Abstract
Context X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene and is the most common form of hereditary rickets. The splice-site mutations account for 17% of all reported PHEX mutations. The functional consequence of these splice-site mutations has not been systemically investigated. Objective: The current study was undertaken to functionally annotate previously reported 22 splice-site mutations in the PHEX gene.
Methods: PHEX mini-genes with different splice-site mutations were created by site-directed mutagenesis and expressed in HEK293 cells. The mRNA transcripts were analyzed by RT-PCR, cloning, and sequencing.
Results: These splicing mutations led to a variety of consequences, including exon skipping, intron retention, and activation of cryptic splice sites. Among 22 splice-site mutations, exon skipping was the most common event accounting for 73% (16/22). Non-canonical splice-site mutations could result in splicing errors to the same extent as canonical splice-site mutations such as c.436+3G > C, c.436 + 4A > C, c.436 + 6T > C, c.437-3C > G, c.850-3C > G, c.1080-3C > A, c.1482 + 5G > C, c.1586 + 6T > C, c.1645 +5G > A, c.1645 + 6T > C, c.1701-16T > A, c.1768 + 5G > A, and c.1899 + 5G > A. Interestingly, non-canonical (c.436 + 6T > C and c.1586 + 6T > C) and canonical splice-site mutations (c.1769-1G > C) could generate partial splicing errors (both wild-type and mutant transcripts were detected), resulting in incomplete inactivation of PHEX gene, which may explain the mild disease phenotype reported previously, providing evidence of genotype-phenotype correlation. c.1645C > T (p.R549*) had no impact on pre-mRNA splicing although it is located next to canonical splice donor site GT.
Conclusions: Exon skipping is the most common outcome due to splice-site mutations. Both canonical and non canonical splice-site mutations can result in either severe or mild RNA splicing defects, contributing to phenotype heterogeneity. Non-canonical splice-site mutations should not be overlooked in genetic screening especially those located within 50 bp from canonical splice site.