Karl P. Schlingmann

Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia

BACKGROUND Vitamin D supplementation for the prevention of rickets is one of the oldest and most effective prophylactic measures in medicine, having virtually eradicated rickets in North America. Given the potentially toxic effects of vitamin D, the recommendations for the optimal dose are still debated, in part owing to the increased incidence of idiopathic infantile hypercalcemia in Britain in the 1950s during a period of high vitamin D supplementation in fortified milk products. We investigated the molecular basis of idiopathic infantile hypercalcemia, which is characterized by severe hypercalcemia, failure to thrive, vomiting, dehydration, and nephrocalcinosis. METHODS We used a candidate-gene approach in a cohort of familial cases of typical idiopathic infantile hypercalcemia with suspected autosomal recessive inheritance. Identified mutations in the vitamin D-metabolizing enzyme CYP24A1 were evaluated with the use of a mammalian expression system. RESULTS Sequence analysis of CYP24A1, which encodes 25-hydroxyvitamin D 24-hydroxylase, the key enzyme of 1,25-dihydroxyvitamin D(3) degradation, revealed recessive mutations in six affected children. In addition, CYP24A1 mutations were identified in a second cohort of infants in whom severe hypercalcemia had developed after bolus prophylaxis with vitamin D. Functional characterization revealed a complete loss of function in all CYP24A1 mutations. CONCLUSIONS The presence of CYP24A1 mutations explains the increased sensitivity to vitamin D in patients with idiopathic infantile hypercalcemia and is a genetic risk factor for the development of symptomatic hypercalcemia that may be triggered by vitamin D prophylaxis in otherwise apparently healthy infants.

Novel TRPM6 Mutations in 21 Families with Primary Hypomagnesemia and Secondary Hypocalcemia

Primary hypomagnesemia with secondary hypocalcemia is a rare autosomal recessive disorder characterized by profound hypomagnesemia associated with hypocalcemia. Pathophysiology is related to impaired intestinal absorption of magnesium accompanied by renal magnesium wasting as a result of a reabsorption defect in the distal convoluted tubule. Recently, mutations in the TRPM6 gene coding for TRPM6, a member of the transient receptor potential (TRP) family of cation channels, were identified as the underlying genetic defect. Here, the results of a TRPM6 mutational analysis of 21 families with 28 affected individuals are presented. In this large patient cohort, a retrospective clinical evaluation based on a standardized questionnaire was also performed. Genotype analysis revealed TRPM6 mutations in 37 of 42 expected mutant alleles. Sixteen new TRPM6 mutations were identified, including stop mutations, frame-shift mutations, splice-site mutations, and deletions of exons. Electrophysiologic analysis of mutated ion channels after heterologous expression in Xenopus oocytes proved complete loss of function of TRPM6. Clinical evaluation revealed a homogeneous clinical picture at manifestation with onset in early infancy with generalized cerebral convulsions. Initial laboratory evaluation yielded extremely low serum magnesium levels, low serum calcium levels, and inadequately low parathyroid hormone levels. Treatment usually consisted of acute intravenous magnesium supplementation leading to relief of clinical symptoms and normocalcemia, followed by lifelong oral magnesium supplementation. Serum magnesium levels remained in the subnormal range despite adequate therapy. This is best explained by a disturbed magnesium conservation in the distal convoluted tubule, which emerged in all patients upon magnesium supplementation. Delay of diagnosis resulted in permanent neurologic damage in three patients.

Bartter- and Gitelman-like syndromes: salt-losing tubulopathies with loop or DCT defects

Salt-losing tubulopathies with secondary hyperaldosteronism (SLT) comprise a set of well-defined inherited tubular disorders. Two segments along the distal nephron are primarily involved in the pathogenesis of SLTs: the thick ascending limb of Henle’s loop, and the distal convoluted tubule (DCT). The functions of these pre- and postmacula densa segments are quite distinct, and this has a major impact on the clinical presentation of loop and DCT disorders – the Bartter- and Gitelman-like syndromes. Defects in the water-impermeable thick ascending limb, with its greater salt reabsorption capacity, lead to major salt and water losses similar to the effect of loop diuretics. In contrast, defects in the DCT, with its minor capacity of salt reabsorption and its crucial role in fine-tuning of urinary calcium and magnesium excretion, provoke more chronic solute imbalances similar to the effects of chronic treatment with thiazides. The most severe disorder is a combination of a loop and DCT disorder similar to the enhanced diuretic effect of a co-medication of loop diuretics with thiazides. Besides salt and water supplementation, prostaglandin E2-synthase inhibition is the most effective therapeutic option in polyuric loop disorders (e.g., pure furosemide and mixed furosemide–amiloride type), especially in preterm infants with severe volume depletion. In DCT disorders (e.g., pure thiazide and mixed thiazide–furosemide type), renin–angiotensin–aldosterone system (RAAS) blockers might be indicated after salt, potassium, and magnesium supplementation are deemed insufficient. It appears that in most patients with SLT, a combination of solute supplementation with some drug treatment (e.g., indomethacin) is needed for a lifetime.

Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD

CONTEXT The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D]. OBJECTIVE We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 μL of serum with a 5-minute run time. DESIGN The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter. SETTING We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models. RESULTS The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia. CONCLUSIONS The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

A critical role of TRPM channel-kinase for human magnesium transport

Hereditary disorders of magnesium homeostasis comprise a heterogenous group of diseases mainly affecting the renal conservation of magnesium. In the past few years, genetic studies in affected individuals disclosed the first molecular components of epithelial magnesium transport: the tight junction protein paracellin‐1 (claudin‐16) was discovered as a key player in paracellular magnesium and calcium reabsorption in the thick ascending limb of Henles loop and the γ‐subunit was identified as a component of renal Na+–K+‐ATPase critical for transcellular magnesium reabsorption in the distal convoluted tubule. However, the molecular identity of proteins directly involved in cellular magnesium transport remained largely unknown until a series of recent studies highlighted the critical role of two members of the transient receptor potential (TRP) family, for body magnesium homeostasis. TRPM6 and TRPM7 belong to the melastatin‐related TRPM subfamily of TRP channels whose eight members exhibit a significant diversity in domain structure as well as cation selectivity and activation mechanisms. Both proteins share the unique feature of an atypical kinase domain at their C‐terminus for which they have been termed ‘chanzymes’ (channels plus enzymes). Whereas electrophysiological and biochemical analyses identified TRPM7 as an important player in cellular magnesium homeostasis, the critical role of TRPM6 for epithelial magnesium transport emerged from the discovery of loss‐of‐function mutations in patients with a severe form of hereditary hypomagnesaemia called primary hypomagnesaemia with secondary hypocalcaemia or HSH. The aim of this review is to summarize the data emerging from molecular genetic, biochemical and electrophysiological studies on these fascinating two new proteins combining ion channel and enzyme functions/properties.

Essential role for TRPM6 in epithelial magnesium transport and body magnesium homeostasis

Magnesium is an important cofactor for many biological processes such as protein synthesis, nucleic acid stability and neuromuscular excitability. The extracellular magnesium concentration is regulated tightly by the extent of intestinal absorption and renal excretion. Despite their critical role in magnesium handling, the molecular mechanisms mediating transepithelial transport are still not understood completely. Recently, genetic studies in patients with primary hypomagnesaemia and secondary hypocalcaemia (HSH), a combined defect of intestinal magnesium absorption and renal magnesium conservation, have identified “transient receptor potential (melastatin) 6” (TRPM6) as the first component involved directly in epithelial magnesium reabsorption. TRPM7, the closest homologue of TRPM6, has a central role in Mg2+ uptake in vertebrate cells since TRPM7-deficient cells become Mg2+ deficient and are not viable. TRPM7 has been characterized functionally as a constitutively active ion channel permeable for a variety of cations including calcium and magnesium and regulated by intracellular concentrations of magnesium and/or magnesium-nucleotide complexes. Both proteins share the unique feature of cation channels fused to serine/threonine kinase domains. This review summarizes recent data that has emerged from molecular genetic, biochemical and electrophysiological studies on these fascinating two new proteins and their involvement in epithelial magnesium transport.

Genetics of hereditary disorders of magnesium homeostasis

Magnesium plays an essential role in many biochemical and physiological processes. Homeostasis of magnesium is tightly regulated and depends on the balance between intestinal absorption and renal excretion. During the last decades, various hereditary disorders of magnesium handling have been clinically characterized and genetic studies in affected individuals have led to the identification of some molecular components of cellular magnesium transport. In addition to these hereditary forms of magnesium deficiency, recent studies have revealed a high prevalence of latent hypomagnesemia in the general population. This finding is of special interest in view of the association between hypomagnesemia and common chronic diseases such as diabetes, coronary heart disease, hypertension, and asthma. However, valuable methods for the diagnosis of body and tissue magnesium deficiency are still lacking. This review focuses on clinical and genetic aspects of hereditary disorders of magnesium homeostasis. We will review primary defects of epithelial magnesium transport, disorders associated with defects in Ca2+/ Mg2+ sensing, as well as diseases characterized by renal salt wasting and hypokalemic alkalosis, with special emphasis on disturbed magnesium homeostasis.

Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy

Hypomagnesemia affects insulin resistance and is a risk factor for diabetes mellitus type 2 (DM2) and gestational diabetes mellitus (GDM). Two single nucleotide polymorphisms (SNPs) in the epithelial magnesium channel TRPM6 (V1393I, K1584E) were predicted to confer susceptibility for DM2. Here, we show using patch clamp analysis and total internal reflection fluorescence microscopy, that insulin stimulates TRPM6 activity via a phosphoinositide 3-kinase and Rac1-mediated elevation of cell surface expression of TRPM6. Interestingly, insulin failed to activate the genetic variants TRPM6(V1393I) and TRPM6(K1584E), which is likely due to the inability of the insulin signaling pathway to phosphorylate TRPM6(T1391) and TRPM6(S1583). Moreover, by measuring total glycosylated hemoglobin (TGH) in 997 pregnant women as a measure of glucose control, we demonstrate that TRPM6(V1393I) and TRPM6(K1584E) are associated with higher TGH and confer a higher likelihood of developing GDM. The impaired response of TRPM6(V1393I) and TRPM6(K1584E) to insulin represents a unique molecular pathway leading to GDM where the defect is located in TRPM6.

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders.