Molecular Genetic Study of Dihydropteridine Reductase Deficient Hyperphenylalaninemia in Chinese

• Main Authors: Pei-Fen Yen, 顏培芬
• Other Authors: Kwang-Jen Hsiao
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/05412331480205815291

碩士 === 國立陽明大學 === 遺傳學研究所 === 89 === Hyperphenylalainemia ( HPA ) is an autosomal recessive disorder of phenylalanine hydroxylation. For the hydroxylation to function normally, phenylalanine hydroxylase ( PAH ) and it’s cofactor, tetrahydrobiopterin ( BH4 ) are required. Dihydropteridine reductase ( DHPR；EC 1.6.99.7 ) plays an important part in the pterin-dependent aromatic amino acid hydroxylation systems. Deficiency of DHPR activity will cause one of tetra-hydrobiopterin deficient forms of phenylketonuria ( PKU ) , which can not be controlled by a low-phenylalanine diet alone. DHPR deficient HPA is characterized by a progressive neurological degeneration and mental retardation. In previous studies, three Chinese HPA patients with DHPR deficiency were found by our laboratory. In the P026 patient, the c.697A>C substitution was found by PCR and sequence reaction. This substitution is not a polymorphism since we know that the c.697A>C could not be found in 110 normal alleles, and this variation is tightly linked with the carrier in the members of patient’s paternal relatives. The data suggests that it may cause DHPR deficiency. To study the structural-functional relationship of the T233P substitution of DHPR, the wild type and c.697A>C mutant DHPR cDNA are expressed in Escherichia coli. The overexpressed DHPR is purified by GST fusion system and characterized the optimal pH value, kinetic constant of NADH and DMPH4, and specific activity. The mutant DHPR enzyme decreases its specific activity to the 65% extent of the normal protein. This indicates that c.697A>C indeed is a disease-causing mutation. Our previous studies indicated that the mutation might influence the mRNA initiation or stability and it maybe something wrong in the upstream of the promoter region. But so far, we only know the coding sequence data and we have little information about DHPR gene. In order to achieve the full sequence information, we picked up a BAC ( Bacteria Artificial Chromosome ) clone by BAC library screening with DHPR specific PCR primers and did the sequence work. A sequence of 130,138 bp was determined in the DHPR con-taining BAC clone 395N09. The DHPR gene ( from start codon to stop codon ) was located at nt 33080~ 57995 of this BAC clone. A STR marker, namely D4S2926, at 20,240 bp down stream to DHPR gene and other eight STS markers were identified in the BAC clone. Since we finish the sequence data, we can get more information from these data by bioinformatic tools. To examine the promoter region of the patient’s allele, which is from the maternal family, compare the precursor RNA expression between known mutant allele ( c.697A>C ) from paternal family and undetermined mutant allele from maternal family. The result displayed that the expression level of the precursor RNA showed no significant difference between two allele types. It needs more investigations to explore the whole DHPR gene to find out the disease-causing factor in the maternal family of the P026 patient.