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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Combined MITOchondrial-NUCLEAR (MITO-NUCLEAR) Analysis for Mitochondrial Diseases Diagnosis: Validation and
Ferdinando Barretta1,2, Fabiana Uomo1, Filomena Caldora1
1Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131 Naples, Italy.
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 3000×, the median coverage was >5000×, and 93.84% of nuclear regions were covered at least 100×.
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.
