Hereditary hypophosphatemic rickets with hypercalciuria and nephrolithiasis-identification of a novel SLC34A3/NaPi-IIc mutation

Abstract

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is characterized by rickets, hyperphosphaturia, hypophosphatemia, elevated 1,25-dihydroxyvitamin-D, increased gastrointestinal calcium absorption and hypercalciuria. Serum calcium, 25-hydroxyvitamin-D and PTH levels are normal. Here we describe a boy with HHRH, nephrolithiasis, and compound heterozygosity for one previously described mutation (g.4225_50del) and a novel splice mutation (g.1226G>A) in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. The patient's mother and grandmother are carriers of g.4225_50del, and both have a history of nephrolithiasis associated with hypercalciuria and elevated 1,25-dihydroxyvitamin-D. His three siblings (2-6 years old), who are also carriers of g.4225_50del, have hypercalciuria but so far their renal ultrasounds are normal. Thus, SLC34A3/NaPi-IIc mutations appear to be associated with variable phenotypic changes at presentation, which can include recurrent nephrolithiasis.

FIG. 1

A: Pedigree. Ages at presentation were: III-1 3 years 8 months old, III-2 and III-3 6 years old, and III-4 2 years old. I-1, II-1, and II-2 are adults. Solid square indicates the index case, who developed rickets during childhood along with renal phosphate-wasting, hypophosphatemia, and hypercalciuria. Open symbols indicate individuals who are healthy. Samples for individuals with dashed lines were unavailable for genotyping. B: Haplotypes for chromosome region 9q34 between markers D9S1826 and D9S1838. Alleles for microsatellite markers are designated as bp or coded. The haplotype associated with g.4225_50del is shaded in light gray, the haplotype associated with g.1226G>A is shaded in dark gray. The mutations and SNPs were identified by nucleotide sequencing analysis in III-1 and by PCR-based assays using restriction enzymatic digests in the other family members.

FIG. 2

RT-PCR of maternal and paternal transcripts from lymphoblastoid cells of III-1. A: One microgram of total RNA from lymphoblastoid cells of III-1 were subjected to nested RT-PCR using primers flanking the mutations g.1226G>A (RT-PCR 1), g.4225_50del (RT-PCR 2) and three SNPs, c.200G>A(p.R67H), c.1140C>T(p.L385L), and c.1538T>A(V513E), as described in the Methods Section. The obtained PCR products were subjected to nucleotide sequence analysis, which indicates that the maternal transcript carrying the SNP haplotype ATT and the mutation g.4225_50del is missing entirely. B: The paternal transcript contained the SNP haplotype GCA and was found to lack the last 22 bp in exon 5, which precede the splice donor site altered by g.1226G>A. The novel transcript is predicted to generate a frame shift, a premature termination codon, and a truncated NaP-IIc protein. RT-PCR 3 uses a reverse primer, which anneal across the 22 bp deletion and was used to confirm presence of the altered paternal transcript in lymphoblastoid cells of III-1 (coding sequence is show in upper case, intronic sequence is shown in lower case).