 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
,,||,,||,§,||
*
Department of Biology, McGill University, Montreal, Canada
Department of Human Genetics, McGill University, Montreal,
Canada
Department of Medicine, McGill University, Montreal, Canada
§
Department of Pediatrics, McGill University, Montreal, Canada
||
McGill University Health Centre Research Institute, Montreal,
Canada
¶
Department of Nephrology, Assaf Harofeh Medical Center, Beer Yaakov,
Israel
Department of Pediatric Nephrology, Northwestern University, Chicago,
Illinois
§§
Department of Pediatrics, University Ziekenhuis Gasthuisberg, Leuven,
Belgium
||||
Department of Pediatrics, Yale University School of Medicine, New Haven,
Connecticut
¶¶
Department of Pediatric Endocrinology, John Hunter Children's Hospital,
Newcastle, Australia.
Correspondence to Dr. Harriet S. Tenenhouse, Montreal Children's Hospital, 2300 Tupper Street, Montreal, Quebec H3H 1P3, Canada. Phone: 514-934-4400, ext. 2342; Fax: 514-934-4331; E-mail: mdht{at}www.debelle.mcgill.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HHRH was first described in a large inbred Bedouin kindred (1), and only a few sporadic cases have been subsequently reported (5,6,7,8). In an extensive study of the Bedouin kindred, 21 members were found to be clinically healthy but displayed "idiopathic" hypercalciuria (IH) and biochemical measurements that were intermediate between normal and affected members of the kindred (2). On the basis of the phenotypic findings in individuals with HHRH and IH, it was suggested that individuals with HHRH and IH are homozygous and heterozygous, respectively, for the same mutant allele (2).
Treatment of HHRH is accomplished by oral Pi supplementation. It increases growth rate, corrects the rickets and osteomalacia, and normalizes all biochemical abnormalities, with the exception of renal Pi wasting (1). On the basis of these findings, it was suggested that HHRH is a primary disorder of renal Pi reabsorption.
The kidney plays a key role in the maintenance of Pi homeostasis (9). Of the three classes of Na/Pi cotransporters identified in mammalian kidney, the type II Na/Pi cotransporter, Npt2, is the most abundant at the mRNA level (10). The Npt2 protein was localized exclusively to the brush border membrane (BBM) of proximal tubule cells (11), where the majority of filtered Pi is reabsorbed, and is a target for regulation by PTH and dietary Pi, major regulators of renal Pi handling (12). The human orthologue, NPT2 (gene SLC34A1 in the Human Gene Nomenclature Database; http://www.gene.ucl.ac.uk/nomenclature) was mapped to chromosome region 5q35 by fluorescence in situ hybridization (13). The NPT2 gene spans 16 kb and consists of 13 exons (14); the initiation codon starts at nucleotide 82 in the second exon (15). The 2.5-kb coding region is predicted to code for a 635 amino acid protein that has eight transmembrane domains (15).
Recent studies in mice in which the Npt2 gene was disrupted by targeted mutagenesis have underscored the importance of Npt2 in the maintenance of Pi homeostasis (16). Mice that are homozygous for the disrupted Npt2 gene (Npt2-/-) exhibit increased urinary excretion of Pi, decreased BBM Na/Pi cotransport, hypophosphatemia, and an elevated serum concentration of 1,25(OH)2D, with attendant hypercalcemia, hypercalciuria, and reduced circulating PTH levels (16). The biochemical features of Npt2-/- mice resemble those of patients with HHRH, and on the basis of these findings, we hypothesized that NPT2 is a candidate gene for HHRH. The present study was undertaken to test this hypothesis in the Bedouin kindred in which the disease was first characterized (1,2), as well as in four small families from different countries.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
DNA Sequencing
Intronic primers 10 to 50 bp from the intron/exon boundaries were designed
to amplify the 13 NPT2 exons
(Table 2). Exons were amplified
as single fragments or amplified together with other exons. PCR was done in a
volume of 50 µl with 100 ng of genomic DNA, 2.5 U of Platinum Taq
(BRL Life Technologies, Burlington, Ontario, Canada), 0.4 µM primer, and 1
µM dNTP. PCR conditions were as follows: 30 s at 94°C, 30 s at the
annealing temperature of the specific primer pair
(Table 2), and 60 s at
72°C, for 35 cycles, with a final extension at 72°C for 10 min. PCR
products were sequenced either using the T7 Sequenase Kit with
[
-33P]dNTP (Amersham Pharmacia Biotech, Baie d'Urfe, Quebec,
Canada) or on an ABI 373A sequencer (Laval University Sequencing Service,
Quebec, Quebec, Canada; or McGill University Sheldon Biotechnology Center,
Montreal, Quebec, Canada).
|
Screening of NPT2 Polymorphisms
Assays based on amplification-created or naturally occurring restriction
sites were developed to screen for the single nucleotide polymorphisms (SNP)
identified by DNA sequencing. The T-to-C substitution in intron 4 creates an
HgaI site when amplified with primer I4R
(5'-GATGTGGAGGTGCTGGAGCTCT-3') and a primer
(5'-GGTTCCTGAAGGGCCTTGGACgA-3') designed with a mismatch (denoted
by lower-case g). The PCR products were digested overnight with 4 U of
HgaI at 37°C. Exon 7 was amplified using the forward primer for
exons 7 to 8 (Table 2) and 7R
(5'-AGGAGGCTGGGTCCCTTGTTAC-3') at an annealing temperature of
61°C. The T-to-C substitution in exon 7 abolishes an NlaIII site,
and overnight digestion of the PCR product was performed with 5 U of
NlaIII at 37°C. The digests were run on 10% acrylamide minigels
and stained with ethidium bromide; genotypes were assigned on the basis of the
digestion pattern. For the intron 4 SNP, the T allele has one fragment of 158
bp; the C allele has two fragments of 131 and 27 bp. For the exon 7 SNP, the T
allele has four fragments of 105, 87, 62, and 36 bp; the C allele has three
fragments of 192, 62, and 36 bp. An allelespecific hybridization assay was
also developed to screen for the exon 7 SNP (details available from the
authors).
Genotyping Microsatellite Markers
DNA samples from the Bedouin kindred were genotyped for markers that flank
NPT2 in chromosome region 5q35 (D5S2108, D5S2058, D5S498,
D5S469, and D5S408) by PCR amplification using the protocol for
[
-32P] end-labeled primers
(17) with minor modifications.
Primer sequences were obtained from the Genome Database
(http://gdbwww.gdb.org). Marker order and sex-averaged distances were obtained
from the Marshfield Medical Research Foundation web site
(http://research.marshfieldclinic.org/genetics). NPT2 was placed
between D5S498 and D5S469 on the basis of radiation hybrid
mapping (http://chrom5.hsis.uci.edu). Thus, our marker order and approximate
intermarker distances are D5S2108—1.33
cM—D5S2058—0.98 cM—D5S498—2.29
cM—NPT2—2.29 cM—D5S469—6.26
cM—D5S408. We manually constructed haplotypes by attempting to
minimize the number of recombinants and assuming that there was no mutation of
marker alleles.
Statistical Analyses
Evidence for linkage between a locus for HHRH and chromosome 5 markers was
analyzed in the Bedouin kindred using a genetic model of an autosomal
recessive trait with either complete penetrance or 75% penetrance, and a
disease allele frequency of 0.025. Although HHRH is a rare disease, we assumed
that it may be more common in the Bedouin population than in the general
population and chose a relatively high carrier prevalence of 5%. Two-point
parametric linkage analysis was done using the MLINK program of the FASTLINK
package (version 4.1P)
(18,19)
of the LINKAGE programs (version 5.2)
(20). Marker allele
frequencies were obtained using the program NEWPREP (Whitehead Institute for
Biomedical Research, Boston, MA). Multipoint nonparametric linkage analysis
was performed with SimWalk2, version 2.60
(21). This program uses Markov
chain Monte Carlo methods to assess the degree of allele sharing that is
identical by descent among affected members of a pedigree. The analysis was
run using 50,000 simulations of the pedigree. We report P values for
statistic A, which measures the number of different paths that lead to marker
alleles that appear among affected individuals. Statistic A was chosen because
it tends to have the most power for detecting linkage to a recessively
inherited trait.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Figure 1) shows a partial pedigree of the Bedouin kindred with 12 individuals having a diagnosis of HHRH. The members of the Bedouin kindred are related in many different ways; there are eight known inbreeding loops. The relationships among the 10 nuclear families with a child affected with HHRH range from parent—offspring to third cousins or third cousins once-removed in two different ways. Not all pairs of nuclear families are known to be related to each other, and there is no single recent ancestor common to all of the parents based on the available genealogical information. The parents of three sibships are consanguineous; two sets of parents are related as first cousins and one set as second cousins onceremoved. Consanguinity is also evident in family A, in which the parents of the affected individual are related as second cousins (Figure 2).
Mutation Analysis
Six individuals with HHRH were selected for DNA sequence analysis: two from
the Bedouin kindred and one each from families A to D (see Figures
1 and
2). Sequencing of the
NPT2 exons in these six individuals did not reveal any putative
disease-causing mutation. In addition, no differences were found in 120 bp of
the promoter region among the individuals with HHRH, control subjects, and
published sequences
(14,23);
available intronic sequence agreed with that reported previously
(23). Two SNP were identified
in intron 4 and exon 7. Sequencing revealed both a T and a C in intron 4, 54
bp from the 3' end of exon 4 (nucleotide 78 in GenBank accession
AH007379). The common name for this SNP is designated as IVS4+54T>C, and
the systematic name is c.469+54T>C. The second SNP was a silent
substitution in codon 258 (CAT or CAC) in exon 7 (T or C at nucleotide 774
where, for numbering purposes, nucleotide 1 is the A in the initiation codon;
nucleotide 855 in GenBank reference sequence NM_003052). The common name for
this SNP is designated as H258H, and the systematic name is c.774T>C.
Single-strand conformation analysis was also used to search for mutations in
the Bedouin kindred and in families B and D. No conformational changes were
observed other than the band shift caused by H258H (data not shown).
DNA samples from all of the available family members were typed for the two NPT2 SNP. The data in Figure 1 show that they do not consistently segregate with HHRH in the Bedouin kindred. Among those with HHRH, there were two genotypic combinations of IVS4+54T>C and H258H: CT/CT (n = 7) and TT/CC (n = 3). Segregation of the SNP in families A to D does not provide any evidence for or against linkage to HHRH (Figure 2).
Both NPT2 SNP were polymorphic in an ethnically diverse sample of 79 individuals. The allele frequencies did not differ significantly among the Chinese, French Canadians, Greeks, and Italians (Fisher's exact test, P = 0.08 for IVS4+54T>C and P = 0.13 for H258H). The predominant allele for IVS4+54T>C was C (allele frequency = 0.78; SD = 0.03) and for H258H was T (allele frequency = 0.71; SD = 0.04). Thirty-four of the 79 individuals were heterozygous for one or both SNP.
Linkage Analysis
Visual inspection of the genotypes in the Bedouin kindred indicated that
two of the individuals with HHRH (604 and 675), who are children of first
cousins, and five others with HHRH (527, 529, 655, 583, and 589) were not
homozygous for the NPT2 SNP
(Figure 1). Furthermore, none
of the three patients who were homozygous for the NPT2 SNP (603, 664,
and 666) were homozygous for the haplotype consisting of the flanking
microsatellite markers. Notably, the two affected siblings who are children of
first cousins (603 and 604) share only one haplotype, and the other pair of
affected siblings shares no haplotype (655 and 664). In families B, C, and D,
none of the individuals with HHRH were homozygous for both NPT2 SNP
(Figure 2). In family A, the
HHRH patient is homozygous for the NPT2 SNP but is not homozygous for
four of the five microsatellite markers (data not shown). Thus, within each of
these kindreds, we infer that individuals with HHRH are not homozygous for an
undetected mutation in NPT2 that was inherited from a recent common
ancestor.
Parametric linkage analysis in the Bedouin kindred, under the assumption that HHRH is a completely penetrant autosomal recessive trait, indicated that there were recombinants between a locus for HHRH and the NPT2 SNP as well as for four of the five microsatellite markers. Linkage to NPT2 within 1% recombination could be excluded (old scores of -2.4 and -2.6 for the intron 4 and exon 7 SNP, respectively). Linkage could be excluded within 5% recombination with D5S2108, and D5S2058, within 2% recombination with D5S498, and within 13% recombination with D5S408 (lod scores < -2). Only two alleles were observed for D5S469, and this marker was not informative in most matings. Reducing the penetrance to 75% gave essentially the same results; lod scores of -3.9 and -4.1 were obtained for linkage of HHRH to the intron 4 and exon 7 SNP, respectively, at 0% recombination.
Because a precise genetic model is not known for HHRH, nonparametic analysis was also performed to test for linkage. The NPT2 SNP are not sufficiently informative for two-point nonparametric linkage analysis; therefore, multipoint analysis was done with SimWalk2. The program was run four times using 50,000 simulations of the pedigree for each run. The average empirical P values for statistic A of the four runs was greater than 0.18 (SD ranged from 0.02 to 0.08) at every marker (D5S2108, D5S2058, D5S498, D5S469, D5S408, and the two NPT2 SNP). In particular, the average P value was 0.23 for both NPT2 SNP (P values ranged from 0.12 to 0.27 over the four runs; SD was 0.07 for both SNP). Clustering among affected individuals of alleles descending from the founders is expected when there is linkage. The multipoint nonparametric analysis of the Bedouin kindred indicates that there is no significant excess marker allele sharing among pedigree members affected with HHRH and provides further evidence against linkage of NPT2 to a locus for HHRH. Linkage analysis was not done on families A to D as the available data do not have sufficient power to test for linkage.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Exclusion of NPT2 as a candidate gene for HHRH in the families studied is consistent with the observed differences in the skeletal phenotype in individuals with HHRH and in mice that are homozygous for the disrupted Npt2 gene. Individuals with HHRH have radiologic and histomorphometric evidence of rickets and osteomalacia (1,22). In contrast, Npt2-/- mice are characterized by a unique age-dependent bone phenotype. At weaning, Npt2-/- mice exhibit poorly developed metaphyseal trabeculae and retarded secondary ossification in the epiphysis when compared with wild-type littermates (16). However, with increasing age, the knockout mice exhibit a reversal and eventual correction of the skeletal abnormalities observed at weaning (16). An age-related improvement of the bone abnormalities is not evident in individuals with HHRH, and treatment with oral Pi supplementation is necessary to heal the rickets and osteomalacia (1).
It has been suggested that HHRH and IH could be the result of mutations in two different genes (2). The strongest support against this hypothesis is that when individuals with HHRH are treated with oral Pi, both the hypophosphatemia and the hypercalciuria are corrected. However, if HHRH is an autosomal recessive disease and individuals with IH are heterozygous for a mutant allele, then IH must be an incompletely penetrant trait because not all obligate heterozygotes have IH. In families A to D, however, the variability in clinical presentation suggests that mutations in different genes may be involved.
Possible candidate genes for HHRH include genes that encode other renal Pi transporters or their regulators. The type I Na/Pi cotransporter accounts for approximately 13% of known Na/Pi cotransporter mRNA in mouse kidney (10) and, as is the case for Npt2, the immunoreactive protein was localized to the BBM of proximal tubule cells (24). The human orthologue NPT1 (also called SLC17A1) was mapped to chromosome region 6p22 (25). Analysis of allele sharing among individuals with HHRH using three microsatellite markers (D6S2238, D6S2237, and D6S2233) in a 500-kb region that includes NPT1 failed to provide evidence of linkage to HHRH in the Bedouin kindred under the assumption that HHRH is a completely penetrant autosomal recessive disease (26).
The type III Na/Pi cotransporters that have a broad tissue distribution (27) account for less than 1% of known Na/Pi cotransporter mRNA in mouse kidney (10). They have been postulated to serve as housekeeping Na/Pi cotransporters, and, thus, their involvement in HHRH is unlikely. Other likely candidate genes for HHRH are the orthologues of diphor-1 (28) and Pius (29), which map to human chromosome region 1q21 (30) and 3p21 (31), respectively. These proteins modify Pi transport, and both have been shown to stimulate Na/Pi cotransport in oocytes co-injected with Npt2 (28,29).
The reabsorption of solutes and ions in the proximal tubule can also occur through tight junction-regulated paracellular pathways. The gene for paracellin-1 was recently identified by positional cloning, and mutations were found in patients with hypomagnesemia and magnesium wasting (32). These findings, as well as functional studies, implicated paracellin-1 in the paracellular transport of Mg2+ (32). Although tight junctions are more selective for cations than anions, further studies are necessary to determine whether a similar mechanism exists for renal Pi reabsorption.
BBM Na/Pi cotransport is extremely sensitive to changes in pH, and this effect is in large part explained by a competition of protons with Na ions for interaction with the cotransporter protein (33). Accordingly, genes involved in the regulation of luminal pH are likely to be important in modifying renal Na/Pi cotransport and may have a role to play in the pathophysiology of HHRH.
XLH, which is phenotypically similar to HHRH, with the exception of inappropriately regulated serum 1,25(OH)2D concentration, is caused by mutations in the PHEX gene, which has significant similarity to members of the M13 endopeptidase family (34). Although the endogenous substrate(s) for PHEX has not yet been identified, it is postulated to be a paracrine/endocrine factor involved in the regulation of skeletal mineralization and Pi homeostasis (34). Thus, mutations in genes involved in the PHEX pathway are possible candidates for HHRH.
The two SNP that were identified in NPT2 may be useful for association studies of multifactorial diseases such as nephrolithiasis and osteoporosis in which NPT2 may play a role. Given the recent study suggesting that SNP can perturb mRNA secondary structure (35), the silent substitution in exon 7 may have an impact on NPT2 gene expression, which in turn could affect Pi homeostasis.
In summary, we identified two SNP in the NPT2 gene and demonstrated that these sequence variants as well as microsatellite markers that flank the NPT2 gene are not linked to HHRH in the families that we studied. Further studies, using candidate gene or genome-wide scan approaches, are necessary to determine the genetic factor(s) responsible for HHRH.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  T. Iwaki, M. J. Sandoval-Cooper, H. S. Tenenhouse, and F. J. Castellino A Missense Mutation in the Sodium Phosphate Co-transporter Slc34a1 Impairs Phosphate Homeostasis J. Am. Soc. Nephrol., September 1, 2008; 19(9): 1753 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Levi Novel NaPi-2c mutations that cause mistargeting of NaPi-2c protein and uncoupling of Na-Pi cotransport cause HHRH Am J Physiol Renal Physiol, August 1, 2008; 295(2): F369 - F370. [Full Text] [PDF]
|
|
|
|
|
|
H. Segawa, S. Yamanaka, A. Onitsuka, Y. Tomoe, M. Kuwahata, M. Ito, Y. Taketani, and K.-i. Miyamoto Parathyroid hormone-dependent endocytosis of renal type IIc Na-Pi cotransporter Am J Physiol Renal Physiol, January 1, 2007; 292(1): F395 - F403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ichikawa, A. H. Sorenson, E. A. Imel, N. E. Friedman, J. M. Gertner, and M. J. Econs Intronic Deletions in the SLC34A3 Gene Cause Hereditary Hypophosphatemic Rickets with Hypercalciuria J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4022 - 4027. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bevilacqua, L. J. Dominguez, V. Righini, V. Valdes, R. Toscano, O. Sangaletti, T. Vago, G. Baldi, M. Barrella, and G. Bianchi-Porro Increased Gastrin and Calcitonin Secretion after Oral Calcium or Peptones Administration in Patients with Hypercalciuria: A Clue to an Alteration in Calcium-Sensing Receptor Activity J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1489 - 1494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. W. Moe and O. Bonny Genetic Hypercalciuria J. Am. Soc. Nephrol., March 1, 2005; 16(3): 729 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K. Frick and D. A. Bushinsky Molecular Mechanisms of Primary Hypercalciuria J. Am. Soc. Nephrol., April 1, 2003; 14(4): 1082 - 1095. [Full Text] [PDF]
|
|
|
|
|
|
H. S. Tenenhouse and H. Murer Disorders of Renal Tubular Phosphate Transport J. Am. Soc. Nephrol., January 1, 2003; 14(1): 240 - 247. [Full Text] [PDF]
|
|
|
|
 |
|
 D. Prie, V. Huart, N. Bakouh, G. Planelles, O. Dellis, B. Gerard, P. Hulin, F. Benque-Blanchet, C. Silve, B. Grandchamp, et al. Nephrolithiasis and Osteoporosis Associated with Hypophosphatemia Caused by Mutations in the Type 2a Sodium-Phosphate Cotransporter N. Engl. J. Med., September 26, 2002; 347(13): 983 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Silve and L. Beck Is FGF23 the long sought after phosphaturic factor phosphatonin? Nephrol. Dial. Transplant., June 1, 2002; 17(6): 958 - 961. [Full Text] [PDF]
|
|
|
|
|
|
S. M. Jan De Beur and M. A. Levine Molecular Pathogenesis of Hypophosphatemic Rickets J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2467 - 2473. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
