Identification of a Pseudogene That Can Masquerade as a Mutant Allele of the PTEN/MMAC1 Tumor Suppressor Gene

Identification of a Pseudogene That Can Masquerade as a Mutant Allele of the PTENTumor Suppressor Gene Alterations of the candidate tumor suppressor phosphatase gene PTEN MMAC1 on chromosome 10q23 have been implicated in a wide range of cancers, including glioblastoma, melanoma, and cancers of the breast, kidney, and endometrium (1–4). In addition, germ-line mutations in PTENhave been described in cancer-prone conditions such as Cowden'ss syndrome (5). In the midst of efforts to determine the frequency of PTENabnormalities in prostate cancer, we discovered a pseudogene closely related to PTEN MMAC1 that can be mistakenly identified as a mutant form of PTEN MMAC1. Oligonucleotide primers specific for the initiating ATG codon in exon 1 (open reading frame [ORF]-5) and the stop codon 404 in exon 9 (ORF-3)of PTENwere designed to amplify the full-length coding region from RNA extracted from human prostate cancer cell lines and xenografts by reverse transcription-polymerase chain reaction. Surprisingly, products of the appropriate size (1226 base pairs [bp]) were amplified from all cell lines tested, including those known to have homozygous deletions of the entire PTEN MMAC1 locus such as in the PC3 cell line (1). To investigate this anomaly, we performed polymerase chain reactions (PCRs) in the absence of reverse transcription and used RNA from the PC3 cell line as well as from DU145, a cell line known to express PTENmessenger RNA (mRNA) (1). A PTEN MMAC1 PCR product was obtained in both cases, but not if the RNA was pretreated with deoxyribonuclease (DNase) I (, A). DNase I treatment, however, did not abrogate the ability to amplify the fragment of correct size after reverse transcription reaction in DU145 cells, indicating that PTENmRNA is expressed. These results could be explained by contamination of the RNA preparations with genomic DNA that contains a sequence homologous to PTEN. Indeed, the same PCR product was obtained by use of genomic DNA as a template from cells with an intact PTENgene (DU145 and LAPC-4) and those with homozygous deletions of PTEN(PC3 and LAPC-9) (, A). Because the PTENsequence corresponding to these primers is separated by approximately 200 kilobases (kb) in the PTENgenomic locus (1,2), we suspected that a highly processed PTENhomologue or pseudo-gene exists.