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Mitochondrial DNA (mtDNA) maintenance disorders embrace a broad range of clinical syndromes distinguished by the evidence of mtDNA depletion and/or deletions in affected tissues. Among the nuclear genes associated with mtDNA maintenance disorders, RNASEH1 mutations produce a homogeneous phenotype, with progressive external ophthalmoplegia (PEO), ptosis, limb weakness, cerebellar ataxia, and dysphagia. The encoded enzyme, ribonuclease H1, is involved in mtDNA replication, whose impairment leads to an increase in replication intermediates resulting from mtDNA replication slowdown. Here, we describe two unrelated Italian probands (Patient 1 and Patient 2) affected by chronic PEO, ptosis, and muscle weakness. Cerebellar features and severe dysphagia requiring enteral feeding were observed in one patient. In both cases, muscle biopsy revealed diffuse mitochondrial abnormalities and multiple mtDNA deletions. A targeted next-generation sequencing analysis revealed the homozygous RNASEH1 mutations c.129-3C>G and c.424G>A in patients 1 and 2, respectively. The c.129-3C>G substitution has never been described as disease-related and resulted in the loss of exon 2 in Patient 1 muscle RNASEH1 transcript. Overall, we recommend implementing the use of high-throughput sequencing approaches in the clinical setting to reach genetic diagnosis in case of suspected presentations with impaired mtDNA homeostasis. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) maintenance disorders embrace a broad range of clinical syndromes distinguished by the evidence of mtDNA depletion and/or deletions in affected tissues. Among the nuclear genes associated with mtDNA maintenance disorders, RNASEH1 mutations produce a homogeneous phenotype, with progressive external ophthalmoplegia (PEO), ptosis, limb weakness, cerebellar ataxia, and dysphagia. The encoded enzyme, ribonuclease H1, is involved in mtDNA replication, whose impairment leads to an increase in replication intermediates resulting from mtDNA replication slowdown. Here, we describe two unrelated Italian probands (Patient 1 and Patient 2) affected by chronic PEO, ptosis, and muscle weakness. Cerebellar features and severe dysphagia requiring enteral feeding were observed in one patient. In both cases, muscle biopsy revealed diffuse mitochondrial abnormalities and multiple mtDNA deletions. A targeted next-generation sequencing analysis revealed the homozygous RNASEH1 mutations c.129-3C>G and c.424G>A in patients 1 and 2, respectively. The c.129-3C>G substitution has never been described as disease-related and resulted in the loss of exon 2 in Patient 1 muscle RNASEH1 transcript. Overall, we recommend implementing the use of high-throughput sequencing approaches in the clinical setting to reach genetic diagnosis in case of suspected presentations with impaired mtDNA homeostasis.

Ribonuclease H1 is a conserved enzyme that cleaves the RNA strand of RNA· DNA heteroduplexes and has important functions in the nuclear and mitochondrial compartments. The therapeutic action of antisense oligodeoxynucleotides involves the recruitment of RNase H1 to cleave disease-relevant RNA targets. Recombinant human ( Hs) RNase H1 was purified from a bacterial expression host, and conditions were identified that provided optimal oligonucleotide-directed RNA cleavage in vitro. Hs- RNase H1 exhibits optimal catalytic activity in pH 7.5 HEPES buffer and a salt ( KCl) concentration of ~ 100-150 m m. Mg2+ best supports Hs- RNase H1 with an optimal concentration of 10 m m, but at higher concentrations inhibits enzyme activity. Mn2+ and Co2+ also support catalytic activity, while Ni2+ and Zn2+ exhibit only modest activities as cofactors. The optimized assay was used to show that an antisense oligonucleotide, added in substoichiometric amounts to initiate RNA cleavage, supports up to 30 rounds of reaction in 30 min. Mutation to alanine of the conserved histidine at position 264 causes an ~ 100-fold decrease in kcat under multiple-turnover conditions, but does not alter Km. Under single-turnover conditions, the H264 A mutant exhibits a 12-fold higher exponential time constant for substrate cleavage. The defective activity of the H264 A mutant is not rescued in either assay condition by higher Mg2+ concentrations. These data implicate the H264 side chain in phosphodiester hydrolysis as well as in product release, and are consistent with a proposed model in which the H264 side chain interacts with a divalent metal ion to support catalysis. [ABSTRACT FROM AUTHOR]