UOSID Genetica Medica e Cardiomiologia, Dipartimento di Medicina di Precisione, Università degli Studi della Campania "Luigi Vanvitelli", 80138 Napoli, Italy.
Telethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy.
Genes (Basel). 2021 Jan 21;12(2):133. doi: 10.3390/genes12020133.
Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16-29 in gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a "non-carrier" status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1-15 and 30-34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16-29 deletion, while the second allele showed a 1-34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16-29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.
下一代测序 (NGS) 改变了我们诊断遗传疾病的方法。如今，NGS 最全面的应用是全基因组测序 (WGS)，它能够检测几乎所有的 DNA 变异。然而，即使在进行了精确的 WGS 后，许多遗传疾病仍然无法得到解决。这可能是由于当前基于 DNA 片段化和短读长的 NGS 方案所致。为了克服这些限制，我们应用了一种链接读取测序技术，该技术将单分子条形码与短读 WGS 相结合。我们能够组装单倍型并沿基因组区分等位基因。作为一个示例案例，我们研究了一位 X 连锁肌营养不良症女性携带者的遗传状况未解之谜。她的家族疾病是由基因中第 16-29 外显子缺失引起的，但她通过多重连接依赖性探针扩增 (MLPA) 和 array CGH 显示这些外显子的正常剂量。这种情况通常被认为与“非携带者”状态相容。出乎意料的是，该女孩还表现出侧翼外显子 1-15 和 30-34 的剂量增加。使用链接读取 WGS，我们能够区分两条 X 染色体。在第一个等位基因中，我们发现了 16-29 缺失，而第二个等位基因显示出 1-34 重复：在这两种情况下，链接读取 WGS 都能以单核苷酸分辨率正确地定位边界。这种 1-34 重复在 trans 中显然恢复了定量检测中看到的 16-29 外显子的正常剂量。这在遗传咨询中产生了巨大影响，将非携带者转变为双重携带者状态的预测。我们得出结论，链接读取 WGS 应被视为一种有价值的选择，以提高我们对未解遗传疾病的理解。
