Analysis Of Phenylalanine Hydroxylase Gene Mutation In Patients With Phenylketonuria In Qingdao

Objective: Phenylketonuria(PKU),also known as hyperphenylalaninemia,is an autosomal recessive genetic disease characterized in phenylalanine metabolic disorders.In order to definite the PKU incidence of Qingdao city,this study aimed at the screening and the genetic diagnosing of PKU in newborns.In combination of multiple molecular-genetic techniques,phenylalanine hydroxylase(PAH)gene analysis has found larger amount of mutations among classical phenylketonuria patients.These findings enriched the genetic spectrum of PAH mutations,and our experience in the screening,the early diagnosing and prenatal diagnosing and consulting of PKU may supply improvement in the birth defect prevention of Qingdao city.Methods:(1)293,735 newborns in Qingdao who were born from January 2016 to December 2017 have underwent PKU screening by ninhydrin fluorescence method.(2)Fluorescence PCR melting curve method,gene capture combined with high-throughput sequencing,multi-junction probe amplification technique(MLPA)and Sanger sequencing technology were performed to search for PAH gene mutations in 38 children and 2 adults.In addition,prenatal diagnosis was performed on 5 fetuses(of which parents are carriers).The positive screening results were initially validated by fluorescence PCR melting curve using patients’ DNA samples.For those with negative results,gene capture combining with high-throughput sequencing was performed for further detection,and the results were verified by Sanger sequencing.For samples with no mutation detected in both the above methods,the multiplexed probe amplification technique(MLPA)method was performed for complementary detection.(3)To confirm the pathogenicity of the detected mutations,bioinformatics analysis were performed with the assistance of searching in The PAH gene database,ClinVar database,HGV database and Pubmed database.Results:(1)Among the 293,735 newborns screened,31 children with phenylketonuria were diagnosed,reaching a PKU incidence of 1/10554.(2)In 40 patients with PKU,74 PAH gene mutations were found,of which 42 were missense mutations,accounting for 56.8%(42/74);19 were nonsense mutations,accounting for 25.8%(19/74).5 splicing mutations,accounting for 6.8%(5/74);5 frameshift mutations,accounting for6.8%(5/74);3 synonymous mutations,accounting for 4.1%(3/74).The mutations mainly located in on exons 1-3,6,7,9-12 and introns 4 and 12,of which exon 7 was 28.4%(21/74).Exon 11 accounted for 20.2%(15/74).(3)Mutation hotspots: In this study,the accumulated frequency of PAH gene mutations is greater than 3%,R243Q(c.728G>A)accounted for 15.0%(12/80),and Y356X(c.1068C>A)accounted for 13.8%(11/80),R53H(c.158G>A)accounted for 8.7%(7/80),R241C(c.721C>T)accounted for 6.3%(5/80),and R111X(c.331C>T)accounted for 5.0%.(4/80),V399V(c.1197A>T)accounted for 3.8%(3/8).(4)Detection of an unreported mutation site c.60+1G>A.(5)The results of prenatal diagnosis: Two fetal patients,one normal and two carriers were detected.Conclusion: The PKU incidence of Qingdao city is close to the national level.Missense mutations were the majority of PAH gene mutations,which mainly located in exon 7.Comparing to other domestic regions,the frequencies of R53 H and Y356 X are higher are relatively higher.A total of 80 PAH genes were detected by multiple techniques for detection of mutation sites.A total of 74 mutations were detected,reaching a detection rate of 92.5%(74/80),which is higher than the detection rate yield by one detection method only.