Kurt Ullrich

Individual blood-brain barrier phenylalanine transport determines clinical outcome in phenylketonuria

Different clinical outcomes in spite of comparable dietary controls are well known in patients with phenylketonuria. Currently, reasons for this phenomenon are unknown. Kinetic investigations in 15 patients with classic phenylketonuria were performed using in vivo nuclear magnetic resonance spectroscopy before and after an oral phenylalanine load (100 mg/kg body weight). Patients brain phenylalanine (Phe) concentrations were quite different in spite of similar blood Phe levels. Interindividual variations of the apparent transport Michaelis constant, Kt,app, covered a range from 0.10 to 1.03mmol/L. The ratio of the maximal transport velocity, Tmax, over the intracerebral consumption rate, Vmet, varied between 2.61 and 14.0. Both parameters as well as the preload brain Phe levels correlated significantly with the degree of cerebral white matter abnormalities on magnetic resonance images. Correlations of Kt,app, Tmax/Vmet, and the preload brain Phe levels with patients intelligence scores approached significance. In conclusion, blood–brain barrier Phe transport characteristics and the resultant brain Phe levels seem to be causative factors for the individual clinical outcome in phenylketonuria. This observation may lead to individual dietary recommendations in the future.

Scoring evaluation of the natural course of mucopolysaccharidosis type IIIA (Sanfilippo syndrome type A).

OBJECTIVE. Mucopolysaccharidosis types IIIA through IIID (Sanfilippo syndrome) are caused by deficiencies of enzymes involved in the degradation of heparan sulfate. The onset and severity of the disease are highly variable. The purpose of this study was to describe the natural course of mucopolysaccharidosis type IIIA in a large cohort of patients. PATIENTS AND METHODS. The natural course of mucopolysaccharidosis type IIIA was assessed in 71 patients by using a detailed questionnaire and a 4-point scoring system and compared with the course of the disease in 14 patients with mucopolysaccharidosis type IIIB and 4 patients with mucopolysaccharidosis type IIIC. RESULTS. In the cohort of patients with mucopolysaccharidosis type IIIA, first symptoms of disease were observed, on average, at 7 months of age. Speech and motor development were delayed in 66.2% and 33.9% of patients, respectively. The median age at diagnosis was 4.5 years. The onset of regression in speech, motor, and cognitive function was observed at an average age of 3.3 years. The loss of all 3 of the assessed abilities was observed at an average age of 12.5 years. Speech was lost before motor and cognitive functions. In a small group of patients who were >12.5 years of age (9.9%), speech, motor, and cognitive skills were partially preserved up to a maximum age of 23.8 years. CONCLUSIONS. To our knowledge, this is the first systematic and comprehensive study on the natural course of mucopolysaccharidosis type IIIA. The 4-point scoring system may be used to classify patients into groups with a rapid or slower course of the disease. This may have an important impact on parental counseling as well as therapeutic interventions.

Glutaric Aciduria Type 1 Metabolites Impair the Succinate Transport from Astrocytic to Neuronal Cells

The inherited neurodegenerative disorder glutaric aciduria type 1 (GA1) results from mutations in the gene for the mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH), which leads to elevations of the dicarboxylates glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in brain and blood. The characteristic clinical presentation of GA1 is a sudden onset of dystonia during catabolic situations, resulting from acute striatal injury. The underlying mechanisms are poorly understood, but the high levels of GA and 3OHGA that accumulate during catabolic illnesses are believed to play a primary role. Both GA and 3OHGA are known to be substrates for Na+-coupled dicarboxylate transporters, which are required for the anaplerotic transfer of the tricarboxylic acid cycle (TCA) intermediate succinate between astrocytes and neurons. We hypothesized that GA and 3OHGA inhibit the transfer of succinate from astrocytes to neurons, leading to reduced TCA cycle activity and cellular injury. Here, we show that both GA and 3OHGA inhibit the uptake of [14C]succinate by Na+-coupled dicarboxylate transporters in cultured astrocytic and neuronal cells of wild-type and Gcdh−/− mice. In addition, we demonstrate that the efflux of [14C]succinate from Gcdh−/− astrocytic cells mediated by a not yet identified transporter is strongly reduced. This is the first experimental evidence that GA and 3OHGA interfere with two essential anaplerotic transport processes: astrocytic efflux and neuronal uptake of TCA cycle intermediates, which occur between neurons and astrocytes. These results suggest that elevated levels of GA and 3OHGA may lead to neuronal injury and cell death via disruption of TCA cycle activity.

Plasma antioxidants in pediatric patients with glycogen storage disease, diabetes mellitus, and hypercholesterolemia

Oxidative modification of lipoproteins in vessel walls plays a key role in atherogenesis. Patients with glycogen storage disease type Ia (GSD Ia) do not develop premature atherosclerosis despite severe hyperlipidemia. We analyzed antioxidative defense and oxidative stress in plasma and serum of patients with GSD Ia (n = 17) compared to patients with type I diabetes mellitus (DMI, n = 17), familial hypercholesterolemia (FH, n = 18), and healthy controls (n = 20). We measured the total radical-trapping antioxidant parameter (TRAP), single antioxidants (sulfhydryl groups, uric acid, vitamin C, alpha-tocopherol, coenzyme Q10), malondialdehyde, oxidized low density lipoprotein (LDL) antibodies, lipid profile [cholesterol, triglyceride, lipoprotein (a)], homocysteine, and hemoglobin (Hb)A(1C). TRAP levels were elevated in the GSD Ia group (p <.01) and correlated with elevated uric acid levels (r = 0.72, p =.001). None of the other plasma antioxidants correlated with TRAP levels. DMI patients showed decreased sulfhydryl groups (p <.01) and a reduced ubiquinol-10 fraction (p <.01). Malondialdehyde (p <.001) and oxidized LDL autoantibodies (p <.05) were increased in the diabetic group. In FH patients, parameters of oxidative stress and TRAP did not differ from controls. We conclude that in GSD Ia an increased antioxidative defense in plasma may protect against lipid peroxidation and thus against premature atherosclerosis. Furthermore, we demonstrated that in DMI increased oxidative mechanisms are already present in childhood.

Reduced cerebral fluoro-L-dopamine uptake in adult patients suffering from phenylketonuria

Deficiency of phenylalanine hydroxylase activity in phenylketonuria (PKU) causes an excess of phenylalanine (Phe) throughout the body, predicting impaired synthesis of catecholamines in the brain. To test this hypothesis, we used positron emission tomography (PET) to measure the utilization of 6-[18F]fluoro-l-dopamine (FDOPA) in the brain of adult patients suffering from PKU and in healthy controls. Dynamic 2-h long FDOPA emission recordings were obtained in seven adult PKU patients (five females, two males; age: 21 to 27 years) with elevated serum Phe levels, but lacking neurologic deficits. Seven age-matched, healthy volunteers were imaged under identical conditions. The utilization of FDOPA in striatum was calculated by linear graphical analysis (k3S, min−1), with cerebellum serving as a nonbinding reference region. The time to peak activity in all brain time—radioactivity curves was substantially delayed in the PKU patients relative to the control group. The mean magnitude of k3S in the striatum of the PKU patients (0.0052±0.0004u2009min−1) was significantly lower than in the control group (0.0088±0.0009u2009min−1) (P<0.001). There was no significant correlation between individual serum Phe levels and k3S. The unidirectional clearance of FDOPA to brain was impaired in adult patients suffering from PKU, presumably reflecting the competitive inhibition of the large neutral amino acid carrier by Phe. Assuming this competition to be spatially uniform, the relationship between striatum and cerebellum time—activity curves additionally suggests inhibition of DOPA efflux, possibly also due to competition from Phe. The linear graphical analysis shows reduced k3S in striatum, indicating reduced DOPA decarboxylase activity.

MR spectroscopy of muscle and brain in guanidinoacetate methyltransferase (GAMT)‐deficient mice: Validation of an animal model to study creatine deficiency

As a model for guanidinoacetate methyltransferase (GAMT) deficiency in humans, a gene knockout mouse model was generated. Here we report on several metabolic abnormalities in these mice, observed by in vivo and in vitro MR spectroscopy. In 1H MR spectra of brain and hindleg muscle a clearly reduced signal of creatine (Cr) was observed in GAMT‐deficient (GAMT–/–) animals. Analysis of the 1H MR spectra of GAMT–/– brain indicated little or no increase of a signal for guanidinoacetate (Gua). In proton MR spectra of muscle, a broad signal of low intensity was observed for Gua. However, substantial Gua accumulation in intact muscle tissue was unequivocally confirmed in high‐resolution magic angle spinning spectra, in which the Gua signal was resolved as one clear sharp singlet. In 31P MR analysis of brain and hindleg muscle a strongly reduced phosphocreatine (PCr) content was shown. In addition, a signal of phosphorylated Gua at 0.5 ppm upfield of PCr was observed, with much higher intensity in muscle than in brain. This signal decreased when ischemia was applied to the muscle and recovered after ischemia was released. Overall, the in vivo 31P and 1H MR spectroscopy of GAMT–/– mice is similar to that of human GAMT deficiency. This opens up new avenues for the fundamental study of tissue‐type dependence of creatine synthesis and transport and for diagnostic and therapeutic aspects of creatine deficiencies in humans. Magn Reson Med 50:936–943, 2003.

Missense mutations in N-acetylglucosamine-1-phosphotransferase α/β subunit gene in a patient with mucolipidosis III and a mild clinical phenotype

Mucolipidosis type III (ML III, pseudo‐Hurler polydystrophy), an autosomal recessive inherited disorder of lysosomal enzyme targeting is due to a defective N‐acetylglucosamine 1‐phosphotransferase (phosphotransferase) activity and leads to the impaired formation of mannose 6‐phosphate markers in soluble lysosomal enzymes followed by their increased excretion into the serum. Mutations in the phosphotransferase γ subunit gene (GNPTAG) have been reported to be responsible for ML III. Here we report on a 14‐year‐old adolescent with a mild clinical phenotype of ML III. He presented with progressive joint stiffness and swelling. Urinary oligosaccharide and mucopolysaccharide excretion was normal. Lysosomal enzyme activities were significantly elevated in the serum and decreased in cultured fibroblasts. Impaired trafficking of the lysosomal protease cathepsin D (CtsD) was confirmed by metabolic labeling of the patients fibroblasts. Neither mutations in the GNPTAG gene nor alterations in the GNPTAG mRNA level were detected whereas the steady state concentration of the 97 kDa GNPTAG dimer was reduced. Most importantly, the patient is homozygous for a pathogenic nucleotide substitution and a polymorphism in the phosphotransferase α/β subunit gene (GNPTA). The data indicate that defects in genes other than GNPTAG can be linked to ML III contributing to the variability of the phenotype.

Tetrahydrobiopterin monotherapy for phenylketonuria patients with common mild mutations

The effect of tetrahydrobiopterin (BH(4)) administration was studied in three infants with BH(4) responsive phenylalanine hydroxylase (PAH) deficiency by correlating different BH(4) doses with plasma phenylalanine levels under defined protein intake.

Excitotoxicity and bioenergetics in glutaryl-CoA dehydrogenase deficiency

Summary: Glutaryl-CoA dehydrogenase deficiency is an inherited organic acid disorder with predominantly neurological presentation. The biochemical hallmark of this disease is an accumulation and enhanced urinary excretion of two key organic acids, glutaric acid and 3-hydroxyglutaric acid. If untreated, acute striatal damage is often precipitated by febrile illnesses during a vulnerable period of brain development in infancy or early childhood, resulting in a dystonic dyskinetic movement disorder. 3-Hydroxyglutaric and glutaric acids are structurally similar to glutamate, the main excitatory amino acid of the human brain, and are considered to play an important role in the pathophysiology of this disease. 3-Hydroxyglutaric acid induces excitotoxic cell damage specifically via activation of N-methyl-D-aspartate receptors. It has also been suggested that secondary amplification loops potentiate the neurotoxic properties of these organic acids. Probable mechanisms for this effect include cytokine-stimulated NO production, a decrease in energy metabolism, and reduction of cellular creatine phosphate levels. Finally, maturation-dependent changes in the expression of neuronal glutamate receptors may affect the vulnerability of the immature brain to excitotoxic cell damage in this disease.

Retention of lysosomal protein CLN5 in the endoplasmic reticulum causes neuronal ceroid lipofuscinosis in Asian Sibship

The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. Ten NCL forms can be distinguished by age at onset, clinicopathologic features, and genetics. In eight of these forms, the underlying genes have been identified. At present, approximately 10% of all patients do not fall into one of the eight known genetic forms of NCL. We have identified two Asian families with two novel homozygous mutations in the CLN5 gene. In the first Pakistani family, two children developed symptoms of an early juvenile NCL. After exclusion of mutations in genes known to be associated with this age of onset in families from many different countries (CLN1, CLN2, CLN3, CLN6, CLN8 and CLN10) SNP array‐based homozygosity mapping led to the identification of a novel homozygous mutation c.1072_1073delTT (p.Leu358AlafsX4) in CLN5. In the second Afghan family, two children developed symptoms of a late infantile NCL. The mutation c.1137G>T (p.Trp379Cys) in CLN5 was identified. The affected children in these families represent the first reported CLN5 patients originating in Asian sibships. Expression analysis showed that mutant p.Leu358AlafsX4 CLN5 is truncated and lacks a used N‐glycosylation site at Asn401. The missense mutation p.Trp379Cys affected neither the size nor glycosylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild‐type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lysosome.