Polymorphism in the PBX1 gene is related to cystinuria in Brazilian families

Abstract The aim of our study was to determine regions of loss of heterozygosity, copy number variation analysis, and single nucleotide polymorphisms (SNPs) in Brazilian patients with cystinuria. A linkage study was performed using DNA samples from six patients with cystinuria and six healthy individuals. Genotyping was done with the Genome‐Wide Human SNP 6.0 arrays (Affymetrix, Inc., Santa Clara, CA, USA). For validation, SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. The homozygote polymorphic genotype of SNP rs17383719 in the gene PBX1 was more frequent (P = 0.015) in cystinuric patients. The presence of the polymorphic allele for this SNP increased the chance of cystinuria by 3.0‐fold (P = 0.036). Pre‐B‐cell leukaemia transcription factor 1 (PBX1) was overexpressed 3.3‐fold in patients with cystinuria. However, when we compared the gene expression findings with the genotyping, patients with a polymorphic homozygote genotype had underexpression of PBX1, while patients with a heterozygote or wild‐type homozygote genotype had overexpression of PBX1. There is a 3‐fold increase in the risk of the development of cystinuria among individuals with this particular SNP in the PBX1 gene. We postulate that the presence of this SNP alters the expression of PBX1, thus affecting the renal absorption of cystine and other amino acids, predisposing to nephrolithiasis.

mutations in SLC3A1 or SLC7A9 in all patients with this disease, and the study results differ according to the geographical location. 3 This study describes novel variants and consists of the molecular characterization of cystinuria in Brazilian families. In this investigation, genomic aberrations associated with cystinuria were examined using genome-wide human single nucleotide polymorphism (SNP) arrays. This approach has the advantages of SNP genotyping and allows the detection of copy number variation (CNV) breakpoints and loss of heterozygosity (LOH), used for surveying segments for allelic losses. Here, we found an association between a SNP in Pre-B-cell leukaemia transcription factor 1 (PBX1) gene and the risk of development of cystinuria.

| Patients
This study included a sample of 18 patients under treatment by a tertiary academic care centre. Eight patients with cystine stones (mean age 27 years, four men and 4 women) and 10 controls (mean age 39 years, three men and seven women) without the evidence of any calculi on urinary tract ultrasound or a previous history of the disease were included in the analysis. Clinical diagnosis of cystinuria was based on the biochemical measurement of cystine in the urine and the presence of cystine renal stones. The serum and urinary metabolic profile of all patients were evaluated by NM-BAPTA method.
For the microarray study, 12 patients out of 18 were selected and divided into two groups: patients with cystine stones (n = 6) and patients without stones and no familial history of cystinuria (n = 6). To validate the results of the genetic polymorphisms by qPCR, we genotyped all 18 patients, 10 without stones and 8 patients with cystinuria.
Patients in both groups provided written informed consent to participate in the study and allowed their biological samples to be genetically analysed. Approval for the study was given by the Institu-

| Affymetrix SNP 6.0 analysis
The whole blood was collected from patients using vacutainer system. Genomic DNA was isolated using a QiaAmp DNA Mini Kit (Qiagen, CA). Total genomic DNA (250 ng) was digested with a restriction enzyme (NspI or StyI) and ligated to adaptors that recognized the cohesive four base pair (bp) overhangs. All fragments resulting from restriction enzyme digestion were substrates for adaptor ligation. A generic primer that recognizes the adaptor sequence was used to amplify adaptor-ligated DNA fragments. PCR conditions were optimized to preferentially amplify fragments in the 200-1100 bp size range. The amplified DNA was then fragmented, labelled, and hybridized to Genome-Wide Human SNP 6.0 arrays (Affymetrix, Inc.), this array including more than 906 600 SNPs and more than 946 000 probes for the detection of CNV. After 16 hours of hybridization at 49°C, the arrays were washed by a Fluidics Station 450 and scanned by a Gene Chip Scanner 3000 (Thermo Fisher Scientific, Waltham, MA, USA).

| SNP validation by qPCR
Seven SNPs were genotyped using a TaqMan ® SNP Genotyping Assay Kit and an ABI 7500 fast system (Applied Biosystems, Foster City, CA, USA). SNP-specific polymerase chain reaction (ss-PCR) primers and fluorogenic probes were designed using Primer Express (Applied Biosystems) ( Table S1). The fluorogenic probes were labelled with a reporter dye (FAM or VIC) and were specific for one of the two possible bases identified for that site in the gene sequence.
The target sequence was amplified in a 10 μL reaction volume that contained 5 μL of TaqMan ® Universal PCR Master Mix, 0.25 μL of SNP Genotyping Assay (primers and probes, Table S1), 1 μL of genomic DNA, and 3.75 μL of DNase-free water. The PCR cycling conditions were 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C.

| Real-time PCR for PBX1 gene expression
We used a mirVana kit (Ambion, Austin, TX, USA) for RNA extraction from whole blood, and cDNA was obtained using a High-Capacity The PCR cycling conditions were 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 1 min at 60°C. All reactions were performed in duplicate, and TaqMan B2M (HS00187842_m1) was utilized as housekeeping gene or endogenous control for gene expression.

| Statistical analysis
The statistical analysis of the microarray slides was performed with plink software (http://pngu.mgh.harvard.edu/~purcell/plink). Categorical variables are expressed as numbers and percentages. The associations between genotype and allelic frequencies in the cases and controls were examined by chi-squared tests and for allelic distribution, the Fisher exact odds ratio (OR) and the corresponding 95% confidence intervals (CIs) were calculated. To compare the urine and serum metabolic profile between cases and controls, we

| Serum and urinary analysis
The mean age of the cystinuric patients and control group cases was 28.5 ± 10.9 and 39.2 ± 15.9 years, respectively (P = 0.126). The serum and urinary metabolic profile of all patients are shown in Tables S2 and S3, respectively. Seven patients with cystinuria were taking citrate at the time of urine collection. The only metabolic difference between the groups was a higher level of serum uric acid in cystinuric patients (6.07 ± 1.88 vs 4.60 ± 0.96 mg/dL; P = 0.047).

| Copy number variation analysis
For the CNV, no statistically significant comparison could be attained between cases and controls due to our small sample size. Nevertheless, the descriptive analysis depicted in Table S4 highlights that the changes found were not specific to either cystinuric patients or controls.

| Loss of heterozygosity analysis
We examined the distribution of LOH on each chromosome. We found 36 changes in patients with cystinuria and only six variations in the control group. When assessing regions with more frequent LOH in cystinuria patients (Table S5), the changes occurred in one or more regions of SLC genes in 83.3% of patients.

| Single nucleotide polymorphism analysis
To confirm the microarray results, qPCR analysis was done for five SNPs associated with the disease in our cohort and in two other cases that have been reported in the literature (Table S1). We also found statistically significant differences for the SNPs For SNPs in the SLC7A9 (rs140134166) and SLC3A1 (rs200248046) genes, no statistical association was observed between cases and controls. Importantly, no case or control patient had the polymorphic allele for SNP in SLC7A9.

| PBX1 gene expression
Due to the significant results found when we evaluated the SNP in

| DISCUSSION
The markers that have arisen for studies of association and linkage are SNPs, the most common and powerful markers due to their abundance and stability. 4 There are more than 10 million SNPs catalogued so far with an allelic frequency of at least 5%. 5 To the best of our knowledge, no other study has searched for SNPs in patients with cystinuria. Our study is a pilot study, and although the sample size is too small for a large-scale analysis, we found few SNPs that were more frequent in cystinuric patients than in patients without kidney stone disease. We have also validated the results through PCR for SNP rs17383719, which is located in the PBX1 gene on chromosome 1. No changes in this gene had been associated with the cystinuria phenotype so far; however, we found that the presence of just one polymorphic allele increased the risk of developing the disease three-fold.  in genes of this specific family. 9 Interestingly, when analysing the frequency of SNPs present in the SLC7A9 (rs140134166) and SLC3A1 (rs200248046) genes, in our cohort the SNP in SLC7A9 was genotyped as wild-type homozygote for all patients. In the SNP SLC3A1, we found a predominance of the heterozygote genotype for cystinuria and control patients. Polymorphisms in these two genes are classic alterations described in the literature in patients with cystinuria. 3,11-13 However, we must emphasize that we have analysed only one change in each of these genes and that recent studies have shown that there are several mutations that may be associated with the phenotype of cystinuria. Furthermore, they might differ according to the geographical location of the studied patients. 14,15 This is the first study to describe LOH and CNVs in patients with cystinuria. LOH was found in regions where the genes of the SLC family usually associated with cystinuria are located. Furthermore, this study describes novel variants, expands the spectrum of cystin-

CONF LICT OF I NTEREST
The authors confirm that there are no conflicts of interest.

AUTHOR CONTRIBU TI ON
NIV, RCAP and RMG performed the research; STR and KRML analyzed the data; STR wrote the paper; STR, KRML and EM designed the research study; GSM, FCT, AD and FCV contributed the essential reagents and tools; WCN, MS and EM supervised the study.