Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing

Abstract

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.

Figure 1. Enrichment of prioritized variants in cases compared to healthy individuals

(A) Mean number of genes containing rare, protein-modifying alleles in cases and healthy controls within the 1034 mitochondrial genes and the 347 auxiliary genes sequenced. Error bars indicate standard error. (B) Percent of cases and controls containing prioritized mitochondrial genes (red), defined as harboring 2 rare, protein-modifying alleles. (C) Percent of cases and controls containing prioritized mitochondrial genes, excluding variants with allele frequency >0.005 in either cases or controls, with enrichment in cases versus controls displayed above. Analysis was restricted to regions well-sequenced in all individuals, and to the 31 cases from non-consanguineous families.

Figure 2. Prioritized variants in 42 patients with OXPHOS disease

(A) 42 patients categorized by presence of prioritized genes (red). The gene names are listed alphabetically at right, with parentheses indicating genes prioritized in two unrelated patients, and triangle indicating support of pathogenicity. (B) 52 prioritized alleles, categorized by type of protein modification.

Figure 3. mtDNA deletion in patient P33

(A) Schematic diagram of mtDNA indicates deletion (black arc) relative to position 0/16,569 (triangle). Arrows indicate long-range PCR primers. (B) mtDNA sequence coverage in 50bp windows. Inset shows Sanger electropherogram of breakpoint. (C) Gel electrophoresis of long-range PCR amplicon displays 14,933 bp fragment in control DNA and 7,681 bp fragment in P33, along with marker II (Roche) and mtDNA from individuals with confirmed single and multiple deletions.

Figure 4. Patient mutations in acylglycerol kinase (AGK)

Schematic diagram shows protein structure with the location of truncating mutations in unrelated patients P41 and P42 shown in red. Conservation of C-terminal region is shown below with identical residues shaded and the C5 domain shared with yeast sphingosine kinases underlined.

Figure 5. NDUFB3 complementation of complex I defects in subject P3 fibroblasts

(A) Barplots show complex I (CI) activity, normalized by complex IV (CIV) activity, in fibroblasts from patient P3, a healthy control individual, and an unrelated complex I patient with a defined C8orf38 mutation P(C8orf38). Enzyme activity was measured before and after transduction with wild-type C8orf38 or NDUFB3 mRNA. Data shown are mean of three biological replicates ± s.e.m. *P < 0.001. (B) Western blot shows protein expression of complex I, complex IV, and loading control complex V (CV).