Original articles
Loss of Heterozygosity of 14q32 in Colorectal Carcinoma

https://doi.org/10.1016/S0165-4608(98)00242-8Get rights and content

Abstract

Previous allelotyping studies on colorectal carcinoma suggest that loss of heterozygosity (LOH) on chromosome 14q may be a common genetic alteration in this tumor type. The purpose of this study was to determine a precise frequency of LOH at 14q32 region in colorectal carcinomas and to define a minimal region of LOH. LOH at 14q32 in 66 primary colorectal carcinomas were analyzed by polymerase chain reaction-based deletion mapping using six highly polymorphic microsatellites. The average rate of informative data was 68.2% in all cases of colorectal carcinomas analyzed. The average rate of LOH at 14q32 was 44.1%, with the highest frequency of 50.0% at D14S267 locus in the informative cases. The minimal region of LOH was defined by markers D14S65 and D14S250 at 14q32. The present data provide useful information for the isolation of tumor suppressor genes from this region.

Introduction

Recent studies on various tumors, such as colorectal carcinoma, suggest that tumorigenesis proceeds from a series of genetic alterations involving both proto-oncogenes and tumor suppressor genes 1, 2. The inactivation of tumor suppressor genes such as p53 [3] or DCC [4], through deletion or mutation, as well as the activation of dominant-acting proto-oncogenes such as Ki-ras [5] is thought to be important. In addition to those genes, the existence of other such genes has been inferred from observations in which specific chromosomal regions were found to be deleted in tumors [6]. These allelic losses have been interpreted as evidence that the regions affected contain tumor suppressor genes. One strategy to search for the involvement of tumor suppressor genes in a particular tumor type is an allelotype analysis using microsatellite DNA markers [7] to determine whether one of the two parental alleles is lost specifically in tumor DNA.

On chromosome 14 at band q32 there is a region where allelic deletions are frequently observed in various tumors. Recently, several groups have reported the presence of allelic loss at the 14q32 region in several solid tumors including renal cell carcinoma [8], neuroblastoma 9, 10, colorectal carcinoma 11, 12, 13, bladder cancer [14], ovarian carcinoma [15], meningioma [16], and breast carcinoma [17]. In colorectal carcinoma, previous analyses reported by Sasaki et al. [11], Young et al. [12] and Ookawa et al. [13] were mostly performed by restriction fragment length polymorphism (RFLP) analysis. However, those were not precise enough to delimit the locus of the tumor suppressor gene and would be expected from a detailed study for a minimal region of deletion.

The purpose of this study was to determine the precise frequency of loss of heterozygosity (LOH) and to define a minimal region of LOH contributing to the eventual localization of potential tumor suppressor gene at 14q32 in colorectal carcinoma. We performed allelotype analysis with Fluorescent PCR 18, 19 and a PCR (polymerase chain reaction)-SSCP (single-strand conformation polymorphism) 20, 21 method using six PCR-amplified microsatellite polymorphic sites from 14q32. We were able to delimit the minimal region of LOH between markers D14S65 and D14S250.

Section snippets

Patients

From 1991 to 1996, 66 patients with colorectal carcinoma underwent surgery at the Second Department of Surgery/Toyama Medical and Pharmaceutical University School of Medicine or satellite hospitals. The tumors used in this study were primary site cancers that had not been previously treated with chemotherapeutic drugs and were representative for grade, stage, histological subtype, and clinical features. Statistical significance was defined as P < 0.05 compared by the χ2 test.

Frozen Specimens

Tumor and paired

Results

LOH in 66 primary colorectal carcinomas was analyzed by a polymerase chain reaction (PCR)-based deletion mapping using six highly polymorphic microsatellites from the 14q32 region. Distinct evidence of LOH at one or more 14q32 markers was observed in 33 of 66 (50%) tumors. The average rate of informative data was 68.2% in all cases of colorectal carcinomas analyzed. Incidence of LOH varied from about 37.5% to 50.0% among loci examined. The highest incidence (50.0%) of LOH was observed at the

Discussion

Our data demonstrate that about half of all colon carcinomas exhibited an allelic deletion at 14q32. This incidence is in agreement with previous reports of Sasaki et al. [11], Young et al. [12] and Ookawa et al. [13], which were mostly studied by RFLP analysis. In this report, we delimit the minimal region of LOH between markers D14S65 and D14S250. These markers have been cytogenetically mapped to 14q32.1 to 14q32.1–32.2, respectively and span approximately 8 centi-Morgans (cM) [7]. Young et

Acknowledgements

This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, and by a grant from Human Frontier Science Program.

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