FRIGE Institute of Human Genetics, FRIGE House, Jodhpur Village Road, Satellite, Ahmedabad, India, 380015.
KLES Prabhakar Kore Hospital, Belgaum, Karnataka, India.
Hum Genomics. 2024 May 10;18(1):46. doi: 10.1186/s40246-024-00613-9.
Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India.
903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood.
We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.
目前用于溶酶体贮积症（LSD）的临床诊断途径包括顺序进行生化酶学检测，然后进行 DNA 测序，该途径具有迭代性，诊断率低，且由于临床表现重叠而成本高昂。在此，我们描述了一种新型的低成本高通量测序检测方法，使用单分子分子反转探针（smMIP）来筛选与印度 29 种常见 LSD 相关的基因中的致病单核苷酸变异（SNV）和拷贝数变异（CNV）。
设计了 903 个 smMIP 以靶向靶向基因的外显子和外显子-内含子边界（n=23; 人类基因组的 53.7 kb），并等摩尔混合以创建测序文库。在 50 名患者的队列中进行了广泛验证后，我们筛选了 300 名具有 LSD 生化诊断（n=187）或临床怀疑（n=113）的患者。在具有 LSD 生化诊断的患者中观察到 83.4%的诊断率。此外，在具有 LSD 高度临床怀疑的患者中观察到 73.9%（n=54/73）的诊断率，而在具有 LSD 低度临床怀疑的患者中观察到 2.4%（n=1/40）。除了检测 SNV 之外，该检测还可以高度置信地检测单和多外显子拷贝数变异。至关重要的是，对于无法进行生化检测的尼曼-匹克病 C 型和神经元蜡样脂褐质沉积症 6 型疾病，可以使用我们的检测进行诊断。最后，我们观察到该检测在与全血相比从干血斑中提取的 DNA 中具有非劣效性能。
我们开发了一种灵活且可扩展的检测方法，可可靠地检测印度 29 种常见 LSD 的遗传原因。与当前的临床范例相比，该检测方法在相同或更低成本下提高了诊断率，同时合并了多种样本类型中多种变异类型的检测。
