Screening and mutation analysis of phenylalanine hydroxylase deficiency in newborns from Jiangxi province

Background: Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder of amino acid metabolism and caused by mutations in the phenylalanine hydroxylase (PAH) gene. Without timely and appropriate dietary management, the disturbance of amino acid metabolism may impair cognitive development and neurophysiological function. Newborn screening (NBS) can aid the early diagnosis of PAHD, which can give accurate therapy to PAHD patients in time. In China, the PAHD incidence and PAH mutation spectrum vary enormously across the provinces. A total of 5,541,627 newborns from Jiangxi province were screened by NBS between 1997 and 2021.

Method: One seventy one newborns from Jiangxi province were diagnosed with PAHD. By Sanger sequencing and the multiplex ligation-dependent probe amplification (MLPA) analysis, mutation analysis was performed in 123 PAHD patients. Using an arbitrary values (AV)-based model, we compared the observed phenotype with the predicted phenotype based on the genotype.

Results: In this study, we speculated the PAHD incidence of Jiangxi province was about 30.9 per 1,000,000 live births (171/5,541,627). We summarized the PAH mutation spectrum in Jiangxi province for the first time. Two novel variants (c.433G > C, c.706 + 2T > A) were found. The most prevalent variant was c.728G > A (14.1%). The overall prediction rate of the genotype-phenotype was 77.4%.

Conclusion: This mutation spectrum is very meaningful to improve the diagnostic rate of PAHD and to increase the accuracy genetic counseling. This study offers data for the genotype-phenotype prediction suitable for Chinese population.

1 Introduction

Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder of amino acid metabolism and its prevalence is about 62.8 per 1,000,000 live births in China (Hillert et al., 2020). It is caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, the PAH Gene Locus-Specific Database (PAHvdb) contains 1584 PAH variants in total. The enzymatic inactivity of PAH variants inhibits the completion of L-phenylalanine (Phe) to L-tyrosine (Tyr) conversion, causing hyperphenylalaninemia (HPA) (Opladen et al., 2012). In addition, its cofactor tetrahydrobiopterin (BH4) deficiency can also lead to HPA (Ye et al., 2018). Without timely and appropriate intervention, the consequent Phe accumulation may impair cognitive development and neurophysiological function (Wang et al., 2019b; Tendi et al., 2022). Based on the degree of Phe accumulation, PAHD is divided into three groups: classic phenylketonuria (cPKU), mild phenylketonuria (mPKU) and mild HPA (mHPA) (Lin et al., 2022). The dietary management is an effective therapeutic approach for PAHD, but the extent of restriction which each group required is different (MacDonald et al., 2020; Firman et al., 2021; Quinn et al., 2022).

Numerous studies have found that the early diagnosis and treatment is essential for the prognosis of PAHD (Huijbregts et al., 2018; van Spronsen et al., 2021; Dobrowolski et al., 2022). The early diagnosis can provide guidelines for a low-phenylalanine diet of PAHD patients (van Spronsen et al., 2021). Newborn screening (NBS) is an effective and low cost method for the early diagnosis (Poloni et al., 2021). Many provinces of China have used NBS for a very long time (Lin et al., 2022). The result of biochemical testing in NBS can provide the early diagnosis of PAHD, whereas the final diagnostic confirmation needs the help of mutational analysis (Opladen et al., 2020).

The clinical phenotype in PAHD patients is significantly related to the residual enzyme activity of PAH and the relationship between enzyme activity and PAH variants has been extensively studied (Blau, 2016; Zhang et al., 2019). Hence, some models have been developed to achieve the genotype-phenotype prediction (Blau, 2016). Practical tests showed that an arbitrary values (AV)-based model was easy to analyze and had a higher predicted rate (Hamilton et al., 2018; Wang et al., 2018). Each PAH variant has an AV and the sum of the two AVs from both variants on the PAH gene is to predict phenotype (Guldberg et al., 1998). In particular, the AV-based model is of better prediction ability in Chinese PAHD patients (Zhu et al., 2017; Li et al., 2018). In China, geographical location and ethnic composition of provinces are significantly different. Therefore, provinces vary enormously in the PAHD incidence and the PAH mutation spectrum (Yan et al., 2019; Tao et al., 2021). Jiangxi province lies in south-central China. To facilitate accurate diagnosis and individualized genetic counseling, it’s necessary to investigate the characteristics of PAH gene variants in local populations.

Here, we successfully extracted genomic DNA from whole blood of 123 PAHD patients and their parents. Then, taking advantage of Sanger sequencing and the multiplex ligation-dependent probe amplification (MLPA) analysis, we summarized the PAH mutation spectrum of Jiangxi province for the first time. Further, the comparison of the phenotype predicted from genotype with the observed phenotype was performed.

2 Materials and methods

2.1 Participants

From October 1997 to December 2021, the newborns were recalled to the Neonatal Screening Center of Jiangxi Maternal and Child health Hospital for further diagnosis, due to high blood Phe concentration in newborn screening. Following criteria for diagnosing HPA in China (Yang et al., 2014), the ratio of blood Phe concentration to tyrosine (Phe/Tyr) was greater than two and blood Phe concentration was above 120 μmol/L. BH4D were excluded by analysis of urinary pterins profile, BH4 loading test and determination of dihydropteridine reductase (DHPR) in red blood cells (PAHD had the normality of pterins profile and normality of DHPR, while BH4D had the abnormity of pterins profile and normality of DHPR). In this study, a total of 123 unrelated patients diagnosed with PAHD were recruited. All participants originated from Jiangxi province, including 73 males and 50 females. According to the consensus about the diagnosis and treatment of hyperphenylalaninemia in China (Yang et al., 2014), PAHD patients were divided into three groups: cPKU (Phe ≥ 1200 μmol/L), mPKU (360 μmol/L ≤ Phe ≤ 1200 μmol/L) and mHPA (120 μmol/L ≤ Phe ≤ 360 μmol/L). This study was approved by the Clinical Research Ethics Committees of Jiangxi Maternal and Child health Hospital, Nanchang, China. All parents/legal guardians of the patients were provided for written informed consent.

2.2 Molecular—Genetics analysis

Genomic DNA was extracted from peripheral blood samples of patients and their parents using a QIAamp DNA Mini Kit (Qiagen). Next, PCR and DNA sequencing of PAH gene were performed to determine the causative variant in each family. Each exon of PAH gene was amplified using the forward primer and the reverse primer designed by Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Supplementary Table S1). The PCR was performed through 2x Taq PCR Master MixII (KT211,TIANGEN) according to the manufacturer’s protocol. Amplification was carried out at 95°C for 5 min for initial denaturing, then 30 cycles at 95°C for 40 s, at 57°C for 30 s and at 72°C for 35 s, followed by a final extension of 8 min at 72°C in a T100 Thermal Cycler for the Classroom (BIO-RAD). Thirteen PCR products were sequenced by a sequencing provider (Tsingke, Changsha). Sequencing results were aligned with the PAH transcript (NM_00027) to precisely identify the nucleotide variants by Seqman Pro. The disease-causing mutations recorded in the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/validate.php) or PAHvdb Database (http://www.biopku.org/home/pah.asp) of variants detected by sequencing were selected as the causative variant in each family. Mutation nomenclature was based on the HGVS guidelines (https://www.HGVS.org/varnomen).

When no variant or one variant were detected in PAH allele by sequencing, the SALSA MLPA Probemix P055-D1PAH kit (MRC-Holland, Netherlands) were subsequently used to analyze large deletions or duplications, according to the manufacturer’s protocol. Amplification products were separated using ABI 3500DXGenetic analyzer and raw data were analyzed with Coffalyser software. The ratio of the peak area of each PAH gene fragment to the corresponding normal controls in the electrophoretograms was calculated to determine the PAH copy-number variants. A ratio of 0 was indicative of the presence of homozygous deletion and 0.45–0.65 for heterozygous deletion, 0.80–1.20 for two copies, and 1.30–1.65 for heterozygous duplication.

2.3 Phenotypic prediction system

According to Guldberg et al. (1998), an AV model was assigned to each PAH variant by referring to the PAHvdb database. The sum of the two AVs from both variants on the PAH gene was to predict phenotype. These predicted phenotypes were classified as the three-phenotype (AV sum of cPKU = 2, 2 < AV sum of mPKU < 9 and AV sum of HPA > 8).

2.4 Statistical analysis

Graph-Pad Prism version 8.0.1 software was used for generating statistical charts in the study. The t-test analysis was applied for differences between groups. Statistical significance was obtained only if the p-value was less than 0.05.

3 Results

3.1 Neonatal PAHD screening

A total of 5,541,627 newborns were screened for neonatal diseases in our center between 1997 and 2021. The screening rate increased from 1.65% (5,391/359,525) in 1997 to 97.68% (339,839/347,910) in 2021 (Table 1). In the past 25 years, 171 newborns were diagnosed as PAHD patients in total. Therefore, we speculated that the prevalence of PAHD in Jiangxi province was about 30.9 per 1,000,000 live births (171/5,541,627). Among 171 PAHD patients, 48 patients diagnosed a long time ago were very difficult to be recalled for further genetic diagnosis and the remaining 123 patients were enrolled to characterize the mutational spectrum (Supplementary Table S2). These patients were comprised of 49 cases of cPKU, 35 cases of mPKU and 39 cases of mHPA (Supplementary Table S2).

TABLE 1

TABLE 1. Newborn screening data of Jiangxi province from 1997 to 2021.

3.2 Mutation analysis of sanger sequencing

We applied Sanger sequencing and MLPA analysis to detect PAH variants in 123 enrolled patients, followed by the phenotype prediction based on the genotype in this study (Figure 1A). A total of 233 disease-causing mutations were identified by Sanger sequencing in the 246 alleles of 123 PAHD patients, with a detection rate of 94.7% (233/246) (Supplementary Table S2). These variants were distributed throughout the coding exons and splicing regions of the PAH gene, and particularly focused on exon 5–8 (Figure 1B). No variants were in exon 1 and 13 (Figure 1B). Subsequently analyzing parental mutations showed that two disease-causing mutations were determined in both alleles of PAH in 111 patients (111/123, 90.2%), 11 patients harbored one mutant allele (11/123, 8.9%) and one patient had no mutation on the PAH allele (1/123, 0.8%) (Supplementary Table S2).

FIGURE 1

FIGURE 1. Mutation analysis of 123 PAHD patients. (A) Workflow of PAHD patient’s enrollment and follow-up testing in the study. (B)The disease-causing mutations were identified by Sanger sequencing in Jiangxi province. Novel mutations were marked red. Abbreviations: E, exon.

3.3 Novel mutations

Two novel variants were not recorded in HGMD and PAHvdb databases in this study: c.433G > C (p.D145H) and c.706 + 2T > A (IVS6+2T > A) (Figure 2). All novel variants mutations were not present in the population frequency databases. Mutation analysis of probands and their parents showed that c.433G > C and c.706+2T > A were in compound heterozygosity with the known variants (c.442-1G > A and c.721C > T), respectively (Supplementary Table S2).

FIGURE 2

FIGURE 2. Two novel mutations identified in 123 PAHD patients. (A) Sanger sequencing results of the c.433G>C mutation on the PAH forward sequence. (B) Sanger sequencing results of the c.706 + 2T>A mutation on the PAH forward sequence.

3.4 Large-scale deletion/duplication analysis

The MLPA analysis was carried out in 12 patients who lacked two disease-causing mutations in both alleles of PAH. The large intragenic deletions were detected in seven patients (Figure 3). These results indicated a heterozygous deletion spanning the 5′-UTR and Exon1 in two patients (Family 3A, F), a heterozygous deletion of Exon5 in three patients (Family 3B), a heterozygous deletion of Exon6 in one patient (Family 3C, D, E), a compound heterozygous deletion of Exon5 and 5′-UTR to Exon1 in one patient (Family 3G) (Figure 2).

FIGURE 3

FIGURE 3. MLPA testing of seven PAHD patients. (A, F) The signal for the area spanning the 5′-UTR and Exon1 was nearly half of the signal observed for the normal sample. (B) The signal for the PAH Exon6 was nearly half of the signal observed for the normal sample. (C, D, E) The signal for the PAH Exon5 was nearly half of the signal observed for the normal sample. (G) Both the signal for the PAH Exon6 and the signal for the area spanning the 5′-UTR and Exon1 was nearly half of the signal observed for the normal sample. (H) normal sample.

3.5 Mutational analysis of 123 PAHD patients

Sanger sequencing of PAH gene showed that 233 variants was positive and MLPA analysis revealed eight large-scale deletions. The combination strategy of Sanger sequencing and MLPA analysis yielded 98.0% positive (241/246) variant findings. Based on mutation types, these variants were grouped in five groups: missense variants (57.3%, 138/241), splicing variants (23.2%, 56/241), non-sense variants (9.1%, 22/241), small deletions (7.1%, 17/241), and large deletions (3.3%, 8/241) (Figure 4A). Among these 241 variants, c.728G > A had the highest frequency (14.1%, 34/241), and other variants, which were just as prevalent, included: c.721C > T (6.2%, 15/241), c.1197A > T (5.0%, 12/241), c.611A > G (5.0%, 12/241), c.442-1G > A (4.1%, 10/241), c.1223G > A (4.1%, 10/241) and c.1174T > A (3.7%, 9/241) (Figure 4B). The seven prevalent variants accounted for 42.3% of all detected variants (Figure 3B).

FIGURE 4

FIGURE 4. Shares of mutational analysis of 123 PAHD patients. (A) The proportion of five different kinds of positive variant findings (%). (B) The relative frequency of various detected variants (%).

3.6 The comparison of phenotypic prediction with the observed data

The two AVs of 84 enrolled patients were both discovered in the PAHvdb database, whereas the AV was not recorded for one or two PAH variants in the 39 remaining cases (Supplementary Table S2). Therefore, 84 patients were enrolled to compare the phenotype predicted with the actual phenotype. The comparison of the predicted phenotype with the observed ones showed that the model had a better prediction ability (65/84, 77.4%), especially in cPKU (39/47, 83.0%) and mHPA patients (21/28, 75.0%) (Table 2). However, the correctly predicted rate of mPKU was only 55.6% (5/9).

TABLE 2

TABLE 2. The accuracy of phenotype prediction based on the genotype.

4 Discussion

Early detection and treatment play a crucial role in the excellent prognosis, resulting in the increased awareness of NBS (Blau, 2016). The screening rate of neonatal screening was on a constant rise in China and reached to 96.1% at 2016 (Xiang et al., 2019). After 25 years of development, we rose the screening rate to 97.68% from 1.65% in Jiangxi province. The NBS is more and more widely used in Jiangxi province, which indicates that we have achieved the promotion of the NBS.

Screening data suggested that the PAHD incidence differed significantly in each Chinese province, ranging from very low in the southern regions (1/188,679) to high in the northern regions (1/3,492) (Xiang et al., 2019). Our results showed that the incidence in Jiangxi province was about 30.9 per 1,000,000 live births, which was a relatively lower than the average prevalence of 62.8 per 1,000,000 live births in China (Xiang et al., 2019). Likewise, analyzing the newborn screening data, the PAHD incidence of Suzhou and Xiamen of southeastern China (1/9,563 and 1/27,922, respectively) were both higher than the Jiangxi province of south-central China (Wang et al., 2019a; Wang et al., 2019b).

As a result, 233 variants and eight large-scale deletions were found in 246 alleles of 123 PAHD patients and the detection ratio achieved 98.0%. Interestingly, the use of MLPA analysis increased the detection ratio, up from 94.7% to 98.0%. Large deletion and duplication account for 12% and 2.1% of the PAH mutations in some ethnic groups (Elhawary et al., 2022). Consistent with previous studies from others, MLPA analysis was a complementary tool to improve the accuracy of diagnosis when no variants were detected in both alleles of PAH by sequencing (Lee et al., 2008; Yan et al., 2019; Tao et al., 2021). In particular, we reported here a patient with the compound heterozygous variants of two large-scale deletions. Similar to other provinces (Zhang et al., 2018; Wang et al., 2019a), c.728G > A was also the highest frequency of variant. But the detection rate of 14.1% was lower than 17.7% of northern China and 16.11% of central China (Liu et al., 2017; Zhang et al., 2018). In addition, the highest frequency of PAH variant in Hainan province was c.611A > G (Zhao et al., 2020), which was also a common variant in Jiangxi province. There are considerable differences in common variants of different countries and provinces (Liu et al., 2017; Zhou et al., 2022). The low incidence and differences of common variants indicated that the concentrated distribution of PAH gene variants in Jiangxi province was less obvious, which was a challenge for genetic counseling. Thus, own spectrum of PAH mutations is an efficient tool for appropriate newborn screening and individualized genetic counseling. Other prevalent variants were also present in other provinces (Zhang et al., 2018; Wang et al., 2019a), probably because the population migration broken the region limits.

Some research applied the AV-based model to analyze the relationship between genotype and phenotype in different regions in China (Zhu et al., 2013; Chen et al., 2015; Wang et al., 2018). By contrast, the overall consistency rate of Shanghai area was 54.4% (Zhu et al., 2013), well below the correctly predicted rate of 77.4% in this study. An 84.21% overall consistency rate in north Jiangsu province was higher than that of this study (Wang et al., 2018). Another study found that the consistency rate of Jiangsu province was 38%, which was lower than the data of this study (Chen et al., 2015). AVs of the milder variant are much higher than the severe one, so the PAHD severity depends largely on the milder variant of two PAH variants (Li et al., 2018; Elhawary et al., 2022). Nevertheless, we and others discovered that the concordance rate for mPKU was very low in China (Zhu et al., 2013). Since patients (Guldberg et al., 1998) studied were all European, the AVs of some variants would be different in Chinese population (Zhu et al., 2013). For example, previous research by Zhu et al. (2013) showed that AVs of R241C, R243Q, R261Q, V388M, V399V, R408Q, A434D and EX6-96A > G could hardly apply to the PAHD classification of Chinese Han population (Zhu et al., 2013). Our research supported this. After the AVs of R241C changing from 8 to 6, the consistency rate of mPKU and mHPA would rise to 73.3% and 95.5% respectively. It indicated that R241C had particularly pernicious effects on Chinese. The phenotypic difference between different populations might be due to different residual enzymatic activity produced by the ubiquitin–proteasome system (UPS) which can regulate cellular protein turnover (Sarodaya et al., 2022).

In summary, for the first time, we successfully constructed the PAH mutation spectrum in Jiangxi province and analyzed the genotypic characteristics of 123 PAHD patients. This mutation spectrum is very meaningful to improve the diagnostic rate of PAHD and to increase the accuracy genetic counseling. We compared the observed phenotype with the predicted phenotype based on the genotype. This study offers data for the genotype-phenotype prediction suitable for Chinese population.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement

The studies involving human participants were reviewed and approved by the Clinical Research Ethics Committees of Jiangxi Maternal and Child health Hospital, Nanchang, China. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Author contributions

BY and YL conceived and designed this study. SC, HG, XX, YZ, FW, and SH collected the written informed consent and analyzed the data. BZ and QL wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Jiangxi Provincial Key Laboratory of Birth Defect for Prevention and Control (No. 20202BCD42017).

Acknowledgments

We would like to thank the patient for participation in this study.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1049816/full#supplementary-material

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