CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia.

Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg(2+) absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg(2+) homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype.

Segmental transport of Ca²âº and Mg²âº along the gastrointestinal tract.

Calcium (Ca(2+)) and magnesium (Mg(2+)) ions are involved in many vital physiological functions. Since dietary intake is the only source of minerals for the body, intestinal absorption is essential for normal homeostatic levels. The aim of this study was to characterize the absorption of Ca(2+) as well as Mg(2+) along the gastrointestinal tract at a molecular and functional level. In both humans and mice the Ca(2+) channel transient receptor potential vanilloid subtype 6 (TRPV6) is expressed in the proximal intestinal segments, whereas Mg(2+) channel transient receptor potential melastatin subtype 6 (TRPM6) is expressed in the distal parts of the intestine. A method was established to measure the rate of Mg(2+) absorption from the intestine in a time-dependent manner by use of (25)Mg(2+). In addition, local absorption of Ca(2+) and Mg(2+) in different segments of the intestine of mice was determined by using surgically implanted intestinal cannulas. By these methods, it was demonstrated that intestinal absorption of Mg(2+) is regulated by dietary needs in a vitamin D-independent manner. Also, it was shown that at low luminal concentrations, favoring transcellular absorption, Ca(2+) transport mainly takes place in the proximal segments of the intestine, whereas Mg(2+) absorption predominantly occurs in the distal part of the gastrointestinal tract. Vitamin D treatment of mice increased serum Mg(2+) levels and 24-h urinary Mg(2+) excretion, but not intestinal absorption of (25)Mg(2+). Segmental cannulation of the intestine and time-dependent absorption studies using (25)Mg(2+) provide new ways to study intestinal Mg(2+) absorption.

The impact of formative testing on study behaviour and study performance of (bio)medical students: a smartphone application intervention study.

BACKGROUND: Formative testing can increase knowledge retention but students often underuse available opportunities. Applying modern technology to make the formative tests more attractive for students could enhance the implementation of formative testing as a learning tool. This study aimed to determine whether formative testing using an internet-based application ("app") can positively affect study behaviour as well as study performance of (bio)medical students. METHODS: A formative testing app "Physiomics, to the next level" was introduced during a 4-week course to a large cohort (n = 461) of Dutch first year (bio)medical students of the Radboud University. The app invited students to complete 7 formative tests throughout the course. Each module was available for 3-4 days to stimulate the students to distribute their study activities throughout the 4-week course. RESULTS: 72% of the students used the app during the course. Study time significantly increased in intensive users (p < 0.001), while no changes were observed in moderate (p = 0.07) and non-users (p = 0.25). App-users obtained significantly higher grades during the final exam of the course (p < 0.05). Non-users more frequently failed their final exam (34%, OR 3.6, 95% CI: 2.0-6.4) compared to moderate users (19%) and intensive users (12%). Students with an average grade <6.5 during previous courses benefitted most from the app, as intensive (5.8 ± 0.9 / 36%) and moderate users (5.8 ± 0.9 / 33%) obtained higher grades and failed their exam less frequently compared to non-users (5.2 ± 1.1 / 61%). The app was also well appreciated by students; students scored the app with a grade of 7.3 ± 1.0 out of 10 and 59% of the students indicated that they would like the app to be implemented in future courses. CONCLUSIONS: A smartphone-based application of formative testing is an effective and attractive intervention to stimulate study behaviour and improve study performance in (bio) medical students.

Expression profiling of claudins in the human gastrointestinal tract in health and during inflammatory bowel disease.

BACKGROUND: Claudins, being part of the tight junction protein family, partially determine the integrity and paracellular permeability of the intestinal epithelium. The aim of this study was twofold. First, the authors set out to create an overview of claudin mRNA expression along the proximal-distal axis of the healthy human intestine. Second, the authors aimed to analyze expression levels of claudins in patients with active and inactive inflammatory bowel diseases (IBD) such as Crohn's disease or ulcerative colitis (UC). METHODS: mRNA expression levels of claudins were determined in gastrointestinal biopsies from healthy patients as well as patients diagnosed with IBD using SybrGreen real-time PCR. RESULTS: Claudins show distinct expression patterns throughout the gastrointestinal tract. Some claudins show a proximal expression pattern, such as CLDN18 which is solely expressed in the stomach, and CLDN2 and -15 that are predominantly expressed in the proximal parts of the gastrointestinal tract. Other claudins, such as CLDN3, -4, -7 and -8, are predominantly expressed in the distal parts of the gastrointestinal tract or show a ubiquitous expression pattern throughout the entire gastrointestinal tract, which is the case for CLDN12. In addition, we show that changes in claudin expression in IBD are dependent on gastrointestinal location and inflammatory activity. CONCLUSIONS: This study provides detailed mRNA expression patterns of various claudins throughout the human gastrointestinal tract. Analysis of expression levels of claudins in patients with CD, active and inactive UC shows that changes in expression are confined to specific intestinal segments and strongly depend on inflammatory activity.

Functional TRPV6 channels are crucial for transepithelial Ca2+ absorption.

TRPV6 is considered the primary protein responsible for transcellular Ca2+ absorption. In vitro studies demonstrate that a negatively charged amino acid (D) within the putative pore region of mouse TRPV6 (position 541) is critical for Ca2+ permeation of the channel. To elucidate the role of TRPV6 in transepithelial Ca2+ transport in vivo, we functionally analyzed a TRPV6D541A/D541A knockin mouse model. After weaning, mice were fed a regular (1% wt/wt) or Ca2+-deficient (0.02% wt/wt) diet and housed in metabolic cages. Blood was sampled for Ca2+ measurements, and the expression of Ca2+ transport proteins was analyzed in kidney and duodenum. Intestinal 45Ca2+ uptake was measured in vivo by an absorption assay. Challenging the mice with the Ca2+-deficient diet resulted in hypocalcemia in wild-type and TRPV6D541A/D541A mice. On a low-Ca2+ diet both mouse strains displayed increased expression of intestinal TRPV6, calbindin-D(9K), and renal TRPV5. TRPV6D541A/D541A mice showed significantly impaired intestinal Ca2+ uptake compared with wild-type mice, and duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice. On a normal diet, serum Ca2+ concentrations normalized in both mouse strains. Under these conditions, intestinal Ca2+ uptake was similar, and the expression levels of renal and intestinal Ca2+ transport proteins were not affected. We demonstrate that TRPV6D541A/D541A mice exhibit impaired transcellular Ca2+ absorption. Duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice, albeit insufficient to correct for the diminished Ca2+ absorption. Under normal conditions, when passive Ca2+ transport is predominant, no differences between wild-type and TRPV6D541A/D541A mice were observed. Our results demonstrate a specific role for TRPV6 in transepithelial Ca2+ absorption.

Drug-induced alterations in Mg2+ homoeostasis.

Magnesium (Mg2+) balance is tightly regulated by the concerted actions of the intestine, bone and kidneys. This balance can be disturbed by a broad variety of drugs. Diuretics, modulators of the EGFR (epidermal growth factor receptor), proton pump inhibitors, antimicrobials, calcineurin inhibitors and cytostatics may all cause hypomagnesaemia, potentially leading to tetany, seizures and cardiac arrhythmias. Conversely, high doses of Mg2+ salts, frequently administered as an antacid or a laxative, may lead to hypermagnesaemia causing various cardiovascular and neuromuscular abnormalities. A better understanding of the molecular mechanisms underlying the adverse effects of these medications on Mg2+ balance will indicate ways of prevention and treatment of these adverse effects and could potentially provide more insight into Mg2+ homoeostasis.

Involvement of claudin 3 and claudin 4 in idiopathic infantile hypercalcaemia: a novel hypothesis?

BACKGROUND: Idiopathic infantile hypercalcaemia (IIH) is a rare disease that generally resolves spontaneously between the age of 1 and 3 years. Similar symptoms may occur in patients suffering from Williams-Beuren syndrome (WBS), which is caused by a microdeletion on chromosome 7. Two of the genes, named CLDN3 and CLDN4, located within this region are members of the claudin family that has been shown to be involved in paracellular calcium (Ca(2+)) absorption. Based on the hemizygous loss of CLDN3 and CLDN4 in WBS and the function of these genes in paracellular Ca(2+) transport, we hypothesized that mutations in CLDN3 or CLDN4 could also be involved in IIH. METHODS: Biochemical characteristics, including calciotropic hormone levels, were obtained from three typical IIH patients. CLDN3 and CLDN4 sequences were also analysed in these patients. The major intestinal Ca(2+) transporter TRPV6 was also screened for the presence of mutations, since hypercalcaemia in IIH and WBS has been shown to result from intestinal hyperabsorption. All three patients were also analysed for the presence of deletions or duplications using a single-nucleotide polymorphism (SNP) array for genomic DNA. RESULTS: The serum Ca(2+) levels of patients were 2.9, 3.3 and 3.8 mmol/L (normal <2.7 mmol/L). Levels of 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) were normal, parathyroid hormone (PTH) and PTH-related peptide (PTHrP) levels were appropriately low. Sequencing of coding regions and intron-exon boundaries did not reveal mutations in CLDN3, CLDN4 and TRPV6. Identified SNPs were not correlated with the disease phenotype. A SNP array did not reveal genomic deletions or duplications. CONCLUSIONS: Biochemical analysis did not reveal inappropriate levels of calciotropic hormones in IIH patients in this study. Furthermore, based on the lack of mutations in CLDN3, CLDN4 and TRPV6, we conclude that IIH is neither caused by mutations in these candidate genes nor by deletions or duplications in the genome of these patients.

Disrupted cell adhesion but not proliferation mediates cyst formation in polycystic liver disease.

The pathogenesis of polycystic liver disease is not well understood. The putative function of the associated proteins, hepatocystin and Sec63p, do not give insight in their role in cystogenesis and their tissue-wide expression does not fit with the liver-specific phenotype of the disease. We designed this study with the specific aim to dissect whether pathways involved in polycystic kidney diseases are also implicated in polycystic liver disease. Therefore, we immunohistochemically stained cyst tissue specimen with antibodies directed against markers for apoptosis, proliferation, growth receptors, signaling and adhesion. We analyzed genotyped polycystic liver disease cyst tissue (n=21) compared with normal liver tissue (n=13). None of the cysts showed proliferation of epithelial cells. In addition, anti-apoptosis marker Bcl-2 revealed slight increase in expression, with variable increase of apoptosis marker active caspase 3. Growth factor receptors, EGFR and c-erbB-2, were overexpressed and mislocalized. We found EGFR staining in the nuclei of cyst epithelial cells regardless of mutational state of the patient. Further, in hepatocystin-mutant polycystic liver disease patients, apical membranous staining of c-erbB-2 and adhesion markers, MUC1 and CEA, was lost and the proteins appeared to be retained in cytoplasm of cyst epithelia. Finally, we found loss of adhesion molecules E-cadherin and Ep-CAM in cyst epithelium of all patients. Nevertheless, we observed normal beta-catenin expression. Our results show that polycystic liver disease cystogenesis is different from renal cystogenesis. Polycystic liver disease involves overexpression of growth factor receptors and loss of adhesion. In contrast, proliferation or deregulated apoptosis do not seem to be implicated. Moreover differential findings for PRKCSH- and SEC63-associated polycystic liver disease suggest a divergent mechanism for cystogenesis in these two groups.

Identification of SLC41A3 as a novel player in magnesium homeostasis.

Regulation of the body Mg(2+) balance takes place in the distal convoluted tubule (DCT), where transcellular reabsorption determines the final urinary Mg(2+) excretion. The basolateral Mg(2+) extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg(2+) extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg(2+) homeostasis using the Slc41a3 knockout (Slc41a3(-/-)) mouse. By quantitative PCR analysis it was shown that Slc41a3 is the only SLC41 isoform with enriched expression in the DCT. Interestingly, serum and urine electrolyte determinations demonstrated that Slc41a3(-/-) mice suffer from hypomagnesemia. The intestinal Mg(2+) absorption capacity was measured using the stable (25)Mg(2+) isotope in mice fed a low Mg(2+) diet. (25)Mg(2+) uptake was similar in wildtype (Slc41a3(+/+)) and Slc41a3(-/-) mice, although Slc41a3(-/-) animals exhibited increased intestinal mRNA expression of Mg(2+) transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3(-/-) mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg(2+) handling.

Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity.

Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.

Importance of dietary calcium and vitamin D in the treatment of hypercalcaemia in Williams-Beuren syndrome.

BACKGROUND: Williams-Beuren syndrome (WBS) is a rare genetic disorder caused by the deletion of 26-28 genes on chromosome 7. Fifteen percent of WBS patients present with hypercalcaemia during infancy, which is generally mild and resolves spontaneously before the age of 4 years. The mechanisms underlying the transient hypercalcaemia in WBS are poorly understood. CASE: We report a case of severe symptomatic hypercalcaemia in a patient with WBS, in which treatment with mild calcium restriction, hyperhydration and repeated bisphosphonate administration only resulted in short-lasting effects. Long-term lowering of serum calcium was only achieved after reducing calcium and vitamin D intake to the bare minimum. CONCLUSIONS: This case illustrates the potential severity of hypercalcaemia in WBS, and demonstrates that both the cause as well as the solution of this problem may be found in the intestinal absorption of calcium. We hypothesise that the phenotypical resemblance between WBS and transient idiopathic infantile hypercalcaemia can be explained by similarities in the underlying genetic defect. Patients suffering from transient infantile hypercalcaemia were recently described to have mutations in CYP24A1, the key enzyme in 1,25-dihydroxyvitamin D3 degradation. In the light of this new development we discuss the role of one of the deleted genes in WBS, Williams syndrome transcription factor (WSTF), in the etiology of hypercalcaemia in WBS.

Hepatocystin is not secreted in cyst fluid of hepatocystin mutant polycystic liver patients.

Autosomal dominant polycystic liver disease (PCLD) is characterized by multiple liver cysts and is caused by mutations in PRKCSH (hepatocystin). Mechanisms of cystogenesis are unknown, but previous studies have shown that hepatocystin is secreted in vitro. The goal of this study was to determine the fate of hepatocystin in vivo. Using immunoprecipitation, we determined that mutant hepatocystin is secreted from both apical and basolateral cell surface of MDCK cells stably transfected with mutant hepatocystin. Analysis of 60 cyst fluid samples from polycystic livers using Western blot, MALDI-TOF MS or nLC-MS/MS did not detect hepatocystin in liver cyst fluid. We did identify 163 ubiquitous serum proteins. No paracrine or autocrine factors were recognized. Although cyst fluids vary greatly in protein concentration, a PCLD specific protein pattern was not established. In conclusion, hepatocystin is not secreted in PCLD liver cyst fluid, suggesting that mutant hepatocystin is either not produced or degraded intracellularly. PCLD cysts develop from intralobular bile ductules and cyst fluid mainly contains common serum proteins comparable to that of other polycystic diseases.