The Detection Of Common Mutations In Phenylketonuria And The Mutation Spectrum Of PAH In Chinese Population

Phenylketonuria (PKU) is a common and devastating autosomal recessive inherited metabolic disorder. It is mainly caused by deficiency or reduced activity of the phenylalanine hydroxylase (PAH) because of the mutations in PAH, which inhibits the normal metabolism of phenylalanine, and leads to an accumulation of phenylalanine (Phe) in the blood and tissue, then resulting in variable severity of clinical onset including emotional disorders, seizures and severe hypophrenia. Fortunately, it can be treated by restricting the intake of Phe, but, the expensive treatment costs bring a heavy economic burden to the family and society. Currently, genetic diagnosis and prenatal diagnosis are considered as the most effective way to solve this problem and reduce the birth rate of PKU. In this dissertation, we established a novel method for rapid and accurate detection of PKU mutations, applied it to investigate the mutation spectrum of PAH in Chinese population, and finally established the mutation spectrum of PAH among Chinese people. This study should be of important clinical significance and helpful for control and prevention of PKU in China.In chapter one, the discovery, classification and molecular basis of PKU and the characteristics of PAH were reviewed, respectively. The advantages and disadvantages of biochemical methods which were commonly used in clinical settings and the existing molecular diagnostic method of PKU were described. Also, epidemiological studies about PKU including newborn screening, prenatal diagnosis and the mutation spectrum of PAH were summerized. Finally, the aim, scope and significance of this dissertation were given.In chapter two, a novel multicolor melting curve analysis (MMCA) method was proposed to detect PAH mutations. Firstly, the principle and application of MMCA were illustrated. Then, according to the mutation types of PAH and their frequencies in China, we established a3-tube,4-color detection system that can simultaneously detect27common mutations based on this technology. The assay could be finished in approximately4h and the limit of detection was100pg human genomic DNA/reaction. Finally, the accuracy and reliability of the method was evaluated by a double-blind test involving112clinical samples. With the exception of17alleles outside of the range of our method, all others207alleles were correctly detected when compared with the DNA sequencing analysis.In chapter three, in order to further investigate the mutation types of PAH and their frequencies in China, we carried out a public welfare joint project named "Kiss one moment:the mutation spectrum of PKU in China" collaborated with the Alliance of PKU in China and the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. We mainly used the MMCA technology, assisted by DNA sequencing, to detect the PAH mutations of929saliva samples collected from307PKU families all over China. Following mutation detection, statistical analysis of the detection results was performed. Eventually, we obtained a mutation spectrum of PAH for Chinese people that features the widest area, largest population, most mutation types, and complete lineage information.