Two missense mutations causing mild hyperphenylalaninemia associated with DNA haplotype 12.

The genetic defects responsible for most phenylketonuria (PKU) and hyperphenylalaninemia (HPA) cases are located in the phenylalanine hydroxylase (PAH) gene. Approximately 50-60 mutations have been reported in Caucasians and are reflected in a wide range of clinical severities. Most mutations are linked to specific haplotypes, as defined by eight polymorphic restriction sites in the PAH gene. We hypothesized that there is at least one mild mutation linked to haplotype 12 in the Swedish PKU/HPA population, since 7 of 8 patients carrying haplotype 12 had mild HPA. Sequence analysis revealed a C-to-G transversion at the second base of codon 322, resulting in a substitution of glycine for alanine, in four mutant haplotype 12 genes, and a G-to-A transition at the second base of codon 408, resulting in a substitution of glutamine for arginine, in another three mutant haplotype 12 genes. These mutations segregated with mutant haplotype 12 alleles in nuclear families but were not present on normal or other mutant alleles. Both mutations were tested in a eukaryotic expression system in which enzyme activities of different mutant PAH enzymes reflect the relative severities of the mutations, although these in vitro activities cannot be translated directly into in vivo hepatic activities. The A322G mutant PAH had about 75% and the R408Q mutant PAH about 55% of the wild-type PAH enzyme activity. These in vitro activities are the highest reported for mutant PAH enzymes produced in the same expression system.(ABSTRACT TRUNCATED AT 250 WORDS)     

The PAH gene, phenylketonuria, and a paradigm shift

"Inborn errors of metabolism," first recognized 100 years ago by Garrod, were seen as transforming evidence for chemical and biological individuality. Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype, was identified in 1934 by Asbj?rn F?lling. It is a disease with impaired postnatal cognitive development resulting from a neurotoxic effect of hyperphenylalaninemia (HPA). Its metabolic phenotype is accountable to multifactorial origins both in nurture, where the normal nutritional experience introduces L-phenylalanine, and in nature, where mutations (>500 alleles) occur in the phenylalanine hydroxylase gene (PAH) on chromosome 12q23.2 encoding the L-phenylalanine hydroxylase enzyme (EC 1.14.16.1). The PAH enzyme converts phenylalanine to tyrosine in the presence of molecular oxygen and catalytic amounts of tetrahydrobiopterin (BH4), its nonprotein cofactor. PKU is among the first of the human genetic diseases to enter, through newborn screening, the domain of public health, and to show a treatment effect. This effect caused a paradigm shift in attitudes about genetic disease. The PKU story contains many messages, including: a framework on which to appreciate the complexity of PKU in which phenotype reflects both locus-specific and genomic components; what the human PAH gene tells us about human population genetics and evolution of modern humans; and how our interest in PKU is served by a locus-specific mutation database (http://www.pahdb.mcgill.ca; last accessed 20 March 2007). The individual Mendelian PKU phenotype has no "simple" or single explanation; every patient has her/his own complex PKU phenotype and will be treated accordingly. Knowledge about PKU reveals genomic components of both disease and health.     

Molecular analysis of phenylketonuria (PKU) in newborns from Texas

This study describes the mutations at the phenylalanine hydroxylase (PAH) locus in patients with the diagnosis of classic PKU (n=18), hyperphenylalaninemia (HPA) variant (n=9) and benign persistent hyperphenylalaninemia (HPA) (n=13) who were identified by the Texas Newborn Screening Program. Blinded studies were done by sequencing of the 13 exons and exon-intron boundaries of the PAH gene in genomic DNA isolated from dry blood spots. Thirty-six different mutations, including 25 missense mutations, six splice mutations, three deletions and two nonsense mutations were detected in 75 of the 80 mutant alleles (94%). The prevalent mutations were R408W (19%), V388M and IVS10nt-11g->a (6% each), Y414C (5%) and H170D, A403V, T380M and IVS7nt1g->a (4% each). Two novel missense mutations were identified in exon 5 (H170D and N167S). There was genotype/phenotype correlation in 33/40 cases (83%). For this population, exons 12, 11, 7, 5 and 8, which carry 78% of the mutations, would have to be screened first. However, the other exons must be studied when either one or no mutations are found in the primary screening. Hum Mutat 17:523, 2001.     

Manipulation of the phenylalanine metabolism in human keratinocytes by retroviral mediated gene transfer

Phenylketonuria (PKU) is an inherited disease causing increased levels of phenylalanine in body fluids due to deficiency of hepatic phenylalanine hydroxylase (PAH) or other enzymes involved in the phenylalanine metabolism. With the long-term goal of using gene transfer to the skin to remove phenylalanine, we have previously shown that overexpression of PAH, catalyzing the hydroxylation of phenylalanine, and GTP cyclohydrolase (GTP-CH), involved in the formation of the necessary cofactor BH4,are required. Here we investigate whether manipulation of additional steps in the phenylalanine clearance pathway can further improve the phenylalanine uptake and metabolism. Transport of phenylalanine into human keratinocytes could be increased by overexpressing the two subunits LAT1 and 4F2hc of the large neutral amino acid transporter. The PAH enzyme activity was titrated by employing mutant PAH enzymes with different specific activity and by increasing the PAH copy number in transduced keratinocytes using a repeated transduction procedure. Finally, the intracellular tyrosine concentration was lowered by overexpression of tyrosinase converting tyrosine to dopaquinone. However, measured over a 24-hour period neither of these manipulations resulted in an increased phenylalanine uptake. These results suggest that other enzymes than GTP-CH, involved in BH4 synthesis and/or regeneration, can be rate-limiting in the genetically modified keratinocytes.     

PAHdb 2003: what a locus-specific knowledgebase can do

PAHdb, a legacy of and resource in genetics, is a relational locus-specific database (http://www.pahdb.mcgill.ca). It records and annotates both pathogenic alleles (n = 439, putative disease-causing) and benign alleles (n = 41, putative untranslated polymorphisms) at the human phenylalanine hydroxylase locus (symbol PAH). Human alleles named by nucleotide number (systematic names) and their trivial names receive unique identifier numbers. The annotated gDNA sequence for PAH is typical for mammalian genes. An annotated gDNA sequence is numbered so that cDNA and gDNA sites are interconvertable. A site map for PAHdb leads to a large array of secondary data (attributes): source of the allele (submitter, publication, or population); polymorphic haplotype background; and effect of the allele as predicted by molecular modeling on the phenylalanine hydroxylase enzyme (EC 1.14.16.1) or by in vitro expression analysis. The majority (63%) of the putative pathogenic PAH alleles are point mutations causing missense in translation of which few have a primary effect on PAH enzyme kinetics. Most apparently have a secondary effect on its function through misfolding, aggregation, and intracellular degradation of the protein. Some point mutations create new splice sites. A subset of primary PAH mutations that are tetrahydrobiopterin-responsive is highlighted on a Curators' Page. A clinical module describes the corresponding human clinical disorders (hyperphenylalaninemia [HPA] and phenylketonuria [PKU]), their inheritance, and their treatment. PAHdb contains data on the mouse gene (Pah) and on four orthologous mutant mouse models and their use (for example, in research on oral treatment of PKU with the enzyme phenylalanine ammonia lyase [EC 4.3.1.5]).     

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。     

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.     

A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia

Hyperphenylalaninemias (HPA) are Mendelian disorders resulting from deficiencies in the conversion of phenylalanine to tyrosine. The vast majority are explained by a primary deficiency of phenylalanine hydroxylase (PAH) activity. The majority of untreated patients experience irreversible impairment of cognitive development. Although it is one of the best known hereditary metabolic disorders, mechanisms underlying the pathophysiology of the disease are still not fully understood; to this end, the availability of an orthologous animal model is relevant. Various mutant hyperphenylalaninemic mouse models with an HPA phenotype, generated by N-ethyl-N'-nitrosourea (ENU) mutagenesis at the Pah locus, have become available. Here we report a new hybrid strain, ENU1/2, with primary enzyme deficiency, produced by cross breeding. The ENU1, ENU1/2, and ENU2 strains display mild, moderate, and severe phenotypes, respectively, relative to the control strain (BTBR/Pas). The Pah enzyme activities of the various models correlate inversely with the corresponding phenylalanine levels in plasma and brain and the delay in plasma clearance response following a phenylalanine challenge. The maternal HPA effect on the fetus correlates directly with the degree of hyperphenylalaninemia, but only the ENU2 strain has impaired learning.     

Positive effect of a simplified diet on blood phenylalanine control in different phenylketonuria variants, characterized by newborn BH4 loading test and PAH analysis

Until today, the mainstay of phenylketonuria (PKU) treatment is a phenylalanine (Phe)-restricted diet. Strict dietary treatment decreases flexibility and autonomy and still has a major impact on patients and their families. Compliance is often poor, particularly in adolescence. The aim of this study was to investigate the effect of the intake of fruits and vegetables containing Phe less than 100 mg/100g ('simplified diet'), as recommended by WHO for all individuals, instead of classical totally restricted diet on the course and treatment control of the disease in a well-characterized PKU cohort (n=80). All individual blood Phe measurements of each patient (1992-2009) were statistically analyzed before and after diet switch. Epidemiological data, age at diagnosis, PAH mutations, BH(4) responsiveness, as well as Phe control measurements and detailed diet information were tabulated in a local database. 62.5% had BH4 loading test and 40% had PAH analysis; 50/80 switched from classical to simplified diet, including 26 classical PKU, 13 moderate PKU, 7 mild PKU and 4 mild hyperphenylalaninemia (HPA). Median Phe levels on a simplified diet did not differ significantly to the median Phe levels on classical diet in all disease groups. Our results indicate that a simplified diet has no negative effect on blood Phe control in patients with hyperphenylalaninemia, independent of severity of the phenotype or the age at diet switch, over the period of 3 years. Thus, a simpler approach to dietary treatment of PKU available to all HPA patients is more likely to be accepted and adhered by patients and might also increase quality of life.     

Molecular Epidemiology of Phenylalanine Hydroxylase Deficiency in Southern Italy: a 96% Detection Rate with Ten Novel Mutations

Hyperphenylalaninemia (HPA) comprises a group of autosomal recessive disorders mainly caused by phenylalanine hydroxylase ( PAH ) gene mutations. We investigated PAH mutations in 126 HPA patients from Southern Italy who were identified in a neonatal screening program. The promoter, coding and exon-flanking intronic sequences of the PAH gene were amplified and sequenced. Mutations were identified in 240/249 alleles (detection rate: 96.4%). We found 60 gene variants; the most frequent were p.R261Q (15.7% of alleles), p.A403V (11.6% of alleles) and c.1066-11G > A (8.8% of alleles). The remaining mutations were rare, and ten are novel. This mutation epidemiology differs from that reported for Northern Italy and other European countries. We also identified several discordant genotype/phenotype correlations. About two-thirds of all mild phenylketonuria patients showed at least one tetrahydrobiopterin (BH₄)-responsive mutation, and are thus candidates for a customized therapeutic approach.     

Excretion of phenol red by the Necturus kidney.

Phenol red (phenolsulfonphthalein, PSP) is thought to be secreted by proximal kidney tubules in all vertebrates. The present study examined PSP transport by the kidney of the salamander, Necturus maculosus. In Necturus kidneys perfused with oxygenated Ringer solution, the PSP/creatinine clearance ratio was unity. Perfusion with 1 mM octanoate converted net p-aminohippurate (PAH) reabsorption to net secretion, but had no effect on PSP. In seven urethan-anesthetized Necturi, the PSP/inulin clearance ratio averaged 0.85 +/- 0.21 (SD), not significantly different from unity. Thin slices from Necturus kidneys incubated in vitro for 2 h failed to accumulate PSP; slice-to-medium (S/M) concentration ratios averaged 0.8 +/- 0.2 (n = 6). With frog kidney slices, (S/M)PSP was 9.6 +/- 1.4 (n = 6). Necturus kidney slices accumulated PAH ((S/M)PAH = 4.1 +/- 0.7) (n = 6), but uptake was not inhibited by 1 mM PSP. We conclude that Necturus kidney tubules transport PAH, but do not transport PSP. These results are consistent with the hypothesis that the organic acid secretory system in most animals involves several carriers.     

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.     

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。     

Genotype and phenotype correlation of phenylalanine hydroxylase deficiency among patients from Henan北大核心CSCD

Objective: To delineate the mutation spectrum of phenylalanine hydroxylase (PAH) gene among patients affected with phenylalanine hydroxylase deficiency (PAHD) in Henan Province of China, and to explore the correlation between the genotype and the phenotype. Methods: A total of 155 affected children were recruited. Potential mutation of the PAH gene were analyzed by direct sequencing. The genotype - phenotype correlation was analyzed by matching the expected and observed phenotypes. Results: Over 72 mutations and 108 genotypes have been identified. There were 7 homozygous mutations, including 1 case with EX6-96A>G/EX6-96A>G, 1 with R241C/R241C, 1 with R413P/R413P, and 4 with R243Q/R243Q. Among these, 6 patients have presented classic PKU phenotypes, except for a R241C/R241C genotype which has led to mild PKU. In 104 patients carrying compound PAH mutations, 52 were classic, 34 were mild and 39 had mild HPA. Patients who were heterozygous for EX6-96A>G/R241C, R243Q/A434D, EX6-96A>G/R413P and EX6-96A>G/ R241C were found with both the classic PKU and mild PKU phenotypes. Common mutations associated with mild HPA have included R53H, R243Q, V399V and H107R. The common mutations associated with mild PKU included R243Q, R241C, EX6-96A>G, and IVS4-1G>A. The prevalent mutations in classic PKU were R243Q, EX6-96A>G and V399V. The consistency between prediction of the biochemical genotype and observed phenotype was 77.78%, especially in classic PKU, the consistency was up to 82.14%. Significant correlations were disclosed between pretreatment levels of phenylalanine and AV sum (r= -0.6729, P<0.01). Conclusion: The mutation spectrum of PAH gene in Henan seems to differ from that of other regions. Independent assortment of mutant alleles may result in a complex genotype-phenotype correlation, but the genotypes of PAHD patients have correlated with the phenotype. © 2016, West China University of Medical Sciences. All rights reserved.     

Two novel genetic lesions and a common BH4-responsive mutation of the PAH gene in Italian patients with hyperphenylalaninemia

Hyperphenylalaninemia (HPA), due to a deficiency of phenylalanine hydroxylase (PAH) enzyme, is caused by mutations in the PAH gene. Molecular analysis in 23 Italian patients with PAH deficiency identified two novel (P281R, L287V) and 20 previously described genetic lesions in the PAH gene. The detection of the A403V amino acid substitution in combination with null mutations in patients with BH4-responsive PAH deficiency leads us to correlate it with BH4 responsiveness.     

DNAJC12 deficiency: A new strategy in the diagnosis of hyperphenylalaninemias

Patients with hyperphenylalaninemia (HPA) are detected through newborn screening for phenylketonuria (PKU). HPA is known to be caused by deficiencies of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH₄). Current guidelines for the differential diagnosis of HPA would, however, miss a recently described DNAJC12 deficiency. The co-chaperone DNAJC12 is, together with the 70 kDa heat shock protein (HSP70), responsible for the proper folding of PAH. All DNAJC12-deficient patients investigated to date responded to a challenge with BH₄ by lowering their blood phenylalanine levels. In addition, the patients presented with low levels of biogenic amine in CSF and responded to supplementation with BH₄, L-dopa/carbidopa and 5-hydroxytryptophan. The phenotypic spectrum ranged from mild autistic features or hyperactivity to severe intellectual disability, dystonia and parkinsonism. Late diagnosis result in permanent neurological disability, while early diagnosed and treated patients develop normally. Molecular diagnostics for DNAJC12 variants are thus mandatory in all patients in which deficiencies of PAH and BH₄ are genetically excluded.     

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co‐chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild‐type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu^1/1 mouse model, homozygous for the V106A‐Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin‐tagged PAH. This form represented a major fraction of PAH in the Enu^1/1 but was also present in liver of wild‐type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin‐dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.     

Pulmonary arterial hypertension in autoimmune diseases: an analysis of 19 cases from a medical center in northern Taiwan.

BACKGROUND AND PURPOSE: Pulmonary arterial hypertension (PAH), a serious complication of autoimmune diseases, has rarely been reported in Taiwan. METHODS: Nineteen patients with various autoimmune diseases diagnosed with PAH at Taipei Veterans General Hospital from 2002 to 2004 were enrolled; the underlying autoimmune diseases included systemic lupus erythematosus (n = 6), primary Sj?gren's syndrome (n = 5), systemic sclerosis (n = 4), adult-onset Still's disease (n = 2), and mixed connective tissue disease (n = 2). The characteristic manifestations of underlying autoimmune diseases and the clinical features of PAH were analyzed. RESULTS: There were 16 female and 3 male patients. The median age at onset of PAH was 44 years and the mean right ventricular systolic pressure (RVSP) was 67.9 mm Hg. Patients without pneumonitis had a significantly higher RVSP value than those with pneumonitis (77.5 +/- 24.3 vs 54.8 +/- 18.4 mm Hg, p=0.041). Four out of 7 patients (57.1%) with RVSP >or=80 mm Hg and 1 out of 12 patients (8.3%) with RVSP <80 mm Hg died. In all of the 19 patients, the severity of RVSP was significantly correlated with serum uric acid (UA) level (r = 0.686, p=0.001). Among the PAH patients without pneumonitis, the severity of RVSP inversely correlated with the diffusion capacity of the lung for carbon monoxide (DLCO) [r = -0.856, p=0.003]. The characteristic manifestations of underlying autoimmune diseases included a high incidence of Raynaud's phenomenon (15/19, 78.9%), a high titer of antinuclear antibody (13/17, 76.5%), positive anti-ribonucleoprotein antibody (8/15, 53.3%), hypergammaglobulinemia (15/19, 78.9%), hyperuricemia (13/19, 68.4%), and less renal involvement. CONCLUSIONS: PAH in autoimmune diseases could be potentially fatal with characteristic manifestations. Moreover, RVSP correlated directly with serum UA level and inversely with DLCO.     

Reduced activity in rats with induced phenylketonuria

Hypoactivity was observed in the open field performance of 28 rats fed a diet containing 3% excess L-phenylalanine and .12% p-chlorophenylalanine between 21–51 days of age (PKU group) compared to both pair-fed and ad lib control groups. Open field testing was conducted 4–11 days following termination of the phenylalanine, p-chlorophenylalanine diet. While on the diet serum phenylalanine levels of the PKU group ranged between 16.6–38.0 mg%. Twenty-four to 48 hours following termination of the PKU diet serum phenylalanine levels had returned to normal (i.e., 2–10 days prior to open field testing). The reduced open field activity of the PKU group is not attributable to differences in food consumption or weight at the time of testing. Hypoactivity is consistent with previous investigations administering chronic doses of phenylalanine, but employing different induction techniques, treatment durations, and activity measures.     

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.