Novel TRPM6 Mutations in 21 Families with Primary Hypomagnesemia and Secondary Hypocalcemia Karl P. Schlingmann,* Martin C. Sassen,* Stefanie Weber,*† Ulla Pechmann,* ʈ Kerstin Kusch,* Lutz Pelken,* Daniel Lotan,‡ Maria Syrrou,§ Jeffrey J. Prebble, David E.C. Cole,¶ Daniel L. Metzger,# Shamima Rahman,** Toshihiro Tajima,†† San-Ging Shu,†† Siegfried Waldegger,* Hannsjoerg W. Seyberth,* and Martin Konrad*§§ *Department of Pediatrics, Philipps-University Marburg, Marburg, Germany; †Pediatric Nephrology, University Children’s Hospital Heidelberg, Heidelberg, Germany; ‡Pediatric Nephrology, The Chaim Sheba Medical Center, Tel- Hashomer, Israel; §Laboratory of General Biology, Medical School, University of Ioannina, Ioannina, Greece; ʈ Department of Pediatrics, Toowoomba Base Hospital, Toowoomba, Australia; ¶Department of Laboratory Medicine & Pathobiology, University of Toronto, Ontario, Canada; #Pediatric Endocrinology, British Columbia Children’s Hospital, Vancouver, British Columbia, Canada; **Hospital for Children Great Ormond Street, London, United Kingdom; ††Department of Pediatrics, Hokkaido University Medical School, Sapporo, Japan; ‡‡Department of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan; and §§Pediatric Nephrology, University Children’s Hospital, Inselspital Bern, Switzerland 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. J Am Soc Nephrol 16: 3061–3069, 2005. doi: 10.1681/ASN.2004110989 rimary hypomagnesemia with secondary hypocalcemia expected finding of hypoparathyroidism is thought to result (HSH) was first described by Paunier et al. (1). Patients from an inhibition of PTH synthesis and secretion induced by P usually present in early infancy with generalized con- extreme hypomagnesemia (2). vulsions or signs of increased neuromuscular excitability. Lab- Contrasting all other known forms of hereditary hypomag- oratory evaluation at manifestation reveals severely reduced nesemia, pathophysiologic studies in affected patients using serum magnesium levels accompanied by hypocalcemia and radioactive magnesium isotopes pointed to a primary defect in barely detectable parathyroid hormone (PTH) levels. The un- intestinal magnesium absorption (3,4). The presence of an ad- ditional renal magnesium leak in HSH was controversially discussed (4–6). By using a DNA pooling strategy, Walder et al. (7) had Received November 22, 2004. Accepted July 11, 2005. mapped a gene locus for HSH on chromosome 9q22. Recently, mutations in the TRPM6 gene have been identified as the Published online ahead of print. Publication date available at www.jasn.org. underlying genetic defect in patients with HSH (8,9). Address correspondence to: Dr. Karl Peter Schlingmann, University Children’s Hospital Marburg, Deutschhausstrasse 12, 35037 Marburg, Germany. Phone ϩ49- TRPM6 codes for TRPM6, a new member of the transient 6421-286-2483; Fax: ϩ49-6421-286-5724; E-mail [email protected] receptor potential (TRP) family of cation channels. Within the Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1610-3061 3062 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 3061–3069, 2005 TRP family, TRPM6 belongs to the TRPM subfamily, whose the only naturally occurring human knockout for a member eight members exhibit a significant diversity in domain struc- of the TRPM family described so far, the mutational analysis ture as well as cation selectivity and activation mechanisms of affected individuals together with the functional analysis (10). TRPM6 is closely related to TRPM7, both sharing the of mutations in heterologous expression systems represents unique feature of a serine/threonine kinase domain c-termi- an attractive approach to gain further insight into TRPM ion nally fused to their ion channel domain (11). channel function. Here, we present comprehensive genotypic The ubiquitously expressed TRPM7 has been characterized data on 28 patients with HSH and provide detailed informa- as a constitutively active ion channel that is permeable for a tion regarding phenotypic presentations and clinical courses variety of divalent cations, including calcium and magnesium, for this genetically characterized cohort. whose basal activity is regulated by intracellular levels of mag- nesium and Mg-ATP (12,13). TRPM7 was shown to play a Materials and Methods crucial role in cellular magnesium homeostasis (14). Patients and Families In contrast to TRPM7, the expression pattern of TRPM6 seems Twenty-one families that have HSH, are of different ethnic origin, and to be more confined, with expression mainly along the gastroin- have 28 affected individuals were analyzed (Table 1). Family pedigrees are testinal tract as well as in kidney predominantly in the distal shown in Figure 1. Parental consanguinity was noted in 12 families, and convoluted tubule (DCT) (8), where it is presumed to be involved six families had two or more affected siblings. Families F1 to F5 were in the apical entry of magnesium into epithelial cells (15). Func- included in the original report on the involvement of TRPM6 in HSH by our group (8). Clinical aspects of families F4, F5, F6, F7, F15, and F17 were tional data on TRPM6 are contradictory. Whereas one study suc- reported previously (6,17–21). For all patients, the diagnosis was based on ceeded in heterologous expression of TRPM6 in mammalian cells manifestation in early infancy with generalized convulsions or muscular and showed channel properties similar to those observed for tetany, profound hypomagnesemia at the time of presentation accompa- TRPM7 (15), another study failed to detect measurable currents nied by hypocalcemia, and relief of clinical symptoms and normocalcemia upon TRPM6 expression (16). Instead, it was demonstrated that upon administration of magnesium salts. heteromultimerization with TRPM7 is essential for correct mem- brane targeting of TRPM6. In this study, TRPM7-induced currents Mutation Analysis were significantly increased by co-expression of TRPM6. Extraction of DNA from blood leukocytes was performed using As TRPM6 mutations that are found in patients with HSH are standard protocols. TRPM6 mutational screening was performed by Table 1. Clinical data of genetically characterized patients with HSHa Initial Initial Oral 2ϩ 2ϩ Mg under Age at Age at Follow- Symptoms at Serum Serum Mg FeMg Additional Patient Gender Manifestation Diagnosis Up (yr) Manifestation Mg2ϩ Ca2ϩ (mmol/ Therapy (%) Diarrhea Findings (mM) (mM) kg per d) (mM) F1.1 F 2 mo 2 mo 4 Seizures 0.21 1.63 1.03 0.59 2.6 Ϫ F2.1 M 3 wk 6 yr 10 Seizures ND 1.29 0.62 0.57 2.8 Ϫ Mental retardation F3.1 F 4 mo 4 mo 3 Seizures 0.10 2.50 1.35 0.55 3.6 ϩ F4.1 M 5 wk 5 wk 25 Seizures 0.41 1.88 3.10 ? ? ϩ F5.1 F 5 wk 5 wk 14 Seizures 0.17 1.50 0.80 0.55 5.1 ? F5.2 F 5 wk 5 wk 13 Seizures 0.22 1.60 0.75 0.55 2.1 ? F6.1 M 5 mo 5 mo 15 Seizures 0.15 1.94 0.49 0.86 3.9 Ϫ F6.2 M 5 wk 5 wk 12 Seizures 0.22 1.73 0.42 0.85 ? Ϫ Cardiac arrhythmia F7.1 M 6 wk 10 mo 25 Seizures 0.21 1.63 3.00 0.60 ? ϩ F8.1 F 3 mo 3 mo 8 Seizures ND 1.74 0.41 0.60 ? Ϫ F9.1 F 2 mo 2 mo 3 Tetany 0.20 1.31 0.56 0.56 ? ϩ Failure to thrive F10.1 F 6 wk 6 wk 15 Seizures ND ? 0.55 0.51 2.4 ϩ F11.1 F 2 mo 2 mo 6 Seizures 0.10 1.66 3.00 0.44 ? ϩ Failure to thrive F11.2 M 4 mo 4 mo 3 Seizures 0.19 ? 3.90 ? ? ϩ Failure to thrive F12.1 M 3 mo b 7 Seizures 0.09 1.60 0.54 0.33 ? Ϫ F12.2 M 4 mo b 3 Asympc 0.16 1.75 0.94 0.53 ? Ϫ F13.1 F 6 mo 6 mo 10 Seizures 0.30 1.75 ? 0.61 3.2 Hyperactivity F13.2 F ? ? 8 Asympc ?? ? ? ?ϩ F13.3 M ? ? 2 Asympc ?? ? ? ?ϩ F14.1 M 7 mo 7 mo 1.5 Seizures 0.29 1.60 0.63 0.50 5.1 ϩ F15.1 F 3 wk 8 wk 28 Seizures 0.20 1.35 1.02 0.78d ? ϩ F16.1 F 5 wk 5 wk 6 Seizures 0.29 1.45 1.73 0.49 4.9 ϩ Failure to thrive F17.1 M 2 wk 2 yr 14 Seizures 0.37 ? ? 0.58 2.5 ϩ Mental retardation F17.2 F 4 mo 4 mo 9 Seizures ? ? ? ? ? ? F18.1 F 4 wk 4 wk 0.5 Seizures 0.44 1.70 2.47 0.45 14.3 ? F19.1 M 3 mo 4 mo 0.5 Seizures 0.10 1.45 2.00 0.50 3.7 ? F20.1 F 2 mo 2 mo 18 Seizures ? ? 0.71 0.62 ? Ϫ F21.1 F 3 wk 3 wk 3 Seizures 0.20 1.72 0.93 0.52 ? Ϫ a F, female; M, male; asymp, asymptomatic; ND, not detectable; FeMg, fractional excretion of magnesium.