A Novel in-Frame 18-bp Microdeletion inMT-CYBCauses a Multisystem Disorder with Prominent Exercise Intolerance

Abstract

A novel heteroplasmic mitochondrial DNA (mtDNA) microdeletion affecting the cytochrome b gene (MT-CYB) was identified in an Italian female patient with a multisystem disease characterized by sensorineural deafness, cataracts, retinal pigmentary dystrophy, dysphagia, postural and gait instability, and myopathy with prominent exercise intolerance. The deletion is 18-base pair long and encompasses nucleotide positions 15,649-15,666, causing the loss of six amino acids (Ile-Leu-Ala-Met-Ile-Pro) in the protein, but leaving the remaining of the MT-CYB sequence in frame. The defective complex III function was cotransferred with mutant mtDNA in cybrids, thus unequivocally establishing its pathogenic role. Maternal relatives failed to show detectable levels of the deletion in blood and urinary epithelium, suggesting a de novo mutational event. This is the second report of an in-frame intragenic deletion in MT-CYB, which most likely occurred in early stages of embryonic development, associated with a severe multisystem disorder with prominent exercise intolerance.

Complex II phosphorylation is triggered by unbalanced redox homeostasis in cells lacking complex III

“…The CIII enzymatic activity was almost completely absent in the homoplasmic MTCYB microdeletion clone, and only a partial activity was present in the 80% heteroplasmic clone (Fig. 1A), in agreement with our previous report [12]. In parallel, we also examined cybrids bearing the pathogenic missense MTCYB p.278Y > C substitution, previously shown by us to cause the almost complete loss of CIII activity, without any significant perturbation of its assembly [13].…”

“…SQR activity was determined in isolated mitochondria by recording spectrophotometrically the reduction of DCIP in the presence of succinate as an electron donor and DB as an electron acceptor. We have previously reported a significant stimulation of SQR activity in human cybrids bearing the 18-bp MTCYB ΔI300-P305 homoplasmic MTCYB micro-deletion [12]. We first asked whether this CII activity stimulation was strictly dependent on the lack of CIII, so we utilized another cybrid clone developed in our lab, bearing the very same MTCYB ΔI300-P305 microdeletion in heteroplasmic conditions, i.e.…”

“…We first asked whether this CII activity stimulation was strictly dependent on the lack of CIII, so we utilized another cybrid clone developed in our lab, bearing the very same MTCYB ΔI300-P305 microdeletion in heteroplasmic conditions, i.e. 80% mutant MTCYB [12] (Fig. 1).…”

“…In this study cybrids bearing the m.15649–15,666 microdeletion (I300-P305) in heteroplasmy or homoplasmy (80% or 100% mutation load, respectively) or bearing isogenic wild type (WT) mtDNA [12] and cybrids with the homoplasmic missense mutation m.15579A > G (p.278Y > C) and isogenic WT mtDNA [13] were used. Cells were grown in high glucose (25 mM) Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FBS) (Gibco, Invitrogen, Italy), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mM sodium-pyruvate, in an incubator with a humidified atmosphere of 5% CO 2 at 37 °C.…”

“…This 18-base pair long, in-frame microdeletion was identified in a patient suffering a severe encephalomyopathy, and leads to the absence of six amino acid (I300-P305) residues in the sixth trans-membrane helix of cytochrome b . Cybrids bearing this MTCYB micro-deletion in homoplasmic state showed an almost complete loss of CIII activity that was associated with a marked increase of CII activity, which is the only respiratory complex fully encoded by nuclear DNA [12]. We inquired whether the stimulation of CII activity in vitro was strictly dependent on CIII dysfunction, and if so, whether it required a severe loss of CIII activity only or also the disruption of CIII assembly.…”