Introduction

Primary hyperoxalurias (PHs) are rare inborn errors of metabolism (glyoxylate) which lead to the overproduction of oxalate in the liver [1, 2]. The accumulation of oxalate results in the calcium oxalate (CaOx) crystals that are deposited in the urinary tract and kidneys [3]. Hyperoxalurias are classified into two main categories (PHs and SHs) [4, 5]. Secondary hyperoxalurias (SHs) generate from an increased oxalate absorption due to various alterations, including intestinal inflammatory diseases, very low calcium diet, bariatric surgery, or undue oxalate precursors intake [6, 7].

PH type I (OMIM 604,285) is the most severe type of hyperoxaluria. It is an autosomal recessive disease resulting in an insufficiency of alanine glyoxylate aminotransferase enzyme (AGT), which takes part in glyoxylate metabolism final step [8]. As a result, glyoxylate is not effectively metabolized to glycine, causing overproduction of both glycolate and oxalate [9]. PH type I is associated with progressive nephrocalcinosis, or recurrent urolithiasis, which leads to CKD stage 5 during the first 2–3 decades of life [10]. Infantile oxalosis is the most severe clinical presentation of PH type I.

PH type II (OMIM 260,000) results from the deficiency of the enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR), which plays a key role in the gluconeogenic pathway and helps in the removal of cytosolic glyoxylate [6, 11]. Lack of GRHPR results in glyoxylate and hydroxypyruvate accumulation that is processed by lactate dehydrogenase to L-glycerate and oxalate. The clinical picture of PH type II is milder compared to PH type I; however, affected individuals may suffer from frequent episodes of urolithiasis that might lead to kidney failure [12].

PH III (OMIM 613,616) results from recessive variants in the HOGA1 gene, which encodes the enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA). Clinical presentations include a less severe form with early onset, a low chronic kidney disease risk, an early disease onset, impaired kidney function and recurrent stone disease [13].

In this study, we sought to characterize the genetic and clinical characteristics of PH patients in Saudi Arabia in order to provide guidance to clinicians and researchers interested in this rare disorder.

Materials and methods

Inclusion criteria

The study included cases with confirmed molecular diagnoses having PHs type I, II, or III. In addition, cases that presented pathogenic or likely pathogenic variants in either AGXT, GRHPR, or HOGA1 genes according to the ACMG criteria were included. Finally, affected individuals having a variant of uncertain significance (VUS) were also included, who had PHs before molecular testing and who had consistent PH phenotypes.

Genetic testing

The molecular diagnosis in PHs patients was performed using single gene testing or WES, as previously reported [14,15,16].

Variant classifications and prioritization

ACMG guidelines were followed for variant classification [17]. Variant filtration was performed using standard methods [18]. Different online databases (ExAC, gnomAD, 1000 genomes, etc.) were used to exclude the polymorphic nature of the identified variants. In addition, the variants were fully segregated within available family members.

Molecular biogenetic testing

Sanger sequencing was performed to segregate the identified variant in all members of the families, followed by WES. Primers were designed using Primer3 and Sanger sequencing was performed using standard methods.

Pathogenicity of the variants

Sanger sequencing confirmed the segregation of all the discovered variants. For missense variants, in silico analysis tools, including Polyphen2, SIFT, Varsome and MutationTaster were used to predict the pathogenicity of the novel variants. Conservation of the variants was investigated using HomoloGene (https://www.ncbi.nlm.nih.gov/homologene).

Results

This retrospective chart review study includes 25 patients diagnosed with PHs from three tertiary centers in Riyadh and Jeddah, Saudi Arabia (October 2019–October 2021). The IRB of KAIMRC, KSU-Medical City Hospital, (Riyadh), and KAU (Jeddah) KSA approved the study. Since the study is based on retrospective data, written informed consent was not required.

Demographic details of patients

The study consisted of 25 patients with 13 males and 12 females, and affected individuals ranged from 3 to 50 years of age (Table 1). The majority (96%) of participants (24/25) were less than 18 years old. All of the affected individuals were from different regions of Saudi Arabia [North (7/25: 28%), central (13/25: 52%), West (5/25: 20%)]. Consanguinity was reported in 88% of the cases (22/25). The age at diagnosis of the affected individuals was between 6 months and 39 years, and a positive family history was observed in 32% (8/25) of cases.

Table 1 Clinical description of 25 PH patients investigated in the present study
Full size table

Initial symptoms

In most cases, the affected individuals presented with vomiting, flank pain, abdominal pain, kidney stones, hematuria, fever, and nephrocalcinosis.

Developmental milestones

Of the 25 patients evaluated, 2 (8%) manifested failure to thrive and developmental delay, including delayed gross and fine motor development, and language issues. All of the other affected individuals achieved normal developmental milestones. Three of the affected individuals were deceased (3/25; 12%) at the ages of 13 years, 39 months and 11 months. Cause of death included burden of CKD stage 5 for the youngest patient — the patient’s parents chose to give the child palliative care at home. Thus, the patient died at home with no documented hospital death report. The second patient died due to septic shock, and the third died at the age of 11 months having primary hyperoxaluria and Down syndrome.

Kidney examination

Detailed clinical investigation showed kidney manifestations in all of the affected individuals, including nephrocalcinosis, hematuria, and multiple kidney stones (Table 1). Nephrolithiasis and/or nephrocalcinosis were present in all patients. Kidney stones were present in (18/25; 72%), while nephrocalcinosis was present in (15/25; 60%), hematuria (8/25; 32%), proteinuria (4/25; 16%), and abdominal pain (9/25; 36%). The age of the patients when kidney stones were observed ranged between 22 months and 13 years, and most of the patients suffered from bilateral kidney stones (18/25; 72%). CKD stage 5 was observed in 28% of the patients (7/25). Skeletal manifestations affected 24% (6/25) of the patients, while other manifestations included recurrent urinary tract infection (12%; 3/25) (Tables 1 and 2).

Table 2 Summary of additional clinical features observed in the present cohort
Full size table

Laboratory examination

Detailed laboratory investigations were performed as summarized in Table 1. Urinary oxalate concentration was investigated in 6 affected individuals (upper normal limit 0.5 mmol/L): all had elevated urinary oxalate level. Plasma oxalate concentration (normal range: 1.3–3.1 μmol/L) was measured in 10 affected individuals and all had high plasma oxalate levels. Three of the affected individuals showed both proteinuria and hematuria, and three affected individuals showed hypercalciuria (Table 1).

Other abnormalities

Two patients revealed additional clinical phenotypes such as hepatomegaly, skeletal malformation and hypotonia (2/25; 8%).

Molecular analysis

All of the 25 affected individuals were subjected to single gene testing or whole-exome sequencing (WES) using standard methods, followed by segregation analysis using Sanger sequencing [15]. Molecular genetic testing revealed a confirmed molecular diagnosis in 19/25 cases (76%), and the majority of them were previously reported disease-causing variants. Nearly all identified variants were homozygous and thus demonstrated an autosomal recessive pattern. In addition, one proband revealed an AGXT-compound heterozygous variant.

AGXT gene variants [c.33dup; p.(Lys12GlnfsX156), c.187G > C; p.(Gly63Arg)] were observed in 14 cases (56%), GRHPR gene variants [c.707A > T; p.(Glu236Val)] were observed in 4 patients (16%), an already reported HOGA1 gene variant (c.860G > T; p.Gly287Val) was observed in 1 patient (4%), and six patients had negative molecular genetics results (24%). The homozygous variant identified in the GRHPR gene (c.707A > T; p.(Glu236Val) was novel. The most frequently identified variants were missense (12/25; 48%), followed by frame-shift duplication variant (7/25; 28%) (Fig. 1). Due to clinical heterogeneity, no clear genotype–phenotype correlations could be concluded. All the identified variants in patients were also screened in all the available family members using standard Sanger sequencing.

Fig. 1
figure 1

Variants identified in the present study

Full size image

The patients with kidney stones and negative molecular results were diagnosed based on the occurrence of kidney stones, type of kidney stones (Ca-oxalate in stone analysis), elevated urine glycolic and oxalic acids in urine organic acid, and elevated urine oxalate /creatinine ratio (hyperoxalemia).

Management

Dialysis was performed for seven patients (7/25; 28%). Six patients were on hemodialysis (HD), and one was initially on peritoneal dialysis then shifted to HD once referred to tertiary hospital. Depending on feasibility, HD was performed almost 5–6 sessions per week for all patients. Every patient was prescribed different treatments depending on the disease severity and condition, including pyridoxine, polystra tacrolimus, amlodipine, atenolol, cholecalciferol, colchicine, folic acid, hydrochlorothiazide, acetaminophen, prednisolone, and potassium citrate (Table 2). These medications improved the overall quality of life of the affected individuals. In addition, liver transplant was performed for two affected individuals (2/25; 8%) and the eGFR values at the time of liver transplant for patient 1 and patient 3 were 7 ml/min/1.73 m2 and 91 ml/min/1.73 m2, respectively.

Vitamin B6 (pyridoxine or pyridoxal phosphate) can help to reduce the production of oxalate in some patients affected with PH1 by enhancing the activity of the pyridoxal phosphate-dependent enzyme alanine: glyoxylate aminotransferase (AGT) [19]. Around 30–50% of PH1-affected individuals are pyridoxine-responsive, and after a minimum of 3 months, therapy resulted in a 30% reduction in urine oxalate concentration [20]. For deficient AGT to respond to pyridoxine, some degree of enzyme activity must be preserved that occurs with certain pathogenic AGXT variants [21]. Most pyridoxine-responsive individuals show maximum benefit at a dose of 5–8 mg/kg/day [22, 23]. Some require higher doses depending on the disease severity; however, a starting pyridoxine dose of 5 mg/kg/day is recommended. The conditions required for pyridoxine follow-up urine collection include stepwise increase in pyridoxine dose (maximum 10–20 mg/kg/day) [24, 25]. Vitamin B6 response can be difficult to determine in patients with CKD at advanced stage since, at that time, the urinary oxalate excretion rates may be influenced by the low GFR.

Discussion

Herein, we present the results of a retrospective study of a pediatric cohort of patients with PH from Saudi Arabia. Genetic variants were identified in 76% of patients (19/25). All patients with PH become symptomatic within the first decade of life. The majority of the PH type I affected individuals developed symptoms by 5 years of age; 1 to 2% of infants with PH type I developed kidney failure [26]. On the other hand, 3.2 years to 8.0 years was the average age for the onset of PH type II [12, 27].

While in European studies, PH type II is estimated to range from 1 to 3 in 1,000,000 people [19], the prevalence of PHs in Saudi Arabia is unknown. PH type I is estimated to have a 1:149,000 prevalence in Europeans and 1:157,000 in African Americans [28]. There is no estimated prevalence for PH type III; however, its prevalence is lower than PH type I and type II, making genotype–phenotype correlations difficult.

PHs type I–III are recessively inherited glyoxylate metabolism disorders, associated with an increased endogenetic oxalate synthesis and thus cause excessive accumulation of glyoxylate in the body. Most cases (70–80%) are classified as PH type I [24], caused by disease-causing variants in the AGXT gene that results in the absence or deficiency of the AGT enzyme. As transamination of glyoxylate to glycine is catalyzed by AGT, its deficiency allows reduction of glyoxylate into glycolate and this is then oxidized into oxalate. PH type II is less severe, resulting from the deficiency of GRHPR enzyme [1, 12, 24]. Garrelfs et al. [29] reported 101 patients from eleven countries exhibiting PH type II. A total of 82.8% of the patients in their cohort revealed urolithiasis and molecular diagnosis revealed 18 novel GRHPR gene mutations. Eight patients had CKD stage 2 and 22 showed CKD stage 5. Fifteen kidney transplantations were also performed in 11 patients [12]. PH type III is caused by disease-causing variants in HOGA1, encoding HOGA responsible for mobilizing the last step in hydroxyproline metabolism [19, 24,25,26,27,28,29,30].

The disease‐causing variants identified in our study are located in three different genes that have been previously implicated in causing PHs. According to the molecular study results, PH type I (OMIM 259,900) caused by homozygous variants in the AGXT gene (OMIM 604,285) was the most common gene mutated. The most common variant and probably the hotspot variant in this region is the frameshift variant in AGXT [c.33dup; p.(Lys12Glnfs*156)].

The present study reports 19 variants in 25 different individuals. The rate of previously reported variants associated with PH was high, while the GRHPR novel variant (c.707A > T; p.(Glu236Val) causing PH type II was identified in 4 different affected individuals. All of the identified variants in patients were also screened in all the available family members using standard Sanger sequencing. The pathogenic effect of the identified variants was confirmed using in silico prediction tools such as PolyPhen, MutationTaster, Varsome, etc. Additionally, the frequency of these variants in the general population was checked in ExAC, gnomAD, 1000 Genome Project databases, and + 2000 exomes/genomes. Additionally, segregation analysis of all the identified variants was performed using Sanger sequencing.

The phenotype in the patients presented here was mild to severe compared to previously reported cases from Saudi Arabia [31]. Indeed, the 16 patients previously reported presented with nephrolithiasis (13/16) and nephrocalcinosis (12/16). Among them, 4 patients revealed CKD stage 5, 3 underwent kidney transplantation, 4 showed nephrocalcinosis and nephrolithiasis and 3 with hyperoxaluria were asymptomatic [31]. In addition, they also observed urinary tract infection, abdominal pain, hematuria, and none of the patients presented an infantile form of PHI [31]. Furthermore, disease-causing AGXT variants [c.33dup; (p.Lys12GlnfsX156), c.187G > C; p.(Gly63Arg)] previously reported, and variants in patients presented here do not cluster within one domain and are distributed in different domains, which might be one of the reasons for heterogeneity in clinical presentation. Although, these regions are crucial for the normal function of AGXT and might be intolerant to different missense and frameshift variations reported in these regions. Consanguineous marriages are very common in Saudi Arabia, accounting for more than 52% of all marriages. Similarly, in the present study consanguineous marriages were observed in 88% of the patients. Interestingly, both consanguineous and non-consanguineous families revealed homozygous variants, showing that the variants are prevalent and hotspots in the Saudi population. The age of the affected individuals at first symptoms was between 1 month and 20 years, and the age at diagnosis was between 6 months and 39 years. In the cohort reported here, the average delay in diagnosis was approximately 3 years and 2 months.

Affected individuals diagnosed with any PHs should be treated immediately with high fluid intake. Urinary CaOx supersaturation can be reduced with alkali citrate treatment [32]. In the liver, citrate is converted to bicarbonate, and the alkali reduces the citrate reabsorption (intratubular) and thus citrate excretion is increased. For PH type I patients, the second treatment strategy includes enhancing the reduced activity of AGT [27]. As a small amount of urinary oxalate is derived from the diet in these patients, dietary oxalate restriction showed limited benefit [33]. In addition, individuals with PH1 have an excellent safety profile with pyridoxine (vitamin B6) treatment.

It was recently reported that the RNA interference (RNAi) therapeutic agent lumasiran targets the glycolate oxidase and thus reduces hepatic oxalate production. It has been reported to reduce urinary oxalate excretion, which is the main cause of kidney failure in PH1 patients. Most patients who received lumasiran treatment revealed normal levels within 6 months [34].

Such studies also highlight the need for periodic reevaluation of variants, either by molecular reanalysis, a certified laboratory, a geneticist, or scientists responsible for variant interpretation. The ACMG has issued standard guidelines for variant classification. A reasonable approach might be to review the molecular findings every 6 to 10 months if there is any reclassification of inherited VUS or likely pathogenic variant. This might help parents planning to have more children through pre-implantation genetic diagnosis (PGD) [35]. Additionally, population-specific allele identification will assist in data sharing among labs and ultimately decrease the burden. Molecular testing results are dynamic and significantly change with new variant reclassification over time that might help reduce VUS burden; however, a significant number of patients with inconclusive test results remain.

Physicians in KSA face many issues associated with disease management and treatment of rare genetic disorders. As the frequency of rare genetic disorders in KSA is more than in any other country, there is a need for a nationwide campaign that might raise awareness. KSA requires its own evidence-based nationwide guidelines for treating such rare disorders and physicians should be equipped with the latest knowledge about the latest therapies and new treatments approved by the FDA. However, prenatal genetic screening including noninvasive prenatal testing (NIPT), preimplantation genetic testing for aneuploidy (PGT-A), and preimplantation genetic testing for monogenic disorders (PGT-M) has been established recently in KSA to tackle such severe rare diseases [35, 36]. However, nationwide systematic neonatal screening and proper awareness is the need of the day [14].

Although we believe this study is the largest cohort from the region, it is limited by the relatively small sample size and the absence of functional studies. Nevertheless, we present clinical, molecular, and genetic data for 25 patients along with identification of common disease-causing variants found in the Saudi population. Such studies might help better understand the disease pathogenesis, which may facilitate evidence-based and well-designed therapies in the future.