5-Oxoprolinuria in Heterozygous Patients for 5-Oxoprolinase (OPLAH) Missense Changes

Abstract

The inherited 5-oxoprolinuria is primarily suggestive of genetic defects in two enzymes belonging to the gamma-glutamyl cycle in the glutathione (GSH) metabolism: the glutathione synthetase (GSS) and the 5-oxoprolinase (OPLAH). The GSS deficiency is the best characterized of the inborn errors of GSH metabolism, whereas the OPLAH deficiency is questioned whether it is a disorder or just a biochemical condition with no adverse clinical effects. Recently, the first human OPLAH mutation (p.H870Pfs) was reported in homozygosis in two siblings who suffered from 5-oxoprolinuria with a benign clinical course. We report two unrelated patients who manifested massive excretion of 5-oxoproline in urine. In both probands, the blood GSH levels were normal and no mutations were found in the GSS gene. The mutational screening of the OPLAH gene, which included the codified sequences, the intronic flanking sequences, the promoter sequence, and a genetic analysis in order to detect large deletions and/or duplications, showed that each patient only harbors one missense mutation in heterozygosis. The in silico analyses revealed that each one of these OPLAH mutations, p.S323R and p.V1089I, could alter the proper function of this homodimeric enzyme. In addition, clinical symptoms manifest in these two probands were not related to GSH cycle defects and, therefore, this study provides further evidence that oxoprolinuria may present as epiphenomenon in several pathological conditions and confound the final diagnosis.

Fig. 1

Genetic findings. (a) Available DNAs from probands and their relatives. (b) Electrophoregrams show the identified mutations in proband ID no. 943 (c. 969C>A, p.S323R) and in proband ID no. 1037 (c.3265G>A, p.V1089I) in the OPLAH gene, together with the corresponding controls’ electrophoregrams. (c) Electrophoresis gel shows the obtained band of 9,0-kb which corresponds to the amplification of the OPLAH gene. No differences were found in any of the analyzed samples ruling out large deletions or duplications (1st and 8th lanes: MWM, Molecular Weight Marker, 1-kb plus DNA ladder; 2nd lane: –Ctrl, PCR negative control; 3rd to 6th lanes: analyzed DNAs, 942, 943, and 944 from family AR-141, and 1037 from family AR-152; 7th lane: +Ctrl, DNA from a healthy subject). (d) Alignment of sequences of the OPLAH protein from human, bovin, mouse, rat, and yeast showing that both mutated residues are conserved amino acids. Identical amino acids in all of these sequences are indicated with an asterisk (*). The closest ATP-binding site to the p.S323R change is shown, which consists of the sequence DMGGT (from residue 324 to 328 in yeast)