Targeted exome sequencing of suspected mitochondrial disorders

Abstract

Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity.

Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines.

Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency.

Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting.

Figure 1. Characteristics of 102 Massachusetts General Hospital patients with suspected mitochondrial disease

ETC = electron transport chain; GI = gastrointestinal; mito. = mitochondrial.

Figure 2. MitoExome sequencing in 102 patients with suspected mitochondrial disease

Number of patients with molecular diagnoses or prioritized variants of unknown significance (pVUS), with the prioritized loci listed at right. Patients who harbored multiple variants were annotated based on their highest priority variant, with all additional pVUS listed in brackets. Parentheses indicate number of patients. Underline indicates experimental support of pathogenicity. ^a m.3243A>G; ^b m.8344A>G; ^c m.8993T>G/C; ^d m.3643_15569del; ^e homozygous; ^f compound heterozygous (phased); ^g potential compound heterozygous (unphased); ^h heterozygous in female; ⁱ not in MitoCarta. DDx = differential diagnosis; mito. = mitochondrial; mtDNA = mitochondrial DNA.

Figure 3. Modeling ATP5A1 mutations in yeast

(A) Family pedigree and genotype at ATP5A1:c.962A>G. (B) Electron transport chain (ETC) biochemistry and mitochondrial DNA (mtDNA) quantitation in muscle. (C) Schematic of ATP5A1 protein, with sequence alignment shown in inset. Vertical bars indicate patient mutation (red) and yeast mitochondrial genome integrity (mgi) mutations (black). (D) Yeast atp1 deletion strains show normal (red) and petite (white) colonies. (E) Mitochondrial physiology of yeast deletion strains report mean values ± SD across 3 replicates. *p < 0.01, 1-tailed t test. ↓ = 20%–30% activity relative to citrate synthase;↓↓ = <20%; ATPase = adenosine triphosphatase; d. = deceased; na = not available; nd = not determined; P/O = phosphate/oxygen; RCR = respiratory control rate.

Figure 4. New molecular diagnoses and prioritized recessive genes

Comparison of MitoExome analysis from our (A) previous study and (B) current study, showing the fraction of patients with firm molecular diagnoses (dark blue) and autosomal recessive candidate genes (light blue). Comparison is restricted to the subset of patients who lacked molecular diagnosis before MitoExome sequencing and to the 1,034 genes analyzed in both studies (thereby excluding 212 differential diagnosis genes from the current study). MGH = Massachusetts General Hospital; mito. = mitochondrial; mtDNA = mitochondrial DNA.