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Novel missense mutation E585K in retinitis pigmentosa leads to compromised RPGR splicing diversity
bioRxiv - Genetics Pub Date : 2020-05-25 , DOI: 10.1101/2020.05.22.109884
Yan-Shan Liu , Ji-Feng Wan , Chun-Yan Ren , Zhou-Heng Xu , Xu-Bin Pan , Jia-Qi Pan , Ruo-Nan Gao , Shao-Qiang Liu , Jia-Li Zhang , Qian-Hao Yao , Ji-Hong Wang , En-Min Li , Jun-Hua Rao , Ping Hou , Jian-Huan Chen

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP). Herein we used whole-exome sequencing to screen possible novel RPGR mutations in RP patients, and identified a novel missense mutation E585K in a patient with early onset but slow disease progression, and a frameshift deletion E998Gfs*78 in a patient with RP sine pigmento and high myopia. Intriguingly, bioinformatic analysis indicated that E585K probably affected RPGR RNA splicing instead of the protein sequence directly. Mini-gene assays in 293T cells revealed that splicing events of the E585K mutant were found to be also exist in wildtype, but with a shifted pattern. In the E585K mini-gene usage of an upstream alternative 5′ splice site (5′ ss) of exon 14 was enhanced, and other splicing events were suppressed, including the canonical 5′ ss of exon 14, skipping of exon 14/15 and retention of intron 14. As a result, RPGR splicing products of the E585K mini-gene were predominated by transcripts containing a 4-bp deletion, with a small fraction of in-frame transcripts containing a retended intron 14, which might explain the slow disease progression in the patient carrying the mutation. RNA-Seq analysis further confirmed existence of these splicing events in endogenous RPGR RNA in human retina, pointing to compromised splicing diversity in the E585K mutant. Our findings thus added to the understanding of genotype-phenotype correlation in RP, and suggested that compromised RPGR splicing diversity might play a role in molecular mechanism of the disease.

中文翻译:

色素性视网膜炎中的新型错义突变E585K导致RPGR剪接多样性受损

色素性视网膜炎GTPase调节基因(RPGR)的突变是X连锁色素性视网膜炎(RP)的主要原因。本文中,我们使用全外显子组测序来筛查RP患者中可能的新RPGR突变,并在发病较早但疾病进展缓慢的患者中鉴定出新的错义突变E585K,并在RP正弦色素沉着患者中鉴定出移码缺失E998Gfs * 78高度近视。有趣的是,生物信息学分析表明E585K可能会影响RPGR RNA剪接,而不是直接影响蛋白质序列。在293T细胞中进行的小基因检测显示,发现E585K突变体的剪接事件也存在于野生型中,但模式发生了改变。在E585K小型基因中,外显子14的上游替代5'剪接位点(5'ss)的使用得到增强,其他剪接事件得到抑制,包括外显子14的规范5's,外显子14/15的跳跃和内含子14的保留。结果,E585K微型基因的RPGR剪接产物主要被含有4 bp缺失的转录本所占,很小一部分包含内含子14的框内转录本,这可能解释了携带突变的患者疾病进展缓慢。RNA-Seq分析进一步证实了人类视网膜内源性RPGR RNA中存在这些剪接事件,表明E585K突变体的剪接多样性受到损害。因此,我们的发现增加了对RP中基因型与表型相关性的了解,并暗示受损的RPGR剪接多样性可能在该疾病的分子机制中起作用。E585K微型基因的RPGR剪接产物主要被含有4 bp缺失的转录本所占,而一小部分的框内转录本却含有被内含的14号内含子,这可能解释了携带突变的患者疾病进展缓慢。RNA-Seq分析进一步证实了人类视网膜内源性RPGR RNA中存在这些剪接事件,表明E585K突变体的剪接多样性受到损害。因此,我们的发现增加了对RP中基因型与表型相关性的了解,并暗示受损的RPGR剪接多样性可能在该疾病的分子机制中起作用。E585K微型基因的RPGR剪接产物主要被含有4 bp缺失的转录本所占,而一小部分的框内转录本却含有被内含的14号内含子,这可能解释了携带突变的患者疾病进展缓慢。RNA-Seq分析进一步证实了人类视网膜内源性RPGR RNA中存在这些剪接事件,表明E585K突变体的剪接多样性受到损害。因此,我们的发现增加了对RP中基因型与表型相关性的了解,并表明受损的RPGR剪接多样性可能在该疾病的分子机制中起作用。这可能解释了携带突变的患者疾病进展缓慢。RNA-Seq分析进一步证实了人类视网膜内源性RPGR RNA中存在这些剪接事件,表明E585K突变体的剪接多样性受到损害。因此,我们的发现增加了对RP中基因型与表型相关性的了解,并表明受损的RPGR剪接多样性可能在该疾病的分子机制中起作用。这可能解释了携带突变的患者疾病进展缓慢。RNA-Seq分析进一步证实了人类视网膜内源性RPGR RNA中存在这些剪接事件,表明E585K突变体的剪接多样性受到损害。因此,我们的发现增加了对RP中基因型与表型相关性的了解,并表明受损的RPGR剪接多样性可能在该疾病的分子机制中起作用。
更新日期:2020-05-25
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