Absence of CHEK2 1100delC, R145W and I157T Mutations in Breast Cancer in a Moroccan Population

Amal ElAmrani, Khalid Moumad, Mohammed Attaleb, Mustapha Benhassou, Asta Försti, Moulay Mustapha Ennaji, Mohammed El Mzibri, Meriem Khyatti

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Absence of CHEK2 1100delC, R145W and I157T Mutations in Breast Cancer in a Moroccan Population

Amal ElAmrani1, 2, Khalid Moumad1, Mohammed Attaleb3, Mustapha Benhassou4, Asta Försti5, 6, Moulay Mustapha Ennaji2, Mohammed El Mzibri3, Meriem Khyatti1,

1Oncovirology Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco

2Laboratory of Virology, Microbiology and Quality/EBQ, Faculty of Sciences & Technics University Hassan II Mohammedia-Casablanca, Mohammedia, Morocco

3Unité de Biologie et Recherche Médicale, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, (CNESTEN), Rabat, Morocco

4Obstetrics Service "A" Maternité Lalla Meryem, CHU Ibn Rochd Casablanca, Morocco

5Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany

6Center for Primary Health Care Research, Clinical Research Center, Lund University, Malmö, Sweden

Abstract

Mutations in the BRCA1 and BRCA2 genes confer a high risk of breast cancer (BC), although they account for only a small fraction of BC susceptibility. Rare mutations in genes conferring moderate risk may contribute to BC risk. Previous studies have shown that mutations in the CHEK2 gene, which encodes for an upstream regulator of BRCA1, may cause a moderately increased BC risk. In the current study we investigated the status of three founder mutations in the CHEK2 gene (c.1100delC, R145W and I157T) using direct sequencing in 50 BC and 50 control samples. No mutations were detected. This result is in line with the postulated existence of a c.1100delC frequency gradient from the North to the South in Europe with higher frequencies in the Northern countries.

Cite this article:

  • ElAmrani, Amal, et al. "Absence of CHEK2 1100delC, R145W and I157T Mutations in Breast Cancer in a Moroccan Population." Journal of Cancer Research and Treatment 2.1 (2014): 6-9.
  • ElAmrani, A. , Moumad, K. , Attaleb, M. , Benhassou, M. , Försti, A. , Ennaji, M. M. , Mzibri, M. E. , & Khyatti, M. (2014). Absence of CHEK2 1100delC, R145W and I157T Mutations in Breast Cancer in a Moroccan Population. Journal of Cancer Research and Treatment, 2(1), 6-9.
  • ElAmrani, Amal, Khalid Moumad, Mohammed Attaleb, Mustapha Benhassou, Asta Försti, Moulay Mustapha Ennaji, Mohammed El Mzibri, and Meriem Khyatti. "Absence of CHEK2 1100delC, R145W and I157T Mutations in Breast Cancer in a Moroccan Population." Journal of Cancer Research and Treatment 2, no. 1 (2014): 6-9.

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1. Introduction

Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide, accounting for 23% of total new cancer cases and 14% of total cancer deaths [1]. BC is a heterogeneous disease in which breast cells become abnormal and multiply to form a malignant tumor. In about 5 to 10% of cases, BC is due to hereditary genetic background caused by mutations in high penetrance susceptibility genes, which enhance familial risk [2]. About 16% of hereditary BCs can be attributed to germline mutations in the breast cancer 1 (BRCA1) and 2 (BRCA2) genes [3]. Mutations in other genes confer moderate risk of BC development. Among them, the Checkpoint kinase 2 (CHEK2) is a low penetrance gene mutated in less than 3% of BC cases [4].

CHEK2 is a functionally related DNA repair gene; it emerges as an important signal transducer that mediates cellular responses to DNA damage. In response to double-strand DNA breaks, CHEK2 activates p53 by phosphorylation [5]. CHEK2 is also a candidate tumor suppressor whose defects contribute to molecular pathogenesis of diverse types of human malignancies. Worldwide, different CHEK2 mutations have been reported to be associated with BC development. The c.1100delC is the first recurrent mutation in the CHEK2 gene to be reported as an important cause of BC [4]. Since then, numerous studies have documented the prevalence of this single founder mutation in various populations, but up to now few studies have been interested in its clinical impact. Thus far, five deleterious recurrent mutations in CHEK2 have been identified that confer about two-fold elevated risk of BC. These include in addition to the truncating mutation c.1100delC, the missense mutations R145W and I157T, the splice site mutation IVS2 + 1G > A and the large genomic 5,395 bp deletion (del5395) [6].

This preliminary study was planned to assess the frequencies of three CHEK2 mutations (c.1100delC, R145W and I157T) in a case-control study of 50 BC patients and 50 breast controls to evaluate the implication of these mutations in predisposition to BC in Morocco.

2. Materials and Methods

A total of 50 BC patients were recruited at the Obstetrics Service of the Ibn Rochd hospital (Casablanca, Morocco) between 2010 and 2012. Tumor tissue samples were obtained from biopsies collected for clinical diagnosis. The characteristics of the studied population and histopathological data are shown in Table 1. Fifty blood samples from healthy women, aged matched with BC patients, were collected to be used as a control group. The study protocol was approved by the Ethical Committee of Pasteur Institute of Morocco, and written informed consent was obtained from each study subject.

All the BC patients and controls were screened for the c.1100delC, R145W and I157T mutations. Mutations detection was done by PCR amplification and direct DNA sequencing as previously described [7]. PCR primers and the amplimers’ sizes are reported in Table 2.

Amplification reaction was performed in a total volume of 25 µl. The amplification mixture contained 50 pmol of each consensus primer, 200 µM of each dNTP, 0.5 units Taq DNA polymerase (Invitrogen / Life Technologies) and 2 µl of DNA sample in 1x Taq polymerase buffer. A touchdown PCR program was used for PCR amplification. PCR conditions were as follows: 94°C for 1 min; seven cycles of 94°C for 30 s, 60°C for 30 s, 68°C for 30 s; twenty eight cycles of 94°C for 30 s, 54°C for 30 s, 68°C for 30 s, followed by 70°C for 5 min and 12°C thereafter. Mutation positive and negative controls as well as a negative control for the PCR reaction were included in the analysis.

For DNA sequencing, the PCR products were purified by the Exo SaP-IT clean up system (USB, USA) and sequenced directly on an ABI 3130XL DNA analyzer (Applied Biosystems, Foster city, CA, USA), using Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster city, CA, USA), according to manufacturer’s protocol. For each sample, PCR amplification and DNA sequencing were performed twice. The sequences obtained were then compared with the non-mutated reference sequence of CHEK2 gene available in the database Genatlas.

Table 1. Characteristics of the Breast cancer cases

3. Results and Discussion

The CHEK2 c.1100delC heterozygosity may be associated with an increased risk of BC; however, it is unclear whether the evidence is sufficient to recommend genotyping in the clinical practice. Thus, the relevance of CHEK2 mutations as a screening target for an elevated risk of BC is of interest. Indeed, it is widely accepted that the risk of BC may be higher for women who have both a CHEK2 gene mutation and a family history of BC [8].

To our knowledge, no study has been conducted on CHEK2 mutations in Moroccan patients with BC. Therefore, we assessed the prevalence of these mutations in a case–control study of BC among Moroccan women. In the present study, the interest was focused on three CHEK2 variants that are known to affect protein function (c.1100delC, R145W, and I175T). The c.1100delC variant is a protein truncating mutation that abrogates CHEK2 kinase activity. R145W has been reported to have disrupted kinase activity and I175T is deficient in binding to BRCA1 and p53 [9].

In our study, neither cancer cases nor controls showed any of the mutations analyzed. Our data on the absence of the c.1100delC mutation in Morocco are in line with those of the previous studies ([10, 11]) and support the notion that c.1100delC does not contribute to BC susceptibility in Mediterranean populations ([12, 13]). In Spain, Osorio et al. did not detect the c.1100delC mutation in 856 samples analyzed [10]. The c.1100delC was also reported to be absent in Asian population. In North American populations, a very low frequency of CHEK2 c.1100delC mutation was reported [14] (Table 3). Interestingly, the highest frequency of CHEK2 1100delC has been found in patients from the North and the West of Europe, as compared to the southern countries exhibiting the lowest frequency (Italy and Spain) ([10, 11]) (Table 3). These findings are in agreement with the hypothesis of the existence of a c.1100delC frequency gradient from the North-West to the South-East of Europe, caused by an ancestral common origin in the North [15]. This gradient may be more accentuated in the Mediterranean countries which may explain the absence of this mutation in the Moroccan population.

Table 3. Population’s frequencies of the studied Mutations in Chek2 gene

The CHEK2 I157T, a missense variant, alters the kinase activity of the CHEK2 protein. This variant has been reported in ethnically diverse populations and has been associated with a modest risk for developing BC among German and Belarusian populations [16] and for developing colon cancer among Polish and Finnish populations [17] (Table 3).

Other rare variants that adversely affect the CHEK2 protein function have been reported in ethnically diverse populations, and seem to marginally contribute to the overall burden of BC [18].

Our findings suggests that CHEK2 mutations is a rare event and play a negligible role in BC risk in Morocco and therefore the clinical relevance of CHEK2 mutations screening as a cancer predisposing gene is very limited.

Acknowledgement

This paper was supported by EU FP7/2007-2013 grant 260715.

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