| Phenylketonuria(PKU,OMIM#261600)is one of the most common autosomal recessive metabolic diseases.The phenylalanine hydroxylase(PAH)gene mutation leads to a decrease or loss of PAH activity,which result in Phenylalanine metabolic disorders in the liver.The occurrence of PKU varies among ethic and geographic regions.The incidence of PKU in northern China is higher than that in the southern region.The incidence of PKU,in the Northwest region of china was significantly higher than the national average.In this study,we investigated the molecular genetic basis,analyzed the mutation spectrum of PAH gene,identified the hotspots and the correlation between mutant sites and phenotype in PKU patients in Northwest China,and laid the basic data for PKU prevention and control.Based on the mutation spectrum of PAH gene,the high-freqencing mutation sites were selected,and a mutation screening program using MassArray genotyping technology was established,and its specificity and accuracy was evaluated.A method for noninvasive prenatal diagnosis of phenylketonuria based on droplet digital PCR(ddPCR)were established,and compared with conventional invasive diagnostic methods to evaluate its accuracy.Through this study,we hope to establish a PKU triple prevention intervention program,include of hot spot mutation screening of PAH gene in pregnant or pre-pregnancy women,noninvasive and invasive prenatal diagnosis and postpartum gene diagnosis of PKU.We collected 475 PKU families in the northwest region of china,analyzed the PAH gene mutation by Sanger sequencing,MLPA method and gap-PCR,and the function of the novel mutation was predicted using bioinformatics software.In the 475 patients of PKU families,PAH gene mutations were identified on 895 of 950 independent alleles,corresponding to a mutation detection rate of 94.21%(95%CI,92.72%-95.70%).A total of 128 different types of mutations were identified.In terms of mutation frequency,c.728G>A was the most prevalent mutation(frequency 14.00%),other mution with relatively high frenquencies were c.611A>G(5.58%),c.1068C>A(4.95%).Seven large deletion/duplication mutations were identified in 74 PKU patients by MLPA.The mutaitons frequency of large scale is 3.02%(27/895),of which c.-4163_-406de13758 was the most common type in the northwest region.The break point of large scale deletion/duplication mutations were analized by gap-PCR.We discovered 20 novel mutaitons in this study,and predicted their function by bioinformation tools.The correlation between phenotype and gene mutations show that c.158G>A and c.1256A>G were most frequently associated with mild hyperphenylalaninemia(MHP).35 hotspot mutaitons were selected for designing the MassArray panel.29 mutations were successfully detected by the panel after optimization.105 PKU patients with clear PAH gene mutations were genotyped by this MassArray panel.All of 29 mutaitons were detected in 105 patients,and the results of MassArray panel were consistent with Sanger sequencing.50 patients newly diagnosed as phenylketonuria were recruited in the double-blind experiment.Mutations of PAH gene of the 50 patients were detected by MassArray panel,and the results were verified with Sanger sequencing and MLPA methods.The result shows that the mutation detection rate by MassArray panel is 74%(95%CI,65%-83%),and the clinical genetic diagnosis rate is 54%(95%CI,40%-68%).We collected 24 PKU families,who needed prenatal diagnosis,in which the proband and parents gene mutations were clear,the traditional invasive prenatal diagnosis were carried out.Invasive prenatal diagnosis results showed that 3 cases were affected fetuses,8 cases were normal fetuses,and 13 cases were pathogenic mutations mixed carriers.Based on the characteristics of PAH gene mutation in prenatal diagnosis family,a novel droplet digital PCR(ddPCR)assay was designed and the sensitivity and specificity of this assay were evaluated.Non-invasive prenatal diagnosis of 8 families was successful performed by ddPCR assay,and the results were consistent with invasive prenatal diagnosis,and no false positive or false negative results were found in this experimants.In this study,the mutation spectrum of PAH gene in Northwest China was idetified,and the distribution of PAH gene hotspot and mutation in Northwest China was charactered.20 novel mutations was identified,and the functional analysis of these mutation sites was carried out,which enriched the PAH gene mutation database.The correlation between some mutant sites and phenotype was clarified by genotype phenotypic analysis.That may lay the foundation for phenotypic prediction of patients.Based on the hotspot analysis of PAH gene mutation in northwest China,we established a novel MassArray panel for detecting hotspot mutation of PAH gene.The MassArray panels can genotype the 29 hotspot mutation sites of PAH gene at the same time,and the detection rate of mutation can reach 74%(95%CI,65%-83%).This panel can be used as a high throughput,low cost and rapid method for screening and diagnosing of PAH gene mutations.The establishment of the program provides a technical guarantee for the future in the PKU high-incidence area to carry out large-scale carrier screening.In this study,a novel droplet digital PCR(ddPCR)assay was established and evaluated.The results showed that ddPCR had high sensitivity and specificity in cfDNA detection,and it was a reliable noninvasive prenatal diagnosis method for PKU families. |
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