Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease

Abstract

Objective: Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) segregation and is associated with multiple mtDNA deletions. We aimed to determine the prevalence of RNase H1 gene (RNASEH1) mutations among patients with mitochondrial disease and establish clinically meaningful genotype-phenotype correlations.

Methods: RNASEH1 was analyzed in patients with (1) multiple deletions/depletion of muscle mtDNA and (2) mendelian progressive external ophthalmoplegia (PEO) with neuropathologic evidence of mitochondrial dysfunction, but no detectable multiple deletions/depletion of muscle mtDNA. Clinicopathologic and molecular evaluation of the newly identified and previously reported patients harboring RNASEH1 mutations was subsequently undertaken.

Results: Pathogenic c.424G>A p.Val142Ile RNASEH1 mutations were detected in 3 pedigrees among the 74 probands screened. Given that all 3 families had Indian ancestry, RNASEH1 genetic analysis was undertaken in 50 additional Indian probands with variable clinical presentations associated with multiple mtDNA deletions, but no further RNASEH1 mutations were confirmed. RNASEH1-related mitochondrial disease was characterized by PEO (100%), cerebellar ataxia (57%), and dysphagia (50%). The ataxia neuropathy spectrum phenotype was observed in 1 patient. Although the c.424G>A p.Val142Ile mutation underpins all reported RNASEH1-related mitochondrial disease, haplotype analysis suggested an independent origin, rather than a founder event, for the variant in our families.

Conclusions: In our cohort, RNASEH1 mutations represent the fourth most common cause of adult mendelian PEO associated with multiple mtDNA deletions, following mutations in POLG, RRM2B, and TWNK. RNASEH1 genetic analysis should also be considered in all patients with POLG-negative ataxia neuropathy spectrum. The pathophysiologic mechanisms by which the c.424G>A p.Val142Ile mutation impairs human RNase H1 warrant further investigation.

Figure 1. Pedigrees and histopathologic findings of patients harboring RNASEH1 c.424G>A p.Val142Ile mutations

(A) Pedigrees of families harboring RNASEH1 mutations. Filled blue symbols represent affected individuals. Arrows indicate probands. Long range PCR (LPCR) and Southern blot (SB) demonstrate multiple deletions of muscle mitochondrial DNA (B-II.8) in patient (P) when compared with control muscle (C). (B) Muscle biopsy histology (A-III.8) demonstrating ragged red fibers (modified Gomori trichrome, GT), ragged blue fibers (succinate dehydrogenase, SDH), and several muscle fibers deficient in cytochrome c oxidase (COX), arrows. Scale bar represents 50 μm in GT and 200 μm in SDH and COX. (C) Ultrastructural examination (A-III.8) showing increased numbers of mitochondria, many of which are structurally abnormal, including the presence of paracrystalline inclusions, arrows. Scale bar represents 1 μm.

Figure 2. Principal component and haplotype analysis of families harboring c.424G>A p.Val142Ile variant and schematic illustrating the newly identified and previously reported RNASEH1 mutations to date

(A) Principal component analysis (top panel). X-axis represents component 1; Y-axis represents component 2. Families A and B cluster to the same ethnic group. Haplotype analysis of individuals harboring the RNASEH1 mutation c.424G>A p.Val142Ile (bottom panel). “1” indicates the presence of the marker, “0” indicates the absence of the marker, and “0/1” is used when haplotype could not be phased. Haplotypes reported are for A-III.8, B-II.8, C-II.1, S-1, and S-3. Green = c.424G>A mutation; gray = markers differing from reference haplotype. Families A and B shared a haplotype of at least 3.57 Mb. However, 2 additional haplotypes were different, suggesting distant recombination events or that the haplotypes arose independently. (B) The RNASEH1 gene has 8 exons encoding 4 protein domains as follows: mitochondrial targeting sequencing domain (M), hybrid-binding domain (HBD), connection domain (CD), and ribonuclease H domain (H-domain). The latter conducts all catalytic activity. Both herein newly identified and previously reported mutations are illustrated in the schematic. To date, all affected individuals harbor the “common” c.424G>A p.Val142Ile mutation either in homozygous or heterozygous states. Brackets = number of families with each mutation. Red = newly reported variant.