title: Late diagnosis of primary hyperoxaluria type 2 in the adult: effect of a novel mutation in GRHPR gene on enzymatic activity and molecular modeling.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine, Nephrology and Hypertension Institute, Tel Aviv University and Sackler School of Medicine, Chaim Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel. lironomy@gmail.com
authors.forename: Nomy
authors.initials: N
authors.lastname: Levin-Iaina
authors.forename: Dganit
authors.initials: D
authors.lastname: Dinour
authors.forename: Lina
authors.initials: L
authors.lastname: Romero
authors.forename: Rotem
authors.initials: R
authors.lastname: Ron
authors.forename: R Leo
authors.initials: RL
authors.lastname: Brady
authors.forename: Scott D
authors.initials: SD
authors.lastname: Cramer
authors.forename: Eli J
authors.initials: EJ
authors.lastname: Holtzman
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000175:diagnosis / Q000201:enzymology* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000429:Alcohol Oxidoreductases; C020830:glyoxylate reductase