name

MUTATION ANALYSIS OF THE POU2F3 PROMOTER REGION. Neil-Jeremy G. Wingkun¹, Zhengyan Zhang², Loan Nguyen², Phyllis C, Huettner, Janet S. Rader². Biology Department, Washington University, St. Louis, MO¹; Department of Obstetrics & Gynecology², Washington University School of Medicine, St. Louis, MO.

Human papillomaviruses (HPVs) are small DNA viruses and major cause of cervical cancer. HPV modifies cellular differentiation to produce skin warts and cancer. The POU domaintranscription factors are a family of more than 40 homeodomain-containingproteins involved in cell differentiation and tissue specification.POU2F3 (OCT11, Skn-1a) plays a major role in the fine-tuning of keratinocyte terminal differentiation is expressed in the superbasal epidermal cells in the skin and can repress the growth of cervical cancer cells by suppressing their differentiation ability. In addition, POU2F3 has been found to regulate HPVs by activating the long control regions of the virus.

In prior research, the Rader lab found that POU2F3 resides in a critical region of nonrandom chromosomal loss on 11q and methylation of the CpG island in the promoter region of POU2F3 correlated with gene silencing in cervical cancer. In addition, initial sequencing of the region showed a somatic mutation in the promoter region of a cervical tumor. My work was to analysis the POU2F3 promoter region for additional genomic changes in a larger sample of tumors. This work would include pathologic evaluation of tumor samples and microdissection of tumor epithelium, DNA extraction and sequencing and use of bioinformatics for analysis.

Thirty OCT embedded cervical cancer biopsies [squamous carcinoma (n=26), adenocarcinoma (n=4)] and matched normal tissue or blood were identified for study due to LOH at 11q23 in the tumor. The slides were prepared using a Leica 1800 Cryostat to cut the tumor tissue into 8 μm slices. After Hematoxylin-Eosin (HE) staining, histology was reviewed with a gynecologic pathologist to mark the areas of tumor epithelium. Next, laser capture microdissection (LCM) was done to isolate tumor cells. For larger sections of tissue, a sterile needle or scalpel was used to resect tumor into DNase-free Eppendorf tubes, and DNA was extracted. DNA was then amplified using primers that encompass a 250 base pair region (-315 to -71) of the promoter region of POU2F3. The PCR setup included 0.2 mmol of dNTPs, 1.5 mmol Mg++, 0.025 units of Platinum Taq polymerase (Invitrogen), and 10 ng of DNA for a 10 μl reaction. The purified products (matched tumor and normal DNAs) were then sequenced using BigDye Terminator v3.1 Sequencing Reagents and an AB 3700 DNA sequencer.

After analyzing the sequences, one known single nucleotide polymorphism (SNP) (rs10892553) was identified. The SNP is T>C transition present in the tumor and normal tissue. If the allele C replaces allele T it will create a new CpG site. While the CpG site is the target of methylation in the promoter region. The heterozygosity of the SNP is 0.17 in our samples. The allele frequency for T is 61.37% and for C, 38.63%. Currently, there is no available frequency data in the SNP databases to compare our results. Another G>A transition was identified in 2 of the 30 matched pairs, which is unknown in current databases. Further work is ongoing to sequence the rest of the region in a larger group of samples.