Intronic deletions in the SLC34A3 gene: a cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria

Abstract

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6-1/2-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24-hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH), and elevated 1,25(OH)2D. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440-1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH.

Keywords: Deletion; HHRH; Hypercalciuria; Hypophosphatemia; Intron; Nephrolithiasis.

Figure 1

SLC34A3 mutations in the HHRH patient. A. Deletion in intron 6. Agarose gel electrophoresis shows three distinct PCR products from the patient. DNA sequencing revealed that top, middle and bottom products represent wild-type/mutant heteroduplex, wild-type, and mutant, respectively. WT, wild type. B. Electropherogram of exon 12. The arrow denotes a deletion of nucleotide “G”. C. Sequence analysis near the intron 6 deletion. The deletion (underline) likely involves four base pair repeats (in blue), which are separated by 26 bp. 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. Upper case, exon; lower case, intron.

Figure 2

Homologous sequences involved in intronic deletions in the SLC34A3 gene. Direct repeats of homologous sequences (gray highlights) likely misalign during meiosis, resulting in deletions of one of the repeats and the sequence flanked by the repeats. Uppercase and lower cases denote exonic and intronic sequences, respectively. Underlines denote deleted sequences. The most 3’ repeats are arbitrarily assigned to be deleted.