Analysis of osteopontin DNA in patients with urolithiasis

We previously reported the importance of osteopontin (OPN) in the formation of urinary calculus. Since OPN protein is present in normal kidneys, we in vestigated the difference in OPN at the DNA level between normal subjects a nd urolithiasis patients. There has not been any genetic investigation of O PN in familial urolithiasis previously reported worldwide. To confirm hered itary predisposing factors for urolithiasis, changes in OPN DNA within a fa mily were investigated in relation to the presence or absence of urinary ca lculus. Leukocyte OPN DNA from two normal subjects and five patients with u rinary calculus was investigated by SSCP analysis: OPN DNA nucleotide seque nce was determined, based on the result of SSCP analysis. As a result, a mu tation of GCC to GCT. encoding amino acid position 250 (Ala-250) was found. To confirm the frequency of mutation at this site, OPN DNA was extracted f rom peripheral blood in 36 normal subjects (Con group), 25 patients with fa milial urolithiasis (FSF), and 40 patients with recurrent urinary calculus and who had had two or more previous episodes (RSF). The degree of mutation at Ala-250 was then examined by restriction fragment length polymorphism ( RFLP) method. As described above. the nucleotide codon encoding the amino a cid sequence position 250, Ala-250, was GCC in two normal subjects. This is the original codon. In five patients with urolithiasis it was GCT, showing a substitution of C with T. On examining the frequency of this mutation, t he ratio of normal homozygous GCC was 11/36 in the Con group, 1/25 in FSF a nd 1/40 in RSF. The ratio of heterozygous GCC/GCT was 16/36 in the Con grou p, 15/25 in FSF and 26/40 in RSF, and the ratio of homozygous GCT was 9/36 in the Con group, 9/25 in FSF and 13/40 in RSF. Furthermore, the gene frequ ency of the normal codon GCC was 0.528 in the Con group, 0.3 in FSF and 0.3 5 in RSF, showing a significantly higher incidence in the Con group (P < 0. 05). The gene frequency of mutated GCT was 0.472 in Con group, 0.7 in FSF a nd 0.65 in RSF, showing a significantly higher incidence in urolithiasis pa tients (P < 0.05). On investigating the inheritance of Ala-250 in five fami lies in which both parent and offspring demonstrated urolithiasis, the nucl eotide substitution in Ala-250 in parents with urolithiasis was inherited b y their offspring. In all five families the offspring developed urinary cal culus. This study showed that there is no difference in OPN structure betwe en the Con group and urolithiasis patients. However, it was predicted that due to the frequency of normally coded GCC being high in the Con group a di fference in the amount of OPN might be caused by a difference in transcript ion velocity between the two groups. Furthermore, it was suggested that exa mining the inheritance of Ala-250 within a family is a diagnostic method fo r identifying the predisposing hereditary factors for urolithiasis patients .