Supplementary Material for: A novel intronic deletion in PDE6B causes autosomal recessive retinitis pigmentosa by interfering with RNA splicing

Introduction: Retinitis pigmentosa (RP) is a rare degenerative retinal disease caused by mutations in approximately seventy genes. Currently, despite the availability of large-scale DNA sequencing technologies, ~30-40% of patients still cannot be diagnosed at the molecular level. In this study, we investigate a novel intronic deletion of PDE6B, encoding the beta subunit of phosphodiesterase 6, in association with recessive RP. Methods: Three unrelated consanguineous families were recruited from the North-Western part of Pakistan. Whole exome sequencing was performed for the probands of each family and the data were analyzed according to an in-house computer pipeline. Relevant DNA variants in all available members of these families were assessed through Sanger sequencing. A minigene-based splicing assay was also performed. Results: The clinical phenotype for all patients was compatible with rod cone degeneration, with onset during childhood. Whole exome sequencing revealed a homozygous 18 bp intronic deletion (NM_000283.3: c.1921-20_1921-3del) in PDE6B, which co-segregated with disease in 10 affected individuals. In-vitro splicing tests showed that this deletion causes aberrant RNA splicing of the gene, leading to the in-frame deletion of 6 codons and likely to disease. Discussion/Conclusion: Our findings further expand the mutational spectrum of the PDE6B gene.

引言：色素性视网膜炎（Retinitis pigmentosa, RP）是一种罕见的视网膜退行性疾病，约由70个基因的突变所引发。当前，尽管大规模DNA测序技术已得到广泛应用，但仍有约30%~40%的患者无法在分子层面获得确诊。本研究针对编码磷酸二酯酶6β亚基的PDE6B基因的一种新型内含子缺失变异与隐性遗传性色素性视网膜炎的关联展开探究。方法：本研究从巴基斯坦西北部地区招募了3个无亲缘关系的近亲婚配家族。对每个家族的先证者进行全外显子组测序（Whole exome sequencing），并通过内部计算机流程分析测序数据。采用桑格测序（Sanger sequencing）对这些家族中所有可获取的成员的相关DNA变异进行验证。同时还开展了基于迷你基因的剪接功能实验（minigene-based splicing assay）。结果：所有患者的临床表型均符合视杆视锥退行性病变特征，发病年龄为儿童期。全外显子组测序显示，PDE6B基因存在纯合型18bp内含子缺失变异（NM_000283.3: c.1921-20_1921-3del），该变异与10名受累个体的疾病表型共分离。体外剪接实验表明，该缺失会导致该基因的RNA剪接异常，引发6个密码子的框内缺失，进而致病。讨论与结论：本研究结果进一步拓展了PDE6B基因的突变谱。