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. 2023 May 15;14(5):1087.
doi: 10.3390/genes14051087.

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

Ferdinando Barretta  1   2 , Fabiana Uomo  1 , Filomena Caldora  1 , Rossella Mocerino  1 , Daniela Adamo  3 , Francesco Testa  4 , Francesca Simonelli  4 , Olga Scudiero  1   2 , Nadia Tinto  1   2 , Giulia Frisso  1   2 , Cristina Mazzaccara  1   2
Affiliations

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

Ferdinando Barretta et al. Genes (Basel). .

Abstract

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.

Keywords: MITO-NUCLEAR; combined sequencing of mitochondrial and nuclear DNA; mitochondrial and nuclear disease panel; mitochondrial diseases; mtDNA; next-generation sequencing (NGS); nuclear genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Post-capture quality profiles of NGS libraries were obtained using an Agilent TapeStation and High Sensitivity D1000 ScreenTape from two different molar dilution ratios, 1:300 and 1:900 (mitochondrial probes: nuclear probes). (A) RUN 1 experiments: S1, S2, and S3 samples were hybridized with a mix of both mitochondrial and nuclear probes at two different dilutions, 1:300 and 1:900 (mitochondrial probes: nuclear probes). Sample S2 was also hybridized with only nuclear probes (CCP17). (B) RUN 2 experiments: S1, S2, S4, S5, TSI, TS2, and TS3 samples were hybridized with a mix of both mitochondrial and nuclear probes at a 1:300 molar dilution ratio. EL1: Electronic Ladder.
Figure 2
Figure 2
Capture efficiency of mtDNA (SSel Custom Constitutional Panel 16.708 Kbp) and nuclear clinic exome (CCP17: custom constitutional panel; Design size: 17 Mb for nuclear clinic exome) in MITO-NUCLEAR blended design into two different molar ratios of mtDNA probes. The total of 10 samples (S1, S2, and S3 from RUN 1 and S1, S2, S4, S5, TS1, TS2, and TS3 from RUN 2) are tested into the 1:300 molar ratios of mitochondrial probes. The three S1, S2, and S3 samples from RUN 1 are also tested into the 1:900 molar ratios of mitochondrial probes. The height of the columns indicates the percentage of nuclear regions (blue) and mtDNA regions (red/green) covered by a minimum number of reads.
Figure 3
Figure 3
Eye fundus examination showing pallor of discs in the right eye (A) and the left eye (B) of the affected proband.
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