Hereditary kidney diseases associated with hypomagnesemia

In the kidney, a set of proteins expressed in the epithelial cells of the thick ascending loop of Henle and the distal convoluted tubule directly or indirectly play important roles in the regulation of serum magnesium levels. Magnesium reabsorption in the thick ascending loop of Henle occurs through a passive paracellular pathway, while in the distal convoluted tubule, the final magnesium concentration is established through an active transcellular pathway. The players involved in magnesium reabsorption include proteins with diverse functions including tight junction proteins, cation and anion channels, sodium chloride cotransporter, calcium-sensing receptor, epidermal growth factor, cyclin M2, sodium potassium adenosine triphosphatase subunits, transcription factors, a serine protease, and proteins involved in mitochondrial function. Mutations in the genes that encode these proteins impair their function and cause different rare diseases associated with hypomagnesemia, which may lead to muscle cramps, fatigue, epileptic seizures, intellectual disability, cardiac arrhythmias, and chronic kidney disease. The purpose of this review is to describe the clinical and genetic characteristics of these hereditary kidney diseases and the current research findings on the pathophysiological basis of these diseases

Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys) generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr) and c.2581G>C; p.(Ala861Pro) abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level

Splicing defects caused by exonic mutations in <i>PKD1</i> as a new mechanism of pathogenesis in autosomal dominant polycystic kidney disease

<div><p>The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the <i>PKD1</i> gene. Recently, we analyzed a substantial number of <i>PKD1</i> missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.</p></div

Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations

Abstract Background Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis type 1 is an autosomal recessive disease characterized by excessive renal magnesium and calcium excretion, bilateral nephrocalcinosis, and progressive chronic renal failure. This rare disease is caused by mutations in CLDN16 that encodes claudin-16, a tight-junction protein involved in paracellular reabsorption of magnesium and calcium in the renal tubule. Most of these variants are located in exons and have been classified as missense mutations. The functional consequences of some of these claudin-16 mutant proteins have been analysed after heterologous expression showing indeed a significant loss of function compared to the wild-type claudin-16. We hypothesize that a number of CLDN16 exonic mutations can be responsible for the disease phenotype by disrupting the pre-mRNA splicing process. Methods We selected 12 previously described presumed CLDN16 missense mutations and analysed their potential effect on pre-mRNA splicing using a minigene assay. Results Our results indicate that five of these mutations induce significant splicing alterations. Mutations c.453G > T and c.446G > T seem to inactivate exonic splicing enhancers and promote the use of an internal cryptic acceptor splice site resulting in inclusion of a truncated exon 3 in the mature mRNA. Mutation c.571G > A affects an exonic splicing enhancer resulting in partial skipping of exon 3. Mutations c.593G > C and c.593G > A disturb the acceptor splice site of intron 3 and cause complete exon 4 skipping. Conclusions To our knowledge, this is the first report of CLDN16 exonic mutations producing alterations in splicing. We suggest that in the absence of patients RNA samples, splicing functional assays with minigenes could be valuable for evaluating the effect of exonic CLDN16 mutations on pre-mRNA splicing

Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors

National audienceThis study deals with the effects of desizing by heat cleaning on the mechanical properties of basalt fibers. The heattreatment at 350°C for 10h, considered in this work, leads to a complete desizing of the fibers. The mechanical propertiesof basalt fibers before and after desizing were investigated by single fiber tensile tests. The tensile strength is reduced of34% after desizing while no evolution of the Young’s modulus is observed. SEM micrographs reveals that the defectslocated on the fibers surface are at the origin of the failure. Moreover, since the basalt structure is not modified during thetreatment, the strength loss is only ascribed to sizing layer removal. In fact, the desizing reveals the surface flaws of thefibers and increases their criticality. Tensile specimens of unidirectional composite materials were produced with an epoxymatrix and tested by considering both fibers states. The differences of mechanical properties observed for the compositeparts corresponds to those observed for single fibers. As a consequence, in the composite, the matrix is not able to playthe protective role of the sizing.Cette étude s’intéresse à l’influence du désensimage par traitement thermique sur les propriétés mécaniques de fibres de basalte. Le traitement thermique à 350°C pendant 10 h, considéré dans ces travaux, permet d’obtenir un désensimage complet des fibres. Les propriétés mécaniques des fibres de basalte avant et après désensimage ont été évaluées par essais de traction sur monofilaments. Une diminution de 34% de la résistance mécanique a été observée suite au désensimage tandis qu’aucune différence significative n’a été constatée sur le module d’Young. L’observation au MEB des faciès de rupture montre que les défauts présents à la surface des fibres sont responsables de la rupture. La structure du basalte n’étant pas modifiée au cours du traitement, la perte de résistance mécanique est exclusivement attribuée au désensimage. Celui-ci a révélé les défauts surfaciques des fibres et aggravé leur criticité. Des éprouvettes en matériaux composites unidirectionnels ont été fabriquées avec une matrice époxy et testées en considérant les deux états de fibres. Les différences de propriétés mécaniques observées sur les composites correspondent à celles observées sur les monofilaments. Autrement dit, la présence de la matrice ne permet pas de « protéger » les défauts surfaciques des fibres révélés par le désensimage, comme pouvait le faire l’ensimage

A Novel Claudin 16 Mutation Associated with Childhood Hypercalciuria Abolishes Binding to ZO-1 and Results in Lysosomal Mistargeting

Mutations in the gene coding for the renal tight junction protein claudin 16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disorder of renal Ca(2+) and Mg(2+) handling that progressively leads to chronic renal failure, with nephrolithiasis having been reported in heterozygous carriers. Screening a cohort of 11 families with idiopathic hypercalciuria identified a novel homozygous mutation in the claudin 16 gene in two families. In contrast to classical symptoms of familial hypomagnesemia with hypercalciuria and nephrocalcinosis, the patients displayed serious but self-limiting childhood hypercalciuria with preserved glomerular filtration rate. The mutation results in inactivation of a PDZ-domain binding motif, thereby disabling the association of the tight junction scaffolding protein ZO-1 with claudin 16. In contrast to wild-type claudin 16, the mutant no longer localizes to tight junctions in kidney epithelial cells but instead accumulates in lysosomes. Thus, mutations at different intragenic sites in the claudin 16 gene may lead to particular clinical phenotypes with a distinct prognosis. Mutations in claudin 16 that affect interaction with ZO-1 lead to lysosomal mistargeting, providing—for the first time, to our knowledge—insight into the molecular mechanism of a disease-associated mutation in the claudin 16 gene