commonly caused by germline mutations in the VHL, MET, and FLCN genes or by constitutional chromosome 3 translocations. However, for many patients with familial RCC, the genetic basis of the disease is undefined. We investigated whether germline mutations in fumarate hydratase (FH) or succinate dehydrogenase subunit genes (SDHB, SDHC, SDHD) were associated with RCC susceptibility in 68 patients with no clinical evidence of an RCC susceptibility syndrome. No mutations in FH, SDHC, or SDHD were identified in probands, but 3 of the 68 (4.4%) probands had a germline SDHB mutation. Patients with a germline SDHB mutation presented with familial RCC (n = 1) or bilateral RCC (n = 2) and no personal or family history of pheochromocytoma or head and neck paraganglioma. Age at diagnosis of RCC in SDHB mutation carriers ranged from 24 to 73 years. These findings 1) demonstrate that patients with suspected inherited RCC should be examined for germline SDHB mutations, 2) suggest that all identified SDHB mutation carriers should be offered surveillance for RCC, and 3) provide a further link between familial RCC and activation of hypoxic-gene response pathways.

Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer.

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumor suppressor gene are common in sporadic conventional renal cell carcinoma (cRCC). Further insight into the clinical significance of these changes may lead to increased biological understanding and identification of subgroups of patients differing prognostically or who may benefit from specific targeted treatments. We have comprehensively examined the VHL status in tissue samples from 115 patients undergoing nephrectomy, including 96 with sporadic cRCC. In patients with cRCC, loss of heterozygosity was found in 78.4%, mutation in 71%, and promoter methylation in 20.4% of samples. Multiplex ligation-dependent probe amplification identified intragenic copy number changes in several samples including two which were otherwise thought to be VHL-noninvolved. Overall, evidence of biallelic inactivation was found in 74.2% of patients with cRCC. Many of the mutations were novel and approximately two-thirds were potentially truncating. Examination of these and other published findings confirmed mutation hotspots affecting codons 117 and 164, and revealed a common region of mutation in codons 60 to 78. Gender-specific differences in methylation and mutation were seen, although not quite achieving statistical significance (P = 0.068 and 0.11), and a possible association between methylation and polymorphism was identified. No significant differences were seen between VHL subgroups with regard to clinicopathologic features including stage, grade, tumor size, cancer-free and overall survival, with the exception of a significant association between loss of heterozygosity and grade, although a possible trend for survival differences based on mutation location was apparent.

Germline SDHD mutation in familial phaeochromocytoma

The genetic basis for familial phaeochromocytoma is unknown in many cases. Since the disorder has been reported in some cases of familial head and neck paraganglioma, which is caused by a mutation in the gene encoding succinate dehydrogenase complex subunit D (SDHD), we investigated this gene in kindreds with familial phaeochromocytoma. A germline SDHD frameshift mutation was identified in a two-generation family consisting of four children with phaeochromocytoma, but somatic mutations were not detected in 24 sporadic phaeochromocytoma tumours. Germline SDHD mutation analysis should be done in individuals with familial, multiple, or early-onset phaeochromocytomas even if a personal or family history of head and neck paraganglioma is absent.

Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility.

Perlman syndrome is a congenital overgrowth syndrome inherited in an autosomal recessive manner that is associated with Wilms tumor susceptibility. We mapped a previously unknown susceptibility locus to 2q37.1 and identified germline mutations in DIS3L2, a homolog of the Schizosaccharomyces pombe dis3 gene, in individuals with Perlman syndrome. Yeast dis3 mutant strains have mitotic abnormalities. Yeast Dis3 and its human homologs, DIS3 and DIS3L1, have exoribonuclease activity and bind to the core RNA exosome complex. DIS3L2 has a different intracellular localization and lacks the PIN domain found in DIS3 and DIS3L1; nevertheless, we show that DIS3L2 has exonuclease activity. DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins. DIS3L2 overexpression suppressed the growth of human cancer cell lines, and knockdown enhanced the growth of these cells. We also detected evidence of DIS3L2 mutations in sporadic Wilms tumor. These observations suggest that DIS3L2 has a critical role in RNA metabolism and is essential for the regulation of cell growth and division.

Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma

Purpose: This study aimed to carry out a comprehensive analysis of genetic and epigenetic changes of the von Hippel Lindau (VHL) gene in patients with conventional (clear cell) renal cell carcinoma and to determine their significance relative to clinicopathologic characteristics and outcome. Experimental Design: The VHL status in 86 conventional renal cell carcinomas was determined by mutation detection, loss of heterozygosity (LOH), and promoter methylation analysis, extending our original cohort to a total of 177 patients. Data were analyzed to investigate potential relationships between VHL changes, clinical parameters, and outcome. Results: LOH was found in 89.2%, mutation in 74.6%, and methylation in 31.3% of evaluable tumors; evidence of biallelic inactivation (LOH and mutation or methylation alone) was found in 86.0% whereas no involvement of VHL was found in only 3.4% of samples. Several associations were suggested, including those between LOH and grade, nodal status and necrosis, mutation and sex, and methylation and grade. Biallelic inactivation may be associated with better overall survival compared with patients with no VHL involvement, although small sample numbers in the latter group severely limit this analysis, which requires independent confirmation. Conclusions: This study reports one of the highest proportions of conventional renal cell carcinoma with VHL changes, and suggests possible relationships between VHL status and clinical variables. The data suggest that VHL defects may define conventional renal cell carcinomas but the clinical significance of specific VHL alterations will only be clarified by the determination of their biological effect at the protein level rather than through genetic or epigenetic analysis alone. (Clin Cancer Res 2009;15(24):7582–92)

The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma.

The VHL gene product (pVHL) forms a multimeric complex with the elongin B and C, Cul2 and Rbx1 proteins (VCBCR complex), which is homologous to the SCF family of ubiquitin ligase complexes. The VCBCR complex binds HIF-1α and HIF-2α, transcription factors critically involved in cellular responses to hypoxia, and targets them for ubiquitin-mediated proteolysis. Germline mutations in the VHL gene cause susceptibility to haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and other tumours. In addition somatic inactivation of the VHL gene occurs in most sporadic clear cell RCC (CC-RCC). However, the absence of somatic VHL inactivation in 30–40% of CC-RCC implies the involvement of other gatekeeper genes in CC-RCC development. We reasoned that in CC-RCC without VHL inactivation, other pVHL-interacting proteins might be defective. To assess the role of elongin B/C, Rbx1 and HIF-1α in RCC tumorigenesis we (a) mapped the genes to chromosomes 8q(cen) (elongin C), 16p13.3 (elongin B) and 22q11.2 (Rbx1) by FISH, monochromosomal somatic cell hybrid panel screening and in silico GenBank homology searching; (b) determined the genomic organisation of elongin C (by direct sequencing of PAC clones), Rbx1 and elongin B (by GenBank homology searching); and (c) performed mutation analysis of exons comprising the coding regions of elongins B, C and Rbx1 and the oxygen-dependent degradation domain of HIF-1α by SSCP screening and direct sequencing in 35 sporadic clear cell RCC samples without VHL gene inactivation and in 13 individuals with familial non-VHL clear cell RCC. No coding region sequence variations were detected for the elongin B, elongin C or Rbx1 genes. Two amino acid substitutions (Pro582Ser and Ala588Thr) were identified in the oxygen-dependent degradation/pVHL binding domain of HIF-1α, however neither substitution was observed exclusively in tumour samples. Association analysis in panels of CC-RCC and non-neoplastic samples using the RFLPs generated by each variant did not reveal allelic frequency differences between RCC patients and controls (P>0.32 by chi-squared analysis). Nevertheless, the significance of these variations and their potential for modulation of HIF-1α function merits further investigation in both other tumour types and in non-neoplastic disease. Taken together with our previous Cul2 mutation analysis these data suggest that development of sporadic and familial RCC is not commonly contributed to by genetic events altering the destruction domain of HIF-1α, or components of the HIF-α destruction complex other than VHL itself. Although (a) activation of HIF could occur through mutation of another region of HIF-a, and (b) epigenetic silencing of elongin B/C, Cul2 or Rbx1 cannot be excluded, these findings suggest that pVHL may represent the sole mutational target through which the VCBR complex is disrupted in CC-RCC. HIF response is activated in CC-RCC tumorigenesis.

RASSF1A promoter region CpG island hypermethylation in phaeochromocytomas and neuroblastoma tumours

Deletions of chromosome 3p are frequent in many types of neoplasia including neural crest tumours such as neuroblastoma (NB) and phaeochromocytoma. Recently we isolated several candidate tumour suppressor genes (TSGs) from a 120 kb critical interval at 3p21.3 defined by overlapping homozygous deletions in lung and breast tumour lines. Although mutation analysis of candidate TSGs in lung and breast cancers revealed only rare mutations, expression of one of the genes (RASSF1A) was absent in the majority of lung tumour cell lines analysed. Subsequently methylation of a CpG island in the promoter region of RASSF1A was demonstrated in a majority of small cell lung carcinomas and to a lesser extent in non-small cell lung carcinomas. To investigate the role of 3p TSGs in neural crest tumours, we (a) analysed phaeochromocytomas for 3p allele loss (n=41) and RASSF1A methylation (n=23) and (b) investigated 67 neuroblastomas for RASSF1A inactivation. 46% of phaeochromocytomas showed 3p allele loss (38.5% at 3p21.3). RASSF1A promoter region hypermethylation was found in 22% (5/23) of sporadic phaeochromocytomas and in 55% (37/67) of neuroblastomas analysed but RASSF1A mutations were not identified. In two neuroblastoma cell lines, methylation of RASSF1A correlated with loss of RASSF1A expression and RASSF1A expression was restored after treatment with the demethylating agent 5-azacytidine. As frequent methylation of the CASP8 gene has also been reported in neuroblastoma, we investigated whether RASSF1A and CASP8 methylation were independent or related events. CASP8 methylation was detected in 56% of neuroblastomas with RASSF1A methylation and 17% without RASSF1A methylation (P=0.0031). These results indicate that (a) RASSF1A inactivation by hypermethylation is a frequent event in neural crest tumorigenesis, particularly neuroblastoma, and that RASSF1A is a candidate 3p21.3 neuroblastoma TSG and (b) a subset of neuroblastomas may be characterized by a CpG island methylator phenotype.

The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas

By positional cloning, we identified two breakpoint-spanning genes in a familial clear cell renal cell carcinoma (CCRCC)-associated t(1;3)(q32.1;q13.3): LSAMP and NORE1 (RASSF1 homolog). Both genes are downregulated in 9 of 9 RCC cell lines. While the NORE1A promoter predominantly presents partial methylation in 6 of the cell lines and 17/53 (32%) primary tumors, the LSAMP promoter is completely methylated in 5 of 9 cell lines and in 14/53 (26%) sporadic and 4 familial CCRCCs. Expression of LSAMP and NORE1A proteins in CCRCC cell lines inhibited cell proliferation. These characteristics indicate that LSAMP and NORE1A may represent new candidate tumor suppressors for CCRCC.

Genetic analysis of mitochondrial complex II subunits SDHD, SDHB and SDHC in paraganglioma and phaeochromocytoma susceptibility

background  Germline mutations in three subunits of mitochondrial complex II (SDHB, SDHC and SDHD) may be associated with susceptibility to phaeochromocytoma (PC) and/or head and neck paraganglioma (HNPGL).