Ronzoni Luisa, Marini Ilaria, Passignani Giulia, Malvestiti Francesco, Marchelli Daniele, Bianco Cristiana, Pelusi Serena, Prati Daniele, Valenti Luca
Biological Resource Centre, Precision Medicine Lab, Transfusion Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Milano, Italy.
Department of Pathophysiology and Transplantation, Università Degli Studi di Milano, Milano, Italy.
Front Genet. 2023 Jun 14;14:1137016. doi: 10.3389/fgene.2023.1137016. eCollection 2023.
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. To validate a customized TS for hereditary CLD diagnosis. 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. The mean depth of coverage of TS-targeted regions was higher with TS than WES (300x vs. 102x; < 0.0001). Moreover, TS yielded a higher average coverage per gene and lower fraction of exons with low coverage ( < 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. 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.
高达30%的成年慢性肝病（CLD）患者的病因仍未得到诊断。全外显子组测序（WES）可以提高遗传疾病的诊断率，但由于成本和结果解读困难，目前尚未广泛应用。靶向基因panel测序（TS）是一种更具针对性的替代诊断方法。为验证用于遗传性CLD诊断的定制TS。我们设计了一个定制panel，包括82个与CLD相关的基因（铁过载、脂质代谢、胆汁淤积性疾病、贮积病、特定遗传性CLD和肝病易感性）。对19例未确诊CLD的无关成年患者的DNA样本进行了TS（HaloPlex）和WES（SureSelect Human All Exon kit v5）分析，并比较了诊断性能。TS靶向区域的平均覆盖深度高于WES（300x对102x；<0.0001）。此外，TS每个基因的平均覆盖率更高，低覆盖率外显子的比例更低（<0.0001）。总体而言，在所有样本中鉴定出374个独特变异，其中98个被分类为“致病性”或“可能致病性”，具有高功能影响（HFI）。大多数HFI变异（91%）可通过两种方法检测到；TS单独鉴定出6个，WES单独鉴定出3个。变异调用的差异主要是由于读取深度的变异性和相应靶区域的覆盖不足。除TS单独检测到的两个变异外，所有变异均通过Sanger测序得到确认。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的检测率和特异性分别为95.8%和100%。TS被确认为一种有效的一级基因检测方法，每个基因的平均深度高于WES，检测率和特异性相当。
