Allelic loss analysis of tumor suppressor genes regardless of heterozygosity: von Hippel-Lindau gene loss in renal cell carcinoma.

Abstract

VHL, the von Hippel-Lindau tumor suppressor gene, has no microsatellites, but rather single nucleotide polymorphisms inside the gene. However, their low heterozygosity is unfavorable for loss of heterozygosity analysis. We examined whether our modified single nucleotide polymorphism genotyping method would be useful for allelic loss analysis of the VHL gene in heterozygous and homozygous genotypes of sporadic renal cell carcinoma. Genomic DNA was extracted from tumor and nontumor tissues in 35 cases of sporadic renal cell carcinoma. The single nucleotide polymorphism (rs1642742), G or A containing region of the VHL gene was amplified from sample DNA and subjected to primer extension reaction with fluorescent dideoxynucleotide triphosphate. Template directed incorporation of fluorescent dideoxyguanosine triphosphate or dideoxyadenosine triphosphate was quantitatively analyzed and the A/G (G/A) signal ratio was compared between tumor and nontumor tissues. We confirmed quantitative template directed incorporation of dideoxyguanosine triphosphate or dideoxyadenosine triphosphate using model templates with various ratios of DNA from the 2 genotypes AA and GG. In 20 heterozygous cases of renal cell carcinoma the A/G signal ratio was significantly differentiated between tumor and nontumor in 9 loss of heterozygosity positive cases but not in 11 loss of heterozygosity negative cases. A total of 15 homozygous renal cell carcinoma cases were tested by adding homozygous control DNA of a different genotype before analysis. Eight of the 15 cases showed a significantly lower signal ratio in tumor than in nontumor, whereas the other 7 showed no significant difference. Our modified single nucleotide polymorphism genotyping is broadly applicable to allelic loss analysis of tumor suppressor genes in heterozygous and homozygous tumors.