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Hindawi Advances in Cell and Gene erapy Volume 2023, Article ID 6344652, 8 pages https://doi.org/10.1155/2023/6344652 Research Article The Association between Genetic Variants and Gene Expression in RAAS Genes Using Captive-Bred Vervet Monkeys (Chlorocebus aethiops) 1,2 2 1 1 Sanele Khoza , Meenu Ghai , Chesa G. Chauke , and Zandisiwe E. Magwebu South African Medical Research Council, Primate Unit and Delft Animal Centre (PUDAC), Cape Town, South Africa University of KwaZulu Natal, School of Life Sciences, Genetics, Westville, South Africa Correspondence should be addressed to Sanele Khoza; firstname.lastname@example.org Received 1 August 2022; Revised 27 December 2022; Accepted 1 February 2023; Published 17 February 2023 Academic Editor: Loree Heller Copyright © 2023 Sanele Khoza et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mendelian genetics contribute largely to the development of hypertension; therefore, the identiﬁcation of genetic variants related to blood pressure (BP) regulation remains crucial and may reveal new therapeutic drug targets. The purpose of the present study was to screen the captive-bred Vervet colony for salt-sensitive sequence variants or single nucleotide polymorphisms (SNPs) in the selected Renin-Angiotensin-Aldosterone System (RAAS) genes associated with salt sensitivity. Blood samples were collected from 16 captive-bred Vervet monkeys for genotyping and gene expression analysis. The impact of the identiﬁed sequence variants was determined using online prediction tools. Sanger sequencing analysis revealed 21 sequence variants in AGT, CYP3A5, GRK4, and SCL4A5, of which 19 were novel and two were previously reported in humans. All novel variants were either predicted to be polymorphic, disease-causing, or possibly damaging by prediction tools. Furthermore, the mRNA expression for AGT was signiﬁcantly higher in the normal BP group (p value = 0.02), and a similar trend was observed for CYP3A5 and GRK4, whereas SCL4A5 was higher in the hypertensive group. The identiﬁed salt-sensitive variants speciﬁcally in GRK4 may be suggestive to be the attributing factor of the elevated BP levels in these captive-bred Vervet monkeys. Therefore, RAAS variants could be considered as a biomarker to identify the potential risk of developing hypertension in both humans and nonhuman primates. 1. Introduction deleterious while a low-sodium diet is regarded as part of a healthy lifestyle and treatment of hypertension . Although Hypertension is a public health concern and can lead to car- salt sensitivity, hypertension, and related cardiovascular dis- diovascular, cerebrovascular, and kidney diseases . The ease (CVDs) result from the interaction of genes with envi- prevalence of hypertension varies with age, sex, and ethnic- ronmental factors such as stress and diet , the ity. Currently, the population data indicates that the inci- underlying mechanism of salt sensitivity is not well under- dence of hypertension ranges from 6.6 to 26 million in stood . Several studies have proved that Mendelian genet- developed countries while in developing countries it is 639 ics contribute largely to the development of hypertension. million, and it is projected that by 2025, the number of Evidence from previous reviews estimated that genes con- patients with hypertension would be 1.56 billion globally tribute 30–50% to the pathogenesis of BP [10, 11]. There- [2, 3]. Excess dietary salt intake has been reported to pre- fore, the identiﬁcation of genetic variants related to BP dominantly contribute to hypertension , and this is asso- regulation remains crucial and may reveal new therapeutic ciated with increased cardiovascular events and mortality drug targets. irrespective of basal blood pressure (BP) levels . In animal Several Renin-Angiotensin-Aldosterone System (RAAS) studies, salt-resistant rats do not develop an elevated BP genes have been suggested to play a vital role in BP response after being fed high-salt diet (8% NaCl), while sensitive rats to salt . These genes include the sodium bicarbonate show elevated BP . This means that a high-sodium diet is cotransporter gene (SLC4A5), G protein-coupled receptor 2 Advances in Cell and Gene Therapy kinase 4 (GRK4), cytochrome P450 3A5 (CYP3A5), and BP and another one was hypertensive while the rest of the angiotensinogen (AGT). SLC4A5 is a transmembrane pro- animals were from independent families (family C, D, E, F, tein that functions as an electrogenic cotransporter of bicar- G, and H). Additionally, three animals were wild caught bonate and sodium , and it is known to be associated from Modderfontein farm, Potchefstroom, in South Africa with hypertension of which ﬁve polymorphisms have been before being housed at PUDAC in 2012 and two of these reported [14, 15]. The GRK4 gene regulates dopamine recep- animals were infertile. tors, which are important in regulating sodium transport and BP . In hypertensive patients, three polymorphisms 2.2. Clinical Assessment, DNA Extraction, and (R65L, A142V, and A486V) have been reported to show an Quantiﬁcation. Animals were handled after chemical increase in GRK4 activity in the renal tubule and cause phos- restraint with Ketamine (Kyron Laboratories, South Africa) phorylation and agonist-independent uncoupling of dopa- at 10 mg/kg. Once an animal was fully unconscious, pheno- mine 1 receptor (D1R) [17, 18]. Additionally, type traits such as BP, peripheral capillary oxygen saturation overexpression of GRK4 causing hypertension has been percentage (SpO2 %), body weight, and heart rate were mea- observed in transgenic mice that were fed high-salt diet sured. Thereafter, blood (2–4 mL) was collected via femoral (A486V) and on a regular diet (A142V) [19, 20]. In South venipuncture into 4 mL Vacutainer® EDTA Tubes for geno- Africa, GRK4 polymorphisms are more common in the Afri- mic DNA extraction using a NucleoSpin Genomic Blood can black population and are associated with impaired DNA puriﬁcation kit (Macherey-Nagel, Germany). The con- sodium excretion . Moreover, the CYP3A5 gene has centration, quantity, and purity of DNA were immediately been reported to play a role in sodium reabsorption and measured using NanoDrop 2000 spectrophotometer analysis BP regulation in the presence of a premature stop codon (Vacutec, South Africa), and DNA quality was conﬁrmed by (rs776746) that inﬂuences salt and water retention and standardizing 2% gel electrophoresis that was stained with reduces its expression . AGT is among the components 2 μL of ethidium bromide (EtBr). that are involved in the activation cascade of the RAAS, which act together to regulate BP by maintaining vascular 2.3. Candidate Gene Selection and Sequence Retrieval for tone and the balance of water and sodium . Genotyping. Bioinformatics research tools such as the NCBI In this study, four RAAS genes (AGT, CYP3A5, GRK4,and GENBANK, Ensembl, and UCSC genomic browser were SLC4A5) were screened for sequence variants followed by gene used to retrieve the genomic sequences for the selected genes expression analysis to investigate the association between salt (AGT, CYP3A5, GRK4, and SLC4A5) using the Green mon- sensitivity and hypertension development using the captive- key (Chlorocebus sabaeus) as a reference sequence bred Vervet monkey model. To date, several studies have dem- (Table S2). Furthermore, NCBI primer BLAST and onstrated nonhuman primates (NHPs) to be eﬀective research PrimerQuest Tool (Whitehead Scientiﬁc, South Africa) models to evaluate the diseases aﬄicting humans. NHP is also were employed to design primers targeting the coding known to be an excellent animal model for various noncom- exons for all the selected genes (Table S3). municable diseases (NCDs) and shares various characteristics with humans . These include the sodium-lithium counter 2.4. PCR Ampliﬁcation and DNA Sequencing. Selected genes transport activity (SLC) in their red blood cells which can lead were ampliﬁed by PCR using the Veriti™ 96-Well Thermal to salt sensitivity . Therefore, the use of the Vervet model Cycler (Applied Biosystems®, USA). Each standard PCR is proposed in this study as a model of choice to better under- reaction (25 μL) consisted of the following reagents: GoTaq stand the genetic contribution of RAAS genes and ﬁnd new PCR Master Mix (2x) (Promega, USA), 2 mmol/L forward −1 therapeutic approaches to combat hypertension. and reverse primer, 50 ng μL DNA, and nuclease-free water. The cycling program was similar to the one that has been previously published . Brieﬂy, cycling conditions 2. Materials and Methods included denaturation at 94 C for 5 minutes, followed by 2.1. Animal Selection. The ethical clearance was obtained 30 cycles at 94 C for 30 seconds, varying annealing (40– ° ° from the Ethics Committee for Research on Animals at the 70 C) for 30 seconds (Table S3), and extension of 72 C for South African Medical Research Council (SAMRC) (ECRA; 1 minute, followed by a ﬁnal extension of 72 C for 5 Ref 10/18). Sixteen captive-bred Vervet monkeys were minutes. The PCR products were puriﬁed using the selected based on age, gender, BP, and lipogram parameters Wizard SV gel and PCR clean-up kit (Promega, USA). (Tables 1, S1). Sample collection procedure and housing Based on the gel electrophoresis results, puriﬁed PCR conditions were according to the Primate Unit Delft Animal products were sequenced and analyzed using ClustalW and Center (SAMRC/PUDAC) standard operating procedures ExPASy translate tool. The impact of the identiﬁed and the revised South African National Standard for the sequence variants was evaluated using online tools such as Care and Use of Animals for Scientiﬁc Purposes (South Afri- Polymorphism Phenotyping (PolyPhen-2)  and Sorting can Bureau of Standards, SANS 10386, 2008). Intolerant from Tolerant (SIFT) website which is provided The pedigree demonstrated two families with half- by Pauline Ng that predict whether a change in amino acid siblings (Figure S1), in which four animals were from the aﬀects protein function. The Ensembl transcript ID of each same family tree, consisting of three half-siblings with gene was selected in MutationTaster , Human Splice normal BP and one half-brother who was hypertensive Site Finder (HSF) , and Variant Eﬀect Predictor (VEP) (family A). Family B had two half-sisters, one with normal  to identify the impact of each variant. Advances in Cell and Gene Therapy 3 Table 1: Clinical parameters. Parameters Normal group Hypertensive group p values Age >3–10 years >3–10 years Gender 4 M and 4 F 4 M and 4 F 103:2±5:343 167:6± 10:53 Systolic BP (mmHg) <0.0001 46:31 ± 8:094 72:81 ± 8:52 Diastolic BP (mmHg) 0.04 4:05 ± 0:2353 3:6± 0:2659 Total cholesterol (mmol/L) 0.23 0:5125 ± 0:1015 0:4025 ± 0:05081 Triglycerides (mmol/L) 0.35 LDL cholesterol (mmol/L) 1:575 ± 0:1688 1:475 ± 0:2016 0.71 2:2±0:1282 1:8± 0:1268 HDL cholesterol (mmol/L) 0.04 Values are mean ± standard deviation; p <0:05 is considered signiﬁcant. BP: blood pressure; LDL: low-density lipoprotein; HDL: high-density lipoprotein; M: males; F: females. 250 150 ⁎⁎⁎ 0 0 Normal Hypertensive Normal Hypertensive Figure 1: Captive-bred Vervet monkeys’ systolic blood pressure (BP) and diastolic BP. The normal group had systolic BP less than ∗∗∗ ∗ 120 mmHg, while the hypertensive group had systolic BP above 130 mmHg. Signiﬁcant diﬀerence (p <0:0001) and signiﬁcant diﬀerence (p <0:05). Total RNA was also extracted from whole blood (2 mL) 2.5. Statistical Analysis. Data were expressed as means ± using a PAXgene blood RNA extraction kit according to standard deviation between the groups. Statistical analysis the manufacturer’s instructions (PreAnalytiX, Qiagen). The was performed using GraphPad Prism, version 7.05. Statisti- qPCR primer assays (Qiagen, Germany) designed for SYBR® cal signiﬁcance was calculated by using the Student t-test, Green-based RT-qPCR detection were used for relative gene and the statistically signiﬁcant diﬀerence was set at p <0:05. expression of the selected genes. Selected primer assays were human-based (Homo sapiens) (Table S4), since the NHPs 3. Results including Green monkey (Chlorocebus sabaeus) and Vervet monkey (Chlorocebus aethiops) assays are not yet available The selected animals showed no symptoms of being unwell on the gene database. The PCR reaction consisted of 5 μL or distressed. As indicated in Table 1, lipogram parameters of 2X Power SYBR Green PCR Mastermix (Applied for triglycerides and LDL cholesterol were not statistically Biosystems, USA), 0.5 μL (1X) of 10X primer stock, 1 μLof diﬀerent except for HDL-C (p =0:04) which was signiﬁ- cDNA, and 3.5 of water. Reactions were prepared in 96- cantly higher in the hypertensive group compared to the well reaction plates. All RT-qPCR standards and cDNA normal group. Phenotype traits such as body weight were samples were ampliﬁed in duplicates using the Applied also not statistically signiﬁcant between the groups. Biosystems universal cycling conditions. A melt curve for Systolic BP was signiﬁcantly high in the hypertensive secondary product detection was included in the RT-qPCR group compared to the normal group (p <0:0001), and a run. Data for relative expression was analyzed with the similar trend was observed in diastolic BP (p =0:04) QuantStudio-3 Real-Time PCR System (Applied (Figure 1). Biosystems, USA), and results were further analyzed using Screening of RAAS genes such as AGT, CYP3A5, GRK4, the delta-delta Ct method (2–ΔΔCt) . The RT-qPCR and SLC4A5 revealed 21 sequence variants in captive-bred data for each gene was normalized to the average of two Vervet monkeys (Table 2). The protein sequence alignment housekeeping genes such as phosphoglycerate kinase 1 of the AGT gene showed that the identiﬁed sequence vari- (PGK1: PPQ09326C) and glyceraldehyde 3-phosphate ants were located in a conserved region and predicted to dehydrogenase (QT01192646). be polymorphisms (V89A, P65P, T76T, and T318T) and Systolic BP (mmHg) Diastolic BP (mmHg) 4 Advances in Cell and Gene Therapy Table 2: Sequence variants identiﬁed in the colony of captive-bred Vervet monkeys (Chlorocebus aethiops). Nucleotide Type of AA Type of PolyPhen- Gene Exon MutationTaster SIFT VEP change change change mutation 2 Exon 2 C195>T Transition P65P Silent Polymorphism ND Neutral Silent variant A228>G Transition T76T Silent Polymorphism ND Neutral Silent variant Missense T266>C Transition V89A Missense Polymorphism Benign Neutral AGT variant Exon 3 C954>A Transversion T318T Silent Polymorphism ND Neutral Silent variant Disease- Exon 5 C1431>T Transition R477R Silent ND Neutral Silent variant causing Missense Exon 5 G373>C Transversion E125Q SNPs Polymorphism PD Neutral variant CYP3A5 Exon Disease- G885>T Transversion L295L Silent ND ND Silent variant 10 causing G888>A Transition V296V Silent ND ND ND Silent variant Missense Exon 3 G305>T Transversion L102R SNP Polymorphism Benign Neutral variant Missense Exon 5 C533>T Transition A178V SNP Polymorphism Benign Neutral variant Missense GRK4 Exon 6 A587>G Transition Q196R SNP ND Benign ND variant Missense G588>C Transversion Q196R SNP ND Benign ND variant Exon Disease- Missense G1241>A Transition S414N SNP Benign Deleterious 11 causing variant Exon 6 A183>G Transition S61S Silent Polymorphism ND Neutral Silent variant Disease- Exon 8 G540>T Transversion T180T Silent ND Neutral Silent variant causing Disease- Exon 9 C705>T Transition R235R Silent Neutral Silent variant causing Exon Disease- G1740>A Transition S580S Silent ND Neutral Silent variant 17 causing SLC4A5 Exon C>1944T Transition G648G Silent ND ND ND Silent variant G1947>C Transversion L649F SNPs ND Benign Neutral Modiﬁer Exon Disease- C2817>T Transition T939T Silent ND Neutral Modiﬁer 24 causing Exon G2892>A Transition V964V Silent ND ND Neutral ND AA: amino acids; PD: possibly damaging; ND: not determined; SNP: single nucleotide polymorphism; SIFT: Sorting Intolerant from Tolerant; VEP: Variant Eﬀect Predictor. disease-causing (R477R) when blasted on the MutationTa- Furthermore, mRNA expression of the selected RAAS ster tool (Table 2). Genotyping ﬁndings further showed that genes was determined between the groups (Figure 2) and the selected captive-bred Vervet monkeys shared the novel gender (Figure 3, Table S5). Statistical signiﬁcance was E125Q sequence variant in the CYP3A5 gene which was only observed for AGT (p =0:04, difference = −0:4±0:1803 regarded as SNP and two silent mutations (L295L and ); however, GRK4 and SLC4A5 had a similar trend V296V) in exon 10, which were predicted to be polymor- whereby the hypertensive was highly expressed while phisms and disease-causing, respectively (Table 2). Out of CYP3A5 was elevated in the normal group (Figure 2). ﬁve sequence variants identiﬁed in the GRK4 gene, two of Moreover, gender analysis only showed a signiﬁcant these variants (L102R and A178V) were predicted by HSF diﬀerence in GRK4 between Vervet males and females to have the potential of interfering with splicing. Moreover, (p =0:004, difference = 0:9288 ± 0:2722), and the same ﬁve silent variants (S61S, T180T, S580S, G648G, and trend was observed for AGT (p =0:82, difference = − T939T) and one SNP were observed in the conserved region 0:08304 ± 0:3598) and SLC4A5 (p =0:06, difference = of the SLC4A5 gene. 0:5764 ± 0:2771), although not signiﬁcant (Figure 3). There Advances in Cell and Gene Therapy 5 AGT CYP3A5 GRK4 SLC4A5 Normal Hypertensive Figure 2: mRNA gene expression analysis of RAAS genes (AGT, CYP3A5, GRK4, and SLC4A5) in captive-bred Vervet monkeys. Fold change data (mean ± SD; n =8) in arbitrary units (a.u) was normalized against two housekeeping genes (PGK1 and GAPDH). The level of signiﬁcance was set at p <0:05 using GraphPad prism software. Signiﬁcant diﬀerence (p <0:05). AGT CYP3A5 GRK4 SLC4A5 Females Males Figure 3: mRNA gene expression analysis of RAAS genes in captive-bred Vervet monkeys. The graph is showing fold-change diﬀerences (mean ± SD; n =8) between females and males using GraphPad prism software. Signiﬁcant diﬀerence (p <0:05) and a.u = arbitrary units. was no signiﬁcant diﬀerence in mRNA expression level observed in GRK4 and SLC4A5, and this can be linked with between animals with R477R variants in AGT and T180T lipid accumulation in the blood vessels that are known to in SLC4A5 against wild-type animals. enhance the expression of renal angiotensin system compo- nents . This may also be related to the fact that elevated RAAS gene expression contributes to an increase in AngII 4. Discussion receptors in the kidney, leading to the development of hypertension [36, 37]. Moreover, AGT variants are associ- About 30–50% of genetic elements contribute to hyperten- ated with a higher prevalence of hypertension . sion , and these include genetic variation in RAAS genes such as AGT, CYP3A5, GRK4, and SLC4A5 which are known Evidence that links genetic variation of CYP3A5 was identiﬁed in this study and all selected animals had E125Q to play a signiﬁcant role in BP regulation . The sequence variants identiﬁed in this study were all located in the con- variant, which was anticipated to be possibly damaging by the PolyPhen-2 (Table 2) and alteration of an exonic splicing served regions and anticipated to be polymorphisms, benign, enhancer (ESE) site by HSF. These predictions suggested and neutral by MutationTaster, PolyPhen-2, and SIFT (Table 2). Furthermore, AGT mRNA expression was signiﬁ- that the E125Q variant might lead to the introduction of a new splice site within the exon, thereby stimulating the cantly higher in the hypertensive compared to the normal group (Figure 2). Consequently, the hypertensive group imbalance of CYP3A5 metabolism activity, which enhances hypertension development. Although the L295L variant had signiﬁcant high level of systolic and diastolic BP com- was regarded as a silent mutation in exon 10 of CYP3A5,it pared to normal groups (Figure 1), and this may be corre- lated with the fact that hypertension is associated with was anticipated to be disease-causing by the MutationTaster. This revealed that silent mutations can aﬀect nucleic acid high AGT expression [33, 34]. A similar trend was also RAAS mRNA expression (a.u) RAAS mRNA expression (a.u) 6 Advances in Cell and Gene Therapy stability, slow down translation rates, and change the struc- Disclosure ture of the protein without causing a change in amino acid Part of this work has been presented at the South African . A study by  which was conducted in the black pop- Association for Laboratory Animal Science (SAALAS) Con- ulation highlighted that CYP3A5 variations linked to higher ference, which was held at Northwest University Sports Vil- BP may inﬂuence salt sensitivity in hypertensive individuals. lage, Potchefstroom, South Africa, from 15 to 18 March 2022. Consequently, CYP3A5 gene expression showed higher expression in Vervet females compared to males, although this change was not signiﬁcant (p =0:09, difference = − Conflicts of Interest 0:4171 ± 0:2346) (Figure 2). This was correlated to the previ- The authors declare that there is no conﬂict of interest ous studies in the human population, which showed that the regarding the publication of this manuscript. CYP3A5 gene is signiﬁcantly higher in females than in males [40, 41]. To further expand on the identiﬁed mutations, two out Acknowledgments of the ﬁve identiﬁed GRK4 variants have been reported in human studies. These variants include L102R (Human: The authors are thankful for the ﬁnancial support from the L65R) and A178V (Human: A142V). A study on average National Research Council Thuthuka (NRF Grant no. African Americans has reported that L65R (Vervet: L102R) 113583, ZE Magwebu), Primate Unit and Delft Animal Cen- and A142V (Vervet: A178V) in GRK4 are associated with tre (SAMRC/PUDAC), and Division of Research Capacity high BP levels especially in men compared to women . Development (RCD) (Grant recipient: Sanele Khoza) under These ﬁndings were also conﬁrmed by a separate study con- internship scholarship program of the South African Medi- ducted in South Africa [16, 21]. Additionally, another study cal Research Council. Extended gratitude is due to the reported that men with A142V mutation showed a statisti- SAMRC/PUDAC Animal Technicians/Technologists: Mr. cally signiﬁcant increase in diastolic BP and an increasing Timothy Collop, Mr. Mbuyiseli Billy, Mrs. Philida Beukes, trend in systolic BP . The GRK4 A142V causes abnor- and Mrs. Joritha van Heerden for their excellent technical malities in dopamine receptors due to high serine phosphor- assistance and expertise in NHP management. ylation . Similarly, the Vervet males had elevated BP levels (133.75/67.13 mmHg) and signiﬁcantly higher GRK4 Supplementary Materials expression compared to females (Figure 2). Therefore, it was speculated that GRK4 A178V might have a similar Table S1: Clinical measurements of the captive-bred Vervet impact in selected Vervet monkeys. The same gene expres- monkeys. Figure S1: the pedigree diagram of captive-bred sion pattern was observed in Vervet males that were highly Vervet monkeys. “*” indicates animals with normal blood expressing SLC4A5. Based on the BP, genotyping, and gene pressure, and “+” indicates hypertensive animals. The circles expression ﬁndings, this study further conﬁrms the role of represent females, and the square represents males. [ ] sequence variants and agrees with previous literature that denotes wild animals that were taken from the Modderfon- RAAS polymorphisms are linked to hypertension and salt tein farm in Potchefstroom before being housed at PUDAC. sensitivity. Table S2: Green monkey (Chlorocebus sabaeus) reference sequence for selected genes. Table S3: Designed PCR primers for AGT, CYP3A5, GRK4, and SLC4A5 genes, Table S4: 5. 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Advances in Cell and Gene Therapy – Hindawi Publishing Corporation
Published: Feb 17, 2023
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