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Burns and Allen Research Institute (V.K., J.S.A.) and Division of Endocrinology, Diabetes and Metabolism (V.K., J.S.A., J.E.G., R.K.R.), University of Southern California, School of Medicine, Los Angeles, California 90033; Division of Human Genetics (X.G., D.H.C.), Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048; and Corning Nichols Institute (M.R.P.), San Juan Capistrano, California 92690
Address all correspondence and requests for reprints to: Robert K. Rude, M.D., University of Southern California, 1975 Zonal Avenue, GN#6602, Los Angeles, California 90089-9317. E-mail: rrude60075{at}aol.com
Abstract
Isolated hereditary renal magnesium (Mg) wasting may result from mutations in the renal tubular epithelial cell tight junction protein paracellin-1 gene or the tubular Na(+),K(+)-ATPase 
-subunit gene FXYD2. The FXYD2 gene mutation was discovered in two Dutch families as an autosomal dominant disorder. It is characterized by isolated renal Mg wasting with resultant symptomatic hypomagnesemia. The defective FXYD2 gene in these families mapped to chromosome 11q23. Here, we describe an American family with a similar phenotype but without linkage to the 11q23 locus; in testing 22 individuals in the pedigree multipoint LOD scores for five different loci from the 11q23 region were equal to -2.97. Compared with unaffected family members and normal controls, affected family members harbored significant reductions in the serum and lymphocyte Mg concentrations and in the serum immunoreactive PTH level with a 4-fold increase in the mean fractional urinary Mg excretion rate during a normomagnesemic clamp. Bone mineral density at the lumbar spine and proximal femur was significantly reduced in affected family members. In conclusion, our data demonstrate locus heterogeneity for the phenotype of isolated renal Mg wasting with hypomagnesemia and suggest that hypomagnesemia, at least in this pedigree, may be associated with low bone mass.
MAGNESIUM (Mg) IS the second most abundant intracellular cation in the body. Its principal functions include regulation of enzyme activity, control of various calcium and potassium channels, and promotion of membrane stabilization. Mg depletion has been associated with a number of clinical conditions including hypocalcemia, hypokalemia, neuromuscular excitability, cardiac arrhythmias, hypertension, atherosclerotic disease, and osteoporosis (1, 2). Hypomagnesemia is a common finding in hospitalized patients, up to 10% of overall hospital admissions and 70% of intensive care unit admissions (2). Mg depletion is usually an acquired disorder resulting either from deficient oral intake or accelerated urinary or intestinal loss. Renal Mg wasting is commonly caused by drug therapy (i.e. diuretics, aminoglycosides, immunosuppressive agents), alcohol, and osmotic diuresis (i.e. diabetes mellitus).
Inherited forms of isolated Mg deficiency are rare and, like acquired forms, may also be of either intestinal or renal origin. Two familial forms of primary renal hypomagnesemia have been described. One form [Mendelian Inheritance in Man (MIM) no. 248250] is inherited in an autosomal recessive fashion and results from mutations in the paracellin-1 gene (PCLN1) on chromosome 3 (3). The second is an autosomal dominant form of hypomagnesemia (MIM no. 154020) termed "isolated renal magnesium wasting." It was originally described in two unrelated Dutch families by Geven et al. (4) in 1987. Meij et al. (5) successfully linked this syndrome to chromosome 11q23 and subsequently identified a specific mutation on the Na+,K(+)-ATPase -subunit of gene FXYD2 (6).
Here, we describe a new family with an apparently autosomal dominant form of primary hypomagnesemia. Although clinically similar to MIM no. 154020 in terms of a renal Mg wasting phenotype, linkage studies have excluded the region of 11q23 containing the FXYD2 gene, providing evidence of locus heterogeneity for this phenotype.
Family and Methods
Clinical assessment
The index case is a 36-yr-old female who first presented at the age of 33 with complaints of paresthesias, muscle weakness, and the onset of seizures. Evaluation of serum and urine biochemical indices showed hypomagnesemia and hypermagnesuria. Despite aggressive therapy with both oral Mg supplements and parenteral Mg administration (up to 48 mEq per day) as well as with 1,25-dihydroxyvitamin D [1,25(OH)2D] or Rocaltrol (0.5 µg per day), serum Mg remained low and the urinary Mg excretion rate high.
After obtaining written informed consent, the index case, 21 members of her family, and five normal volunteers were screened for the presence of Mg depletion. In addition to the index case, frankly low serum Mg levels were found in a total of six additional subjects in the family of the index case. Fifteen family members (including all seven hypomagnesemic subjects) and five normal volunteers were admitted and subjected to provocative Mg infusion testing in the General Clinical Research Center (GCRC) at the USC+LAC Medical Center in Los Angeles, California. Subjects were admitted to the GCRC for 2 d. Renal ultrasound, knee radiographs, and bone mineral density of the lumbar spine, determined by dual-energy x-ray absorptiometry using a Hologic 2000 bone densitometer (Hologic, Inc., Waltham, MA), were performed in all subjects.
On the morning of the first in-hospital day, a 24-h urine collection was begun for Mg, calcium, creatinine, amino acids, and type I collagen fragments. On the morning of the second day, fasting blood samples were drawn for serum Mg, lymphocyte Mg, calcium, bicarbonate, potassium, chloride, immunoreactive PTH (iPTH), 25-hydroxyvitamin D [25(OH)D], 1,25(OH)2D, bone-specific alkaline phosphatase, and osteocalcin and for plasma amino acids. A baseline, 2-h second void urine collection was also obtained on the second in-hospital day. Fractional urinary Mg excretion was then calculated before and during a 4-h iv Mg infusion (0.2 mEq/kg body weight as MgSO4·7H2O). Four 1-h urine samples were collected, and blood for serum Mg was drawn at baseline and at the end of each 1-h urine collection period. Fractional urinary Mg excretion measurement as described by us (7) commenced when the serum ultrafilterable Mg at baseline and/or during the Mg infusion was within the normal range. Ultrafilterable serum Mg was presumed to be 74% of the total serum Mg concentration (7). Vital signs (blood pressure and pulse) were obtained at baseline and hourly during the infusion. An electrocardiogram was obtained before and at the end of the Mg infusion.
Serum, urine, and lymphocyte Mg was determined by atomic absorption absorptiometry (8). Lymphocyte protein was determined by the method of Lowry. Serum and urine creatinine and electrolytes were determined by Autoanalyzer (Lexington, MA). Serum iPTH was determined by a two-site immunoradiometric assay for intact PTH (Quest Laboratories, San Juan Capistrano, CA). The serum 25(OH)D was assessed by competitive protein binding assay and serum 1,25(OH)2D by radioreceptor assay (Quest Laboratories). Serum osteocalcin and bone-specific alkaline phosphatase was determined by immunoradiometric assay (Quest Laboratories). The concentration of N-telopeptide of type I collagen in the urine was determined by enzyme immunoassay and urine deoxypyridinoline by high-performance liquid chromatography (Quest Laboratories). Plasma and urine amino acids were separated and analyzed on a Beckman 6300 analyzer (Beckman Coulter, Inc., Fullerton, CA) in the Harbor-UCLA GCRC Mass Spectrometry Core Facility (9).
Genetic analysis
In addition to those 15 family members who underwent the Mg loading test, blood cell samples were obtained from seven other unaffected family members who resided elsewhere. Hence, DNA was available for genotyping from 22 individuals in the pedigree. Genotypes were determined at markers for loci from the primary hypomagnesemia region at chromosome 11q23 (6). Marker order and distances within the region were determined using information from the genome databases: cen - D11S4092 - 0.1 cM - D11S4127 - 1.1 cM - D11S4195 - 0.2 cM - D11S1356 - 0.8 cM - D11S4104 - tel. Multipoint LOD scores were calculated using the program GENEHUNTER 2.0 (Wartzman Group Inc., Frederick, MD) (10). We assumed an autosomal dominant mode of inheritance, and the disease allele frequency was set at 0.001 (i.e. a disease prevalence of about 2/1000). LOD scores were calculated under six different penetrance models with the penetrance ranging from 0.5–1.0.
Statistical analysis
Comparisons among groups were made using t test. Values are expressed as the mean ± SD.
Results
Biochemistry
The serum Mg concentration was significantly reduced in the seven affected family members compared with the nine unaffected family members studied in the GCRC (Table 1
); this included the index case, three of her four children, her sister, and two of her sister’s offspring (see pedigree, Fig. 1). The mean serum Mg concentration of those family members whose blood samples were obtained elsewhere were all normal; the mean of all 15 unaffected family members was 0.81 ± 0.07. Total lymphocyte Mg content was determined in the seven affected and four unaffected subjects (Table 1). Compared with unaffected subjects, lymphocyte Mg was lower in affected subjects, suggesting a total body Mg deficit in those individuals. Fractional urinary Mg excretion was assessed in 15 family members. The mean fractional clearance of Mg was significantly elevated in the seven hypomagnesemic family members compared with unaffected family or normal subjects (Table 1), documenting renal Mg wasting as the cause of the hypomagnesemia. No difference was observed in the mean serum calcium (Table 1) or 25(OH)D and 1,25(OH)2D concentrations (data not shown) between affected and unaffected family members. The mean serum iPTH level was lower in affected subjects compared with unaffected family members. There was a trend for lower 24-h urinary calcium in the affected family members, however, no difference was observed when urinary calcium excretion was expressed as mg/100 ml/GFR/1.73 m2 or mg/kg body weight (Table 1). No difference was noted either in the serum or the urine values of chloride, bicarbonate, potassium, and amino acids; all were within the range of normal.
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Markers of bone turnover were assessed in the seven affected and in eight unaffected family members. Values for serum osteocalcin and serum bone-specific alkaline phosphatase, as well as for the fractional urinary excretion rates of deoxypyridinoline and N-telopeptides of type I collagen, were not different between affected and unaffected family members; children and adolescents in both groups had appropriately elevated levels of all markers of bone turnover compatible with age-appropriate skeletal growth and development. Dual-energy x-ray absorptiometry Z-score (SD from the mean for age- and gender-matched controls) at the lumbar spine in affected family members was significantly lower than in unaffected family members (Table 1
). Because a normative database for proximal femur for age less than 20 yr was not available at the time of this study, Z-scores were not available for five affected and one unaffected family members. However, in two affected adults the mean Z-score of -0.69 ± 0.11 was significantly less than three unaffected members, Z = -0.09 ± 0.23 (P = 0.04). There were no spine fractures noted in the affected family members.
Renal ultrasound and knee radiographs
Renal ultrasound was performed on all seven affected and eight unaffected family members. There was no evidence of nephrocalcinosis in any subject. Duplication of the collecting system was noted in one affected and two unaffected members of the pedigree. X-Ray of the knees was performed in the seven affected and in eight unaffected family members. Chondrocalcinosis was observed only in the index case.
Cardiovascular
There was no significant difference in supine blood pressure between affected and unaffected subjects. No subject was hypertensive. The electrocardiogram was normal in all subjects. Parenteral Mg infusion resulted in shortening of the Q–T interval in affected subjects.
Genetic analysis
Examination of the pedigree (Fig. 1) suggested that the hypomagnesemia phenotype was likely to be inherited in an autosomal dominant fashion. However, because we did not observe male to male transmission, an X-linked dominant mode of inheritance cannot be ruled out. In addition, neither I-1 nor I-2 were affected. Their unaffected status was supported by normal Mg infusion studies. Under an autosomal dominant model, the best explanations for the unaffected status of I-1 and I-2 include nonpenetrance or germ-line mosaicism for a dominant mutation.
Genotypes were determined at markers for loci from the primary hypomagnesemia region on chromosome 11, and haplotypes were constructed by parsimony (Fig. 1). Assuming an autosomal dominant model, inheritance of different, apparently nonrecombinant, parental haplotypes by the two affected individuals in generation II excluded linkage to the region. Similarly, the two affected offspring of II-9 inherited different maternal haplotypes, thereby excluding linkage to the region. Multipoint LOD scores for markers at loci from the primary hypomagnesemia region at chromosome 11q23 are shown in Table 2. An autosomal dominant inheritance pattern, at various levels of penetrance, was assumed. Because the LOD scores for markers D11S4092, D11S4127, D11S4195, D11S1356, and D11S4104 were always less than -2.97, regardless of the level of penetrance specified, the region of chromosome 11 containing these loci was excluded.
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Despite the abundance and physiological importance of the cation, Mg metabolism is poorly understood. Only 3% of filtered Mg appears in the urine, suggesting that renal reabsorption is the primary mechanism for Mg homeostasis (11). The major sites of Mg reabsorption in the kidney are the proximal tubule, the thick ascending loop of Henle, and the distal tubule, accounting for 25–30%, 65–70%, and up to 5% of the Mg reabsorbed, respectively (12). Renal handling of Mg is influenced by a combination of extracellular volume, extracellular cation concentration, and changes in voltage across the tubules (13). The relative state of Mg directly affects its own reabsorptive processes in the kidney (12). Excess Mg intake (i.e. beyond normal dietary requirements) leads to increased excretion of Mg into the urine, whereas Mg deprivation leads to a marked decrease in urinary Mg excretion. These findings have led to the description of a renal tubular maximum for Mg (7). Alteration of this renal Mg threshold can lead to renal Mg wasting (i.e. a Mg leak).
Both intestinal and renal forms of familial Mg deficiency have been described. Of these, an autosomal recessive disorder due to a defect in intestinal Mg absorption (MIM no. 307600) was linked to chromosome 9 (14). This form is associated with severe hypomagnesemia and secondary hypocalcemia and presents early in life. It is characterized by normal renal Mg excretion. On the other hand, inherited forms of renal Mg wasting are beginning to inform us of the actual transport mechanisms for Mg. Based on their mode of inheritance and biochemical phenotype, several different forms of primary renal Mg wasting have been described. The first of the following is the only one with truly isolated renal Mg wasting. The second disorder is associated with hypercalciuria. In the remainder, renal Mg wasting is associated with multiple defects in renal transport.
The first form of renal Mg wasting, primary isolated renal hypomagnesemia, can be familial or sporadic (4). Hypomagnesemia appears to be the major finding, although low serum potassium has been reported. The familial cases exhibit an autosomal dominant inheritance pattern (MIM no. 154020). Clinically, this is the mildest of the three groups, with tetany and convulsions reported in only the most severely affected individuals. Onset is usually in childhood to early adult life. In one family the gene defect has been mapped to chromosome 11q23 (5) and determined to be due to mutations in the Na+,K(+)-ATPase -subunit gene FXYD2 (6). The Na+,K(+)-ATPase resides in the basolateral membrane of the epithelial cells in the distal renal convoluted tubule and functions to maintain an electrochemical gradient favoring the paracellular and transcellular transport of Mg from the urine to the blood.
The second form of renal Mg wasting is inherited in an autosomal recessive fashion (MIM no. 248250). It is characterized clinically by hypomagnesemia, hypercalciuria, and secondary hyperparathyroidism (3); the latter may lead to nephrocalcinosis and renal failure (15). The clinical phenotype in these patients is rescued with renal transplantation, suggesting an endogenous defect in the kidney (15). Other clinical problems observed in the first group include tetany, polydipsia and polyuria, ocular abnormalities, defects in renal acidification, oligospermia, and chondrocalcinosis. Onset occurs in infancy or early childhood. A recent report demonstrated that a genetic defect in paracellin-1 on chromosome 3q, a renal tight junction protein required for paracellular Mg resorption in the thick ascending loop of Henle, is responsible for this disorder (3, 15).
The third form of renal Mg wasting has been coined the "familial hypokalemia-hypomagnesemia syndrome" or "Gitelman syndrome" (MIM no. 263800). This is an autosomal recessive disorder due to a genetic defect of the thiazide-sensitive NaCl cotransporter gene on chromosome 16 (16); mutations in this cotransporter result in the failure to reclaim NaCl from the distal nephron and its loss in the urine. The clinical phenotype in these patients is very similar to that described for patients with hypovolemic, hypokalemic alkalosis (Bartter syndrome), excepting that Gitelman subjects also suffer from hypocalciuria and hypermagnesuric hypomagnesemia (17). About 30% of patients with Bartter syndrome (MIM no. 241200) may also show hypomagnesemia, but it is usually associated with hypercalciuria (reviewed in Ref. 11).
The family described here is distinguished from patients with the autosomal recessive paracellin-1 gene mutation; patients with the latter disease suffer from ocular abnormalities and hypercalciuria, leading to nephrolithiasis and nephrocalcinosis (3). Furthermore, the mode of inheritance appears to be autosomal dominant, not recessive, and neither ocular defects nor hypercalciuria are a feature of the pedigree reported here. The family studied here is also phenotypically distinct from families with Gitelmen’s syndrome. Neither the index case nor affected family members were hypotensive, hypovolemic, hypokalemic, or alkalotic. Finally, although it is possible that the family reported here represents an unusual variant of familial hypomagnesemia with secondary hypocalcemia (MIM no. 307600) (14), in subjects with this disorder Mg is presumed eliminated in the bowel, with Mg excretion in the urine being normal. The family described here is most similar to the previously described first group of families with the autosomal dominant form of primary hypomagnesemia. The index case presented with symptomatic hypomagnesemia complicated by seizures. Because of similar phenotypes between the described family and families with autosomal dominant isolated renal hypomagnesemia, we evaluated this family for linkage to the region of chromosome 11 containing the FXYD2 gene. Linkage studies (see Fig. 1) excluded the primary isolated renal hypomagnesemia locus on chromosome 11, including the genetic interval containing the FXYD2 gene.
The data presented here demonstrate locus heterogeneity for the hypomagesemia phenotype and suggest that there is yet another gene, in addition to the already recognized paracellin-1 gene; Na+,K(+)-ATPase -subunit FXYD2 gene, and the thiazide-sensitive NaCl cotransporter gene, the product of which participates in the reclamation of Mg from the urine. The finding of lower bone mineral density in our patients with renal Mg wasting is a new observation. Epidemiological studies have linked a low dietary Mg intake with low bone mass (1, 18), and dietary Mg depletion in animal models has resulted in osteoporosis due to decreased bone formation and increased bone resorption (19). The mechanism(s) responsible for these alterations in bone metabolism is unclear. Whether the low bone mass observed in the family presented here is unique or part of the hypermagnesiuric phenotype remains to be determined. Nonetheless, the collective data also underscore the principal role of the kidney in maintenance of human Mg homeostasis.
Acknowledgments
We are grateful to the nursing staff of the USC+LAC GCRC for care of the index case and her family members.
Footnotes
This work was supported by General Clinical Research Center Grants RR00043 and RR00425 from the National Institute for Research Resources.
Abbreviations: 1,25(OH)2D, 1,25-Dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D; GCRC, General Clinical Research Center; iPTH, immunoreactive PTH; Mg, magnesium; MIM, Mendelian Inheritance in Man.
Received March 28, 2001.
Accepted August 21, 2001.
References
-subunit. Nat Genet 26:265–266[CrossRef][Medline]
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