PDF(Pubmed)
Abstract：
In 2018, our center started a program to offer genetic diagnosis to patients with kidney and liver monogenic rare conditions, potentially eligible for organ transplantation. We exploited a clinical exome sequencing approach, followed by analyses of in silico gene panels tailored to clinical suspicions, obtaining detection rates in line with what reported in literature. However, a percentage of patients remains without a definitive genetic diagnosis. This work aims to evaluate the utility of NGS data re-analysis for those patients with an inconclusive or negative genetic test at the time of first analysis considering that (i) the advance of alignment and variant calling processes progressively improve the detection rate, limiting false positives and false negatives; (ii) gene panels are periodically updated and (iii) variant annotation may change over time.
114 patients, recruited between 2018 and 2020, with an inconclusive or negative NGS report at the time of first analysis, were included in the study. Re-alignment and variant calling of previously generated sequencing raw data were performed using the GenomSys Variant Analyzer software.
21 previously not reported potentially causative variants were identified in 20 patients. In most cases (n = 19), causal variants were retrieved out of the re-classification from likely benign to variants of unknown significance (VUS). In one case, the variant was included because of inclusion in the analysis of a newly disease-associated gene, not present in the original gene panel, and in another one due to the improved data alignment process. Whenever possible, variants were validated with Sanger sequencing and family segregation studies. As of now, 16 out of 20 patients have been analyzed and variants confirmed in 8 patients. Specifically, in two pediatric patients, causative variants were de novo mutations while in the others, the variant was present also in other affected relatives. In the remaining patients, variants were present also in non-affected parents, raising questions on their re-classification.
Overall, these data indicate that periodic and systematic re-analysis of negative or inconclusive NGS data reports can lead to new variant identification or reclassification in a small but significant proportion of cases, with benefits for patients\' management.
114 patients, recruited between 2018 and 2020, with an inconclusive or negative NGS report at the time of first analysis, were included in the study. Re-alignment and variant calling of previously generated sequencing raw data were performed using the GenomSys Variant Analyzer software.
21 previously not reported potentially causative variants were identified in 20 patients. In most cases (n = 19), causal variants were retrieved out of the re-classification from likely benign to variants of unknown significance (VUS). In one case, the variant was included because of inclusion in the analysis of a newly disease-associated gene, not present in the original gene panel, and in another one due to the improved data alignment process. Whenever possible, variants were validated with Sanger sequencing and family segregation studies. As of now, 16 out of 20 patients have been analyzed and variants confirmed in 8 patients. Specifically, in two pediatric patients, causative variants were de novo mutations while in the others, the variant was present also in other affected relatives. In the remaining patients, variants were present also in non-affected parents, raising questions on their re-classification.
Overall, these data indicate that periodic and systematic re-analysis of negative or inconclusive NGS data reports can lead to new variant identification or reclassification in a small but significant proportion of cases, with benefits for patients\' management.
摘要：
背景：2018年，我们中心开始了一项计划，为患有肾脏和肝脏单基因罕见疾病的患者提供基因诊断，可能有资格进行器官移植。我们利用了一种临床外显子组测序方法，随后是针对临床怀疑量身定制的计算机基因面板分析，获得的检测率与文献报道的一致。然而,仍有一定比例的患者没有明确的基因诊断.这项工作旨在评估NGS数据重新分析的实用性，对于那些在首次分析时具有不确定或阴性遗传测试的患者，考虑到（i）对齐和变体调用过程的进步逐步提高了检出率，限制假阳性和假阴性;(ii)基因面板定期更新和(iii)变体注释可能随时间变化。
方法：114名患者，在2018年至2020年之间招募，在首次分析时具有不确定或负面的NGS报告，包括在研究中。使用GenomSys变体分析仪软件进行先前生成的测序原始数据的重新比对和变体调用。
结果：在20例患者中发现了21例以前未报告的潜在致病变异。在大多数情况下（n=19），从可能的良性变异到未知显著性变异(VUS)的重新分类中检索到因果变异.在一个案例中,该变异之所以被包括在内，是因为它包含在一个新的疾病相关基因的分析中，不存在于原始基因面板中，和另一个是由于改进的数据对齐过程。只要有可能,通过Sanger测序和家族分离研究验证了变异.截至目前,对20名患者中的16名进行了分析，并在8名患者中证实了变异。具体来说,在两名儿科患者中，致病变异是从头突变，而在其他变异中，该变体也存在于其他受影响的亲属中。在剩下的病人中，变异也存在于未受影响的父母中，对他们的重新分类提出了质疑。
结论：总体而言，这些数据表明，对阴性或不确定的NGS数据报告进行定期和系统的重新分析，可以在一小部分但很大一部分病例中导致新的变异识别或重新分类。对患者管理有好处。
方法：114名患者，在2018年至2020年之间招募，在首次分析时具有不确定或负面的NGS报告，包括在研究中。使用GenomSys变体分析仪软件进行先前生成的测序原始数据的重新比对和变体调用。
结果：在20例患者中发现了21例以前未报告的潜在致病变异。在大多数情况下（n=19），从可能的良性变异到未知显著性变异(VUS)的重新分类中检索到因果变异.在一个案例中,该变异之所以被包括在内，是因为它包含在一个新的疾病相关基因的分析中，不存在于原始基因面板中，和另一个是由于改进的数据对齐过程。只要有可能,通过Sanger测序和家族分离研究验证了变异.截至目前,对20名患者中的16名进行了分析，并在8名患者中证实了变异。具体来说,在两名儿科患者中，致病变异是从头突变，而在其他变异中，该变体也存在于其他受影响的亲属中。在剩下的病人中，变异也存在于未受影响的父母中，对他们的重新分类提出了质疑。
结论：总体而言，这些数据表明，对阴性或不确定的NGS数据报告进行定期和系统的重新分析，可以在一小部分但很大一部分病例中导致新的变异识别或重新分类。对患者管理有好处。
