HLA Class I and Class II Associations with ESRD in Saudi Arabian Population

Background Chronic renal failure (CRF) leads in the majority of instances to end stage renal disease (ESRD) requiring renal replacement therapy. Our interest was to evaluate the possible associations of HLA class I and class II antigens with ESRD independent of other factors, in Saudi Arabia population. Methodology A retrospective study to determine the HLA class I and class II polymorphisms and their association with ESRD, was performed on 350 patients with ESRD, and 105 healthy unrelated control. Patients and control groups were typed by SSOP lumenix techniques. The alleles positively associated to the ESRD were: HLA-B*15, B*18, B*49 - DRB1*03, negatively associated alleles were A*26, HLA-B*39, B*50. The haplotypes positively associated with ESRD were: HLA-A*01-DRB1*13 and HLA-A*30-DRBI*03. The negatively associated haplotypes were: HLA-A*02-B*39, A*02-B*50, A*24-B*35, A*24-B*58, A*24-DRB1*16, A*68-DRB1*04, A*02-DQB1*03, A*29-DQB1*02, A*29-DOB1*05 and B*27-DRB1*07 and the last one is the most significant protective haplotypes. Conclusion The high Relative Risk (RR) observed and its statistical correlation reflect the strength of the described association between HLA antigens and ESRD.


Introduction
Chronic Kidney Disease (CKD) is now one of the major health problems globally and early diagnosis is vital to prevent the development of end-stage renal failure (ESRD) which is defined as the need for renal replacement therapy such as dialysis or Kidney Transplant [1]. Various factors have been shown to be associated with ESRD such as age, gender, genetics, race, hypertension, and smoking etc. [2]. Although, the exact reasons for the growth of the ESRD program are unknown, it is postulated that changes in the demographics of the population, differences in disease burden among racial groups and under-recognition of earlier stages of CKD and of risk factors for CKD may partially explain this growth [3]. Genome-wide strategies have been used to pinpoint genes that contribute to the risks of development of these disorders [4].
In a recent systemic review based on 44 studies from Gulf Cooperation Council (GCC) showed that the incidence of ESRD has increased while the prevalence and mortality rate of ESRD in the GCC has not been reported sufficiently. The leading primary causes of ESRD recorded in the countries of GCC is diabetes which co-morbid conditions being hypertension and hepatitis C virus infection, the most common death was cardio vascular disease and sepsis [5]. The prevalence of CKD in the young Saudi population is around 5.7% [1]. According to the Saudi Center for Organ Transplantation (SCOT) annual report in 2012, the incidence of dialysis in the Kingdom of Saudi Arabia was 129 new cases per million population (PMP) with 3666 new patients in 2012 [6].
This compares to 360 PMP, 585 PMP and in the United States and Europe, respectively [7,8]. While the prevalence of dialysis patients in the Kingdom of Saudi Arabia in 2012 was 499 new cases PMP with 14,171 dialysis patients [6]. This country showed higher prevalence that other developing countries which, it is vary from less than 100 PMP and 163 PMP in sub-Saharan Africa and India respectively, to about 330 PMP in Jordan and 360 PMP in Iran [9][10][11].
The rising prevalence is due largely to two main factors; the ageing of the population and the global epidemic of diabetes. An increasing prevalence of the most common causes of ESRD in the GCC: the prevalence of obesity that is associated with multichronic diseases have been reported, which exceeds that in the developed countries because of their rapid economic growth and associated changes in lifestyle [12,13]. The International Diabetes Federation reports that five of the countries of the GCC ranked globally among the top ten countries in the world for diabetes prevalence [14].
The most common causes in Saudi Arabia is diabetic nephropathy (DN) prevalence as a cause of ESRD is 17.27%, glomerulonephritis 12.68%, hypertensive nephropathy prevalence is 7.75%, Secondary glomerulonephritis/Vasculitis haemolyticureamic syndrome (HUS) is 1.7%, uncertain aetiology, and other conditions, that are rarely reported as causes of ESRD, such as schistosomiasis, tuberculosis, sickle cell nephropathy and contrast nephropathy 19.59% [15]. Co-morbidities associated with ESRD diabetes prevalence is 47.85%, hypertension prevalence is 77.88%, cardiovascular disease prevalence is 14.51% and vascular disease as a co-morbidity condition as 11.7% [15].
Genes of human leukocyte antigens (HLA) (major histocompatability complex (MHC)) are located on short arm of chromosome six which are inherited in a co-dominant manner from parents to the offspring [16]. The HLA system is being paid more and more attention because it is very significant in polymorphous immunological reactions [17]. Histocompatibility testing has great importance in the selection of kidney recipient candidates and donors for transplantation, particularly HLA-DRB1, HLA-A, and HLA-B alleles, which code for molecules with a central role in the immune response and an essential role in the control of self recognition and consequently defense against microorganisms [18]. Previous studies have shown that certain polymorphisms of HLA class I and class II may influence the susceptibility to ESRD [19].
This study was conducted to determine protective and susceptible role of HLA class I and class II alleles and haplotypes in end stage renal disease patients. DNA extraction and DNA typing of HLA loci 10 mL of blood was collected from the patients and normal donors after obtaining prior informed consent. DNA was isolated from blood samples by a fully automated system (MagNA Pure Compact, Roche) according to the manufacturer's instructions. The isolated DNA was stored at 280uC to be used for PCR. HLA-A, HLA-B HLA-DRB1 and DQRB1genotyping was performed by using LABType polymerase chain reaction reversed sequence-specific oligonucleotide (PCR-rSSO) (Luminex, One Lambda, Canoga Park, CA, USA) according to the manufacturer's instructions. Briefly, target DNA was amplified using specific primer sets that are biotinylated at 5 terminus for labeling the PCR product to allow its detection by R-Phycoerythrin-conjugated Streptavidine (SAPE). PCR product was denatured, neutralized, and rehybridized to oligonucleotide probes bound to fluorescently coded microbeads. Then biotinylated hybridized amplicons on microbeads were allowed to be labeled with SAPE. Luminex 100 flow analyzer detected the fluorescent intensities of Phycoerythrin (PE) on each microbeads. The retrieved output was analyzed by HLA Fusion software (ONE LAMBDA, INC, USA) for allele identification.

Statistical analysis
HLA alleles and haplotype frequencies were estimated by direct counting in patients and in healthy controls to compare the differences between the allele frequencies in the control and ESRD groups, a 262 contingency table analysis was performed using the Fisher exact test. The strength of association between HLA alleles was estimated by Relative Risk (RR) and 95% confidence intervals (CI). P,0.05 was considered to be statistically significant and was considered sufficient to reject the null hypothesis. Values with relative risk higher than 1 were considered positive association with ESRD risk. Other alleles which express relative risk lowers than 1 were considered negative association with ESRD protection (A line on the software used for statistical analysis like SPSS, OpenEpi with Version no. 20).

Results and Discussion
Since the mid-1980's, there has been marked increase in the incidence and prevalence of ESRD around the world [20]. Various pathologies may be associated to HLA alleles and/or haplotypes such as idiopathic membranous nephropathy, minimal change nephritic syndrome, immunoglobulin A (IgA) nephropathy, post-streptococcal glomerulonephritis and familiar focal glomerulonephritis [21,22]. Family studies of the HLA class II system in acute post-streptococcal glomerulonephritis in the population of Zulia State confirm in the association between HLA alleles and the disease [23].
Idiopathic membranous glomerulonephritis is associated with HLA-DR2 in Japanese patients and with HLA-DR3 in Caucasians [24]. Similarly, compared to the Greek population, a higher frequency of HLA-DRB1*0301 has been reported in British patients with idiopathic membranous nephropathy [25]. Also antiglomerular basement membrane nephritis in Japanese patients has a strong association with DRB1*1501 [26]. Although the presence of certain HLA alleles may be associated with increased susceptibility to renal disease, other HLA alleles may be protective, for example, DRB1*07 is negatively associated with membranous glomerulonephritis in Caucasians [27].
In a study at the King Khalid University Hospital, which investigated 235 Saudi patients with ESRD and 60 normal, healthy individuals, the most common alleles in both groups were HLA-A2, A68, A24, A3, HLA-B51, B50, B7, B8 with no any significant difference between the two groups [20] [20]. Another report from the Azerbaijan republic, which investigated 26 patients with ESRD and 51 healthy controls, HLA-A2, A3, A24, and HLA-B35, B51 and B18 were the most frequently detected HLA antigens in all subjects. A significant association was found between susceptibility to ESRD and HLA-A11, A33 and HLA-B49 (P,0.05) [28].
Diabetic nephropathy (DN) is leading cause of ESRD worldwide. It affects approximately 30% of patients with long-standing type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Also familiar clustering of DN suggests genetic predisposition factor plays a role in the pathogenesis of this disease [29] [29]. Of the 12844 Saudi patients on dialysis, about 24% are in the active waiting list for transplantation with another 20% being evaluated for inclusion in the waiting list. The data showed that 31% of the dialysis patients are hypertensive, 14% are diabetic in addition to 30% suffer of both diabetes and hypertension [6].
The antigen frequencies were then compared to other studies on populations with close ethnicity Arab population. A recent study with Sudanese subjects showed that for the HLA-A loci, HLA-A2, 30, 3, 24, 1 and A68 were the most frequent antigens and for HLA-B loci HLA-B51, B41, B39, B57, B35, B50 and B52, were the most frequent antigens. HLA-DR13 and DR15 showed the highest antigen frequencies in the DR alleles. In HLA-DQ, DQ1 showed the highest gene frequency [30] [30]. In the Jordanian population A*0201 and B*0713, were the most frequent HLA class I alleles found and DRB1*0704, DRB1*0401 and DRB1*1501 were the most frequent [31] [31]. Another study on the HLA distribution in Egyptian patients with ESRD (n = 100) and normal healthy controls (n = 100), showed that the highest frequency for class I HLA-B35 (28%), B21 (18%) and class II HLA-DRB1*03 (29%) and DRB1*04 (29%). The number of individuals with HLA-B35 was significantly higher in the patient group (P = 0.025), whereas individuals with HLA-B5, B18 were significantly lower in the patient group (P = 0.044 and 0.048 respectively) [32].
This study was carried out without regard to primary renal disease leading to ESRD and focused on the final outcomes. This would help to avoid unexplained causes contributed by many known or unknown diseases. However, several studies provided the evidence that genetic polymorphism and epigenetic variations determine the individual susceptibility of patients to develop rapid progressive kidney disease [33]. A negative association between most A locus antigens and chronic glomerulonephritis and hypertensive nephrosclerosis were found In Turkish patients. And positive association were found between the HLA-B58 and HLA-DRB1*03 with amyloidosis and diabetic nephropathy, respectively [27]. Another Egyptian study among 457 individuals comprising 207 ESRD patients and 250 healthy controls showed that HLA-A2, -B8 and DRB1*3 and HLA-DRB1*11 were significantly correlated with diabetic nephropathy, in addition the B8-DR3 haplotype was shown to be susceptible to DM [34].
There is large variability in the frequency of HLA alleles present in the population ethnic groups. These differences in the results published by different authors are due to the variability in the frequency of HLA alleles present in the population ethnic groups. Also, may be due to the difference in the linkage disequilibrium between the HLA alleles and genes within or flanking the MHC region involved in immune response may play a role in ESRD progression. For instance the polymorphism in genes encoding the cytokines IL-6, IL-4 TNF-a and INF-c have been shown that may be involved in the progression to ESRD [35][36][37].
A study investigating the incidence of kaposi's sarcoma after renal transplantation reported a remarkably high frequency of HLA-A2 (83.3%) among patients compared to normal individuals (43.6%) [38]. In addition, a Canadian study found a higher percentage (69%) of the HLA-A2 allele in young patients with diabetic ESRD compared to normal, healthy individuals (36%), with the majority of patients showing either the HLA-A2-DR4 or HLA-DR8 haplotypes.
In developing countries like Saudi Arabia and other Gulf Cooperation Council (GCC) countries, which share a similar cultural background, ethnicity, socio-demographic distributions and socioeconomic development, many reports, has been shown evidence of increasing prevalence of the most common causes of ESRD [5]. Also there is increasing in the prevalence of obesity in these countries and its associated multichronic diseases, exceeds that in the developed countries because of their rapid economic growth and associated changes in lifestyle [12,13]. Since certain haplotypes particularly two loci and three loci are common to some of the racial groups, it would be possible to find small disparity between population within these countries in the HLA frequencies and haplotype. Hence, there is a need to compare haplotype frequencies among the populations from different regions of Saudi Arabia and GCC. This would be important for creating a national registry and organ-sharing network for GCC people.
In conclusion, this study throws light on the frequency and distribution of HLA class I and class II alleles and haplotypes that confer susceptibility and protection in the Saudi Arabian patients with end stage renal disease (ESRD). However a larger study encompassing more number of patients and controls would be needed to assess the clinical relevance of the results obtained. In the future, molecular diagnostics on HLA polymorphisms may serve to identify patients with survival advantages and could be used by physicians to plan their treatment in the GCC.

Recommendation
The association of HLA alleles and/or haplotype could be used as a risk classification marker and thereby progression to much more severe disease could be protected or diagnosed earlier. This would require further investigation with larger number of patients and healthy individuals and with every disease may cause ESRD. A great need to compare haplotype frequencies among the populations from different regions of Saudi Arabia and GCC populations to develop a national registry and organ-sharing network for GCC people. Table 5. The frequency and associations of HLA DQB1 alleles in ESRD patients and healthy controls.