key: cord-0955600-mz9ldlwv authors: Said, Rahma; Jenni, Rim; Boussetta, Sami; Ammous, Feryel; Zouari, Skander; Zaghbib, Selim; Chakroun, Marouene; Derouiche, Amine; Chebil, Mohamed; Ouerhani, Slah title: Association of a common genetic variant (insertion/deletion) in ACE gene with prostate cancer susceptibility in a Tunisian population date: 2021-11-19 journal: J Clin Lab Anal DOI: 10.1002/jcla.24129 sha: 457febc52c73204d4de70b39f0fae24c385aba47 doc_id: 955600 cord_uid: mz9ldlwv BACKGROUND: Angiotensin‐converting enzyme (ACE) plays a pivotal role in several pathologies including cancers. The association of insertion/deletion (I/D) polymorphism of the ACE gene with prostate cancer (PC) risk remains controversial. We aimed to investigate for the first time, to our Knowledge, in North Africa the potential relationship between ACE I/D polymorphism with PC susceptibility and clinical outcomes of PC patients. METHODS: This case‐control study included 143 healthy individuals and 124 patients diagnosed with PC. Using genomic DNA, the samples were genotyped for ACE I/D polymorphism by polymerase chain reaction (PCR). RESULTS: We found that The D allele is significantly associated with an increased risk of PC and D/D + D/I genotypes were at 3 times increased risk of PC ([p = 0.005], OR = 2.95, IC 95% = 1.26–7.09) compared with I/I genotype (p = 0.003, OR = 0.3, IC 95% = 0.12–0.74). We observed an association between D/D and D/I genotypes with advanced age (≥70 years) (p = 0.014; r (2) = 0.22). Furthermore, there is a significant prediction of advanced Gleason score ≥8 based on epidemiological parameters and ACE genotype (p = 0.000; R(2) = 0.349), although no significant association was observed with stage and metastasis. CONCLUSION: The ACE I/D polymorphism is likely to predispose to PC and could play a role in PC progression and aggressiveness. Prostate cancer (PC) is the most frequent cancer diagnosed among men aged more than 40 years. 1 In 2018, this malignancy affects 1.6 million new cases per year in the world. 2 Despite the PC mortality rates have been steadily declining in most western countries including North America, as well as in Western and North Europe causing 358,989 deaths (3.8% of all deaths caused by cancer in men) in 2018, the PC death still important. [2] [3] [4] In Tunisia, PC is considered the third diagnosed cancer in men aged between 40 and 85 years with an incidence of 810 new cases per year. 3 Epidemiological and environmental factors appear to play an important role in the development of this Neoplasm. 3, [5] [6] [7] [8] Moreover, several studies highlight the importance of single nucleotide polymorphisms (SNPs) in the genetic susceptibility, development, and/ or progression of PC. 1, 5, 6, 9, 10 Among these polymorphisms, the genetic variants of the Renin-Angiotensin System (RAS) were well studied in association with cancers. [11] [12] [13] [14] [15] [16] RAS includes Angiotensin-Converting Enzyme (ACE), the key effector peptide of RAS, converts angiotensin-I to angiotensin-II and splits bradykinin into inactive fragments. 11 The alteration of ACE enzyme activity was associated with the development of many diseases such as COVID, 17, 18 hypertension, and cardiovascular diseases (CVDs), 19, 20 renal pathology, 21 Alzheimer disease, 22, 23 polycystic ovarian syndrome, 24 and cancers. 25 An Insertion/Deletion polymorphism in the ACE gene is well described; it consists of either presence or absence of 287 bp Alu repetitive sequences (rs4646994) inserted in the intron 16 of ACE gene, which was reported to account for nearly half of the phenotypic variance of enzyme serum concentration. [26] [27] [28] This genetic variation results in three genotypes I/I, ID, and D/D. 26, 28 ACE I/D polymorphism has widely been studied in association with genetic susceptibility of various cancers including lung, gastric, breast, malignant glioma, laryngeal, hepatocellular carcinoma, renal, and prostate. [29] [30] [31] [32] [33] [34] [35] Previous studies demonstrated the association between this polymorphism and increasing PC risk development in Asiatic and Latino populations. 29, 36 However, its impact on the Caucasian population is still debated. 29 There is a sizeable body of evidence that delineates the implication of ACE I/D polymorphism in the pathogenesis as well as its potential prognosis value in several cancers. [37] [38] [39] It has also been reported that ACE genetic polymorphism in PC is associated with disease progression. 40 To our knowledge, the relationship between ACE I/D polymorphism and PC in North Africa has yet to be explored. Thus, in the present study, we aimed to examine the relationship between ACE I/D gene polymorphism and PC susceptibility, clinicopathological features, and clinical outcomes of a Tunisian population. All participants included in this research provided written informed consent, and the study was approved by the local committee of Charles Nicolle Hospital (17-03-2016). Rigorous inclusion and exclusion criteria were adopted to ensure the credibility of the results (Table S1) Peripheral blood samples were obtained in Ethylenediaminetetraacetic acid (EDTA) at pH = 8 tubes. Genomic DNA was extracted using the standard phenol-chloroform protocol. 41 ACE (I/D) genotype was determined using a polymerase chain reaction, as described previously, 42 followed by electrophoresis. The PCR primers; that target the region of the Alu insertion or deletion of the gene ACE were designed as described 42 and listed in Table S2 . PCR reaction was performed The Hardy Weinberg (HW) was determined using the software package Arlequin (version 3.01). The difference between genotypes and alleles frequencies between PC patients and controls was determined by the χ2 test. Furthermore, odds ratios (ORs) and their 95% Confidence Intervals (IC 95%) were calculated as a measure of the association of the polymorphic sites with PC risk. A p-value was considered significant at <0.05. All statistical analyses except H.W were carried out with version 21 of the SPSS software. Genotype frequencies were in Hardy-Weinberg equilibrium (p > 0.05) ( Table 3 ). The frequencies of the ACE*D allele demonstrate a different distribution of this allele between controls and cases (0.608 and 0.723, respectively) ( Table 3 ). The analysis of the genotypic distribution showed that, in the recessive model, carries of (D/D + D/I) genotypes were found to be at increased risk of PC compared to control subjects harboring the ACE I/I genotype (p = 0.005, OR = 2.95, IC 95% = 1.26-7.09) ( Table 3) (Table 3) . Furthermore, the relationship between ACE genotypes and epidemiological parameters was explored. We found a positive association between D/D or D/I genotypes and advanced age (≥70 years) (p = 0.014; r 2 = 0.22). However, D/D or D/I genotypes in PC patients were not associated with the increased risk of PC in smokers compared to non-smokers (Table 4 ). Furthermore, we investigate the potential prognostic role (Table 5 ). However, the association between epidemiological parameters, genotypes, and advanced stage or metastasis did not yield significant results (p > 0.05). Activation of RAS plays a crucial role in many tissues including, nor- for Tunisian population suggest that the RAS could be a good regulator of prostatic function. Moreover, it has been revealed that the level of RAS particular elements, and sensitivity of tissues on angiotensin could be modulated by the steroid hormones such as androgens. These androgens are responsible for the maintenance and growth of prostate gland. In addition, an inverse association has been shown between the risk of a PC incident and the use of any antihypertensive medication. 48 Moreover, a significant association has been established between ACE I/D gene polymorphism and serum enzyme concentration. 49 Thus, questioning the biological rationale underlying the selection of this candidate polymorphism gene. Our study aimed to investigate whether ACE I/D gene polymorphism is associated with the occurrence of PC and with clinical and epidemiological parameters in a Tunisian population. This is to our knowledge, the first study performed in North Africa to this concern. In this Tunisian cohort, the frequencies of the D allele were 0.608 and 0.723 in controls and patients, respectively, which were different from Latino and Asian ethnicities. 29 We found that the D allele frequency of Tunisian men controls is close to The Iranian men controls and that the D allele frequency of PC Tunisian patients is near to Turkish PC patients. 29 Indeed, carrying of the D allele was as- level. 49 Thus, we think that D allele could play a major role on angiogenesis, proliferation and progress of PC disease. Moreover, we explore the potential prognostic value of ACE polymorphism in PC. We found a positive association between D/D or D/I genotypes and advanced age (≥70 years). We were also able to detect a significant correlation between advanced Gleason grade (≥8) and epidemiological parameters with D/D or D/I genotypes. However, we failed to demonstrate any association between ACE of environmental and lifestyle, which could interact with ACE genetic variants. A limitation of our study is the relatively small sample size in part due to the low incidence of the disease in Tunisia. Further analysis on a larger cohort could help better to understand the significance of ACE I/D polymorphism in the pathobiology of PC and its contribution to the aggressiveness of the disease. In summary, our study provides the first evidence of a correlation between ACE I/D gene polymorphism and PC susceptibility in a Tunisian population and its potential prediction of clinical behavior. ACE I/D polymorphism may improve PC prognostication. Further studies are needed to explore other ACE gene polymorphisms and their expression level, in the Tunisian population, and the mechanisms by which they contribute to PC development and prognosis. The teamwork would like to express their thanks and gratitude to patients and controls for their participation. This work is supported by the Ministry of Higher Education and Scientific Research in Tunisia. The authors all report that they have no conflicts of interest in this study. Data sharing does not apply to this article as no datasets were generated or analyzed during the current study. 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