Molecular analysis of dihydropteridine reductase deficiency

二氢蝶呤还原酶(DHPR)在四氢生物蝶呤的代谢中起着至关重要的作用，四氢生物蝶呤是苯丙氨酸、酪氨酸和色氨酸羟化的辅因子。DHPR缺乏症是一种常染色体隐性遗传性疾病，以高苯丙氨酸血症和各种神经系统症状为特征，如严重的精神发育迟缓和惊厥。我们研究了三名患有DHPR缺乏症的日本患者。Northern印迹分析表明，病例1的细胞培养物中DHPR mRNA正常表达，而病例2和病例3的培养物中DHPR mRNA的表达明显减少。用逆转录/聚合酶链式反应(RT-PCR)对病例1的DHPR mRNA进行测序分析，发现130位核苷酸发生t-to-c改变，导致36位氨基酸(W36R)上的色氨酸被精氨酸取代。含有W36R的DHRP基因的表达分析没有显示DHPR活性，表明它是一个致病突变。病例1是近亲婚姻的产物，是突变的纯合子。例2的RT-PCR分析显示DHPR基因异常剪接，在外显子3和4之间有152个碱基的插入，该插入编码异亮氨酸和亮氨酸，随后是一个终止密码子。基因组DNA分析显示，内含子3的a-g替换产生了一个新的剪接供体位点。该突变可能激活了位于152bp上游的一个潜在剪接受体位点，在内含子3中产生了一个新的外显子。该突变在病例2和他的患病兄弟中以纯合子形式存在，而母亲是杂合子。例3在DHPR cDNA编码区没有核苷酸替换，可能存在编码区外的突变，影响了mRNA的表达或稳定性。

Dihydropteridine reductase (DHPR) plays an essential role in the metabolism of tetrahydrobiopterin, which is required as a cofactor for hydroxylation of phenylalanine, tyrosine and tryptophane. Deficiency of DHPR is inherited as an autosomal recessive disorder characterized by hyperphenylalaninemia and various neurological symptoms such as severe mental retardation and convulsion. We have studied three Japanese patients with DHPR deficiency. Northern blot analysis showed that DHPR mRNA was normally expressed in a cell culture obtained from case 1, whereas marked reduction of DHPR mRNA was observed in cultures from case 2 and 3. Sequencing analysis of DHPR mRNA from case 1 by reverse transcription/polymerase chain reaction (RT-PCR) identified a t-to-c change at nucleotide position 130, which resulted in a substitution of tryptophane with arginine at amino acid position 36 (W36R). Expression analysis of DHRP cDNA harboring W36R did not show DHPR activity, indicating that it is a disease-causing mutation. Case 1, a product of a consanguineous marriage, was homozygous for the mutation. RT-PCR analysis of case 2 revealed an abnormally spliced DHPR mRNA with a 152 bp-insertion between exon 3 and 4. The insertion encoded isoleucine and leucine followed by a stop codon. The analysis of genomic DNA revealed an a-to-g substitution in intron 3 which created a novel splicing donor site. The mutation probably activated a potential splicing acceptor site located 152 bp upstream, producing a "new" exon in intron 3. The mutation was present in homozygous form in case 2 as well as in his affected brother, while the mother was a heterozygote. Case 3 did not carry nucleotide substitution in the coding region of DHPR cDNA.The case probably has a mutation outside the coding region, which affects the expression or the stability of mRNA.