Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy.
Mei Yao, Liya Jiang, Yue Yan, Yicheng Yu, Yuwei Chen, Xiaoyi Wang, Yijie Feng, Yiqin Cui, Dongming Zhou, Feng Gao, Shanshan Mao
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引用次数: 0
Abstract
Objectives: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA.
Methods: A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probe amplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing.
Results: A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. No difference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations.
Conclusions: The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce the time frame for diagnosis, facilitating early intervention and preventing birth defects.
目的:脊髓性肌萎缩症(SMA)是由存活运动神经元1(SMN1)的同基因缺失和复合杂合突变引起的神经肌肉疾病,其严重程度与存活运动神经元2(SMN2)的拷贝数有关。本研究旨在开发一种快速、全面的 SMA 诊断方法:方法:采用扩增难治性突变系统聚合酶链反应-毛细管电泳(ARMS-PCR-CE)方法检测了292名临床疑似SMA患儿和394名家庭成员,该方法针对19种已报道的突变,并将检测结果与多重连接依赖性探针扩增(MLPA)结果进行了比较。通过SMN1长程PCR和桑格测序进一步确认了点突变个体:结果:本研究共发现了 202 名 SMA 患儿、272 名携带者和 212 名正常人。在这些群体中,SMN1和SMN2第7和第8外显子的R值分布没有差异,变异系数始终低于0.08。在检测SMN1和SMN2的外显子7和8拷贝数时,ARMS-PCR-CE结果与MLPA结果一致。研究中约有4.95%(10/202)的患者存在复合杂合突变:ARMS-PCR-CE检测是一种全面、快速、准确的SMA诊断方法,可同时检测SMN1/SMN2中外显子7和8的拷贝数,以及SMN1中的19个点突变和SMN2中的2个增强子。这种方法可有效缩短诊断时间,便于早期干预和预防出生缺陷。
期刊介绍:
Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. CCLM welcomes contributions on the progress in fundamental and applied research and cutting-edge clinical laboratory medicine. It is one of the leading journals in the field, with an impact factor over 3. CCLM is issued monthly, and it is published in print and electronically.
CCLM is the official journal of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) and publishes regularly EFLM recommendations and news. CCLM is the official journal of the National Societies from Austria (ÖGLMKC); Belgium (RBSLM); Germany (DGKL); Hungary (MLDT); Ireland (ACBI); Italy (SIBioC); Portugal (SPML); and Slovenia (SZKK); and it is affiliated to AACB (Australia) and SFBC (France).
Topics:
- clinical biochemistry
- clinical genomics and molecular biology
- clinical haematology and coagulation
- clinical immunology and autoimmunity
- clinical microbiology
- drug monitoring and analysis
- evaluation of diagnostic biomarkers
- disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
- new reagents, instrumentation and technologies
- new methodologies
- reference materials and methods
- reference values and decision limits
- quality and safety in laboratory medicine
- translational laboratory medicine
- clinical metrology
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