Cysteamine (Cystagon®) adherence in patients with cystinosis in Spain: successful in children and a challenge in adolescents and adults.

BACKGROUND: Cysteamine has improved survival and prognosis in cystinosis. Increasing numbers of patients reach adulthood and face new challenges such as compliance that wanes over time. The aim of this study was to evaluate adherence to cysteamine treatment in a group of cystinotic patients in Spain in an attempt to identify potential therapy pitfalls and improve the overall care of affected individuals. Despite the impact of cysteamine on prognosis, there is a paucity of data regarding adherence. METHOD: Thirty-four cystinotic patients (21 male) 38% ≥18 years were enrolled in a voluntary, anonymous survey. Replies were obtained from patients (15/34), mothers (11/34), fathers (4/34) and both parents (4/34). RESULTS: Patient age (median and interquartile range) at diagnosis was 1 year (0.57-1), and patient age at Cystagon® initiation was also 1 year (0.8-1.8). Sixteen (47%) were kidney transplant (KTx) recipients; six were retransplanted. Age at first KTx 10 years (8.7-13.7). Patient understanding of multiorgan involvement in cystinosis: 4.1 organs reported; eye 97% and kidney 91%. Cysteamine was given by mother (100%) and father (83%) in <11 year olds, or self-administered (94%) in ≥11 year olds. Four daily doses in 89% versus 56% in <11 year olds or ≥11 year olds, with fixed schedule in 94% versus 50% in <11 or ≥11 year olds and progressive loss of reminders over time. Furthermore, 44% complained of unpleasant smell. Motivation for treatment compliance was 100% versus 40% in <11 versus ≥11 year olds, respectively. Disease impact in patients <18 years is as follows: school (29%), social (14%), 'feeling different' (10%); in patients ≥18 years: 'feeling different' (62%), professional (39%) and job absenteeism (31%). Referring physician: paediatric nephrologist (94%) and nephrologist (63%) in <11 versus ≥11 year olds. Ophthalmological follow-up: 83% versus 38% in <11 versus ≥11 year olds. Patient opinion of physician expertise: paediatric nephrologist (94%) and nephrologist (44%). New treatment options (65%) and better information (42%) were demanded to improve adherence. CONCLUSION: Treatment with Cystagon is effective in young patients. However, adherence diminishes over time in adolescents and adults despite disease impact. Strategies such as better information on the disease, patient self-care promotion and facilitated transition to adult healthcare services are required to improve compliance and the clinical management of cystinosis.

Nephrin mutations cause childhood- and adult-onset focal segmental glomerulosclerosis.

Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type presenting before the first 3 months of life. Recently, NPHS1 mutations have also been identified in childhood-onset steroid-resistant nephrotic syndrome and milder courses of disease, but their role in adults with focal segmental glomerulosclerosis remains unknown. Here we developed an in silico scoring matrix to evaluate the pathogenicity of amino-acid substitutions using the biophysical and biochemical difference between wild-type and mutant amino acid, the evolutionary conservation of the amino-acid residue in orthologs, and defined domains, with the addition of contextual information. Mutation analysis was performed in 97 patients from 89 unrelated families, of which 52 presented with steroid-resistant nephrotic syndrome after 18 years of age. Compound heterozygous or homozygous NPHS1 mutations were identified in five familial and seven sporadic cases, including one patient 27 years old at onset of the disease. Substitutions were classified as 'severe' or 'mild' using this in silico approach. Our results suggest an earlier onset of the disease in patients with two 'severe' mutations compared to patients with at least one 'mild' mutation. The finding of mutations in a patient with adult-onset focal segmental glomerulosclerosis indicates that NPHS1 analysis could be considered in patients with later onset of the disease.

A missense mutation in the chloride/proton ClC-5 antiporter gene results in increased expression of an alternative mRNA form that lacks exons 10 and 11. Identification of seven new CLCN5 mutations in patients with Dent's disease.

Mutations in the voltage-gated chloride/proton antiporter ClC-5 gene, CLCN5, are associated with Dent's disease, an X-linked renal tubulopathy. Our interest is to identify and characterize disease-causing CLCN5 mutations, especially those that alter the splicing of the pre-mRNA. We analyzed the CLCN5 gene from nine unrelated Spanish Dent's disease patients and their relatives by DNA sequencing. Pre-mRNA splicing analysis was performed by RT-PCR. Seven new mutations were identified, consisting of three missense mutations (C219R, F273L, and W547G), one splice-site mutation (IVS-2A > G), one deletion (976delG), and two non-sense mutations (Y140X and W314X). We found that missense mutation W547G also led to increased expression of a new alternative isoform lacking exons 10 and 11 that was expressed in several human tissues. In addition, we describe another novel CLCN5 splicing variant lacking exon 11 alone, which was expressed only in human skeletal muscle. We conclude that missense mutation W547G can also alter the expression levels of a CLCN5 mRNA splicing variant. This type of mutation has not been previously described in the CLCN5 gene. Our results support the importance of a routine analysis at the pre-mRNA level of mutations that are commonly assumed to cause single amino acids alterations.

Region-specific alterations of global protein expression in the remodelled rat myocardium.

We applied the novel ProteinChip technology (SELDI-MS) to investigate and identify differentially regulated proteins upon myocardial remodelling in different heart regions. Tissue samples were isolated from the atria, the interventricular septum, and the right and left ventricles three months after surgically-induced myocardial infarction (MI) in rats. Corresponding protein extracts from control versus MI hearts were analysed on two different ProteinChip surfaces. In each of the functionally distinct cardiac regions, we obtained specific protein profile alterations upon myocardial remodelling. Most alterations were observed in the non-infarcted right ventricle, where down-regulation occurred more frequently than up-regulation of protein expression. Three of the differentially regulated proteins were identified: the metabolic enzyme triosephosphate isomerase (TIM), the cell signalling protein Raf-1 kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine binding protein (PEBP), and the small heat shock protein alphaB-crystallin. These proteins showed a pronounced tissue-dependent regulation. TIM was down-regulated only in the atrium and in the left ventricle, RKIP/PEBP was down-regulated only in the right ventricle and in the interventricular septum, and alphaB-crystallin was up-regulated only in the right and in the left ventricle. A simple correlation of peak intensity changes using two of the identified peaks demonstrated the diagnostic potential of SELDI-MS. We conclude that this novel proteomic method is a powerful high-throughput tool for the fast detection of region-specific cardiac protein profiles in small biopsy samples, and that SELDI-MS may become a useful complementary technique for the diagnosis and prognosis of cardiac diseases.

Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region.

In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.

Expression pattern of neuronal and skeletal muscle voltage-gated Na+ channels in the developing mouse heart.

In the mammalian heart, a variety of voltage-gated Na(+) channel transcripts and proteins have been detected. However, little quantitative information is available on the abundance of each transcript during development, or the contribution of TTX-sensitive Na(+) channels to the cardiac sodium current (I(Na)). Using competitive and real-time RT-PCR we investigated the transcription of six Na(+) channels (Na(v)1.1-Na(v)1.6) and the beta1 subunit during mouse heart development. Na(v)1.5 was predominantly expressed in the adult heart, whereas the splice variant Na(v)1.5a was the major Na(+) channel isoform in embryonic hearts. The TTX-resistant Na(+) channel transcripts (Na(v)1.5 and Na(v)1.5a) increased 1.7-fold during postnatal development. Transcripts encoding TTX-sensitive Na(+) channels (Na(v)1.1-Na(v)1.4) and the beta1 subunit gradually increased up to fourfold from postnatal day (P)1 to P126, while the Na(v)1.6 transcript level remained low and constant over the same period. In adults, TTX-sensitive channel mRNA accounted for 30-40% of the channel pool in whole-heart preparations (Na(v)1.3 > Na(v)1.4 > Na(v)1.2 >> Na(v)1.1 approximately Na(v)1.6), and 16% in mRNA from isolated cardiomyocytes (Na(v)1.4 > Na(v)1.3 > Na(v)1.2 > Na(v)1.1 > Na(v)1.6). Confocal immunofluorescence on ventricular myocytes suggested that Na(v)1.1 and Na(v)1.2 were localized at the intercalated disks and in the t tubules. Na(v)1.3 labelling predominantly produced a diffuse but strong intracellular signal. Na(v)1.6 fluorescence was detected only along the Z lines. Electrophysiological recordings showed that TTX-sensitive and TTX-resistant Na(+) channels, respectively, accounted for 8% and 92% of the I(Na) in adult ventricular cardiomyocytes. Our data suggest that neuronal and skeletal muscle Na(+) channels contribute to the action potential of cardiomyocytes in the adult mammalian heart.