Association between Human Prothrombin Variant (T165M) and Kidney Stone Disease

<div><p>We previously reported the association between <em>prothrombin</em> (<em>F2</em>), encoding a stone inhibitor protein - urinary prothrombin fragment 1 (UPTF1), and the risk of kidney stone disease in Northeastern Thai patients. To identify specific <em>F2</em> 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%) (<em>P = </em>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%) (<em>P = </em>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 (<em>P = </em>0.033 and 0.003, respectively). The effect of amino-acid change on UPTF1 structure was also examined by homologous modeling and <em>in silico</em> 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.</p> </div

Genotype and allele frequencies of SNP rs5896 in <i>F2</i> in male group (77 patients with kidney stone disease and 90 control subjects).

<p>CI = confidence interval; OR = odd ratio.</p

<i>F2</i> gene structure, SNP rs5896, alignment of prothrombin amino acid sequences, and 3D structure of UPTF1.

<p>A: Gene structure of <i>F2</i>. Exons 1–14 and intervening sequences (introns) are indicated by boxes and line, respectively. Fragment numbers 1 to 12 represent expected PCR products. B: DNA sequencing profile showing a SNP (rs5896, c.494 C>T) in exon 6, resulting in a substitution of threonine (T) by methionine (M) at position 165 (T165M). Vertical arrows indicate the positions of nucleotide variations. SNP genotypes are indicated by bold capital letters above the vertical arrows. C: Multiple alignment of amino acid sequences in a highly conserved region of prothrombin (F2), residues 152–185 (human sequence numbering) from eleven species. The symbols “*” and “:” under the alignment indicate the positions with conserved and conservative-changed amino acids, respectively. The T165 position is indicated by an arrow. D: Three dimensional (3D) structure of urinary prothrombin fragment 1 (UPTF1), showing an amino acid alteration, T165M. Wild-type T165 is indicated as green, which is superimposed by the altered amino acid, M165, indicated as gray. Wild-type residue is shown as green letters while the variant residue is presented as black letters. The dash line implies the predicted H-bond between T165 and E180 residues. The oxygen and sulfur atoms are shown with red and yellow, respectively.</p

Genotype and allele frequencies of SNP rs5896 in <i>F2</i> in female group (132 patients with kidney stone disease and 126 control subjects).

<p>CI = confidence interval; OR = odd ratio.</p

Analysis of rs5896 (c.494 C>T) by polymerase chain reaction - restriction fragment length polymorohism (PCR-RFLP).

<p>A: Locations of SNP rs5896 and restriction sites of <i>Nco</i>I on amplified DNA fragment (733 bp), containing exons 5 and 6. The expected RFLP patterns for genotyping of SNP rs5896 are indicated as lines with the lengths of 426, 140, 93 and 74 bp for allele T, and 426, 233 and 74 bp for allele C. B: <i>Nco</i>I-digested DNA patterns separated by electrophoresis on 3% agarose: lane 1, 100-bp ladder markers; lane 2, undigested PCR product (733 bp); lane 3, homozygote TT (426, 140, 93 and 74 bp); lane 4, heterozygote CT (426, 233, 140, 93 and 74 bp), and lane 5, homozygote CC (426, 233 and 74 bp).</p

Genotype and allele frequencies of SNP rs5896 in <i>F2</i> in 209 patients with kidney stone disease and 216 control subjects.

<p>CI = confidence interval; OR = odd ratio.</p