Chronic kidney disease (CKD) affects over 10% of the adult population worldwide and is now recognized as the most rapidly increasing contributor to the global burden of disease (Thomas et al., 2017). Recent predictions by the Institute for Health Metrics and Evaluation indicate that CKD will become the fifth cause of years of life lost globally by 2040 (Foreman et al., 2018). In Saudi Arabia, end-stage renal disease (ESRD), the terminal manifestation of CKD, affects more than 6% of the population, resulting in substantial morbidity, mortality, and high healthcare costs (Alsuwaida et al., 2010). At present, there are approximately 17,000 patients on dialysis, and this number is increasing at an exponential rate of 8% annually, with a national dialysis incidence of 140 new cases per million population (PMP) (Wang et al., 2016).

Despite the enormous economic and societal burden of CKD, its pathogenesis remains elusive, impeding specific diagnosis and targeted therapy (Romagnani et al., 2017). CKD is a complex disorder comprising numerous pathophysiologically distinct conditions, which share the common feature of leading to persistent anomalies in the kidney structure and/or function (O’Seaghdha and Fox, 2011). Prior research has shown that CKD results from a combination of genetic and environmental factors (Satko and Freedman, 2005). Moreover, familial aggregation of CKD in diverse ethnic groups is well-documented, confirming the role of heritable factors in predisposition to CKD (Cañadas-Garre et al., 2018a). Although CKD can be identified by well-established clinical biomarkers including the estimated glomerular filtration rate (eGFR), albuminuria, or serum creatinine (SCr) levels, the early prediction of individual risk for CKD or the likelihood of later progression to ESRD remains a challenge (Satko and Freedman, 2005; Cañadas-Garre et al., 2018b).

Genetic studies, initially using candidate gene approaches, and more recently, genome-wide association studies (GWAS) have identified numerous genetic biomarkers conferring susceptibility and disease progression in CKD (Köttgen et al., 2012; Sveinbjornsson et al., 2014; Prokop et al., 2018). However, these genetic biomarkers do not account for all the susceptibility to CKD and explain a minority of the overall heritability. Although there are several causes of kidney failure in Saudi patients on dialysis, as reported in the registry of the Saudi Centre for Organ Transplantation (Bullich et al., 2018), genetic and congenital anomalies of the urinary system are listed as the causes of only 2% and 1.6% of cases, respectively. However, this is an underestimated percentage, and many of the causes listed as “unknown” (7%) or hypertension (38%) may well be due to genetic factors. Globally, a 20% prevalence of family history of kidney disease was reported by incident dialysis patients in the USA (of them, 22.2% in diabetes mellitus; 18.9% in hypertension; 22.7% in glomerulonephritis; and 13.0% in patients with other etiologies) (Fallerini et al., 2014). In another US study, 23% of incident dialysis patients had close relatives with ESRD, while 21% of patients over 55 years old had a family history of ESRD. The prevalence values of family history among patients with diabetes, glomerulonephritis, hypertensive nephrosclerosis, and “other” causes of their CKD were 24.4%, 22.5%, 23.2%, and 17.5%, respectively (Riedhammer et al., 2020). Additionally, a study from Norway reported that individuals with a first-degree relative with ESRD had a 7.2 times higher risk of ESRD than individuals without a first-degree relative with ESRD (Cañadas-Garre et al., 2018a). Importantly, heritability studies of eGFR in twin studies reported an estimate of 50% (Cañadas-Garre et al., 2019). The familial aggregation studies demonstrated that heritability ranged from 36 to 75% for eGFR and from 16% to 49% for albuminuria (Cañadas-Garre et al., 2018a; Adam et al., 2020).

In this study, we used a genetic testing approach based on targeted next-generation sequencing of 104 genes causative or associated with CKD in Saudi patients with ESRD. We sought to elucidate the genetic causes of CKD and identify genetic variants associated with CKD and related traits in the Saudi population, supporting precision medicine for Saudi patients with kidney disease.

Herein, we screened a relatively large cohort of ESRD patients to identify genetic factors contributing to extremely severe forms of CKD. We applied an advanced genetic testing approach using a targeted NGS gene panel including 102 genes causative or associated with CKD to characterize a cohort of 534 Saudi patients. We identified 13 statistically significant variants within genes implicated in kidney function. All 13 variants were not previously reported to be associated with kidney diseases. Among these 13 variants, four were novel variants that were not previously reported in any other population. Unlike most GWASs that explored the role of genetic factors in kidney function among the general population, this study uniquely reported the association between genetic variants and severe forms of kidney disease among a homogenous disease-specific population.

Several GWASs were performed for variable kidney function traits. The Chronic Kidney Disease Genetic Consortium (CKDGen) performed a GWAS on more than 67,000 individuals of the European ancestry and reported 20 loci associated with renal function and CKD (Prokop et al., 2018). The study showed that a genetic risk score (GRS) including 16 SNPs explained a 1.4% variation in the renal function, as explained by serum creatinine (eGFRcrea). Later GWASs which included larger sample sizes were able to bring the number of variants that are associated with eGFRcrea up to 63, explaining up to 3.99% of the phenotypic variance (Fallerini et al., 2014; Bullich et al., 2018). As expected, variants identified from these GWAS were of modest effect size and common with allele frequency above 5%. Since these studies were performed on the general population with the majority of study participants having eGFR greater than 60 ml/min/1.732, transferring such results into concrete risk estimation, for the development of ESRD remains uncertain. Hence, genotyping these common variants provides minimal clinically relevant information to individual patients. Importantly, Parsa et al. (2013) examined systematically the association of eGFR with common variants in 258 genes responsible for Mendelian forms of kidney abnormalities using the CKDGen Consortium GWAS data [30]. This study, in addition to others, has been largely unsuccessful in identifying common variants in genes that cause rare monogenic forms of CKD, except for a few genes such as UMOD, LPR2, and SLC7A4 [30–32]. Although such an approach has proven its success in other complex traits such as diabetes and lipid levels, it suggests that this failure might be a result of relying on the GWAS panel and subjects from the general population [30]. This is specifically true as CKDGen encompasses a general population that may not easily identify new variants beyond what has already been identified by their GWAS approach. These challenges imply that further research is needed by performing targeted sequencing of these genes or whole-exome sequencing in a disease-specific cohort.

As such, our study has targeted patients with ESRD and identified 13 novel genetic variants associated with ESRD in the Saudi population. The top significant variant (NM_016341.4:c.4724G>C) is a missense variant within the PLCE1 gene that results in the substitution of arginine for proline at position 1,575 (p.R1575P) and is classified by ClinVar as uncertain significance for association with nephrotic syndrome type 3. When the same variant was not reported before for association with nephrotic syndrome type 3, several other variants within PLCE1 have been reported in familial and sporadic variants within PLCE1 (O'Seaghdha et al., 2014). The gene product of PLCE1 is expressed in the developing kidney in glomerular podocytes, and sequence alteration may lead to abnormal protein products that cause an arrest in normal glomerular development (O'Seaghdha et al., 2014). The second top variant (NM_001127898.4:c.163 + 72T>A) is a rare intronic variant within the CLCN5 gene, 0.00101 allele frequency in gnomAD, which has not been reported before for association with any phenotype. Several variants within CLCN5 are associated with different types of renal tubular disorders, such as Dent’s disease (Santín et al., 2011). The third variant (NM_001692.4:c.2T>C) causes a missense mutation, resulting in the substitution of methionine for threonine at position 1 (p.M1T) within the ATP6V1B1 gene, which encodes a component of vacuolar ATPase (V-ATPase), a multisubunit enzyme that mediates acidification of intracellular organelles. ATP6V1B1 variants are associated with an autosomal recessive form of distal renal tubular acidosis 2 (Santín et al., 2011). In addition, we have identified variants in LAMB2, INVS, FRAS1, C5orf42, SLC12A3, COL4A6, SLC3A1, RET, WNK1, and BICC1; which all play major pathophysiological roles in the pathogenesis of kidney functions (Kestilä et al., 1998).

From both clinical and economic viewpoints, understanding the genetic etiology behind CKD is essential before opting for renal transplantation. Such information may provide novel insights into disease recurrence risk post-transplantation. This study was performed in a unique and novel (Philippe et al., 2008) population that has not been previously explored—the uniqueness of the Saudi population lay in the high consanguinity rate—which provides a perfect population to study the genetic basis of diseases. This has been seen previously in a study that detected the highest rate of causative genes in the Saudi population (45.2%) compared to only 13.7% in Al-Hamed et al. (2016), which showed a strong correlation between the rate of consanguinity and the detection rate of disease-causing genes (R2 = 0.9414). Nonetheless, findings from this study might be limited by some important factors; first, the results are reported from a very homogenous population originating from the Saudi population, and it must be replicated in other diverse ethnic groups to validate the reported association. Second, the control group was younger and healthier than the case group, which could confound the reported results. However, given the fact that these genes are implicated in kidney function, they have pathophysiological roles that could mitigate some of these concerns. Third, we have only tested the disease status as a binomial trait, and future studies should be performed to test other kidney-related functions.

In summary, utilizing a comprehensive kidney-disease gene panel using a case-control study in a Saudi Arabian cohort, we identified 13 genetic variants significantly associated with ESRD in this cohort. Since this study was conducted on a disease-specific population and not on a general population, like most of the previously conducted GWAS, it provides more concrete evidence of the roles of these variants in the pathogenesis of ESRD. Furthermore, studies to replicate the findings of this study, as well as functional analysis of the identified variants, may provide measures to reduce the burden of CKD in KSA. In particular, that elucidating the genetic etiology of CKD may have important ramifications when considering disease recurrence risk post-transplantation. Further studies both to replicate these findings in larger sample sizes and among individuals of different ethnic groups and to functionally validate these candidate genetic variants are imperative. This may provide a comprehensive priority list of molecular targets for translational research and eventually help reduce the burden of CKD in KSA (Franceschini et al., 2006).