Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases

Abstract

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.

Figure 1. Genetic analysis of family with early onset spastic ataxia-neuropathy syndrome.

(A) Pedigree of family. (B) Sequencing of the c.1847G>A; p.Y616C AFG3L2 mutation in family members. (C) Alignment of amino acid sequences from AFG3L2 of several vertebrate species indicates that Y616 and the flanking residues are highly conserved.

Figure 2. Neuroimaging of family members.

T1 sagittal magnetic resonance imaging of the brain of IV-2 (A) reveals notable cerebellar atrophy. His asymptomatic mother, III-2 (B), has mild cerebellar atrophy, while his asymptomatic father, III-1 (C) has normal imaging.

Figure 3. MrpL32 maturation and yeast complementation assays for the evaluation of AFG3L2^Y616C activity.

(A) Protein expression of AFG3L2 in Δyta10Δyta12 cells was analyzed by SDS-PAGE and immunoblotting using AFG3L2-specific antibodies. Maturation of MrpL32, a substrate of m-AAA proteases, was monitored in isolated mitochondria by immunoblotting using polyclonal antisera directed against MrpL32. The outer membrane protein Tom20 was used as a loading control. (B) Respiratory growth of Δyta10Δyta12 cells expressing human m-AAA protease subunits. To assess the functional activity of homo-oligomeric m-AAA proteases, AFG3L2^Y616C was expressed in Δyta10Δyta12 cells alone or co-expressed with AFG3L2, AFG3L2^E575Q (proteolytic site mutant) or AFG3L2^K354A (ATPase domain-Walker A motif mutant) where indicated. Cell growth was analyzed at 30°C on glucose- (YPD) or glycerol containing (YPG) media. (C) Protein expression and maturation of MrpL32 were monitored in Δyta10Δyta12 cells expressing the indicated variants of human m-AAA protease subunits as in (A). (D) To monitor the activity of hetero-oligomeric m-AAA complexes, AFG3L2^Y616C and AFG3L2^Y616C/E575Q were expressed with paraplegin (SPG7) or SPG7^K355A (Walker A motif mutant). Cell growth was analyzed at 30°C on glucose- (YPD) or glycerol-containing (YPG) media.

Figure 4. Assembly of AFG3L2, AFG3L2^Y616C, and AFG3L2^Y616C/E575Q in mitochondria.

(A) Mitochondrial extracts (100 µg protein) harbouring AFG3L2 (and variants thereof) and paraplegin (SPG7) as indicated were isolated from Δyta10Δyta12 cells and solubilized with digitonin (1% (w/v)) at a concentration of 5 mg/ml. Extracts were analyzed by BN-PAGE, transferred onto a PVDF membrane and stained with Coomassie blue G-250 (right panel). After destaining, the membrane was used for immunoblotting using AFG3L2- or SPG7-specific antibodies. The outer membrane protein Tom40 was used as a loading control. Thyroglobulin (669 kDa) and apoferritin (440 kDa) were used for size calibration. (B) Mitoplasts were prepared from primary fibroblasts of family members and solubilized with digitonin. Soluble and pellet fractions were analysed by SDS-PAGE using AFG3L2- and paraplegin-specific antibodies. No alteration of the steady-state levels of the two proteins was observed in the proband (IV.1). (C) To detect assembled m-AAA proteases in the patient cell line, the same preparations were analyzed by BN-PAGE using AFG3L2- or paraplegin-specific antibodies. HSP60 was used for calibration. WT: wild-type; ΔΔ: Δyta10Δyta12; CVmon: complex V monomer; CVdim: complex V dimer; CIIIdim: complex III dimer.