New Variations in the Promoter Regions of Human DOCK4 and RAP1A Genes, and Coding Regions of RAP1A in Sporadic Breast Tumors

Abstract

Breast cancer is the most common cancer among women in developed countries. The prevalence of the disease is increasing in the world. Its annual incidence among Iranian women is about 7000 cases. RAP1A, a tumor suppressor gene, is located at 1p13.3 and plays an important role in the cellular adhesion pathway and is involved in the pathogenesis of breast cancer. The DOCK4 gene, which is located at 7q31.1, specifically activates RAP1A gene. In the present study, DNA samples from 64 cases of sporadic breast tumors (referred to Mehrad Hospital in Tehran) were screened using PCR-SSCP method and the number of observed variations compared with the control group (100 normal women). Mutation detection for coding exons of RAP1A gene and the 500 bp upstream of transcription initiation site as promoters of both DOCK4 and RAP1A were carried out and compared with the control group. The promoter region of DOCK4 showed a heterozygous mutation with G>A transition at nucleotide -303 in a fibroadenoma case. With regard to RAP1A we found a heterozygous mutation, G>A transition in an adenoid cystic carcinoma case, and another heterozygous mutation, G>T transversion in an intraductal papilloma case both at nucleotide +45. A homozygous variation, T>A transversion was also found at nucleotide +29 of a fibroadenoma case. The differences in the frequency of variations mentioned above were not statistically significant. However Fisher's exact showed significant difference for T>A transversion. Although, the higher frequency of these mutations and variations may be related to the disease, a larger sample size is needed for the confirmation of our findings.

Keywords: DOCK4; Loss of heterozygosity (LOH); PCR-SSCP; RAP1A; Sporadic breast tumor.

Figure 1

PCR amplified products of DOCK4 and RAP1A genes, M: 100 bp DNA ladder; PRO1, PRO2 and PRO3: Different regions of DOCK4 gene promoter; EX3, EX4 and EX5: PCR amplified fragments (exons 1, 2 and 3) of RAP1

Figure 2

DOCK4 and PRO3 PCR products of patient's samples, M: 100 bp DNA Ladder; S1-S7: samples; The PCR product size is 284 bp and single strand is separated in 500 bp and 560 bp. In sample 5, an obvious band shift is observed

Figure 3

RAP1A. EX3 PCR products from patient's samples, M: 100 bp DNA Ladder marker; S1-S11: RAP1.EX3 PCR products from different samples; the size of PCR products are 174 bp. Two band shifts in single-strand sequences are observed at positions around 290 and 340 bp. There are 2 faint bands of single-strand sequences at positions around 265 bp and 315 bp which are not found in S4 and S8. All bands are more intense in S6