Abstract：
CNGA3 encoding the main subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is one of the major disease-associated genes for achromatopsia. Most CNGA3 variants are missense variants with the majority being functionally uncharacterized and therefore hampering genetic diagnosis. In light of potential gene therapy, objective variant pathogenicity assessment is essential.
We established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant re-classification.
We provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology-based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%.
Our aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.
We established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant re-classification.
We provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology-based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%.
Our aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.
摘要：
目的：编码视锥细胞光感受器环核苷酸门控离子通道主亚基的CNGA3是色盲的主要疾病相关基因之一。大多数CNGA3变体是错义变体,其中大多数是功能上未表征的并且因此妨碍遗传诊断。鉴于潜在的基因治疗，客观的变异致病性评估至关重要。
方法：我们建立了基于中等通量水母发光蛋白的发光生物测定法，允许通过量化细胞培养系统中CNGA3通道介导的钙内流来评估突变型CNGA3通道功能，从而实现基于ACMG/AMP的变体重新分类。
结果：我们提供了150个尚未表征的CNGA3错义替换的功能读出，其中55个以前被归类为不确定意义（VUS）的变体，鉴定25个为功能正常，125个为功能异常。这些数据使基于ACMG/AMP的52/55VUS变异重新分类为良性，可能良性或可能致病，达到94.5%的VUS重新分类率。
结论：我们基于水母发光蛋白的生物测定允许对150个CNGA3错义变体进行功能上确保的临床变体解释，从而实现并支持VUS重新分类，并确保对患有CNGA3相关色盲的患者进行分子诊断。特此确定有资格对该疾病进行未来基因治疗试验的患者。
方法：我们建立了基于中等通量水母发光蛋白的发光生物测定法，允许通过量化细胞培养系统中CNGA3通道介导的钙内流来评估突变型CNGA3通道功能，从而实现基于ACMG/AMP的变体重新分类。
结果：我们提供了150个尚未表征的CNGA3错义替换的功能读出，其中55个以前被归类为不确定意义（VUS）的变体，鉴定25个为功能正常，125个为功能异常。这些数据使基于ACMG/AMP的52/55VUS变异重新分类为良性，可能良性或可能致病，达到94.5%的VUS重新分类率。
结论：我们基于水母发光蛋白的生物测定允许对150个CNGA3错义变体进行功能上确保的临床变体解释，从而实现并支持VUS重新分类，并确保对患有CNGA3相关色盲的患者进行分子诊断。特此确定有资格对该疾病进行未来基因治疗试验的患者。
