Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, 72076, Tübingen, Germany.
Sci Rep. 2023 Feb 18;13(1):2896. doi: 10.1038/s41598-023-29452-9.
Achromatopsia is an autosomal recessive cone photoreceptor disease that is frequently caused by pathogenic variants in the CNGA3 gene. Here, we present a systematic functional analysis of 20 CNGA3 splice site variants detected in our large cohort of achromatopsia patients and/or listed in common variant databases. All variants were analyzed by functional splice assays based on the pSPL3 exon trapping vector. We demonstrated that ten variants, both at canonical and non-canonical splice sites, induced aberrant splicing, including intronic nucleotide retention, exonic nucleotide deletion and exon skipping, resulting in 21 different aberrant transcripts. Of these, eleven were predicted to introduce a premature termination codon. The pathogenicity of all variants was assessed based on established guidelines for variant classification. Incorporation of the results of our functional analyses enabled re-classification of 75% of variants previously classified as variants of uncertain significance into either likely benign or likely pathogenic. Our study is the first in which a systematic characterization of putative CNGA3 splice variants has been performed. We demonstrated the utility of pSPL3 based minigene assays in the effective assessment of putative splice variants. Our findings improve the diagnosis of achromatopsia patients, who may thus benefit from future gene-based therapeutic strategies.
全色盲是一种常染色体隐性圆锥感光细胞疾病，通常由 CNGA3 基因中的致病性变异引起。在这里，我们对我们的大型全色盲患者队列中检测到的或常见变异数据库中列出的 20 个 CNGA3 剪接位点变异进行了系统的功能分析。所有变体均通过基于 pSPL3 外显子捕获载体的功能剪接测定进行分析。我们证明，十个变体，无论是在规范的还是非规范的剪接位点，均诱导异常剪接，包括内含子核苷酸保留、外显子核苷酸缺失和外显子跳跃，导致 21 种不同的异常转录本。其中十一个被预测会引入过早终止密码子。所有变体的致病性均基于变体分类的既定准则进行评估。将我们的功能分析结果纳入其中，使得先前分类为意义不明的变异体中的 75%能够重新分类为可能良性或可能致病性。我们的研究是首次对潜在的 CNGA3 剪接变异体进行系统表征。我们证明了 pSPL3 基于小基因的测定在有效评估潜在剪接变异体中的实用性。我们的发现改善了全色盲患者的诊断，他们可能因此受益于未来基于基因的治疗策略。
