Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2)

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GR HPR) has been characterized, and a single mutation has been detected in fou r PH2 patients. In this report, we have identified five novel mutations. On e nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (M et322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanin e in the 3' splice site of intron G. The functional consequences of the mis sense transversion and transition mutations have been investigated by trans fection of cDNA encoding the mutated protein into COS cells. Cells transfec ted with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). T hese results further confirm that mutations in the GRHPR gene form the gene tic basis of PH2. Ten of the 11 patients that we have genotyped are homozyg ous for one of the six mutations identified to date. Because of this high p roportion of homozygotes, we have used microsatellite markers in close link age with the GRHPR gene to investigate the possibility that the patients ar e the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related pers ons. Furthermore, genotyping has revealed the possible presence of a founde r effect for the two most common mutations and the location of the gene nea r the marker D9S1874.