Mutational and phenotypic spectrum of phenylalanine hydroxylase deficiency in Zhejiang Province, China

Phenylalanine hydroxylase deficiency (PAHD), one of the genetic disorders resulting in hyperphenylalaninemia, has a complex phenotype with many variants and genotypes among different populations. Here, we describe the mutational and phenotypic spectrum of PAHD in a cohort of 420 patients from neonatal screening between 1999 and 2016. The observed phenotypes comprised 43.57% classic phenylketonuria, 33.10% mild PKU, and 23.33% mild hyperphenylalaninemia, with an overall PAHD incidence of 1 in 20,445. Genetic testing was performed for 209 patients and 72 variants including seven novel variants were identified. These included two synonymous and five pathogenic nonsynonymous variants (p.S36*, p.T186I, p.L255W, p.F302V and p.R413H). The most common variant among all patients was p.R243Q, followed by p.R241C, p.Y204C, p.R111* and c.442-1G > A. Variants p.R53H and p.F392I occurred only in MHP with 19.3% and 8.0% of the observed alleles respectively. The genotypes p.[R241C];[R243Q], p.[R243Q];[R243Q], and p.[Y204C];[R243Q] were abundant across all PAHD patients. The distributions of the null allele and the three defined genotypes, null/null, null/missense, and missense/missense, were significantly different between the cPKU and mPKU patients. However, no significant differences were found between mPKU and MHP patients, indicating that other modifier factors influence the phenotypic outcome in these patients. The data presented here will provide a valuable tool for improved genetic counseling and management of future cases of PAHD in China.

Seven novel variants were identified (Supplementary Fig. 1.), including two synonymous variants: c.285C > T (p.I95I) in a cPKU patient and c.441T > C (p.P147P) in an MHP patient. Besides the conservation analysis of amino acid, other five pathogenic nonsynonymous variants were evaluated with bioinformatic programs and predicted as putative functional variants (Supplementary Table S2). Of these, c.107C > A (p.S36*) and c.1238G > A (p.R413H) were observed for cPKU; c.764T > G (p.L255W) and c.904T > G (p.F302V) were observed for mPKU; and c.557C > T (p.T186I) was observed for MHP. According to the PAH protein domains, p.S36* variant localized in the regulatory domain, p.T186I, p.L255W and p.F302V in the catalytic domain, and the p.R413H in the oligomerization domain. The pathogenicity of novel variants should be proved by more PAHD cases and their functional analysis in vitro.
Genotypic distribution for PAHD. In total, biallelic mutations were genotyped in 183 individuals, and of which 85.8% (157/183) were compound heterozygous alleles, and 78.38% of these observed for cPKU, 90.28% for mPKU, and 91.89% for MHP patients. Monoallelic mutations were found and confirmed for the remaining 26 (Table 3). Strikingly, none of these genotypes were observed to coexist in any of the three phenotypes. However, seven genotypes were concurrent in both cPKU and mPKU patients, and four genotypes associated with not only the mPKU phenotype but also the MHP phenotype (Supplementary Table S3).
We hypothesized that the genotypes and null alleles would exhibit significant differences in distribution among the three phenotypes. Thus, 29 null PAH alleles were sorted into three defined genotype classes (null/ null, null/missense, and missense/missense) as described in previous reports (Supplementary Table S4) 8,18 . Of the cPKU patients, 77.03% carried at least one null allele, with 54.79% observed for mPKU patients. Furthermore, the null/null genotype was identified in 26 cPKU patients, but only in six mPKU patients ( Table 4). The distribution of the three defined genotypes, null/null, null/missense, and missense/missense, were significantly different (p < 0.001) between cPKU and mPKU patients. The null allele frequency also differed remarkably between cPKU and mPKU (p < 0.001). No significant difference was found between mPKU and MHP patients in either the distribution of genotypes or the null allelic frequency (p = 0.21 and p = 0.57, respectively).

Discussion
Here, we carried out a retrospective study on samples obtained via neonatal screening for HPA since 1999 in Zhejiang Province of southeast China, illustrating the mutational and phenotypic spectrum of PAHD for a greater understanding of PAH gene variants and their association with PAHD phenotypes.
It is confirmed that the incidence of PAHD for south China is lower than that for Northern China alone (1/3,425-1/7,849) 19,20 . Compared with the data from the BIOPKU database, the comprising of the three PAHD phenotypes in the present study is different that more than 50% of PAHD cohort from Zhejiang Province comprises mPKU and MHP patients as reported in Denmark PAHD group 20 .
The identified variants included 63.89% missense mutations, 13.89% splice site mutations, and 11.11% nonsense mutations as previously observed 21 . Exons 3, 6, 7, 11, and 12 appeared to be hotspots for PAHD-associated  Table 2. PAH variants and allele distribution in PKU and MHP patients from Zhejiang Province. Italic, novel variants; common mutations are in bold. Allele frequency in PKU is a ratio as the amount of one variant in cPKU and mPKU to 304 alleles occurred in PKU patients; Allele frequency in MHP is a ratio as the amount of one variant in MHP cases to 88 alleles found in all MHP cases. Allele frequency in PAHD is a ratio as the amount of one variant to all 392 alleles in PAHD. present in 27% of the alleles and 49% of the MHP patients (Table 2). While p.R53H is known to be harbored by Japanese PKU patients 25 , it is more common in the general Korean population with a frequency of 2.57% 26,27 .
Since these two mutations are observed in healthy subjects and they retain >70% residual PAH activity, p.R53H and p.F392I are classified here as mutations associated with MHP. Another notable finding was that 30% of the MHP patients (16/53) with abnormal Phe values observed during newborn screening were carriers of a pathogenic allele. Genetic counseling should be provided to such patients prior to conception. The genotypes in our PAHD cohort were highly heterogeneous as >60% of the patients presented with a unique genotype, and 85.8% were compound heterozygotes compared with 76% reported in the BIOPKU database 16 8 . Interestingly, two p.R241C homozygous patients showed MHP, which was also detected in Korean PAHD population before 23 . The distribution of null alleles and the genotypes null/null, null/missense, and missense/missense showed significant differences between cPKU and mPKU patients as well as between cPKU and MHP patients. However, no significant differences were observed for the distribution of either between mPKU and MHP patients. This indicates that other modifier factors may influence PAHD phenotype, especially for mPKU and MHP. It is therefore unsurprising that several genotypes are shared by both mPKU and MHP phenotypes.
Certainly, the specific PAH genotype is key for determining the metabolic phenotype. More than 80% (26/32) of the double-null genotypes correlated to cPKU phenotype in the present study. Interestingly, MHP and mPKU phenotypes have similar frequencies of null alleles in genotypes. This implies that the interaction between compound heterozygous alleles play a major role in phenotypic outcome 28 . Additionally, we confirmed that in MHP patients, mild mutations determine disease severity. Genotypes comprising a combination of p.R58H with one of the null alleles p.R111*, c.442-1G > A, p.Y204C, p.Y356*, or p.L255S, resulted in the MHP phenotype. A similar phenomenon was observed for the p.F392I/null genotype associated with the MHP phenotype, although further samples are needed to confirm this, and the effect on PAH structure and function needs to be considered for further insights. In conclusion, the data presented in this study will provide a valuable tool for improved genetic counseling and management of future patients of PAHD in China.   Table 4. The three classes of defined genotypes and null allele frequencies associated with the PAHD phenotypes. p1, difference between cPKU and mPKU patients; p2, difference between mPKU and MHP. 3 × 2 contingency analysis for genotype, 2 × 2 contingency analysis for allele frequencies.