Exclusion of mutations in FXYD2, CLDN16 and SLC12A3 in two families with primary renal Mg2+ loss

Iwan C. Meij1, Lambert P. W. J. van den Heuvel1, Sies Hemmes2, Walter A. van der Vliet1, Johannes L. Willems3, Leo A. H. Monnens1 and Nine V. A. M. Knoers4,

1 Department of Pediatrics, 3 Department of Clinical Chemistry and 4 Department of Human Genetics, University Hospital Nijmegen, Nijmegen and 2 Department of Pediatrics, Gelderse Vallei Ziekenhuis Ede, The Netherlands



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion and conclusions
 References
 
Background. Based on genetic studies in families with hereditary renal Mg2+ reabsorption disorders, several genes were shown to be involved in renal Mg2+ transport. Mutations in the CLDN16 gene were found to underlie autosomal recessive hypomagnesaemia associated with hypercalciuria and nephrocalcinosis. The FXYD2 gene was implicated in autosomal dominant renal Mg2+ wasting associated with hypocalciuria. Mutations in the SLC12A3 gene, also known as NCC, cause Gitelman's syndrome. In addition to hypokalaemic metabolic alkalosis, hypomagnesaemia associated with hypocalciuria is considered to be a hallmark feature of this latter disorder.

Methods. We have characterized a new family with presumed dominant renal hypomagnesaemia by detailed clinical examination and mutation analysis of CLDN16, FXYD2 and SLC12A3. In addition, we have performed mutation analysis of these three genes in a previously described family with autosomal recessive renal Mg2+ wasting. In this family, linkage analysis was performed with polymorphic markers in the vicinity of the FXYD2 gene.

Results. The phenotype of the new family closely resembles that of the known dominant families with a mutation in FXYD2, but mutations in this gene were not identified in the new family. No mutations were found in CLDN16 and SLC12A3 either. Sequencing of the three genes in the patients of the recessive family revealed no mutations. In addition, haplotype analysis excluded linkage to the FXYD2 region on chromosome 11q23.

Conclusion. Our results indicate that, in addition to the currently known loci involved in renal Mg2+ handling, at least one other gene must be involved.

Keywords: CLDN16; FXYD2; hypomagnesaemia; magnesium; renal; SLC12A3



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion and conclusions
 References
 
Hereditary renal Mg2+ wasting comprises a group of disorders characterized by hypomagnesaemia. The first of these is associated with hypercalciuria and nephrocalcinosis and is inherited as an autosomal recessive trait (HHN, OMIM 248250). Ocular abnormalities are often seen in patients; other symptoms reported in HHN patients are urinary tract infections, nephrolithiasis, hyperuricaemia and incomplete tubular acidosis [1]. In HHN, mutations in the CLDN16 gene encoding the tight junction protein CLAUDIN16 have been found to be responsible for the renal magnesium loss [2].

The second disorder features a dominant mode of inheritance of primary renal Mg2+ loss (OMIM 154020). It is associated with lowered urinary Ca2+ excretion and, at adult age, chondrocalcinosis is frequently seen. In serum, Mg2+ concentrations can be as low as 0.39 mmol/l. Electrolyte concentrations, including Ca+, Na+, K+, Cl-, bicarbonate are in the normal range, as are plasma renin activity and plasma aldosterone [3]. In a neonate with this disorder the serum Mg2+ concentration was even found to be below 0.2 mmol/l [4]. In dominant renal Mg2+ loss the FXYD2 gene, encoding the Na+,K+-ATPase {gamma}-subunit, was shown to be mutated [5]. The Na+,K+-ATPase maintains both concentration and electrochemical gradients along the kidney nephron, which are very important for ion transport. The {gamma}-subunit shows tissue-specific expression and has a modulatory role in Na+,K+-ATPase function [68]. {gamma}-Subunit expression was shown to be most abundant in the thick ascending limb and distal convoluted tubule [8].

In addition to CLDN16 and FXYD2, mutations in the SLC12A3 gene give rise to a phenotype with lowered serum Mg2+ concentrations as well. In humans, these mutations lead to autosomal recessive Gitelman's syndrome [9] (GS, OMIM 263800) which is characterized by hypokalaemic metabolic alkalosis and hypomagnesaemia with hypocalciuria. Sometimes, similar to the situation in autosomal dominant hypomagnesaemia, chondrocalcinosis is seen in these patients.

The current knowledge on genes involved in renal Mg2+ handling definitely facilitates the genetic screening of patients with a hereditary Mg2+ reabsorption disorder.

We recently recruited a new family (Pedigree I, Figure 1AGo) with renal magnesium loss associated with hypocalciuria. This family and another family with primary renal Mg2+ loss, first described by Geven et al. [10], were submitted to genetic analysis (Pedigree II, Figure 1BGo). Here the results are presented of mutation analysis of CLDN16, FXYD2 and SLC12A3 in these two families.



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Fig. 1.  (A) Pedigree of Family I. (B) Pedigree of Family II (from Geven et al. [10]). The probands are indicated with an arrow. Consanguinity is indicated by double lines connecting the parents.

 



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion and conclusions
 References
 
Families
Informed consent was obtained from all family members involved in this study.

Family I (Figure 1AGo). The proband is the second child of non-consanguinous parents. She was evaluated during the first 2 years of life after having had three short attacks of loss of consciousness accompanied by alternating increase or loss of muscle tone, and staring. The EEG showed no signs of epilepsy. Close examination revealed a serum magnesium concentration of 0.51 mmol/l (normal 0.70–1.15 mmol/l).

At the age of 1 year and 3 months with Mg2+ supplementation, serum Mg2+ was 0.71 mmol/l and serum Ca2+ was 2.52 mmol/l (normal 2.15–2.60 mmol/l). Serum Na+ was 142 mmol/l (normal 136–146 mmol/l), serum K+ was 4.6 mmol/l (normal 3.5–5.1 mmol/l) and serum parathormone was 3 pmol/l (normal <10 pmol/l). Renal function was also normal as reflected in creatinine (44 µmol/l) (normal 45–90 µmol/l).

At 2 years of age and with oral Mg2+ supplementation, her serum Mg2+ was 0.64 mmol/l. No seizure-like symptoms have occurred since Mg2+ supplements were given and growth and development were normal. At nearly 6 years of age, urinary Ca2+ excretion was measured and found to be 0.02 Ca2+/Creat molar ratio (normal 0.04–0.8). Urinary Mg2+ excretion was 0.5 Mg2+/Creat molar ratio (normal 0.3–1.0) which, considering the low serum Mg2+ value (0.58 mmol/l), indicates a renal loss of Mg2+. Investigation of the parents revealed normal serum Mg2+ in the mother (0.73 mmol/l) but severely lowered serum Mg2+ in the father (0.46 mmol/l). Detailed study of the father under non-supplemented conditions, revealed a urinary magnesium excretion of 0.1–0.2 Mg2+/Creat molar ratio (normal 0.2–0.3), which, considering the low serum Mg2+ value, indicates a renal loss of Mg2+. Urinary Ca2+ was consistently in the low normal range (0.05–0.1 Ca2+/Creat molar ratio) (normal 0.06–0.45) as was also seen for some of the patients in the dominant renal hypomagnesaemia family described by Geven et al. [3]. Serum Ca2+ (2.31 mmol/l) and PO42- (1.03 mmol/l) were normal.

At 15 years of age, the father once had an ‘attack’ resulting in stiffening of leg and facial muscles. At the age of 34 years he had a second attack, this time accompanied by headache, tremor, difficulty with talking (wrong words) and palpitations for about an hour. The family of the father was investigated in more detail, and both his two sisters and his parents had normal serum magnesium values. It appears this is a new case of renal hypomagnesaemia associated with hypocalciuria, which started in the father of the proband. The inheritance appears to be dominant, but since the grandparents and the mother of the proband might be carriers, pseudodominance can not be excluded.

Family II (Figure 1BGo). In this family, two Dutch sisters suffered from primary renal Mg loss associated with normocalciuria (OMIM 248250). These girls were the offspring of a consanguinous mating and since both parents were healthy, the inheritance pattern was likely to be autosomal recessive. The clinical data of this family have previously been described in detail by Geven et al. [10]. Briefly, the two patients of this family had serum Mg2+ levels of 0.56 and 0.53 mmol/l and urinary Mg2+ values of 2.9–3.7 mmol/h and 3.9–6.6 mmol/24 h, respectively. Considering the low serum Mg2+ levels, the urinary Mg2+ excretion is too high, indicating a renal Mg2+ reabsorption defect. They both had convulsions in their first year of life and showed mild psychomotor retardation during childhood. Both serum and urinary Ca2+ were in the normal range, as were serum Na+, K+, Cl-, bicarbonate and blood pH. Plasma renin activity and plasma aldosterone concentrations were in the normal range in both patients.

Mutation detection
Samples of peripheral blood were taken and genomic DNA was isolated using standard procedures.

FXYD2 was amplified by EXPAND PCR (Boehringer Mannheim) according to the manufacturers' instructions at 68°C for 30 cycles using the forward primer for exon 1b and the reverse primer for exon 6 (Table 1Go) using 50 ng of genomic DNA as template. From the resulting 4.5 kb genomic fragment, exons 1–6 were sequenced using the primers listed in Table 1Go. The alternative first exon 1a [11] was amplified separately using the primers listed in Table 1Go at 55°C for 35 cycles at 1.5 mM MgCl with an elongation step of 30 s at 72°C and denaturation for 30 s at 94°C using Taq polymerase (Gibco). The obtained PCR product includes ~200 bp of putative promotor sequence prior to exon 1a. Using the same conditions, ~1000 bp of putative promotor sequence before exon 1b were investigated (for primers, see Table 1Go). Each of the exons of the CLDN16 and the SLC12A3 gene were amplified separately from genomic DNA, using the primers listed in Tables 2Go and 3Go, respectively. Sequencing was carried out using the same primers. The conditions for the amplification of CLDN16 exons were: annealing for 30 s at the temperature listed in Table 2Go for 35 cycles in the presence of 1.5 mM MgCl2 with an elongation step of 30 s at 72°C and denaturation for 30 s at 94°C using Taq polymerase (Gibco). For the SLC12A3 gene: annealing for 45 s under the conditions listed in Table 3Go for 35 cycles in the presence of 2 mM MgCl2 (unless otherwise mentioned in the table) with an elongation step of 45 s at 74°C and denaturation for 30 s at 94°C using AmpliTaq Gold polymerase (Perkin-Elmer).


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Table 1.  Primers and conditions used for mutation detection of the FXYD2 gene

 

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Table 2.  Primers and conditions used for mutation detection of the CLDN16 gene

 

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Table 3.  Primers and conditions used for mutation detection of the SLC12A3 gene

 

DNA sequencing
Automatic dideoxy sequencing of all these genes was performed on an ABI377 using the ABI Prism Taq DyeDeoxy terminator cycle sequencing ready reaction kit (Perkin-Elmer).

Marker typing
Individuals from Family II were genotyped using several markers from the 11q23 region encompassing the FXYD2 gene as described previously [12]. Semi-automated genotyping was performed on an ABI 377 sequencer. Data were analysed by Genescan 2.1 software and Genotyper 2.0 software (Perkin-Elmer). The marker data were obtained from the 1996 Généthon map [13].



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion and conclusions
 References
 
In the probands of both families, the Na+,K+-ATPase {gamma}-subunit gene, FXYD2, was screened for mutations, including the alternatively spliced first exon. The 5'-UTR, exon–intron boundaries and the putative promotor sequence prior to each alternatively spliced first exon were included as well as the internal 83 bp repeat sequence. No mutations were found. Additionally, the CLDN16 and SLC12A3 genes were screened, also yielding no mutations in either family. Linkage of hypomagnesaemia to chromosome 11q23 could be excluded in Family II since the healthy and affected siblings shared identical haplotypes in this region (Figure 2Go).



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Fig. 2.  Linkage analysis in Family II with markers from the chromosome 11q23 region. Only the direct family of the proband is depicted. Affected and non-affected siblings share the same haplotype.

 



   Discussion and conclusions
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion and conclusions
 References
 
We have described a new family with renal hypomagnesaemia associated with hypocalciuria (Pedigree I). The inheritance pattern of the disorder appeared dominant, but could be pseudodominant as well. Pseudodominant inheritance has been shown previously for families with presumed dominant Gitelman's syndrome [14].

In addition to the genetic analysis of this family we also included a previously described family with renal recessive hypomagnesaemia associated with normocalciuria [10] in our study. In both families, we have performed mutation analysis in FXYD2, CLDN16 and SLC12A3 and did not find any mutations.

The symptoms of affected individuals in Pedigree I are very similar to those seen in the dominant renal hypomagnesaemia family in which a mutation in the Na+,K+-ATPase {gamma}-subunit gene, FXYD2, was identified. For this reason FXYD2 was the logical candidate for Family I. The only gene known to be involved in an autosomal recessive disease with primary renal Mg2+ loss is the CLDN16 gene and was therefore, a candidate for Family II.

The last gene included in the mutation analysis of both our families was SLC12A3. The main reason for this was the phenotype of a mouse lacking the mouse homologue of SLC12A3. In this animal, hypomagnesaemia and hypocalciuria were observed without hypokalaemia and metabolic alkalosis [15].

Although mutations in FXYD2, CLDN16 and SLC12A3 are associated with a specific mode of inheritance, it is not uncommon that different kinds of mutations in the same gene can cause both dominant and recessive phenotypes. Examples of this are mutations in the Ca2+/Mg2+ sensing receptor which can cause either autosomal dominant hypocalciuric hypercalcaemia, autosomal dominant hypocalcaemia (gain of function) or autosomal recessive neonatal severe hyperparathyroidism [reviewed in 16]. Similarly, mutations in the aquaporin 2 gene AQP2 have been shown to cause both recessive and dominant nephrogenic diabetes insipidus [reviewed in 17].

Using genomic primers, we have ruled out mutations in the coding sequence of FXYD2, CLDN16 and SLC12A3, including the exon–intron boudaries in both families and part of the putative promotor regions. In addition, in Pedigree II, the complete FXYD2 gene region on chromosome 11q23 was excluded by linkage analysis, ruling out the possibility of a mutation in nearby gene expression control elements or in alternatively spliced exons. Very recently, a third splice-variant of the Fxyd2 gene was found in mouse [18], however, no human homologue was found in the human genomic sequence.

Our mutation analysis in the two families with primary renal Mg2+ wasting has excluded all of the genes known or suspected to play a (primary) role in renal Mg2+ reabsorption. Therefore, we conclude that another gene or genes must be involved in the renal handling of Mg2+.

According to a model postulated by Quamme et al. [19], at least two other proteins must be involved in active Mg2+ transport in the kidney. On the luminal side, a channel through which Mg2+ enters the cell and on the basal side, a (co-)transporter or exchanger or both are responsible for Mg2+ extrusion into the interstitium. Each of these would be excellent candidates for the as yet unsolved hereditary hypomagnesaemia cases. However, the genes encoding the apical Mg2+ entry channel and the basolateral Mg2+ transporter or exchanger, still remain to be cloned even though the properties of the Mg2+ channel have been well characterized [reviewed in 20]. In the past, genetic (linkage) analysis of families suffering from rare electrolyte reabsorption disorders, has proved a very powerful tool in identifying such genes. Families such as the ones described in this paper, will be a great aid in the attempts to identify these genes and to further elucidate the mechanism and pathology of renal magnesium reabsorption.



   Acknowledgments
 
The work presented in this paper was supported by the Dutch Kidney Foundation grant C95.5001 and by a Biomed II grant PL950260 from the European Economic Community for the ‘Identification of gene defects in Bartter's syndrome and Gitelman's syndrome’.



   Notes
 
Correspondence and offprint requests to: N. V. A. M. Knoers, MD, PhD, Department of Human Genetics 417, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Email: n.knoers{at}antrg.azn.nl Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion and conclusions
 References
 

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Received for publication: 11. 8.01
Accepted in revised form: 13. 9.02





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