A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice

Mitochondrial respiratory chain complex III (cytochrome bc1 complex, CIII) oxidizes coenzyme Q, reduces cytochrome c, and translocates protons to generate membrane potential for ATP synthesis. The mitochondrial inner membrane AAA-family translocase BCS1L, frequently mutated in CIII deficiency is required for the topogenesis of the electron-transferring RISP (Rieske iron-sulphur protein, UQCRFS1) subunit and its assembly into CIII. Homozygous Bcs1lc.A232G (Bcs1lp.S78G) knock-in mice, carrying a GRACILE syndrome patient mutation, recapitulate many manifestations of human CIII deficiency. They display post-weaning growth failure, hepatopathy, renal tubulopathy, and, in a C57BL/6JBomTac-derived genetic background (Lund colony), deterioration due to metabolic crisis with extreme hypoglycemia by 35 days of age.

When the mutant strain was brought to another facility via embryo transfer and bred it with the closely related C57BL/6JCrl background (Helsinki colony), the homozygotes developed similar early visceral and systemic manifestations but, unexpectedly, survived the early metabolic crisis and lived fivefold longer, to up to 200 days. Suspecting genetic drift in the isolated Lund colony, it was hypothesized that (a) homozygous genetic change(s) underlie the highly consistent survival difference between the two colonies. Here a whole-genome sequencing (WGS) was performed to identify candidate variants, followed by a simple genetic experiment to show that a spontaneous mitochondrial DNA (mtDNA) variant underlies the survival difference. Mouse phenotyping, computational, and spectroscopic data was combined to show how the effects of this non-pathogenic variant and the disease-causing Bcs1l mutation converge to exacerbate CIII deficiency and disease progression.

Short-lived Bcs1l p.S78G mice carry a novel mtDNA variant WGS (n = 3 for Lund C57BL/6JBomTac and n = 2 for Helsinki C57BL/6JCrl) revealed 844 homozygous single-nucleotide polymorphisms and 3655 small insertion/deletions between the strains, only 8 of which were in coding regions of genes. One of these was an mtDNA variant (m.G14904A) not present in any Mus musculus sequence in GenBank. Genotyping of approximately 80 mice throughout past generations using archived genomic DNA from ear biopsies revealed that the variant was introduced from wild-type (WT) C57BL/6JBomTac females repeatedly after 2008, when congenization of the Bcs1lc.A232G knock-in allele was started. Inspection of the pedigrees of the mt-Cyb-genotyped mice showed that two early-generation females had given birth to both WT and variant-carrying progeny, suggesting initial heteroplasmy. However, sequencing of 346 bacterial clones of mt-Cyb PCR amplicon from the liver, kidney, heart, and skeletal muscle DNA showed no sign of heteroplasmy in somatic tissues in later generations. The fact that the variant was homoplasmic in an apparently healthy WT mouse colony suggested that it must be non-pathogenic. Indeed, analysis of mtDNA sequences deposited to GenBank showed that the three-toed sloth species (Bradypus) of South America, known for their very low metabolic rate16,17 naturally carry this variant. Intriguingly, the variant affects the mtDNA-encoded CIII subunit cytochrome b, changing a conserved aspartate 254 to asparagine (mt-Cybp.D254N). A negatively charged amino acid in this position is highly conserved across eukaryotes and aerobic prokaryotes, with limited conservancy in archaea. Therefore, as mt-Cybp.D254N potentially directly affects CIII function, it appeared as a most likely genetic modifier of the survival of Bcs1lp.S78G mice.

Original article:

Purhonen J, Grigorjev V, Ekiert R, Aho N, Rajendran J, Pietras R, Truvé K, Wikström M, Sharma V, Osyczka A, Fellman V, Kallijärvi J.  A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice. Nat Commun 11: 322, 2020 (doi: 10.1038/s41467-019-14201-2).