No abstract

Mutations in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been associated with a spectrum of epilepsies and neurodevelopmental disorders. Here, we report the phenotypes of 71 patients and review 130 previously reported patients. We found that (i) encephalopathies with infantile/childhood onset epilepsies (≥3 months of age) occur almost as often as those with an early infantile onset (<3 months), and are thus more frequent than previously reported; (ii) distinct phenotypes can be seen within the late onset group, including myoclonic-atonic epilepsy (two patients), Lennox-Gastaut not emerging from West syndrome (two patients), and focal epilepsies with an electrical status epilepticus during slow sleep-like EEG pattern (six patients); and (iii) West syndrome constitutes a common phenotype with a major recurring mutation (p.Arg853Gln: two new and four previously reported children). Other known phenotypes include Ohtahara syndrome, epilepsy of infancy with migrating focal seizures, and intellectual disability or autism without epilepsy. To assess the response to antiepileptic therapy, we retrospectively reviewed the treatment regimen and the course of the epilepsy in 66 patients for which well-documented medical information was available. We find that the use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening. Regarding the genetic findings, truncating mutations were exclusively seen in patients with late onset epilepsies and lack of response to sodium channel blockers. Functional characterization of four selected missense mutations using whole cell patch-clamping in tsA201 cells-together with data from the literature-suggest that mutations associated with early infantile epilepsy result in increased sodium channel activity with gain-of-function, characterized by slowing of fast inactivation, acceleration of its recovery or increased persistent sodium current. Further, a good response to sodium channel blockers clinically was found to be associated with a relatively small gain-of-function. In contrast, mutations in patients with late-onset forms and an insufficient response to sodium channel blockers were associated with loss-of-function effects, including a depolarizing shift of voltage-dependent activation or a hyperpolarizing shift of channel availability (steady-state inactivation). Our clinical and experimental data suggest a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy.

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We treated 93 children with nephropathic cystinosis with oral cysteamine (mean dose, 51.3 mg per kilogram of body weight per day) for up to 73 months. This agent is known to be effective in depleting cells of cystine. In our study, the mean cystine depletion from leukocytes was 82 percent. A historical control group of 55 children received either ascorbic acid (27 children) or placebo (28). At age six, 2 of 17 controls had a serum creatinine level less than 1.0 mg per deciliter, as compared with 17 of 27 patients treated with cysteamine for at least one year (odds ratio, 12.8; 95 percent confidence interval, 2.1 to 33.9). At the end of the study, creatinine clearance was higher in the cysteamine group than in the control group (38.5 vs. 29.7 ml per minute per 1.73 m2; 95 percent confidence limits on the difference, 1.8 and 15.8), even though the cysteamine group was on average 1.4 years older than the control group. Cysteamine also improved growth; those in the cysteamine group between two and three years of age grew at 93 percent of the normal velocity, as compared with 54 percent in the control group. Fourteen percent of the patients could not tolerate the taste and smell of cysteamine. Concurrent controls treated in a blinded fashion with a placebo were not included in this study. With this limitation in mind, we conclude that oral cysteamine, by depleting cells of cystine, helps maintain renal glomerular function, improves growth, and constitutes the current treatment of choice for nephropathic cystinosis.

Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb "European" deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G-->A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.

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Abstract. Lysosomes constitute only 4% of the intracellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 t~M [asS]cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactivity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A lysosomal transport system, highly specific for cysteine, appears to facilitate this rapid lysosomal cysteine sequestration. Time courses of [35S]cysteine uptake into isolated, Percoll-purified fibroblast lysosomes at pH 7.0 and 37°C are linear for the first 4-5 min and attain a steady state by 10 min. Lysosomal cysteine uptake displays a Km of 0.05 mM at pH 7.0 and an activation energy of 21 kcal/mol, corresponding to a Qt0 of 3.2. The role of this transport system in delivering cysteine into lysosomes is supported by its pH curve showing a slow rate of cysteine transport at the acidic pHs between 5 and 6, but then increasing sevenfold between pH 6 and 7.5 to be maximally active near the cytosolic pH of 7. Carrier mediation by this lysosomal transport route demonstrates a high specificity for cysteine as indicated by the inability of the following amino acids to significantly inhibit at 5 mM the lysosomal uptake of 0.035 mM [3sS]e-cysteine: ala, ser, pro, val, gly, homocysteine, D-or e-penicillamine, arg, asp, or leu. Similarly, D-cysteine and 3-mercaptopropionate were poor inhibitors, suggesting that both the L-isomer and a-amino group of cysteine appear to be required for recognition by the cysteine-specific transport system. In contrast, cysteamine, which lacks an a-carboxyl group, was able to strongly inhibit lysosomal cysteine uptake. The physiological importance of this cysteinespecific lysosomal transport system may be to aid lysosomal proteolysis by delivering cysteine into the lysosomal compartment to (a) maintain the catalytic activity of the thiol-dependent lysosomal enzymes and (b) break protein disulfide bridges at susceptible linkages, thereby allowing proteins to unfold, facilitating their degradation.VSOSOMES are a major intracellular site for the degradation of a wide variety of macromolecules which are delivered to this organelle by receptor-mediated endocytosis, pinocytosis, or autophagy. Many of the metabolites formed in the lysosome as a consequence of the breakdown of macromolecules appear to egress from the lysosomal compartment by way of carrier-mediated transport systems. Specific lysosomal transport systems for cystine (13)(14)(15)19), the cationic amino acids (29, 31), small neutral amino acids (30), large neutral amino acids (6, 47), nucleosides (28), acidic monosaccharides (24, 32), and the sugars N-acetyl galactosamine and N-acetyl glucosamine (20) have been described. The importance of transport systems in mediating the escape of metabolites from the lysosomal compartment becomes especially obvious in the lethal genetic disease, nephropathic cystinosis. In this disorder, defective A preliminary report of this work has appe...

Abstract. Nephropathic cystinosis is a lethal disorder of lysosomal cystine storage due to defective lysosomal cystine transport. How lysosomal cystine causes this multisystemic disorder culminating in end-stage renal disease is not known, because the cystine is isolated from cellular metabolism by the lysosomal membrane. It is here reported that in both normal and nephropathic cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, increased lysosomal cystine causes an increased rate of apoptosis. In nephropathic cystinotic fibroblasts, the rate of apoptosis is 14.8% after exposure to TNF-␣ versus 7.8% in control normal fibroblasts. Anti-Fas antibodies and UV exposure induced apoptosis in 18.1% and 17.4% of nephropathic cystinotic fibroblasts, respectively, versus 5.2% and 7.1% in normal fibroblasts when analyzed by CaspACE (P Ͻ 0.05). Similar results were found when the cells were analyzed by TdT-mediated dUTP nick end labeling (TUNEL). When the cystine content of normal fibroblasts is increased by exposure to cystine dimethylester (CDME), the apoptotic rate is increased to the rate seen in nephropathic cystinotic cells.Decreasing the cystinotic cells' cystine content by use of cysteamine results in normalization of the apoptotic rate. Renal proximal tubule epithelial (RPTE) cells are much more sensitive to CDME than fibroblasts, reaching 43.8% apoptosis 6 h after exposure to CDME alone, compared with 38.2% when exposed to TNF-␣ alone. Serum withdrawal causes an apoptotic rate of 8.7% in nephropathic cystinotic fibroblasts, compared with 6.1% in normal fibroblasts. That rate increases to 37.3% in normal fibroblasts after CDME exposure.