Adult Leigh Syndrome Associated with the m.15635T>C Mitochondrial DNA Variant Affecting the Cytochrome b (MT-CYB) Gene

Abstract

We report on a sporadic patient suffering Leigh syndrome characterized by bilateral lesions in the lenticular nuclei and spastic dystonia, intellectual disability, sensorineural deafness, hypertrophic cardiomyopathy, exercise intolerance, and retinitis pigmentosa. Complete sequencing of mitochondrial DNA revealed the heteroplasmic nucleotide change m.15635T>C affecting a highly conserved amino acid position (p.Ser297Pro) in the cytochrome b (MT-CYB) gene on a haplogroup K1c1a background, which includes a set of four non-synonymous polymorphisms also present in the same gene. Biochemical studies documented respiratory chain impairment due to complex III defect. This variant fulfils the criteria for being pathogenic and was previously reported in a sporadic case of fatal neonatal polyvisceral failure.

Keywords: Leigh syndrome; MT-CYB; Respiratory complex III; mtDNA.

Figure 1

Pedigree and genetic analysis: (A) Pedigree of the family. Asterisks indicate the DNA available. (B) Heteroplasmy evaluation of the m.15635C>T variant, by NGS approach, in all tissues and family members available. (C) Graphical timeline showing the evolution of the symptoms as they appeared throughout the patient’s life.

Figure 2

Clinical data of the proband: (A) Optical coherence tomography (OCT). The fundus photography images of the patient’s eyes display a moderate swelling of the optic disc fibers (asterisks) without cup and the presence of peripapillary microangiopathy (arrows) (top panel); structural OCT performed in the macular area confirmed a thinning of the GC-IPL in the central area around the fovea (bottom panel); GC-IPL: ganglion cell inner plexiform layers. (B,C) Muscle biopsy from the proband: histoenzymatic staining for both COX and SDH activities revealed increased subsarcolemmal staining (arrows), indicative of a moderate increase in mitochondrial mass, which is usually a compensatory feature. No COX-deficient or ragged red fibers were observed. (D–G) Brain MRI: bilateral striatal necrosis (arrows) (D,E), severe cerebellar atrophy and mild brainstem atrophy with consensual 4th ventricle dilation are evident (asterisk) (F,G).

Figure 3

Biochemical studies on cybrids: (A) Time course of cell viability in glucose-free medium supplemented with galactose. Two syngeneic cybrid clones, 100% wild-type and bearing the homoplasmic m.15635T>C (p.297S>P) variant, were used. The SRB absorbance value at time zero corresponds to 100% viable cells. Each data point represents the mean ± SD of at least three experiments. Statistical analysis was performed by t-test; ** p < 0.01. (B) Respiratory complex III and citrate synthase activities were assessed as described in [21]. Data are the means ± SD of at least three experiments. Statistical analysis was performed by t-test; * p < 0.05. (C) The rate of ATP synthesis driven by CI (malate/pyruvate), CII (succinate), CIII (DBH₂), and glycerol-3-phosphate dehydrogenase (G3P) was measured as previously described in [21]. Data are the means ± SD of at least four experiments. (D) SDS-PAGE and Western blot analyses of representative respiratory chain subunits from 40 μg of crude mitochondria were performed as previously reported [21]. Western blot analysis was carried out using the antibodies against the CI (NDUFA9, NDUFS3, and NDUFB8) subunits, the CII-SDHA subunit, the CIII-UQCRC2 subunit, the CIV-COX IV subunit, and the CV-α subunit. (E) Digitonin-solubilized (2 g/g protein) mitoplasts, isolated from WT and homoplasmic mutated cybrids, were separated by BN-PAGE. One gel was used for the CI In-Gel Activity (CI-IGA), as described in the Materials and Methods. The other gels were transferred to nitrocellulose membranes for Western blot analysis, using antibodies against the indicated subunits of OXPHOS complexes.

Figure 4

In silico analysis of human monomeric mutated cytochrome b in different haplogroup backgrounds: The highly conserved amino acid positions Pro297 and Tyr358 are indicated in red and orange, respectively. Different missense variants of haplogroup K1c (A) and haplogroup J2b (B) are indicated by the green and blue arrows. The two missense variants common to the two haplogroups in MT-CYB (Ile7 and Ala194) are highlighted in green, while the other variants carried specifically by each haplogroup are shown in blue.