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Intronic Deletions in the SLC34A3 Gene Cause Hereditary Hypophosphatemic Rickets with Hypercalciuria

Departments of Medicine (S.I., A.H.S., E.A.I., M.J.E.), Pediatrics (E.A.I.), and Medical and Molecular Genetics (M.J.E.), Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Pediatrics (N.E.F.), Duke University Medical Center, Durham, North Carolina 27710; and Department of Medicine (J.M.G.), Division of Endocrinology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Michael J. Econs, M.D., Department of Medicine, Indiana University School of Medicine, 541 North Clinical Drive, Clinical Building 459, Indianapolis, Indiana 46202-5121. E-mail: mecons{at}iupui.edu.

	Abstract

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Context: Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia and rickets/osteomalacia with increased serum 1,25-dihydroxyvitamin D [1,25-(OH)₂D] resulting in hypercalciuria.

Objective: Our objective was to determine whether mutations in the SLC34A3 gene, which encodes sodium-phosphate cotransporter type IIc, are responsible for the occurrence of HHRH.

Design and Setting: Mutation analysis of exons and adjacent introns in the SLC34A3 gene was conducted at an academic research laboratory and medical center.

Patients or Other Participants: Members of two unrelated families with HHRH participated in the study.

Results: Two affected siblings in one family were homozygous for a 101-bp deletion in intron 9. Haplotype analysis of the SLC34A3 locus in the family showed that the two deletions are on different haplotypes. An unrelated individual with HHRH was a compound heterozygote for an 85-bp deletion in intron 10 and a G-to-A substitution at the last nucleotide in exon 7. The intron 9 deletion (and likely the other two mutations) identified in this study causes aberrant RNA splicing. Sequence analysis of the deleted regions revealed the presence of direct repeats of homologous sequences.

Conclusion: HHRH is caused by biallelic mutations in the SLC34A3 gene. Haplotype analysis suggests that the two intron 9 deletions arose independently. The identification of three independent deletions in introns 9 and 10 suggests that the SLC34A3 gene may be susceptible to unequal crossing over because of sequence misalignment during meiosis.

	Introduction

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HEREDITARY HYPOPHOSPHATEMIC rickets with hypercalciuria (HHRH; OMIM 241530) is a rare genetic disorder of phosphate homeostasis, first described in a large Bedouin kindred (1). Similar to other genetic phosphate wasting disorders such as X-linked hypophosphatemia and autosomal dominant hypophosphatemic rickets, HHRH is characterized by decreased renal phosphate reabsorption, hypophosphatemia, and rickets/osteomalacia (1, 2). In addition, individuals with HHRH often suffer from bone pain, muscle weakness, and growth retardation (1). However, HHRH is distinguished from X-linked hypophosphatemia and autosomal dominant hypophosphatemic rickets by the fact that patients with HHRH have an appropriately elevated level of serum 1,25-dihydroxyvitamin D [1,25-(OH)₂D; calcitriol] concentration, resulting in attendant hypercalciuria and suppression of PTH secretion (1, 2).

Sodium-phosphate cotransporters type II, encoded by the solute carrier family 34 (SLC34A1–3) genes, play a major role in phosphate homeostasis (3). Mice homozygous for the disrupted sodium-phosphate cotransporter type IIa (Slc34a1; NaPi-IIa or Npt2a) gene exhibit hypophosphatemia, increased urinary phosphate excretion, increased serum 1,25-(OH)₂D, increased serum alkaline phosphatase activity, hypercalciuria, and reduced circulating PTH levels (4). Despite the similar biochemical features seen in NaPi-IIa knockout mice and individuals with HHRH, disease-causing mutations were not found in the SLC34A1 gene in the HHRH kindreds (5). Recently, mutations in the SLC34A3 gene, which encodes the sodium-phosphate cotransporter type IIc (NaPi-IIc or NPT2c), were identified as the molecular cause for HHRH by positional cloning (6, 7). In the present study, we performed mutation analysis of the SLC34A3 gene in two unrelated families with HHRH.

	Subjects and Methods

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Study subjects

Both children in family A (AII-1 and AII-2 in Fig. 1A) (studied at the Children’s Clinical Research Center of the New York Hospital-Cornell Medical College) had classical manifestations of HHRH: renal phosphate wasting associated with consistently elevated levels of 1,25-(OH)₂D and hypercalciuria. Representative laboratory test values of AII-1 at 6 yr of age and AII-2 at 3.5 yr of age are summarized in Table 1. The proband (patient AII-2) was diagnosed at 2 yr of age with hypophosphatemic rickets and hypercalciuria. She had severe bowing of her lower extremities with a height at the 10th percentile for age. She was treated with leg bracing and 500–750 mg oral potassium phosphate per day in divided doses. She has not had nephrolithiasis or nephrocalcinosis. However, calcium oxalate crystals were noted on urinalysis. She did not have glycosuria or amino aciduria. During the course of her treatment, she sustained several fractures. By 8 yr of age, bowing of her legs had improved but not resolved. Her height was at the 25th percentile for age.

View larger version (34K): [in this window] [in a new window]   FIG. 1. Family pedigree and mutation analysis. A, Pedigrees of two families with HHRH. Circles denote female subjects; squares denote male subjects. Black symbols denote affected individuals. An arrow indicates the proband in each family. The generations are labeled in Roman numerals, and the individuals within each generation are designated with Arabic numerals. B, Mutation analysis of the SLC34A3 gene. The SLC34A3 genomic region flanking intron 9 (in family A) and intron 10 (in family B) were amplified by PCR. The G-to-A inversion in exon 7 (g.1702GA; Gln252Gln) disrupts one of four HphI restriction sites present in the PCR amplicon. Therefore, after digestion with HphI, the normal allele (WT) generates five fragments (19, 54, 158, 176, and 289 bp), whereas the disease allele has an additional fragment (465 bp) instead of the 176- and 289-bp fragments. The 19-bp fragment is not shown. WT denotes normal control sample. C, Sequence homologies in the SLC34A3 introns. Genomic DNA sequence of exons 9 through 11 is shown. Exons are in uppercase; introns are in lowercase. Highlighted sequences denote direct repeats likely involved in deletions of intron 9 (yellow) and intron 10 (light blue) (see Results for details). Deleted sequences are in red. Nucleotides present in the deletion allele (intron 10) are highlighted in red. Because the precise position of the deletions cannot be determined due to the presence of direct repeats, most 3' nucleotides are arbitrarily assigned to have been deleted. The nucleotide in yellow denotes single nucleotide polymorphism g.2704TA.

The siblings’ father (AI-1) is of German/French/Jewish descent, and mother (AI-2) is of Italian descent. Parental heights are at the 50th percentile (father is 178 cm, and mother is 163 cm). There is an extensive history of nephrolithiasis on both sides of the family and also a history of rickets and bowed legs on the paternal side. Two family members reportedly had sialolithiasis (salivary gland stones).

The affected Caucasian girl in family B (BII-1 in Fig. 1A) was referred for failure to thrive with growth arrest at 9.5 yr of age. Her laboratory test results at 11 yr of age are shown in Table 1. She was noted to have hypophosphatemia and elevated alkaline phosphatase. Additional testing revealed normal PTH and 25-hydroxyvitamin D concentrations, with an elevated 1,25-(OH)₂D. Hypercalciuria was demonstrated when taking approximately 1200 mg calcium/d in her diet. However, with a low-calcium diet, her urine calcium declined. Initial radiographs suggested osteopenia, without obvious rachitic changes. She was treated with oral phosphate supplementation without vitamin D but returned for follow-up inconsistently. At 12.5 yr of age, her height was at the fifth percentile. Radiographs showed persistent osteopenia with development of rickets at the distal femoral epiphyses.

Her family history was notable for a maternal history of lower extremity bowing as a child, which had been treated with leg braces. At the time of the patient’s initial evaluation, her mother’s calcium, phosphate, and PTH concentrations were normal. Her maternal grandmother had osteoarthritis and osteopenia with a reportedly low phosphate. Her father had a normal phosphate and no family history of bone diseases.

The study was approved by the Institutional Review Boards of Duke University, Indiana University-Purdue University Indianapolis, and Weill-Cornell Medical College. Written informed consent was obtained from all study subjects and/or their parents before their participation in the study.

Mutation analysis

Genomic DNA was extracted from blood obtained from study subjects, using standard procedures. All 13 exons (one noncoding and 12 coding), as well as adjacent intronic sequences, were amplified in eight fragments using Multiplex PCR Kit (QIAGEN Inc., Valencia, CA) or AmpliTaq DNA Polymerase (Applied Biosystems, Foster City, CA). Primer sequences and PCR conditions are available upon request. PCR products were electrophoresed in a 2% agarose gel and purified using DNA Gel Extraction Kit (QIAGEN). Approximately 100 ng of each PCR amplicon was directly sequenced from forward and/or reverse PCR primer, using Big-Dye Terminator Cycle Sequencing Kit and the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

Sequence variations identified by DNA sequencing were verified in control DNA samples. Single nucleotide substitutions were analyzed by PCR-restriction fragment length polymorphism (RFLP). Genomic DNA fragments were amplified by PCR and then incubated with 5 U of respective restriction enzymes (New England Biolabs, Beverly, MA) listed in Table 2. The digested PCR products were electrophoresed in a 2% agarose gel and visualized under UV light. Deletions were directly analyzed by agarose gel electrophoresis after PCR.

Total RNA was isolated from whole blood collected in Tempus Blood RNA Tubes (Applied Biosystems), using Versagene RNA Purification Kit (Gentra Systems, Minneapolis, MN). First-strand cDNA was synthesized from 1 µg each of total RNA, using Advantage RT-for-PCR Kit (Clontech, Mountain View, CA). PCR amplification was performed with forward primer in exon 8 (5'-CGC CAC TAA CAG CAG TCT CA-3') and reverse primer in exon 12 (5'-TGT TGG AGC CCA GTA AGA GG-3'). The RT-PCR products were purified and sequenced as described above.

	Results

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Mutation screening of the SLC34A3 gene revealed that both affected siblings (AII-1 and AII-2) in family A were homozygous for a 101-bp deletion in intron 9 (g.2259–2359del) (Fig. 1B). Their parents were heterozygous for the deletion, as indicated by the presence of long and short PCR amplicons. The deletion truncates intron 9 from 167 to 66 bp (Fig. 1C).

The affected individual (BII-1) in family B was a compound heterozygote for a G-to-A inversion at the last nucleotide in exon 7 (g.1702GA, Gln252Gln) and an 85-bp deletion in intron 10 (g.2615–2699del) (Fig. 1B). The PCR-RFLP analysis of the inversion showed that this mutation was transmitted from her mother (BI-2) to the proband (Fig. 1B). DNA from the father was not available for testing. However, the absence of the intron 10 deletion in the mother suggests that the deletion in the proband was paternally transmitted. Similar to the intron 9 deletion, the intron 10 deletion results in truncation of intron 10 from 139 to 54 bp (Fig. 1C).

The two intronic deletions and one silent mutation identified in these families were not found in 100 unrelated healthy Caucasian individuals (Table 2). In addition, seven polymorphisms were identified in the mutation screening of SLC34A3 (Table 2). Most polymorphisms were found in control samples, and some were previously described in other populations (6, 7). However, the g.970CT (Asp91Asp) polymorphism was unique to family A and not found in 100 individuals tested. Haplotype analysis of family A showed that the rare T nucleotide is cosegregating with the paternal intron 9 deletion (Fig. 2). Furthermore, the C-to-T substitution does not affect exonic splicing enhancer predicted by RESCUE-ESE (http://genes.mit.edu/burgelab/rescue-ese/) (8, 9). Therefore, g.970CT is likely a rare base pair change with no significant effect.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Haplotype analysis of the SLC34A3 locus in family A. The presence and absence of the intron 9 deletion (g.2259–2359del) are denoted by symbols + and –, respectively. Paternally and maternally inherited deletion alleles are indicated by dark and light gray backgrounds, respectively.

Sequence analysis of the deleted regions revealed that direct repeats of homologous sequences are present in the proximity (Fig. 1C). Exon 8 through intron 9 harbors two identical sequences (51 bp each), which are separated by 50 bp. Intron 10 through exon 11 also contains highly similar sequences (47 bp each) flanking a 38-bp internal sequence. These short direct repeats are predicted to promote sequence misalignment during meiosis, leading to deletion of one of the repeats along with internal sequence between the repeats. To determine whether the intron 9 deletions in family A originated from the same ancestral haplotype, haplotypes of the SLC34A3 locus were constructed, using single nucleotide polymorphisms identified in this study. Both affected siblings were heterozygous at all polymorphic sites tested except the intron 9 deletion and g.4452TA (Fig. 2), indicating that these two deletions arose in two independent unequal crossing over events.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Recently, mutations in the SLC34A3 gene were identified in families with HHRH (6, 7). In the present study, we identified two intronic deletions and one silent mutation in SLC34A3 in two unrelated families with HHRH. The intron 9 deletion in family A causes retention of truncated intron 9, which inserts 22 amino acid residues in the encoded NaPi-IIc protein. Although analysis of mRNA could not be performed for mutations found in another patient, BII-1, her mutations are also expected to cause exon skipping, cryptic splice site activation, or intron retention, which likely leads to alteration in mRNA sequence. There are multiple cis-acting elements in or surrounding exons to ensure proper splicing of RNA. Therefore, splicing defects could result from the removal of cis-acting elements by the deletions in introns 9 and 10. However, because minimum intron size in humans centers around 92 bp (12) and no apparent cis-acting elements are removed by the deletions, it is likely that deletions reducing intron sizes to 66 and 54 bp constrain the intron length required for spliceosome assembly. There are numerous human diseases caused by deletions of 1 or 2 bp in an intron. However, substantial deletions in already small introns are rather uncommon. To our knowledge, only three similar cases have been reported in the literature: autosomal dominant polycystic kidney disease caused by deletions (18 and 20 bp) in the same 75-bp intron in the PKD1 gene (13) and Rothmund-Thomson syndrome caused by 11-bp deletion found in a 77-bp intron in the RECQL4 gene (14).

Because HHRH is an autosomal recessive disease, biallelic mutations are required for full-scale disease manifestations. However, both families studied here are known to have a strong history of renal and/or bone diseases, indicating that carriers of a single mutation may also show some clinical features of HHRH. The finding is consistent with previously described families with HHRH, in which heterozygotes for the mutant allele sometimes have clinical manifestations (6, 7). However, the manifestation of clinical phenotypes in carriers of SLC34A3 mutations (both homozygotes and heterozygotes) is rather intriguing because the closely related NaPi-IIa (encoded by SLC34A1) is unable to compensate for the loss of NaPi-IIc even though both cotransporters are expressed at apical membranes of the proximal tubules in the kidney and are targets for the adjustment of renal reabsorption of phosphate (3, 15). Similarly, NaPi-IIc cannot correct phenotypes resembling HHRH in NaPi-IIa knockout mice, although the skeletal abnormalities are eventually reversed with increasing age (4, 16). Furthermore, NaPi-IIc is suggested to be a growth-related renal NaPi cotransporter because its expression is significantly higher in weaning rats compared with adults (15). Therefore, inability to compensate for the loss of each other is likely because of a temporal and/or spatial expression difference of these cotransporters, which may be controlled by different regulatory mechanisms.

Patient AII-1 had recurrent nephrolithiasis in his early childhood. There is a strong history of nephrolithiasis in both his maternal and paternal sides. In fact, mice homozygous for disrupted NaPi-IIa develop nephrocalcinosis (16), and mutations in the human homolog (i.e. SLC34A1) have been implicated in nephrolithiasis and osteoporosis associated with hypophosphatemia (17). Because increased urinary phosphate excretion and hypercalciuria are a hallmark of HHRH, it is not surprising to find stone formation in SLC34A3 mutation carriers. However, stone formation in HHRH has been reported in only a few cases: one of several patients in the extended Bedouin kindred (1), an affected patient in a small kindred (6), and an affected patient with a familial history of nephrolithiasis in a Turkish kindred (18). Identification of another patient with nephrolithiasis in our study suggests that stone formation should also be considered a more common manifestation of HHRH than recognized.

Both children in family A are homozygous for the intron 9 deletion. However, their parents are nonconsanguineous and of different ethnicity. The deletions are on two completely different haplotypes. The very rare T allele at Asp91Asp was found on the paternal deletion allele but was absent from the maternal deletion allele. These data suggest that the two intron 9 deletions likely arose independently. Furthermore, the same deletion mutation was also found in another HHRH family of Irish/French/German origin (6). The deletion in this family was on a different haplotype (G-C-T-del-T-T) (Dr. Clemens Bergwitz, personal communication) from the present cases, lending support for frequent deletion events at the SLC34A3 locus. In addition to the intron 9 deletion, we identified a novel deletion in intron 10 of the SLC34A3 gene. Although these deletions were not found in 100 control samples tested, the presence of two different homologous sequences makes this gene particularly susceptible to unequal crossing over during meiosis. Because the aforementioned deletions in PKD1 and RECQL4 are also facilitated by intronic direct repeats (13, 14), the presence of direct repeats in small introns may be a common feature of substantial intronic deletions.

In conclusion, we identified three different mutations in the SLC34A3 gene in families with HHRH. Two of the mutations were disease-causing deletions in short introns, which may have deleterious consequences in RNA splicing because of constraint on the intronic length or removal of cis-acting regulatory elements. The present study confirms that mutations in the SLC34A3 gene are responsible for appearance of HHRH and suggests that the gene may be particularly susceptible to deletions because of the existence of intragenic homologous sequences.

	Acknowledgments

 
We are indebted to families with HHRH for their participation in this study. We thank Oun Kheav at the Biochemistry Biotechnology Facility of the Indiana University School of Medicine for his technical assistance with DNA sequencing. We also thank Dr. Ana L. Paredes at Miami Children’s Hospital for sending us blood samples for RNA analysis.

	Footnotes

 
This work was supported by National Institutes of Health Grants R01 AR42228, P01 AG18397, and M01 RR00047.

First Published Online July 18, 2006

Abbreviations: HHRH, Hereditary hypophosphatemic rickets with hypercalciuria; NaPi-IIa, sodium-phosphate cotransporter type IIa; 1,25-(OH)₂D, 1,25-dihydroxyvitamin D; RFLP, restriction fragment length polymorphism.

Received December 30, 2005.

Accepted July 12, 2006.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ichikawa, S. Articles by Econs, M. J. Search for Related Content PubMed PubMed Citation Articles by Ichikawa, S. Articles by Econs, M. J. Related Collections Pediatric Endocrinology Calcium and Bone Metabolism