Identification and in vitro expression of mutations causing dihydropteridine reductase deficiency

Six mutations resulting in the recessive inherited disorder dihydropteridine reductase deficiency are reported, five of which are previously unknown. Two are nonsense mutations, resulting in premature termination of the protein, with the remaining four being missense mutations. The mutations found lie in the middle to 3' end of the dihydropteridine reductase reading frame, with the exception of one mutation which lies at codon 23, which is the only mutation found in more than one patient. The mutation pattern can be described as heterogeneous. The wild type and several of the mutant DHPR cDNA's were expressed in E. coli and the proteins purified and examined by a variety of techniques, including calculation of kinetic constants. One mutation (Gly23-->Asp) results in completely inactive protein, while a second (Trp108-->Gly) has substantial activity but does not completely dimerize. Both this mutant and a third, His158-->Tyr, are extremely susceptible to in vitro protease digestion, indicating that their three-dimensional structure has been altered. The protein studies underline the heterogeneous nature of DHPR mutations, in that the effects of different amino acid substitutions on the DHPR enzyme are varied.