Combined MITOchondrial-NUCLEAR (MITO-NUCLEAR) Analysis for Mitochondrial Diseases Diagnosis: Validation and Implementation of a One-Step NGS Method

Background: Next-generation sequencing (NGS) technology is revolutionizing diagnostic screening for mitochondrial diseases (MDs). Moreover, an investigation by NGS still requires analyzing the mitochondrial genome and nuclear genes separately, with limitations in terms of time and costs. We describe the validation and implementation of a custom blended MITOchondrial-NUCLEAR (MITO-NUCLEAR) assay for the simultaneous identification of genetic variants both in whole mtDNA and in nuclear genes included in a clinic exome panel. Furthermore, the MITO-NUCLEAR assay, implemented in our diagnostic process, has allowed us to arrive at a molecular diagnosis in a young patient. Methods: Massive sequencing strategy was applied for the validation experiments, performed using multiple tissues (blood, buccal swab, fresh tissue, tissue from slide, and formalin-fixed paraffin-embedded tissue section) and two different blend-in ratios of the mitochondrial probes: nuclear probes; 1:900 and 1:300. Results: Data suggested that 1:300 was the optimal probe dilution, where 100% of the mtDNA was covered at least 3000x, the median coverage was >5000x, and 93.84% of nuclear regions were covered at least 100x. Conclusions: Our custom Agilent SureSelect MITO-NUCLEAR panel provides a potential one-step investigation that may be applied to both research and genetic diagnosis of MDs, allowing the simultaneous discovery of nuclear and mitochondrial mutations.

Automated summary

This study presents a custom MITO-NUCLEAR assay that enables simultaneous identification of genetic variants in both mtDNA and nuclear genes, providing a potential one-step investigation for the genetic diagnosis of mitochondrial diseases.