DataSheet1_Validation of a targeted gene panel sequencing for the diagnosis of hereditary chronic liver diseases.pdf

Background: The cause of chronic liver diseases (CLD) remains undiagnosed in up to 30% of adult patients. Whole-Exome Sequencing (WES) can improve the diagnostic rate of genetic conditions, but it is not yet widely available, due to the costs and the difficulties in results interpretation. Targeted panel sequencing (TS) represents an alternative more focused diagnostic approach.Aims: To validate a customized TS for hereditary CLD diagnosis.Methods: We designed a customized panel including 82 CLD-associated genes (iron overload, lipid metabolism, cholestatic diseases, storage diseases, specific hereditary CLD and susceptibility to liver diseases). DNA samples from 19 unrelated adult patients with undiagnosed CLD were analyzed by both TS (HaloPlex) and WES (SureSelect Human All Exon kit v5) and the diagnostic performances were compared.Results: The mean depth of coverage of TS-targeted regions was higher with TS than WES (300x vs. 102x; p < 0.0001). Moreover, TS yielded a higher average coverage per gene and lower fraction of exons with low coverage (p < 0.0001). Overall, 374 unique variants were identified across all samples, 98 of which were classified as “Pathogenic” or “Likely Pathogenic” with a high functional impact (HFI). The majority of HFI variants (91%) were detected by both methods; 6 were uniquely identified by TS and 3 by WES. Discrepancies in variant calling were mainly due to variability in read depth and insufficient coverage in the corresponding target regions. All variants were confirmed by Sanger sequencing except two uniquely detected by TS. Detection rate and specificity for variants in TS-targeted regions of TS were 96.9% and 97.9% respectively, whereas those of WES were 95.8% and 100%, respectively.Conclusion: TS was confirmed to be a valid first-tier genetic test, with an average mean depth per gene higher than WES and a comparable detection rate and specificity.

背景：慢性肝病（CLD）的病因在高达30%的成年患者中仍未确诊。全外显子组测序（WES）能够提高遗传性疾病的诊断率，但由于成本和结果解释的困难，其尚未得到广泛应用。靶向面板测序（TS）代表了一种更为集中的诊断方法。目标：验证一种用于遗传性CLD诊断的定制化TS。方法：我们设计了一个包含82个与CLD相关的基因（包括铁过载、脂质代谢、胆汁淤积性疾病、储存性疾病、特定遗传性CLD和易患肝病的风险）的定制化面板。对19例未确诊CLD的无相关成年患者的DNA样本进行了TS（HaloPlex）和WES（SureSelect Human All Exon kit v5）分析，并比较了其诊断性能。结果：TS靶向区域的平均覆盖深度高于WES（300x对比102x；p < 0.0001）。此外，TS产生了每个基因的平均覆盖深度更高和低覆盖度外显子比例更低的结果（p < 0.0001）。总体而言，在所有样本中鉴定出374个独特的变异，其中98个被分类为“致病性”或“可能致病性”，具有高度功能影响（HFI）。大多数HFI变异（91%）被两种方法均检测到；6个由TS独特识别，3个由WES识别。变异调用中的差异主要归因于读取深度的变异性以及相应目标区域覆盖不足。所有变异均经Sanger测序确认，除TS独特检测到的两个变异外。TS靶向区域变异的检测率和特异性分别为96.9%和97.9%，而WES的相应指标分别为95.8%和100%。结论：TS被证实为一种有效的首级遗传检测方法，其每个基因的平均覆盖深度高于WES，且检测率和特异性与WES相当。