Joint Detection Of HPA Gene Mutations Based On Multicolor Melting Curve Analysis

Hyperphenylalaninemia(HPA)is a common genetic disorder of amino acid metabolism.According to the different pathogenesis,it can be divided into PAH(phenylalanine hydroxylase)deficiency and BH4(tetrahydrobiopterin)deficiency,both of which are autosomal recessive diseases.PAH deficiency is caused by mutations in the PAH gene encoding phenylalanine hydroxylase,which causes the biological activity of phenylalanine hydroxylase to decrease or even be lost.BH4 deficiency is caused by a decrease or lack of activity of the enzymes responsible for BH4 biosynthesis or regeneration in the body.At present,there are many biochemical methods for detecting HPA in clinical practice,but most of them are tedious,time-consuming and have a certain false positive rate,and the genetic diagnosis method still needs to be used for subsequent verification.In this dissertation,the PTS gene and PAH gene mutations are used as detection targets.A complete and rapid HPA detection system suitable for the Chinese population from dry blood spot extraction to genetic detection was established.The accuracy and feasibility of the system were verified by clinical specimens.In Chapter 1,introduced the pathogenesis,clinical symptoms,clinical diagnosis and treatment of HPA.At the same time,the methods of extracting dried blood spots and the deletion of large fragments of PAH gene were also reviewed.Finally,the research content and significance of this paper were put forward.In Chapter 2,a simple,efficient,and cost-effective method for DNA extraction from dried blood spots was established.This method can extract genomic DNA at a concentration of 1 to 4 ng/μL from a blood spot specimen with a diameter of 3 mm.The entire extraction process taken only 25 minutes and the cost of a single sample was about 0.5 yuan.Finally,in order to verify the feasibility of the extraction method,a total of 669 qualified HPA clinical specimens were successfully extracted using this method.In Chapter 3,based on the Multicolor Melting Curve Analysis(MMCA)technology,established a detection system for 10 types of large PAH gene deletions common in Chinese population,and examined the sensitivity and stability.Finally,120 clinical specimens were detected using the system,and the detection results were compared with the Sanger sequencing results.The results were completely consistent,which verifies the accuracy and feasibility of the system.In Chapter 4,669 clinically-screened HPA-positive specimens from 7 regions were detected using the HP A joint detection system established in the laboratory,and all results were verified by Sanger sequencing.During the entire process,a total of 1190 allelic mutations were detected,including 50 PTS gene point mutations,1104 PAH gene point mutations,36 PAH gene large fragment deletions,and 3 unreported PAH gene mutations were found.Respectively c.1256delA,c.976T>G,c.210T>C.The HPA joint detection system has 8 reaction tubes,covering 20 PTS gene point mutations,more than 125 PAH gene point mutations,and 10 large PAH gene deletions.It is a new type of HPA detection method,which has the advantages of high coverage,excellent specificity,high accuracy,simple operation,and short time consumption.Combined with our established method for rapid extraction of dried blood spot DNA and automatic interpretation software,we can achieve rapid detection of HPA from blood spot extraction to interpretation of test results,and it is expected to conduct HPA large-scale newborn screening in China.