Tetrahydrobiopterin responsiveness in phenylketonuria differs between patients with the same genotype

Recently, BH(4)-responsive phenylalanine hydroxylase (PAH) deficiency was reported in patients with specific mutations in the PAH gene, and it was suggested that BH(4) responsiveness may be determined by the respective genotypes. We now report on three patients with PAH deficiency and the same genotype but different responses to standardized BH(4) loading. Our results suggest that BH(4) responsiveness in PAH deficiency is at least partly independent from PAH genotype.     

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.     

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.     

Tetrahydrobiopterin responsiveness: results of the BH4 loading test in 31 Spanish PKU patients and correlation with their genotype

Tetrahydrobiopterin (BH4) responsiveness in patients with mutations in the phenylalanine hydroxylase (PAH) gene is a recently recognized subtype of hyperphenylalaninemia characterized by a positive BH4 loading test. According to recent estimates, this phenotype may be quite common, suggesting that a large group of individuals may benefit from BH4 substitution, eliminating the need of life-long dietary restrictions. This underscores the importance of identifying BH4-responsive patients in each population, establishing the association with specific PAH mutations. In this work, we describe the results of a pilot study performed with 31 Spanish PAH-deficient patients subjected to a BH4 loading test. Overall, 11/31 (37%) showed a positive response with a 30% decrease in blood Phe levels 8 h after the BH4 challenge, and three additional patients, considered slow responders, showed this decrease only after 12-16 h. We report for the first time a patient homozygous for a splicing mutation with a slow response, suggesting an effect of BH4 supplementation on PAH gene expression. Most of the responsive patients belong to the mild hyperphenylalaninemia (MHP) or mild phenylketonuria phenotypic groups. In MHP patients we report for the first time the results of parallel single Phe doses confirming the utility of these analyses for a better evaluation of the response. Genotype analysis confirms the involvement in the response of specific mutations (D415N, S87R, R176L, E390G, and A309V) present in hemizygous patients, and provide relevant information for the discussion of the potential mechanisms underlying BH4 responsiveness.     

Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: an implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

We previously proposed a novel disease entity, tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency, in which administration of BH4 reduced elevated levels of serum phenylalanine [J. Pediatr. 135 (1999) 375-378]. Subsequent reports indicate that the prevalence of BH4-responsive PAH deficiency is much higher than initially anticipated. Although growing attention surrounds treatment with BH4, little is known about the mechanism of BH4 responsiveness. An early report indicates that BH4 concentration in rat liver was 5 microM where Km for BH4 of rat PAH was estimated to be 25 microM in an oxidation experiment using a liver slice, suggesting relative insufficiency of BH4 in liver in vivo. In the present study, we developed a breath test for mice using [1-13C]phenylalanine in order to examine the BH4 responsiveness of normal PAH in vivo. The reliability of the test was verified using BTBR mice and its mutant strain lacking PAH activity, Pahenu2. BH4 supplementation significantly enhanced 13CO2 production in C57BL/6 mice when phenylalanine was pre-loaded. Furthermore, BH4 apparently activated PAH in just 5 min. These observations suggest that submaximal PAH activity occurs at the physiological concentrations of BH4 in vivo, and that PAH activity can be rapidly enhanced by supplementation with BH4. Thus, we propose a possible hypothesis that the responsiveness to BH4 in patients with PAH deficiency is due to the fact that suboptimal physiological concentrations of BH4 are normally present in hepatocytes and the enhancement of the residual activity may be associated with a wide range of mutations.     

Tetrahydrobiopterin sensitivity in German patients with mild phenylalanine hydroxylase deficiency

We report the results of tetrahydrobiopterin (BH4) loading tests in 10 German patients with mild phenylketonuria. A significant decline of phenylalanine values after application of BH4 was observed in all but one patients. Molecular genetic analyses revealed a range of different PAH gene mutations. Re-testing of one patient previously reported as non-responsive to BH4 loading showed a moderate response with a higher dose of BH4. Nevertheless, there appear to be kinetic differences in phenylalanine hydroxylation in patients with the same genotype. Non-responsiveness to 20 mg/kg BH4 was observed only in a single patient who was compound heterozygous for the novel mutation R176P (c.527G>C) and the common null-mutation P281L. In summary, our data are in line with recent reports indicating that BH4 sensitivity is a normal feature of most mild forms of PAH deficiency but may be influenced by other factors.     

Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.     

儿童运动及智能障碍者四氢生物蝶呤代谢病筛查及基因研究

目的探讨四氢生物蝶呤（tetrahydrobiopterin，BH4）代谢中各酶缺乏在儿童运动及智能发育障碍者中发生率及基因突变。方法对100例运动及智能障碍患者进行苯丙氨酸（phenylalanine，Phe）及BH4负荷试验、尿蝶呤谱分析、红细胞二氢蝶啶还原酶测定，并对部分患者进行多巴治疗性诊断；对诊断为多巴反应性肌张力障碍（dopa-responsive dystonia，DRD）及6-丙酮酰四氢蝶呤合成酶（6-pyruvoyl tetrahydropterin synthase，PTS）缺乏者做基因突变检测。结果100例中70例基础血Phe浓度正常，6例（6％）诊断为DRD；30例有高苯丙氨酸血症[Phe（1022±290）μmol／L]，8例（8％）诊断为VIS缺乏症，22例（22％）诊断为苯丙氨酸羟化酶缺乏症。发现2例DRD患者其三磷酸鸟苷环化酶基因（GTP cyclohydrolase 1 gene，GCHI）突变为IVS5＋3insT，8例FIS缺乏症患者存在PTS基因7种突变类型，其中259C→T，286G→A，155A→G最常见，占75％。结论一些肌张力障碍或智能障碍者是由于BH4代谢障碍所致，有必要做筛查诊断以明确诊断。     

四氢生物蝶呤反应性苯丙氨酸羟化酶缺乏症在我国南北地区差异的临床初步研究

目的通过对不同类型高苯丙氨酸血症（hyperphenylalaninemia,HPA）临床特点的分析,探讨我国南、北方四氢生物蝶呤（tetrahydrobiopterin,BH4）反应性苯丙氨酸羟化酶（phenylalanine hydroxylase,PAH）缺乏症患者对BH4的反应性。方法（1）108例HPA患儿,男63例、女45例,平均年龄7.05个月。所有患者都进行口服BH4负荷试验,同时进行尿蝶呤谱分析、红细胞二氢蝶啶还原酶测定。对其中血苯丙氨酸（phenylalanine,Phe）浓度〈600μmol/L者给予口服Phe-BH4联合负荷试验。（2）根据患儿父母双方祖籍,以长江为界将诊断为BH4反应性PAH缺乏症的患儿分为南、北两组。比较南、北方组BH4反应性PAH缺乏症患儿在BH4负荷试验中血Phe浓度的变化。结果（1）HPA中诊断BH4反应性PAH缺乏症36人（33.3%）,BH4无反应性苯丙酮尿症（phenlketonuria,PKU）49人（45.4%）,四氢生物蝶呤缺乏症（BH4D）23人（21.3%）。BH4反应性PAH缺乏症血Phe浓度8h、24h时分别平均下降了49.24%和65.35%。（2）36例BH4反应性患者分为南方组23人、北方组13人。南、北方组BH4反应性患儿服药后24h时血Phe浓度均值分别为（217.02±189.03）μmol/L和（458.75±342.54）μmol/L（P〈0.05）,而两者在服药后2h、4h、8h、24h时血Phe浓度下降的百分数差异均无统计学意义（P〉0.05）。结论部分因PAH缺乏引起的PKU患儿口服BH420mg/kg后24h,血Phe浓度较服药前下降30%以上,其中绝大多数为轻、中度HPA（血Phe120～1200μmol/L）,少数为经典型PKU（血Phe〉1200μmol/L）。本研究中我国南方组BH4反应性PAH缺乏症服药24h时血Phe浓度较北方组低,但是南、北方患者对药物的总体反应性差异无统计学意义。     

中国南方四氢生物蝶呤缺乏症筛查、临床及基因突变的系列研究

目的 得出我国南方高苯丙氨酸血症（hyperphenylalaninemia,HPA)者四氢生物蝶呤（tetrahydrobiopterin,BH4)缺乏症的发病率,总结BH4缺乏症者基因突变和临床转归的研究。方法 对87例HPA者做尿新蝶呤（N）和生物蝶呤（B）分析;对BH4缺乏症者进行基因突变检测及治疗随访。结果 11例诊断为6－丙酮酰四氢蝶呤合成酶（PTPS）缺乏所致BH4缺乏症,其尿N／B＞38,B％＜5％,在HPA中其发病率为12％。5例中发现4种PTPS基因突变类型,即P87S、N52S、D96N及G144R（新突变类型）。5例经BH4、L－DOPA及5－羟色氨酸治疗后体格发育良好,4例智商（IQ）70－80分。结论 对所有HPA者需进行BH4缺乏症的筛查,以降低误诊率。     

Response of patients with phenylketonuria in the US to tetrahydrobiopterin

Tetrahydrobiopterin (BH4) responsive forms of phenylketonuria (PKU) have been recognized since 1999. Subsequent studies have shown that patients with PKU, especially those with mild mutations, respond with lower blood phenylalanine (Phe) concentrations following oral administration of 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). To determine the incidence of BH4 responding PKU patients in the United States and characterize their phenylalanine hydroxylase (PAH) mutations, a study was undertaken at UTMB in Galveston and the Children's Hospital of Los Angeles on 38 patients with PKU. Patients were screened by a single oral dose of BH4, 10 mg/kg and blood Phe and tyrosine were determined at 0, 4, 8, and 24 h. Twenty-two individuals (58%) responded with marked decrease in blood Phe (>30%) at 24h. Some of the patients that responded favourably were clinically described as having Classical PKU. Blood tyrosine concentrations did not change significantly. Twenty subjects with PKU, responsive and non-responsive to BH4, were enrolled in a second study to evaluate blood Phe response to ascending single doses of BH4 with 10, 20, and 40 mg/kg and to evaluate multiple daily doses, for 7 days each, with 10 and 20 mg/kg BH4. The 7-day trial showed a sustained decrease in blood Phe in 14 of 20 patients taking 20 mg/kg BH4 (70%). Of these 14 patients, 10 (71%) responded with a significant decrease in blood Phe following 10 mg/kg BH4 daily. To understand the mechanism of response to BH4, the kinetics and stability of mutant PAH were studied. We found that mutant PAH responds with increase in the residual enzyme activity following BH4 administration. The increase in activity is multi-factorial caused by increased stability, chaperone effect, and correction of the mutant Km. These studies indicate that BH4 can be of help to patients with PKU, including some considered to have Classical PKU. The PKU population in US is heterogeneous and mutations can be varied so mutations need to be characterized and response to BH4 tested. It is more likely that mutations with residual activity should respond to BH4, therefore the clinical definition of "Classical PKU" should be reconciled with the residual activity of PAH mutations.     

Stimulation of hepatic phenylalanine hydroxylase activity but not Pah-mRNA expression upon oral loading of tetrahydrobiopterin in normal mice

Tetrahydrobiopterin (BH4) supplementation in patients with BH4-responsive phenylalanine hydroxylase (PAH) deficiency is an alternative to low-phenylalanine diet. To further investigate hepatic BH4-responsiveness, oral administration of 50 mg BH4/kg/day for 5 weeks was performed in wild-type mice. We observed a 2-fold increase in PAH protein by quantitative Western blot analysis and a 1.7-fold increase in enzyme activity, but no change in Pah-mRNA expression by quantitative real-time PCR analysis in treated mice compared to controls. Our findings support the proposed chemical-chaperone effect of BH4 to protect PAH.     

The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response

The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.     

Plasma biopterin levels and tetrahydrobiopterin responsiveness

Tetrahydrobiopterin (BH4) responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) gene was found during neonatal screening for PKU. This study determined blood BH4 and phenylalanine in two patients with hyperphenylalaninemia following oral load with BH4 10 mg/kg. Our patients underwent neonatal screening for PKU, had normal biopterin metabolism and their PAH mutations were determined. Peak plasma biopterin levels in Case 1, which were reached at between 2 and 4h after loading, were 612, 297, and 178 nmol/L at age 30 days, 55 days, and 19 months, respectively, and the maximum phenylalanine decreasing rates, which were found at 24h, were 54, 16, and 4%, respectively. In Case 2, peak plasma biopterin levels were 747 and 327 nmol/L at age 20 and 55 days, respectively, and the maximum phenylalanine decreasing rates were 39 and 32%, respectively. In the BH4 loading test, the peaks of BH4 in both patients lowered ( approximately 50%), on the same dose schedule of BH4, as patients got older.     

四氢生物喋呤负荷试验在高苯丙氨酸血症鉴别诊断中的临床应用

目的探讨四氢生物喋呤（BH4）负荷试验在高苯丙氨酸血症（HPA）鉴别诊断中的应用价值。方法自2005年5月到2007年4月，51例HPA患儿采用口服BH4（20mg／kg）负荷试验。对其中血苯丙氨酸（Phe）浓度小于600μmol／L患儿采用口服Phe—BH4联合负荷试验，结合尿喋呤分析、血红细胞二氢喋呤还原酶（DHPR）活性测定。结果（1）在BH4负荷试验中，不同类型HPA患儿的血Phe浓度表现出各不相同的改变。51例HPA患儿中，共鉴别出5例BH4缺乏症，10例BH4反应性苯丙氨酸羟化酶（PAH）缺乏症，36例BH4无反应性苯丙氨酸羟化酶（PAH）缺乏症。（2）在17例中度苯丙酮尿症（PKU）患儿中，9例（52．9％）为BH4反应性PAH缺乏症。结论BH4负荷试验在HPA早期鉴别诊断中十分重要，部分中度PKU对BH4有反应，可使用BH4替代治疗。     

Molecular characterization of phenylketonuria and tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Japan

Phenylketonuria (PKU) is a heterogeneous metabolic disorder caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). On the basis of phenotype/genotype correlations, determination of phenylketonuric genotype is important for classification of the clinical phenotype and treatment of PKU, including tetrahydrobiopterin therapy. We characterized the genotypes of 203 Japanese patients with PKU and hyperphenylalaninemia using the following systems: (1) denaturing high-performance liquid chromatography with a GC-clamped primer; (2) direct sequencing; and, (3) multiplex ligation-dependent probe amplification. Of 406 mutant alleles, 390 (96%) were genotyped; 65 mutations were identified, including 22 new mutations. R413P, R241C, IVS4-1g>a, R111X and R243Q were prevalent mutations. Mutations prevalent in the Japanese cohort are also common in Korean and Northern Chinese populations, suggesting same origin. The spectrum of prevalent mutations was not significantly different among six Japanese districts, indicating that Japan comprises a relatively homogeneous ethnic group. We classified the mutations by clinical phenotypes and in vivo PAH activity and estimated the mutations with potential tetrahydrobiopterin (BH(4)) responsiveness. The frequency of BH(4) responsiveness based on the genotype was 29.1% in Japanese PKU patients. A catalog of PKU genotypes would be useful for predicting clinical phenotype, deciding on the subsequent treatment of PKU including BH(4) therapy, and genetic counseling in East Asia.     

Molecular basis of mild hyperphenylalaninaemia in Poland.

The major cause of the different forms of hyperphenylalaninaemia (HPA) is mutations in the gene encoding phenylalanine hydroxylase (PAH). The aim of this study was to determine the mutations responsible for mild forms of HPA and to relate different clinical phenotypes of HPA patients to their PAH genotypes. Four "mild" mutations, including the most frequent A403V and R297H mutations, occurred exclusively in mild hyperphenylalaninaemia (MHP). Mutations A104D, R243Q, R241H, and Y414C were detected in patients with mild phenylketonuria (mild PKU) only. These results may be useful in establishing a molecular differential diagnosis for PAH deficiency in Poland.     

Effects of tetrahydrobiopterin and phenylalanine on in vivo human phenylalanine hydroxylase by phenylalanine breath test

BH(4) administration results in the reduction of blood phenylalanine level in patients with tetrahydrobiopterin (BH(4))-responsive phenylalanine hydroxylase (PAH) deficiency. The mechanism underlying BH(4) response remains unknown. Here, we studied the effects of BH(4) and phenylalanine on in vivo PAH activity of normal controls using the phenylalanine breath test (PBT) by converting l-[1-(13)C] phenylalanine to (13)CO(2). Phenylalanine oxidation rates were expressed as Delta(13)C ((13)CO(2)/(12+13)CO(2), per thousand) and cumulative recovery rates over 120min (CRR(120), %; total amount of (13)CO(2)/the administered dose of (13)C-phenylalanine). Under physiological conditions of blood phenylalanine, BH(4) administration reduced the Delta(13)C peak from 40.8 per thousand to 21.6 per thousand and CRR(120) from 16.9% to 10.2%. Under high blood phenylalanine conditions, administration of BH(4) increased the Delta(13)C peak from 30.7 per thousand to 46.0 per thousand, while the CRR(120) was similar between phenylalanine (19.9%) and phenylalanine+BH(4) (21.1%) groups. Corrected Delta(13)C and CRR(120) were calculated against serum phenylalanine levels to remove the effects of phenylalanine loading. After BH(4) administration, the corrected Delta(13)C peak increased from 82.7 per thousand to 112.6 per thousand, while the corrected CRR(120) was similar (47.6% and 45.6%). These results indicate that phenylalanine worked as a regulator of in vivo PAH by serving as both a substrate and an activator for the enzyme. Excessive dosages of BH(4) inhibited PAH under normal phenylalanine conditions and activated PAH under conditions of high phenylalanine. The regulation system is therefore designed to maintain phenylalanine levels in the human body. Appropriate BH(4) supplementation must be reviewed in patients with BH(4)-responsive PAH deficiency.     

Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles

Phenylketonuria (PKU, MIM 261600; EC 1.14.16.1) results from mutations in the phenylalanine hydroxylase (PAH) gene. Newborn metabolic disease screening uses blood dried on filter paper (DBS) to prospectively identify candidate newborns affected with PKU via an elevated concentration of phenylalanine. However, it is then important to confirm the specific category of PKU since classical PKU requires a stringent diet while milder categories may not require diet and a very important BH4-responsive category may be treated with the PAH cofactor 6R-tetrahydrobiopterin (BH4). Since there is a close genotype-phenotype correlation in PKU, determining the PAH genotype can be extremely important for therapy as well as prognosis. A simple and rapid method of accurately determining the PAH genotype would be a valuable addition to the diagnosis of PKU. Described herein is a means to identify variants in the PAH gene using high-resolution melt profiling, which compares the thermal denaturation profile of a patient sample to that of a control. Regions where the patient and control samples produce a common profile were not further evaluated, while those regions where the patient profile deviates from the control were assessed by DNA sequencing. Additionally described is a scheme utilizing redundant analysis with melt profile controls and a novel multiplex genotyping assay to triage deviation owing to known polymorphisms. Two mutations were identified in 93 of the 95 patients assessed and in the remaining two patients a single mutation was identified. Melt profiling provided 99% sensitivity to identify sequence variants in the PAH gene.     

Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. PAH requires the cofactor BH(4) to function and the rate-limiting step in the synthesis of BH(4) is GTP cyclohydrolase I (GTP-CH). The skin is a potential target tissue for PKU gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed a higher copy number of transgenes in keratinocytes and also a higher metabolic capacity.