The Impact of ACE Gene Variants on Acute-Phase Reactants in Children with Rheumatic Heart Disease

Rheumatic heart disease (RHD) is the most important sequela of upper respiratory group A Streptococcus (GAS) infection. The role of the common angiotensin-converting enzyme (ACE) insertion/deletion (I/D) variant in the disease and its subtypes remains uncertain. The acute-phase reactants (APRs) C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) form part of the Jones criteria for diagnosing RHD, and genetic factors are known to influence baseline CRP and ESR levels. Therefore, here, we investigated the relationship between the ACE I/D polymorphism and APR levels in RHD. A total of 268 individuals were recruited, including 123 RHD patients and 198 healthy controls. There was a trend toward a higher D allele frequency in RHD patients. The ACE I/D polymorphism genotype frequency and DD+ID allelic carriage were significantly associated with a high APR level (p = 0.04 and p = 0.02, respectively). These results highlight the importance of ACE I/D polymorphisms in RHD for disease stratification, but not for disease predisposition. Further studies in larger cohorts and different populations are now required to confirm this association and to explore the mechanism of this effect.


Introduction
Rheumatic heart disease (RHD) usually occurs during childhood after group A streptococcal infection. In response to the infection, an uncontrolled immune response in genetically susceptible hosts causes rheumatic fever (RF) and inflammation that severely damages the heart valves, thus causing RHD [1]. RHD is often associated with poverty and poor healthcare services, so it disproportionately affects low-and middle-income countries (LMICs) whilst having been almost completely eradicated from high-income countries [2]. The burden of RHD increased from 15 million cases in 2005 to 40.5 million cases in 2019 [3]. Although penicillin is used to treat group A streptococcal infections and as a preventative medicine, the disease still affects many young people in LMICs [4].
The genetic predisposition to RHD is well documented [5]. Genome-wide association and case-control studies have identified many important genes significantly associated with RHD, many of which have immunological functions such as immunoglobulin heavy chain alleles (IGHV4-61), IL10, and macrophage migration inhibitory factor (MIF) (for recent reviews, see [5,6]). Moreover, there is a reported association between RHD subtypes and clinical laboratory findings. Some genes have been reported to influence disease severity based on the subgrouping of patients according to valve involvement, e.g., MVLs (mitral valve lesions) and CVLs (combined valve lesions). For example, in a cohort of patients from

Patients and Laboratory Testing
This was a case-control study of 123 unrelated Saudi Arabian RHD patients recruited from the Pediatric Cardiology Clinic at the Maternity and Children Hospital, Medina, Saudi Arabia. The patient and control groups were described previously [7,24,27]. Laboratory measurements, including baseline ESR and CRP, were performed on all patients as part of the clinical assessment according to the Maternity and Children Hospital laboratory standard operating procedure. RF was diagnosed according to the modified Jones criteria at initial diagnosis [8] and confirmed via echocardiography. Elevated APR was defined as patients with an ESR > 22 mm/h for men and 27 mm/h for women and elevated CRP > 20 mg/L. Exclusion criteria were patients with other heart complications, inflammatory conditions, and RF without valve involvement.
The Centre for Genetics and Inherited Diseases (CGID) research ethics committee and the Maternity and Children Hospital ethics committee approved the study, which followed the World Medical Association Declaration of Helsinki. All adult patients and donors and the parents/guardians of participants <18 years old signed a fully informed and written consent approved by the committees. The control group comprised 198 age-, gender-, and ethnicity-matched unrelated healthy blood donor volunteers without evidence or a family history of cardiac diseases or any autoimmune diseases.

Sample Preparation and Genotyping
DNA samples were extracted from 2 mL peripheral blood using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Extracted DNA was quantified via spectrophotometry (MaestroNano, MaestroGen, Las Vegas, NV, USA). DNA samples were stored at −80 • C until use. The genotyping method was previously reported [28]. Briefly, primers flanking the insertion/deletion region (forward primer 5 -CTG GAG ACC ACT CCC ATC CTT TCT-3 and reverse primer 5 -GAT GTG GCC ATC ACA TTC GTC AGA T-3 ) were used to detect the presence or absence of the 287 bp sequence in intron 16 of ACE [28]. Amplification was carried out in a 12.5 µL reaction volume containing 30 ng genomic DNA added to 2× Go-Taq master mix including MgCl 2 , 10× PCR buffer, dNTPs, and 10 units of Taq DNA polymerase (Cat #M7132, Promega, Madison, WI, USA). A Veriti thermal cycler was used to amplify the PCR product (Life Technologies, Carlsbad, CA, USA). PCR cycles were as follows: initial denaturation at 95 • C for 2 min, followed by 35 cycles with denaturation at 95 • C for 30 s, annealing at 58 • C for 15 s, and extension at 72 • C for 30 s. PCR products were visualized using 2% agarose gels and ethidium bromide staining under UV light (G:BOX, Syngene, Bangalore, India). Samples with an insertion allele (I) produced a 490 bp PCR fragment; a deletion allele (D) produced a 190 bp product without the 287 bp Alu insertion. In heterozygous samples, two PCR fragments (490 and 190 bp) were detected. Randomly selected samples were repeated to confirm the accuracy of the method. Genotypes were determined by direct counting and labeled DD homozygous when only the 190 bp fragment was present, II homozygous when only the 490 bp fragment was present, and ID heterozygous when both fragments were present.

Statistical Analysis
The genotype and allele frequencies were determined by direct counting in patients and controls. Differences in genotype distributions of the polymorphism between cases and controls were analyzed using chi-squared contingency table analysis or Fisher's exact test, as appropriate. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated. A p-value < 0.05 was considered statistically significant. All genotyping data were checked for any deviation from the Hardy-Weinberg equilibrium via chi-squared testing. Statistical analysis of the data was performed using SPSS v.24 (IBM, Chicago, IL, USA).

Patient Characteristics
The clinical records of 123 RHD patients attending the Pediatric Cardiology Clinic at the Maternity and Children Hospital, Medina, Saudi Arabia, were reviewed. Seventy-five patients had an elevated baseline APR (ESR > 22 mm/h for men, 27 mm/h for women; CRP > 20 mg/L). The clinical characteristics of all patients and the control group are shown in Table 1. There was no significant difference in age and gender between the control and patient groups. Abbreviations: CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; n, number; SD, standard deviation; NA, not available; PR, retrograde P waves. There was no significant difference in the sex distribution between the control and patient groups (p = 0.24). Table 2 shows the distribution of ACE I/D polymorphism genotypes, allele frequencies, and allelic carriages in the RHD patients and control group. All frequencies were in Hardy-Weinberg equilibrium. The genotype distribution of ACE I/D was not significantly different between patients and controls. However, the DD+ID allelic carriage was higher in patients than controls, with patients having a 2.9-fold increased risk of RHD than those with the II genotype (p = 0.04, OR = 2.9, 95% CI: 1.1-7.6).

Association with ARP
The RHD group was divided into two subgroups according to APR levels (high and normal), which were compared with the control cohort. The genotypes, allele frequencies, and allele carriage distributions of these three groups are shown in Table 3. There was a significant difference in the distribution of the ACE I/D genotype frequency, D allele frequency, and DD+ID allele frequency between patients with high APR levels and controls (p = 0.04, p = 0.04, and p = 0.02, respectively) but not between patients with normal APR levels and controls. There were no differences in allele frequency between patients with high and normal APR levels. Figure 1 presents a bar chart showing the distribution of the ACE I/D polymorphism frequencies in the RHD and control groups. and allele carriage distributions of these three groups are shown in Table 3. There was a significant difference in the distribution of the ACE I/D genotype frequency, D allele frequency, and DD+ID allele frequency between patients with high APR levels and controls (p = 0.04, p = 0.04, and p = 0.02, respectively) but not between patients with normal APR levels and controls. There were no differences in allele frequency between patients with high and normal APR levels. Figure 1 presents a bar chart showing the distribution of the ACE I/D polymorphism frequencies in the RHD and control groups.

Association with Disease Subtypes
To examine whether the ACE D allele was associated with disease severity, we investigated the association between the ACE I/D allele and valvular heart involvement. Table 4 shows the distribution of ACE I/D genotypes in patient subgroups and controls. Carrying the ACE D allele was significantly associated with MVLs compared with controls (p = 0.02). However, there was no significant difference in the CVL subgroup.

Discussion
The rationale for studying the ACE I/D polymorphism is based on strong evidence that it plays a role in modulating immune responses and modifies clinical outcomes in other diseases [29]. Previously, we reported a weak association between RHD and the ACE I/D polymorphism [24]. However, in this study, we investigated the association between the ACE I/D polymorphism and baseline APR levels in RHD patients. RHD patients tended to have higher D/D genotype frequencies than controls. Interestingly, D allele carriage (D/D+I/D genotypes) was significantly increased in patients compared with controls, and there was a strong association between D allele carriage and elevated APR. Conversely, there was no difference in ACE polymorphisms in patients with normal APR levels compared with controls. Moreover, there was no difference in the distribution of ACE I/D polymorphism frequencies in RHD patients with high and normal baseline APR levels. D allele carriage was also associated with mitral valve involvement but not CVLs in patients with high APR levels.
The association between the ACE I/D polymorphism and RHD has previously been reported in several populations [22], but the results were inconsistent. With respect to Arab populations, a study from Egypt reported an association between the DD genotype and RHD [25], and another study from Saudi Arabia reported a weak association with RHD [24]. Two studies from Turkey and Taiwan reported an association with the I/I genotype. Our current results therefore confirm that the association between the ACE I/D polymorphism and disease stratification is stronger than the association with the disease as a whole. Therefore, ACE may play an important role as a modifier rather than a causative gene in RHD.
The relationship between ACE polymorphisms and APR levels has been investigated in other diseases. For instance, in chronic obstructive pulmonary disease, the ACE I/D polymorphism was associated with high CRP levels [30]. However, whether this represents a direct or an indirect association between D allele carriage and increased inflammatory responses remains unclear. Cross-talk between the RAS system and CRP is well described [31]; ACE is responsible for the conversion of Ang I to the vasoconstrictor Ang II, and in turn, Ang II can induce CRP in vascular smooth muscle cells to stimulate CRP production in aortic endothelial cells [32,33]. In turn, CRP induces T cells to express high levels of interleukin-1β (IL-1 β) [34]. IL-1 β plays an important role in activating inflammasomes, which has been shown to play an important role in RHD [35]. Another pathogenic effect of high CRP is its ability to induce heart tissue damage by activating complement [36], which is known to play a role in RHD pathogenicity as well. Therefore, our results may indicate a direct effect of the D allele on CRP levels, although other studies have failed to detect this association, such as in coronary artery disease, where no association was detected between RAS polymorphisms (including ACE I/D) and CRP levels [37]. Figure 2 presents a schematic that shows the possible role of CRP in the pathogenicity of RHD.
The effect of ACE polymorphisms on other inflammatory markers has been widely reported. For example, in myocardial infarction patients, the ACE ID/DD genotype was independently associated with high IL-6 and kallikrein, a protease that plays a significant role in initiating and maintaining systemic inflammatory responses [38]. In another study, the ACE D/D allele was associated with low nitric oxide metabolites (nitrite and nitrate) in healthy men with high blood pressure [39]. Interestingly, high blood pressure was also associated with the ACE D/D genotype in obese youths [40]. In complicated cases with COVID-19, D-dimer levels were significantly higher in patients with D/D and I/D genotypes, indicating a potential biomarker of poor disease outcome [41]. In addition, COVID-19 severity was associated with the ACE D/D genotype and with high CRP levels [42]. Moreover, the I/D polymorphism was reported to show sexual dimorphism in young adults, with the DD genotype associated with higher maximal fat oxidation values than the ID/II genotype in men, and the ID/II genotype having higher maximal fat oxidation values in women [43]. In ischemic stroke patients, the ACE D allele was associated with high total cholesterol and low-density lipoprotein levels, indicating a possible biomarker for early prevention [44]. In cardiovascular disease patients, the ACE I/I genotype and I allele were associated with increased circulating glucose levels and hence a 2-fold increased risk of diabetes [45]. These studies indicate that the ACE I/D polymorphism has a biological influence.
[31]; ACE is responsible for the conversion of Ang I to the vasoconstrictor Ang II, and in turn, Ang II can induce CRP in vascular smooth muscle cells to stimulate CRP production in aortic endothelial cells [32,33]. In turn, CRP induces T cells to express high levels of interleukin-1β (IL-1 β) [34]. IL-1 β plays an important role in activating inflammasomes, which has been shown to play an important role in RHD [35]. Another pathogenic effect of high CRP is its ability to induce heart tissue damage by activating complement [36], which is known to play a role in RHD pathogenicity as well. Therefore, our results may indicate a direct effect of the D allele on CRP levels, although other studies have failed to detect this association, such as in coronary artery disease, where no association was detected between RAS polymorphisms (including ACE I/D) and CRP levels [37]. Figure 2 presents a schematic that shows the possible role of CRP in the pathogenicity of RHD.

Figure 2.
A schematic showing the possible role of C-reactive protein (CRP) in the pathogenicity of rheumatic heart disease (RHD) after group A streptococcal (GAS) infection. (A) Rheumatic fever (RF) and RHD are thought to be initiated by infection with rheumatogenic strains of GAS in susceptible hosts [5]. (B) An increased level of ACE leads to high angiotensin II, which induces high CRP [32,33]. In turn, CRP induces T cells to express high levels of interleukin-1β (IL-1 β) [34,38]. IL-1 β plays an important role in activating inflammasomes which has been shown to play an important role in RHD [35]. (C) Another pathogenic effect of high CRP is its ability to induce heart tissue damage by activating complement [36], which is known to play a role in RHD pathogenicity as well.  [5] and Carlus et al. 2020 [39]. The figure was originally designed using Biorender.com (please see reference [5]). and RHD are thought to be initiated by infection with rheumatogenic strains of GAS in susceptible hosts [5]. (B) An increased level of ACE leads to high angiotensin II, which induces high CRP [32,33]. In turn, CRP induces T cells to express high levels of interleukin-1β (IL-1 β) [34,38]. IL-1 β plays an important role in activating inflammasomes which has been shown to play an important role in RHD [35]. (C) Another pathogenic effect of high CRP is its ability to induce heart tissue damage by activating complement [36], which is known to play a role in RHD pathogenicity as well. Figure reprinted/adapted with permission from Ref. Abdallah et al., 2021 [5] and Carlus et al., 2020 [39]. The figure was originally designed using Biorender.com (please see reference [5]).
Baseline ESR and CRP levels have been reported to be influenced by different genetic factors in different diseases. In familial Mediterranean fever, APR levels were significantly higher in children with homozygous and heterozygous M694V mutations compared with those with the wildtype genotype; therefore, monitoring APR levels in patients harboring the mutation may help to prevent disease complications [46]. In rheumatoid arthritis, an intronic variant of ANKRD55 was associated with the disease, and there was a significant correlation between CRP levels and the variant [47]. Elevated ESR and CRP levels are an important marker for the acute stage of Kawasaki disease (KD) [48], and a polymorphism in ITPR3, whose protein product mediates the release of intracellular calcium, was associated with increased serum CRP levels in KD patients. Interestingly, the polymorphism was not associated with the risk of KD, but rather the disease complications [49]. In a GWAS, two polymorphisms were associated with CRP levels in KD patients in both the test and validation cohorts [50]. The first variant (rs4786091) was an intronic polymorphism found in an RNA-binding protein gene, RNA binding Fox-1 homolog 1 (RBFOX1), which plays a role in tissue-specific alternative splicing [51]. RBFOX1 has been associated with the risk of allergy [52], neurodevelopmental disorders [53], and aggressive behavior [54]. The second variant (rs120687530) was a common intergenic polymorphism in the promoter region of CRP. However, more recent studies have found that different genotypes and haplotypes in the promoter region of CRP are associated with its protein expression [55]. In addition, in a familial study, the heritability of serum CRP levels was found to be quartile-specific, namely age and sex, which may explain the different associations between CRP levels and different genes in different diseases [56].
Baseline ESR levels are also influenced by different genetic factors in different diseases. A promoter polymorphism in IL-10 (rs1800871) was associated with increased CRP and ESR levels in patients with spinal tuberculosis, a complication of Mycobacterium tuberculosis infection [57]. Moreover, an angiopoietin 2 (ANGPT2) rs3020221 polymorphism was found to influence both CRP and ESR levels in rheumatoid arthritis patients. Interestingly, rs3020221 was associated with elevated CRP and ESR levels and Disease Activity Score-28 (DAS-28) ESR scores [58,59]. Similarly, the rs562929801 polymorphism in miRNA-5189 was significantly associated with DAS-28-ESR scores in rheumatoid arthritis patients treated with methotrexate [60]. Another polymorphism in SPRED2 (rs934734) was associated with an elevated ESR in rheumatoid arthritis patients [61]. Vitamin D receptor (VDR) polymorphisms also impact ESR levels, and in systemic sclerosis patients, BglI and ApaI polymorphisms were associated with an increased ESR [62], while the BsmI polymorphism was associated with an increased ESR in rheumatoid arthritis patients [63].
Our study has a few limitations. One limitation is the limited sample size. In addition, we analyzed APR as a whole rather than analyzing CRP and ESR individually, as these data were not available in the patient records. Finally, we did not investigate the exact mechanisms underlying this association in RHD.

Conclusions
In conclusion, the role of the ACE I/D genotype in different phenotypes is important to consider in the context of complex, multi-factorial diseases. Our results revealed a strong association between the ACE I/D polymorphism and increased baseline APR levels in children with RHD. These results confirm the importance of ACE I/D polymorphisms in RHD disease stratification, although they carry less influence in disease predisposition. However, whether this association is direct or indirect remains unclear. Given the scarcity of data in this area, further studies in larger cohorts and in different populations are required to confirm this association and to explore the mechanism underlying this effect. Informed Consent Statement: All adult patients and donors and the parents/guardians of participants <18 years old signed a fully informed and written consent approved by the committees.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.