Hereditary renal diseases associated with magnesium loss comprise a set of rare tubular disorders with different modes of inheritance (reviewed in reference 1 2 3 et al. (4 5 6 , 7 , 8 , 9
In contrast to primary hypomagnesemia and Gitelman syndrome, FHHNC is generally complicated by chronic renal failure (CRF) in early childhood or adolescence. Recurrent urinary tract infections (UTI) and polyuria/polydipsia are frequent initial symptoms. In addition to marked hypomagnesemia, all affected individuals exhibit hypercalciuria and nephrocalcinosis. Additional symptoms at manifestation include nephrolithiasis, abdominal pain, convulsions, muscular tetany, failure to thrive, incomplete distal renal tubular acidosis (dRTA), and hypocitraturia (10 , 11 , 12 12 12 , 13 , 14 6 12
Clinical observations and clearance studies performed with patients with FHHNC point to impaired reabsorption of magnesium and calcium in the thick ascending limb of Henle's loop (TAL) as the primary tubular defect (14 15 PCLN-1 gene were recently identified as the underlying genetic defects in FHHNC (7 PCLN-1 encodes paracellin-1 (claudin-16), a newly identified, tight junction protein of the claudin multigene family that is expressed in the TAL and the distal convoluted tubule. The claudin family comprises a set of structurally related proteins involved in the formation of tight junction strands in various tissues (16
Materials and Methods
Patients and Families
Twenty-five FHHNC families of different ethnic origins, with 33 affected individuals who were treated in 12 European pediatric departments, were analyzed in this study (Table 1 ). Family pedigrees are shown in Figure 1 . Clinical aspects of families F1 (10 9 8
Table 1: Characteristics of the patient cohorta
Figure 1: Family pedigrees for families F1 to F25 and results of family studies. Family members affected by hypercalciuria (HC), nephrolithiasis (NL), nephrocalcinosis (NC), and/or recurrent urinary tract infections (UTI) are indicated by arrows. Filled symbols, affected individuals; half-filled symbols, heterozygous mutation carriers. Individuals for whom blood samples were not available are labeled nd (no data). Two families were not examined (indicated by * ).
Parental consanguinity was noted for four families. Eight families were multiplex, with two affected siblings. In one of these families (F14), there were uniovular twins. This study was approved by the local ethics committee, and informed consent was obtained from the patients and/or their parents.
Clinical and Laboratory Data
Serum levels of magnesium, calcium, potassium, creatinine, PTH, and uric acid and acid-base status were analyzed using standard techniques. Additional parameters evaluated included urinary excretion of magnesium, calcium, and citrate and urinary acidification capability. Hypomagnesemia was defined as repeated serum magnesium levels of <0.65 mM (17 18 et al. (19 et al. (20 2 .
The onset and presence of polyuria/polydipsia, UTI, nephrolithiasis, and other symptoms of FHHNC were documented. In addition, the results of renal sonography, radiography, and/or computer tomography were recorded.
Family histories were analyzed for 23 families, focusing on hypercalciuria, nephrocalcinosis, nephrolithiasis, and recurrent UTI. For first-degree family members and FHHNC-unaffected siblings, urinary calcium excretion was examined and renal ultrasonography was performed. For adult individuals, hypercalciuria was defined as a urinary calcium/creatinine ratio of >0.6 mmol/mmol.
Mutation Analysis
PCLN-1 mutation screening was performed by single-strand conformation polymorphism analysis (21 7 PCLN-1 gene was used to amplify the coding sequence (exons 1 to 5) of genomic DNA by PCR. Amplified products were separated on polyacrylamide gels by electrophoresis (Multiphor II; Pharmacia Biotech, Uppsala, Sweden). Subsequently, exons with conformational variants were directly sequenced using corresponding sequencing primers (ALFexpress DNA sequencer; Pharmacia Biotech).
Haplotype Analysis
The microsatellite markers D3S1294, D3S1314, D3S1288, D3S2747 (22 7 PCLN-1 gene on chromosome 3q27, were amplified by PCR using primer pairs as reported (with Cy5-labeled forward primers). Fragments were separated on 6% polyacrylamide gels run under denaturing conditions in an ALFexpress DNA sequencer (Pharmacia Biotech), and data were analyzed using Fragment Manager software version 1.2 (Pharmacia Biotech). Alleles were numbered according to their order in gel electrophoresis, and haplotypes were constructed from the genotypic data. The most likely haplotypes were inferred by minimizing the number of cross-over events in each family.
Statistical Analyses
Statistical analyses of the paracellin-1 structure and amino acid composition, sequence alignments, and similarity searches were conducted using software provided by Infobiogen (http://www.infobiogen.fr) http://www.ncbi.nlm.nih.gov)
Results
Clinical Presentation
Table 1 summarizes the clinical and genotypic data. There were 20 female patients and 13 male patients in the cohort. Ages at the time of the initial manifestation ranged from 2 mo to 18 yr, with a median age of 3.5 yr (n = 30). Recurrent UTI, polyuria/polydipsia, hematuria, and abacterial leukocyturia were the most frequent presenting symptoms (Table 2 ). Few patients presented with nephrolithiasis, cerebral convulsions, muscular tetany, or abdominal pain. In multiplex families, diagnosis of the disease for one sibling generally prompted the search for FHHNC in the other sibling. For one patient (patient F21-1), nephrocalcinosis was detected incidentally by abdominal sonography during evaluation of a gastrointestinal infection at the age of 3 mo; further clinical evaluation revealed hypomagnesemia, hypercalciuria, and polyuria.
Table 2: Initial clinical presentation
Ten patients were affected by different eye lesions (Table 1 ). Hearing impairment was not noted for any of the patients in this study cohort. Differential diagnoses included idiopathic hypercalciuria, medullary sponge kidneys, Dent's disease, and hyperprostaglandin E syndrome/antenatal Bartter syndrome.
Biochemical Data and Renal Function
The minimal serum magnesium value for each patient is presented in Table 1 . The lowest serum magnesium level noted was <0.65 mM for all patients. The median value of the lowest serum magnesium levels was 0.40 mM (n = 32). Serum calcium levels were <1.8 mM at any time for 8 patients, 1.8 to 2.2 mM for 8 patients, and >2.2 mM for 14 patients. Two hypocalcemic patients exhibited signs of rickets in the neonatal period. The median maximal urinary calcium excretion value was 10.0 mg/kg per 24 h (n = 27). When measured for the first time, GFR was >80 ml/min per 1.73 m2 for 7 patients, 60 to 80 ml/min per 1.73 m2 for 13 patients, and <60 ml/min per 1.73 m2 for 11 affected children (Figure 2 ).
Figure 2: Changes in GFR for 30 patients affected by familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). The GFR of FHHNC-affected patients during their clinical courses are presented. The GFR was calculated as described (
20 ). At the onset of end-stage renal disease, dialysis was initiated or renal transplantation was performed.
Renal Sonographic and Histologic Results
For all patients, advanced bilateral nephrocalcinosis with characteristic medullary distribution was observed early in the course of the disease, independently of GFR. Renal cysts were observed for five patients. Renal histologic findings, which were documented for 11 patients, demonstrated calcium deposits, glomerular sclerosis, immature glomeruli, tubular atrophy, and interstitial fibrosis to a variable extent.
Clinical Course
Clinical symptoms and laboratory data recorded during the follow-up period are summarized in Table 3 . The median of follow-up was 7 yr, with a range of 0.5 to 25.5 yr. The majority of patients developed polyuria, polydipsia, and UTI. For most patients examined, we observed elevated serum PTH levels, incomplete dRTA, hypocitraturia, and hyperuricemia.
Table 3: Clinical and biochemical findings observed during the follow-up perioda
Figure 2 demonstrates that progression to CRF was variable. Patient F8-1 exhibited the most rapid decline in renal function, beginning with a GFR of 33 ml/min per 1.73 m2 at the age of 3 yr and reaching end-stage renal disease (ESRD) at 5.5 yr of age. In contrast, patients F1-2 and F2-1 attained ESRD late, at 34 and 37.5 yr of age, respectively. Several other patients also exhibited protracted courses.
By the end of the follow-up period, 12 patients (36%) had reached ESRD. The median age at the time of ESRD was 14.5 yr (range, 5.5 to 37.5 yr; one patient's age missing). Four patients were receiving dialysis treatment, and eight patients had undergone transplantation. Eleven patients (33%) exhibited GFR of <60 ml/min per 1.73 m2 , whereas ten patients (31%) still exhibited GFR of >60 ml/min per 1.73 m2 . Treatment with magnesium salts (to supplement renal loss) and thiazides (to reduce urinary calcium excretion rates) for the majority of patients apparently failed to prevent the progression of the disease.
Mutation Analysis of PCLN-1
Mutation analysis of PCLN-1 by single-strand conformation polymorphism analysis and direct sequencing revealed 15 different mutations among our cohort of 25 families (Table 1 ). Both mutant PCLN-1 alleles were detected in all affected individuals, with two exceptions; only a heterozygous splice site mutation was observed for patient F19, although the complete coding region and the adjacent intronic sequences were analyzed, and no PCLN-1 mutation was detected for patient F24. Figure 3 depicts affected amino acid residues in the paracellin-1 protein.
Figure 3: Paracellin-1 protein model deduced from hydrophilicity plots. Amino acid residues affected by PCLN-1 mutations in the cohort of families F1 to F25 are depicted in black (novel PCLN-1 mutations are underlined). Two newly identified splice site mutations are not shown. Charged wild-type amino acids in the extracellular loops are marked. On the basis of the results of mutation analysis and sequence comparisons, the paracellin-1 protein seems to be shorter than reported previously (the 70 amino-terminal amino acids that are presumably lacking are depicted in gray). Both translational start sites are shown.
In addition to the mutations described previously, eight novel PCLN-1 mutations were identified, including five missense mutations, one frameshift mutation, and two splice site mutations. All mutations cosegregated with the phenotype, and none of the single-nucleotide exchanges was observed in at least 100 control chromosomes.
Of the mutant alleles, 31 of 46 (67%) exhibited a missense mutation affecting the first extracellular loop of paracellin-1. Amino acid sequence alignments with other members of the claudin multigene family revealed the highest degree of homology in this part of the protein, and Leu145Pro, Arg149Leu, Leu151Thr, and Leu151Phe mutations all affected highly conserved amino acid residues in this loop. In total, 22 of 46 mutant alleles (48%) were affected by a Leu151Phe exchange. Haplotype analysis with microsatellite markers flanking the PCLN-1 gene [D3S1294, D3S1314, D3S1288, D3S2747 (22 7 PCLN-1 gene (intron 1/2, splice 325+61T→C) were subsequently performed. Results revealed a common extended haplotype that was shared by 50% (nine of 18) of the Leu151Phe-affected alleles but was not present in 72 unaffected control alleles (Figure 4 ). The highly polymorphic marker D3S1314 was mapped to the genomic clone of EMBL/GenBank/DDBJ accession no. AC009520, which also comprises the 5′ end of the human PCLN-1 gene, defining a physical distance between D3S1314 and PCLN-1 of 13.7 kb. All Leu151Phe-affected chromosomes exhibited the same allele (numbered as allele 6 ) for marker D3S1314. In contrast, only 17% of control chromosomes exhibited this allele. This finding strongly suggests a founder effect for the Leu151Phe mutation.
Figure 4: Founder effect of Leu151Phe, demonstrated by extended haplotype analysis. The prevalence of an extended haplotype in Leu151Phe-affected chromosomes, compared with unaffected control chromosomes, is presented. Alleles are numbered according to their order after gel electrophoresis. The numbers of different alleles for each marker were as follows: D3S1294, n = 11; 539-5, n = 9; D3S1314, n = 12; D3S1288, n = 10; D3S2747, n = 8. The intronic polymorphism (PM) of the PCLN-1 gene (intron 1/2; splice 325+61T→C) was numbered 2 , compared with number 1 for wild-type alleles. cen, centromere; tel, telomere.
In three families (F4, F8, and F12), a frameshift mutation affecting amino acid residue Arg55 was identified, in addition to a missense mutation further 3′ downstream on the same chromosome. The Arg55fs mutation was caused by deletion of two guanine residues (G165/G166) and insertion of a single cytosine residue (C165). This mutation affects the amino-terminal part of the protein, leading to premature termination at position 90. Unexpectedly, analysis of 72 control chromosomes revealed a frequency of the G165/G166 deletion-C165 insertion of 16.7% (12 of 72 chromosomes), indicating a common polymorphism rather than a pathogenic mutation.
Genotype-Phenotype Relationship and Intrafamilial Variability
Phenotypic evaluation with respect to the results of the mutation analysis revealed no obvious genotype-phenotype relationship. However, with respect to the progression of renal failure, we observed a rather close intrafamilial phenotypic concordance, with a mild clinical course for the siblings of family F5, an intermediate course for patients of families F1, F9, and F21, and a more severe course for patients of families F7 and F14 (uniovular twins) (Figure 2 ). In contrast, the siblings of family F2 differed in disease severity, reaching ESRD at the ages of 13.5 and 37 yr. Similarly, the younger sibling of patient F8 began dialysis treatment at 5.5 yr of age, whereas his sister, who was 8 yr of age at the end of the follow-up period, maintained a stable GFR of 60 ml/min per 1.73 m2 .
Family Histories
Histories of nephrolithiasis, nephrocalcinosis, and/or hypercalciuria were observed for 13 of 23 families (56%), with nephrolithiasis being the most frequent finding. Detailed results are presented in Figure 1 .
Discussion
Phenotype Analysis
In cooperation with other European pediatric departments, we assembled a study cohort consisting of 25 FHHNC families, with 33 affected individuals, for the study of genotypes and phenotypes in FHHNC. Phenotypic evaluation of affected individuals revealed disease onset in early childhood, with recurrent UTI, polyuria/polydipsia, hematuria, and abacterial leukocyturia being the most frequently observed symptoms. In general, the final diagnosis of FHHNC was made by recognition of the triad of severe hypomagnesemia, marked hypercalciuria, and nephrocalcinosis. According to our data, 33% of the patients presented with reduced GFR (<60 ml/min per 1.73 m2 ) at the time of diagnosis. At the end of the follow-up period, 36% of the patients exhibited ESRD and 33% exhibited GFR of <60 ml/min per 1.73 m2 .
Although much progress in understanding FHHNC has been made with the identification of the genetic defect, there is still uncertainty regarding the pathophysiologic basis of renal failure in this disorder. It seems that loss or impairment of paracellin-1 function results in decreased reabsorption of magnesium in the TAL, leading to profound hypermagnesiuria and hypomagnesemia (7 15 14 23 23 , 24
With the interfamilial variability but frequent intrafamilial concordance observed for our study cohort, additional factors influencing the rate of progression of renal insufficiency might be related to the genetic backgrounds of the affected patients. Further factors include aspects of medical surveillance and patient compliance with supportive treatment.
In general, therapeutic success is poor in FHHNC. Despite continuous administration of magnesium supplements to most patients, serum magnesium levels usually remain low, with urinary magnesium excretion increasing proportionally because of the disease-specific alterations in the renal magnesium threshold and transport maximum (14 et al. (25
Evaluation of family histories, determination of urinary calcium excretion rates, and renal sonography revealed striking incidences of hypercalciuria, nephrolithiasis, and UTI among family members not affected by FHHNC (in 13 of 23 families). A comparable observation was reported by Praga et al. (12 PCLN-1 mutations) was affected by nephrolithiasis, nephrocalcinosis, and/or hypercalciuria. It seems reasonable to propose a relationship between isolated hypercalciuria and mutations in the PCLN-1 gene. With the exception of the uncle of patient F5, further results of mutation analysis support this hypothesis; heterozygous PCLN-1 mutations were also observed for the hypercalciuric brother of patient F18 and the grandparents of patients F12, F22, and F25, who were affected by nephrolithiasis. It might be expected that mutation analysis of kindreds affected by familial hypercalciuria with nephrocalcinosis and/or nephrolithiasis (with an apparently dominant mode of inheritance) would demonstrate heterozygous mutations in the PCLN-1 gene in a number of families.
Genotype Analysis
Genotyping revealed PCLN-1 mutations in all mutant alleles of our study cohort, except for three alleles. Fifteen different mutations were found in PCLN-1 , including eight novel mutations. Therefore, 24 different PCLN-1 mutations have been identified to date (7 , 8 8 Table 1 and Figure 3 ). Because amino acid sequence alignments of paracellin-1 with other members of the claudin multigene family revealed a high degree of homology in this region of the protein, a special importance for paracellin-1 function must be proposed. Because of the large number of neutral amino acids, the first extracellular loop is predicted to bridge the intercellular space (26 Figure 3 ), which is unique to paracellin-1 (in contrast to other claudins), is proposed to contribute to the cation sensitivity of the paracellular pathway in the TAL (7
Of the mutant alleles, 48% were affected by a Leu151Phe substitution. The predominance of Leu151Phe-affected families originating from Germany or eastern Europe (Czech Republic, Slovenia, Bosnia, Macedonia, or Poland) is striking; 14 of 18 such families bore this mutation on at least one allele, whereas it was not observed among the families that originated from France, Algeria, Tunisia, or Turkey. Moreover, the Leu151Phe mutation was not observed in the study by Simon et al. (7 Figure 4 ). Allele 6 of the microsatellite marker D3S1314, which is 13.7 kb distant from PCLN-1 and is highly polymorphic (with 12 different alleles), cosegregated with the Leu151Phe mutation in 100% of the cases, whereas only 17% of the control chromosomes bore this allele. The extended haplotype covering the PCLN-1 region for a genetic distance of 2.3 cM was shared by 50% of the Leu151Phe-affected chromosomes but was not observed in any of the unaffected chromosomes. Recombination events in previous generations that are not detectable in our study cohort could explain the lack of complete concordance of the extended haplotype. Because all Leu151Phe-affected patients originated from Germany or eastern Europe and the Leu151Phe exchange was not observed in patients who originated from other countries, a founder effect seems very likely. Only four of the 19 families originating from Germany or eastern Europe were not affected by a Leu151Phe exchange. This observation will simplify genetic analysis among individuals with suspected FHHNC who originate from these countries.
With respect to the protein structure of paracellin-1, the following aspect of PCLN-1 mutation analysis seems to be important: in three families, one mutant allele exhibited a frameshift mutation affecting amino acid residue Arg55, in addition to a missense mutation further 3′ downstream. Although two mutations per affected allele have also been described for other hereditary diseases (27 28 et al. (7 PCLN-1 gene encodes a second methionine (Met71) in the context of a more suitable Kozak consensus sequence, compared with Met1 (70 amino acids 3′ downstream), that is in a position analogous to the start site of other claudins (16 , 26 PCLN-1 mRNA. Further support for this theory arises from sequence alignments of human paracellin-1 with bovine paracellin-1, which was recently identified (29 , 30 Figure 5 ). This finding strongly indicates that human paracellin-1 is considerably shorter than previously reported and lacks a major part of the amino terminus, similar to other members of the claudin family. This finding supports the hypothesis that the second methionine is the original translational start site. Alternatively, two different isoforms might exist in the kidney, resulting from two different translational start sites. It might be speculated that the shorter protein is able to prevent renal disease in individuals who are affected by the G165/G166 deletion-C165 insertion. It is hoped that additional studies on the translational regulation of paracellin-1 will resolve this controversy.
Figure 5: Amino acid sequence alignment of human paracellin-1 (EMBL/GenBank/DDBJ accession no. AF152101) and bovine paracellin-1 (EMBL/GenBank/DDBJ accession no. AB030082). Identical amino acids are shadowed in gray. Human and bovine translational start methionines, as reported in the GenBank submissions, are indicated by asterisks and thin black boxes. The proteins share a methionine in a suitable Kozak consensus sequence (human Met71/bovine Met20, marked by a thick black box), followed by a sequence of high homology (identity, 91%; similarity, 95%). hs, Homo sapiens ; bt, Bos taurus . The one-letter amino acid code is used.
In summary, although much has been learned with respect to FHHNC pathophysiologic processes and the underlying genetic defect, new questions regarding aspects of the gene and protein structures have arisen. Additional basic studies on tight junction assembly and the proteins involved might answer some of these questions. With respect to the rapid deterioration of renal function in a great number of affected children, our understanding of the development of renal insufficiency in FHHNC must be extended. Therapeutic strategies, especially those targeting the prevention of ESRD, will hopefully improve in the future.
We are indebted to the participating patients and their families for their cooperation. We thank Nora Alffen and Ulla Pechmann for excellent technical assistance. Drs. Konrad and Seyberth were supported by the Deutsche Forschungsgemeinschaft (Ko1480/3-2). Dr. Weber was supported by the Stiftung P. E. Kempkes (Kennziffer 30/99).
1. Cole DE, Quamme GA: Inherited disorders of renal magnesium handling. J Am Soc Nephrol 11: 1937–1947, 2000
2. Geven WB, Monnens LA, Willems HL, Buijs WC, ter Haar BG: Renal magnesium wasting in two families with autosomal dominant inheritance. Kidney Int 31: 1140–1144, 1987
3. Gitelman HJ, Graham JB, Welt LG: A new familial disorder characterized by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians 79: 221–235, 1966
4. Michelis MF, Drash AL, Linarelli LG, De Rubertis FR, Davis BB: Decreased bicarbonate threshold and renal magnesium wasting in a sibship with distal renal tubular acidosis: Evaluation of the pathophysiological role of parathyroid hormone. Metabolism 21: 905-920, 1972
5. Castrillo JM, Rapado A, Traba ML, Esbrit P, Hernando L: Nefrocalcinosis con Hipomagnesemia. Nefrologia, 3: 159-165, 1983
6. Benigno V, Canonica CS, Bettinelli A, von Vigier RO, Truttmann AC, Bianchetti MG: Hypomagnesaemia-hypercalciuria-nephrocalcinosis: A report of nine cases and a review. Nephrol Dial Transplant 15: 605-610, 2000
7. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP: Paracellin-1, a renal tight junction protein required for paracellular Mg
2+ resorption. Science (Washington DC) 285: 103-106, 1999
8. Weber S, Hoffmann K, Jeck N, Saar K, Boeswald M, Kuwertz-Broeking E, Meij II, Knoers NV, Cochat P, Sulakova T, Bonzel KE, Soergel M, Manz F, Schaerer K, Seyberth HW, Reis A, Konrad M: Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis maps to chromosome 3q27 and is associated with mutations in the PCLN-1 gene. Eur J Hum Genet 8: 414-422, 2000
9. Gregoric A, Bracic K, Novljan G, Marcun-Varda N: Pseudotumor cerebri in a child with familial hypomagnesemia-hypercalciuria. Pediatr Nephrol 14: 269–270, 2000
10. Manz F, Scharer K, Janka P, Lombeck J: Renal magnesium wasting, incomplete tubular acidosis, hypercalciuria and nephrocalcinosis in siblings. Eur J Pediatr 128: 67–79, 1978
11. Rodriguez-Soriano J, Vallo A: Pathophysiology of the renal acidification defect present in the syndrome of familial hypomagnesaemia-hypercalciuria. Pediatr Nephrol 8: 431-435, 1994
12. Praga M, Vara J, Gonzalez-Parra E, Andres A, Alamo C, Araque A, Ortiz A, Rodicio JL: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis. Kidney Int 47: 1419-1425, 1995
13. Ulmann A, Hadj S, Lacour B, Bourdeau A, Bader C: Renal magnesium and phosphate wastage in a patient with hypercalciuria and nephrocalcinosis: Effect of oral phosphorus and magnesium supplements. Nephron 40: 83–87, 1985
14. Rodriguez-Soriano J, Vallo A, Garcia-Fuentes M: Hypomagnesaemia of hereditary renal origin. Pediatr Nephrol 1: 465-472, 1987
15. Quamme GA: Renal magnesium handling: New insights in understanding old problems. Kidney Int 52: 1180–1195, 1997
16. Morita K, Furuse M, Fujimoto K, Tsukita S: Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA 96: 511-516, 1999
17. Geven WB, Monnens LA, Willems JL: Magnesium metabolism in childhood. Miner Electrolyte Metab 19: 308-313, 1993
18. Moxey-Mims MM, Stapleton FB: Hypercalciuria and nephrocalcinosis in children. Curr Opin Pediatr 5: 186–190, 1993
19. Matos V, van Melle G, Boulat O, Market M, Bachmann C, Guignard JP: Urinary phosphate/creatinine, calcium/creatinine, and magnesium/creatinine ratios in a healthy pediatric population. J Pediatr 131: 252-257, 1997
20. Schwartz GJ, Brion LP, Spitzer A: The use of plasma creatinine concentration for estimating glomerular filtration rate in infants, children, and adolescents. Pediatr Clin North Am 34: 571-590, 1987
21. Orita M, Suzuki Y, Sekiya T, Hayashi K: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874–879, 1989
22. Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau P, Marc S, Hazan J, Seboun E, Lathrop M, Gyapay G, Morissette J, Weissenbach J: A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature (Lond) 380: 152-154, 1996
23. Wrong O: Nephrocalcinosis. In: Oxford Textbook of Clinical Nephrology, 2nd Ed., edited by Davison AM, Cameron JS, Grünfeld J-P, Kerr DNS, Ritz E, Winearls CG, Oxford, UK, Oxford University Press, 1998, pp 1376–1396
24. Köckerling A, Reinalter SC, Seyberth HW: Impaired response to furosemide in hyperprostaglandin E syndrome: Evidence for a tubular defect in the loop of Henle. J Pediatr 129: 519-528, 1996
25. Monnens L, Starremans P, Bindels R: Great strides in the understanding of renal magnesium and calcium reabsorption. Nephrol Dial Transplant 15: 568–571, 2000
26. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S: Claudin-1 and -2: Novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 1539-1550, 1998
27. Lemmink HH, Knoers NV, Karolyi L, van Dijk H, Niaudet P, Antignac C, Guay-Woodford LM, Goodyer PR, Carel JC, Hermes A, Seyberth HW, Monnens LA, van den Heuvel LP: Novel mutations in the thiazide-sensitive NaCl cotransporter gene in patients with Gitelman syndrome with predominant localization to the C-terminal domain. Kidney Int 54: 720-730, 1998
28. Kozak M: Interpreting cDNA sequences: Some insights from studies on translation. Mamm Genome 7: 563–574, 1996
29. Hirano T, Kobayashi N, Itoh T, Takasuga A, Nakamaru T, Hirotsune S, Sugimoto Y: Null mutation of PCLN-1/claudin-16 results in bovine chronic interstitial nephritis. Genome Res 10: 659-663, 2000
30. Ohba Y, Kitagawa H, Kitoh K, Sasaki Y, Takami M, Shinkai Y, Kunieda T: A deletion of the paracellin-1 gene is responsible for renal tubular dysplasia in cattle. Genomics 68: 229-236, 2000
