Genotype-phenotype associations in French patients with phenylketonuria and importance of genotype for full assessment of tetrahydrobiopterin responsiveness

Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive disorder caused by a deficient hepatic phenylalanine hydroxylase (PAH) activity [1]. To date, no less than 890 disease causing mutations have been registered to the BioPKU database [2]. In general, the genotype dictates the phenotype [3]. Individuals with simultaneous presence of two null alleles exhibit the classic PKU (cPKU) phenotype, whereas individuals with mutations bearing residual enzyme activity display the mild PKU (mPKU) or the mild hyperphenylalaninemia (mHP).

PAH is a homotetrameric or homodimeric enzyme, whose activity is regulated by the binding of its substrate, Phenylalanine (Phe), and its cofactor, tetrahydrobiopterin (BH4). Each subunit of this multimeric enzyme is composed of the regulatory, the catalytic and the oligomerization domains [4].

The mainstay of the treatment for PKU remains the low-phenylalanine diet [5]. Additional strategy involving the use of sapropterin, a specific pharmacological chaperone similar to the physiologic cofactor tetrahydrobiopterin, is being introduced [6]. Sapropterin enhances PAH enzymatic activity, through multiple mechanisms including (1) stabilization of PAH dimer/tetramer, (2) up-regulation of gene expression, and (3) stabilization of mRNA [4, 7, 8]. However, sapropterin is not efficient in all patients. The degree of sapropterin-responsiveness depends on the presence of responsive mutations [9] having still residual PAH activity [10]. Since PAH is a multimeric protein, it depends also on the allelic complementation of the particular genotype [11, 12].

Numerous studies have investigated mutations in the PAH gene as well as prediction of BH4-responsiveness based on genotypes. However, only few have additionally performed BH4-loading tests [13‐20]. Thus the status of several genotypes is unknown and whether they are BH4-responsive or not needs to be clarified.

We have performed a systematical research of mutations in French PKU patients and systematically evaluated the response to oral BH4-loading test. The aim was to improve the prediction of BH4-responsiveness by relating genetic information such as type, location and combination of mutations, to BH4-loading test responses.

In the present study, patients were classified according to pre-treatment Phe concentration following French and European recommendations into three phenotype categories i.e. mHP (180–600 μmol/L), mPKU (600–1200 μmol/L) and cPKU (>1200 μmol/L Phe) [5, 22]. While recent European consensus publication is still pending, NIH American conference consensus, which was published at a time when our study was completed, proposed 5 categories, namely mHP not requiring treatment (<360 μmol/L), mild HP-gray zone (360–600 μmol/L), mPKU (600–900 μmol/L), moderate PKU (900–1200 μmol/L) and cPKU(>1200 μmol/L Phe) [26]. This different classification system has to be kept in mind when considering the findings and conclusions in our mHP group.

Our multicentric cohort study represents respectively 17.4 % of global cPKU and mPKU and 2.6 % of global mHP diagnosed in France since the beginning of the neonatal screening in the seventies (data from the French neonatal screening association AFDPHE, [27]). In this study, in which we included only 364 patients with both genetic testing and BH4 testing, frequency of mHP is 9 %. Frequency of mHP in all genotyped individuals (n = 701) in our center is 19.3 %, which is higher than the frequency in the 364 patients included in this study (9 %), but still not representative of the 39.4 % of mHP observed among the whole PKU patients screened in metropolitan France since the beginning of the neonatal screening ([27]. Underrepresentation of patients with the milder phenotypes can be partly explained by the fact that there is no recommendation for BH4-testing in patients with Phe concentration under 480 μmol/L. Several mHP are lost to follow-up, in a center dependent manner, and they miss either molecular analysis and/ or BH4 testing.

Investigation of the PAH gene combining dHPLC, sequencing and MLPA allowed us to reach a 99.05 % mutation detection rate, in agreement with results found in Australia: 99.0 % [28], Austria: 98.6 %, [29] or Turkey: 97.3 % [30]. dHPLC is a useful method for screening a large set of samples. It proved to be reliable in our hands and cost effective by narrowing down the number of exons needed to be sequenced. However, because of the chemistry of the method, the genetic variants may not be clearly separated. Now several factors argue that direct sequencing should be the first-line test to identify mutations in the PAH gene. Sequencing is carried out faster and costs have dropped down. In addition, next generation sequencing is spreading quickly and allows analyzing a large number of patients in a short time and at costs going lower and lower. Next generation sequencing also allows analyzing the whole gene including introns and regulatory regions. A spectrum of 127 mutations is reported in this study, including 82 mutations with unknown BH4-responsiveness, according to information available in the literature. This study enables us to classify 5 of them as responsive (c.434A > T, c.500A > T, c.529G > C, c.1045 T > G and c.1196 T > C). The mutation c.1196 T > C (p.Val399Ala) is positioned four nucleotides upstream the exon/intron junction in exon 11 and one base upstream the 5’ splice-site. Exon 11 is particularly vulnerable to mutations affecting splicing regulatory elements [31]. Thus, c.1197A > T and c.1199G > A have been shown to lead to exon skipping [32]. Minigene assays are needed to determine if the mutation c.1196 T > C could or not disrupt the balance between exonic splicing enhancers and silencers [31]. The c.434A > T (p.Asp145Val) mutation involves the substitution of a charged amino acid by a hydrophobic residue. The c.500A > T (p.Asn167Ile) mutation involves the substitution of a polar residue by a hydrophobic residue. The c.529G > C (p.Val177Leu) mutation leads to the substitution of a hydrophobic residue, mostly buried in the loop between C2 and C3 alpha helices [33], by a residue with similar physical and chemical properties. Another mutation affecting the same nucleotide, c.529G > A (p.Val177Met), had previously been described as BH4-responsive [10]. Residue Ser349 is an essential amino acid in the active site of the gene. The substitution of Serine349 by Alanine (c.1045 T > G) removes the hydroxyl group within hydrogen bonding distance of the iron-chelating H285 required for PAH catalytic activity. Missense mutations leading to modifications of either the protein overall hydrophobicity or the ability to form hydrogen bonds within the polypeptide backbone or between side chains may impact on the folding/conformation of PAH. Gersting and colleagues suggested that the consequences of these missense mutations might be compensated for by pharmacological doses of BH4 [7]. However, further functional studies are needed to verify that restoration of the enzymatic activity is possible by the addition of BH4 [7, 34, 35]. Another group of five mutations fitted the criteria for classification as responsive mutations but were not recognized as such. Indeed, there were observed in patients with a BH4-responsive rate comprised between 30 and 40 percent and we do believe that there responsiveness should be subsequently assessed by the long-term achievement of blood Phe target concentration under BH4 treatment.

The position and the nature of the mutation influence the activity of PAH enzyme, which in turn determines the patient phenotype [21]. Our study suggests that for any genotype to be potentially BH4-responsive it needs to contain one missense mutation. Indeed, 98.2 % of the responsive patients in this study carried at least one missense mutation. Conversely, homozygous or compound heterozygous patients carrying any other type of mutations are likely not to be BH4-responders, and administering a long term BH4-therapy to them will not yield an appropriate cost-effectiveness ratio. It is true that combinations of some splice-site mutations were reported previously to be associated with a partial BH4-responder phenotype. The explanation was that those mutations were not fully penetrant, determining a phenotype with persistent residual activity [36]. However, for these patients, based on our experience, BH4 would have rather limited effect in improving dietary Phe tolerance.

In this study, the frequency of BH4-responders reached 31.6 %. It is of note that, if the threshold of responsiveness was more severe (decrease of Phe concentration >40 %), the overall responsive rate would be 22.9 %, which is actually a more reasonable estimate of patients treated with BH4 in France (139 patients: data may 2013). Zurfluh and colleagues [10] postulated that the average frequency of patients potentially BH4-responsive in European populations is 55 %. However, these authors considered the BH4-responsive mutations to be dominant in compound heterozygous patients and classified carriers of one BH4-responsive allele as systematically responsive. This is not always true. Two mutant monomers interact to constitute the holoenzyme and there may be unexpected negative as well as positive inter-allelic complementation in compound heterozygous [4]. Indeed, we found that genotypes carrying 1 or 2 responsive mutations cannot be expected to be always BH4-responsive. Actually, only 66.7 % of patients carrying 2 responsive mutations, 35.4 % of patients carrying 1 responsive mutation and 1 non-responsive mutation and 35.2 % of patients carrying 1 responsive mutation and 1 unclassified mutation were effective BH4-responders. It must be stressed out that BH4-responsiveness depends on the disease phenotype, since 100 % of patients with mHP carrying two responsive alleles were found to be BH4-responders whereas this frequency was only 42.3 % for patients with cPKU.

Fourteen genotypes were assigned to both responsive and non-responsive patients. Similar discordances involving some of these genotypes were already described in the BioPKU database [2], as reported in Table 1. Indeed, while the c. [1066-11G > A]; [1066-11G > A] genotype, associated with a null predicted residual activity, was observed in five PKU non-responsive patients, one tPKU patient carrying this genotype showed a positive BH4 responsive test (H0 1356 μmol/L). However, this patient showed a Phe decrease limited to 31 %. A second example is c. [194 T > C]; [1222C > T] (p.[Ile65Thr]; [Arg408Trp]) genotype, associated both with one non-responsive tPKU (H0 2760 μmol/L) and one responsive tPKU (H0 1380 μmol/L) with a Phe decrease limited to 32 %. This dual BH4-responsiveness may be due to a BH4-loading test performed under a rather strict low-Phe diet with subnormal Phe concentrations. Indeed, in the case of some mutations, the stimulation effect of BH4 on residual enzymatic activity is weaker at low Phe concentrations, as previously described for c.782G > A (p.Arg261Gln) [4, 9, 34]. In our study, patients with p.[Arg261Gln]; [Arg261Gln] genotype and low Phe concentrations at the beginning of BH4-loading test were found to be BH4-unresponsive whereas patients with the same genotype but higher Phe concentrations were found to be responsive (Phe decrease of minus 66 %, minus 59 %, minus 38 %, minus 37 % and 0 % for basal Phe concentration before testing of 1080, 1236, 768, 960 and 430 μmol/L respectively). Another reason for dual responsiveness to BH4 is the intraday fluctuation during the period of the loading test [10]. This is especially true if the decrease in blood Phe concentration at 24 hour is around 30 to 40 %, like in two cPKU patients in this study. This causes to mis-conclude that the patient is BH4-responsive while his genotype is predicted to be unresponsive. However, only failure of long-term Phenylalanine dietary tolerance improvement would definitively confirm that the patient is unresponsive. A third reason is an interindividual variability in BH4 intestinal absorption or in concentration of cofactor required for PAH activity [9]. Lastly, a dual BH4-responsiveness can be due to possible variations in the protocol of the test [12, 20]. However, since the procedure was standardized (all centers used the same BH4 dose, the same time of follow-up and a common definition of responsiveness), discrepancies cannot be explained here by differences in protocols. To note, recent data from the European literature [14] showed that a 48 h-loading test would allow a better recognition of late responders . Here, a 24 h-BH4 loading test was used. Some late-responsive patients may thus have been misclassified as non-responders and some genotypes left unclassified could have been assigned a responsiveness status.

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975. Informed consent for genotype assessment was obtained from patients (or parents) for being included in the study.