BACKGROUND Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization. RESULTS Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the RPGR gene, 353-bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified. CONCLUSIONS Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing.

中文翻译：

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以眼底镜检查为导向的基因测试，可重新评估阴性的整个外显子组测序结果。

背景技术全外显子组测序（WES）允许公正地寻找疾病的遗传原因。与随后在诊断途径中使用时相比，将其用作第一级基因测试可能会受到青睐，因为与每次诊断相关的增量成本较低。但是，WES存在技术限制，可能导致不正确的否定变体调用。我们的研究通过使用主要由眼底镜检查结果驱动的后续测试对WES阴性结果进行重新评估，提出了这些局限性。这些测试包括靶向基因测试，遗传的视网膜基因板，全基因组测序（WGS）和阵列比较基因组杂交。结果在眼底镜检查结果的指导下进行的随后的基因检测确定了以下导致WES未能检测到的导致色素性视网膜炎的变体类型：RPGR基因的移码缺失和无义变异，MAK基因的353 bp Alu重复插入以及EYS和PRPF31基因的大外显子缺失变异。还确定了导致Star​​gardt病的ABCA4基因和导致Leber先天性黑病的GUCY2D基因的深度内含子变异体。结论与表型不一致的阴性WES分析应引起临床怀疑。随后的基因测试可能会检测WES遗漏的遗传变异，并使患者有资格进行基因替代治疗和即将进行的临床试验。如果表型发现支持遗传病因，则WES结果阴性后应进行靶向基因测序，基于阵列的方法或全基因组测序。和EYS和PRPF31基因中的大外显子缺失变异体。还确定了导致Star​​gardt病的ABCA4基因和导致Leber先天性黑病的GUCY2D基因的深度内含子变异体。结论与表型不一致的阴性WES分析应引起临床怀疑。随后的基因测试可能会检测WES遗漏的遗传变异，并使患者有资格进行基因替代治疗和即将进行的临床试验。如果表型发现支持遗传病因，则WES结果阴性后应进行靶向基因测序，基于阵列的方法或全基因组测序。和EYS和PRPF31基因中的大外显子缺失变异体。还确定了导致Star​​gardt病的ABCA4基因和导致Leber先天性黑病的GUCY2D基因的深度内含子变异体。结论与表型不一致的阴性WES分析应引起临床怀疑。随后的基因测试可能会检测WES遗漏的遗传变异，并使患者有资格进行基因替代治疗和即将进行的临床试验。如果表型发现支持遗传病因，则WES结果阴性后应进行靶向基因测序，基于阵列的方法或全基因组测序。结论与表型不一致的阴性WES分析应引起临床怀疑。随后的基因测试可能会检测WES遗漏的遗传变异，并使患者有资格进行基因替代治疗和即将进行的临床试验。如果表型发现支持遗传病因，则WES结果阴性后应进行靶向基因测序，基于阵列的方法或全基因组测序。结论与表型不一致的阴性WES分析应引起临床怀疑。随后的基因测试可能会检测WES遗漏的遗传变异，并使患者有资格进行基因替代治疗和即将进行的临床试验。如果表型发现支持遗传病因，则WES结果阴性后应进行靶向基因测序，基于阵列的方法或全基因组测序。