Learning Physiology From Inherited Kidney Disorders.

The identification of genes causing inherited kidney diseases yielded crucial insights in the molecular basis of disease and improved our understanding of physiological processes that operate in the kidney. Monogenic kidney disorders are caused by mutations in genes coding for a large variety of proteins including receptors, channels and transporters, enzymes, transcription factors, and structural components, operating in specialized cell types that perform highly regulated homeostatic functions. Common variants in some of these genes are also associated with complex traits, as evidenced by genome-wide association studies in the general population. In this review, we discuss how the molecular genetics of inherited disorders affecting different tubular segments of the nephron improved our understanding of various transport processes and of their involvement in homeostasis, while providing novel therapeutic targets. These include inherited disorders causing a dysfunction of the proximal tubule (renal Fanconi syndrome), with emphasis on epithelial differentiation and receptor-mediated endocytosis, or affecting the reabsorption of glucose, the handling of uric acid, and the reabsorption of sodium, calcium, and magnesium along the kidney tubule.

Extracellular vesicles contribute to early cyst development in autosomal dominant polycystic kidney disease by cell-to-cell communication.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1-/- mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1-/- mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1-/- iMCD3 cells to EVs derived from Pkd1-/- mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1-/- cells to mDCT15 Pkd1-/- EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.

Fluid shear stress stimulates ATP release without regulating purinergic gene expression in the renal inner medullary collecting duct.

In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.

Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

Possible role for rare TRPM7 variants in patients with hypomagnesaemia with secondary hypocalcaemia.

BACKGROUND: Hypomagnesaemia with secondary hypocal-caemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase transient receptor potential melastatin type 6. Patients have very low serum magnesium (Mg2+) levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without a diagnosis. METHODS: In this study, two families with an HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7-identified variants on Mg2+ transport was examined. RESULTS: For the first time, variants in TRPM7 were identified in two families as a potential cause for hereditary HSH. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcaemia. In the first family, a splice site variant caused the incorporation of intron 1 sequences into the TRPM7 messenger RNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit decreased cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. CONCLUSIONS: We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesaemia and secondary hypocalcaemia. Screening of unresolved patients with hypocalcaemia and secondary hypocalcaemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.

Colonic expression of calcium transporter TRPV6 is regulated by dietary sodium butyrate.

Dietary fibers have been shown to increase the intestinal absorption of calcium (Ca2+) and magnesium (Mg2+). However, the mechanisms that explain the enhanced electrolyte absorption remain unknown. Therefore, this study aims to investigate the short-term and long-term effects of 5% (w/w) sodium butyrate (Na-butyrate), an important end-metabolite of bacterial fermentation of dietary fibers, on Ca2+ and Mg2+ homeostasis in mice. Serum Ca2+ levels were only significantly increased in mice treated with Na-butyrate for 1 day. This was associated with a twofold increase in the mRNA expression levels of Trpv6 in the proximal and distal colon. Contrary, Na-butyrate did not affect serum Mg2+ concentrations at either of the intervention periods. However, we observed a reduction in urinary Mg2+ excretion, although not significantly, after 1 day of treatment. A significant reduction of 2.5-fold in urinary Mg2+ excretion was observed after 14 days of treatment. Indeed, 14-day Na-butyrate supplementation increased colonic Trpm7 expression by 1.2-fold compared to control mice. In conclusion, short-term Na-butyrate supplementation increases serum Ca2+ levels in mice. This was associated with increased mRNA expression levels of Trpv6 in the colon, suggesting that Na-butyrate regulates the expression of genes involved in active intestinal Ca2+ absorption.

Structural insight into TRPV5 channel function and modulation.

TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.

Low gut microbiota diversity and dietary magnesium intake are associated with the development of PPI-induced hypomagnesemia.

Proton pump inhibitors (PPIs) are used by millions of patients for the treatment of stomach acid-reflux diseases. Although PPIs are generally considered safe, about 13% of the users develop hypomagnesemia. Despite rising attention for this issue, the underlying mechanism is still unknown. Here, we examine whether the gut microbiome is involved in the development of PPI-induced hypomagnesemia in wild-type C57BL/6J mice. After 4 wk of treatment under normal or low dietary Mg2+ availability, omeprazole significantly reduced serum Mg2+ levels only in mice on a low-Mg2+ diet without affecting the mRNA expression of colonic or renal Mg2+ transporters. Overall, 16S rRNA gene sequencing revealed a lower gut microbial diversity in omeprazole-treated mice. Omeprazole induced a shift in microbial composition, which was associated with a 3- and 2-fold increase in the abundance of Lactobacillus and Bifidobacterium, respectively. To examine the metabolic consequences of these microbial alterations, the colonic composition of organic acids was evaluated. Low dietary Mg2+ intake, independent of omeprazole treatment, resulted in a 10-fold increase in formate levels. Together, these results imply that both omeprazole treatment and low dietary Mg2+ intake disturb the gut internal milieu and may pose a risk for the malabsorption of Mg2+ in the colon.-Gommers, L. M. M., Ederveen, T. H. A., van der Wijst, J., Overmars-Bos, C., Kortman, G. A. M., Boekhorst, J., Bindels, R. J. M., de Baaij, J. H. F., Hoenderop, J. G. J. Low gut microbiota diversity and dietary magnesium intake are associated with the development of PPI-induced hypomagnesemia.

TRPV5 in renal tubular calcium handling and its potential relevance for nephrolithiasis.

Nephrolithiasis or renal stone disease is an increasingly common problem, and its relatively high recurrence rate demands better treatment options. The majority of patients with nephrolithiasis have stones that contain calcium (Ca2+), which develop upon "supersaturation" of the urine with insoluble Ca2+ salts; hence processes that influence the delivery and renal handling of Ca2+ may influence stone formation. Idiopathic hypercalciuria is indeed frequently observed in patients with kidney stones that contain Ca2+. Genetic screens of nephrolithiasis determinants have identified an increasing number of gene candidates, most of which are involved in renal Ca2+ handling. This review provides an outline of the current knowledge regarding genetics of nephrolithiasis and will mainly focus on the epithelial Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), an important player in Ca2+ homeostasis. Being a member of the TRP family of ion channels, TRPV5 is currently part of a revolution in structural biology. Recent technological breakthroughs in the cryo-electron microscopy field, combined with improvements in biochemical sample preparation, have resulted in high-resolution 3-dimensional structural models of integral membrane proteins, including TRPV5. These models currently are being used to explore the proteins' structure-function relationship, elucidate the molecular mechanisms of channel regulation, and study the putative effects of disease variants. Combined with other multidisciplinary approaches, this approach may open an avenue toward better understanding of the pathophysiological mechanisms involved in hypercalciuria and stone formation, and ultimately it may facilitate prevention of stone recurrence through the development of effective drugs.

Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.

The NaCl cotransporter (NCC) is essential for electrolyte homeostasis and control of blood pressure. The human SLC12A3 gene, which encodes NCC, gives rise to 3 isoforms, of which only the shortest isoform [NaCl cotransporter isoform 3 (NCC3)] has been studied extensively. All NCC isoforms share key phosphorylation sites at T55 and T60 that are essential mediators of NCC function. Recently, a novel phosphorylation site at S811 was identified in isoforms 1 and 2 [NaCl cotransporter splice variant (NCCSV)], which are only present in humans and higher primates. The aim of the current study, therefore, is to investigate the role of S811 phosphorylation in the regulation of NCC by a combination of biochemical and fluorescent microscopy analyses. We demonstrate that hypotonic low-chloride buffer increases S811 phosphorylation, whereas phosphorylation-deficient S811A mutant hinders phosphorylation at T55 and T60 in NCCSV and NCC3. NCCSV S811A impairs NCC3 activity in a dominant-negative fashion, although it does not affect plasma membrane abundance. This effect may be explained by the heterodimerization of NCCSV with NCC3. Taken together, our study highlights the dominant-negative effect of NCCSV on T55 and T60 phosphorylation and NCC activity. Here, we reveal a new function of NCCSV in humans that broadens the understanding on NCC regulation in blood pressure control.-Tutakhel, O. A. Z., Bianchi, F., Smits, D. A., Bindels, R. J. M., Hoenderop, J. G. J., van der Wijst, J. Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.

TRP channels in calcium homeostasis: from hormonal control to structure-function relationship of TRPV5 and TRPV6.

Maintaining plasma calcium levels within a narrow range is of vital importance for many physiological functions. Therefore, calcium transport processes in the intestine, bone and kidney are tightly regulated to fine-tune the rate of absorption, storage and excretion. The TRPV5 and TRPV6 calcium channels are viewed as the gatekeepers of epithelial calcium transport. Several calciotropic hormones control the channels at the level of transcription, membrane expression, and function. Recent technological advances have provided the first near-atomic resolution structural models of several TRPV channels, allowing insight into their architecture. While this field is still in its infancy, it has increased our understanding of molecular channel regulation and holds great promise for future structure-function studies of these ion channels. This review will summarize the mechanisms that control the systemic calcium balance, as well as extrapolate structural views to the molecular functioning of TRPV5/6 channels in epithelial calcium transport.

Effects of a high-sodium/low-potassium diet on renal calcium, magnesium, and phosphate handling.

The distal convoluted tubule (DCT) of the kidney plays an important role in blood pressure regulation by modulating Na+ reabsorption via the Na+-Cl- cotransporter (NCC). A diet containing high salt (NaCl) and low K+ activates NCC, thereby causing Na+ retention and a rise in blood pressure. Since high blood pressure, hypertension, is associated with changes in serum calcium (Ca2+) and magnesium (Mg2+) levels, we hypothesized that dietary Na+ and K+ intake affects Ca2+ and Mg2+ transport in the DCT. Therefore, the present study aimed to investigate the effect of a high-Na+/low-K+ diet on renal Ca2+ and Mg2+ handling. Mice were divided in four groups and fed a normal-Na+/normal-K+, normal-Na+/low-K+, high-Na+/normal-K+, or high-Na+/low-K+ diet for 4 days. Serum and urine were collected for electrolyte and hormone analysis. Gene and protein expression of electrolyte transporters were assessed in kidney and intestine by qPCR and immunoblotting. Whereas Mg2+ homeostasis was not affected, the mice had elevated urinary Ca2+ and phosphate (Pi) excretion upon high Na+ intake, as well as significantly lower serum Ca2+ levels in the high-Na+/low-K+ group. Alterations in the gene and protein expression of players involved in Ca2+ and Pi transport indicate that reabsorption in the proximal tubular and TAL is affected, while inducing a compensatory response in the DCT. These effects may contribute to the negative health impact of a high-salt diet, including kidney stone formation, chronic kidney disease, and loss of bone mineral density.

Interspecies differences in PTH-mediated PKA phosphorylation of the epithelial calcium channel TRPV5.

The epithelial calcium (Ca2+) channel TRPV5 (transient receptor potential vanilloid 5) is expressed in the distal convoluted tubule of the kidney and facilitates active Ca2+ reabsorption. This process is instrumental for the maintenance of Ca2+ homeostasis. Therefore, all aspects of TRPV5 function are tightly regulated by the calciotropic parathyroid hormone (PTH). Rabbit (rb)TRPV5 channel activity was shown to be stimulated upon PTH-mediated protein kinase A (PKA) phosphorylation. Since there is incomplete conservation of the PKA consensus motif (RR/QxT) across species, the aim of this study was to extend these findings to humans and characterize the expression and function of human (h)TRPV5. Functional differences between rbTRPV5 and hTRPV5 upon PTH stimulation were investigated using 45Ca2+ uptake assays, Fura-2 Ca2+ imaging, and cell surface biotinylation. While PTH treatment enhanced rbTRPV5 channel activity, it did not stimulate hTRPV5 activity. Mutation of the human RQxT motif into rabbit RRxT (hTRPV5 Q706R) partially restored the sensitivity to PTH. An ancestral sequence reconstruction of TRPV5 orthologues demonstrated that the change in the RRxT motif coincides with the creation of another putative PKA motif (RGAS to RRAS) in the amino terminus of hTRPV5. Interestingly, a constitutively phosphorylated hTRPV5 mutant (hTRPV5 S141D) displayed significantly decreased channel function, while its plasma membrane abundance was increased. Taken together, PTH-mediated stimulation of TRPV5, via PKA, is not conserved in humans. Our data suggest that PTH regulation of TRPV5 is altered in humans, an important observation for future studies that may add to new concepts on the role of PTH in renal Ca2+ handling.

Renal Handling of Circulating and Renal-Synthesized Hepcidin and Its Protective Effects against Hemoglobin-Mediated Kidney Injury.

Urinary hepcidin may have protective effects against AKI. However, renal handling and the potential protective mechanisms of hepcidin are not fully understood. By measuring hepcidin levels in plasma and urine using mass spectrometry and the kidney using immunohistochemistry after intraperitoneal administration of human hepcidin-25 (hhep25) in C57Bl/6N mice, we showed that circulating hepcidin is filtered by the glomerulus and degraded to smaller isoforms detected in urine but not plasma. Moreover, hepcidin colocalized with the endocytic receptor megalin in proximal tubules, and compared with wild-type mice, megalin-deficient mice showed higher urinary excretion of injected hhep25 and no hepcidin staining in proximal tubules that lack megalin. This indicates that hepcidin is reaborbed in the proximal tubules by megalin dependent endocytosis. Administration of hhep25 concomitant with or 4 hours after a single intravenous dose of hemoglobin abolished hemoglobin-induced upregulation of urinary kidney injury markers (NGAL and KIM-1) and renal Interleukin-6 and Ngal mRNA observed 24 hours after administration but did not affect renal ferroportin expression at this point. Notably, coadministration of hhep25 and hemoglobin but not administration of either alone greatly increased renal mRNA expression of hepcidin-encoding Hamp1 and hepcidin staining in distal tubules. These findings suggest a role for locally synthesized hepcidin in renal protection. Our observations did not support a role for ferroportin in hhep25-mediated protection against hemoglobin-induced early injury, but other mechanisms of cellular iron handling may be involved. In conclusion, our data suggest that both systemically delivered and locally produced hepcidin protect against hemoglobin-induced AKI.

Urinary ß-galactosidase stimulates Ca2+ transport by stabilizing TRPV5 at the plasma membrane.

Transcellular Ca(2+)transport in the late distal convoluted tubule and connecting tubule (DCT2/CNT) of the kidney is a finely controlled process mediated by the transient receptor potential vanilloid type 5 (TRPV5) channel. A complex-type-N-glycan bound at the extracellular residue Asn358 of TRPV5 through post-translational glycosylation has been postulated to regulate the activity of TRPV5 channels. Using in vitro Ca(2+)transport assays, immunoblot analysis, immunohistochemistry, patch clamp electrophysiology and total internal reflection fluorescence microscopy, it is demonstrated that the glycosidase ß-galactosidase (ß-gal), an enzyme that hydrolyzes galactose, stimulates TRPV5 channel activity. However, the activity of the non-glycosylated TRPV(N358Q)mutant was not altered in the presence of ß-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, ß-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. ß-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, ß-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.

Kinase and channel activity of TRPM6 are co-ordinated by a dimerization motif and pocket interaction.

Mutations in the gene that encodes the atypical channel-kinase TRPM6 (transient receptor potential melastatin 6) cause HSH (hypomagnesaemia with secondary hypocalcaemia), a disorder characterized by defective intestinal Mg2+ transport and impaired renal Mg2+ reabsorption. TRPM6, together with its homologue TRPM7, are unique proteins as they combine an ion channel domain with a C-terminally fused protein kinase domain. How TRPM6 channel and kinase activity are linked is unknown. Previous structural analysis revealed that TRPM7 possesses a non-catalytic dimerization motif preceding the kinase domain. This interacts with a dimerization pocket lying within the kinase domain. In the present study, we provide evidence that the dimerization motif in TRPM6 plays a critical role in regulating kinase activity as well as ion channel activity. We identify mutations within the TRPM6 dimerization motif (Leu1718 and Leu1721) or dimerization pocket (L1743A, Q1832K, A1836N, L1840A and L1919Q) that abolish dimerization and establish that these mutations inhibit protein kinase activity. We also demonstrate that kinase activity of a dimerization motif mutant can be restored by addition of a peptide encompassing the dimerization motif. Moreover, we observe that mutations that disrupt the dimerization motif and dimerization pocket interaction greatly diminish TRPM6 ion channel activity, in a manner that is independent of kinase activity. Finally, we analyse the impact on kinase activity of ten disease-causing missense mutations that lie outwith the protein kinase domain of TRPM6. This revealed that one mutation lying nearby the dimerization motif (S1754N), found previously to inhibit channel activity, abolished kinase activity. These results provide the first evidence that there is structural co-ordination between channel and kinase activity, which is mediated by the dimerization motif and pocket interaction. We discuss that modulation of this interaction could comprise a major regulatory mechanism by which TRPM6 function is controlled.

Mg2+ homeostasis: the balancing act of TRPM6.

PURPOSE OF REVIEW: The tight control of blood magnesium (Mg) levels is of central importance for numerous physiological processes. A persistent low Mg status (hypomagnesemia) is associated with severe health risks and is involved in the pathogenesis of type 2 diabetes mellitus, osteoporosis, asthma, and heart and vascular diseases. The current view has expanded significantly as a result of the identification of novel genes and regulatory pathways involved in hypomagnesemic disorders. This review aims to give an up-to-date overview of transient receptor potential melastatin 6 (TRPM6) regulation and its role in the maintenance of Mg homeostasis. RECENT FINDINGS: The epithelial Mg channel TRPM6 is considered to be the Mg entry pathway in the distal convoluted tubule of the kidney, where it functions as gatekeeper for controlling the body's Mg balance. Various factors and hormones contribute not only to the function, but also to the dysregulation of TRPM6, which has a substantial impact on renal Mg handling. Recent genetic and molecular studies have further elucidated the signaling processes of epithelial Mg transport, including their effect on the expression and function of TRPM6. SUMMARY: Knowledge of TRPM6 functioning is of vital importance to decipher its role in Mg handling and will, in particular, provide a molecular basis for achieving a better understanding of Mg mal(re)absorption and hence systemic Mg balance.

Decreased calcium permeability caused by biallelic TRPV5 mutation leads to autosomal recessive renal calcium-wasting hypercalciuria.

Hypercalciuria is the most common metabolic risk factor in people with kidney stone disease. Its etiology is mostly multifactorial, although monogenetic causes of hypercalciuria have also been described. Despite the increased availability of genetic diagnostic tests, the vast majority of individuals with familial hypercalciuria remain unsolved. In this study, we investigated a consanguineous pedigree with idiopathic hypercalciuria. The proband additionally exhibited severe skeletal deformities and hyperparathyroidism. Whole-exome sequencing of the proband revealed a homozygous ultra-rare variant in TRPV5 (NM_019841.7:c.1792G>A; p.(Val598Met)), which encodes for a renal Ca2+-selective ion channel. The variant segregates with the three individuals with hypercalciuria. The skeletal phenotype unique to the proband was due to an additional pathogenic somatic mutation in GNAS (NM_000516.7:c.601C>T; p.(Arg201Cys)), which leads to polyostotic fibrous dysplasia. The variant in TRPV5 is located in the TRP helix, a characteristic amphipathic helix that is indispensable for the gating movements of TRP channels. Biochemical characterization of the TRPV5 p.(Val598Met) channel revealed a complete loss of Ca2+ transport capability. This defect is caused by reduced expression of the mutant channel, due to misfolding and preferential targeting to the proteasome for degradation. Based on these findings, we conclude that biallelic loss of TRPV5 function causes a novel form of monogenic autosomal recessive hypercalciuria, which we name renal Ca2+-wasting hypercalciuria (RCWH). The recessive inheritance pattern explains the rarity of RCWH and underscores the potential prevalence of RCWH in highly consanguineous populations, emphasizing the importance of exploration of this disorder within such communities.

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

Beyond current treatment of Fanconi Anemia: What do advances in cell and gene-based approaches offer?

Fanconi anemia (FA) is a rare inherited disorder that mainly affects the bone marrow. This condition causes decreased production of all types of blood cells. FA is caused by a defective repair of DNA interstrand crosslinks and to date, mutations in over 20 genes have been linked to the disease. Advances in science and molecular biology have provided new insight between FA gene mutations and the severity of clinical manifestations. Here, we will highlight the current and promising therapeutic options for this rare disease. The current standard treatment for FA patients is hematopoietic stem cell transplantation, a treatment associated to exposure to radiation or chemotherapy, immunological complications, plus opportunistic infections from prolonged immune incompetence or increased risk of morbidity. New arising treatments include gene addition therapy, genome editing using CRISPR-Cas9 nuclease, and hematopoietic stem cell generation from induced pluripotent stem cells. Finally, we will also discuss the revolutionary developments in mRNA therapeutics as an opportunity for this disease.