ACE I/D polymorphism is not a genetic modifier of renal features in sickle cell anemia patients

Introduction: Sickle cell anemia (SCA) exhibits a host of complications that contribute to increased morbidity and mortality at the youngest ages. Objectives: The aim of this investigation is to look into the association between ACE I/D polymorphism and renal function in Indian patients with SCA. Patients and Methods: About 190 SCA patients confirmed by hemoglobin (Hb) electrophoresis were selected for this study. The severity of the disease was determined using anemia, clinical complications, total white blood cells count, and scores of blood transfusion. To define different renal function phases, estimated glomerular filtration rate (eGFR) was computed in adults and children using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) and Schwartz equations respectively. The ACE I/D polymorphism was conducted using polymerase chain reaction (PCR) and separation through agarose electrophoresis. Results: The risk of impaired renal function was not statistically distinct between ACE I/D genotypes and alleles. Further, the genotypes of ACE I/D and the risk of disease severity was not found to be associated with each other. Conclusion: This investigation found that ACE I/D is an insignificant genetic modifier of renal function or severity of disease in patients with SCA.


Implication for health policy/practice/research/ medical education
In a study on190 sickle cell anemia patients, we found the risk of impaired renal function was not statistically distinct between ACE I/D genotypes and alleles.

Objectives
The aim of this investigation is to look into the association between ACE I/D polymorphism and renal function in Indian patients with SCA.

Study design
This infirmary-based cross-sectional investigation was conducted at the outpatient clinic of Sickle Cell Institute Chhattisgarh (SCIC), Raipur, and the Institutional ethics committee of SCIC approved this study. About 190 SCA patients (validated by Hb electrophoresis) were appended in this investigation. Adult subjects signed written informed consent, and minors were accompanied by their parents or guardians who signed a written consent on their behalf. Information related to hematological variables and hemoglobin (Hb) fractions was obtained from the individual patient's record. From each participant, 3 ml of plasma sample was collected in an EDTA vacutainer. An Ilab 650 automatic analyzer was used for quantifying the serum creatinine and blood urea. The estimated glomerular filtration rate (eGFR) was measured in adults and children (17 years) using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation (20) and the Schwartz equation, respectively (21,22). Further, the stage of kidney disease was determined by using eGFR and all the SCA patients were divided into four groups: glomerular hyperfiltration (GHF: eGFR >140 mL/min/1.73 m 2 ), chronic kidney disease 1 (CKD 1: eGFR<140 to 90 mL/min/1.73 m 2 ), chronic kidney disease 2 (CKD 2: eGFR<89 to 60 mL/min/1.73 m 2 ) and chronic kidney disease 3 (CKD 3: eGFR<59 to 30 mL/min/1.73 m 2 ) (23). The severity of the disease was determined using anemia, complications, total leukocyte count, and transfusion scores (24). The standard procedure was used to extract DNA from all samples (25).

Determination of ACE I/D genotypes
Polymerase chain reaction (PCR) as well as agarose electrophoresis were used to genotype the ACE I/D polymorphism.
The subsequent oligonucleotide primers; 5′-CTGGAGACCACTCCCATCCTTCT-3′ and 5′-GATGTGGCCATCACATTCGTCAGAT-3′ were used to perform amplifications. PCR amplifications were carried with the following conditions: 94°C (5 minutes) for 1 cycle, 94°C (1 minute), 58.5°C (40 seconds) and 72°C (30 seconds) for 35 cycles, and a final extension step of 72°C (7 minutes). The PCR product was resolved on 2% agarose gel. The DD and II genotypes were assigned to PCR reactions that produced single bands of 190 and 490 base pairs (bp), respectively, whereas the ID genotype was assigned to PCR reactions that produced two bands of 190 and 490 bp. To avoid mistyping the ID genotype as DD genotype, all samples with DD genotype were subjected to an additional PCR using insertion specific primers (26). A 335bp product produced by this PCR is also considered the I allele.

Statistical analysis
The distribution of clinical and biochemical variables among ACE I/D genotypes was analyzed using ANOVA. To evaluate the association between ACE I/D and renal function or disease severity, the chi-squared test was conducted. SPSS version 22 was used for all analyses (IBM Corp., Armonk, NY.). A two-tailed P value of 0.05 is deemed as statistical significance.

Results
There were 190 SCA patients investigated, including 106 men (55.8%) and 84 women (44.2%). The average age of the participants in the study was 16.5±9.3 years. According to the outcome of this investigation, ID genotype was the most common among patients with SCA, followed by the II and DD genotypes. Figure 1 depicts the distribution of ACE I/D genotypes in SCA patients based on kidney function. SCA patients with various ACE I/D genotypes had almost similar hematological profile ( Table 1). The risk of impaired renal function (GHF, CKD 2 and CKD 3 stages) among SCA patients with distinct ACE I/D genotypes in codominant, dominant, and allelic models was shown in Table 2. No statistically significant variation in the risk of renal impairment among ACE genotypes and alleles was found; suggesting that ACE I/D is an insignificant modifying factor of renal function in SCA patients. Participants with normal kidney function (CKD 1 stage) and different stages of kidney damage had almost similar hematological profile (Supplementary file 1,  Table S1). Individuals with CKD 3 stage had higher HBF levels (22.0 ± 7.4%) than those with normal renal function (18.3 ± 6.3%; P = 0.025). Additionally, CKD 2 stage and CKD 3 stage patients had substantially higher blood urea and creatinine levels than the GHF and CKD 1 groups. Higher SGOT and SGPT levels were found in CKD 2 stage and CKD 3 stage patients than CKD 1 and GHF patients.  (Table 3).

Discussion
The current study found that the ACE DD genotype is the most common in patients with SCA, followed by the II Figure 2. The incidence of ACE I/D genotypes according to SCA severity groups.  and DD genotypes. No significant variation in the risk of CKD among ACE I/D genotypes and alleles. Further, the genotypes of ACE I/D polymorphism are not linked to the disease severity.
Sickle nephropathy, characterized by persistent proteinuria, develops early in life, and is linked to disease severity (27). In adults, CKD stage 3 (renal insufficiency) is a primary source of morbidity and fatality. Several lines of evidences indicated that the ACE I/D gene polymorphism is a major risk factor for thrombotic diseases (18,19,28). According to some studies, the ACE polymorphism could be a genetic susceptibility factor in the advancement of CKD. To date, there are only few case-control study that tried to establish the link the ACE I/D polymorphism with the SCA complications. The ACE I/D polymorphism is not associated with the early sickle cell glomerulopathy (29). In African SCA patients, no statistically significant correlation between ACE I/D polymorphism and SCA complications was revealed (30). Rennin-angiotensinaldosterone system (RAAS) inhibition, reduce proteinuria and slow kidney disease progression in patients with various clinical conditions (12,31). Although this strategy has not been thoroughly tested in patients with SCA, these agents were recommended based on their general efficacy in decreasing the intraglomerular pressure in SCA-related CKD (32).
Treatment with ACE inhibitor, enalapril was shown to decrease the urinary protein excretion as well as controlled serum albumin level in infants and children with sickle nephropathy. Addition of hydroxyurea therapy stabilized the urine protein/creatinine ratio levels in these patients (33). A year ACEIs or ARB therapy in SCA patients was associated with trends for reducing urine albumin and systolic blood pressure (34, 35). ACEIs are safe and effective in providing cardio-renal protection by decreasing albuminuria in SCA patients (36). However, ACEIs have been linked to some side effects, including a dry, irritating cough and a higher risk of lung cancer (37). According to American Society of Hematology guideline, ACEIs and ARBs use require proper follow-ups and observing toxic effects such as hyperkalemia, cough, and hypotension in SCA patients (38).

Conclusion
In summary, this investigation demonstrated that ACE I/D polymorphism is an insignificant genetic modifier of renal function or severity of the disease in patients with SCA.

Limitations of the study
The scope of the present study is limited, as we have not measured creatinine in SCA patients based on isotope dilution mass spectrometry. In addition, the nested study design adopted results in selection bias. However, unlike previous studies, the present study used well-characterized SCA patients.