Mitochondrial single-stranded DNA binding protein novel de novo SSBP1 mutation in a child with single large-scale mtDNA deletion (SLSMD) clinically manifesting as Pearson, Kearns-Sayre, and Leigh syndromes

Abstract

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband’s fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1 . Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication.

Mammalian mitochondrial DNA replication and mechanisms of deletion formation

“…Interestingly, disease-causing mutations have recently been identified in the gene coding for mtSSB. The mutant proteins bind ssDNA less efficiently and mtDNA is depleted in affected individuals (Gustafson et al 2019;Piro-Megy et al 2019;Del Dotto et al 2020) An indepth analysis of replication intermediates in patientderived cell lines may provide important insights about possible priming outside the OriL region.…”

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging

“…Pathogenic variants of at least 24 nuclear genes whose protein products are responsible for mtDNA maintenance co-segregate with mitochondrial disease [7][8][9][10]. Two such genes (POLG and POLG2, respectively) encode the catalytic and accessory subunits of DNA polymerase γ (Pol γ).…”

Mitochondrial diseases: expanding the diagnosis in the era of genetic testing

“…SSBP1 is required to stabilize single-stranded mtDNA and stimulates DNA synthesis by POLG. As described above, dominant pathological variants in SSBP1 have recently been shown to induce optic atrophy, hearing loss, and foveopathy with mtDNA depletion, and recessive variants express features of Pearson, Kearns-Sayre, and Leigh syndrome [105,116,129] .…”

“…The exact mechanisms of producing single large deletions during development remain unclear. Recently, an inherited autosomal recessive variant in the mitochondrial single-stranded DNA-binding protein 1 (SSBP1) has been shown to produce a single large mtDNA deletion [105] . Duplications of mtDNA have not, to date, been reported to cause disease with the exception of a single case report [106] .…”

“…However, other than the presence or absence of peripheral neuropathy, the clinical phenotype of CPEO is similar between single and multiple deletions in mtDNA [113] . There has recently been a patient who has a single large 5-kb mtDNA deletion caused by a nuclear-encoded gene involved in mitochondrial DNA replication, SSBP1 [105] . This gene's product binds and protects single-stranded DNA during mtDNA replication.…”