Original article
Prevalence of mutations in APC, CTNNB1, and BRAF in Tunisian patients with sporadic colorectal cancer

https://doi.org/10.1016/j.cancergencyto.2008.06.016Get rights and content

Abstract

Sporadic colorectal tumorigenesis is caused by alterations in the Wnt (APC, CTNNB1) and Ras pathways. Our objective was to analyze the occurrence of these genetic alterations in relation to tumor and patient characteristics. The prevalence of somatic alteration in the hot-spot regions of the APC, BRAF, and CTNNB1 genes was investigated in 48 unselected and unrelated Tunisian patients with sporadic colorectal cancer, and the association between the molecular features at these genes in relation to tumor and patient characteristics (age at diagnosis, sex, tumor localization, stage, and differentiation) was analyzed. Loss of heterozygosity was observed at the APC locus in 52% of the analyzed tumors. 6 novel mutations were detected by polymerase chain reaction sequencing in the mutation cluster region of the APC gene. No mutations were observed in the CTNNB1 gene in any tumor, but 8% of tumors harbored mutation in the BRAF gene. Clinicopathological analyses showed an association between APC point mutations and the earliest occurrence of sporadic colorectal cancer. The findings confirm the heterogeneity of APC gene alteration and also reveal a particular profile of this pathology among Tunisian patients that confirms the epidemiological data for this country.

Introduction

Colorectal cancer (CRC), the second most prevalent cancer worldwide [1], is a consequence of a combination of environmental factors and an accumulation of specific genetic alterations. The molecular pathology of CRC is the most studied among cancers, and cancer-related genetic alterations have been discovered. Based on these results, Fearon and Vogelstein [2] proposed an adenocarcinoma sequence model of colorectal carcinogenesis.

Mutations in the adenomatous polyposis coli gene (APC) (OMIM 175100; http://www.ncbi.nlm.nih.gov/sites/entrez) have been reported to be the earliest and most frequent mutational event in this adenocarcinoma sequence [3], [4], [5], [6]. APC is considered to be a tumor suppressor gene. Located on chromosome region 5q21∼q22 [7], APC is composed of 15 exons and encodes a multifunctional protein (the APC protein) involved in the Wnt signaling pathway (the wingless signal transduction pathway) [8]. APC is mutated not only in familial adenomatous polyposis, but also in 80% of sporadic colorectal cancer cases [9], [10], [11]. Loss of heterozygosity (LOH) at the APC locus seems to be the predominant mutational event in the colorectal carcinogenesis [[10], [12], [13]]. Until today, >700 somatic mutations have been cited in this gene [14]. The majority of them (95%) are nonsense or frame-shift mutations leading to a truncated protein product with an abnormal function.

The CTNNB1 gene (OMIM 116806), which encodes the β-catenin protein, is another Wnt pathway component described as a target for mutations in sporadic colorectal cancer [15], [16]. Mutations of either CTNNB1 or APC genes render the β-catenin resistant to degradation and so contribute to tumorigenesis [15].

Novel activating mutations in sporadic CRC have recently been identified that suggest other genetic pathway alterations for CRC. Alterations involving the BRAF gene (OMIM 164757) which is an element of the RAS/RAF/MEK/MAPK kinase pathway are the cause of an increased kinase activity, leading to excessive activation of the above mitogenic pathway and to uncontrolled proliferation of colorectal cancer cells [17], [18]. The most prevalent point mutation, which accounts for ∼80% of all mutations in the BRAF gene in CRC, is V600E, corresponding to a conversion of valine 600 to glutamic acid [17], [18], [19].

In Tunisia, CRC is considered to be the prime digestive cancer, with an incidence of 7.4 new cases per 100,000 inhabitants annually [20]. It affects with a predilection young adults (the mean age is 10 years younger than reported for occidental series), and 14.2% of Tunisian CRC patients are affected before age 40. All these data suggest a profile of this pathology particular to Tunisia.

Our objective was to analyze the mutation spectra in APC, CTNNB1, and BRAF genes in relation to tumor and patient characteristics.

Section snippets

Patients

The study sample was 48 tumors from unrelated sporadic colorectal cancer patients (25 women, 23 men; median age 61 years ± 13) with no family history of cancer. Patients were recruited from the Charles Nicolle Hospital of Tunis. They were classified on the bases of their histopathological profiles.

Genomic DNA isolation

Genomic DNA was extracted from tumor and corresponding normal tissue in paraffin-embedded blocks using the DNeasy tissue kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Only

Results

We investigated 48 colorectal tumor cases to evaluate LOH at the APC gene using three informative markers described by Kapitanović et al. [21]. This analysis showed 17 of 48 cases (35%) with evidence for APC LOH in at least one marker. Three harbored with the APC LOH a truncating mutation in the mutation cluster region (codon 1240–1513). Comparison of LOH-negative and LOH-positive genotypes between the available clinicopathological data of our patients shows no significant differences (Table 2

Discussion

The present study established the somatic mutation profiles of sporadic CRC in tumors of Tunisian patients, focusing on three genes (APC, BRAF, and CTNNB1).

The major alterations in the tumor tissue of sporadic CRC cases involved the APC gene in 25 of 48 cases (52%). The main mutations in this locus were the loss of heterozygosity and truncating point mutations. Although our mutation analysis was restricted to the mutation cluster region of the APC, this result confirms that most somatic

Acknowledgments

The author would like to thank Dr. Houssein Khodjet El Khil, Dr. Chiraz Bouchlaka (Laboratoire de Génétique, Immunologie et Pathologies Humaines de la Faculté des Sciences de Tunis), and Prof. Hamza Nejla (Professor of English at the faculty of sciences of Tunis) for their kind assistance during the writing of this manuscript.

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    Both authors contributed to the work equally, and each should be considered first author.

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