Mutation- and tissue-specific alterations of RPGR transcripts.
OBJECTIVE
The majority of patients with X chromosome-linked retinitis pigmentosa (XlRP) carry mutations in the RPGR gene. The authors studied whether patients with RPGR mutations show additional splice defects that may interfere with RPGR properties.


METHODS
Patient-derived cell lines with RPGR mutations were raised in suspension. To verify mutations, direct sequencing of PCR products was performed. Patient-specific alterations in RPGR splicing were analyzed by RT-PCR and confirmed by sequencing. Tissue-specific expression levels of RPGR splice variants were quantified by real-time PCR using pools of different human donor tissues.


RESULTS
The authors analyzed the splicing of RPGR in seven RP patient-derived lymphoblastoid cell lines carrying hemizygous RPGR mutations. In three patient cell lines, they identified and characterized splice defects that were present in addition to a mutation. These splice defects were likely to interfere with normal RPGR properties. Furthermore, they identified four novel RPGR transcripts, either containing a new exon termed 11a or skipping the constitutive exons 12, 14, and 15. Novel and known RPGR isoforms were found to be differentially regulated in several human tissues. In human retina, approximately 10% of RPGR transcripts are alternatively spliced between exons 9 and 15.


CONCLUSIONS
These findings show that splicing of RPGR is precisely regulated in a tissue-dependent fashion and suggest that mutations in RPGR frequently interfere with the expression of alternative transcript isoforms. These results implicate the importance of RPGR transcript analysis in patients with RP. The authors further discuss RPGR splicing as a modifier of different disease phenotypes described in patients with XlRP.
Schmid
Fabian
F
Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland.
Glaus
Esther
E
Cremers
Frans P M
FP
Kloeckener-Gruissem
Barbara
B
Berger
Wolfgang
W
Neidhardt
John
J
D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D005136:Eye Proteins; D020033:Protein Isoforms; D022821:RNA Splice Sites; C099929:RPGR protein, human