The Research Of Phenylalanine Hydroxylase Mutation In Patients With Phenylketonuria

BackgroundPhenylketonuria(PKU;261600 and 261630)is a common autosomal recessive disorder caused by phenylalanine hydroxylase(PAH)or BH4 deficient.There are classical and non classical phenylketonurias according to concentration of phenylalanine in PKU patients.Classical phenylketonuria about 90%of patients of PKU which had been characterized by high blood phenylalanine(Phe)concentrations and cognitive and behavioural deficiencies unless treated with a phenylalanine(Phe)restricted diet in newborns is belong to median terms.Newborns diagnosed with PKU should be treated immediately,and this dietary treatment of PKU children should continue until at least an adult age.However,dietary treatment of PKU is very expensive compared to patients' income,so some of them give up the treatments because of the high cost of treatment.Every year,there have been 40 million RMB cost for therapy of PKU in Henan province according to the morbidity of the national statistics with 1:11400.PKU,especially classical PKU,not only harm health but is also of considerable harm to society stabilization,so this study foucs on PAH mutations in classical PKU.Mutation ocours on the PAH gene located on chromosome 12.There would be a 25%risk for Phenylketonuria major in each pregnancy,if both parents are carriers of Phenylketonuria-associated gene mutations.so far,over 500 different PAH mutations have been identified to date,the majority being single nucleotide substitutions,deletions or insertions of nucleotides leading to a frame-shift.The distribution of PAH gene mutations is uneven in the location of each gene,and a variety of hotspots are concentrated in several adjacent exons.The incidence of phenylketonuria is mainly caused by the mutation of the phenylalanine hydroxylase gene and the abnormal structure of the enzyme.The structural complexity of the enzyme gene determines the diversity of its mutations.Traditionally,the detection methods of PAH gene causing phenylketonuria mainly include PCR-Denaturing Gradient Gel Electrophoresis,PCR-Single-strand conformation polymorphism,High resolution melting and High Performance Liquid Chromatography.However,with the rapid development of genomics,especially gene sequencing technology,especially the popularity of large-scale parallel sequencing,gene sequencing is regarded as the preferred method for detecting phenylketonuria and related genetic disease gene mutations.Therefore,it is necessary to establish a set of mutant sites with diagnostic values associated with regions,populations,etc.Although thousands of mutant sites on mutant genes can now be obtained by liquid-phase hybrid capture,capture chips are expensive and not suitable for a wide range of clinical applications.It is very important to analyze and summarize PAH gene mutations in various provinces and cities.Therefore,there are three sections in this study:1.Systematic analysis the gene mutation site of phenylalanine hydroxylase in 17 provinces in China.2.establish the PAH gene mutation in Henan province.3.Sequence all of exons in PAH gene by next generation squencing and investigate the advantage of NGS.Section ? Research and analysis of phenylalanine hydroxylase mutation in 17 provinces in ChinaObjectiveTo improve the database of phenylalanine hydroxylase gene mutation,explore the characteristics of the mutation of phenylalanine hydroxylase gene among different provinces and nations in China.Method1.Literature retrieval(phenylketonuria or phenylalanine hydroxylase gene mutation)and mutation frequency(exon or splice region),retrieval related literature2.Literature screeningThe retrieved literature is screened according to the criteria for inclusion or exclusionInclusion criteria:(1)The content of the study is to report the mutation of PAH genes;(2)The PAH gene mutations were collected from patients or their families,and the number of studies was more than 10 cases.(3)The research method is similar,the research crowd is not limited to race(4)The original literature has sufficient information expressionExclusion criteria:(1)Unclear grouping of subjects(2)Incomplete data(3)Case reports or review articles.3.Data analysisThe SPSS 19.0 software is used to analyze the counting data and the X2 test is used to test the standard of a =0.05.Results1 Research status of phenylketonuria gene mutation in various regions of China37 studies on PAH gene-related mutations in patients with phenylketonuria were studied nationwide.The study covers 17 provinces,autonomous regions and municipalities across the country.The study period ranges from 1994 to 2017,including PAH exon 1,2,3,4,5,6,7,10,11,12 and the two sides of the splicing site.The total number of samples was 1455,and 2929 mutations were detected in all the literatures.2 Nationwide mutation of PAH gene in patients with phenylketonuriaAccording to the collected survey results,the hot spots of PAH genes have 14 mutations,in which the wrong mutation 7,nonsense mutation 4,splicing mutation,silent mutation,missing mutations each one.Hotspot mutation frequency at 2%of the mutation sites have 6,respectively:R243Q,Y204C,Y356X,R111X,V399V,R413P other hot spots are:R241C,IVS4-1G>A,S70del,A434D,R53H,W326X,Y166X,R408W.3 The distribution of PAH gene hotspot mutations in patients with phenylketonuria in ChinaR243Q,Y204C,Y356X,R111X,V399V and R413P.The distribution of these six high frequency hotspots is different in the north,south and west regions of the country.Among them,the frequency of mutation of R243Q,V399V and R413P in northern China,southern and western regions was statistically significant.4 The hotspots mutation of PAH gene in the Han,Hui and uygur patients with phenylketonuriaR243Q,Y204C,Y356X,R111X,V399V,R413P,these 6 high-frequency hotspots mutation frequency in the national Han,Hui and uygur people in the distribution of the difference has significant statistical significance.ConclusionR243Q is the most important mutation point of PAH gene in Chinese population,and the mutation of Y204C,Y356X,R111X,V399V,R413P is widely distributed among our population,but it has certain mutation difference in different nations and regions.Section ? The research of phenylalanine hydroxylase mutation phenylketonuria in Phenylketonuria childrenObjectiveThe aim of this study is to explore the situation of PAH gene mutation in the classical PKU patients in Henan province,to establish the PAH gene mutation database for the nationalities in the region,to provide important reference data for genetic counseling and prenatal diagnosis,and to further explore the function of PAH gene.MethodsThis study was based on the eighth edition of the "Genetic pathology" diagnostic criteria published by the People's Health publishing house,25 cases of phenylketonuria who diagnosed by genetic counseling clinic in Zhengzhou University Third Affiliated Hospital of genetic counseling clinic,Zhengzhou University Third Affiliated Hospital,Henan Province neonatal screening center at January 2015-January 2016 were used as subjects.1.Genomic DNA extraction was carried out by the rapid extraction kit of human peripheral blood genome.2.The use of PCR methods to capture and increase the mutation site of PAH gene 13 exogenous primers by primers.3.Application of Sanger sequencing method to sequencing the exon DNA of the captured PAH Gene 13.4.See the first part of the statistical methodology.Results1A new mutation in patients with PKU in Henan provinceA new gene mutation that occurred on the Exon3 which is c.251 A>G of the was detected.2 Detection of PAH gene mutation in children with phenylketonuriaIn 50 chromosomes,28 species were detected,53 mutations,and mutation detection rate was 92%.The mutation sites were not detected in 2 cases in 25 children,7 cases were detected 3 site mutations,11 children detected a 2 point mutation,5 children detected 1 site mutations.3 The distribution of mutation frequencies of PAH gene site in children with phenylketonuria53 mutation sites were distributed in the PAH genes 3rd,6,7,11,12,13 exon and 4th,5,6,12 in the Sub region(see table 8);The region with the highest mutation frequency is the 6th,7 Exon region,which occupies 53.7%of the total mutation,where the most mutation frequency is C.1238G>C(P.R413P),the mutation frequency is 17%,followed by C.617A>G(P.Y206C)and C.728G>A(p).R243Q accounted for 7.5%of the total mutation frequency respectively.Conclusion1.c.251 A>G(p.D84G)on the exon3 of PAH gene is a new mutation in the provincial PKU patients.2.c.1238G>C(P.R413P),c.617A>G(P.Y206C)and c.728G>A(P.R243Q)are the hotspots of PAH gene mutation in PKU patients in the province.The section ? phenylalanine hydroxylase mutation mutation testing by Next Generation SequencingObjectiveThis study intends to use the second generation gene sequencing technology for phenylketonuria patients to make genetic diagnosis.The accuracy of the first generation mutation and PAH gene mutation in patients with phenylketonuria was compared with that of the second generation,to establish a diagnostic platform for the diagnosis of the disease using the second generation gene sequencing technology.MethodsIn this study,the research subjects are 25 phenylketonuria patients who came for diagnosis in Obstetrics and gynecology of the Third Affiliated Hospital of Zhengzhou University between January 2014 and January 2016 and planned to receive genetic diagnosis published according to the eighth edition of "genetic disease" diagnostic criteria published by People's Health Publishing department.1.Human peripheral blood genomic DNA should be used to extract kits quickly and ten extract genomic DNA.2.Long PCR primers are used to capture and amplify the PAH gene by multiplex PCR methods.3.The Next generation gene sequencing platform should be used to the 13 exon DNA of captured PAH gene is sequenced by using the second generation gene sequencing platform.4.Statistical analysis method is the same with that in the first partResults1 The construction of the library should be sequencedThe success construction of the library for capture methods of long PCR primers,and the library fragment is basically distributed around 200bp,reaching the sequence analysis standard.2 Detection of PAH gene mutation in PKU patients by the next generation gene sequencingThe results of sequencing analysis of 13 exons of PAH gene and their bilateral splice region in 25 children with phenylketonuria were detected by the second generation gene sequencing.29 species were detected in 50 chromosomes,with 54 mutations.One of the 25 children had no detectable mutation,and the mutation rate was 96%.There was no statistically significant difference in the accuracy of the same generation.ConclusionCompared with the traditional Sanger sequencing,the next generation gene sequencing technique is used to analyze the mutation method of PAH gene mutation in patients with phenylketonuria,and the results are reliable and the cost is low,which is more suitable for the sequencing of mutations with a wide range of samples.