e-Methods

Cell lines and mutation analyses

Blood samples were collected from nine members of the family after informed consent was obtained. DNA was PCR-amplified (primer sequences available on request) to screen the complete sequence of GDAP1, P0, PMP22, EGR2,MFN2 and GJB1 genes. PCR products were sequenced using the ABI Prism BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).

Primary dermal fibroblasts were obtainedfrom skin biopsies taken after getting written consent frompatient II-5. Fibroblasts were maintained in DMEM/10% bovine calf serum (BCS) at 37°C in a humidified atmosphere with 5% CO2. All fibroblast cultures were mycoplasma-free, as shown by the DAPI/Hoechst in situ coloration and by PCR (Venor®Gem, BioValley, Marne-la-Vallée, France). All experiments were carried out on cells with similar passage numbers, ranging from 5 to 15, so as to avoid artefacts due to senescence, i.e. at passage numbers >30.

Deconvolution microscopy and mitochondrial network analysis

Mitochondria were labelled using Mitotracker® Green (Molecular Probes, CA, USA).Fibroblasts were pulse-loaded with 100nM Mitotracker® Green for 15 min in a humidified atmosphere(95% air, 5% CO2) at 37°C. After loading, cells were thoroughly washed with DMEM without red phenol, 1% FCS.For imaging, coverslips were placed on the stage of an inverted microscope.Images were acquired with a inverted wide-field Leica (DMI6000B, Microsystems, Wetzlar, Germany) equipped with a Roper CoolSnap HQ2 camera (Roper Scientific, Tucson, AZ, USA) a high-sensitivity CCD camera for quantitative fluorescence microscopy. A precision, piezoelectric driver mounted underneath the objective lens allows faster Z-step movements, keeping the sample immobile while shifting the objective lens. An average of 30 planes images were acquired along the Z-axis at 0.2 µm increments. Metamorph® 7.6 software (Molecular Devices, Sunnyvale, CA, USA) were used for image acquisition. We use point-spread function (PSF)-Speck microspheres for testing the optical stability of our system. Image restoration algorithms can be used to reassign the optical blur to its original location (Huygens software, Scientific Volume Imaging, Hilversum, The Netherlands). Each successive iteration leads to a new estimation closer to the real object. At the end of the iterative process, the blur is reduced and the signal considerably increased in the regions of the structures of interest. Imaris 6.3® software (Bitplane, Zurich, Switzerland) for 3D processing and morphometric analysis.

Mitochondrial biochemistry

Enzymatic Activities. Mitochondrial respiratory chain complex activities were measured on cell homogenates using a Beckman DU 640 spectrophotometer (Beckman coulter, Fullerton, CA, USA) as described elsewhere.4

Efficiency of Mitochondrial ATP Production in Permeabilized Cells. The rate of mitochondrial ATP synthesis and the ATP/O ratio were determined in cells permeabilized by digitonin exposure as previously described.4 Cells were resuspended in the respiratory buffer (10 mM KH2PO4, 300 mM mannitol, 10 mM KCl and 5 mM MgCl2, pH 7.4) supplemented with 2 mM iodoacetate and 2 mM EDTA, so as to prevent glycolytic ATP synthesis and ATP hydrolysis by cellular ATPases. The respiratory rates of 3-5*106 cells were recorded at 37°C in 1.5 ml glass chambers using a two-channel, high-resolution Oxygraph respirometer (Oroboros, Innsbruck, Austria). ATP synthesis was started by addition of 5 mM malate and 5 mM pyruvate and the subsequent addition of 1.5 mM ADP. Four aliquots were sampled each minute and quenched with an equal volume of 1% TCA solution and neutralized by adding a 25 mM HEPES, 2 mM EDTA, pH 7.75 buffer. The ATP synthesized in situ was measured using the Enliten ATP assay (Promega, Madison, WI, USA). Luminescence was measured on a multidetection reader for microplates Xenius XML (SAFAS, Monaco) using a 10-second integration period. Standardization was performed with known quantities of ATP measured under the same conditions.

The results obtained in patient II-5 were compared with those obtained from seven normal controls, from three patients heterozygous for the p.C240Y mutation in GDAP1andfrom ten patients harbouring seven different MFN2mutations (p.M21V, p.R94Q, p.T105M, p.A166T, p.R364Q, p.E744A and p.R468H). The biochemical findings obtained for the patient II-8 carrying the p.R468Hmutation were previously published.7

Statistical analysis

Mann-Whitney tests were used for statistical comparisons with differences considered significant at p < 0.05.

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