Variability in Kidney Stone Incidence Between Black and White South Africans: AGT Pro11Leu Polymorphism Is Not a Factor

Abstract

Background and Purpose:

Kidney stone disease is rare in the South African black (B) population and more prevalent in the white (W) population. Genetic studies have not previously examined this anomaly. The AGT Pro11Leu polymorphism in the alanine:glyoxylate aminotransferase (AGT) enzyme has been suggested as possibly playing a role in the pathogenesis of idiopathic calcium oxalate kidney stones. The present study was undertaken to investigate whether differences occur in the frequency of this polymorphism in subjects of both race groups.

Materials and Methods:

Healthy B (n=60) and W (n=60) male subjects each provided early morning spot urine, blood, and buccal cell samples. The AGT Pro11Leu locus was amplified using the polymerase chain reaction and polymorphism was genotyped using a restriction fragment length polymorphism.

Results:

There was no difference in the frequency of the AGT Pro11Leu polymorphism, and the major allele (C) was present at a frequency of 0.82 in B and 0.83 in W. Thus, the most common genotype homozygous normal CC genotype was observed at similar frequencies in both groups (0.68 and 0.65 in B and W, respectively), as were the heterozygous CT genotype (CT) and the homozygous variant TT genotype (TT) genotypes (0.33 & 0.02 and 0.28 & 0.03 in B and W, respectively). Neither urinary oxalate nor any other component in the two groups was correlated with the frequency of the AGT Pro11Leu polymorphism.

Conclusions:

Our data imply that the AGT Pro11Leu polymorphism is not directly responsible for the low incidence of stone formation in B. We conclude that other factors must be instrumental in protecting the B population from urolithiasis.

Introduction

In South Africa, the incidence of kidney stone disease has been reported as being much lower in the black (B) population than in the white (W) population.^1,2 The reasons for this disparity are unknown, in spite of several previous physicochemical and biochemical investigations,^1,3

–6 Somewhat surprisingly, genetic studies in South African populations have not been undertaken in attempts to explain this anomaly, even though it is commonly accepted that calcium oxalate (CaOx) stone formation may be influenced by such factors.^7,8 Indeed, the analyses of several South African stone collections from W patients during the past 30 years have shown this component to be the major one, comprising 60%–75% of all stones, in all but one of the studies.⁹ Furthermore, in those rare cases in which stones from B patients were obtained and analyzed, the composition thereof was not significantly different to that obtained for stones from the W group.⁹ Stone severity in either group has not been reported, presumably because it is not different to that which occurs in other race groups.

AGXT is a gene that encodes the intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT), a pyridoxal-phosphate-dependent enzyme that metabolizes glyoxylate by converting it to glycine.^10,11 Several mutations (>50) have been identified in this gene and it has been postulated that these mutations ultimately lead to severe hyperoxaluria.^12

–15 One mutation is the AGT Pro11Leu polymorphism in AGXT, which results in two variants: the major allele (C) and minor allele (T).^10,16 It is possible that the activity of the protein encoded by the minor allele of AGT has a lower activity compared with the major allele protein in vivo and results in an increased oxalate synthesis in hepatocytes.¹⁷ In addition, this polymorphism has also been reported to reduce the specific catalytic activity of AGT.¹⁸

It has been speculated that the presence or absence of the Pro11Leu polymorphism might be a contributory factor in an individual's susceptibility to idiopathic CaOx stone formation.¹⁵ We undertook to investigate whether differences occur in the frequency of this AGT polymorphism in healthy B and W South Africans with a view to establishing whether it may contribute to the difference in stone incidence in the two groups.

Materials and Methods

Study groups

Healthy South African males (60 B and 60 W) aged 18–30 years participated in the study. These study groups were limited in size due to financial constraints. The subjects were recruited from the staff and student cohorts of the University of Cape Town. The study was confined to males to avoid possible gender conflicts, given that the incidence of stone disease is different between males and females.¹⁹ The lower age limit approximates the age at which stone incidence begins to rise in males,¹⁹ while the upper limit was determined by the cohort of subjects available to us. All participants signed an informed consent form after receiving written and verbal information about the study. The study was approved by the Faculty of Human and Health Sciences Research Ethics Committee of the University of Cape Town (REC REF 390/2007). Studies were performed in accordance with the ethical standards as originally laid down in the 1964 Declaration of Helsinki and updated in October 2001.

Subjects were excluded if they or their first-degree relatives had a history of stone disease or any renal disorder, in accordance with our hypothesis that differences in the polymorphism frequency may differ in healthy subjects of the two race groups. Similarly, subjects were excluded if they had chronic digestive disease and/or gut disease or if they were taking any supplements and/or minerals. Imaging procedures were not performed to identify asymptomatic stone formers.

Sample collection

Total genomic deoxyribonucleic acid (DNA) was extracted from either blood or buccal cells using standard protocols. DNA from blood was extracted using a DNeasy^® Blood and Tissue Kit (QIAGEN, Hilden, Germany). Buccal cells were collected using sterile swabs (Epicenter Biotechnologies, Madison, WI) and the DNA was extracted using a standard cell lysis followed by a salting out procedure with an isopropanol precipitation.²⁰ The final DNA pellets were left to air-dry overnight, and then resuspended in 50 μL of TE (Tris-EDTA) buffer [10 mM Tris (tris(hydroxymethyl)aminomethane), 1 mM EDTA (ethylene diamine tetra acetic acid), pH 7.6].

Detection of Pro11Leu polymorphism

The AGT Pro11Leu locus was amplified using polymerase chain reaction (PCR) followed by a restriction enzyme digestion.¹⁰ PCR amplification was performed in thin-walled 0.2-mL PCR tubes containing the following reagents in a 20 μL reaction volume: 10 to 50 ng genomic DNA, 0.25 units of Super-Therm DNA polymerase (Hoffman-La-Roche, NJ), 1× reaction buffer (final concentration: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT (DL-dithiothreitol; Cleland's reagent), 50% glycerol and stabilizers), 0.4 μM of primers MIT 2 (5′-GCACAGATAAGCTTCAGGGA-3′) and EX-2R (5′-CTTGAAGGATGGATCCAGGG-3′), 1 mM MgCl₂, and 0.2 mM deoxynucleotide triphosphates. The PCR cycling parameters consisted of one denaturing step at 95°C for 3 minutes, followed by 35 cycles of 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute, and a final incubation step of 72°C for 10 minutes. For all samples, 15 μL of the PCR product was digested overnight at 37°C using 10 units of Eco130I restriction enzyme (StyI; Fermentas, Helsingborg, Sweden) in 10× Buffer orange (Fermentas, Helsingborg, Sweden). Restriction enzyme digest products were electrophoresed on a 2% agarose gel and visualized using ethidium bromide staining. Gels were electrophoresed for 80 minutes at 100 voltage.

Spot urine collection and treatment

As mentioned previously, financial constraints limited the number of analyses that could be performed. Accordingly, a subset (20 B and 20 W) of the original cohort of subjects provided urine samples (freshly voided early morning spot samples) on the same day that the blood samples were collected. Spot urine samples rather than 24-hour samples were preferred because collection and transport of these were more convenient for many of the B subjects who lived in township areas. Spot samples were deemed acceptable, as reliable correlations between them and 24-hour urine samples have been previously reported for several urinary components, although these were in the urine samples from females.²¹ Urine samples were tested for hematuria and nitrite using urinalysis test strips (Medi Test Combi 5N, Macherey-Nagel; Düren). All urine samples tested negative. Urine pH and volume were measured. Urine aliquots were filtered through a 0.74-μm filter to remove cellular debris and proteinaceous material. Sodium (Na), potassium (K), calcium (Ca), magnesium, Ox, citrate, chloride, creatinine, phosphate, and urate were measured using standard protocols.²²

Statistical analysis

Genetic data analysis was performed using GENEPOP (version 4), while urinary data were analyzed using one-way analysis of variance, the Pearson's chi-squared risk test method, and the student's t-test using STATISTICA. Data were considered significant if p≤0.05.

Results

Estimation of AGT Pro11Leu polymorphism

Restriction Fragment Length Polymorphism analysis confirmed the Pro11Leu polymorphism in our study groups with the CC homozygotes corresponding to 512 base pair DNA fragments of the major AGXT allele (C) and TT homozygotes to 619 base pair DNA fragments of the minor AGXT allele. The allele frequency of C, the major allele was not different in the two populations (0.82 in B and 0.83 in W, respectively). Consequently, the genotype frequencies did not differ significantly between the two populations either. The genotype frequencies for CC, CT, and TT were 0.65, 0.33, and 0.02 in B and 0.69, 0.28, and 0.03 in W, respectively. Both groups were in the Hardy–Weinberg equilibrium (p=0.667±0.0017 and p=1.00±0.0 for B and W, respectively). To examine whether the two populations were distinct, we calculated the fixation index, F_ST and this value was effectively zero (0.0078), again confirming that there is no genetic diversity between these two population groups and the AGT Pro11Leu locus.

Urinary analysis

Two urinary parameters, Na and K, were significantly higher in B relative to W (Table 1). Neither mean urinary Ox nor mean urinary excretion (mg/mg creatinine) of the aforementioned parameters or any of the other components in the two groups could be correlated with the frequency of the AGT Pro11Leu polymorphism.

Table 1.

Mean Urinary Compositions (SE) from Spot Urine Samples of Black and White Subjects and the Correlations with the CC and CT AGXT Genotype Distributions

				Genotype
Parameter ^a	Black (n=20) ^b	White (n=20) ^b	p-value	CC	CT	p-value
pH	5.72 (0.11)	5.73 (0.06)	0.9479	5.74	5.70	0.8088
Volume (mL)	261 (29)	260 (36)	0.9889	282	197	0.1087
Citrate	0.3073 (0.06)	0.1634 (0.06)	0.1136	0.2669	0.14	0.2329
Oxalate	0.0095 (0.003)	0.0116 (0.003)	0.5936	0.0123	0.0052	0.1122
Calcium	0.0537 (0.01)	0.0412 (0.01)	0.4945	0.0534	0.0296	0.2556
Magnesium	0.0227 (0.01)	0.0230 (0.01)	0.9722	0.0255	0.0149	0.2993
Sodium	2.1771 (0.50)	0.7949 (0.42)	0.0566^c	1.7159	0.7964	0.2798
Potassium	0.7932 (0.13)	0.1832 (0.12)	0.0022^d	0.5308	0.3601	0.4840
Uric acid	0.2297 (0.03)	0.2801 (0.02)	0.1890	0.2675	0.2170	0.2562
Phosphate	1.5260 (0.27)	1.8135 (0.20)	0.4637	1.8327	1.1811	0.1462

Units are mg/mg creatinine.

Combination of CC and CT groups.

Approaching significance.

Significance at p≤0.05.

SE=standard error of the mean; CC=homozygous normal CT genotype; CT=heterozygous CT genotype; AGXT=gene encoding metabolic enzyme alanine:glyoxylate aminotransferase.

Discussion

It has been previously reported that the AGT Pro11Leu polymorphism seems to vary among human populations, with the highest frequency reported in the Middle East and Europe (0.14–0.28), intermediate frequency in Africa (0.08–0.10), and the lowest in Eastern and Southern Asia (0.02–0.06).^10,23,24 It is worth noting that a higher incidence of stone formation has also been reported in the Middle East (20.1%), whereas it is 5%–9% in Europe, variable in Africa, and the lowest in Asia (1%–5%).^25
–27 As such, there seems to be a correlation between the frequency of this polymorphism and stone disease.

In the present study, the frequency of this polymorphism was 0.33 in B and 0.28 in W. Homozygosity for the major allele (C) Pro11 was the most common genotype in both B and W groups (0.68 and 0.65, respectively). Thus, homozygosity of the minor allele (T) was rare in both population groups (B: 0.02 and W: 0.03). Since there were no significant differences in the frequency of this polymorphism between the B and W groups, we conclude that it does not contribute toward the disparate stone incidence in the two groups. As far as we are aware, the present study is the first to document and compare the presence of this genotype in subjects of the B and W race groups in South Africa. As such, our findings are of interest.

There are other aspects of the present study that are noteworthy. A compelling qualitative relationship between AGT distribution and diet has been reported.²⁸ Caldwell et al.¹⁰ have shown that the frequency of the Pro11Leu polymorphism is much higher in populations, in which the ancestral diet is extremely meat-rich relative to those in which it is more mixed or more vegetarian. However, a recent study by Ségurel et al.²⁹ found a lower frequency of this detrimental mutation in herders, whose diet is more meat-rich, as compared with agriculturalists. These findings therefore challenge the veracity of Caldwell et al's¹⁰ claim. In the present study, we found similar frequencies of this polymorphism in two population groups, one of which has a history of traditional diet (B) and the other a history of western diet (W).³⁰ Interestingly, one of us (AR) has previously reported a significantly higher intake of animal protein in B relative to W, and a diet that is relatively hyperoxalurogenic in the former group.²⁷ These findings support the conclusion of Ségurel et al.²⁹ that the distribution of the observed AGXT gene variation could be due to demographic history rather than local adaptation to diet. According to Vester et al.,³¹ almost two-thirds of protein intake of the South African B population (especially in rural B) comes from plant sources.

With regard to urinary analysis, it is well recognized that during deficient hepatic AGT activity, glyoxylate may be oxidized to Ox, a major risk factor for primary hyperoxaluria type 1 (PH1) and CaOx stone formation.^15,32 In the present study, mean urinary Ox and indeed the mean urinary excretion of all the other components in the two groups could not be correlated with the frequency of the AGT Pro11Leu polymorphism. Correlations might have occurred in 24-hour urine collections rather than the spot samples, which we used and this should be addressed in the future studies of this nature.

Two recognized urinary risk factors for stone formation (K and Na) were found to be significantly higher in B than in W (K: p=0.0022, Na: p=0.0566). These findings are incidental to the focus of the present study and do not warrant discussion here. Nevertheless, we recognize that these differences suggest variations in the diet and that they could have potentially affected the results.

Our study had several limitations. Our study groups were small; larger cohorts are needed for more rigorous investigations.³³ In addition, the small number of urine samples for analysis and the absence of 24-hour samples could have influenced the results. In addition, our study involved subjects in a relatively small age group, so applying our findings across all age ranges might be tenuous. Furthermore, we did not include CaOx stone formers as a distinct group, as this was beyond the scope of the study.

Additionally, our study was certainly not exhaustive as we investigated only one single-nucleotide polymorphism of the AGXT gene. The latter consists of 11 exons covering about 10,000 base pairs of DNA, which codes for the 392 amino acid protein AGT. Given the size of this gene, more than 160 distinct mutations have been identified, resulting in a spectrum of clinical severity. A recent article by Kanoun et al.³⁴ identified the double mutation in five unrelated Tunisian families and they report an Ile244Thr mutation in addition to the Pro11Leu polymorphism. As with many diseases, finding a clear genotype–phenotype relationship is difficult given that it may be the result of numerous interactions among mutations, often of more than one gene, and coupled with epigenetic factors. Thus, it is not surprising that we found no clear difference in the allele frequency at the single locus we examined in our study.

Although nephrolithiasis is widely regarded as a complex, multifactorial disease resulting from an interaction between environmental and genetic factors,^35,36 the relative contributions of genetic factors and environmental factors in stone formation are not well established. A multivariate study by Saborio and Scheinman³⁷ concluded that genetic background accounts for 56% of the risk of stone formation, while in a later study, Baggio³⁸ noted that the environmental factors in renal stone disease seem to diminish the role of genetic factors. More recently, Goldfarb et al.³⁹ observed that despite data favoring a heritable component of stone disease in the general population, the genetic basis for Ca stones in general remains unknown. Whatever its contribution in causing stone formation, the present study investigated a possible converse role for genetics in which it might contribute to stone protection rather than stone pathogenesis. We believe that it is the first study in the urolithiasis field to adopt such an approach. Further studies that adopt this shift in thinking are possibly warranted.

Conclusions

Footnotes

Acknowledgments

We would like to thank Prof. Danpure CJ (University College, London) for helpful discussions. Dr. Natalie Roetz and 3rd year Molecular and Cell Biology students: A Davidson, L Geldenhuys, M. Herzog, O. Nair, M. Rahim, N. Thawer, S. Thawer, and E Wilson Poe (University of Cape Town) for assisting with genetic aspects of the study and Dr Ian Durbach (University of Cape Town) for statistical consultations.

Disclosure Statement

No competing financial interests exist.

Abbreviations Used

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