Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

Applying next-generation sequencing to 42 infants with mitochondrial disease highlights both the potential and the challenge of using this technology in clinical diagnosis. Getting to the Genetic Root of Mitochondrial Disease Next-generation DNA sequencing is being applied with great success in research settings to uncover new disease genes. Despite these successes, it is unclear how useful the technology will be for routine clinical diagnosis given the challenge of interpreting DNA variations in individual patients. In a new study, Calvo and colleagues apply next-generation sequencing to infants with mitochondrial disorders, a large collection of inherited diseases that are notoriously difficult to diagnose because of the multitude of candidate genes and the highly variable nature of the clinical presentation. First, the authors selected 42 unrelated infants with mitochondrial diseases that were refractory to standard clinical genetic testing. Then, for each child, they sequenced the DNA of the mitochondrial genome, the 100 genes previously linked to mitochondrial disease, and the ~1000 additional genes that are known to participate in mitochondrial biology. Of all the DNA differences present in the patients, the researchers prioritized those that were rare in the general population, predicted to disrupt protein function, and inherited in a recessive fashion. Such variants showed fivefold enrichment in the patients compared to that in healthy control individuals. In 10 patients (24%), firm molecular diagnoses were made in genes previously linked to mitochondrial diseases; 13 patients (31%) had prioritized recessive mutations in genes not previously linked to disease. For two of these genes, the authors were able to show that the mutations caused the mitochondrial disorder. These results suggest that next-generation sequencing may be able to provide a molecular diagnosis for ~25% of currently unsolved cases of infantile mitochondrial disease. An additional 25% of cases could be solved in the coming few years as more genes are formally proven to be linked to mitochondrial disease. The remaining 50% of patients in whom diagnosis was not possible underscores the challenge of interpreting DNA sequence data for clinical diagnosis. Nevertheless, the study by Calvo and colleagues will help to calibrate clinicians’ expectations regarding the diagnostic use of next-generation sequencing. Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed “MitoExome” sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.