von Hippel-Lindau exonic methylation analysis using MALDI-TOF mass spectrometry.
OBJECTIVE
Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.


METHODS
In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation.


RESULTS
Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated.


CONCLUSIONS
To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future.
School of Medicine, Emory University, Atlanta, Georgia.
Fei
F
Lian
Department of Urology, School of Medicine, Emory University, Atlanta, Georgia.
Shilpa
S
Sreedharan
Department of Urology, School of Medicine, Emory University, Atlanta, Georgia.
Rebecca S
RS
Arnold
Department of Urology, School of Medicine, Emory University, Atlanta, Georgia.
Viraj A
VA
Master
Department of Urology, School of Medicine, Emory University, Atlanta, Georgia.
Kenneth
K
Ogan
Department of Urology, School of Medicine, Emory University, Atlanta, Georgia.
John G
JG
Pattaras
Division of General Medicine and Geriatrics, Department of Internal Medicine, School of Medicine, Emory University, Atlanta, Georgia.
David L
DL
Roberts
Department of Urology, School of Medicine, Emory University, Atlanta, Georgia; Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, Atlanta, Georgia; Department of Hematology and Medical Oncology, School of Medicine, Emory University, Atlanta, Georgia; Department of Urology, Atlanta Veterans Affairs Medical Center, Atlanta, Georgia. Electronic address: jpetros@emory.edu.
John A
JA
Petros
D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000235:genetics; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005091:Exons / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000096:biosynthesis / Q000235:genetics*
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human
carcinoma; gene expression; kidney; mass spectrometry; methylation; renal cell