Objective To assess the use of Fok I polymorphism (the most frequent polymorphism, at the start codon of the vitamin D receptor gene, VDR) as a convenient genetic marker in identifying the cause of urolithiasis.
Patients, subjects and methods A normal control group of 90 healthy subjects and 146 patients with calcium oxalate stones were examined. Using polymerase chain reaction (PCR)-based restriction analysis, the relationship between Fok I polymorphism and urolithiasis was evaluated. An unexcisable length of 265 bp was identified (allele CC) and two fragments (169 bp and 96 bp) identified as excisable lengths (allele TT).
Results There was a statistically significant difference between the groups (chi-square test, P < 0.05) for the genotype of the VDR Fok I start codon polymorphism. The odds ratio (95% confidence interval) for the C allele in those at risk of stone disease was 1.672 (1.149–2.432).
Conclusions These results suggest that the VDR Fok I start codon polymorphism may be a good candidate for a genetic marker in calcium oxalate stone disease.
Urolithiasis is a multifactorial disease; the cause of calcium oxalate stones is heterogeneous and might involve both genetic and environmental factors. Although the genetic causes have been studied extensively, no chromosomal mapping has been conducted in patients with stones and idiopathic hypercalciuria (IH) [1,2]. The only conclusive evidence through genetic studies  is that urolithiasis is a polygenic defect and partly penetrative. Hypercalciuria is a well-recognized risk factor associated with calcium oxalate urolithiasis, and is usually treated to prevent stone formation. However, no well-documented disorder has been detected to support this clinical occurrence and treatment has variable results. Recently, single nucleotide polymorphisms (SNPs) were used as a tool to map the disease-causing gene, the result of which provided evidence that vitamin D receptor (VDR) gene polymorphism was associated with urolithiasis, making possible a search for the candidate genes [4,5].
The VDR gene has been used as a genetic marker in the prediction of bone mass density in menopausal women . The SNP sites most frequently used were the start codon (Fok I) and intron 8 (Bsm I) polymorphisms. In a study of postmenopausal Mexican-American women, Gross et al. found a marked association between Fok I polymorphism and bone marrow density (BMD). Subsequent epidemiological studies confirmed the association between VDR gene polymorphism and BMD in premenopausal black and white women . However, diverse ethnic backgrounds may have yielded discrepant results. Nevertheless, VDR gene polymorphisms have been used as genetic markers to determine their association with calcium oxalate disease [9,10]. Because both the control and experimental groups consisted of ethnically homogeneous patients, the current study used a PCR-based restriction analysis to investigate the distribution of VDR gene start codon polymorphisms between healthy control subjects and patients with stone.
Patients, subjects and methods
The control group comprised 90 healthy volunteers aged > 40 years (42 men and 48 women, mean 55.5 years, range 42–73) who had no history of familial stone disease, or of renal calcification. All patients in the control group also underwent renal ultrasonography and an assessment for urinary microscopic haematuria to ensure that they were urologically healthy. The stone formers comprised 146 patients (101 men and 45 women, mean age 44.2 years, range 24–73) with calcium oxalate stone who had been treated between 1998 and 2000 (in the department of urology). Serial blood and urine biochemistry tests were undertaken to exclude possible hypercalcaemia, hyperuricaemia or hyperuricosuria. Patients who had symptoms of UTI during their treatment for stones were excluded from the analysis. Informed consent was obtained from all patients and subjects. Stone composition was verified using infrared spectroscopy and showed either calcium oxalate monohydrate or dihydrate, or a combination of the two. The genomic DNA was prepared from peripheral blood using a genomic DNA isolation reagent kit (Genomaker, Taiwan).
PCRs for the VDR gene start codon polymorphism were carried out in a total volume of 25 µL, containing genomic DNA (2–6 pmol of each primer), 1 × Taq polymerase buffer (1.5 mmol/L MgCl2) and 0.25 units of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA, USA). The primers of the VDR gene were:
5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′, according to the report by Harris et al.. PCR products were amplified in a programmable thermal cycler (GeneAmp PCR System 2400, Perkin Elmer). The cycling condition for start codon polymorphism was set as follows: one cycle at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 20 s, and one final cycle of extension at 72 °C for 7 min
The PCR product of the 265 bp band was mixed with two units of Fok I (Novel, Beverly, MA, USA) and with the reaction buffer, according to the manufacturer's instructions. The restriction site was designed to be located at the recognisable allele of ATG to form an excision site. Two fragments of 169 bp and 96 bp, respectively, will be present if the product is excisable. The reaction was incubated at 37°C overnight; 10 µL of the product was then loaded into 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was then divided into three groups; excisable (TT), unexcisable (CC) and heterozygote (TC). The allelic frequency distribution of polymorphisms between the control and patient groups was compared using the chi-square test. The odds ratios (OR) with 95% CI were calculated for the polymorphism of the VDR gene. Results were considered statistically significant when the probability of findings occurring by chance was < 5% (P < 0.05).
The bands on the gel showed the three distinct groups, i.e. two excisable (TT) and unexcisable (CC) homozygotes and one heterozygotic group (TC) (Fig. 1). The frequencies of the genotypes in the patient and control groups are shown in Table 1, and for each gender. There were significant differences between the groups in the distribution of Fok I polymorphism (chi-square, P < 0.05). The frequency of the CC allele in the patients (37%) was higher than in the control group. There was an apparent correlation between Fok I polymorphism and the risk of calcium oxalate stone disease. From the allelic distribution of C/T polymorphism in subjects and patients, the OR in patients was 1.672 (1.149–2.432).
Table 1. The distribution of the VDR gene Fok I polymorphism between healthy control subjects and the calcium oxalate stone formers, and by gender in each group
The activation of VDR after ligand binding could regulate the expression of osteocalcin , which has been proposed to be involved in the formation of stone matrices . Furthermore, VDR is not only upregulated by vitamin D but also by protein kinase A, parathyroid hormone and growth factors. VDR can also down-regulate protein kinase C . These functions related to the VDR indicate that the VDR gene associated with stone disease is not limited to the regulation of calcium but is affected by other signalling pathways, thus potentially complicating its use in mapping calcium oxalate stone disease.
Hypercalciuria is common among patients with calcium oxalate nephrolithiasis and is thought to contribute to stone formation by increasing the state of urine supersaturation. However, a genetic mechanism for hypercalciuria has not yet been defined from current evidence. Mutations either in CLCN5 or in the calcium-sensing receptor gene are not a common cause of IH [14,15]. The only promising genetic locus for hypercalciuria is that associated with intestinal absorptive hypercalciuria, located at chromosome 1q23.3-q24, but there remains a 4.3 Mbase region to be studied . Although hypercalciuria may be associated with stone formation, 80–90% of IH is asymptomatic and not associated with lithiasis . The restriction of calcium intake (based on a diagnosis of hypercalciuria) to prevent stone recurrence has the reverse effect [18,19]. Problems of decreased bone mineral density, reciprocal hyperoxaluria and the increased risk of stone formation are also reported . Therefore, calcium oxalate disease is complex and not clearly associated with calcium per se.
Using the restriction endonuclease Fok I to identify a single base difference of the VDR gene start codon among individuals, the distribution of SNPs was determined in the present patients. The results indicate that Fok I polymorphism might be a useful genetic marker for mapping calcium oxalate stone disease. The C allele was prominent in the patient group; individuals with the CC or CT genotypes of the VDR gene are at high risk of calcium oxalate stone disease. Interestingly, the allelic distribution of Fok I was lower in Taiwanese subjects (23% CC) than in the population assessed by Harris et al.; in the latter, half the subjects were CC homozygotes.
In a study of the distribution of the VDR allele in patients with stones and normal healthy subjects, Zerwekh et al. found no link between mutations of the VDR and the common VDR genotype. They also found no significant differences between VDR genotypes and serum or urine biochemical variables in their subgroup of patients with absorptive hypercalciuria. They concluded that absorptive hypercalciuria, the most common cause of kidney stones reported by Pak et al., may be a heterogeneous disorder . As both a different genetic site and different population were assessed in the present study, the results differed from those of Zerwekh et al.; there was a significant association between VDR polymorphism and calcium oxalate stone disease, based on selecting patients with idiopathic calcium oxalate recurrent stones. Therefore, different selection criteria for patients and the use of different genetic markers may give different associations. Furthermore, the VDR gene may have alternative pathways involving the formation of calcium oxalate stone; further studies of the mechanism are needed.
Although it is currently not possible to identify the gene responsible for stone disease, with the accumulation of mapped genes, the likelihood that these regions will contain a candidate gene is promising . The candidate genes might provide further analyses for tissue expression or clinical presentations in a variety of groups. The polymorphism of Fok I may be a suitable genetic marker for the further study of the possible causes of urolithiasis. Because the present results are preliminary and obtained from a homogeneous group of Taiwanese patients, the findings should be confirmed.
This study was supported by a grant from China Medical College Hospital (DMR-90–008).