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.
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.
Authors: Minjing Zou; Ayla Guven; Huda A BinEssa; Roua A Al-Rijjal; Brian F Meyer; Ali S Alzahrani; Yufei Shi Journal: Front Genet Date: 2020-11-27 Impact factor: 4.599
Authors: Chaozhe Yang; Naoe Harafuji; Amber K O'Connor; Robert A Kesterson; Jacob A Watts; Amar J Majmundar; Daniela A Braun; Monkol Lek; Kristen M Laricchia; Hanan M Fathy; Shrikant Mane; Shirlee Shril; Friedhelm Hildebrandt; Lisa M Guay-Woodford Journal: Sci Rep Date: 2021-09-14 Impact factor: 4.996
Authors: Melissa M Formosa; Dylan J M Bergen; Celia L Gregson; Antonio Maurizi; Anders Kämpe; Natalia Garcia-Giralt; Wei Zhou; Daniel Grinberg; Diana Ovejero Crespo; M Carola Zillikens; Graham R Williams; J H Duncan Bassett; Maria Luisa Brandi; Luca Sangiorgi; Susanna Balcells; Wolfgang Högler; Wim Van Hul; Outi Mäkitie Journal: Front Endocrinol (Lausanne) Date: 2021-08-13 Impact factor: 5.555