An RNA-First approach identifies deep intronic PHEX variants in children with X-Linked hypophosphatemic rickets

Abstract

Objectives: To identify intronic variants causing mis-splicing of PHEX in patients with clinically-diagnosed X-linked hypophosphatemic rickets (XLH) in whom no variant was identified on clinical molecular diagnostic testing. Methods: Urine samples were obtained from 5 patients (5-12 years, 3 females) with clinically-diagnosed XLH where no candidate variant was identified on rickets gene panel sequencing of genomic DNA. Urine-derived cells (UDC) were cultured as previously described (1). PHEX was expressed at low levels in UDC, though expression was superior to fibroblasts and blood (1). PCR amplification of mRNA was performed, followed by next-generation sequencing, as described (2). Abnormalities were confirmed using Sanger sequencing. In addition, DNA sequencing of PHEX intronic regions was performed to identify candidate variants potentially causing mis-splicing seen on RNA analysis. Results: Abnormal mRNA splicing was identified in 3 of 5 patients, and a novel candidate variant was identified on sequencing of PHEX introns in each of the 3 patients with splicing defects. Pseudoexons were seen in 2 patients. An 84 bp pseudoexon in PHEX intron 21 caused by a deep intronic variant (c.2147+1197A>G) was identified in an 11-year-old girl. This variant was de novo (not present in either of the unaffected parents) and corresponded to a variant that we had previously published (2). We subsequently found the same variant in two more families with XLH, suggesting that this variant is a common but hitherto overlooked cause of XLH. In addition, a pseudoexon in intron 17 of PHEX secondary to a deep intronic variant (c.1768+173A>G) was detected in another 11-year-old female with XLH. Finally, RNA analysis identified skipping of PHEX exon 13 in a 12-year-old boy. An 18 bp deletion in intron 13 was detected on whole gene sequencing, which was not seen in the boy's unaffected mother, suggesting that the deep intronic deletion in this boy was causing the exon skipping and thereby giving rise to XLH.