Residual Phenylalanine Hydroxylase Activity Research Articles

CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model.

The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells – an in vitro model for PKU – corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases.

In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU

Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme.To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups.Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH4 responsiveness).The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH4) responsiveness.

Genetics of Phenylketonuria: Then and Now.

More than 950 phenylalanine hydroxylase (PAH) gene variants have been identified in people with phenylketonuria (PKU). These vary in their consequences for the residual level of PAH activity, from having little or no effect to abolishing PAH activity completely. Advances in genotyping technology and the availability of locus-specific and genotype databases have greatly expanded our understanding of the correlations between individual gene variant, residual PAH activity, tetrahydrobiopterin (BH4 ) responsiveness, and the clinical PKU phenotype. Most patients (∼76%) have compound heterozygous PAH gene variants and one mutated allele may markedly influence the activity of the second mutated allele, which in turn may influence either positively or negatively the activity of the biologically active heterotetrameric form of the PAH. While it is possible to predict the level of BH4 responsiveness (∼71%) and PKU severity (∼78%) from the nature of the underlying gene variants, these relationships remain complex and incompletely understood. A greater understanding of these relationships may increase the potential for individualized management of PKU in future. Inherited deficiencies in BH4 metabolism account for about 1%-2% of all hyperphenylalaninemias and are clinically more severe than PKU. Almost 90% of all patients are deficient in 6-pyruvoyl-tetrahydropterin synthase and dihydropteridine reductase.

Co-expression of phenylalanine hydroxylase variants and effects of interallelic complementation on in vitro enzyme activity and genotype-phenotype correlation.

In phenylketonuria (PKU) patients, the combination of two phenylalanine hydroxylase (PAH) alleles is the main determinant of residual enzyme activity in vivo and in vitro. Inconsistencies in genotype-phenotype correlations have been observed in compound heterozygous patients and a particular combination of two PAH alleles may produce a phenotype that is different from the expected one, possibly due to interallelic complementation. A dual eukaryotic vector system with two distinct PAH proteins N-terminally fused to different epitope tags was used to investigate the co-expression of PAH alleles reported in patients with inconsistent phenotypes. PAH variant proteins were transiently co-transfected in COS-7 cells. PAH activity was measured by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), and protein expression was measured by Western blot. Genotypes were compared with predicted PAH activity from the PAH locus-specific database (PAHvdb) and with phenotypes and tetrahydrobiopterin (BH4) responsiveness from more than 10,000 PKU patients (BIOPKU database). Through the expression and co-expression of 17 variant alleles we demonstrated that interallelic interaction could be both positive and negative. The co-expressions of p.[I65T];[R261Q] (19.5% activity; predicted 43.5%) and p.[I65T];[R408W] (15.0% vs. 26.8% activity) are examples of genotypes with negative interallelic interaction. The co-expressions of p.[E178G];[Q232E] (55.0% vs.36.4%) and p.[P384S];[R408W] (56.1% vs. 40.8%) are examples of positive subunit interactions. Inconsistencies of PAH residual enzyme activity in vitro and of PKU patients' phenotypes were observed as well. The PAH activity of p.[R408W];[A300S] is 18.0% of the wild-type activity; however, 88% of patients with this genotype exhibit mild hyperphenylalaninemias (MHPs). The co-expression of two distinct PAH variants revealed possible dominance effects (positive or negative) by one of the variants on residual PAH activity as a result of interallelic complementation.

Correlation between genotype and the tetrahydrobiopterin-responsive phenotype in Chinese patients with phenylketonuria.

Background:A growing body of research has suggested that tetrahydrobiopterin (BH4) responsive phenotype can be predicted by the phenylalanine hydroxylase (PAH) genotype in patients with phenylketonuria (PKU), but data concerning the association between genotype and BH4 responsiveness are scarce in China.Methods:A total of 165 PKU patients from China who had undergone a 24-h loading test with BH4 administration were recruited. Genotyping was performed by the next-generation sequencing (NGS) technique. Using the predicted residual PAH activity, we analyzed the association between genotype and BH4-responsiveness.Results:Among the 165 patients, 40 patients (24.24%) responded to BH4. A total of 74 distinct mutations were observed, including 13 novel mutations. The mutation p.R241C was most frequently associated with response. Two known mutations (p.A322T and p.Q419R) and two novel mutations (p.L98V and IVS3-2A>T) were first reported as responsive to BH4. Residual PAH activity of at least 12.5% was needed for responsive genotypes.Conclusion:Genotype-based predictions of BH4-responsiveness are only for selecting potential responders. Accordingly, it is necessary to test potential responders with a long-term BH4 challenge.

Safety of extended treatment with sapropterin dihydrochloride in patients with phenylketonuria: Results of a phase 3b study

Background Phenylketonuria (PKU) results from impaired breakdown of phenylalanine (Phe) due to deficient phenylalanine hydroxylase (PAH) activity. Sapropterin dihydrochloride (sapropterin, Kuvan®) is the only US- and EU-approved pharmaceutical version of naturally occurring 6R-BH 4, the cofactor required for PAH activity. Sapropterin enhances residual PAH activity in sapropterin-responsive PKU patients and, in conjunction with dietary management, helps reduce blood Phe concentrations for optimal control. Approval was based on the positive safety and efficacy results of four international clinical studies, the longest of which was 22 weeks in duration. Objective To evaluate the safety of long-term treatment with sapropterin in PKU subjects who participated in previous Phase 3 sapropterin trials. Methods PKU-008 was designed as a Phase 3b, multicenter, multinational, open-label, 3-year extension trial to evaluate the long-term safety of sapropterin in patients with PKU who were classified as sapropterin responders and participated in prior Phase 3 sapropterin studies: 111 subjects aged 4–50 years completed prior studies and were subsequently enrolled in study PKU-008. Routine safety monitoring was performed at 3-month intervals and included adverse event reporting, blood Phe monitoring, clinical laboratory evaluations, physical examinations and vital sign measurements. Results Average exposure during PKU-008 was 658.7 ± 221.3 days (range, 56–953; median, 595). The average total duration of participation in multiple studies (PKU-001, PKU-003, PKU-004, and PKU-008; or PKU-006 and PKU-008) was 799.0 ± 237.5 days (range, 135-1151). The mean sapropterin dose was 16.2 ± 4.7 mg/kg/day. Most adverse events were considered unrelated to treatment, were mild or moderate in severity, and were consistent with prior studies of sapropterin. No age-specific differences were observed in adverse event reporting. Three subjects discontinued treatment due to adverse events that were considered possibly or probably related to study treatment (one each of difficulty concentrating, decreased platelet count, and intermittent diarrhea). No deaths were reported. Of seven reported serious adverse events, one was considered possibly related to study treatment (gastroesophageal reflux). There were no laboratory or physical examination abnormalities requiring medical interventions. For most subjects, blood Phe concentrations were consistently within target range, confirming the durability of response in subjects undergoing extended treatment with sapropterin. Conclusion Sapropterin treatment was found to be safe and well tolerated at doses of 5 to 20 mg/kg/day for an average exposure of 659 days. This study supports the safety and tolerability of sapropterin as long-term treatment for patients with PKU.

Molecular genetics and impact of residual in vitro phenylalanine hydroxylase activity on tetrahydrobiopterin responsiveness in Turkish PKU population

Background The prevalence of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU) in Turkey is high (1 in 6500 births), but data concerning the genotype distribution and impact of the genotype on tetrahydrobiopterin (BH 4) therapy are scarce. Objective To characterize the phenotypic and genotypic variability in the Turkish PKU population and to correlate it with physiological response to BH 4 challenge. Methods We genotyped 588 hyperphenylalaninemic patients and performed a BH 4 loading test (20 mg/kg bw) in 462 patients. Residual PAH activity of mutant proteins was calculated from available in vitro expression data. Data were tabulated in the BIOPKU database ( www.biopku.org). Results Eighty-eight mutations were observed, the most common missense mutations being the splice variant c.1066-11G>A (24.6%). Twenty novel mutations were detected (11 missense, 4 splice-site, and 5 deletion/insertions). Two mutations were observed in 540/588 patients (91.8%) but in 9 patients atypical genotypes with > 2 mutations were found (8 with p.R155H in cis with another variant) and in 19 patients mutations were found in BH 4-metabolizing genes. The most common genotype was c.1066-11G>A/c.1066-11G>A (15.5%). Approximately 22% of patients responded to BH 4 challenge. A substantial in vitro residual activity (average > 25% of the wild-type enzyme) was associated with response to BH 4. In homozygous genotypes ( n = 206), both severity of the phenotype ( r = 0.83) and residual PAH activity ( r = 0.85) correlate with BH 4 responsiveness. Conclusion Together with the BH 4 challenge, these data enable the genotype-based classification of BH 4 responsiveness and document importance of residual PAH activity. This first report of a large-scale genotype assessment in a population of Turkish PKU patients also documents a high prevalence (47%) of the severe classic phenotype.

Blood phenylalanine concentrations in patients with PAH‐deficient hyperphenylalaninaemia off diet without and with three different single oral doses of tetrahydrobiopterin: Assessing responsiveness in a model of statistical process control

Tetrahydrobiopterin (BH(4)) cofactor loading is a standard procedure to differentiate defects of BH(4) metabolism from phenylalanine hydroxylase (PAH) deficiency. BH(4) responsiveness also exists in PAH-deficient patients with high residual PAH activity. Unexpectedly, single cases with presumed nil residual PAH activity have been reported to be BH(4) responsive, too. BH(4) responsiveness has been defined either by a >or=30% reduction of blood Phe concentration after a single BH(4) dose or by a decline greater than the individual circadian Phe level variation. Since both methods have methodological disadvantages, we present a model of statistical process control (SPC) to assess BH(4) responsiveness. Phe levels in 17 adult PKU patients of three phenotypic groups off diet were compared without and with three different single oral dosages of BH(4) applied in a double-blind randomized cross-over design. Results are compared for >or=30% reduction and SPC. The effect of BH(4) by >or=30% reduction was significant for groups (p < 0.01) but not for dose (p = 0.064), with no interaction of group with dose (p = 0.24). SPC revealed significant effects for group (p < 0.01) and the interaction for group with dose (p < 0.05) but not for dose alone (p = 0.87). After one or more loadings, seven patients would be judged to be BH(4) responsive either by the 30% criterion or by the SPC model, but only three by both. Results for patients with identical PAH genotype were not very consistent within (for different BH(4) doses) and between the two models. We conclude that a comparison of protein loadings without and with BH(4) combined with a standardized procedure for data analysis and decision would increase the reliability of diagnostic results.

Genotype-predicted tetrahydrobiopterin (BH 4)-responsiveness and molecular genetics in Croatian patients with phenylalanine hydroxylase (PAH) deficiency

Specific mutations in the gene encoding phenylalanine hydroxylase ( PAH), located on chromosome 12q22-24.1, are linked to tetrahydrobiopterin (BH 4; sapropterin)-responsive phenylketonuria (PKU). Diagnosis is usually done through the newborn screening for PKU, followed by a BH 4 loading test. So far, more than 60 mutant alleles, presenting with a substantial residual PAH activity (average ∼47%), were identified in more than 500 patients worldwide. We investigated the predictive value of BH 4-responsive PAH mutations in Croatian population. From a group of 127 PKU patients, 62 were selected (based on the genotype) as potentially BH 4-responsive and 39 loaded with BH 4 (20 mg/kg). The overall frequency of BH 4-responsiveness (>30% blood phenylalanine reduction within 24 h) was 36% (14 out of 39 patients with 23 different genotypes), significantly less than expected. The best responders were patients with mild hyperphenylalaninemia (4/4; 100%), followed by mild PKU (8/9; 89%), and classical PKU (2/26; 8%). The most common BH 4-responsive genotypes were p.E390G/p.R408W and p.P281L/p.E390G. These genotypes correspond for approximately >30% residual PAH activity. The p.E390G mutation was 100% associated with BH 4-responsiveness, regardless of the second allele (p.R408W, p.P281L, p.F55Lfs, p.L249P). With regard to the predicted relative PAH activity of recombinantly expressed mutant alleles, there was a significant ( p < 0.002) difference between BH 4-responders and non-responders. In a general Croatian PKU population, disease-causing mutations were identified on 226 alleles (99%). There were 35 different mutations: 21 missense, 8 splice site, 3 nonsense, 2 single nucleotide deletions, and 1 in-frame deletion. Four mutations are reported for the first time: p.E76D, p.L333P, p.G346E, and IVS8-2A > G. Five mutations accounted for over two-thirds of investigated alleles: p.L48S, p.R261Q, p.P281L, p.E390G, and p.R408W. Thus, the Croatian PKU population seems to be more homogenous than some other Mediterranean or Central European populations. This study reveals the importance of a full genotype for the prediction of BH 4-responsiveness. In contrast to previous assumption and with exception of the p.E390G mutation, single allele mutations are not reliable for the selection of potential PKU candidates for pharmacological therapy with BH 4.

The activity of wild type and mutant phenylalanine hydroxylase with respect to the C-oxidation of phenylalanine and the S-oxidation of S-carboxymethyl- l-cysteine

The involvement of the enzyme, phenylalanine hydroxylase (PAH), in the S-oxidation of S-carboxymethyl- l-cysteine (SCMC) is now firmly established in man and rat. However, the underlying role of the molecular genetics of PAH in dictating and influencing the S-oxidation polymorphism of SCMC metabolism is as yet unknown. In this work we report that the S-oxidation of SCMC was dramatically reduced in the tetrahydrobiopterin (BH 4) responsive mutant PAH proteins (I65T, R68S, R261Q, V388M and Y414C) with these enzymes possessing between 1.2% and 2.0% of the wild type PAH activity when SCMC was used as substrate. These same mutant proteins express between 23% and 76% of the wild type PAH activity when phenylalanine was used as the substrate. The PAH mutant proteins (R158Q, I174T and R408W) that result in the classical phenylketonuria (PKU) phenotype expressing 0.2–1.8% of the wild type PAH activity when using phenylalanine as substrate were found to have <0.1% of the wild type PAH activity when SCMC was used as the substrate. Mutations that result in PAH proteins retaining some residual PAH activity with phenylalanine as substrate have <2.0% residual activity when SCMC was used as a substrate. This investigation has led to the hypothesis that the S-oxidation polymorphism in man is a consequence of an individual carrying one mutant PAH allele which has resulted in the loss of the ability of the residual PAH protein to undertake the S-oxidation of SCMC in vivo.

Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Mutations in the phenylalanine hydroxylase (PAH) gene result in phenylketonuria (PKU). Tetrahydrobiopterin (BH(4))-responsive hyperphenylalaninemia has been recently described as a variant of PAH deficiency caused by specific mutations in the PAH gene. It has been suggested that BH(4)-responsiveness may be predicted from the corresponding genotypes. Data from BH(4) loading tests indicated an incidence of BH(4)-responsiveness of >40% in the general PKU population and >80% in mild PKU patients. The current project entailed genotype analysis of 315 BH(4)-responsive patients tabulated in the BIOPKUdb database and comparison with the data from the PAHdb locus-specific knowledgebase, as well as with previously published PAH mutations for several European countries, Northern China, and South Korea. We identified 57 mutations, presenting with a substantial residual PAH activity (average approximately 47%), presumed to be associated with BH(4)-responsiveness. More than 89% of patients are found to be compound heterozygotes. The three most common mutations found in >5% of BH(4)-responsive patients are p.A403 V, p.R261Q, and p.Y414C. Using the Hardy-Weinberg formula the predicted average frequency of BH(4)-responsiveness in European populations was calculated to be 55% (range 17-79%, lowest in Baltic countries and Poland and highest in Spain), 57% in Northern China, and 55% for South Korea. The genotype-predicted prevalence of BH(4)-responsiveness was higher than prevalence data obtained from BH(4) loading tests. Inconsistent results were observed for mutations p.L48S, p.I65 T, p.R158Q, p.R261Q, and p.Y414C. Our data suggest that BH(4)-responsiveness may be more common than assumed and to some extent may be predicted or excluded from the patient's genotype.

Effects and clinical significance of tetrahydrobiopterin supplementation in phenylalanine hydroxylase‐deficient hyperphenylalaninaemia

In recent years several studies on tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency have been published. The molecular mechanisms of BH4 responsiveness are not conclusively understood, but there is evidence that BH4 responsiveness in hyperphenylalaninaemia (HPA) depends on the patient's genotype and residual PAH activity. As a BH4 preparation will soon obtain marketing approval as an alternative treatment for phenylketonuria (PKU), it is particularly important to evaluate this treatment and to define criteria to identify patients with a potential benefit from it. Most of the patients found to be BH4-responsive suffered from mild PKU or mild hyperphenylalaninaemia (MHP) and some of these would not be treated at all in many countries. Of patients with moderate and classic forms of PKU, only a few were classified as responders and the clinical significance of the effect size may be small.

Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria.

Phenylalanine homeostasis in mammals is primarily controlled by liver phenylalanine hydroxylase (PAH) activity. Inherited PAH deficiency (phenylketonuria or PKU) leads to hyperphenylalaninemia in both mice and humans. A low level of residual liver PAH activity ensures near-normal dietary protein tolerance with normal serum phenylalanine level, but the precise threshold for normal phenylalanine clearance is unknown. We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice. Serum phenylalanine levels remained normal in mice exhibiting nearly complete liver repopulation with PAH-deficient hepatocytes (<5% residual wild-type liver PAH activity). Conversely, transplantation of PAH-positive hepatocytes into PAH-deficient Pah(enu2) mice, a model of human PKU, yielded a significant decrease in serum phenylalanine (<700 muM) when liver repopulation exceeded approximately 5%. These data suggest that restoration of phenylalanine homeostasis requires PAH activity in only a minority of hepatocytes.

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency, state of the art

Since 1999 an increasing number of patients with phenylalanine hydroxylase (PAH) deficiency are reported to be able to decrease their plasma phenylalanine (Phe) concentrations after a 6R-tetrahydrobiopterin (BH 4) challenge. The majority of these patients have mild PKU or MHP (mild hyperphenylalaninemia) and harbour at least one missense mutation in the PAH gene associated with this phenotype. The rate of decrease and the lowest achieved Phe level vary between patients with different genotypes but appears to be similar in patients with the same genotype. A number of the mutations associated with BH 4-responsiveness have been studied in an ‘in vitro’ eukaryotic cell expression system leading to biosynthesis of a mutant PAH enzyme with some residual activity. Patients bearing mutations that cause severe structural distortion in the expressed protein (loss of function mutations), leading to undetectable PAH activity, are not responsive to BH 4. These observations suggest that residual PAH activity (in vitro) is a prerequisite for BH 4-responsiveness. However, an in vitro residual PAH activity is not a guarantee for in vivo BH 4-responsiveness. Mechanisms behind this responsiveness could be relieve of decreased binding affinity for BH 4, BH 4-mediated increase of PAH gene expression or stabilization of the mutant enzyme protein by BH 4. BH 4-responsive PAH-deficient patients have only been reported since 1999. For the western countries this is explained by the fact that the manufacturer changed the diastereoisomeric purity of the BH4 preparation from 69% of the natural 6R-BH4 (31% of 6S-BH4) to 99.5% 6R-BH4. The new findings on BH 4-responsiveness may be of clinical relevance because these patients can be treated with BH 4 with concomitant relief or withdrawal of the burdensome PKU diet. These observations warrant further clinical studies to assess efficacy, optimal dosage, and safety of BH 4 treatment in this group. The data strongly emphasize the necessity of the BH 4 loading test in patients detected in the newborn PKU screening.

Frequency of 12 mutations in 114 children with phenylketonuria in the Midwest region of the USA.

Phenylalanine hydroxylase (PAH) is a liver-specific enzyme involved in the hydroxylation of phenylalanine (phe) to tyrosine. The deficiency of PAH results in excessive accumulation of phe in body fluids that leads to phenylketonuria (PKT). Among Caucasians, PKT affects 1 in 10000 live births and the carrier frequency of this inherited metabolic disorder is 1 in 50 (Scriver et al 1989). Clinically, this disorder is accompanied by neurological impairment and mental retardation of varying degree that seem to bear a relationship to the residual PAH activity in these patients (Okano et al 1991; Ramus et al 1993)

Effect of cotrimoxazole on the response to phenylalanine loading in man

Cotrimoxazole impairs the L-phenylalanine tolerance of control subjects as judged by the time course of the plasma phenylalanine concentration and the plasma ratios [phenylalanine] / [tyrosine] after oral and intravenous loading with phenylalanine. These subjects are presumed not to be heterozygous for any of the abnormal recessive genes which cause hyperphenylalaninaemia when they are present in the homozygous state. Patients with classical phenylketonuria did not show this effect. Cotrimoxazole did not affect the renal clearance of phenylalanine. The effect appears to be virtually entirely due to the trimethoprim component as opposed to the sulphamethoxazole component of Cotrimoxazole. The evidence presented is compatible with the view that the drug inhibits phenylalanine hydroxylation in the patients' tissues, presumably by inhibiting dihydrobiopterin reductase. We suggest that it may be possible to utilise the observed effect of cotrimoxazole on phenylalanine hydroxylation to identify in vivo those hyperphenylalaninaemic patients who have residual phenylalanine hydroxylase activity.