Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland.
Invest Ophthalmol Vis Sci. 2010 Mar;51(3):1628-35. doi: 10.1167/iovs.09-4031. Epub 2009 Oct 15.
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.
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.
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.
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.
大多数 X 连锁性视网膜炎色素变性（XlRP）患者携带 RPGR 基因突变。作者研究了 RPGR 突变患者是否存在其他剪接缺陷，这些缺陷可能会影响 RPGR 的性质。
建立 RPGR 基因突变的患者源性悬浮细胞系。通过直接对 PCR 产物进行测序来验证突变。通过 RT-PCR 分析 RPGR 剪接的患者特异性改变，并通过测序进行验证。通过使用不同的人类供体组织池，通过实时 PCR 定量 RPGR 剪接变体的组织特异性表达水平。
作者分析了七个携带 RPGR 半合子突变的 RP 患者源性淋巴母细胞系的 RPGR 剪接。在三个患者细胞系中，他们发现并鉴定了除突变外还存在的剪接缺陷。这些剪接缺陷可能会干扰正常的 RPGR 性质。此外，他们还鉴定了四个新的 RPGR 转录本，它们要么包含一个新的外显子 11a，要么跳过组成性外显子 12、14 和 15。新的和已知的 RPGR 同工型在几种人类组织中存在差异调节。在人类视网膜中，大约 10%的 RPGR 转录本在 9 号和 15 号外显子之间进行选择性剪接。
这些发现表明 RPGR 的剪接是一种组织依赖性的精确调节方式，并提示 RPGR 突变经常干扰替代转录本同工型的表达。这些结果表明在 RP 患者中进行 RPGR 转录本分析的重要性。作者进一步讨论了 RPGR 剪接作为 XlRP 患者描述的不同疾病表型的修饰因子。
