纳米孔测序结合自适应采样和NanoExpansion可准确表征重复扩增疾病。

文献信息

中文摘要

重复扩增疾病由短串联重复序列（STR）的异常扩增引起。我们评估了靶向长读长纳米孔测序在132名参与者中对STR扩增的全面表征，包括已通过Southern印迹表征的强直性肌营养不良1型（DM1，n=51）和DM2（n=3）患者、通过重复引物聚合酶链反应（RP-PCR；验证组）评估的C9orf72相关肌萎缩侧索硬化（ALS n=11）、疑似脊髓小脑共济失调（SCA，n=46）、小脑性共济失调伴神经病和前庭反射消失综合征（CANVAS；n=13）、亨廷顿病（1例）、肯尼迪病（1例）、弗里德赖希共济失调（FRDA，2例）以及4名健康对照（测试组）。使用Oxford Nanopore GridION或PromethION-2Solo平台上的自适应采样（AS）对天然DNA进行测序。进行长PCR测序以作比较。我们还开发了NanoExpansion，一种基于Python的工具，用于确定扩增等位基因的大小并检测序列中断。我们正确表征了所有DM1、DM2、ALS、FRDA、亨廷顿病和肯尼迪病病例，并识别出8例SCA27B、2例SCA37、3例SCA31、1例SCA3和9例CANVAS病例。与AS方法相比，长PCR低估了超过800个重复的等位基因。在27/51（52.9%）的DM1病例和1/10（10%）的ALS中检测到重复中断，但在CANVAS、FRDA、亨廷顿病、肯尼迪病或SCA病例中未检测到。这种集成的测序分析流程可对重复扩增疾病进行准确、全面的表征，并有可能改善常规遗传诊断。

英文摘要

Expansion repeat disorders are caused by abnormal expansions of short tandem repeats (STRs). We evaluated targeted long-read nanopore sequencing for comprehensive characterization of STR expansions in 132 participants, including patients with myotonic dystrophy type 1 (DM1, n = 51) and DM2 (n = 3) already characterized by Southern blot, C9orf72-associated amyotrophic lateral sclerosis (ALS n = 11) evaluated by repeat primed polymerase chain reaction (RP-PCR; validation group), suspected spinocerebellar ataxia (SCA, n = 46), cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS; n = 13), Huntington's disease (1), Kennedy disease (1), Friedreich ataxia (FRDA, 2), and 4 healthy controls (test group). Native DNA was sequenced using adaptive sampling (AS) on Oxford Nanopore GridION or PromethION-2Solo platforms. Long-PCR sequencing was performed for comparison. We also developed NanoExpansion, a Python-based tool for sizing expanded alleles and detecting sequence interruptions. We correctly characterized all DM1, DM2, ALS, FRDA, Huntington's disease, and Kennedy disease cases, and identified eight SCA27B, two SCA37, three SCA31, one SCA3, and nine CANVAS cases. Compared with the AS approach, long-PCR underestimated alleles exceeding 800 repeats. Repeat interruptions were detected in 27/51 (52.9%) DM1 cases and 1/10 (10%) ALS, but not in CANVAS, FRDA, Huntington's disease, Kennedy disease, or SCA cases. This integrated sequencing analysis pipeline provides accurate, comprehensive characterization of repeat expansion disorders and has the potential to improve routine genetic diagnosis.