背景:这项研究的目的是创建一个分子诊断平台,并为高度怀疑线粒体疾病的患者建立一个诊断管道。三种方法的有效性,即,传统限制性片段长度多态性-聚合酶链反应(RFLP-PCR),用于热点检测和完整线粒体DNA(mtDNA)的Sanger测序,第三代(纳米孔)全mtDNA测序,将在诊断疑似原发性线粒体疾病(PMDs)的患者中进行比较。还讨论了不同方法的优点和局限性。
方法:进行了一项单中心前瞻性队列研究,以验证疑似线粒体疾病的诊断流程。在第一阶段,使用五组引物的基于PCR的方法筛选八个热点(m.3243A>G,m.3460G>A,m.834A>G,m.8993T>G,m.9185T>C,m.11778G>A,m.13513G>A,和m.4977缺失)使用RFLP或直接Sanger测序。Sanger测序也用于确认RFLP阳性样品。在第二阶段,对于八个热点筛查结果为阴性的样本,使用Sanger测序或第三代纳米孔测序进行线粒体全基因组测序.
结果:在2020年6月至2023年5月之间,招募了30名年龄在0至63岁之间的临床可疑线粒体疾病患者。PMD诊断的阳性率为8/30=26.7%,基于RFLP的方法的异质水平灵敏度约为5%。其余22例第一阶段检测为阴性的患者使用Sanger测序或第三代测序纳米孔进行检测,所有检测结果均为病理性mtDNA突变阴性.与Sanger测序法相比,RFLP-PCR的结果受到RFLP酶消化不完全的限制。对于mtDNA的全基因组测序,桑格测序,而不是纳米孔测序,是我们机构的首选,因为它具有成本效益。
结论:在我们的高选择性队列中,在8个热点屏幕的第一阶段,大多数测试呈阳性。Sanger测序是一种常规、准确的线粒体疾病筛查方法,至少在该地区最常见的热点地区。结果表明,Sanger测序是一种准确的方法,具有更高的成本效益。这种完整的分子诊断平台具有成本相对较低、报告时间较短的优点,促进具有此类疾病临床证据的患者的关键识别。该诊断流程图也已转化为三级医院的常规临床使用。
BACKGROUND: The aim of this study was to create a molecular diagnostic platform and establish a diagnostic pipeline for patients highly suspected of mitochondrial disorders. The effectiveness of three methods, namely, traditional restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), Sanger sequencing for hotspot detection and whole mitochondrial DNA (mtDNA), and third-generation (Nanopore) whole mtDNA sequencing, will be compared in diagnosing patients with suspected primary mitochondrial diseases (PMDs). The strengths and limitations of different methods are also discussed.
METHODS: A single-center prospective cohort study was conducted to validate the diagnostic pipeline for suspected mitochondrial diseases. In the first stage, a PCR-based method with five sets of primers was used to screen for eight hotspots (m.3243A>G, m.3460G>A, m.8344A>G, m.8993T>G, m.9185T>C, m.11778G>A, m.13513G>A, and m.4977deletion) using either RFLP or direct Sanger sequencing. Sanger sequencing was also used to confirm the RFLP-positive samples. In the second stage, for samples with negative screening results for the eight hotspots, mitochondrial whole-genome sequencing was performed using Sanger sequencing or third-generation nanopore sequencing.
RESULTS: Between June 2020 and May 2023, 30 patients from ages 0 to 63 with clinically suspected mitochondrial disease were enrolled. The positive yield for the diagnosis of PMDs was 8/30=26.7%, and the sensitivity of the heteroplasmy level for the RFLP-based method was approximately 5%. The remaining 22 patients who tested negative at the first stage were tested using Sanger sequencing or the third-generation sequencing Nanopore, and all tested negative for pathological mtDNA mutations. Compared to the Sanger sequencing method, the results of RFLP-PCR were compromised by the limitations of incomplete RFLP enzyme digestion. For whole-genome sequencing of mtDNA, Sanger sequencing, instead of nanopore sequencing, is preferred at our institution because of its cost-effectiveness.
CONCLUSIONS: In our highly selective cohort, most tested positive in the first stage of the 8 hot spots screen. Sanger sequencing is a conventional and accurate method for mitochondrial disease screening, at least for the most common hot spots in the region. The results revealed that Sanger sequencing is an accurate method with the benefit of being more cost-effective. This integral platform of molecular diagnosis bears the advantages of being relatively low cost and having a shorter reporting time, facilitating crucial identification of patients with clinical evidence of such disorders. This diagnostic flowchart has also been translated into routine clinical use in the tertiary hospital.