Sapropterin dihydrochloride, 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, in the treatment of phenylketonuria.

Sapropterin dihydrochloride, 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is being introduced in the US for treatment of phenylketonuria (PKU). This compound has been in use in Europe to treat mild forms of PKU. Tetrahydrobiopterin is the cofactor in the hydroxylation reaction of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. It is also involved in other reactions, which are not the focus of this review. The cofactor BH4 is synthesized in many tissues in the body. The pathway of BH4 biosynthesis is complex, and begins with guanosine triphosphate (GTP). The first reaction that commits GTP to form pterins is GTP cyclohydrolase. Several reactions follow resulting in the active cofactor BH4. During the hydroxylation reaction BH4 is oxidized to quinonoid-BH2, which is recycled by dihydropteridine reductase, resulting in the active cofactor. It was discovered that some patients with PKU had a decline in blood phenylalanine after oral intake of BH4. This response to BH4 is not the result of change in the synthesis or regeneration of the cofactor, but rather an effect on the mutant enzyme phenylalanine hydroxylase either by accommodating the higher K(m) of the mutant enzyme or by acting as a chaperone for the mutant enzyme. This response has become of intense interest in the treatment of PKU.

Mutations in the regulatory domain of phenylalanine hydroxylase and response to tetrahydrobiopterin.

Tetrahydrobiopterin (BH4) is a co-factor that enhances the activity of other enzymes, and this co-factor level is found to be affected in phenylketonuria (PKU), an amino acid metabolism disorder. The present study was aimed at understanding the effect of BH4 on mutations in the regulatory domain of phenylalanine hydroxylase (PAH). Among 14 patients, 5 patients were classical PKU, 3 were atypical PKU, and 6 were mild PKU. All of these patients had at least one mutation in the regulatory domain. Patients were given 10 mg/kg BH4, and the response of blood phenylalanine (Phe) levels was monitored following treatment. The level of blood Phe decreased after BH4 treatment in all of the patients. These studies suggest that mutations in the regulatory domain also responded to BH4 even if the patient had classical PKU.

Response of phenylketonuria to tetrahydrobiopterin.

A favorable response, indicated by decline of blood phenylalanine (Phe) in patients with phenylketonuria (PKU), to orally administered 6-R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) has been reported in many countries following the first publication in 1999. In this review, we describe the experience in the United States with PKU patients and their response to BH4. A significant response to BH4 is arbitrarily considered as a decrease of 30% or greater of blood Phe concentration 24 h after administration of BH4. In our studies, 18 of 37 patients with PKU (49%) responded to oral BH4 by >30% decrease in blood Phe concentration. Four PKU patients responded with a decrease of blood Phe concentration between 17.3 and 26.3%. It is suggested that patients with sufficient response to BH4 are candidates who will benefit from BH4 as it becomes available for PKU management. In a separate trial, 20 patients with PKU were screened with ascending doses of BH4: 10, 20, and 40 mg/kg. A favorable response was found in 10 subjects (50%) after 10 mg/kg BH4 and 14 subjects (70%) after 20 mg/kg BH4. There was no additional advantage to 40 mg/kg BH4. A 1-wk trial with 10 and 20 mg/kg BH4 in the same 20 patients showed blood Phe concentrations lowest after 7 d of BH4. The BH4-responsive patients were genotyped and most were compound heterozygotes with 1 mild mutation on 1 allele, responsible for the increase of the residual activity of Phe hydroxylase when BH4 was added. Individuals with the same genotype exhibit different responses upon administration of BH4, attributed to epigenetic factors, such as the metabolic makeup of the individual. Patients with PKU, regardless of their genotype or classification, need to be screened for response to BH4. The majority of patients are identified by 10 mg/kg BH4.

Hyaluronidase increases the biodistribution of acid alpha-1,4 glucosidase in the muscle of Pompe disease mice: an approach to enhance the efficacy of enzyme replacement therapy.

Pompe disease (glycogen storage disease type II) is a glycogen storage disease caused by a deficiency of the lysosomal enzyme, acid maltase/acid alpha-1,4 glucosidase (GAA). Deficiency of the enzyme leads primarily to intra-lysosomal glycogen accumulation, primarily in cardiac and skeletal muscles, due to the inability of converting glycogen into glucose. Enzyme replacement therapy (ERT) has been applied to replace the deficient enzyme and to restore the lost function. However, enhancing the enzyme activity to the muscle following ERT is relatively insufficient. In order to enhance GAA activity into the muscle in Pompe disease, efficacy of hyaluronidase (hyase) was examined in the heart, quadriceps, diaphragm, kidney, and brain of mouse model of Pompe disease. Administration of hyase 3000 U/mouse (intravenous) i.v. or i.p. (intraperitoneal) and 10 min later recombinant human GAA (rhGAA) 20 mg/kg i.v. showed more GAA activity in hyase i.p. injected mice compared to those mice injected with hyase via i.v. Injection of low dose of hyase (3000 U/mouse) or high dose of hyase (10,000 U/mouse) i.p. and 20 min or 60 min later 20 mg/kg rhGAA i.v. increased GAA activity into the heart, diaphragm, kidney, and quadriceps compared to hyase untreated mice. These studies suggest that hyase enhances penetration of enzyme into the tissues including muscle during ERT and therefore hyase pretreatment may be important in treating Pompe disease.

Canavan disease: studies on the knockout mouse.

Canavan disease (CD) is an autosomal recessive disorder, characterized by spongy degeneration of the brain. Patients with CD have aspartoacylase (ASPA) deficiency, which results accumulation of N-acetylaspartic acid (NAA) in the brain and elevated excretion of urinary NAA. Clinically, patients with CD have macrocephaly, mental retardation and hypotonia. A knockout mouse for CD which was engineered, also has ASPA deficiency and elevated NAA. Molecular studies of the mouse brain showed abnormal expression of multiple genes in addition to ASPA deficiency. Adenoassociated virus mediated gene transfer and stem cell therapy in the knockout mouse are the latest attempts to alter pathophysiology in the CD mouse.

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.

Mouse neural progenitor cells differentiate into oligodendrocytes in the brain of a knockout mouse model of Canavan disease.

Canavan disease (CD) is an autosomal recessive disorder that leads to spongy degeneration in the white matter of the brain. Aspartoacylase (ASPA) synthesizing cells, oligodendrocytes, are lost in CD. Transplantation of neural progenitor cells (NPCs) offers an interesting therapeutic approach for treating neurodegenerative diseases by replacing the lost cells. Therefore, the NPCs transplantation to the brain of the CD mouse was studied. Injection of mouse NPCs to the striatum and cerebellum of juvenile CD mouse showed numerous BrdU positive cells at 1 month after injection. The same result was also observed in the adult CD mouse brain after 5 weeks of post-transplantation period. The implanted cells differentiated into oligodendrocytes and fibrous astrocytes, as observed using glial cell marker. This is the first report to describe the survival, distribution and differentiation of NPCs within the brain of CD mouse and a first step toward the potential clinical use of cell therapy to treat CD.

Aspartoacylase deficiency does not affect N-acetylaspartylglutamate level or glutamate carboxypeptidase II activity in the knockout mouse brain.

Aspartoacylase (ASPA)-deficient patients [Canavan disease (CD)] reportedly have increased urinary excretion of N-acetylaspartylglutamate (NAAG), a neuropeptide abundant in the brain. Whether elevated excretion of urinary NAAG is due to ASPA deficiency, resulting in an abnormal level of brain NAAG, is examined using ASPA-deficient mouse brain. The level of NAAG in the knockout mouse brain was similar to that in the wild type. The NAAG hydrolyzing enzyme, glutamate carboxypeptidase II (GCP II), activity was normal in the knockout mouse brain. These data suggest that ASPA deficiency does not affect the NAAG or GCP II level in the knockout mouse brain, if documented also in patients with CD.

High level of orexin A observed in the phenylketonuria mouse brain is due to the abnormal expression of prepro-orexin.

Orexins/hypocretins are recently discovered neuropeptides, synthesized mainly in the lateral hypothalamus of the brain. Orexins regulate various functions including sleep and apetite. We recently reported increased amount of orexin A in the phenylketonuria (PKU) mouse brain. Whether this is caused by overexpression of the precursor for orexins, prepro-orexin was studied in the PKU mouse brain. Microarray expression analysis revealed overexpression of orexin gene in the brain of PKU mouse. Quantitative real-time RT-PCR showed increased level of prepro-orexin mRNA in the PKU mouse brain. In addition, expression of genes associated with cell signal and growth regulation was also affected in the PKU mouse brain, as observed by microarray analysis. These data suggest that up-regulation of orexin mRNA expression is the possible factor for inducing high orexin A in the brain of PKU mouse. The metabolic environment in the brain of PKU mouse affects normal expression of other genes possibly to result in pathophysiology seen in the PKU mouse, if documented also in patients with PKU.

Biopterin responsive phenylalanine hydroxylase deficiency.

PURPOSE: Phenylketonuria (PKU) is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. There have been more than 400 mutations identified in the PAH gene leading to variable degrees of deficiency in PAH activity, and consequently a wide spectrum of clinical severity. A pilot study was undertaken to examine the response to 6-R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) in patients with atypical and classical PKU. METHODS: PAH gene mutation analysis was performed using denaturing gradient gel electrophoresis and gene sequencing. Patients with classical, atypical, or mild PKU were orally given BH4 10 mg/kg. Blood phenylalanine and tyrosine levels were determined using tandem MS/MS at 0 hours, 4 hours, 8 hours, and 24 hours intervals. RESULTS: Thirty-six patients were given a single oral dose of 10 mg/kg of BH4. Twenty one patients (58.33%) responded with a decrease in blood phenylalanine level. Of the patients that responded, 12 were classical, 7 atypical, and 2 mild. The mean decline in blood phenylalanine at 24 hours was > 30% of baseline. There were 15 patients who did not respond to the BH4 challenge, 14 of those had classical and one had atypical PKU. Mapping the mutations that responded to BH4 on the PAH enzyme showed that mutations were in the catalytic, regulatory, oligomerization, and BH4 binding domains. Five patients responding to BH4 had mutations not previously identified. CONCLUSION: The data presented suggest higher than anticipated number of PKU mutations respond to BH4, and such mutations are on all the domains of PAH.

Canavan disease: a monogenic trait with complex genomic interaction.

Canavan disease (CD) is an inherited leukodystrophy, caused by aspartoacylase (ASPA) deficiency, and accumulation of N-acetylaspartic acid (NAA) in the brain. The gene for ASPA has been cloned and more than 40 mutations have been described, with two founder mutations among Ashkenazi Jewish patients. Screening of Ashkenazi Jews for these two common mutations revealed a high carrier frequency, approximately 1/40, so that programs for carrier testing are currently in practice. The enzyme deficiency in CD interferes with the normal hydrolysis of NAA, which results in disruption of myelin and spongy degeneration of the white matter of the brain. The clinical features of the disease are macrocephaly, head lag, progressive severe mental retardation, and hypotonia in early life, which later changes to spasticity. A knockout mouse for CD has been generated, and used to study the pathophysiological basis for CD. Findings from the knockout mouse indicate that this monogenic trait leads to a series of genomic interaction in the brain. Changes include low levels of glutamate and GABA. Microarray expression analysis showed low level of expression of GABA-A receptor (GABRA6) and glutamate transporter (EAAT4). The gene Spi2, a gene involved in apoptosis and cell death, showed high level of expression. Such complexity of gene interaction results in the phenotype, the proteome, with spongy degeneration of the brain and neurological impairment of the mouse, similar to the human counterpart. Aspartoacylase gene transfer trial in the mouse brain using adenoassociated virus (AAV) as a vector are encouraging showing improved myelination and decrease in spongy degeneration in the area of the injection and also beyond that site.

Expression of glutamate transporter, GABRA6, serine proteinase inhibitor 2 and low levels of glutamate and GABA in the brain of knock-out mouse for Canavan disease.

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by spongy degeneration of the brain. The clinical features of CD are hypotonia, megalencephaly, and mental retardation leading to early death. While aspartoacylase (ASPA) activity increases with age in the wild type mouse brain, there is no ASPA activity in the CD mouse brain. So far ASPA deficiency and elevated NAA have been ascribed with the CD. Other factors affecting the brain that result from ASPA deficiency may lead pathophysiology of CD. The NMR spectra and amino acid analysis showed lower levels of glutamate and gamma-aminobutyric acid in the CD mouse brain compared to the wild type. Microarray gene expression on CD mouse brain showed glutamate transporter-EAAT4 and gamma-aminobutyric acid-A receptor, subunit alpha6 (GABRA6) were lower 9.7- and 119.1-fold, respectively. Serine proteinase inhibitor 2 (Spi2) was 29.9-fold higher in the CD mouse brain compared to the wild type. The decrease of GABRA6 and high expression of Spi2 in CD mouse brain were also confirmed by real-time RT-PCR. This first report showing abnormal expression of EAAT4, GABRA6, Spi2 combined with lower levels of glutamate and GABA are likely to be associated with the pathophysiology of CD.

Founder mutation R245H of Sanfilippo syndrome type A in the Cayman Islands.

Sanfilippo A syndrome is an autosomal recessive lysosomal storage disease. This disease was reported in the Cayman Islands population with carrier frequency of 1/7 to 1/10 in the West Bay district of Grand Cayman. The carrier testing of Sanfilippo A disease for families at risk was carried out using the thermal characteristics of sulfamidase activity. In the present study, a search for mutations in the sulfamidase gene in an index family was performed. In addition, 77 individuals, relatives of children with Sanfilippo A syndrome, were also studied by single-strand conformation polymorphism (SSCP), restriction fragment-length polymorphism (RFLP) analyses, and sequencing. A single mutation, G746A (R245H), was found in the family, with the patient being homozygous and both parents and 1 of the 3 siblings being carriers. Among the 77 family members of the patient with Sanfilippo syndrome, the same mutation was found among carriers of the disease. The finding of a single mutation supports the idea of a founder effect, which facilitates accurate carrier identification of Sanfilippo A syndrome in the population of Cayman Islands.

DOOR syndrome: deficiency of E1 component of the 2-oxoglutarate dehydrogenase complex.

Four patients from three families with the clinical features of DOOR syndrome (onycho-osteodystrophy, dystrophic thumbs, sensorineural deafness, and increased urinary levels of 2-oxoglutarate) are the subjects of this report. Our report deals with the autosomal recessive form of the disease, wherein the activity of 2-oxoglutarate decarboxylase (E1(0)) in fibroblasts and white blood cells of the patients is decreased. The activity of E1(0) in all patients' fibroblasts and white blood cells was significantly lower compared to the controls. This study demonstrates for the first time that E1(0) deficiency is an important biochemical marker for the autosomal recessive form of DOOR syndrome.

Nutrient intake and congenital heart defects in maternal phenylketonuria.

OBJECTIVE: The purpose of this study was to determine whether nutritional components other than high maternal blood phenylalanine levels (> or = 10 mg/dL) are associated with congenital heart defects in the offspring of women with hyperphenylalaninemia. STUDY DESIGN: Of the 414 subjects who had live births, 249 women (60.1%) started diet treatment before 8 weeks of gestation and had nutritional assessments and infant outcome data. Maternal phenylalanine levels, protein intake, and the incidence of congenital heart defects were determined. Simple contingency table analysis was done by chi(2) and Fisher exact test. RESULTS: A significantly increased incidence of congenital heart defects was observed in offspring of mothers with hyperphenylalaninemia who had an elevated blood phenylalanine level >10 mg/dL at 0 to 8 weeks of gestation and a protein intake of < or = 50% of the recommended dietary allowance (P <.0013). CONCLUSION: An inadequate intake of protein during pregnancy in conjunction with elevated blood phenylalanine levels appear to have an additive effect in the incidence of congenital heart defects in the offspring of women with hyperphenylalaninemia.