IDENTIFICATION AND IN-VITRO EXPRESSION OF MUTATIONS CAUSING DIHYDROPTERIDINE REDUCTASE DEFICIENCY

Six mutations resulting in the recessive inherited disorder dihydropte ridine reductase deficiency are reported, five of which are previously unknown. Two are nonsense mutations, resulting in premature terminati on of the protein, with the remaining four being missense mutations. T he mutations found lie in the middle to 3' end of the dihydropteridine reductase reading frame, with the exception of one mutation which lie s at codon 23, which is the only mutation found in more than one patie nt. The mutation pattern can be described as heterogeneous. The wild t ype and several of the mutant DHPR cDNA's were expressed in E. coli an d the proteins purified and examined by a variety of techniques, inclu ding calculation of kinetic constants. One mutation (Gly23-->Asp) resu lts in completely inactive protein, while a second (Trp108-->Gly) has substantial activity but does not completely dimerize. Both this mutan t and a third, His158-->Tyr, are extremely susceptible to in vitro pro tease 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 substit utions on the DHPR enzyme are varied.