Introduction

Phenylketonuria (PKU, OMIM #261600) is an inherited disorder characterized by increased level of phenylalanine in the blood. PKU is frequently caused by functional deficiency of phenylalanine hydroxylase (PAH), an enzyme that converts phenylalanine to other compounds in the body. More than 900 mutations have been identified in PAH and recorded in the locus-specific database (LSD) known as PAHvdb (http://www.biopku.org/pah/). The occurrence of PKU varies among ethic and geographic regions, reaching approximately 1 in 15,000 newborns1. In mainland China, the average incidence is 1in 11,6142 and in Taiwan 1in 55,0573.

The frequencies and distributions of PAH mutations also differs among different populations4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32. The wide variability of common mutations between ethnic groups and geographical areas makes PAH deficiency a genetic disease of great allelic heterogeneity. Comprehensive catalog of PAH mutation spectrum will be of value to evaluate genotype-phenotype correlations, to provide genetic consultations to patients’ families as well as prenatal diagnosis and to refine diagnoses in and anticipate the dietary requirements of affected patients33,34,35.

A large-scale, unbiased comprehensive survey of PAH mutations in Chinese population was not available. Most previous analyses concerning the Chinese population were limited to a few common mutations, or were confined to a certain region of PAH, resulting in a selective bias3,12,36,37,38,39,40,41. One report carried out survey of entire PAH in a small-scale survey including 212 patients in mainland China12.

In the present study, we report a spectrum of PAH mutations complied from a large cohort of 796 PKU patients in mainland China. We determined the sequence of entire PAH gene using next-generation sequencing (NGS). Among 194 mutations identified, 41 were not reported in literature and 101 not reported in Chinese population. We believe that these results will facilitate the development of appropriate genetic counseling for PKU patients in China.

Results

Mutation spectrum

In the included cohort of Chinese patients, potential disease-causing mutations were identified on 1516 of 1592 independent alleles, corresponding to a mutation detection rate of 95.23%.A total of 720 patients were completely genotyped, whereas in the remaining 76 individuals only one causative mutation was identified; the other mutation site could not be identified by this platform. Among the fully genotyped patients, two mutations were detected in 683 of the patients, who had either compound heterozygous (n = 622) or homozygous (n = 61) genotypes, three mutations were found in 35 of the patients and four mutations were revealed in 2 of the patients. The gene analysis results were summarised in Table 1.

Table 1 The spectrum of PAH mutations in Chinese Mainland PKU patients.
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A total of 194 different types of mutations were identified, including 134 missense mutations (69.07%), 25 splice-site mutations (12.89%), 18 deletions (9.28%), 14 nonsense mutations (7.22%), 2 insertions (1.03%) and 1 silence/splice (0.52%). 76.33% of the total mutations are found in exons. Most mutations was localized in exon7 (33.44%), exon11 (13.18%), exon6 (10.48%), exon12 (10.29%), exon3 (8.94%). Interestingly, no mutations were identified in exon13.

In terms of mutation frequency, p.R243Q was the most prevalent mutation (frequency 17.53%). Other mutations with relatively high frequencies were p.EX6-96A > G, p.V399V, p.R241C, p.R111*, p.Y356*, p.R413P and IVS4-1G > A (7.66%, 5.84%, 5.40%, 4.77%, 4.46%, 4.33%, 3.77%, respectively). Eight mutations, including p.R53H, p.A434D, p.R408Q, IVS7 + 2T > A, p.G247V, p.S70del, p.R261Q and p.Y166* were found with relatively lower frequencies (ranging from 1% to 3%). The remaining 178 mutations were found at relative frequencies of less than 1%.

Comparison between northern and southern Chinese populations

The frequencies of each PAH mutations were compared among geographical regions in China. We used sixteen mutations with frequency more than 1% for this comparison. Six mutations showed obvious local mutation clustering: p.V399V, p.R413P and IVS7 + 2T > A were found to be clustered in northern Chinese populations and p.R241C, p.R408Q and p.Y166* were clustered in southern Chinese populations. Among the remaining 178 mutations that exhibited a relative frequency of <1%, thirteen exhibited significant differential distribution in northern and southern China.

Novel sequence variants

Forty-one novel nucleotide lesions that have not been registered in the BIOPKU database (http://www.biopku.org/pah/) were identified in this study: p.V5Sfs*34, IVS1 + 5_ + 6delGC, IVS1-3T > C, p.F39del, p.E43*, p.N58*, p.L62Pfs*7, p.E78Q, p.E78V, p.I95del, p.D101N, p.G103D, IVS3-2A > G, p.F121V, p.E127K, p.E127G, p.G148R, p.R169S, p.R169G, p.H170P, p.E178K, p.N223I, p.F260I, p.E280Nfs*61, p.V291M, p.V291L, p.P292S, p.D296H, p.Q304K, p.T328N, p.K335E, p.A342Hfs*59, p.Y343*, IVS10-12delT, IVS10-1G > C, IVS10-1G > T, p.Y356D, IVS11-1G > C, p.T372R, p.I406V, p.Q429K.

Among these mutations, the majority were missense mutations (n = 25), followed by small deletions (n = 8), nonsense (n = 2), splice (n = 5) and insertions (n = 1). Thirty-four of the novel mutations were detected in coding regions and the remaining were located in introns.

The prediction results of novel mutations are listed in Table 2. A total of 25 novel mutations were predicted; of these, 20 mutations were predicted to be probably damaging, 5 mutations were tolerated and the remainder of the 16 mutations could not be predicted using this tool. The frequencies of the novel mutations were relatively low, which indicates that they are rare mutations.

Table 2 Pathologic analysis of 25 novel mutations of the PAH gene.
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Discussion

A comprehensive survey of the mutation spectrum of the protein of interest in a given population not only can provide insight into the structural and functional aspects of the protein as well as genotype-phenotype correlations14,15,20,22,23,26,28,42, but also facilitate genetic counseling in patients’ families. In this study, we described the molecular basis of PKU in a mainland Chinese population by analysing mutations in the PAH gene using NGS. Among a cohort of 796 patients, mutations were detected on 1516 of the 1592 independent alleles, representing a mutation detection rate of 95.23% (Table 1). A total of 194 distinct mutations were found, demonstrating the high genetic heterogeneity that is inherent in PKU.

The number of different mutations in a given population is usually high and is typically comprised of a few prevalent mutations and a large number of private mutations43. In comparing our study with previous reports, 83 of the mutations that we identified have been previously reported; however, the remaining 101 mutations (including 41 novel mutations) were reported for the first time in a Chinese mainland population12,36,37,38,39,41. As shown in Table 3, eight mutations including p.R243Q, p.EX6-96A > G, p.V399V, p.R241C, p.R111*, p.Y356*, p.R413P and IVS4-1G > A are common mutations in the mainland Chinese population, although the rank order of these mutations was different. Among them, R241C, p.EX6-96A > G, p.V399V, R413P, R243Q and R111* are also considered to be prevalent in the Chinese Taiwanese population40. The epidemiology of phenylketonuria in China is complicated. The prevalence of PKU in northern China (1/11,000) is close to what has been documented in Caucasian populations, but the prevalence in southern China is much lower44. A comparison between northern and southern China indicated marked differences in the relative frequencies of mutations. Among eight common mutations, V399V, R241C and R413P gave significant p values between two regions, with respective p values of 0.037, 0.007 and 0.002. The result that p.R413P clustered in northern China is consistent with what has been reported by Gu. et al.12. Furthermore, p.V399V has been previously detected primarily in populations of Xinjiang and northern China36,37, whereas p.R241C has been primarily detected in populations in southern China and in Taiwan patients3,45. The majority of the population in Taiwan has descended from southeast China. Based on our data, we hypothesize that the uniform distribution of V399V, R241C and R413P is a result of migration and the founder effect.

Table 3 The distribution of common PAH gene mutations within China.
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It is well known that different ethnic groups have their own distinctive and diverse PAH mutant allele series that include either one or a few prevalent founder alleles46. When comparing PAH mutational data between different ethnic groups, correlations between the mutations in and the genetic histories of the investigated populations were found. Marked differences were identified when comparing PAH mutations with ≥ 3% frequency (totaling 34) between Asian and European countries (Table 4). Five mutations, including p.R243Q, p.EX6-96A > G, p.R241C, p.R413P and IVS4-1G > A, were found to be common mutations in East Asian countries such as China, Japan14 and Korea8, accounting for 53.76%, 69.70% and 62.10% of the total mutations respectively. Three mutations, including p.R111*, p.Y356* and p.T278I, were frequently detected in China and Japan14, China and Korea8, Japan14 and Korea8 respectively. The remaining mutations, including p.V399V, p.A259T, p.R252W, p.Y325* and p.V388M, were found to be common in only one country. In sharp contrast, these mutations except for p.R252W, the above mentioned mutations were either rarely detected or undetected in West Asia and Europe countries. For example in Iran17 and Turkey18, three common mutations including p.R261Q, p.P281L and IVS10-11G, A were shared, However these mutations were either rare or did not occur in East Asia. Instead, they were prevalent in select Europe countries. In Europe, p.R408W was found to be the common mutation, ranking first in the Czech Republic28 (East Europe) and Germany5 (West Europe) and second in Danemark4 (North Europe) and Serbia32(South Europe). These results suggest that p.R408W was the most prevalent founder allele in the European population46. In contrast, p.R408W was either rarely detected or undetected in populations of East Asia, whereas it was the most common mutation in Turkish populations18. The remaining mutations were only found to be prevalent in subsets of the four countries. For example, p.L48S was the most prevalent mutation in Serbian populations32 and it was also common in Turkish populations18. The p.R158Q, p.A403V, p.Y414C and IVS12 + 1G > A mutations were relatively common in only two countries. Based on the above comparison, we identified that there were several overlaps of mutant allele distributions between West Asia and Europe and that the mutations that were common in East Asia were different from these.

Table 4 Relative frequencies (%) of common PAH gene mutations detected in Asian and European populations.
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In the present study, the high mutation detection rate of 95.23% was similar to previous studies in which sequencing analysis of the PAH gene was conducted12,15, but it was relatively lower than the results from studies that employed exon analysis combined with multiplex ligation-dependent probe amplification (MLPA)14,20,24,28. This is because NGS is able to detect small deletions and insertions, whereas it is not able to detect large deletions or duplications. Despite scanning the entirety of the PAH coding region and its exon–intron boundaries, no mutations were detected in 76 alleles. The most likely explanation behind this is that the mutations are located in regions that were not detectable in this study (for example, in the promoter regions, the 5′ and 3′ UTRs, in non-coding RNA binding sites, or in the intronic sequences far away from exon–intron boundaries). Alternatively, the mutations may have been large deletions or duplications.

Using NGS as a routine genetic diagnostic tool enables thousands of DNA sequences to be simultaneously obtained in notably reduced turnaround times and at a significantly reduced cost. Furthermore, this technique provides high sensitivity, specificity and coverage (including all coding regions of the involved exons and adjacent intronic regions). However, the biggest limitation to using NGS is the need to analyse and interpret complicated data.

We believe our study will provide guidance for future medical practice such as prenatal screening and early diagnosis of PKU. Diagnostic methods can be developed based on the known characteristics of a population. Currently, PKU screening is performed in newborn babies as a part of the tertiary prevention in birth defects preventive network in China. Developing a new method for screening might enhance primary prevention. Based on the mutational spectrum presented in this study, our hope is that carrier screening can be conducted preceding gestation, which would offer timely guidance with respect to prenatal diagnosis for couples who are both carriers.

Methods

Subjects

A total of 796 unrelated patients from 29 separate newborn screening centres of China were enrolled. These patients were diagnosed at birth either through a neonatal screening program or based on clinical presentation. Demographic data, including age, consanguinity, family history and geographical origin and biochemical testing data, including plasma phenylalanine (Phe) levels, dihydropteridine reductase activity, urinary biopterin and neopterin ratio and tetrahydrobiopterin loading, were collected. The ages of the included patients ranged from 6- months to 5-years old. In families with more than one patient, only one member of each sibling pair was included in the study of mutation frequency. The numbers of patients in northern China and southern China that were divided by the Qinling Mountains and the Huaihe River were 557 and 239, respectively. Both parents of the included patients were native.

These patients were classified into one of three separate phenotype categories according to their pretreatment plasma Phe levels, including mild hyperphenylalaninaemia (Phe 120–600 umol/L), mild PKU(Phe 600-1200 μmol/L) and classic PKU (Phe >1200 μmol/L)47. Among the 796 patients, 145 (18.22%) were mild hyperphenylalaninemia(MHP), 215 (27.01%) were mild PKU(mPKU) and 418 (52.51%) were classic PKU(cPKU), the remaining 18 patients (2.26%) could not be classified because the pretreatment Phe levels were not available(Table 5). The entire list of patients’ phenotype and blood Phe levels can be found as Supplementary Table S1. The parental permissions and informed consents were obtained from the parents of all patients. The research was approved by the Ethics Committee of West China Second University Hospital, Sichuan University (No: 2014018) and followed the tenets of the Declaration of Helsinki.

Table 5 Patients’ phenotype and blood Phe levels.
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DNA extraction

Blood samples (4 ml) were collected from each patient and their parents by venipuncture in EDTA. Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp® DNA Blood Mini Kit (Qiagen, Cat.No.51106, Germany) according to the recommended protocol.

Next-generation sequencing

A series of unique primers were designed to amplify all 13 exons and their surrounding introns, which covered 200bp upstream and 200bp downstream of the exons of the PAH gene. A pair of primers that were designed to amplify approximately 150 bp of the human β-globin gene (HBB) (accession number AY260740) was used as an internal quality control to identify false negatives caused by inadequate DNA or failed PCR.

To improve the throughput of the assay, the PCR primers were designed to not only amplify the target DNA but also to provide a unique primer index for each of the 96 samples in each of the plates (multiple index PCR). This strategy resulted in 96 sets of 10-bp-long nucleic acid tags that were individually included at the 5′ ends of each of the PAH and HBB primers. The second index that was used for each sample was the 8-bp-long nucleic acid tag from the library adapter sequence, which identified the specific 96-well plate that each sample was included in. This index was attached to the amplicons of the samples through an adapter preparation process. Using this “double index system,” hundreds of samples can be mixed together and detected in one sequencing chip at the same time.

PCR was performed on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA), with a cycling protocol that consisted of denaturation at 94 °C for 30 s, 56° C for 30 s and 72 °C for 1 min. After 35 cycles, gel electrophoresis was used to verify the quality of the amplified DNA, only eligible DNAs was included in the library preparation. The PCR amplification products were prepared for DNA pooling and the diverse adapter library was added onto the amplification products. After a concentration of DNA was obtained that could satisfy the requirements of the library preparation method, gene mutations were sequenced using an Illumina Hiseq 2000 (Illumina Inc, San Diego, CA, USA) sequencing instrument.

After sequencing the samples, the raw sequence data were analysed using in-house software. First, all sequence data were traced back to the specimens from which they arose according to the sequences of the primer and adapter indices. Second, the amplicon sequences of each of the samples were aligned with standard reference PAH sequences from the database PAHvdb (http://www.biopku.org/pah/); SNPs were found in target areas and relevant information was noted.

All of the PAH gene sequencing reactions and analyses were performed in the Centre of BGI Health clinical laboratory, Shenzhen, China.

Validation tests of Sanger sequencing

When a given patient’s mutation locus was detected, it was amplified by polymerase chain reaction (PCR) from a parent’s sample and then sequenced bidirectionally in an ABI-3730 DNA analyser. This not only validated the locus but also confirmed the carrier status of the parents. The PCR cycling protocol consisted of an initial denaturation at 95 °C for 3 min, followed by 35 cycles, of 95 °C for 40 s, 55 °C for 30 s and 72 °C for 30 s, with a final extension at 72 °C for 10 min.

Pathologic analysis of novel mutations

A SIFT prediction was performed (http://sift.jcvi.org/) using the “SIFT Human SNPs” tool to obtain predictions for nonsynonymous SNPs. The annotation version was Homo sapiens GRCh37 Ensembl 63. A list of the chromosomal positions and alleles corresponding to the 41 novel mutations were uploaded into the import the web-site.

Statistical analysis

Statistical analysis was performed using statistical package for social science software (SPSS version 16.0). Mutational frequencies were calculated by the counting method. An x2 analysis was performed to test for differences between two geographic populations. A p value <0.05 was considered statistically significant.

Additional Information

How to cite this article: Li, N. et al. Molecular characterisation of phenylketonuria in a Chinese mainland population using next-generation sequencing. Sci. Rep. 5, 15769; doi: 10.1038/srep15769 (2015).