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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 and Shanshan Mao ORCID logo EMAIL logo
Published/Copyright: June 12, 2024
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 62 Issue 12

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.

Keywords: polymerase chain reaction-capillary electrophoresis; spinal muscular atrophy; survival motor neuron 1; survival motor neuron 2; compound heterozygous mutations
Corresponding author: Shanshan Mao, Department of Neurology, Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, No. 3333, Binsheng Road, Binjiang District, Hangzhou, 310052, P.R. China, E-mail:
MY, LYJ and YY contributed equally to this work and share first authorship.

Funding source: The Fundamental Research Funds for the Central Universities

Award Identifier / Grant number: 226-2023-00153

Funding source: National Natural Science Foundation of China

Award Identifier / Grant number: 82271735

Funding source: The Key R&D Program of Zhejiang Province

Award Identifier / Grant number: 2022C03167

Acknowledgments

The authors acknowledge all the subjects for participating in this study. We would like to appreciate Ifan Chiu for his instruction.

  1. Research ethics: The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Children’s Hospital of Zhejiang University School of Medicine (Study Reference Number: 2022-IRB-199).

  2. Informed consent: Informed consent was obtained from all individuals included in this study.

  3. Author contributions: MY, LYJ and YY wrote the manuscript draft. MY and YWC performed the analysis; YCY, XYW, YJF and YQC collected the samples and clinical information. YWC, DMZ and FG revised the manuscript. SSM supervised the study and revised the manuscript. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Competing interests: Authors state no conflict of interest.

  5. Research funding: This study was supported by the Key R&D Program of Zhejiang Province (2022C03167), the National Natural Science Foundation of China (82271735), and the Fundamental Research Funds for the Central Universities (226-2023-00153).

  6. Data availability: The datasets used and/or analyzed in this study are available from the corresponding authors upon reasonable request.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-0334).

Received: 2024-03-12
Accepted: 2024-05-26
Published Online: 2024-06-12
Published in Print: 2024-11-26

© 2024 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2024-0334
Keywords for this article
polymerase chain reaction-capillary electrophoresis; spinal muscular atrophy; survival motor neuron 1; survival motor neuron 2; compound heterozygous mutations

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. External quality assurance (EQA): navigating between quality and sustainability
  4. Reviews
  5. Molecular allergology: a clinical laboratory tool for precision diagnosis, stratification and follow-up of allergic patients
  6. Nitrous oxide abuse direct measurement for diagnosis and follow-up: update on kinetics and impact on metabolic pathways
  7. Opinion Papers
  8. A vision to the future: value-based laboratory medicine
  9. Point-of-care testing, near-patient testing and patient self-testing: warning points
  10. Navigating the path of reproducibility in microRNA-based biomarker research with ring trials
  11. Point/Counterpoint
  12. Six Sigma – is it time to re-evaluate its value in laboratory medicine?
  13. The value of Sigma-metrics in laboratory medicine
  14. Genetics and Molecular Diagnostics
  15. Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy
  16. Can we identify patients carrying targeted deleterious DPYD variants with plasma uracil and dihydrouracil? A GPCO-RNPGx retrospective analysis
  17. General Clinical Chemistry and Laboratory Medicine
  18. Comparison of ChatGPT, Gemini, and Le Chat with physician interpretations of medical laboratory questions from an online health forum
  19. External quality assessment performance in ten countries: an IFCC global laboratory quality project
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