A whole genome SNP genotyping by DNA microarray and candidate gene association study for kidney stone disease.

BACKGROUND: Kidney stone disease (KSD) is a complex disorder with unknown etiology in majority of the patients. Genetic and environmental factors may cause the disease. In the present study, we used DNA microarray to genotype single nucleotide polymorphisms (SNP) and performed candidate gene association analysis to determine genetic variations associated with the disease. METHODS: A whole genome SNP genotyping by DNA microarray was initially conducted in 101 patients and 105 control subjects. A set of 104 candidate genes reported to be involved in KSD, gathered from public databases and candidate gene association study databases, were evaluated for their variations associated with KSD. RESULTS: Altogether 82 SNPs distributed within 22 candidate gene regions showed significant differences in SNP allele frequencies between the patient and control groups (P < 0.05). Of these, 4 genes including BGLAP, AHSG, CD44, and HAO1, encoding osteocalcin, fetuin-A, CD44-molecule and glycolate oxidase 1, respectively, were further assessed for their associations with the disease because they carried high proportion of SNPs with statistical differences of allele frequencies between the patient and control groups within the gene. The total of 26 SNPs showed significant differences of allele frequencies between the patient and control groups and haplotypes associated with disease risk were identified. The SNP rs759330 located 144 bp downstream of BGLAP where it is a predicted microRNA binding site at 3'UTR of PAQR6 - a gene encoding progestin and adipoQ receptor family member VI, was genotyped in 216 patients and 216 control subjects and found to have significant differences in its genotype and allele frequencies (P = 0.0007, OR 2.02 and P = 0.0001, OR 2.02, respectively). CONCLUSIONS: Our results suggest that these candidate genes are associated with KSD and PAQR6 comes into our view as the most potent candidate since associated SNP rs759330 is located in the miRNA binding site and may affect mRNA expression level.

Association between human prothrombin variant (T165M) and kidney stone disease.

We previously reported the association between prothrombin (F2), encoding a stone inhibitor protein - urinary prothrombin fragment 1 (UPTF1), and the risk of kidney stone disease in Northeastern Thai patients. To identify specific F2 variation responsible for the kidney stone risk, we conducted sequencing analysis of this gene in a group of the patients with kidney stone disease. Five intronic SNPs (rs2070850, rs2070852, rs1799867, rs2282687, and rs3136516) and one exonic non-synonymous single nucleotide polymorphism (nsSNP; rs5896) were found. The five intronic SNPs have no functional change as predicted by computer programs while the nsSNP rs5896 (c.494 C>T) located in exon 6 results in a substitution of threonine (T) by methionine (M) at the position 165 (T165M). The nsSNP rs5896 was subsequently genotyped in 209 patients and 216 control subjects. Genotypic and allelic frequencies of this nsSNP were analyzed for their association with kidney stone disease. The frequency of CC genotype of rs5896 was significantly lower in the patient group (13.4%) than that in the control group (22.2%) (P = 0.017, OR 0.54, 95% CI 0.32-0.90), and the frequency of C allele was significantly lower in the patient group (36.1%) than that in the control group (45.6%) (P = 0.005, OR 0.68, 95% CI 0.51-0.89). The significant differences of genotype and allele frequencies were maintained only in the female group (P = 0.033 and 0.003, respectively). The effect of amino-acid change on UPTF1 structure was also examined by homologous modeling and in silico mutagenesis. T165 is conserved and T165M substitution will affect hydrogen bond formation with E180. In conclusion, our results indicate that prothrombin variant (T165M) is associated with kidney stone risk in the Northeastern Thai female patients.

Prothrombin haplotype associated with kidney stone disease in Northeastern Thai patients.

OBJECTIVE: To evaluate genetic variations associated with kidney stone disease in Northeastern Thai patients. METHODS: Altogether, 67 single nucleotide polymorphisms (SNP) distributed within 8 candidate genes, namely TFF1, S100A8, S100A9, S100A12, AMBP, SPP1, UMOD, and F2, which encode stone inhibitor proteins, including trefoil factor 1, calgranulin (A, B, and C), bikunin, osteopontin, tamm-Horsfall protein, and prothrombin, respectively, were initially genotyped in 112 individuals each and in additional subjects to consist of 164 patients and 216 control subjects in total. RESULTS: We found that minor allele and homozygous genotype frequencies of 8 of 10 SNPs distributed within the F2 gene were significantly higher in the control group than in the patient group. Two F2 haplotypes were found to be dually associated with kidney stone risk, one (TGCCGCCGCG) with increased disease risk and the other (CGTTCCGCTA) with decreased disease risk. However, these 2 haplotypes were associated with the disease risks in only the female, not the male, group. CONCLUSIONS: The results of our study indicate that genetic variation of F2 is associated with kidney stone risk in Northeastern Thai female patients.

Aerobic vanillate degradation and C1 compound metabolism in Bradyrhizobium japonicum.

Bradyrhizobium japonicum, a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome and is able to use low concentrations of vanillate, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of vanA1B, pcaG1H1, and genes for C(1) compound catabolism was upregulated in B. japonicum USDA110 cells grown in vanillate (N. Ito, M. Itakura, S. Eda, K. Saeki, H. Oomori, T. Yokoyama, T. Kaneko, S. Tabata, T. Ohwada, S. Tajima, T. Uchiumi, E. Masai, M. Tsuda, H. Mitsui, and K. Minamisawa, Microbes Environ. 21:240-250, 2006). To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption in vanA1B, pcaG1H1, and mxaF mutants of USDA110. The vanA1B and pcaG1H1 mutants were unable to grow in minimal media containing 1 mM vanillate and protocatechuate, respectively, although wild-type USDA110 was able to grow in both media, indicating that the upregulated copies of vanA1B and pcaG1H1 are exclusively responsible for vanillate degradation. Mutating mxaF eliminated expression of gfa and flhA, which contribute to glutathione-dependent C(1) metabolism. The mxaF mutant had markedly lower cell growth in medium containing vanillate than the wild-type strain. In the presence of protocatechuate, there was no difference in cell growth between the mxaF mutant and the wild-type strain. These results suggest that the C(1) pathway genes are required for efficient vanillate catabolism. In addition, wild-type USDA110 oxidized methanol, whereas the mxaF mutant did not, suggesting that the metabolic capability of the C(1) pathway in B. japonicum extends to methanol oxidation. The mxaF mutant showed normal nodulation and N(2) fixation phenotypes with soybeans, which was not similar to symbiotic phenotypes of methylotrophic rhizobia.