Gain-of-function variants in CLCN7 cause hypopigmentation and lysosomal storage disease.

Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.

Airway management and perioperative adverse events in children with mucopolysaccharidoses and mucolipidoses: A retrospective cohort study.

BACKGROUND: Children suffering from mucopolysaccharidoses (subtypes I, II, III, IV, VI, and VII) or mucolipidoses often require anesthesia, but are at high risk for perioperative adverse events. However, the impact of the disease subtype and the standard of care for airway management are still unclear. AIMS: This study aimed to assess independent risk factors for perioperative adverse events in individuals with mucopolysaccharidoses/mucolipidoses and to analyze the interaction with the primary airway technique implemented. METHODS: This retrospective study included individuals with mucopolysaccharidoses/mucolipidoses who underwent anesthesia at two high-volume centers from 2002 to 2016. The data were analyzed in a multivariate hierarchical model, accounting for repeated anesthesia procedures within the same patient and for multiple events within a single anesthesia. RESULTS: Of 141 identified inpatients, 67 (63 mucopolysaccharidoses and 4 mucolipidoses) underwent 269 anesthesia procedures (study cases) for 353 surgical or diagnostic interventions. At least one perioperative adverse event occurred in 25.6% of the cases. The risk for perioperative adverse events was higher in mucopolysaccharidoses type I (OR 8.0 [1.5-42.7]; P = .014) or type II (OR 8.8 [1.3-58.6]; P = .025) than in type III. Fiberoptic intubation through a supraglottic airway was associated with the lowest risk for perioperative adverse events and lowest conversion rate. Direct laryngoscopy was associated with a significantly higher risk for airway management problems than indirect techniques (estimated event rates 47.8% vs 10.1%, OR 24.05 [5.20-111.24]; P < .001). The risk for respiratory adverse events was significantly higher for supraglottic airway (22.6%; OR 31.53 [2.79-355.88]; P = .001) and direct laryngoscopy (14.8%; OR 14.70 [1.32-163.44]; P = .029) than for fiberoptic intubation through a supraglottic airway (2.1%). CONCLUSIONS: The disease subtype and primary airway technique were the most important independent risk factors for perioperative adverse events. Our findings indicate that in MPS/ML children with predicted difficult airway indirect techniques should be favored for the first tracheal intubation attempt.

Hippocampal synaptic connectivity in phenylketonuria.

In humans, lack of phenylalanine hydroxylase (Pah) activity results in phenylketonuria (PKU), which is associated with the development of severe mental retardation after birth. The underlying mechanisms, however, are poorly understood. Mutations of the Pah gene in Pah(enu2)/c57bl6 mice result in elevated levels of phenylalanine in serum similar to those in humans suffering from PKU. In our study, long-term potentiation (LTP) and paired-pulse facilitation, measured at CA3-CA1 Schaffer collateral synapses, were impaired in acute hippocampal slices of Pah(enu2)/c57bl6 mice. In addition, we found reduced expression of presynaptic proteins, such as synaptophysin and the synaptosomal-associated protein 25 (SNAP-25), and enhanced expression of postsynaptic marker proteins, such as synaptopodin and spinophilin. Stereological counting of spine synapses at the ultrastructural level revealed higher synaptic density in the hippocampus, commencing at 3 weeks and persisting up to 12 weeks after birth. Consistent effects were seen in response to phenylalanine treatment in cultures of dissociated hippocampal neurones. Most importantly, in the hippocampus of Pah(enu2)/c57bl6 mice, we found a significant reduction in microglia activity. Reorganization of hippocampal circuitry after birth, namely synaptic pruning, relies on elimination of weak synapses by activated microglia in response to neuronal activity. Hence, our data strongly suggest that reduced microglial activity in response to impaired synaptic transmission affects physiological postnatal remodelling of synapses in the hippocampus and may trigger the development of mental retardation in PKU patients after birth.

Acute renal proximal tubule alterations during induced metabolic crises in a mouse model of glutaric aciduria type 1.

The metabolic disorder glutaric aciduria type 1 (GA1) is caused by deficiency of the mitochondrial glutaryl-CoA dehydrogenase (GCDH), leading to accumulation of the pathologic metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in blood, urine and tissues. Affected patients are prone to metabolic crises developing during catabolic conditions, with an irreversible destruction of striatal neurons and a subsequent dystonic-dyskinetic movement disorder. The pathogenetic mechanisms mediated by GA and 3OHGA have not been fully characterized. Recently, we have shown that GA and 3OHGA are translocated through membranes via sodium-dependent dicarboxylate cotransporter (NaC) 3, and organic anion transporters (OATs) 1 and 4. Here, we show that induced metabolic crises in Gcdh(-/-) mice lead to an altered renal expression pattern of NaC3 and OATs, and the subsequent intracellular GA and 3OHGA accumulation. Furthermore, OAT1 transporters are mislocalized to the apical membrane during metabolic crises accompanied by a pronounced thinning of proximal tubule brush border membranes. Moreover, mitochondrial swelling and increased excretion of low molecular weight proteins indicate functional tubulopathy. As the data clearly demonstrate renal proximal tubule alterations in this GA1 mouse model during induced metabolic crises, we propose careful evaluation of renal function in GA1 patients, particularly during acute crises. Further studies are needed to investigate if these findings can be confirmed in humans, especially in the long-term outcome of affected patients.

Combined D2-/L2-hydroxyglutaric aciduria (SLC25A1 deficiency): clinical course and effects of citrate treatment.

Combined D,L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182) is a rare neurometabolic disorder clinically characterized by muscular hypotonia, severe neurodevelopmental dysfunction, and intractable seizures associated with respiratory distress. Biochemically, DL-2HGA patients excrete increased amounts of D- and L-2-hydroxyglutarate (D2HG and L2HG, respectively), with predominance of D2HG, and α-ketoglutarate, and show a decrease in urinary citrate. Impaired function of the mitochondrial citrate carrier (CIC) due to pathogenic mutations within the SLC25A1 gene has been identified as the underlying molecular cause of the disease. CIC mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Thus, depletion of cytosolic citrate as well as accumulation of citrate inside mitochondria have been considered to play a role in the pathophysiology of DL-2HGA. Here, we report for the first time on a patient with a genetically confirmed diagnosis of DL-2HGA and treatment with either malate or citrate. During malate treatment, urinary malate concentration increased, but beyond that, neither biochemical nor clinical alterations were observed. In contrast, treatment with citrate led to an increased urinary excretion of TCA cycle intermediates malate and succinate, and by trend to an increased concentration of urinary citrate. Furthermore, excretion of D2HG and L2HG was reduced during citrate treatment. Clinically, the patient showed stabilization with regard to frequency and severity of seizures. Treating DL-2HGA with citrate should be considered in other DL-2HGA patients, and its effects should be studied systematically.

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 [(14)C]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 [(14)C]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.

Proteolytic processing of the gamma-subunit is associated with the failure to form GlcNAc-1-phosphotransferase complexes and mannose 6-phosphate residues on lysosomal enzymes in human macrophages.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

Analysis of potential biomarkers and modifier genes affecting the clinical course of CLN3 disease.

Mutations in the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis, a pediatric neurodegenerative disorder characterized by visual loss, epilepsy and psychomotor deterioration. Although most CLN3 patients carry the same 1-kb deletion in the CLN3 gene, their disease phenotype can be variable. The aims of this study were to (i) study the clinical phenotype in CLN3 patients with identical genotype, (ii) identify genes that are dysregulated in CLN3 disease regardless of the clinical course that could be useful as biomarkers, and (iii) find modifier genes that affect the progression rate of the disease. A total of 25 CLN3 patients homozygous for the 1-kb deletion were classified into groups with rapid, average or slow disease progression using an established clinical scoring system. Genome-wide expression profiling was performed in eight CLN3 patients with different disease progression and matched controls. The study showed high phenotype variability in CLN3 patients. Five genes were dysregulated in all CLN3 patients and present candidate biomarkers of the disease. Of those, dual specificity phosphatase 2 (DUSP2) was also validated in acutely CLN3-depleted cell models and in CbCln3(Δex7/8) cerebellar precursor cells. A total of 13 genes were upregulated in patients with rapid disease progression and downregulated in patients with slow disease progression; one gene showed dysregulation in the opposite way. Among these potential modifier genes, guanine nucleotide exchange factor 1 for small GTPases of the Ras family (RAPGEF1) and transcription factor Spi-B (SPIB) were validated in an acutely CLN3-depleted cell model. These findings indicate that differential perturbations of distinct signaling pathways might alter disease progression and provide insight into the molecular alterations underlying neuronal dysfunction in CLN3 disease and neurodegeneration in general.

Residual activity and proteasomal degradation of p.Ser298Pro sulfamidase identified in patients with a mild clinical phenotype of Sanfilippo A syndrome.

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo syndrome) is a fatal inherited lysosomal storage disease accompanied by progressive neurologic degeneration. The gene underlying MPS IIIA, SGSH, encodes a lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (sulfamidase). Mutational analysis of a large cohort of MPS IIIA patients showed a correlation of the missense mutation p.Ser298Pro and a slowly progressive course of the disease. We report here on the expression of the mutant p.Ser298Pro sulfamidase in BHK cells retaining low residual activity. Pulse-chase experiments showed that rapid degradation is responsible for the low steady state level of the mutant protein. Processing and secretion of p.Ser298Pro sulfamidase suggests that small amounts of the newly synthesized enzyme are transported to lysosomes. Most of the mutant sulfamidase exits the endoplasmic reticulum for proteasomal degradation. The ability to predict the clinical course of MPS IIIA in patients with the p.Ser298Pro mutation, as well as the residual enzymatic activity, and the reduced stability of the mutant sulfamidase suggest that this subgroup of patients is especially well suited to early sulfamidase replacement therapy or treatment with selective pharmacological chaperones.

A charitable access program for patients with lysosomal storage disorders in underserved communities worldwide.

BACKGROUND: Lysosomal storage disorders (LSDs) are rare genetic disorders, with heterogeneous clinical manifestations and severity. Treatment options, such as enzyme replacement therapy (ERT), substrate replacement therapy, and pharmacological chaperone therapy, are available for several LSDs, including Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (mucopolysaccharidosis type II [MPS II]). However, patients in some countries face challenges accessing treatments owing to limited availability of locally licensed, approved drugs. METHODS: The Takeda LSD Charitable access program aims to meet the needs of individuals with GD, FD or MPS II with the greatest overall likelihood of benefit, in selected countries, through donation of ERT to nonprofit organizations, and support for medical capacity-building as well as family support via independent grants. Long-term aims of the program are to establish sustainable healthcare services delivered by local healthcare providers for patients with rare metabolic diseases. Patients receiving treatment through the program are monitored regularly, and their clinical data and progress are reviewed annually by an independent medical expert committee (MEC). The MEC also selects patients for enrollment completely independent from the sponsoring company. RESULTS: As of 31 August, 2019, 199 patients from 13 countries were enrolled in the program; 142 with GD, 41 with MPS II, and 16 with FD. Physicians reported improvements in clinical condition for 147 (95%) of 155 patients with follow-up data at 1 year. CONCLUSIONS: The response rate for follow-up data at 1 year was high, with data collected for > 90% of patients who received ERT through the program showing clinical improvements in the majority of patients. These findings suggest that the program can benefit selected patients previously unable to access disease-specific treatments. Further innovative solutions and efforts are needed to address the challenges and unmet needs of patients with LSDs and other rare diseases around the world.

Disease-causing missense mutations affect enzymatic activity, stability and oligomerization of glutaryl-CoA dehydrogenase (GCDH).

Glutaric aciduria type 1 (GA1) is an autosomal recessive neurometabolic disorder caused by mutations in the glutaryl-CoA dehydrogenase gene (GCDH), leading to an accumulation and high excretion of glutaric acid and 3-hydroxyglutaric acid. Considerable variation in severity of the clinical phenotype is observed with no correlation to the genotype. We report here for the first time on expression studies of four missense mutations c.412A > G (p.Arg138Gly), c.787A > G (p.Met263Val), c.1204C > T (p.Arg402Trp) and c.1240G > A (p.Glu414Lys) identified in GA1 patients in mammalian cells. Biochemical analyses revealed that all mutants were enzymatically inactive with the exception of p.Met263Val which showed 10% activity of the expressed wild-type enzyme. Western blot and pulse-chase analyses demonstrated that the amount of expressed p.Arg402Trp protein was significantly reduced compared with cells expressing wild-type protein which was due to rapid intramitochondrial degradation. Upon cross-linkage the formation of homotetrameric GCDH was strongly impaired in p.Met263Val and p.Arg402Trp mutants. In addition, GCDH appears to interact with distinct heterologous polypeptides to form novel 97, 130 and 200 kDa GCDH complexes. Molecular modeling of mutant GCDH suggests that Met263 at the surface of the GCDH protein might be part of the contact interface to interacting proteins. These results indicate that reduced intramitochondrial stability as well as the impaired formation of homo- and heteromeric GCDH complexes can underlie GA1.

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.

Transport and distribution of 3-hydroxyglutaric acid before and during induced encephalopathic crises in a mouse model of glutaric aciduria type 1.

Glutaric aciduria type 1 (GA1) is caused by the deficiency of glutaryl-CoA dehydrogenase (GCDH). Affected patients are prone to the development of encephalopathic crises during an early time window with destruction of striatal neurons and a subsequent irreversible movement disorder. 3-Hydroxyglutaric acid (3OHGA) accumulates in tissues and body fluids of GA1 patients and has been shown to mediate toxic effects on neuronal as well as endothelial cells. Injection of (3H)-labeled into 6 week-old Gcdh(-/-) mice, a model of GA1, revealed a low recovery in kidney, liver, or brain tissue that did not differ from control mice. Significant amounts of 3OHGA were found to be excreted via the intestinal tract. Exposure of Gcdh(-/-) mice to a high protein diet led to an encephalopathic crisis, vacuolization in the brain, and death after 4-5 days. Under these conditions, high amounts of injected 3H-3OHGA were found in kidneys of Gcdh(-/-) mice, whereas the radioactivity recovered in brain and blood was reduced. The data demonstrate that under conditions mimicking encephalopathic crises the blood-brain barrier appears to remain intact.

Growth charts for patients with Sanfilippo syndrome (Mucopolysaccharidosis type III).

BACKGROUND: Mucopolysaccharidosis (MPS) type III (Sanfilippo syndrome) comprises a group of rare, lysosomal storage diseases caused by the deficiency of one of four enzymes involved in the degradation of heparan sulfate. The clinical hallmark of the disease is severe neurological deterioration leading to dementia and death in the second decade of life. Adult MPS patients are generally of short stature. To date there is no clear description of the physical development of MPS III patients. The aim of this study was to document growth reference data for MPS III patients. We collected growth data of 182 German MPS III patients and were able to develop growth charts for this cohort. Growth curves for height, weight, head circumference, and body mass index were calculated and compared to German reference charts. RESULTS: Birth height, weight and head circumference were within the physiological ranges. Both genders were significantly taller than healthy children at 2 years of age, while only male patients were taller at the age of four. Growth velocity decelerated after the ages of 4.5 and 5 years for female and male patients, respectively. Both genders were significantly shorter than the reference group at the age of 17.5 years. Head circumference was larger compared to healthy matched controls within the first 2 years of life and remained enlarged until physical maturity. CONCLUSION: MPS III is a not yet treatable severe neuro-degenerative disease, developing new therapeutic strategies might change the course of the disease significantly. The present charts contribute to the understanding of the natural history of MPS III. Specific growth charts represent an important tool for families and physicians as the expected height at physical maturity can be estimated and therapeutic effects can be monitored.

The mutation p.Ser298Pro in the sulphamidase gene (SGSH) is associated with a slowly progressive clinical phenotype in mucopolysaccharidosis type IIIA (Sanfilippo A syndrome).

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is caused by mutations in the N-sulfoglucosamine sulfohydrolase (SGSH) gene and the resulting defective lysosomal degradation of the glycosaminoglycan heparan sulfate. The onset and progression of the disease are highly variable. Seventy-five mutations distributed over the SGSH gene have been described. We here report on the analysis of the natural course of the disease in 54 MPS IIIA patients through the use of a detailed questionnaire and four-point scoring system and an examination of the underlying mutations. By assessing the degree of developmental regression over time a group of seven patients with a slowly progressive course of the disease were identified. In these seven patients and in 3 other mildly affected patients the missense mutation c.892T>C (p.Ser298Pro) was found on one allele. These patients showed a lower frequency and later onset of the typical symptoms of the disease. The onset of regression in speech abilities and cognitive functions were delayed by 0.7 and 0.8 years, respectively, and the onset of regression of motor functions occurred 6.1 years later than in all other MPS IIIA patients. Severe regression in speech, cognitive and motor functions were delayed by 5, 5.9, and 11.2 years, respectively. These data suggest that in MPS IIIA patients carrying the mutation p.Ser298Pro a slowly progressive phenotype can be predicted and this may have an important impact on parental counselling and therapeutic interventions.

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-DOPA [corrected] (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+/-0.0004 min(-1)) was significantly lower than in the control group (0.0088+/-0.0009 min(-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.

High concentrations of phenylalanine stimulate peroxisome proliferator-activated receptor gamma: implications for the pathophysiology of phenylketonuria.

If left untreated, the common inherited metabolic disorder phenylketonuria (PKU) presents with mental retardation and reduced brain weight. The underlying molecular reasons for these deficits are unknown so far. Using human neuroblastoma cells as a model for normal human neuroblasts, elevated phenylalanine concentrations suppressed proliferation of these cells in culture. Furthermore, microarray and functional assays of these cells revealed that both phenylalanine and the known PPARgamma agonist rosiglitazone regulated the same set of genes causing subsequently similar changes in the functional assays. The lowered brain weight of PKU patients may thus be the result of reduced neuroblast proliferation caused by phenylalanine-induced stimulation of PPARgamma receptors. The observation that high concentrations of small substrates can activate receptors may serve as a new paradigm for other metabolic diseases and provides a new approach for the treatment of these disorders by application of specific receptor antagonists.

Pregnancies in glycogen storage disease type Ia.

OBJECTIVE: Reports on pregnancies in women with glycogen storage disease type Ia (GSD-Ia) are scarce. Because of improved life expectancy, pregnancy is becoming an important issue. We describe 15 pregnancies by focusing on dietary treatment, biochemical parameters, and GSD-Ia complications. STUDY DESIGN: Carbohydrate requirements (milligrams per kilogram per minute), triglyceride and uric acid levels, liver ultrasonography, and creatinine clearance were investigated before, during, and after pregnancy. Data from the newborn infants were obtained from the records. RESULTS: In the first trimester, a significant increase in carbohydrate requirements was observed (P = .007). Most patients had acceptable triglyceride and uric acid levels during pregnancy. No increase in size or number of adenomas was seen. In 3 of 4 patients, a decrease in glomerular filtration rate was observed after pregnancy. In 3 pregnancies, lactic acidosis developed during delivery with severe multiorgan failure in 1. All but 1 of the children are healthy and show good psychomotor development. CONCLUSION: Successful pregnancies are possible in patients with GSD-Ia, although specific GSD-Ia-related risks are present.

3-Hydroxyglutaric acid is transported via the sodium-dependent dicarboxylate transporter NaDC3.

Patients with glutaryl-CoA dehydrogenase (GCDH) deficiency accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3OH-GA) in their blood and urine. To identify the transporter mediating the translocation of 3OH-GA through membranes, kidney tissue of Gcdh-/- mice have been investigated because of its central role in urinary excretion of this metabolite. Using microarray analyses of kidney-expressed genes in Gcdh-/- mice, several differentially expressed genes encoding transporter proteins were identified. Real-time polymerase chain reaction analysis confirmed the upregulation of the sodium-dependent dicarboxylate cotransporter 3 (NaDC3) and the organic cation transporter 2 (OCT2). Expression analysis of NaDC3 in Xenopus laevis oocytes by the two-electrode-voltage-clamp technique demonstrated the sodium-dependent translocation of 3OH-GA with a K (M) value of 0.95 mM. Furthermore, tracer flux measurements in Chinese hamster ovary cells overexpressing OCT2 showed that 3OH-GA inhibited significantly the uptake of methyl-4-phenylpyridinium, whereas 3OH-GA is not transported by OCT2. The data demonstrate for the first time the membrane translocation of 3OH-GA mediated by NaDC3 and the cis-inhibitory effect on OCT2-mediated transport of cations.

Elevated phenylalanine levels interfere with neurite outgrowth stimulated by the neuronal cell adhesion molecule L1 in vitro.

Elevated levels of phenylalanine (Phe) as observed in patients with phenylketonuria interfere with proper neuronal development, leading to severe psychomotor deficits and mental retardation. We have analyzed the effects of Phe on neurite outgrowth in vitro. When expressed in fibroblasts, the neuronal cell adhesion molecules L1 and plexin B3 strongly increase the length of neurites emanating from cerebellar neurons in co-culture experiments. Elevated Phe blocks L1-mediated, but not plexin B3-mediated outgrowth, whereas tyrosine is ineffective. Elevated Phe also interferes with aggregation of fibroblasts overexpressing L1, suggesting that the pathological effect of elevated Phe occurs by interfering with L1-mediated cell adhesion.