4. Discussion
This study analyzed the general situation of neonatal disease screening and PKU incidence in Shaanxi province, China from 2010 to 2018. The screening rate of neonatal diseases increased from 58% in 2010 to 95% in 2018.In the past 9 years, 569 cases of PKU were diagnosed. Through treatment with a low phenylalanine diet, the prognosis was good, and the ultimate goal of early detection and treatment was achieved. Based on this, the incidence of PKU in our province was approximately 1.5/10000, which is basically consistent with the literature(7). but higher than the national average (0.9/10000). Therefore, it is of great importance to carry out a three-level prevention strategy for PKU in our province to lower its incidence.
In this study, 13 exons and their flanking sequences of the PAH gene were studied in 84 children with PKU in Shaanxi province, and the mutation spectrum of the PAH gene in Shaanxi province was revealed for the first time. A total of 51 kinds of 159 mutant alleles were found, and the most common type of mutation was missense, accounting for 62.89% (100/159) of all mutations. Exon 7 was the most widely mutated exon, accounting for 32.08% (51/159) of all cases; p.R243Q had the highest mutation frequency, accounting for 20.13% (32/159) of all mutations. In conclusion, the mutations of the PAH gene in Shaanxi province found in this study were basically consistent with previous reports from China and abroad: exon 7 is a mutation hotspot, especially the p.R243Q mutation, which should be given more attention (8).
A deficiency of PAH caused by gene mutation is the main cause of PKU (9). Gene detection is an important method to determine the cause of PKU and a guide for family reproduction. Although PAH gene mutations were detected in 84 children with PKU, only 1 mutation was detected in 14 of them. Considering the genetic characteristics of PKU, generally 2 or more mutations in the gene locus are required to cause disease (10). Therefore, additional gene detection in these 14 children needs to be performed, and it cannot be used to provide reproductive guidance for their families. Reasons for this situation include the possible deletion or duplication of large segments of the PAH gene, which needs to be detected by MLPA. Due to the limitation of detection methods, this study did not use MLPA to analyze these 14 children, which is one of the shortcomings of this study. We plan to complete this analysis in a future study. In addition, there may be mutations in other areas of the PAH gene in addition to those analyzed in this study, and the limitations of the detection methods cannot be determined at the present time. Thus, further research is needed.
As stated earlier, the most common molecular basis of PKU is mutation of the PAH gene, so molecular diagnosis using the PAH gene is key to reducing the number of children born with this disease. According to research in China and abroad, 1101 mutations have been identified in the PAH gene, with obvious heterogeneity. There are significant differences in the location and distribution of PAH loci among different races and regions (11). We detected PAH gene mutations in 84 children with PKU diagnosed in our center and created a gene mutation map of PAH in Shaanxi province. In this study, ARMS-PCR combined with fluorescent probe technology was used to detect the mutation sites. We designed detection methods using monochromatic and bichromatic fluorescence. Based on the results of this study, together with the conclusions of studies in other regions in China, we selected the mutation hotspots of the PAH gene with a high incidence in China and developed a detection kit targeting these mutation sites suitable for clinical use. We selected 9 sites with a high mutation rate, which contained more than 50% of the mutations identified in Shaanxi province. The identification of 126 positive samples showed that the consistency between the two-color fluorescence amplification technology and NGS was 100%.
On the basis of the common mutations of the PAH gene in Shaanxi province, through many tests, optimization of the conditions, and performance verification, we have developed a PAH gene mutation screening kit, which lays the foundation for a clinical pathway of the three-level prevention system of “premarital, pre-pregnancy, pregnancy health care / prenatal diagnosis and neonatal genetic metabolic disease screening / and treatment.” However, the complexity of PAH gene mutations means that the detection of all PAH gene mutation sites cannot be realized by the current ARMS-PCR combined with fluorescent probe technology detection kit. With the deepening of our understanding of the mutation spectrum, we will analyze the common mutation sites of the PAH gene again, with an aim to optimize the mutation detection sites and further improve the mutation detection rate.