Genotyping Of Large Fragment Deletions In PAH

Hyperphenylalaninemia（HPA）is an autosomal recessive genetic disease with a high incidence in China.HPA divided into PAH deficiency and BH4 deficiency.PAH deficiency is mainly caused by deficiency or reduced activity of phenylalanine hydroxylase（PAH）,which inhibits the normal metabolism of phenylalanine（Phe）,and leads to a series of clinical symptoms.PAH deficiency is an inherited metabolic disease that can be treated with diet.The more earlier of diagnosis and treatment,the patient’s intelligence can develop normally.The incidence and mutation types of HPA are obviously different in different populations and regions.The incidence rate in China is approximately I:10,397.Now,more than 1,000 mutations have been reported in PAH,and most of the mutations are missense mutations.However,about 5%of PAH large fragment deletions can not detected by conventional sequencing methods.This article focused on the PAH large fragment deletions,analysed the hot spot mutation of PAH large fragment deletion in China,and established a new method to quickly and accurately detect PAH large fragment deletions.The accuracy and feasibility of the method was verified by clinical specimen verification.In chapter one,we firstly introduced the pathogenesis and clinical symptoms of PAH deficiency.Then,we systematically introduced the methods used for PAH deficiency screening and diagnosis,and the treatment strategies.We introduced the types and frequencies of PAH large fragment deletions in different regions and populations,and summarized the detection methods for PAH large fragment deletions.In the end,we listed the purpose,content and significance of this paper.In chapter two,we identified the breakpoint of Ex4₇del by Gap-PCR and Sanger sequencing.We combined with six types of PAH large fragment deletions and counted all large fragment deletion alleles.There are 41 large fragment deletion alleles in 39 PAH deficiency patients by MLPA.Among them,Exldel3.7kb（36.59%,15/41）,Ex6de17793（26.83%,11/41）,Ex4₅de116271（19.51%,8/41）are the hot spot mutations of PAH large fragment deletion in China,Ex4de111612,Exldel5.3kb,Ex5de17750 are unique types of large fragment deletion in Chinese population.In chapter three,to study the ability of detecting large fragment deletions by multiplex mediator probe melting analysis,the PAHlarge fragment deletions detection system was established to genotype the 10 types of PAH large fragment deletions in Chinese population.Then we evaluated the stability,repeatability,sensitivity and accuracy of the genotyping system by plasmid standards and clinical specimens.The results showed that the sensitivity of the large fragment deletions genotyping system can reach 50 pg/reaction human genomic DNA.In addition,222 clinical samples were also genotyped in this system.Compared with MLPA,more than 2 samples were detecded large fragment deletions by multiplex mediator probe melting analysis,and all the results were verified by sequencing.The genotyping system is simple,rapid,accurate,and can be used to screen low-concentration genomic DNA templates such as neonatal blood spots,which is suitable for large-scale screening of PAH deficiency in China.