Tracking functional changes in the cancer genome : a molecular genetic analysis of renal and prostatic carcinomas using PCR based techniques by a candidate chromosome and candidate gene approach

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Surgical Science

Sammanfattning: Renal cell carcinoma (RCC) is the most common malignancy occurring in the adult kidney. Prostatic carcinoma is the most common male malignancy in most Western countries. Several environmental factors have been suggested to contributed to the tumor genesis but the involvement of genetic factors is also recognized. Here we applied a variety of molecular genetic techniques to characterize somatic and constitutional genetic alterations in patients with these types of tumors. Von Hippel-Lindau disease (VHL) is an autosomal dominant disorder predisposing to the development of RCC. The disease is caused by the germline mutations of the VHL gene located in chromosomal region 3p25. Using PCR /direct DNA sequencing and Southern blot analysis, 7 germline mutations were identified in 10 VHL families (Paper 1). We further showed the importance of VHL mutation analysis both in prognosis of patient and presymptomatic diagnosis of VHL mutation carriers. Particular note was emphasized on the selection of kidney donors from relatives of a possible VHL patient affected by bilateral RCCs. Somatic deletions of the long arm of chromosome 10 were demonstrated in 24% of the RCC analyzed (Paper 2). As the deletion included the PTEN/MMAC 1 gene, mutation analysis was carried out using PCR/direct DNA sequencing. Three single base changes were identified in 3/6 tumors showing loss of the PTEN/MMAC1 region. A homozygous deletion coupled with absent PTEN/MMAC1 expression was detected in the RCC line UOK147. The result suggests the possible importance of PTEN/MMAC1 gene mutation in clinical prognosis of a subset of RCCs. Using PCR/LOH analysis we detected LOH of 13q markers in 18/36 prostatic carcinomas analyzed (Paper 3). Two important regions of deletion were defined, which did not included the Rb I locus at 13q14 or the BRCA2 locus at 13q12. Mutation analysis of the BRCA2 gene in six tumors did not reveal any important mutations. Immunohistochemical analysis of the same tumor materials showed that frequent aberrant pRb expression was correlated with a tetraploid DNA content but not concordant with the loss of the Rb1 gene. The data suggest the existence of yet unidentified tumor suppressor genes on 13q that are important for prostate cancer development. In the same set of prostatic carcinomas, 16q deletions were demonstrated in 75% of tumors (Paper4). Extensive deletions were found in all the metastases while small deletions were also present in 54% of primary tumors. Four important deleted regions were identified and the presence of the terminal 16q deletion was closely correlated with grade, stage and metastases. The location of tandemly arranged classical cadherin genes was coincident with the four important deleted regions. We chose the CDH I gene at 16q22.1 that encodes the E-cadherin protein as the first candidate gene. Mutation analysis of all the 16 coding exons did not find any mutations in the ten tumors showing LOH at the CDH I region. In addition, we also detected frequent reduced or absent E-cadherin expression in the same set of materials. The finding suggests that chromosome 16q deletions are important in the development of prostate cancer metastasis and could potentially be used as a prognostic marker. Using a modified PCR/LOH analysis in sporadic prostatic carcinomas, we found two possible deletions at the putative prostate cancer susceptibility locus HPCX on Xq27-28 (Paper 5). These deletions caused a complete loss of the DNA sequences in the region. The findings further support the importance of this locus in sporadic tumors. The association between the CAG repeats polymorphism in the androgen receptor gene and prostatic carcinomas was analyzed in Swedish and Japanese populations (Paper 6). Both sporadic and familial prostatic carcinomas in Sweden showed association with short CAG repeats whereas sporadic prostatic carcinomas in Japan showed association with long repeats. In both populations the allele AR(CAG)22 showed negative correlation with prostatic carcinomas, and the alleles AR(CAG)21 and AR(CAG)23 showed positive correlation. Compared to Swedish populations, Japanese males more frequently carried AR(CAG)22 and less frequently AR(CAG)21 and AR(CAG)23. This difference in allelic distribution may be part of the genetic background that determines a dramatically lower incidence of clinical prostate cancer in Japan. The present study may contribute to a better understanding of the genetic mechanism for the development of renal and prostatic carcinoma. The results also present the potential of molecular genetic analysis in clinical diagnosis and prognosis of these two common types of urological malignancies.

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