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.