|Year : 2018 | Volume
| Issue : 4 | Page : 793-800
|The prevalence of APOL1 gene variants in a cohort of renal disease patients in Western Saudi Arabia
Soheir Adam1, Maha Badawi1, Galila Zaher1, Bandar Alshehri2, Ahmed Basaeed3, Musharraf Jelani2, Abdullah Kashqari4
1 Department of Hematology, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia
2 Princess Al Jawhara Center for Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia
3 Department of Medicine, International Medical Center, Jeddah, Kingdom of Saudi Arabia
4 Department of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia
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|Date of Submission||03-Sep-2017|
|Date of Acceptance||03-Oct-2017|
|Date of Web Publication||28-Aug-2018|
| Abstract|| |
Two variants for APOL1; the gastrointestinal (G1) variant (S342G and 1384M substitutions) and the G2 variant (N388 and Y389 deletions) have been previously described to be associated with renal disease. The prevalence of APOL1 variants in Saudi Arabia is unknown. We aimed to determine the prevalence of APOL1 variants in a cohort of patients with renal disease in Saudi Arabia. Patients with renal disease followed up at King Abdulaziz University Hospital were approached consecutively at the out patient clinic, and unaffected controls were approached at the blood donation area. Clinical and laboratory data were collected from electronic medical records. Laboratory variables in controls were obtained on enrollment. This is a cross-sectional, cohort study. One hundred and one patients with a mean age of 54.5 (±19) years, and 119 unaffected controls with a mean age of 31.9 (±7.89) years, were enrolled. Seventy-four patients (68.5%) had hypertension and 62 (57.4%) had diabetes. The mean estimated glomerular filtration rate was 22.47 (± 27.6) mL/min. Two patients were heterozygous for G1 allele. Among the control group, two were heterozygous for G1 allele, and three were heterozygous for G2. All five controls had no evidence of renal disease and no family history of renal disease. The prevalence of APOL1 genetic risk variants in the study cohort was very low. Larger studies are needed to determine the prevalence among renal disease patients in Saudi Arabia.
|How to cite this article:|
Adam S, Badawi M, Zaher G, Alshehri B, Basaeed A, Jelani M, Kashqari A. The prevalence of APOL1 gene variants in a cohort of renal disease patients in Western Saudi Arabia. Saudi J Kidney Dis Transpl 2018;29:793-800
|How to cite this URL:|
Adam S, Badawi M, Zaher G, Alshehri B, Basaeed A, Jelani M, Kashqari A. The prevalence of APOL1 gene variants in a cohort of renal disease patients in Western Saudi Arabia. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2019 Sep 21];29:793-800. Available from: http://www.sjkdt.org/text.asp?2018/29/4/793/239658
| Introduction|| |
A strong association has been described between APOL1 genetic alleles and focal and segmental glomerulosclerosis (FSGS) and chronic kidney disease (CKD).,, The APOL1 gene lies on chromosome 22 and two common genetic variants were identified in association with CKD; a missense variant gastrointestinal (G1) (rs73885319) (S342G and 1384M substitutions) and a six-base pair deletion labeled G2 (rs71785313)(N388 and Y389 deletions)., Two copies of the risk alleles or double heterozygosity of the risk alleles (i.e., G1/G1, G1/G2, or G2/G2) greatly increase the risk of CKD.
The population in Western Saudi Arabia is ethnically diverse. In addition to individuals of Saudi indigenous tribal origins, there are Saudi individuals of Asian, African, and European descents. This study was designed to determine the prevalence of APOL1 alleles in a cohort of patients with renal disease and in unaffected controls.
| Patients and Methods|| |
The study was conducted at the King Abdulaziz University Hospital (KAUH) and approved by the Ethical Committee.
Adult patients with renal disease attending the outpatient renal disease clinics at KAUH were approached sequentially and briefed on the study. On agreement to participate and after signing a written consent, blood samples were drawn from participants. Data were collected by interview and review of medical records including history of hypertension, diabetes, renal replacement therapy, history of renal transplant, and comorbidities. Body mass index (BMI) was calculated using the most recent weight and height readings and estimated glomerular filtration rate (eGFR) was calculated for both patients and controls, using the CKD-EPI creatinine equation, adjusting for body surface area. Laboratory tests were performed including; recent hemoglobin level, serum creatinine, urinary creatinine, cystatin C in urine, proteinuria, 24 h protein in urine and cause of renal disease.
Similarly, unaffected healthy individuals were approached sequentially in the blood donation area at KAUH and if agreeable to participate, they signed a written consent and samples were drawn for molecular testing and other laboratory tests including; urea, creatinine, sodium, potassium and albumin. Urine samples were collected for the following tests; albuminuria, albumin/creatinine ratio and creatinine in urine. Other data collected from controls included number of previous blood donations, personal and family history of renal disease, hypertension and diabetes. The BMI and eGFR were calculated for all participants.
Molecular analysis methodology
Molecular testing was performed at Princess Al Jawhara Center of Excellence in Research of Hereditary Disorders. Based on the uniqueness of the Saudi Arabian population, it is difficult to surmise the prevalence of the relevant alleles for our genetic association analysis. We hypothesized the prevalence of APOL1 variants of at least 10%.
Blood samples were processed within 24–48 h after collection to isolate the DNA using DNA isolation kit from Applied Biosystems (USA).
The primer sets for each single nucleotide polymorphism (SNP) were selected based on the unique SNP location in the genome by Primer3 software (http://frodo.wi.mit.edu/). The human sequences were retrieved from online databases of Ensemble Genome Browser (http://www.ensembl.org/Homo_sapiens) and National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/gene).
The polymerase chain reaction (PCR) for DNA amplification was carried out using 1 μL of human genomic DNA (40 ng) in a 50 μL reaction mixture which contained 1 unit of Taq DNA polymerase, 20 to 30 pmol of forward and reverse primers, 1 mM of magnesium chloride and 2.5 of ×10 ammonium sulfate buffer. Agarose gel 2% was used to analyze the PCR products. Amplified products were mixed with an equal volume of bromophenol blue dye. To assess the size of amplified products, a 100-bp DNA ladder was used. To visualize DNA on Agarose gel, the gel was placed in an UVitec Gel Documentation system 232 (UVitec, Cambridge, UK) for imaging the gel and was visualized under the UV light and photographed on thermal paper.
For targeted DNA sequencing, the PCR products were purified using QIA quick PCR Purification Kit. The purified PCR products were used as DNA template for sequencing PCR cycle sequencing reaction with ABI Prism Big-Dye Terminator Cycle Sequencing Ready Reaction Kit v1.1 (Life Technologies, USA).
Sequencing raw data were analyzed through ABI Sequence Scanner software version 1.0 (ABILife Technologies, USA. BioEdit sequence alignment program, version 6.0.7) was used for aligning normal sequences against each and every test sample sequence individually to genotype SNPs in their unique locations [Figure 1]. The genotypes of the SNPs were recorded in an excel sheet for further statistical analysis.
| Statistical Analysis|| |
This study used the Statistical Package for Social Sciences (SPSS) version 23.0 (SPSS Inc., Chicago, IL, USA). Variables are presented through counts and percentages and well as means and standard deviation using a simple descriptive analysis. To determine relationships of both categorical variables, this study used Chi-square test for continuous variables, and t-test for two groups means. All tests were under the assumption of normal distribution; otherwise, alternative corresponding tests were used. Finally, a P <0.05 was considered the criteria for rejecting the null hypothesis.
| Results|| |
One hundred and one renal disease patients were included in the study. Mean age at enrollment was 54.5 (±19) years and 68 (63%) were male [Table 1] and [Table 2]. Seventy-four (68.5%) were of Arab origin and 27 (25%) were nonArab.
Diabetes mellitus (DM) was present in 59 patients (54.5%) and hypertension in 74 patients (68.5%). The mean eGFR was 22.47 (±27.6) mL/min.
Other comorbidities associated with renal disease included systemic lupus erythema-tosus, present in three patients, and Alport's syndrome in two patients. Seven patients (6.9%) had a family history of CKD and one patient had a positive family history of sickle cell disease.
One hundred and nineteen unaffected controls were enrolled in the study. Of those, 108 were male (90.8%) and 11 (9.2%) were female. The mean age at enrollment was 31.9 (±7.89, range; 17–55) years [Table 3] and [Table 4].
The mean BMI was 29.4 (±5.43), and mean serum creatinine was 84.5 umol/L (± 12.44). Calculation of the eGFR showed a mean of 117.64 (± 21.6) mL/min and the mean BUN was 4.14 (±1.21) umol/L. Nine individuals (7.6%) had a positive family history of CKD and five individuals (4.2%) had a positive family history of sickle cell disease.
APOL1 Allele testing
Target alleles were positive in two male patients with renal disease, one was an Arab and Saudi national and the other was non-Arab. Both patients were heterozygous for G1 allele, and they had no family history of renal disease. Their laboratory results are shown in [Table 5].
|Table 5: Characteristics of patients heterozygous for APOL1 risk alleles.|
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Three controls were found to have one G1 allele and two control subjects had one G2 allele. Out of the five heterozygous control subjects, four were Saudi nationals and one was Palestinian. All five controls were male and had no family history of renal disease. Laboratory screening for renal disease was negative, and their laboratory values are shown in [Table 6].
|Table 6: Characteristics of controls heterozygous for APOL1 risk alleles.|
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| Discussion|| |
Renal disease represents a significant healthcare burden in Saudi Arabia. However, the allele frequency of APOL1 gene variants in Saudi Arabia is unknown. We found only two cases of heterozygous G1, G2 alleles among 101 renal disease patients.
In Africa, the allele frequency of APOL1 gene variants, G1 and G2 is as high as 38%, while in Europe it is as low as 4%. Furthermore, the prevalence of APOL1 alleles is variable among African populations; G1 is more common, especially in West Africa but G2, while less common, is more uniformly distributed. The population in Western Saudi Arabia is of diverse ethnic descent. The results thus reflect the prevalence of APOL1 alleles in individuals of all ethnicities and not specifically in those of tribal origins.
Identifying individuals at risk for developing CKD is imperative to facilitate follow-up and early referral to nephrologists. Furthermore, the risk of death from cardiovascular disease in patients with CKD is considerable. Screening for CKD among high-risk individuals, revealed a prevalence of 20.8%. Among the general population, CKD was found to be as high as 4.7%. Interestingly, APOL1 variants-induced cytotoxicity was found to be dose-dependent in vitro,, which may be part of the reason why only a subset of patients with two copies of APOL 1 variants develop nephropathy.
A previous report from Saudi Arabia found that 0.84% of 10,601 Saudi Arabian males with normal creatinine, were in CKD stage 3 when assessed by eGFR. Of 14,695 females included in the same study, 19.24% were in CKD stage 2. Thus, we used eGFR to screen for CKD in our study population.
An association was described between non-muscle myosin heavy chain 9 (MYH9) and HIV-associated nephropathy and FSGS.,
Polymorphisms of MYH9 gene, present on chromosome 22 were proposed as genetic risk factors for end-stage renal disease (ESRD), described as MYH9 nephropathies. Evidence then gradually evolved that APOL1 gene variants G1 and G2, are strongly associated with the risk of developing non-diabetic nephropathy, earlier age at starting renal replacement therapy and faster progression of renal disease. Furthermore, the expression of these variants led to an increased risk for development of FSGS, lupus nephritis, renal disease secondary to hypertension and HIV-associated nephropathy.,,,
The APOL 1 gene is part of a set of six genes on chromosome 22 and it is thought to play a role in innate immunity., Till date, the mechanism of development of renal disease in the presence of G1 and G2 variants has not been entirely elucidated. APOL 1 expression is exclusive to humans and some higher primates, which may obscure in vivo studies. In vitro, the expression of G1 and G2 variants in human embryonic kidney cells was found to increase the rate of cell swelling and cell death. This was notably preceded by intra-cellular potassium efflux and activation of stress activated protein kinases.
Anyaegbu et al reported that on screening family members of African American patients with ESRD for APOL1 variants, 60% were found to have two APOL1 risk variants. However, in our study cohort only five control subjects were heterozygous for either allele and none of them had clinical evidence of renal disease. Moreover, family history of renal disease was negative in all five individuals.
This study has some limitations; the size of the cohort was determined based on the allele frequency in other populations. It may well be that we need larger numbers to effectively determine the allele frequency among CKD patients in Saudi Arabia.
| Conclusion|| |
The prevalence of APOL1 allele variants in our study cohort was very low and likewise in our control group. While larger studies are needed to screen for APOL1 variants in Saudi patients with renal disease, screening for other polymorphisms that may be inherent to the Saudi population is warranted.
Conflict of interest: None declared.
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Dr. Soheir Adam
Department of Hematology, King Abdulaziz University, P. O. Box 80215, Jeddah
Kingdom of Saudi Arabia
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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