Sudrié-Arnaud Bénédicte, Snanoudj Sarah, Dabaj Ivana, Dranguet Hélène, Abily-Donval Lenaig, Lebas Axel, Vezain Myriam, Héron Bénédicte, Marie Isabelle, Duval-Arnould Marc, Marret Stéphane, Tebani Abdellah, Bekri Soumeya
Department of Metabolic Biochemistry, Rouen University Hospital, 76000 Rouen, France.
Normandie Univ, UNIROUEN, CHU Rouen, INSERM U1245, 76000 Rouen, France.
Diagnostics (Basel). 2021 Feb 12;11(2):294. doi: 10.3390/diagnostics11020294.
Diagnosis of lysosomal disorders (LDs) may be hampered by their clinical heterogeneity, phenotypic overlap, and variable age at onset. Conventional biological diagnostic procedures are based on a series of sequential investigations and require multiple sampling. Early diagnosis may allow for timely treatment and prevent clinical complications. In order to improve LDs diagnosis, we developed a capture-based next generation sequencing (NGS) panel allowing the detection of single nucleotide variants (SNVs), small insertions and deletions, and copy number variants (CNVs) in 51 genes related to LDs. The design of the LD panel covered at least coding regions, promoter region, and flanking intronic sequences for 51 genes. The validation of this panel consisted in testing 21 well-characterized samples and evaluating analytical and diagnostic performance metrics. Bioinformatics pipelines have been validated for SNVs, indels and CNVs. The clinical output of this panel was tested in five novel cases. This capture-based NGS panel provides an average coverage depth of 474× which allows the detection of SNVs and CNVs in one comprehensive assay. All the targeted regions were covered above the minimum required depth of 30×. To illustrate the clinical utility, five novel cases have been sequenced using this panel and the identified variants have been confirmed using Sanger sequencing or quantitative multiplex PCR of short fluorescent fragments (QMPSF). The application of NGS as first-line approach to analyze suspected LD cases may speed up the identification of alterations in LD-associated genes. NGS approaches combined with bioinformatics analyses, are a useful and cost-effective tool for identifying the causative variations in LDs.
溶酶体疾病（LDs）的诊断可能会受到其临床异质性、表型重叠以及发病年龄各异的影响。传统的生物学诊断程序基于一系列连续的检查，且需要多次采样。早期诊断有助于及时治疗并预防临床并发症。为了改善LDs的诊断，我们开发了一种基于捕获的下一代测序（NGS）检测板，可检测与LDs相关的51个基因中的单核苷酸变异（SNV）、小插入和缺失以及拷贝数变异（CNV）。LD检测板的设计涵盖了51个基因的至少编码区、启动子区和侧翼内含子序列。该检测板的验证包括测试21个特征明确的样本并评估分析和诊断性能指标。生物信息学流程已针对SNV、插入缺失和CNV进行了验证。该检测板的临床应用在5个新病例中进行了测试。这种基于捕获的NGS检测板提供了平均474倍的覆盖深度，可在一次综合检测中检测SNV和CNV。所有靶向区域的覆盖深度均高于所需的最低30倍深度。为了说明其临床实用性，已使用该检测板对5个新病例进行了测序，并使用Sanger测序或短荧光片段定量多重PCR（QMPSF）对鉴定出的变异进行了确认。将NGS作为分析疑似LD病例的一线方法可能会加快LD相关基因改变的鉴定。NGS方法与生物信息学分析相结合，是一种用于识别LDs致病变异的有用且具有成本效益的工具。
