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Abstract：
BACKGROUND: Pathogenic variants in BRCA genes play a crucial role in the pathogenesis of ovarian cancer. Intronic variants of uncertain significance (VUS) may contribute to pathogenicity by affecting splicing. Currently, the significance of many intronic variants in BRCA has not been clarified, impacting patient treatment strategies and the management of familial cases.
METHODS: A retrospective study was conducted to analyze BRCA intronic VUS in a cohort of 707 unrelated ovarian cancer patients at a single institution from 2018 to 2023. Three splicing predictors were employed to analyze detected intronic VUS. Variants predicted to have splicing alterations were selected for further validation through minigene assays. Patient and familial investigations were conducted to comprehend cancer incidence within pedigrees and the application of poly (ADP-ribose) polymerase inhibitors (PARPi) by the patients. In accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG), the intronic VUS were reclassified based on minigene assay results and clinical evidence.
RESULTS: Approximately 9.8% (69/707) of patients were identified as carriers of 67 different VUS in BRCA1/2, with four intronic variants accounting for 6% (4/67) of all VUS. Splicing predictors indicated potential splicing alterations in splicing for BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C variants. Minigene assays utilizing the pSPL3 exon trapping vector revealed that these variants induced changes in splicing sites and frameshift, resulting in premature termination of translation (p. Ala1453Glyfs and p. Pro143Glyfs). According to ACMG guidelines, BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C were reclassified as pathogenic variants. Pedigree investigations were conducted on patients with BRCA1 c.4358-2A>G variant, and the detailed utilization of PARPi provided valuable insights into research on PARPi resistance.
CONCLUSIONS: Two intronic VUS were reclassified as pathogenic variants. A precise classification of variants is crucial for the effective treatment and management of both patients and healthy carriers.
METHODS: A retrospective study was conducted to analyze BRCA intronic VUS in a cohort of 707 unrelated ovarian cancer patients at a single institution from 2018 to 2023. Three splicing predictors were employed to analyze detected intronic VUS. Variants predicted to have splicing alterations were selected for further validation through minigene assays. Patient and familial investigations were conducted to comprehend cancer incidence within pedigrees and the application of poly (ADP-ribose) polymerase inhibitors (PARPi) by the patients. In accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG), the intronic VUS were reclassified based on minigene assay results and clinical evidence.
RESULTS: Approximately 9.8% (69/707) of patients were identified as carriers of 67 different VUS in BRCA1/2, with four intronic variants accounting for 6% (4/67) of all VUS. Splicing predictors indicated potential splicing alterations in splicing for BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C variants. Minigene assays utilizing the pSPL3 exon trapping vector revealed that these variants induced changes in splicing sites and frameshift, resulting in premature termination of translation (p. Ala1453Glyfs and p. Pro143Glyfs). According to ACMG guidelines, BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C were reclassified as pathogenic variants. Pedigree investigations were conducted on patients with BRCA1 c.4358-2A>G variant, and the detailed utilization of PARPi provided valuable insights into research on PARPi resistance.
CONCLUSIONS: Two intronic VUS were reclassified as pathogenic variants. A precise classification of variants is crucial for the effective treatment and management of both patients and healthy carriers.
摘要：
背景：BRCA基因的致病变异体在卵巢癌的发病机制中起着至关重要的作用。意义不确定的内含子变体（VUS）可能通过影响剪接而导致致病性。目前,许多内含子变体在BRCA中的意义尚未得到澄清，影响患者治疗策略和家族病例管理。
方法：进行了一项回顾性研究，以分析2018年至2023年在单个机构中707名无关卵巢癌患者的BRCA内含子VUS。使用三个剪接预测因子来分析检测到的内含子VUS。选择预测具有剪接改变的变体用于通过小基因测定进一步验证。进行了患者和家族调查，以了解家系中的癌症发生率以及患者使用聚（ADP-核糖）聚合酶抑制剂（PARPi）的情况。根据美国医学遗传学和基因组学学院（ACMG）的指导方针，根据小基因检测结果和临床证据对内含子VUS进行了重新分类.
结果：大约9.8%(69/707)的患者被鉴定为BRCA1/2中67种不同VUS的携带者，其中4种内含子变异占所有VUS的6%(4/67)。剪接预测因子表明BRCA1c.4358-2A>G和BRCA2c.475+5G>C变体的剪接中潜在的剪接改变。利用pSPL3外显子捕获载体的Minigene分析显示，这些变体诱导剪接位点和移码的变化，导致翻译提前终止(p.Ala1453Glyfs和p.Pro143Glyfs)。根据ACMG指南,BRCA1c.4358-2A>G和BRCA2c.475+5G>C被重新分类为致病变体。对BRCA1c.4358-2A>G变异的患者进行了谱系调查，PARPi的详细利用为PARPi抗性的研究提供了有价值的见解。
结论：两个内含子VUS被重新分类为致病性变异。变体的精确分类对于患者和健康携带者的有效治疗和管理至关重要。
方法：进行了一项回顾性研究，以分析2018年至2023年在单个机构中707名无关卵巢癌患者的BRCA内含子VUS。使用三个剪接预测因子来分析检测到的内含子VUS。选择预测具有剪接改变的变体用于通过小基因测定进一步验证。进行了患者和家族调查，以了解家系中的癌症发生率以及患者使用聚（ADP-核糖）聚合酶抑制剂（PARPi）的情况。根据美国医学遗传学和基因组学学院（ACMG）的指导方针，根据小基因检测结果和临床证据对内含子VUS进行了重新分类.
结果：大约9.8%(69/707)的患者被鉴定为BRCA1/2中67种不同VUS的携带者，其中4种内含子变异占所有VUS的6%(4/67)。剪接预测因子表明BRCA1c.4358-2A>G和BRCA2c.475+5G>C变体的剪接中潜在的剪接改变。利用pSPL3外显子捕获载体的Minigene分析显示，这些变体诱导剪接位点和移码的变化，导致翻译提前终止(p.Ala1453Glyfs和p.Pro143Glyfs)。根据ACMG指南,BRCA1c.4358-2A>G和BRCA2c.475+5G>C被重新分类为致病变体。对BRCA1c.4358-2A>G变异的患者进行了谱系调查，PARPi的详细利用为PARPi抗性的研究提供了有价值的见解。
结论：两个内含子VUS被重新分类为致病性变异。变体的精确分类对于患者和健康携带者的有效治疗和管理至关重要。
