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Autosomal Recessive Renal Proximal Tubulopathy andHypercalciuria: A New Syndrome

Daniella Magen, MD, Lior Adler, BSc, Hana Mandel, MD, Edna Efrati, PhD,and Israel Zelikovic, MD

Background: The best described primary inherited proximal tubulopathies include X-linked hypercalciuricephrolithiasis (XLHN), caused by a mutation in the chloride channel gene CLCN5, and classic Fanconi’s syndrome,

he genetic basis of which is unknown. The aim of this study is to examine the clinical, biochemical, and geneticharacteristics of a highly consanguineous Druze family with autosomal recessive proximal tubulopathy andypercalciuria (ARPTH), a syndrome not reported previously. Methods: Three children (2 girls, 1 boy) of the familyeferred for evaluation of renal glycosuria and hypercalciuria and 10 of their close relatives were evaluated clinicallynd biochemically. All study participants underwent genetic analysis to exclude involvement of the CLCN5 gene.esults: Evaluation of the 3 affected children showed glycosuria, generalized aminoaciduria, hypouricemia,ricosuria, low molecular weight (LMW) proteinuria, and hypercalciuria in all 3 children and phosphaturia in 2hildren. They had no metabolic acidosis or renal insufficiency. One affected girl had nephrocalcinosis. Twohildren had a history of growth retardation and radiological findings of metabolic bone disease. Parathyroidormone and 1,25-dihydroxyvitamin D [1,25(OH) 2Vit D] blood levels in affected children were normal. Unaffected

amily members examined had no renal tubular defects or LMW proteinuria. Genetic linkage analysis excludedosegregation of the ARPTH phenotype with the CLCN5 locus. Conclusion: ARPTH is a new syndrome character-zed by nonacidotic proximal tubulopathy, hypercalciuria, metabolic bone disease, and growth retardation. It can beistinguished from XLHN by its autosomal recessive mode of inheritance and normal serum levels of calciotropicormones, as well as the absence of LMW proteinuria in obligate carriers. The gene mutated in ARPTH remains toe identified. Am J Kidney Dis 43:600-606.2004 by the National Kidney Foundation, Inc.

NDEX WORDS: Fanconi’s syndrome; proximal kidney tubule; hypercalciuria; nephrocalcinosis; chloride channel;

ow molecular weight (LMW) proteinuria.

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ENAL FANCONI’S syndrome is a proxmal tubular transport disorder that, in

lassic form, is characterized by renal glycria, aminoaciduria, hyperuricosuria, metabcidosis, electrolyte imbalance, dehydration,ophosphatemic rickets, and growth retar

ion.1 Fanconi’s syndrome may be accompany hypercalciuria; however, renal calcium losually is not a primary manifestation of tyndrome, but rather a secondary phenomeesulting from either bone resorption causedhronic acidosis or therapy for rickets with cium and vitamin D. Fanconi’s syndrome may

From the Pediatric Nephrology Unit; Laboratory of Devel-pmental Nephrology; and Department of Pediatrics, Meyerhildren’s Hospital, Rambam Medical Center; and Facultyf Medicine, Technion-Israel Institute of Technology, Haifa,srael.

Received July 30, 2003; accepted in revised form Decem-er 8, 2003.Address reprint requests to Daniella Magen, MD, Pediat-

ic Nephrology Unit, Meyer Children’s Hospital, Rambamedical Center, PO Box 9602, Haifa 31096, Israel. E-mail:

anmag@tx.technion.ac.il© 2004 by the National Kidney Foundation, Inc.0272-6386/04/4304-0002$30.00/0

gdoi:10.1053/j.ajkd.2003.12.024

American Journal o00

primary disease or may occur secondararious hereditary or acquired disorders.enetic basis of primary Fanconi’s syndromnknown.X-Linked hypercalciuric nephrolithias

XLHN) is a group of inherited proximal tubuopathies in which hypercalciuria appears arimary and cardinal manifestation. Variants

he syndrome include X-linked recessive nephithiasis,2 Dent’s disease,3,4 X-linked recessivypophosphatemic rickets,5 and low moleculaeight (LMW) proteinuria with hypercalciurnd nephrocalcinosis.6,7 All these syndromehare several clinical and biochemical manifeions, including multiple proximal tubular tranort defects of varying severity, hypercalciuith nephrolithiasis and/or nephrocalcinosis,resence of metabolic bone disease, decreerum parathyroid hormone (PTH) levels alevated serum 1,25-dihydroxyvitamin

1,25(OH)2Vit D] levels, progressive renal insuciency, and male predominance. Most caremales have LMW proteinuria. All 4 syndromave an X-linked recessive mode of inheritannd all are caused by mutations in CLCN5,

ene encoding the kidney-specific voltage-gated

f Kidney Diseases, Vol 43, No 4 (April), 2004: pp 600-606

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hloride channel CLC-5.8,9 “Dent’s disease” isow accepted as the generic term for all disor-ers caused by mutations in CLCN5. Recenttudies using mouse models of Dent’s diseaseave shown that defective function of CLC5,hich resides at the endosomal membrane of theroximal tubular cell, leads to impaired endocy-osis of LMW proteins and calciotropic hor-ones.10-14 This, in turn, results in LMW protein-

ria and hypercalciuria.10,11

There are 2 previous reports on nonacidoticroximal tubulopathy and hypercalciuria inher-ted in a non–X-linked fashion.15,16 The geneticefect in these syndromes has not been eluci-ated.The purpose of the current study is to examine

he clinical, biochemical, and genetic characteris-ics of a highly inbred Druze family in northernsrael affected with a new syndrome consistingf renal proximal tubulopathy and hypercalci-ria. This syndrome appears to be transmitted asn autosomal recessive trait.

METHODS

atientsA highly consanguineous Druze family residing in a small

illage in central Galilee was investigated. Three children in

Fig 1. Pedigree of a Druze family with proximal tuumerals, numbers of generations; Arabic numerals, sircles (females), affected children; hatched and empamily members, respectively; arrow, index case.

he family were identified as having proximal tubulopathy w

nd hypercalciuria. The pedigree of the kindred is shown inig 1. The fathers (II1 and II7 in the pedigree) are 2 brothersho married twin sisters (II2 and II8) who are their first-egree cousins. The pattern of inheritance of the disease inhe family is compatible with an autosomal recessive trait.hree children affected with proximal tubulopathy (III3,

II9, and III10) and 10 close relatives were included in thetudy. At the time of enrollment, no patient had receivedherapy.

Patient 1. A 4-year-old girl (III3; index case) was re-erred for evaluation of renal glycosuria and findings ofephrocalcinosis on renal ultrasonography. Medical historyncluded growth retardation and 1 documented febrile uri-ary tract infection at the age of 1 year, which was the causeor initiation of renal evaluation by her primary physician.here was no history of flank pain or macroscopic hematu-

ia. No bone pain or fractures were reported. Psychomotornd intellectual development was normal. On examination,he girl appeared small for her age, but well nourished. Hereight was 13 kg (3rd percentile for her age), and height was5 cm (3rd to 10th percentile). She had no dysmorphiceatures or skeletal deformities. Blood pressure was 85/60m Hg. The rest of her physical examination was unremark-

ble.Patient 2. A 10.5-year-old boy (III9; cousin of patient 1)

resented in infancy with failure to thrive, polyuria, andolydipsia. Evaluation in another hospital showed evidencef proximal tubulopathy with significant hypercalciuria. Noetabolic acidosis was documented. He was managed with

igh fluid intake and administered no specific medications.is growth rate gradually improved, and he sought noedical advice for several years. At the age of 10.5 years, he

athy and hypercalciuria; see text for details. Romanumbers of tested individuals; fi lled square (male) anduares and circles, investigated and not investigated

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as referred to our clinic for re-evaluation, at which time he

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MAGEN ET AL602

till had polyuria and polydipsia, but no enuresis. He had noistory of bone pain or deformities, renal calculi, or urinaryract infections. On physical examination, he appeared wellourished and had no evidence of hypovolemia. His weightas 28.6 kg (25th percentile for his age), and height was 137

m (25th to 50th percentile). Blood pressure was normal. Head no dysmorphic features or skeletal abnormalities.Patient 3. A 5-year-old girl (III10; sister of patient 2)

as referred to our clinic for further evaluation of renallycosuria, hypercalciuria, and generalized aminoaciduria.hese urinary abnormalities were found during an evalua-

ion triggered by the findings in her family members. Sheas asymptomatic, with no history of growth retardation,one deformities, polyuria, polydipsia, or dehydration. Hereight and height were 15.6 kg and 102 cm, respectively

both at the 10th percentile for her age). The rest of herhysical examination was unremarkable.

henotypic AnalysisClinical data were obtained from patients, their parents,

nd their care providers. Blood and urine samples for bio-hemical analyses were collected after appropriate informedonsent was obtained from the subjects or their parents.erum glucose, electrolyte, creatinine, liver enzyme, albu-in, lipid, calcium, phosphorus, uric acid, and alkaline

hosphatase levels, as well as plasma pH and bicarbonateevels, were analyzed by means of standard automatedaboratory techniques. Freshly voided urine samples werebtained for analysis of glucose and pH. A 24-hour urineample was collected for analysis of calcium, phosphorous,ric acid, creatinine, protein, and �2-microglobulin. Electro-horesis of urinary proteins was performed. Urinary aminocids levels were determined by means of a high-perfor-ance liquid chromatography method. Blood levels of intactTH (iPTH), 25-hydroxyvitamin D [25(OH)Vit D], and,25(OH)2Vit D were analyzed in affected patients by meansf radioimmunoassay. Renal sonography, wrist X-ray study,nd ophthalmologic slit-lamp examination were performedn affected patients. Calcium excretion is expressed as therinary calcium-creatinine ratio (in milligrams per milli-ram). Uric acid (Ua) excretion per deciliter of glomerularltration rate was calculated according to the formula:UUa � SCr�/Ucr, where Cr is creatinine and U and S areoncentrations in milligrams per deciliter in urine and se-um, respectively.17 The ratio of maximum reabsorption ofhosphate to glomerular filtration rate was determined asreviously described.18,19

Patients had no dietary restrictions at the time of the study.

enetic Linkage AnalysisGenomic DNA was extracted from peripheral-blood lym-

hocytes of family members by using standard techniques.20

inkage analysis was performed using microsatellite poly-orphic markers linked to the CLC-5 voltage-gated chloride

hannel gene (CLCN5) on chromosome X. The following 6arkers from the Geneton microsatellite panel21 were exam-

ned: DXS8334, DXS7589, DXS8016, DXS676, DXS8113,nd DXS453. Genotyping was performed by means ofolymerase chain reaction with genomic DNA and 5� fluo-

22

escently labeled primers for these markers. Products were c

esolved on an ABI 310 sequencer system, and allele sizesere determined using Genescan 3.1 and Genotyper 2.1

oftware (PE Applied Biosystems, Foster City, CA).

RESULTS

linical and Biochemical Characteristics

Tables 1 and 2 list laboratory findings andhenotypic features in the 3 affected family mem-ers. Findings in all 3 affected children includedenal glycosuria, generalized aminoaciduria,MW proteinuria, hypouricemia, hyperuricos-ria, and hypercalciuria. Hypophosphatemia wasound in patient 1, and hyperphosphaturia washown in patients 1 and 3. Metabolic acidosisas not found in any patient. Glomerular filtra-

ion rate and serum iPTH, 25(OH)Vit D,,25(OH)2Vit D, serum liver enzyme, albumin,nd lipid levels were within normal range. Aenal sonogram showed marked bilateral nephro-alcinosis in patient 1. Patients 1 and 2 had aistory of growth retardation in early infancy,nd both had radiological evidence of metabolicone disease by wrist X-ray (patient 1 showedigns of healed rickets, and patient 2 showedeneralized osteopenia). Eye examination by slitamp was unremarkable in all affected children.he parents and other siblings examined had nolinical or biochemical abnormalities. No LMWroteinuria was found in unaffected family mem-ers.

inkage Analysis

To exclude the remote possibility that theisease in this family was transmitted in X-inked fashion and the affected girls were carriersf a mutation in the gene encoding the CLC5hloride channel (CLCN5), analysis of linkageo this gene was performed. Haplotype analysissing the appropriate microsatellite markers (seeethods) flanking the locus of CLCN5 did not

how cosegregation of the phenotype with thisocus (Fig 2). It should be noted that the findingf partially informative markers in this highlynbred family precluded us from using some ofhe markers tightly linked to the CLCN5 locus.evertheless, our linkage analysis data appear toe sufficient to exclude CLCN5 as the mutatedene in this family.

DISCUSSION

This study describes clinical and biochemical

haracteristics and presents genetic linkage anal-

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sis of a highly consanguineous kindred affectedith a syndrome consisting of proximal tubulopa-

hy and hypercalciuria. Diagnosis of this newyndrome in this family was based on the constel-ation of several features, including generalizedubular dysfunction without metabolic acidosis,

Table 1. Results of Labora

1

loodCreatinine (mg/dL) 0.4Calcium (mg/dL) 9.9Phosphorus (mg/dL) 2.8Alkaline phosphatase (IU/L) 158Uric acid (mg/dL) 1.0pH 7.41Bicarbonate (mEq/L) 22.6iPTH (pg/ml) 2825(OH)Vit D (ng/mL) 211,25(OH)2Vit D (pg/mL) 31rineCalcium-creatinine ratio (mg/mg) 0.9TP/GFR (mg/dL) 2.5Uric acid/dL of GFR (mg/dL) 0.76�2-Microglobulin (ng/mL) 18,249

NOTE. To convert serum creatinine in mg/dL to �mol/Lhosphorus in mg/dL to �mol/L, multiply by 0.323; uric acimol/L, multiply by 1; PTH in pg/mL to ng/L, multiply by 1; 2

n pg/mL to pmol/L multiply by 2.6; urinary calcium-creatininAbbreviations: TP, tubular phosphate reabsorption; GFR*Reference values are for children aged 4-11 years.

Table 2. Phenotypic Features in Affected Patients

Patient No.

1 2 3

ge (y) 4 10.5 5ex F M Flycosuria � � �eneralized aminoaciduria � � �ypouricemia � � �MW proteinuria � � �ricosuria � � �ypophosphatemia � � �hosphaturia � � �etabolic acidosis � � �ypercalciuria � � �ephrocalcinosis � � �istory of growth retardation � � �etabolic bone disease* � � �

c*Diagnosis by X-ray.

arked LMW proteinuria, significant hypercalci-ria and/or nephrocalcinosis, metabolic bone dis-ase, inappropriately “normal” levels of 1,25(OH)2

it D and iPTH, and, finally, an autosomal reces-ive mode of inheritance.

This new syndrome, which we have termedutosomal recessive proximal tubulopathy withypercalciuria (ARPTH), appears to be distinctrom previously described proximal tubulopa-hies. The nature of the tubulopathy and therominent hypercalciuria do not seem to repre-ent primary classic Fanconi’s syndrome. Theelatively benign nature of the disease in affectedamily members; the absence of clinical or labo-atory abnormalities in liver, gastrointestinal,eart, skeletal muscle, central nervous system, orye function; and the absence of metabolic acido-is make the presence of an inborn error ofetabolism or mitochondrial cytopathy in this

amily very unlikely. Also, no evidence of ancquired disorder known to cause Fanconi’s syn-rome was found in any of the affected familyembers. Several manifestations in the family

eported here clearly distinguish ARPTH from

ndings in Affected Patients

Patient No.Reference

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ly by 88.4; calcium in mg/dL to �mol/L, multiply by 0.25;/dL to �mol/L, multiply by 59.48; bicarbonate in mEq/L to

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tory Fi

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lassic XLHN (or Dent’s disease). These include

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MAGEN ET AL604

he absence of progressive renal insufficiency (ateast in the young patients reported here), normalevels of iPTH and calciotropic hormones (de-pite renal phosphate wasting and hypophos-hatemia), the absence of LMW proteinuria inbligate carriers, and, most importantly, the auto-omal recessive mode of inheritance. Our conclu-ion is strengthened by our linkage analysis data,hich excluded the CLCN5 gene as responsible

or the disease in this family.Two previous reports described hereditary

roximal tubulopathy with hypercalciuria.15,16

ieder et al15 described a consanguineous Arab-sraeli family in which 2 siblings (male andemale) presented with primary Fanconi’s syn-rome, severe rickets with bone deformities,ypercalciuria, and no acidosis at the ages of 17nd 11 years, respectively. As in ARPTH, theode of inheritance in the family reported byieder et al15 appears to be autosomal recessive.owever, the hypophosphatemic bone disease in

his family was much more severe than inRPTH. Furthermore, calcitriol levels in both

iblings reported by Tieder et al15 were signifi-antly elevated. These increased levels are in

Fig 2. Linkage analysis of the 2 subfamilies with prnd circles (females), affected individuals; numbersaplotype analysis using microsatellite markers linkedosegregation of the ARPTH phenotype with thisicrosatellite repeat length in marker 453, probably be

ontrast to calcitriol levels found in patients with e

lassic Fanconi’s syndrome23-25 and the patientsith ARPTH reported here, which are eitherormal or inappropriately low considering theegree of hypophosphatemia. The investigatorsostulated that their patients showed a normalesponse of the renal 25(OH)Vit D-1 �-hydroxy-ase system to hypophosphatemia, leading tolevated calcitriol levels, which, in turn, resultedn absorptive hypercalciuria. It is plausible thatatients with ARPTH, similar to those with clas-ic Fanconi’s syndrome,23,24 have abnormal regu-ation of 1�-hydroxylation of vitamin D.

Vezzoli et al16 reported a kindred from Lombar-ia, Italy, with nonacidotic proximal tubulopathynd hypercalciuria. As in ARPTH, the phenotypef these patients included glycosuria, aminoacid-ria, tubular proteinuria, hyperuricosuria, andhosphaturia, without renal tubular acidosis. Pa-ients also had hypercalciuria and urolithiasis.owever, as opposed to the autosomal recessiveode of inheritance in the family reported here,

he disorder described by Vezzoli et al16 wasransmitted as an autosomal dominant trait, indi-ated by male-to-male transmission of the dis-ase. In addition, the patients reported by Vezzoli

16

l tubulopathy and hypercalciuria. Filled square (male)ath symbols, individuals within the kindred. (Left)CLCN5 locus on chromosome Xp11.23 did not showIndividuals 11 and 27 do not show the expected

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PROXIMAL TUBULOPATHY AND HYPERCALCIURIA 605

We suggest that in contrast to the secondaryypercalciuria observed in classic Fanconi’s syn-rome and the cases reported by Tieder et al15

nd Vezzoli et al,16 renal calcium wasting inRPTH, which cannot be explained by meta-olic acidosis or elevated calcitriol levels, is arimary phenomenon resulting from impairedubular handling of calcium.

The genetic basis and pathophysiologic mecha-isms underlying the solute wasting in generalnd the hypercalciuria in particular in patientsith ARPTH remain to be established. A wideenome screen in members of families affectedith ARPTH may be required to initially map the

ocus for ARPTH and subsequently identify theutosomal gene mutation that causes this newyndrome.

Defects in adenosine triphosphate generation,ransmembrane Na� gradient, or membrane per-eability in the proximal tubule have been pro-

osed as potentially responsible for the abnor-alities in solute reabsorption in Fanconi’s

yndrome.1 Nevertheless, in view of the pheno-ypic similarity between ARPTH and XLHN, its tempting to speculate that derangement in therafficking and/or acidification-dependent endo-ytosis of apical proteins, proposed to be defec-ive in XLHN,10-14 is responsible for the impairedubular handling of solutes and LMW proteinurian ARPTH, as well. Such an abnormality in therafficking machinery in ARPTH may involve aenetic defect in 1 of the multiple proteins otherhan CLC5 chloride channel participating in thisrocess. These proteins may include the protonump V-type H� ATPase, the Na�/H� ex-hanger-3, and the endocytic receptors megalinnd cubilin, among others.11

Another possible mechanism for the develop-ent of hereditary proximal tubulopathy may

nvolve mutations in genes encoding transcrip-ion factors, which regulate the expression ofenes for various proximal tubular transporterroteins. Hepatocyte nuclear factor-1� (HNF-�) recently was shown to regulate expression ofhe gene encoding the murine proximal tubularigh-capacity/low-affinity sodium-glucose co-ransporter-2,26 and both HNF-1�27 and HNF-�28 regulate expression of the murine sodium-hosphate cotransporter gene. HNF-1� knockoutice have severe renal Fanconi’s syndrome.26 In

umans, mutations in the gene encoding HNF-1� t

esult in maturity-onset diabetes of the youngype 3, which also is characterized by defects inubular reabsorption of glucose and amino ac-ds.29 Thus, it is possible that mutations in a genencoding a transcription factor with such anction are responsible for ARPTH.

In conclusion, ARPTH is a new syndromeharacterized by nonacidotic proximal tubulopa-hy, hypercalciuria, metabolic bone disease, androwth retardation. It can be distinguished fromLHN by its autosomal recessive mode of inher-

tance, inappropriately normal serum levels ofalciotropic hormones, and absence of LMWroteinuria in obligate carriers. ARPTH mostikely is caused by a mutation in a gene otherhan the renal chloride channel gene CLCN5,hich is mutated in XLHN. The genetic defectnderlying ARPTH and molecular mechanismsesponsible for renal solute wasting in this syn-rome are subjects for additional studies.

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ueber P, Schroeder ET: X-Linked recessive nephrolithiasisith renal failure. N Engl J Med 325:681-686, 19913. Dent CE, Friedman M: Hypocalciuric rickets associ-

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MAGEN ET AL606

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yte nuclear factor 1� controls renal expression of thept1-Npt4 anionic transporter locus. J Mol Biol 322:929-41, 200228. Soumounou Y, Gauthier C, Tenenhouse HS: Murine

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001