Levin-Iaina Nomy, Dinour Dganit, Romero Lina, Ron Rotem, Brady R Leo, Cramer Scott D, Holtzman Eli J
Department of Medicine, Nephrology and Hypertension Institute, Tel Aviv University and Sackler School of Medicine, Chaim Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel.
J Urol. 2009 May;181(5):2146-51. doi: 10.1016/j.juro.2009.01.045. Epub 2009 Mar 17.
Genetic causes of nephrolithiasis are underestimated. Primary hyperoxaluria type 2 is a rare autosomal recessive disease caused by mutations in the GRHPR gene, leading to an accumulation of oxalate and L-glycerate with recurrent kidney stone formation and nephrocalcinosis, and the later development of renal failure and systemic oxalate depositions. We studied the effects of a novel GRHPR mutation on GRHPR enzymatic activity and molecular modeling.
Genomic DNA from a 50-year-old male with a late diagnosis of primary hyperoxaluria type 2 was extracted, analyzed and compared with the established human GRHPR gene sequence. Restriction enzyme analysis of the patient, 30 healthy controls and 30 patients with nephrolithiasis of various causes was done to confirm the presence of the mutation. GRHPR activity was analyzed by site directed mutagenesis of WT and mutant clones. We studied the effects of the mutation on enzymatic molecular modeling.
We found the novel homozygous single missense mutation A975G in exon 9, creating an amino acid change from asparagine to aspartic acid in position 312. No mutations were detected in restriction enzyme analysis in all 30 healthy controls and 30 patients with nephrolithiasis of various causes. Transfected cells with the mutant clone showed abolished GRHPR activity. Molecular modeling studies revealed that the mutation was likely to disrupt the correct folding of the GRHPR substrate binding domain, hence affecting the enzyme active site.
Primary hyperoxaluria type 2 should be considered in patients at adult stone clinics who have had a history of nephrolithiasis since childhood, especially in those with consanguineous parents. Biochemical analysis followed by mutation identification should be the approach for making the definitive diagnosis of primary hyperoxaluria type 2.
肾结石的遗传病因被低估。原发性高草酸尿症2型是一种罕见的常染色体隐性疾病，由GRHPR基因突变引起，导致草酸盐和L-甘油酸盐蓄积，伴有复发性肾结石形成和肾钙质沉着症，后期会发展为肾衰竭和全身性草酸盐沉积。我们研究了一种新型GRHPR突变对GRHPR酶活性和分子模型的影响。
提取一名50岁男性的基因组DNA，该男性原发性高草酸尿症2型诊断较晚，对其进行分析并与已确立的人类GRHPR基因序列进行比较。对该患者、30名健康对照者和30名各种病因的肾结石患者进行限制性酶切分析，以确认突变的存在。通过对野生型和突变型克隆进行定点诱变来分析GRHPR活性。我们研究了该突变对酶分子模型的影响。
我们在外显子9中发现了新型纯合单错义突变A975G，导致第312位氨基酸由天冬酰胺变为天冬氨酸。在所有30名健康对照者和30名各种病因的肾结石患者的限制性酶切分析中均未检测到突变。转染突变型克隆的细胞显示GRHPR活性丧失。分子模型研究表明，该突变可能会破坏GRHPR底物结合域的正确折叠，从而影响酶活性位点。
对于成年结石门诊中自幼有肾结石病史的患者，尤其是父母为近亲的患者，应考虑原发性高草酸尿症2型。生化分析后进行突变鉴定应是明确诊断原发性高草酸尿症2型的方法。
