Novel somatic mutations in the VHL gene in Swedish archived sporadic renal cell carcinomas

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Abstract

Frequent loss-of-function somatic mutations of the VHL gene have been detected in sporadic renal cell carcinoma (RCC), indicating that inactivation of the VHL gene plays a critical role in RCC. In this study, we collected 35 archived Swedish sporadic RCCs identified from an epidemiological study on occupational exposure and kidney cancer to test how well stored pathological specimens could be retrieved and analyzed for VHL mutations. Thirty specimens were successfully analyzed with PCR-SSCP and sequencing. Aberrant SSCP bands were detected in 16 out of the 30 samples (53%). Sequencing analysis of the aberrant bands revealed seven deletions, one insertion, one base substitution on a splicing site, six missense mutations, one silent mutation and several base substitutions in the 5′ non-coding region and intron 1. Most were novel somatic mutations that have not been reported in sporadic RCC. The mutations were found in three types of non-papillary RCC cases, i.e. 14 clear cells, one granular chromophilic and one sarcomatoid RCC. Interesting multiple mutations were found in three cases (5, 3, 2 mutations, respectively).

Introduction

Kidney cancer is a common disease which has been estimated to afflict about 127 000 individuals world-wide each year, accounting for 1.7% of all malignant diseases. There is an increasing trend in incidence in both men and women from almost all areas in the world [1]. About 80–90% of all adult kidney neoplasms are renal cell carcinomas (RCC) which originate from the epithelial cells of proximal tubules of renal parenchyma. The etiology of kidney cancer is still poorly understood, although a number of environmental factors, such as tobacco and occupational exposures to gasoline and other petroleum products, asbestos and dry cleaning solvents have been associated with sporadic RCC [2], [3].

Inherited RCC occurs frequently in families of the von Hippel-Lindau disease, a rare hereditary cancer-prone syndrome predisposing to a variety of tumors most commonly angiomas of the retina, hemangioblastoma of the cerebellum, pheochromocytoma and RCC [4]. The disease gene, von Hippel-Lindau (VHL), is located in human chromosome 3p25 and has been identified recently [5]. The VHL gene is considered a tumor suppressor gene since the frequencies of mutation and loss of heterozygosity (LOH) of the VHL gene in sporadic clear cell renal carcinomas have been reported to be 57% and 98%, respectively [4], consistent with the ‘two-hits’ theory of tumor suppression [6]. The VHL gene product appears to be a multifunctional protein that has been shown to be involved in regulation of transcriptional elongation [7], control of cell cycle [8] and regulation of transcription [9].

To date, more than 500 mutations of the VHL gene have been identified. Most are germline mutations found in the VHL families. Only 143 somatic mutations in sporadic RCC have been reported [4], [10], [11], [12], [13], [14], [15]. Analysis of mutations in tumor suppressor genes not only can implicate their involvement in specific cancer and help to predict aspects of clinical behavior and prognosis, but may also provide clues to cancer etiology. Successful examples of the latter are p53 mutation analyses in which specific mutation patterns in skin carcinoma, hepatocellular carcinoma and lung carcinoma were associated with the predominant environmental risk factors, i.e. UV light, aflatoxin B and tobacco smoking, respectively [16]. In order to investigate the possible carcinogens underlying the occurrence of RCC by the strategy of mutation analysis, a large mutation database with detailed exposure and personal information is required. A database on VHL mutations is under construction but it still has no epidemiological data [15]. Here we analyze somatic VHL mutations in 35 Swedish sporadic RCCs identified from an epidemiological study on occupational exposure and kidney cancer.

We wanted to test how well stored pathological specimens could be retrieved and analyzed for VHL mutations.

Section snippets

Samples

A multicenter collaborative case-control study of renal cell cancer was carried out previously in six centers in five countries to evaluate possible etiological factors for RCC [2], [3]. The Swedish part of the study was defined as all individuals of ages 20–79 years, born in Sweden and resident in any of eight counties in central Sweden during the time period from June 1, 1989 to December 31, 1991. All incident cases of RCC (ICD-9 189.0) notified to the Regional Cancer Registry were included.

Results

PCR amplification of fragments of the VHL gene was carried out successfully in 30 out of total 35 archived samples. Aberrant bands on single-strand conformation polymorphism (SSCP) gel were detected in the PCR product of 16 out of the 30 samples (53%) analyzed and in all the PCR segments except exon 1A (Fig. 1). Sequencing analysis of the aberrant bands revealed seven deletions (one of the deletions including donor site of splicing), one insertion, one base substitution in the splicing site,

Discussion

It is assumed that most human cancers originate from environmental causes including life style factors and exposure to environmental carcinogens. However, many such factors remain unknown and pose a challenge to molecular epidemiology to track down the responsible mutagens deductable from the spectra of mutations found in different tumors [22]. The accumulation of valid mutation data with proper information of exposure is necessary for achieving this purpose. Archived cancer cases should be a

Acknowledgements

The study was supported by the Swedish Cancer Fund.

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