Simultaneous MFN2 and GDAP1 mutations cause major mitochondrial defects in a patient with CMT

Mutations in the MFN2 gene are associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a dominant axonal CMT, whereas mutations in GDAP1 are associated with recessive demyelinating CMT (CMT4A), recessive axonal CMT (AR-CMT2), and dominant axonal CMT (CMT2K). Both proteins are involved in energy metabolism and dynamics of the mitochondrial network.1,–,3 We have previously reported that, in fibroblasts from patients with CMT, MFN2 mutations resulted in a mitochondrial energy coupling defect,4,5 whereas dominant mutation in GDAP1 resulted in defective complex I activity.6

In this study, we investigated mitochondrial bioenergetics from a severely affected patient with CMT harboring combined mutations in both GDAP1 and MFN2 genes.

### Methods.

For details, see e-Methods on the Neurology ® Web site at www.neurology.org.

#### Patients.

Patient II-5 (figure 1A), a 71-year-old woman of Spanish origin, had severe distal muscle weakness from the age of 3, becoming wheelchair-bound during her third decade. Clinical examination showed severe weakness of limbs with proximal and distal amyotrophy, tactile and nociceptive hypoesthesia with a gloves-and-socks distribution, and abolition of the limb reflexes. She had pes cavus and moderate vocal cord paresis. Electrophysiologic studies (table e-1) indicated a severe axonal neuropathy characterized by a …

Mutations in the MFN2 gene are associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a dominant axonal CMT, whereas mutations in GDAP1 are associated with recessive demyelinating CMT (CMT4A), recessive axonal CMT (AR-CMT2), and dominant axonal CMT (CMT2K). Both proteins are involved in energy metabolism and dynamics of the mitochondrial network. [1][2][3] We have previously reported that, in fibroblasts from patients with CMT, MFN2 mutations resulted in a mitochondrial energy coupling defect, 4,5 whereas dominant mutation in GDAP1 resulted in defective complex I activity. 6 In this study, we investigated mitochondrial bioenergetics from a severely affected patient with CMT harboring combined mutations in both GDAP1 and MFN2 genes.

Methods.
For details, see e-Methods on the Neurology ® Web site at www.neurology.org.
Patients. Patient II-5 ( figure 1A), a 71-year-old woman of Spanish origin, had severe distal muscle weakness from the age of 3, becoming wheelchairbound during her third decade. Clinical examination showed severe weakness of limbs with proximal and distal amyotrophy, tactile and nociceptive hypoesthesia with a gloves-and-socks distribution, and abolition of the limb reflexes. She had pes cavus and moderate vocal cord paresis. Electrophysiologic studies (table e-1) indicated a severe axonal neuropathy characterized by a major reduction of motor action potential in the left median nerve (0.1 mV) with a slightly reduced motor conduction velocity (43 m/s).
Patient II-8, her 56-year-old brother, presented with a mild CMT2 clinical phenotype. Electrophysiologic examination showed a sensory axonal neuropathy (table e-1). His 2 daughters, aged 19 and 25 years, are currently asymptomatic. Patient II-2, who had a phenotype compatible with CMT, had died of respiratory failure.
Results. Mutation analysis. Patient II-8 and his asymptomatic daughter (III-15) were found to be heterozygous for the pathogenic p.R468H mutation in MFN2, previously described. 7 Individuals II-3, II-4, and II-7 were heterozygous for the p.Q163X mutation in GDAP1. Patient II-5 was found to be heterozygous for the p.R468H mutation in MFN2 and homozygous for the p.Q163X mutation in GDAP1 ( figure 1A).
Mitochondrial imaging and biochemistry. No alteration of the mitochondrial network was revealed in MFN2: p.R468H fibroblasts and in MFN2: In patient II-5 fibroblasts, a more severe energy coupling defect than in MFN2 patients was discovered with 85% reduction of the ATP/O ratio compared to controls (figure 1Ba). Mitochondrial uncoupling was associated with a 65% decrease of mitochondrial ATP production that was absent in fibroblasts with a single MFN2 mutation (figure 1Bb). Similarly to patients with dominant mutation in GDAP1, fibroblasts carrying mutations in both MFN2 and GDAP1 showed a 40% reduction of complex I activity compared with MFN2 patients and controls (see malate pyruvate complex I substrates value on figure 1Bc, and enzymatic complex measurements on figure 1Bd).

Discussion.
We report a patient with CMT carrying simultaneous mutations in GDAP1 and MFN2. Initially, this patient was found to be heterozygous for the p.R468H mutation in MFN2. Individuals carrying this mutation usually have mild CMT phenotype or are asymptomatic. 7 Hence the sole presence of this mutation could not be responsible for the severe clinical phenotype, 7 suggesting the need for further genetic analysis, which revealed the existence of the p.Q163X homozygous mutation in GDAP1 as an explanation of the clinical severity.
We have previously reported that MFN2 mutations resulted in an energy coupling defect with normal mitochondrial ATP production, 4,5 whereas dominant mutation in GDAP1 resulted in defective complex I activity with a decrease in ATP production when complex I substrates (malate and pyruvate) were used. 6 A compensatory mechanism was postulated since the production of ATP was normal when succinate, the complex II substrate, was used (figure 1Bb). In a patient carrying mutations in GDAP1 and MFN2, we showed defective complex I activity as well as severe mitochondrial uncoupling. Interest-Supplemental data at www.neurology.org ingly, the reduction of complex I activity was similar to that of fibroblasts with the dominant GDAP1 mutation. In addition, the ATP production with complex I and complex II substrates was significantly reduced compared to fibroblasts harboring either the MFN2 or GDAP1 mutations alone (figure 1Bb). These findings underscore the role of GDAP1 in the function of respiratory complex I and suggest that this combination of mutations has biochemical deleterious synergistic effects. In MFN2 patients, the mitochondrial uncoupling was associated with a higher respiratory rate involving complex II, serving as a compensatory mechanism (MPS value, figure 1Bc). 5 However, in MFN2-GDAP1 double mutant fibroblasts, the complex I defect may have limited such compensation, leading to a decrease in ATP production.
Finally, our study suggests that the clinical heterogeneity of CMT may be related to the simultaneous presence of mutant alleles in different CMT genes, emphasizing the need for extensive genetic investigation to provide accurate diagnosis.  Genetic Diagnosis Center of Inherited Disease-IDIBELL (C.C.,