Carriage of Ser217Leu and Ala541Thr Variants of ELAC2 Gene and Risk Factors in Patients with Prostate Cancer in Burkina Faso

Background Genetic factors are one of the significant contributors to prostate cancer (PCa) development, and hereditary prostate cancer 2 (HPC2) locus gene ELAC2 is considered a PCa susceptibility region. The HPC2/ELAC2 gene has been identified by linkage analysis in familial prostate cancer patients in the United States but has never been studied in Burkina Faso. The objective of the present study was to analyze the carriage of the C650T (Ser217Leu) and G1621A (Ala541Thr) mutations of the ELAC2 gene and the risk factors in prostate cancer patients in Burkina Faso. Methods This case-control study included 76 participants, including 38 histologically confirmed prostate cancer cases and 38 healthy controls without prostate abnormalities. PCR combined with restriction fragment length polymorphism (RFLP) was used to characterize the genotypes of the Ser217Leu and Ala541Thr polymorphisms of the ELAC2 gene. The correlations between the different genotypes and risk factors for prostate cancer were investigated. Results The C650T mutation was present in 44.73% of prostate cancer cases and 47.37% of controls. The G1621A mutation was present in 26.32% of prostate cancer cases and 15.79% of controls. We did not detect an association between prostate cancer risk and the Ser217Leu (p=0.972) and Ala541Thr (p=0.267) variants of the ELAC2 gene. Also, the two ELAC2 SNPs did not correlate with clinical stage, prostate-specific antigen (PSA) level at diagnosis, or the Gleason score on biopsies. However, we found that 100% of homozygous carriers of the T650 mutation have an A1621 mutation (p ≤ 0.001). Conclusion Ser217Leu and Ala541Thr polymorphisms of ELAC2, considered alone or in combination, are not associated with prostate cancer risk.


Introduction
Worldwide, approximately, 19.3 million new cancer cases and nearly 10 million cancer deaths occurred in 2020 [1]. Prostate cancer (PCa) is the second most common malignancy (after lung cancer) [1], with 1,414,259 new cases and 375,304 deaths (3.8% of all cancer deaths in men) in 2020. In Burkina Faso, it is the most common malignant tumor in men. In the same year, PCa accounted for 8.30% of incident cancer cases (997 cases) and 7.00% of cancer deaths (608) [2]. Although the exact etiology of prostate cancer remains elusive, well-established risk factors include advanced age, ethnicity, and family history [3]. Populations of African ancestry, such as African Americans, Caribbeans, and blacks in Europe, had the highest incidences, earlier disease, and more aggressive form compared with other racial and ethnic groups [4]. Men of African ancestry are estimated to have a relative risk of 9.7 versus 3.9 for Caucasians and 1.6 for Asians, when two or more frst-degree relatives have PCa [5]. Regarding family history, more than 20% of patients with prostate cancer report a family history [6]. Tis is partly due to shared genes but also due to a similar pattern of exposure to certain environmental carcinogens and common lifestyles [3,6]. Te prostate-specifc antigen (PSA) test and digital rectal exam (DRE) are currently the main methods of noninvasive prostate cancer screening [7]. Tese tests are not specifc because some of the PSA levels tested may result in false positives due to infection or hyperplasia. With the ultimate goal of developing new, more accurate, and benefcial biomarkers in the detection, prevention, and treatment of this disease, several genome-wide linkage and association studies (GWAS) have been conducted to elucidate the molecular mechanisms involved in the genesis and progression of prostate cancer [8]. Tavtigian et al. in 2001 [9] identifed the HPC2/ELAC2 gene on chromosome 17p12 as a prostate cancer susceptibility gene in large, high-risk pedigrees in Utah. Subsequently, several other loci associated with inherited forms of PCa were identifed, including HPC1 (1q24-25), HPC20 (20q13), HPCX (Xq27-28), PG1 (prostate cancer susceptibility gene 1, 8p22-23), and CAPB (1p36) [10]. Two additional inherited PCa susceptibility genes have been identifed at two of these loci. Tese are the RNASEL (2'-5'-oligoadenylate-dependent ribonuclease L) and MSR1 (macrophage scavenger receptor 1) genes [11][12][13]. However, mutations in these diferent genes have low to moderate penetrance. Te ELAC2 gene located at position 17p12, with a size of 26.454 kb, having 25 exons, encodes a metal-dependent hydrolase of 826 amino acids (of 92 kilo-Dalton) potentially involved in the repair of DNA interstrand cross-linking and mRNA editing [14,15]. Sequence analysis of HPC2/ ELAC2 identifed four sequence variants, including a rare frameshift and three missense changes, two of which were common in the study populations [9,[16][17][18]. Tese two missense mutations C650T and G1621A result in a change of the amino acid serine to leucine at position 217 and a change of alanine to threonine at position 541, respectively. However, conficting results regarding the association of ELAC2 gene polymorphisms and the occurrence of PCa have been obtained in diferent populations around the world, testifying to the genetic complexity and heterogeneity of prostate cancer susceptibility [17,[19][20][21][22][23][24][25]. In West Africa, and specifcally in Burkina Faso, several studies on the epidemiological and therapeutic aspects of PCa have been carried out, but only a few studies on the genetic and molecular aspects. It is with this in mind that this preliminary study was conducted to determine the involvement of the Ser217Leu (C650T) and Ala541Tr (G1621A) variants of the ELAC2 gene in prostate cancer in the Burkinabe population. Tis could provide additional information that could potentially be exploited for early screening and diagnoses of high-risk individuals for early therapeutic intervention or ease of management.

Design of Study.
Tis is a case-control study that was conducted from September, 2019 to January, 2021. It included men, regardless of ethnicity, at least 60 years old (for controls), with a positive (cases) or negative (controls) histological diagnosis of prostate adenocarcinoma who consented to participate in the study. Tis study involved a population of all professions and social categories. Seventysix (76) men were selected, including 38 cases and 38 controls. All patients were followed at the urology department of the Saint Camille Hospital in Ouagadougou (HOSCO) or the Nina clinic in Ouagadougou. Biomolecular analyses were performed at the Laboratory of Molecular and Genetic Biology (LABIOGENE) of the Joseph KI-ZERBO University in Ouagadougou and the Pietro Annigoni Biomolecular Research Center (CERBA) in Ouagadougou (Burkina Faso).

Sample Collection.
After consent was obtained from the patients (cases and controls), a questionnaire was distributed to collect sociodemographic and clinical data from the participants. Ten, for each consenting participant, venous blood (5 ml) was collected in tubes impregnated with ethylenediaminetetraacetic acid (EDTA). After centrifugation at 3500 g for 15 minutes, the plasma and pellets were collected in cryotubes and stored at −20°C at CERBA, pending nucleic acid extraction.

Quantifcation of Total PSA.
Te sera were used for the determination of total PSA in the HOSCO laboratory on the Cobas ® 6000 Analyzer (Basel, Switzerland) using the Elecsys ® Total PSA reagent (Roche, Basel, Switzerland) employing the electrochemiluminescence (ECLIA) method.

Genotyping.
Genomic DNA was extracted from the pellet using the "Rapid Salting Out" technique, described by Miller et al. in 1988 [26]. Te two single-nucleotide polymorphisms (SNPs) in the HPC2/ELAC2 gene were genotyped for all subjects using PCR-RFLP, as described by Xu et al. [20]. In brief, conventional PCR was performed for the amplifcation of regions carrying the Ser217Leu and Te resulting PCR products were subjected to enzymatic digestion with the enzyme Taq α I, 20,000 units/mL (New England Biolabs, Paris, France) at 65°C for 3 hours for the Ser217Leu polymorphism (with one restriction site on the amplifed fragment) and Fnu4HI, 20,000 units/mL (New England Biolabs, Paris, France) at 37°C for 3 hours for the Ala541Tr variant (with three restriction sites on the amplifed fragment). Te total reaction medium of 25 μL included 5 μL of 1X enzyme bufer, 0.5 μL of the enzyme, 14.5 μL of sterile PCR water, and 5 μL of PCR product. Digestion products were subjected to 2% agarose gel electrophoresis for 45 minutes and visualized under UV light at 132 nm using an image analyzer VILBER (Baden-Württemberg, Germany). Te PCR-RFLP patterns for the Ser21Leu and Ala541Tr polymorphisms are indicated in Figure 1.

Statistical
Analysis. Data were entered into Excel 2016 spreadsheet and analyzed using Stata version 13.0 (https:// www.Stata.com) and IBM ® SPSS software (https://www. IBM.com). Association of categorical variables was performed using the chi-square test and considered signifcantly diferent at p < 0.05. Diferences in genotype frequencies between cases and controls were tested using standard chisquare tests. Odd ratio (OR) and 95% confdence interval (CI) were calculated by genotype with Epi infoTM 7 software (Center for Disease Control and Prevention, Atlanta, Georgia, USA). Table 2 summarizes the sociodemographic characteristics and behavioral risk factors selected for PCa. Te age of the cases ranged from 55 to 84 years (mean age 69.81 ± 8.05 years), while the age of the controls ranged from 60 to 90 years (mean age 69.11 ± 6.46 years), indicating no diference in the mean age of the case and control groups (p � 0.668). Te age distribution at diagnosis showed that 59.46% of the cases were diagnosed between 50 and 70 years of age and 40.54% over 70 years of age. Te mean age at diagnosis was 67.46 ± 8.03 years. A total of 63.15% of PCa patients and 50.00% of controls were alcohol users. Tere was no diference in the fractions of alcohol consumers and non-consumers among the case and control groups (p � 0.354). Regarding the presence of a family history of PCa, 36.84% of cases answered yes versus 28.95% of controls, indicating a non-statistically signifcant diference (p � 0.551).

Patients' Plasma PSA Level and Gleason
Score of the Prostate Gland at Diagnosis. Te range of patients' diagnostic PSA at diagnosis levels and the Gleason scores of the prostate is shown in Table 3. Te majority of cases (81.6%) had a total PSA level at diagnosis greater than 20 ng/mL (range 22.5 ng/mL to 4028.33 ng/mL). Te Gleason score of 7 was the most represented (60.5%).

Allelic Frequencies.
We frst compared the allele frequencies regarding Ser217Leu and Ala541Tr polymorphisms in HPC2/ELAC2 gene in Burkinabe men with PCa and control subjects. Ser217Leu and Ala541Tr polymorphisms were in the Hardy-Weinberg equilibrium in our study population. Te diferences in the allele frequencies for Ser217Leu polymorphisms (C and T alleles) and Ala541Tr polymorphisms (G and A alleles) are shown in Table 4. Tere was no signifcant diference in the frequency of C or Talleles (p � 1.000) or G and A alleles (p � 0.289) between the case and control groups (Table 4).

Genotypic Frequencies of the ELAC2 Gene and Association
with Prostate Cancer. Te diferences in genotypic frequencies of the Ser217Leu polymorphisms (C and T alleles) and the Ala541Tr polymorphisms (G and A alleles) are shown in Table 5. Te genotypic frequencies of the Ser217Leu variant were 55.27% for the wild-type CC, 39.47% for the heterozygous CT, and 5.26% for the homozygous mutant TT in cases, and 52.63%, 42.11%, and 5.26% in controls, respectively (p � 0.972). For the Ala541Tr variant, the genotypic frequencies for the wild-type GG, heterozygous GA, and homozygous mutant AA were 73.68%, 26.32%, and 0.0% in patients and 84.21%, 15.79%, and 0.0%, respectively, in controls (p � 0.267). No correlation between Ser217Leu and Ala541Tr polymorphisms and the occurrence of prostate cancer was obtained. However, for the Ala541Tr polymorphism, the GA genotype was associated with a family history of PCa (OR � 4.57 (1.13-18.47); p � 0.050).  [20].

Variant
Primers Size (pb) Prostate Cancer 3 Also, 87.5% of GA genotype carriers had at least one T allele of the Ser217Leu polymorphism.

ELAC2 Genotypes and Gleason Score of the Disease.
Te diferences in the allele frequencies for Ser217Leu polymorphisms (C and T alleles) and Ala541Tr polymorphisms (G and A alleles) are shown in Table 4. Tere was no signifcant diference in the frequency of C or Talleles (p � 1.000) or G and A alleles (p � 0.293) among the case and control groups (  Ala541Tr polymorphism. PCR-amplifed DNA fragments containing the Ala541Tr polymorphisms were digested with Fnu4HI and then subjected to 2% agarose gel electrophoresis. Te AA mutant genotype (not found in the population), reportedly generated DNA fragments of 165 bp, the GA genotype generated three DNA fragments of sizes 165 bp, 110 bp, and 55 bp, and the GG wild type generated DNA fragments of sizes 110 bp and 55 bp. .44%, and 0.00% of carriers of the GG, GA, and AA genotypes, respectively, had a Gleason score ≥7 (Figure 2). No signifcant association was found between these variants and the Gleason score (Ser217Leu: p � 0.725, Ala541Tr: p � 0.072).

Associations of Ser217Leu and Ala541Tr
Polymorphisms with PSA at Diagnosis. All patients (100%) with the TT mutated genotype of Ser217Leu had a PSA >20 ng/ml. In contrast, carriers of the CC (19.05%) and CT (21.43%) genotypes had PSA levels between 5.38 and 20 ng/ml. For Ala541Tr, 81.48% of GA heterozygous patients had PSA levels above 20 ng/ml. In contrast, the majority (55.56%) of patients with the wild-type GG genotype had the lowest PSA levels at diagnosis (Figure 3). However, no association was found between these mutations and a PSA level at diagnosis greater than 20 ng/ml (Ser217Leu: p � 0.773; Ala541T: p � 0.925).

Discussion
Many studies have reported that black men are diagnosed with prostate cancer at a younger age [27,28]. We obtained a mean age of patients at diagnosis of 67.46 ± 8.03 years with an age range of 51 to 81 years, similar to the age range found in Burkina Faso in 2022 and Cameroon (67.8 ± 7.44 years) in 2019 [25,29]. Tese results could be attributed to late diagnosis and the low rate of prostate cancer screening programs in Burkina Faso, highlighting the high PSA levels at diagnosis obtained in our study (mean 642.12 ± 1153.42 ng/ml). Tese levels are almost similar to those obtained (mean PSA of 537 ng/ml et 627.85 ng/ ml) in a previous study in Burkina Faso [29,30]. Regarding the Gleason score, 73.65% of the patients had a Gleason score of 7 or higher. One of the established risk factors for prostate cancer is the presence of a family history. Te risk would be increased when there are more than three 1 st and/or 2 nd -degree relatives with PCa [31]. In our study, only 36.84% of patients reported a family history of prostate cancer. Te ELAC2 gene was genotyped in 76 study participants and the frequency of each genotype was determined. For the Ser217Leu polymorphism, the frequencies of the genotypes, CC, CT, and TT were 55.27%, 39.47%, and 5.26% in cases, and 52.63%, 42.11%, and 5.26% in controls, respectively (p � 0.972). For the Ala541Tr variant, the frequencies of the GG, GA, and AA genotypes were 73.68%, 26.32%, and 0.0% in cases and 84.21%, 15.79%, and 0.0% in controls, respectively (p � 0.267). Te AA mutated genotype was not found in our study population. Te HPC2/ELAC2 locus was frst discovered as a prostate cancer susceptibility gene through a genetic linkage study by Tavtigian et al. in 2001 [9]. However, most subsequent studies have focused on its role as a common gene with low penetrance [32,33]. In our study, we did not fnd a direct correlation between the Ser217Leu (p � 1.000) and Ala541Tr (p � 0.293) polymorphisms in the ELAC2 gene and the occurrence of prostate cancer. Our results are similar to those found by Suzuki et al. [23] in Japan, Stanford et al. [17] in the United States, Shea et al. [34] in the Afro-Caribbean population of Tobago, and Meitz et al. [35] in the United Kingdom. In general, the meta-analysis by Xu et al. [21] obtained evidence of an association between the Ser217Leu polymorphism and prostate cancer risk in Caucasians and Asians, but not in Africans. In addition, Xu et al. [21] found that the Ala541Tr polymorphism was associated with an increased risk of prostate cancer in Asians but not in Caucasians and Africans in all genetic models (somatic and inherited). However, several other studies show rather contradictory results regarding the association between Ser217Leu and Ala541Tr variants and prostate cancer risk. Indeed, Yokomizo et al. [19] in Japan found that only the Tr541 allele was associated with an increased risk of PCa; they obtained a signifcantly higher frequency of this allele in PCa patients (8.4%) compared to the control group (2.1%) (p � 0.003). In contrast, Robbins et al. [36] found that only the Leu217 allele was signifcantly associated with prostate cancer risk in the African American population (p � 0.030). Comparing the frequencies of carriers of both mutations simultaneously in cases and controls, we found that 100% of homozygous carriers of the T650 mutation have an A1621 mutation (p ≤ 0.001). Rebbeck et al. [32] found that the A1621 mutation was only observed in men who also carried T650 and that the probability of developing prostate cancer was increased in men who carried the Leu217/Tr541 variants simultaneously (OR � 2.37, 95% CI 1.06-5.29, and p � 0.040). To determine whether the Ser217Leu and Ala541Tr mutations might be involved in increased disease severity, we compared PSA levels and prostate Gleason scores to genotype frequencies. Te result showed that the mutations were not involved in the increased disease severity (p > 0.050).
Te diferences between our results and those of other studies could be attributed to the diference in sample size on the one hand and on the other hand, to the diferent ethnic origins and geographical locations of the various

Conclusion
Our preliminary study found no direct association between the Ser217Leu and Ala541Tr variants of the HPC2/ELAC2 locus and prostate cancer risk. Te patients included in this study had advanced disease. A larger study is needed to better understand the role of diferent SNPs of ELAC2, other risk factors, and other genes in prostate cancer predisposition, which may beneft early diagnosis of the disease in at-risk individuals and management of the disease.

Data Availability
All data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Te CERBA/LABIOGENE institutional ethics committee approved the protocol. Te data were processed in strict compliance with anonymity. Te study conforms to the ethical guidelines of the 1975 Declaration of Helsinki.

Consent
Participants gave their free and informed consent on a consent form.

Conflicts of Interest
Te authors declare that they have no conficts of interest regarding the publication of this work.

Authors' Contributions
Aïda and Jacques conceived and designed the study. Aïda, Bienvenu, Abdou, and Zohoncon were involved in patient recruitment, data generation, collection, and assembly. Aïda, Albert, Lassina, Hermann, Bapio, Tovo and Pegdwendé, Essona, Kalifou, and Clarisse were involved in data extraction, analysis, and interpretation. Aïda and Abdou were involved with drafting or revising the manuscript. Florencia and Jacques provided administrative, technical, and material support. Jacques supervised the study. All authors critically revised and approved the fnal version of this publication.