Cysteamine revisited: repair of arginine to cysteine mutations.

Cysteamine is a small aminothiol endogenously derived from coenzyme A degradation. For some decades, synthetic cysteamine has been employed for the treatment of cystinosis, and new uses of the drug continue to emerge. In this review, we discuss the role of cysteamine in cellular and extracellular homeostasis and focus on the potential use of aminothiols to reconstitute the function of proteins harboring arginine (Arg) to cysteine (Cys) mutations, via repair of the Cys residue into a moiety that introduces an amino group, as seen in basic amino acid residues Lys and Arg. Cysteamine has been utilized in vitro and ex vivo in four different genetic disorders, and thus provides "proof of principle" that aminothiols can modify Cys residues. Other aminothiols such as mercaptoethylguanidine (MEG) with closer structural resemblance to the guanidinium moiety of Arg are under examination for their predicted enhanced capacity to reconstitute loss of function. Although the use of aminothiols holds clinical potential, more studies are required to refine specificity and treatment design. The efficacy of aminothiols to target proteins may vary substantially depending on their specific extracellular and intracellular locations. Redox potential, pH, and specific aminothiol abundance in each physiological compartment are expected to influence the reactivity and turnover of cysteamine and analogous drugs. Upcoming research will require the use of suitable cell and animal models featuring Arg to Cys mutations. Since, in general, Arg to Cys changes comprise about 8% of missense mutations, repair of this specific mutation may provide promising avenues for many genetic diseases.

Folinic acid treatment for schizophrenia associated with folate receptor autoantibodies.

BACKGROUND: Auto-antibodies against folate receptor alpha (FRα) at the choroid plexus that block N(5)-methyltetrahydrofolate (MTHF) transfer to the brain were identified in catatonic schizophrenia. Acoustic hallucinations disappeared following folinic acid treatment. Folate transport to the CNS prevents homocysteine accumulation and delivers one-carbon units for methyl-transfer reactions and synthesis of purines. The guanosine derivative tetrahydrobiopterin acts as common co-factor for the enzymes producing dopamine, serotonin and nitric oxide. METHODS: Our study selected patients with schizophrenia unresponsive to conventional treatment. Serum from these patients with normal plasma homocysteine, folate and vitamin B12 was tested for FR autoantibodies of the blocking type on serial samples each week. Spinal fluid was analyzed for MTHF and the metabolites of pterins, dopamine and serotonin. The clinical response to folinic acid treatment was evaluated. RESULTS: Fifteen of 18 patients (83.3%) had positive serum FR auto-antibodies compared to only 1 in 30 controls (3.3%) (χ(2)=21.6; p<0.0001). FRα antibody titers in patients fluctuated over time varying between negative and high titers, modulating folate flux to the CNS, which explained low CSF folate values in 6 and normal values in 7 patients. The mean±SD for CSF MTHF was diminished compared to previously established controls (t-test: 3.90; p=0.0002). A positive linear correlation existed between CSF MTHF and biopterin levels. CSF dopamine and serotonin metabolites were low or in the lower normal range. Administration of folinic acid (0.3-1mg/kg/day) to 7 participating patients during at least six months resulted in clinical improvement. CONCLUSION: Assessment of FR auto-antibodies in serum is recommended for schizophrenic patients. Clinical negative or positive symptoms are speculated to be influenced by the level and evolution of FRα antibody titers which determine folate flux to the brain with up- or down-regulation of brain folate intermediates linked to metabolic processes affecting homocysteine levels, synthesis of tetrahydrobiopterin and neurotransmitters. Folinic acid intervention appears to stabilize the disease process.

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.

Sepiapterin reductase deficiency in a 2-year-old girl with incomplete response to treatment during short-term follow-up.

Sepiapterin reductase (SR) catalyses the last step in the tetrahydrobiopterin biosynthesis pathway; it converts 6-pyruvoyl-tetrahydropterin (6-PTP) to BH(4) in an NADPH-dependent reaction. SR deficiency is a very rare autosomal recessive disorder with normal phenylalanine (Phe) concentration in blood and diagnostic abnormalities are detected in CSF. We present a 16-month-old girl with SR deficiency. From the newborn period she presented with an adaptation regulatory disorder. At the age of 3 months, abnormal eye movements with dystonic signs and at 4.5 months psychomotor retardation were noticed. Since that time axial hypotonia with limb spasticity (or rather delayed reflex development), gastro-oesophageal reflux and fatigue at the end of the day has been observed. Brain MRI was normal; EEG was without epileptiform discharges. Analysis of biogenic amine metabolites in CSF at the age of 16 months showed very low HVA and 5-HIAA concentrations. Analysis of CSF pterins revealed strongly elevated dihydrobiopterin (BH(2)), slightly elevated neopterin and elevated sepiapterin levels. Plasma and CSF amino acids concentrations were normal. A phenylalanine loading test showed increased Phe after 1 h, 2 h and 4 h and very high Phe/Tyr ratios. SR deficiency was confirmed in fibroblasts and a novel homozygous g.1330C>G (p.N127K) SPR mutation was identified. On L-dopa and then additionally 5-hydroxytryptophan, the girl showed slow but remarkable progress in motor and intellectual ability. Now, at the age of 3 years, she is able to sit; expressive speech is delayed (to 1 1/2 years), passive speech is well developed. Her visual-motor skills, eye-hand coordination and social development correspond to the age of 2 1/2 years.

Pharmacokinetics of orally administered tetrahydrobiopterin in patients with phenylalanine hydroxylase deficiency.

The oral loading test with tetrahydrobiopterin (BH(4)) is used to discriminate between variants of hyperphenylalaninaemia and to detect BH(4)-responsive patients. The outcome of the loading test depends on the genotype, dosage of BH(4), and BH(4) pharmacokinetics. A total of 71 patients with hyperphenylalaninaemia (mild to classic) were challenged with BH(4) (20 mg/kg) according to different protocols (1 x 20 mg or 2 x 20 mg) and blood BH(4) concentrations were measured in dried blood spots at different time points (T(0), T(2), T(4), T(8), T(12), T(24), T(32) and T(48 h)). Maximal BH(4) concentrations (median 22.69 nmol/g Hb) were measured 4 h after BH(4) administration in 63 out of 71 patients. Eight patients presented with maximal BH(4) concentrations approximately 44% higher at 8 h than at 4 h. After 24 h, BH(4) blood concentrations dropped to 11% of maximal values. This profile was similar using different protocols. The following pharmacokinetic parameters were calculated for BH(4) in blood: t (max) = 4 h, AUC (T(0-32)) = 370 nmol x h/g Hb, and t (1/2) for absorption (1.1 h), distribution (2.5 h), and elimination (46.0 h) phases. Maximal BH(4) blood concentrations were not significantly lower in non-responders and there was no correlation between blood concentrations and responsiveness. Of mild PKU patients, 97% responded to BH(4) administration, while one was found to be a non-responder. Only 10/19 patients (53%) with Phe concentrations of 600-1200 mumol/L responded to BH(4) administration, and of the patients with the severe classical phenotype (blood Phe > 1200 mumol/L) only 4 out of 17 patient responded. An additional 36 patients with mild hyperphenylalaninaemia (HPA) who underwent the combined loading test with Phe+BH(4) were all responders. Slow responders and non-responders were found in all groups of HPA.

The -24/-12 promoter comes of age.

A new bacterial promoter type has been identified in the last few years. Originally designated as nif (= nitrogen fixation) or ntr (= nitrogen regulation) consensus promoter, it is now evident that this promoter occurs in many different bacterial species and is used not only for genes involved in nitrogen assimilation but also for genes determining many other unrelated metabolic functions. The general features of this type of promoter are (i) the conserved -24(GG)/-12(GC) consensus sequence, (ii) its recognition by a specific RNA polymerase sigma factor, sigma 54, which is encoded by the ntrA gene (synonyms: glnF, rpoN, rpoE), and (iii) the requirement for a transcriptional regulatory protein to activate the expression of the associated genes. In addition, many (but not all) of these genes possess a promoter-upstream activator sequence (enhancer) which is the target site for the binding of the activating protein and is required for maximal expression. In some cases, in which gene expression does not appear to be dependent on the presence of upstream binding sites, the activating protein may interact directly with the RNA polymerase-promoter complex. In conclusion, the expression from all -24/-12 consensus promoters known to date is positively controlled.

6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding.

6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases. The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers. A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit. We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity. Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity. The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction. We altered these residues and found a complete loss of enzymatic activity for the mutant C42A. The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar KM values for the substrate as compared to wild-type PTPS. Based on these results we propose a model of the substrate fitted into the active site and we described a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88. Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons. Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server.

Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase.

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.

Retrovirus-mediated gene transfer of 6-pyruvoyl-tetrahydropterin synthase corrects tetrahydrobiopterin deficiency in fibroblasts from hyperphenylalaninemic patients.

Tetrahydrobiopterin (BH4) deficiency, a variant form of hyperphenylalaninemia with progressive neurological dysfunction, is primarily caused by autosomal recessive mutations in the gene encoding the 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS is a biosynthetic enzyme for the BH4 co-factor, and its deficiency is associated with a malfunction of the phenylalanine catabolism in the liver and a lack of biogenic amine neurotransmitters dopamine and serotonin in the brain. We have previously isolated the wild-type PTPS cDNA and identified several mutations responsible for a decreased enzyme in patients. This study reports the in vitro correction of BH4 deficiency by using retrovirus mediated transfer of the PTPS cDNA into primary fibroblast cultures established from different patients. The Bing packaging cell line was used for amphotropic virus production. Following PTPS gene transfer, stimulation with cytokines restored biosynthesis of BH4 in originally defective cells to values comparable to those of heterozygous fibroblasts from clinically healthy subjects. These results not only provide a direct proof that the mutations in PTPS were causative for the mutant phenotype, but they are also the first step toward gene therapy as a potential alternative approach to treat BH4 deficiency.

A new Bradyrhizobium japonicum gene required for free-living growth and bacteroid development is conserved in other bacteria and in plants.

In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, a new DNA region, orf74, was discovered which is required for optimal free-living growth and, by consequence, also necessary for the formation of an effective symbiosis. A Tn5-233 insertion of orf14 resulted in a mutant, strain 74, that has a reduced growth rate in free-living cultures under all conditions tested and less than 1% residual symbiotic nitrogen fixation activity as compared with the wild type. Nodule distribution and nodule morphology are severely affected similarly as in a previously characterized B. japonicum nifA mutant. Protein databank searches revealed that the 142-amino-acid protein encoded by orf74 is homologous to a 161-amino-acid protein encoded by orf17 of Bacillus subtilis (approximately 46% identity; J. C. R. Struck, R. Kretschmer-Kazemi Far, W. Schröder, F. Hucho, H. Y. Toschka, and V. A. Erdmann, Biochim. Biophys. Acta, 1050:80-83, 1990) as well as to a 178-amino-acid protein encoded by orf178 of Escherichia coli (approximately 45% identity; K. L. Poulsen, N. W. Larsen, S. Molin, and P. Andersson, Mol. Microbiol., 6:895-905, 1992). Significant similarity was also found with unknown ORFs of two plant species. When expressed from a strong constitutive promoter, orf17 of B. subtilis could partially complement B. japonicum mutant 74. By contrast, orf74 of B. japonicum was unable to functionally complement an E. coli orf178 mutant. The conservation of this DNA region in gram-negative and gram-positive bacteria suggests that the gene is essential for a fundamental cellular process which is required in B. japonicum for both free-living and symbiotic growth.

Epidermal H(2)O(2) accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: identification of a general mechanism in regulation of all 6BH4-dependent processes?

It has been shown in vivo that patients with the depigmentation disorder vitiligo accumulate hydrogen peroxide (H(2)O(2)) accompanied by low catalase levels and high concentrations of 6- and 7-biopterin in their epidermis. Earlier it was demonstrated that epidermal 4a-OH-tetrahydrobiopterin dehydratase, an important enzyme in the recycling process of 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH(4)), has extremely low activities in these patients concomitant with a build-up of the abiogenic 7-isomer (7BH(4)), leading to competitive inhibition of epidermal phenylalanine hydroxylase. A topical substitution for the impaired epidermal catalase with a pseudocatalase effectively removes epidermal H(2)O(2), yielding a recovery of epidermal 4a-OH-tetrahydrobiopterin dehydratase activities and physiologic 7BH(4) levels in association with successful repigmentation demonstrating recovery of the 6BH(4) recycling process. Examination of recombinant enzyme activities, together with 4a-OH-tetrahydrobiopterin dehydratase expression in the epidermis of untreated patients, identifies H(2)O(2)-induced inactivation of this enzyme. These results are in agreement with analysis of genomic DNA from these patients yielding only wild-type sequences for 4a-OH-tetrahydrobiopterin dehydratase and therefore ruling out the previously suspected involvement of this gene. Furthermore, our data show for the first time direct H(2)O(2) inactivation of the important 6BH(4) recycling process. Based on this observation, we suggest that H(2)O(2) derived from various sources could be a general mechanism in the regulation of all 6BH(4)-dependent processes.

E. coli minichromosome replication: regulation of initiation at oriC.

The initiation of Escherichia coli DNA replication is a highly regulated event with many parameters exerting positive and negative effects. The activity of the dnaA protein (the initiator protein) is profoundly influenced by the tight binding of the adenine nucleotides ATP and ADP. Further regulation of dnaA protein activity may occur through dnaA protein-cell membrane associations. A replicatively inactive form of dnaA protein is found aggregated with phospholipids; enzymatic treatment of the aggregates with phospholipase A2 or dnaK protein liberates dnaA protein with restored replication activity. Proper DNA structure is essential for replication. The energy stored in the DNA's supercoiling is crucial for dnaA protein's ability to initiate replication. Under conditions where strand-opening by dnaA protein is inhibited, such as low free superhelicity, an R-loop formed by RNA polymerase activates the origin at a distance by aiding strand-opening. A novel protein has been identified as a specific inhibitor of the initiation of DNA replication. This 33-kDa protein binds to the AT rich region of oriC and inhibits strand-opening by dnaA protein.

Pterin-4alpha-carbinolamine dehydratase in rat brain. I. Patterns of co-localization with tyrosine hydroxylase.

The bifunctional protein, PCD/DCoH, is both a pterin-4alpha-carbinolamine dehydratase (PCD) and a dimerization cofactor of the hepatic nuclear factor 1alpha (DCoH). In association with brain tyrosine hydroxylase (TH), which is required for dopamine synthesis, PCD catalyses dehydration and thus recycling of the cofactor tetrahydrobiopterin (BH(4)). PCD immunoreactivity in the catecholaminergic system of the rat brain was studied using a rabbit polyclonal antibody. Double immunofluorescence was performed to establish intracellular co-localization with TH. PCD immunoreactivity was found to be high and consistently present in all the neuron groups expressing TH. More than 90% of the TH+ cells were also expressing PCD. The highest co-expression (99-100% of TH+ cells) was observed in pontine catecholaminergic cell groups including locus coeruleus. Lower co-expression was observed in substantia nigra (17% of TH+ cells without PCD) and particularly in arcuate nucleus (41% of TH+ cells without PCD). Our results argue in favor of a generalized recycling of BH(4) in catecholaminergic neurons except when the neuron terminal field is located outside the blood-brain barrier. The respective roles of synthesis and recycling of BH(4) in the control of TH activity are discussed.

Tetrahydrobiopterin and inherited hyperphenylalaninemias.

Tetrahydrobiopterin deficiency, a variant of hyperphenylalaninemia, may be caused by deficiency of one of the following enzymes: guanosine triphosphate cyclohydrolase 1,6-pyruvoyltetrahydropterin synthase, dihydropteridin reductase and pterin-4a-carbinolamine dehydratase. The first two enzymes are involved in the biosynthesis of tetrahydrobiopterin, the last two in its regeneration. Although these diseases are rare, early detection by selective screening is essential for the treatment and outcome. Tetrahydrobiopterin deficiencies are very heterogenous ranging from mild forms requiring only marginal if any treatment to severe forms which are in some cases very difficult to treat. All variants of tetrahydrobiopterin deficiency can be differentiated from the classical phenylketonuria (PKU) by measurement of pterin metabolites in patients' urine, tetrahydrobiopterin loading test, and by dihydropteridine reductase activity in erythrocytes from the Guthrie card.

Guanosine triphosphate cyclohydrolase I deficiency: a rare cause of hyperphenylalaninemia.

Tetrahydrobiopterin (BH4) deficiencies are a heterogeneous group of disorders caused by a defect in two of the three enzymes involved in its biosynthesis or in the two recycling enzymes. Except for the deficiency of dehydratase, an enzyme catalyzing a reaction in the recycling pathway, all other variants of BH4 deficiency are characterized by developmental delay, progressive neurological deterioration, hypokinesis, drooling, swallowing difficulty, truncal hypotonia, increased limb tone, myoclonus and brisk deep tendon reflexes. A deficiency of guanosine triphosphate cyclohydrolase I (GTPCH), the first enzyme in the biosynthetic pathway of BH4, is described in a 14-month-old male infant with hyperphenylalaninemia, developmental delay, hypertonia of the extremities, seizures, feeding difficulties, and vomiting. Urinary pteridine screening revealed very low levels of neopterin and biopterin which was highly suggestive of GTPCH deficiency. Low cerebrospinal fluid concentrations of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid concentrations, together with no detectable neopterin and decreased concentrations of biopterin and folate, agreed with the diagnosis of GTPCH deficiency. Subsequently measured neopterin and biopterin synthesis in cytokine-stimulated skin fibroblasts confirmed GTPCH deficiency, albeit indirectly. The patient showed marked improvement on a low-protein low-phenylalanine diet with neurotransmitter precursor administration. The favorable outcome in this patient clearly shows that not only newborns with elevated phenylalanine levels but also older children with neurological signs and symptoms should be screened for a BH4 deficiency in order to have maximum benefit of the treatment.

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer.

Phenylketonuria (PKU) is an inborn error of metabolism caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) which leads to high blood phenylalanine (Phe) levels and consequent damage of the developing brain with severe mental retardation if left untreated in early infancy. The current dietary Phe restriction treatment has certain clinical limitations. To explore a long-term nondietary restriction treatment, a somatic gene transfer approach in a PKU mouse model (C57Bl/6-Pahenu2) was employed to examine its preclinical feasibility. A recombinant adeno-associated virus (rAAV) vector containing the murine Pah-cDNA was generated, pseudotyped with capsids from AAV serotype 8, and delivered into the liver of PKU mice via single intraportal or tail vein injections. The blood Phe concentrations decreased to normal levels (< or =100 microM or 1.7 mg/dl) 2 weeks after vector application, independent of the sex of the PKU animals and the route of application. In particular, the therapeutic long-term correction in females was also dramatic, which had previously been shown to be difficult to achieve. Therapeutic ranges of Phe were accompanied by the phenotypic reversion from brown to black hair. In treated mice, PAH enzyme activity in whole liver extracts reversed to normal and neither hepatic toxicity nor immunogenicity was observed. In contrast, a lentiviral vector expressing the murine Pah-cDNA, delivered via intraportal vein injection into PKU mice, did not result in therapeutic levels of blood Phe. This study demonstrates the complete correction of hyperphenylalaninemia in both males and females with a rAAV serotype 8 vector. More importantly, the feasibility of a single intravenous injection may pave the way to develop a clinical gene therapy procedure for PKU patients.

Mutations in the GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase genes.

Tetrahydrobiopterin deficiencies are highly heterogeneous disorders, with more than 30 molecular lesions identified in the past 2 years in the GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase genes. The spectrum of mutations causing a reduction of these two biosynthetic enzymes is reviewed. Only three mutations, two present homozygously, are reported in the GTP cyclohydrolase I gene to cause the rare autosomal recessively inherited form of hyperphenylalaninemia. Most of the other mutations, which are scattered over the entire coding region for the six exon-containing GTP cyclohydrolase I gene, are observed in a heterozygous state with the wild-type allele and are associated with the dominant DOPA-responsive dystonia. Compound heterozygous or homozygous mutations spread over all six exons encoding the 6-pyruvoyl-tetrahydropterin synthase cause an autosomal recessively inherited variant of hyperphenylalaninemia, mostly accompanied by a deficiency of dopamine and serotonin.