letter

166 nature genetics • volume 31 • june 2002

Hypomagnesemia with secondary hypocalcemia iscaused by mutations in TRPM6, a new member ofthe TRPM gene family

Karl P. Schlingmann1*, Stefanie Weber1*, Melanie Peters1, Lene Niemann Nejsum2, Helga Vitzthum3, KarinKlingel4, Markus Kratz5, Elie Haddad6, Ellinor Ristoff7, Dganit Dinour8, Maria Syrrou9, Søren Nielsen2,Martin Sassen1, Siegfried Waldegger1, Hannsjörg W. Seyberth1 & Martin Konrad1

* These authors contributed equally to this work.

Magnesium is an essential ion involved in many biochemicaland physiological processes. Homeostasis of magnesium levelsis tightly regulated and depends on the balance betweenintestinal absorption and renal excretion. However, little isknown about specific proteins mediating transepithelial mag-nesium transport. Using a positional candidate gene approach,we identified mutations in TRPM6 (also known as CHAK2),encoding TRPM6, in autosomal-recessive hypomagnesemiawith secondary hypocalcemia (HSH, OMIM 602014)1,2, previ-ously mapped to chromosome 9q22 (ref. 3). The TRPM6 proteinis a new member of the long transient receptor potential chan-nel (TRPM) family4 and is highly similar to TRPM7 (also knownas TRP-PLIK), a bifunctional protein that combines calcium- andmagnesium-permeable cation channel properties with proteinkinase activity5−7. TRPM6 is expressed in intestinal epitheliaand kidney tubules. These findings indicate that TRPM6 is cru-cial for magnesium homeostasis and implicate a TRPM familymember in human disease.The disease HSH is characterized by very low magnesium andlow calcium serum levels. Affected individuals show neurologicsymptoms of hypomagnesemic hypocalcemia, includingseizures and muscle spasms, during infancy. Untreated, the dis-order may be fatal or may result in neurological damage.Hypocalcemia is secondary to parathyroid failure resulting from

magnesium deficiency8. Normocalcemia and relief of clinicalsymptoms can be assured by oral administration of high dosesof magnesium2. Pathophysiology of HSH is largely unknown,but physiological studies indicate a primary defect in intestinalmagnesium transport9. In some individuals, an additional renalmagnesium leak, caused by altered magnesium reabsorption inthe distal convoluted tubule (DCT), was suspected10,11.

A gene locus (HOMG1) for HSH was previously mapped tochromosome 9q22 (ref. 3) and further refined to a critical inter-val between markers D9S1115 and D9S175 (ref. 12). We studiedfive families with typical HSH (Table 1). Extended disease haplo-types in the three consanguinous families were compatible withlinkage, but did not allow further narrowing of the critical inter-val (Fig. 1). Flanking markers D9S1115 and D9S175 define aphysical interval of approximately 1.7 Mb (Fig. 2a).

A search for candidate genes within this region revealed fivegenes (Fig. 2a) and various expressed sequence tag (EST) clones(see Methods). Focusing on sequences expressed in intestine,we identified two overlapping human ESTs with identity toparts of TRPM6, recently cloned from kidney cDNA4. The geneTRPM6 encodes a putative ion channel that is highly similar tothe transient receptor potential (TRP) channel family. TRP ionchannels are characterized by six transmembrane segments, aconserved pore-forming region and a Pro-Pro-Pro motif fol-

Published online: 28 May 2002, DOI: 10.1038/ng889

Table 1 • Clinical characteristics and TRPM6 mutations in individuals with HSH

Age at First Initial Mg2+ FE Mg2+ Parental Nucleotide Patient onset symptoms (mM) (%) Origin consanguinity exchange Exon Consequence

F1.1 9 wk seizures 0.21 4.8 Turkey yes 1769C→G 15 Ser590X

F2.1 3 wk seizures 0.08 2.8 Turkey yes 2667+1G→A 5′ss19 exon skipping

F3.1 4 mo seizures 0.10 4.0 Sweden no [3537–1G→A] + 3′ss25 exon skipping[422C→T] 4 Ser141Leu

F4.1 4 wk seizures 0.41 high Israel no [1280delA] + 10 His427fsX429[3779−91del] 25 Glu1260fsX1283

F5.1 5 wk seizures 0.17 4.5 Albania yes 2207delG 16 Arg736fsX737

F5.2 5 wk seizures 0.22 2.6 Albania yes 2207delG 16 Arg736fsX737

FE Mg2+, fractional excretion of magnesium; fs, frameshift; del, deletion. Nucleotide positions correspond to the coding sequence as deposited in GenBank.

1Department of Pediatrics, Philipps University of Marburg, Deutschhausstrasse 12, D-35037 Marburg, Germany. 2Water and Salt Research Center, Institute ofAnatomy, University of Aarhus, Denmark. 3Department of Physiology I, University of Regensburg, Germany. 4Department of Molecular Pathology, UniversityHospital, Tübingen, Germany. 5University Children’s Hospital, Mannheim, Germany. 6Robert Debré Hospital, Paris, France. 7Department of Pediatrics,Karolinska Institute, Huddinge University, Stockholm, Sweden. 8Department of Nephrology, The Chaim Sheba Medical Center, Ramat-Gan, Israel. 9Laboratory ofGeneral Biology-Genetics, University of Ioannina, Greece. Correspondence should be addressed to M.K. (e-mail: konradm@mailer.uni-marburg.de).

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://g

enet

ics.

nat

ure

.co

m

letter

nature genetics • volume 31 • june 2002 167

lowing the last transmem-brane segment13. The TRPM6protein belongs to the TRPMsubfamily, whose membersshare a long amino-terminaldomain of unknown function.This protein shows highestsimilarity with TRPM7 (52% overall identity; Fig. 2b), recentlyidentified as a magnesium- and calcium-permeable ion chan-nel5. In addition to their ion channel domain, TRPM6 andTRPM7 have a carboxy-terminal region with sequence similar-ity to the atypical α-kinase family (Fig. 2c)7. The high similarityof TRPM6 to TRPM7, along with its partial identity with ESTscloned from intestine, prompted further examination ofTRPM6 in HSH.

Aligning TRPM6 cDNA with genomic clones revealed 38exons spanning 134 kb (Fig. 2d). As the translation start site indi-cated by the TRPM6 mRNA sequence originally reported was not

represented in the genomic sequence database (TAG instead ofATG), we carried out 5′ RACE–PCR on cDNA from human smallintestine and kidney. PCR fragments from both tissues containedan upstream in-frame methionine within a suitable Kozaksequence. Comparison with genomic clones indicated an addi-tional exon (termed exon 1A), located 29 kb upstream of exon 1(Fig. 2d), encoding the 5′ untranslated region and ten additionalamino acids. Thus, the full-length TRPM6 encodes a protein of2,022 amino acids encoded by 39 exons.

Single-strand conformational polymorphism (SSCP) analysisand subsequent direct sequencing revealed seven different

F 1

F 2

F2.1

8227221

2522411

7415411

2522411

D9S1799D9S1115

D9S284

23938-194

8580-154023938-470

D9S175

F1.1

9123131

63231511

632315

1123111

2522411

2522411

6323151 1

632315

F1.2

F 5

F5.2

7123211 1

712321

1

712321

3124212

4225242

7123211

F5.1

7123211 1

712321

Fig. 1 Haplotype analysis of familiesF1, F2 and F5. Filled symbols indicateaffected individuals. A slash markindicates deceased individuals, anddouble lines indicate consanguinity.Genotyping data and schematic seg-regating haplotype bars for chromo-some 9q22 are shown below thesymbol for each individual. Black barsindicate the haplotype segregatingwith the gene underlying HSH; whitebars show the wildtype haplotypes.

cen tel

NT_008580 NT_023953 NT_023938 NT_029358

D9S1115

8580-1540

D9S175

D9S284

23938-470

23938-194

OSTF1

TRPM6

RORB

1

5' 3'

38

Ser590XSer141Leu

4

D9S1799

loss of ss loss of ss

FLJ10110

FLJ20559

UTR3'1A

ATG TCC CAG AAA ATG AAA GAA CAA CCT GTC TTG GAG CGC TTG CAG

ctttttag TCC CAG AAA . . . . . 29 kb. . . . .

TCC CAG AAA

ATG AAA GAA CAA CCT GTC TTG GAG CGC TTG CAG gtaagctc

. . . . .

. . . . .

. . .

M K

AF350881

5' RACE fragment

aaag5' UTR . . .

E Q P V L E R L Q S Q K

M S Q K

aaag5' UTR . . .

genomic sequence

kinasedomain

TRPM6

NH2

COOH

extracellular

intracellularTRPM1

TRPM6

TRPM7

TRPM2

TRPM4

TRPM5

α

TRPM6 α

TRP domain

coiled coiltransmembrane

1 2 3 4

conserved N terminus kinase

Leu429X Leu737X

1516 1910

Leu1283X

fs fs fs

TRPM6

25

Fig. 2 Characterization of TRPM6. a, Phys-ical map of the HOMG1 critical region(filled horizontal bar) between previouslyidentified polymorphic markers D9S1115and D9S175. Human genome sequencesegments (white horizontal boxes) arederived from the NCBI database. Theposition of polymorphic markers ismarked by vertical bars. Arrows belowindicate the location and orientation ofknown and putative genes annotated bythe NCBI RefSeq project. cen, cen-tromeric; tel, telomeric. b, Phylogenetictree of the TRPM subfamily (human full-length protein sequences) showing theclose vicinity of TRPM6 to TRPM7. c, Pro-posed model of TRPM6, which shows thetypical properties of the TRPM family: along conserved intracellular N terminusand six transmembrane domains with apore-forming loop between the last twosegments. The intracellular C terminus ofTRPM6 is highly homologous to the α-kinase family (adapted from ref. 23).d, Genomic organization and mRNAstructure of TRPM6. The gene consists of39 exons (white boxes) spanning approxi-mately 163 kb of genomic DNA includingan additional exon (1A) and the 5′untranslated region (UTR) locatedapproximately 29 kb upstream of theoriginal start site were identified.Remaining exon numbers are derivedfrom alignment of TRPM6 mRNA withgenomic sequence, which showed theabsence of the published start methion-ine in the genomic sequence. Functionaldomains were deduced from the TRPM7model as previously described5. Themutations detected in HSH, along withthe corresponding exon number andtheir functional consequences for theprotein, are indicated. fs, frameshift.

a

b c

d

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://g

enet

ics.

nat

ure

.co

m

letter

168 nature genetics • volume 31 • june 2002

mutations in TRPM6 (Table 1 and Fig. 2d). Examples ofsequence analysis are given in Fig. 3a. We detected a homozy-gous in-frame stop codon at Ser590, which truncates the proteinprior to the first transmembrane domain in family F1. In familyF2, the affected individual bears a homozygous donor splice-sitemutation adjacent to exon 19. One heterozygous mutation infamily F3 affects the acceptor splice site of exon 25. Both dinu-cleotide splice-site sequences (GT, AG) are highly conserved andcrucial for mRNA splicing14. The second mutation in family F3is a nonconservative amino-acid exchange of a highly conservedserine residue (Ser141Leu) within the first N-terminal region.The affected individual of family F4 is compound heterozygouswith respect to two frameshift mutations both truncating sub-stantial parts of the TRPM6 protein (Fig. 2d and Table 1). Infamily F5, we identified a deletion of 1 bp that results in proteintruncation prior to the transmembrane domains (Table 1). Allmutations cosegregate with the phenotype and were notdetected in 102 control chromosomes.

To confirm intestinal expression of TRPM6, we carried outRT–PCR on various rat tissues. We obtained PCR amplification

products in intestine and kidney (Fig. 3b,c). We also analyzedsegmental localization of TRPM6 in microdissected nephrons(Fig. 3d), which showed the strongest signal in the DCT, themain site of active transcellular magnesium reabsorption alongthe nephron15. We obtained weak signals in proximal tubule andcollecting duct.

Expression of TRPM6 was also assessed by in situ hybridiza-tion in various human tissues. We observed TRPM6 mRNA incolon epithelial cells (Fig. 4a), duodenum (Fig. 4b), jejunumand ileum. We obtained no specific signals using sense probes(Fig. 4c). We also detected TRPM6 mRNA in single distal renaltubule cells (Fig. 4d). No signals were observed in stomach, lungand heart (data not shown).

Together with the disease phenotype, these findings indicatethat TRPM6 is essential in epithelial magnesium absorption. Invertebrates, intestinal magnesium absorption is related to lumi-nal magnesium in a curvilinear manner (Fig. 5)16. The hyper-bolic curve reflects a saturable active transcellular transport atlow intraluminal magnesium concentrations mediated by anelectrogenic apical entry and an active basolateral transport. The

linear function at higher concentrations reflects a pas-sive paracellular magnesium absorption. Alternatively,this absorption pattern might result from a progressive‘tightening’ of the junction complex induced by increas-ing luminal magnesium17. The observation that indi-viduals with HSH achieve normal serum magnesiumlevels by high oral magnesium intake although theyshow impaired intestinal magnesium absorption sup-ports the theory of two independent pathways. Ourresults suggest that TRPM6 represents a molecular com-ponent of the active transcellular pathway and that, inHSH, high oral magnesium doses are sufficient to over-come the phenotype of mutant TRPM6 by increasingparacellular magnesium absorption.

As we also identified TRPM6 expression in kidney, anadditional renal phenotype in HSH could be expected.Urinary magnesium excretion rates in individuals withHSH have been examined10,11. With respect to their lowserum magnesium levels, the individuals studied hereshowed inappropriately high fractional magnesiumexcretion rates (Table 1), indicating an additional role of

Fig. 3 Sequence analysis and tissue distribution of TRPM6. a, Muta-tion analysis of TRPM6 by direct sequencing. Genomic sequenceanalysis in a control individual (top), one parent (middle) and theaffected individual (bottom) from families F1−F3. Vertical lines divideexonic from intronic sequence. Mutated nucleotides and resultingamino-acid changes are shown under the affected individual’ssequence. Bold letters indicate the mutated nucleotides. IndividualF1.1 bears a homozygous stop mutation (X) at aa S590; individualF2.1 is homozygous with respect to a G→A exchange disrupting thedonor splice site after exon 19. Individual F3.1 is compound heterozy-gous with respect to an amino-acid substitution (Ser141Leu) and anacceptor splice-site mutation at position −1 of exon 25. Consensussplice-site sequences are indicated in red. b, TRPM6 mRNA expressionin rat intestine and kidney. We detected a TRPM6 fragment of 415 bpalong the entire intestinal tract and kidney. No signal was detectedin liver. The presence of cDNA in all samples was confirmed by detec-tion of β-actin mRNA. c, Detection of TRPM6 mRNA in mucosal cellsfrom rat intestine. TRPM6 expression in intestinal epithelia could beconfirmed after mechanical removal of submucosal layer and muscletissue in rat small (sm) intestine and colon. d, TRPM6 RT−PCR onmicrodissected rat nephron segments. We detected the strongest sig-nal in DCT. Weak TRPM6 mRNA was present in proximal convolutedtubule (PCT), connecting tubule/cortical collecting duct (CT/CCD),outer medullary collecting duct (OMCD) and inner medullary collect-ing duct (IMCD). No TRPM6 expression was detected in glomeruli(glom), proximal straight tubule (PST), descending thin limb (dTL),ascending TL (aTL), medullary thick ascending limb (mTAL) and corti-cal TAL (cTAL). Parallel amplifications of marker genes served as con-trols for segment specificity of the preparation (data not shown).

F1 F2 F3

exon 15 intron 19exon 19 exon 4 intron 24 exon 25

control

parent

patient

G G A G N T G A G

G G A G T G A GA

C T A N G G T TA

C T A G G G T TA

C T AA G G T TNA T C T A G T CN

A T C T A G T CN

G G A G G T G A G

T N AA A A AAG

A TA AAAAGG

TG AC A A AAA

motherfather

A A G C A A AG

Ser590X

A T C G T CCT

Ile140 Ser141Leu

GAGG G T TGt g g a g gaa

aT AtT A

Lys589 Lys591 Val142Glu889 Val1180

A T C T A G T CC

a c

415 bp

TRPM6

duod

enum

ileum

jeju

num

colo

nliv

er

kidn

ey

+ _ + _ + _ + _ + _ + _

415 bp

TRPM6

G3PDH

sm. i

ntes

tine

colo

n

_

415 bp

TRPM6

microdissected rat nephron segments

glom

PCT

PST

dTL

dTL/

aTL

mTA

LcT

ALD

CT

CT/

CC

DO

MC

DIM

CD

mucosatissue

HO

2

RT

_

_

HO

2

β actin_

β actin_

a

b c

d

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://g

enet

ics.

nat

ure

.co

m

letter

nature genetics • volume 31 • june 2002 169

impaired renal magnesium reabsorption in HSH11. This is con-firmed by the characterization of a considerable renal leak ofmagnesium in HSH patients in an accompanying report18.

This study highlights new aspects of the molecular basis ofmagnesium transport in humans. Mutations in CLDN16 werepreviously identified19 in familial hypomagnesemia with hyper-calciuria and nephrocalcinosis (OMIM 248250). The geneCLDN16, a newly discovered member of the claudin multigenefamily, mediates paracellular magnesium reabsorption throughtight junctions in renal tubular epithelia. In isolated renal mag-nesium loss (OMIM 154020), a dominant-negative mutationwas reported in FXYD2 that results in a trafficking defect of the γ-subunit of the Na+/K+-ATPase20.

The identification of TRPM6 mutations in HSH suggests thatTRPM6 is the first component of intestinal magnesium absorp-tion identified at the molecular level. Moreover, the involvementof TRPM6 in magnesium absorption in intestine and kidneyraises the question of whether its homolog TRPM7, which isexpressed in many tissues, is also involved in cellular magnesiumhomeostasis. These findings emphasize the growing functionaldiversity of TRPM family members, which have previously beenshown to be important in cellular processes such as cell survival,progression of tumors and intracellular signaling5,21–23.

MethodsSubjects. Diagnosis of HSH in the five families studied was based on acharacteristic history of neurologic symptoms, including tetany, musclespasms and seizures due to hypomagnesemic hypocalcemia during thefirst six months of life. Families F4 and F5 have been described previous-ly10,24. The most prominent biochemical findings were hypomagne-semia (< 0.4 mmol L−1), hypocalcemia and suppressed parathyroid hor-mone levels. In all affected individuals, hypomagnesemia could be con-trolled by high oral magnesium supplements, which is typical of HSHand contrasts the phenotype seen in individuals with exclusive renalmagnesium loss. The first-grade cousin of individual F1.1 also has aclinical history typical of HSH; however, we could not obtain DNA fromthis individual. The study was carried out with the approval of the EthicsCommittee of the Philipps University of Marburg, Germany. Weobtained informed consent from all subjects.

Genotyping. We isolated genomic DNA from peripheral blood by stan-dard methods. We carried out PCR and haplotype analysis as describedpreviously25. Microsatellite markers were generated by dinucleotide repeatsearches in genomic clones mapping to the critical region or were derivedfrom public databases.

Physical map, candidate genes. We obtained genomic data for the con-struction of a physical map of the critical region by a search in genomicdatabases. We compared cDNA with genomic sequences using the BLASTprogram. Start codon prediction was carried out with the ATGpr program.

Five genes mapped to the critical interval (TRPM6, RORB, OSTF1 and thegenes encoding FLJ20559 and FLJ10110). The gene RORB is an orphannuclear receptor; RORB knockout mice show a phenotype (atypical behav-ior, blindness) completely different from that of HSH26. The gene OSTF1encodes an intracellular protein produced by osteoclasts that indirectlyinduces osteoclast formation and bone resorption27. FLJ20559 is a hypo-thetical protein highly similar to uridine kinases, which are known to beinvolved in the salvage pathway of pyrimidine synthesis28; thus, the geneencoding this protein is probably not involved in HSH. FLJ10110 is a hypo-thetical protein of unknown function that seems to have a broad tissue dis-tribution; we excluded mutations of the gene encoding this protein as thecause of HSH by direct sequencing in families F1–F3.

RACE analysis. To obtain the complete TRPM6 cDNA, we carried out 5′RACE of human small intestine and kidney marathon-ready cDNA(Clontech). Primer sequences are available upon request. Fragmentswere cloned into the pCR-II-TOPO vector (Invitrogen) and sequencedfrom both strands with M13 vector primers using an ABI Prism 310sequencer (Applera).

SSCP analysis and direct sequencing. We screened for TRPM6 mutationsby SSCP analysis. Based on the sequence of the human gene, we used over-lapping sets of primers to amplify the coding sequences of genomic DNAby PCR. We designed TRPM6 primers with Primer3 software; sequencesare available upon request. Amplified products were separated on poly-acrylamide gels by electrophoresis, and exons with conformational vari-ants were directly sequenced using corresponding sequencing primers asdescribed previously25.

RNA extraction and RT−PCR. We extracted RNA by conventional meth-ods. Reverse transcription of total RNA and mRNA was done using theSuperscript First Strand Synthesis System for RT−PCR (Invitrogen). Wecarried out reverse-transcription negative control reactions on half of theextracted mRNA. PCR was carried out for 30 cycles. Primer sequences areavailable upon request. We included controls to confirm the presence ofcDNA and carried out a PCR negative-control reaction.

Fig. 4 In situ hybridization analysis of TRPM6 expression. a,b, TRPM6 mRNAexpression in the surface and crypt epithelial cells of the colon (a) and in villousepithelial cells of the duodenum (b). c, Control hybridization with a senseprobe on consecutive tissue sections. d, In the kidney, TRPM6 mRNA expressionis restricted to a few distal nephron segments, probably representing distalconvoluted tubules.

paracellular

transcellular

combined

Mg intake2+

net M

g

abs

orpt

ion

2+

Fig. 5 Intestinal magnesium absorption. The curvilinear absorption of magne-sium in human intestine (black line) is proposed to result from two transportmechanisms: (i) a transcellular transport, saturable at high luminal magnesiumconcentrations, which is of functional importance at low luminal magnesiumconcentrations (dotted line) and (ii) a paracellular passive transport (dashedline) linearly rising with elevated luminal magnesium concentrations. Theobservation that the defect in intestinal absorption in individuals with HSHmay be compensated by high oral magnesium intake suggests that the para-cellular pathway is intact. This, in turn, indicates that TRPM6 is involved in thetranscellular active magnesium absorption pathway.

a b

c d

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://g

enet

ics.

nat

ure

.co

m

letter

170 nature genetics • volume 31 • june 2002

Microdissection of rat nephron segments. We obtained nephron segmentsby using a modified collagenase digestion protocol as previously described29.At least 11 mm of each segment were pooled for RNA extraction. After cDNAsynthesis (as described above), we carried out PCR on cDNA samples corre-sponding to an initial tubule length of 1 mm derived from at least three differ-ent sets of nephron segments from different animals. Parallel amplificationsof marker genes (SLC12A1, SLC12A3, AQP1, AQP4, PTHR1, SLC5A2) servedas controls for segment-specificity of the preparation.

In situ hybridization. Human tissue specimens from stomach, duodenum,jejunum, ileum, colon, lung and kidney were fixed in 4% paraformaldehyde,0.1 M sodium phosphate buffer (pH 7.2) for 4 h and embedded in paraffin.Tissue sections (4 µm) were dewaxed and hybridized essentially asdescribed30. The hybridization mixture contained either the 35S-labeled RNAantisense or sense control TRPM6 probe (500 ng ml−1) in buffer (10 mM TrisHCl, pH 7.4; 50% (vol/vol) deionized formamide; 600 mM NaCl; 1 mMEDTA; 0.02% polyvinylpyrrolidone; 0.02% Ficoll; 0.05% bovine serumalbumin; 10% dextran sulfate; 10 mM dithiothreitol; denatured sonicatedsalmon sperm DNA at 200 µg ml−1 per rabbit liver tRNA at 100 µg ml−1).We carried out hybridization with RNA probes at 42 °C for 18 h. Slides werewashed as described30, and then for 1 h at 55 °C in 2 × standard saline cit-rate. Nonhybridized single-stranded RNA probes were digested by RNAse A(20 µg ml−1) in 10 mM Tris HCl, pH 8.0; 0.5 M NaCl for 30 min at 37 °C.Tissue slide preparations were autoradiographed and stained with hema-toxylin and eosin.

Accession numbers. Genomic clones mapping to the critical interval:NT_008580, NT_023953, NT_023938, NT_029358; TRPM6, AF350881;EST clones representing parts of TRPM6: colon, AK000094; small intes-tine, AK026281. The sequence of the full-length human TRPM6 cDNAsequence was submitted to GenBank (AF 448232).

AcknowledgmentsWe thank the patients and their families for participating in this study, U.Pechmann and P. Barth for excellent technical assistance, C. Antignac, R.Preisig-Müller, C. Derst and N. Jeck for helpful discussions and C. Loirat, D.Lotan, W. Scheurlen, A. Siamopoulou, S. Alfandaki, G. Celsi and A. Kernellfor providing clinical data. S.W., H.W.S. and M.K. were supported by theDeutsche Forschungsgemeinschaft. S.W. was supported by the Kempkes-Stiftung, University of Marburg. L.N.N. and S.N. were supported by theDanish National Research Foundation.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 28 November 2001; accepted 4 April 2002.

1. Paunier, L., Radde, I.C., Kooh, S.W., Conen, P.E. & Fraser, D. Primaryhypomagnesemia with secondary hypocalcemia in an infant. Pediatrics 41,385–402 (1968).

2. Shalev, H., Phillip, M., Galil, A., Carmi, R. & Landau, D. Clinical presentation andoutcome in primary familial hypomagnesaemia. Arch. Dis. Child. 78, 127–130(1998).

3. Walder, R.Y. et al. Familial hypomagnesemia maps to chromosome 9q, not to theX chromosome: genetic linkage mapping and analysis of a balanced translocationbreakpoint. Hum. Mol. Genet. 6, 1491–1497 (1997).

4. Ryazanova, L.V., Pavur, K.S., Petrov, A.N., Dorovkov, M.V. & Ryazanov, A.G. Noveltype of signaling molecules: protein kinases covalently linked with ion channels.Mol. Biol. 35, 271–283 (2001).

5. Nadler, M.J. et al. LTRPC7 is a Mg-ATP-regulated divalent cation channel requiredfor cell viability. Nature 411, 590–595 (2001).

6. Runnels, L.W., Yue, L. & Clapham, D.E. TRP-PLIK, a bifunctional protein withkinase and ion channel activities. Science 291, 1043–1047 (2001).

7. Ryazanov, A.G. et al. Identification of a new class of protein kinases representedby eukaryotic elongation factor-2 kinase. Proc. Natl Acad. Sci. USA 94, 4884–4889(1997).

8. Anast, C.S., Mohs, J.M., Kaplan, S.L. & Burns, T.W. Evidence for parathyroid failurein magnesium deficiency. Science 177, 606–608 (1972).

9. Milla, P.J., Aggett, P.J., Wolff, O.H. & Harries, J.T. Studies in primaryhypomagnesaemia: evidence for defective carrier-mediated small intestinaltransport of magnesium. Gut 20, 1028–1033 (1979).

10. Matzkin, H., Lotan, D. & Boichis, H. Primary hypomagnesemia with a probabledouble magnesium transport defect. Nephron 52, 83–86 (1989).

11. Cole, D.E. & Quamme, G.A. Inherited disorders of renal magnesium handling. J.Am. Soc. Nephrol. 11, 1937–1947 (2000).

12. Walder, R.Y. et al. Hypomagnesemia with secondary hypocalcemia (HSH):narrowing the disease region on chromosome 9. Am. J. Hum. Genet. 65, 451(1999).

13. Harteneck, C., Plant, T.D. & Schultz, G. From worm to man: three subfamilies ofTRP channels. Trends Neurosci. 23, 159–166 (2000).

14. Shapiro, M.B. & Senapathy, P. RNA splice junctions of different classes ofeukaryotes: sequence statistics and functional implications in gene expression.Nucleic Acids Res. 15, 7155–7174 (1987).

15. Quamme, G.A. Renal magnesium handling: new insights in understanding oldproblems. Kidney Int. 52, 1180–1195 (1997).

16. Fine, K.D., Santa Ana, C.A., Porter, J.L. & Fordtran, J.S. Intestinal absorption ofmagnesium from food and supplements. J. Clin. Invest. 88, 396–402 (1991).

17. Kayne, L.H. & Lee, D.B. Intestinal magnesium absorption. Miner. ElectrolyteMetab. 19, 210–217 (1993).

18. Walder, R.Y. et al. Mutation of TRPM6 causes familial hypomagnesemia withsecondary hypocalcemia. Nature Genet. 31 (2002); advance online publication, 28May 2002 (DOI:10.1038/ng901).

19. Simon, D.B. et al. Paracellin-1, a renal tight junction protein required forparacellular Mg2+ resorption. Science 285, 103–106 (1999).

20. Meij, I.C. et al. Dominant isolated renal magnesium loss is caused by misroutingof the Na(+),K(+)-ATPase γ-subunit. Nature Genet. 26, 265–266 (2000).

21. Duncan, L.M. et al. Melastatin expression and prognosis in cutaneous malignantmelanoma. J. Clin. Oncol. 19, 568–576 (2001).

22. Perraud, A.L. et al. ADP-ribose gating of the calcium-permeable LTRPC2 channelrevealed by Nudix motif homology. Nature 411, 595–599 (2001).

23. Cahalan, M.D. Cell biology. Channels as enzymes. Nature 411, 542–543 (2001).24. Challa, A., Papaefstathiou, I., Lapatsanis, D. & Tsolas, O. Primary idiopathic

hypomagnesemia in two female siblings. Acta Paediatr. 84, 1075–1078 (1995).25. Konrad, M. et al. Mutations in the chloride channel gene CLCNKB as a cause of

classic Bartter syndrome. J. Am. Soc. Nephrol. 11, 1449–1459 (2000).26. Andre, E. et al. Disruption of retinoid-related orphan receptor β changes

circadian behavior, causes retinal degeneration and leads to vacillans phenotypein mice. EMBO J. 17, 3867–3877 (1998).

27. Reddy, S. et al. Isolation and characterization of a cDNA clone encoding a novelpeptide (OSF) that enhances osteoclast formation and bone resorption. J. CellPhysiol. 177, 636–645 (1998).

28. Koizumi, K. et al. Cloning and expression of uridine/cytidine kinase cDNA fromhuman fibrosarcoma cells. Int. J. Mol. Med. 8, 273–278 (2001).

29. Weber, S. et al. Primary gene structure and expression studies of rodentparacellin-1. J. Am. Soc. Nephrol. 12, 2664–2672 (2001).

30. Klingel, K. et al. Ongoing enterovirus-induced myocarditis is associated withpersistent heart muscle infection: quantitative analysis of virus replication, tissuedamage, and inflammation. Proc. Natl Acad. Sci. USA 89, 314–318 (1992).

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://g

enet

ics.

nat

ure

.co

m