RNA-first Approach Identifies Deep Intronic PHEX Variants in X-linked Hypophosphatemic Rickets

Context: Up to 20% of patients with X-linked hypophosphatemic rickets (XLH) have no causative variant identified on routine molecular diagnostic testing. Objective: To identify intronic variants causing PHEX mis-splicing in patients with XLH. Setting: The metabolic bone clinic of a pediatric orthopedic hospital. Participants: Four patients (age 6 to 12 years; 3 girls) with clinically diagnosed XLH and no PHEX variant on routine testing. Main Outcome Measures: RNA and DNA sequence analysis of PHEX. Methods: Urine-derived cells were cultured, and mRNA was extracted and transcribed to cDNA. PHEX cDNA was amplified by PCR, followed by sequencing of PCR products. Sequencing of PHEX intronic DNA regions was performed to identify variants causing mis-splicing observed on RNA analysis. Results: PHEX mis-splicing was identified in 3 of the 4 participants, and an intronic variant was identified in all 3 cases. In a 12-year-old boy, transcript analysis showed skipping of PHEX exon 13, while sequencing of PHEX intronic regions revealed a de novo 18 bp deletion in intron 13. In a 7-year-old girl, a pseudoexon in PHEX intron 17 was found, associated with a de novo deep intronic variant (c.1768 + 173A > G) that activated a cryptic splice donor site. Finally, an 84 bp pseudoexon in PHEX intron 21 caused by a recurrent de novo deep intronic variant (c.2147 + 1197A > G) was identified in an 11-year-old girl. Conclusion: Analysis of RNA from urine-derived cells combined with sequencing of PHEX introns can identify deep intronic variants in individuals with XLH without a detectable PHEX variant in routine exon-centric molecular diagnosis.