Subjects
This study enrolled two autosomal recessive Korean families with axonal sensorimotor polyneuropathy (FC26 and FC355, Fig. 1a). After careful clinical and electrophysiological examinations, 300 healthy controls were recruited from the neurological department. All participants provided written, informed consents according to the protocol approved by the Institutional Review Board for Ewha Womans University, Mokdong Hospital (ECT 11-58-37). In addition, the patients provided written, informed consent for the publication of individual clinical details, and for the publication of family trees.
Clinical assessments
Two independent neurologists evaluated each patient, and collected clinical information including assessments of motor and sensory impairments, deep tendon reflexes, and muscle atrophy. Muscle strength of flexor and extensor muscles were assessed manually using the standard medical research council (MRC) scale. In order to detect any physical disability we used a nine-point functional disability scale (FDS) [12], which was based on the following criteria: 0: normal; 1: normal but with cramps and fatigability; 2: an inability to run; 3: walking difficulty but still possible unaided; 4: walking with a cane; 5: walking with crutches; 6: walking with a walker; 7: wheelchair bound; and 8: bedridden. Sensory impairments were assessed for the level and severity of pain, temperature, vibration and position. Age at onset was determined by asking patients for their ages, when symptoms first appeared.
Electrophysiological study
Motor and sensory conduction velocities of median, ulnar, peroneal, tibial, and sural nerves were determined. Motor conduction velocities (MCVs) of the median and ulnar nerves were determined by stimulating at the elbow and wrist, while recording compound muscle action potentials (CMAPs) over the abductor pollicis brevis and adductor digiti quinti, respectively. In the same way, the MCVs of peroneal and tibial nerves were determined by stimulating at the knee and ankle, while recording CMAPs over the extensor digitorum brevis and adductor hallucis, respectively. Sensory conduction velocities (SCVs) were obtained over a finger-wrist segment from the median and ulnar nerves by orthodromic scoring, and were also recorded for sural nerves. Sensory nerve action potential (SNAP) amplitudes were measured from positive peaks to negative peaks.
Sural nerve biopsy
Distal sural nerve was biopsied from patient 1 (Fig. 1a, II-1) at 34 years, and pathological examination included light and electron microscopic analyses. Formalin-fixed sections were stained with hematoxylin and eosin (H&E), modified Masson’s trichrome, and Luxol fast blue. For electron microscopic study, the specimen was fixed in 2 % glutaraldehyde in 25 mM cacodylate buffer. Semithin sections were stained with toluidine blue and ultra-thin cut samples were contrasted with uranyl acetate and lead citrate.
Vastus lateralis muscle biopsy
Cross-sections of the biopsy of the vastus lateralis muscle from patient two (Fig. 1b, III-2) at 22 years were stained with H&E, modified Gomori-trichrome, NADH-tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), periodic acid Schiff (PAS), Oil-red-O and adaenosine triphosphatase reaction, and immunostained for myosin heavy chain (Vision Biosystems, Newcastle, UK). Samples were also examined using electron microscopy.
MR images of brain, hip, thigh and lower leg
Brain, hip, thigh and lower leg of both patients were evaluated using a 1.5-T system (Siemens Vision; Siemens, Erlangen, Germany). Whole brains were scanned using a slice thickness of 7 mm and 2-mm interslice gap, to produce 16 axial images. The imaging protocol consisted of T2-weighted spin echo (SE) (TR/TE = 4,700/120 ms), T1-weighted SE (TR/TE = 550/12 ms), and fluid-attenuated inversion recovery (FLAIR) (TR/TE = 9,000/119 ms, inversion time 2,609 ms) images. Images of the hip, thigh and lower leg were obtained in axial [field of view (FOV) 24–32 cm, slice thickness 6 mm, and slice gap 0.5–1.0 mm] and coronal planes (FOV 38–40 cm, slice thickness 4–5 mm, slice gap 0.5–1.0 mm). xThe following protocol was used: T1-weighted SE (TR/TE 570–650/14–20, 512 matrices), T2-weighted SE (TR/TE 2800–4000/96–99, 512 matrices), and fat-suppressed T2-weighted SE (TR/TE 3090–4900/85–99, 512 matrices).
Exome sequencing and filtering
Whole exome sequencing (WES) was performed for six samples (three from each family), according to a previous study [13]. Briefly, WES was performed using the Human SeqCap EZ Human Exome Library v3.0 (Roche/NimbleGen, Madison, WI, USA), and the HiSeq 2000 Genome Analyzer (Illumina, San Diego, CA, USA). The UCSC assembly hg19 was used as the reference sequence and variant calling was achieved in cases with >20 single nucleotide polymorphisms (SNP). We collected functionally significant variants (missense, nonsense, exonic indel and splicing site variants) from about 70 peripheral neuropathy genes and 15 mitochondrial DNA depletion syndrome (MTDPS)-related genes, and then variants agreeing with autosomal recessive inheritance were selected. Causative variants were confirmed by the Sanger’s sequencing method, and conservation analysis of mutation sites was performed using the MEGA5 program, ver 5.05 (http://www.megasoftware.net/). In silico analyses were performed using the prediction algorithms SIFT (http://sift.jcvi.org) and MUpro (http://www.ics.uci.edu/~baldig/mutation).
Construction of wild-type and mutant MPV17
To obtain the MPV17 transcript, cDNA was synthesized using Superscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) from total mRNA of HEK293. Then polymerase chain reaction (PCR) was performed using the cDNA as a template. The amplified PCR product was cloned into the expression vector, pCMV-myc (Clontech, Mountain View, CA, USA). Mutant MPV17 transcript were generated by QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). All primers’ sequences are listed in Additional file 1: Table S1.
Transfection and knockdown of MPV17
NSC34 cells were cultured in a 10 % FBS, 1 % PS and high glucose Dulbecco’s modified eagle medium (DMEM; Biowest, Nuaille, France). To express MPV17 transcript in the motor neuron, NSC34 cells were transfected with MPV17 DNA-containing vectors using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s recommendation. Knockdown of MPV17 was performed using MPV17-specific siRNA and Lipofectamine 2000 reagent (Invitrogen) (Additional file 1: Table S1). Cells were harvested after overexpression and knockdown of MPV17 at 24 and 72 h.
Measurement of proliferation and cell viability
After 3 days of knockdown, NSC34 cells were transferred to 24-well plates. Then, the proliferation of the cells was determined by direct counting under a microscope at 24 h intervals. For the overexpression model, NSC34 cells, cultured on 24-well plates, were transfected with wild-type or mutant MPV17. Cells were counted at 24 h intervals. Sensitivity to H2O2 was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells treated with H2O2 were incubated with 10 mM MTT solution for 2 h, then the cells were lysed with dimethyl sulfoxide. Relative numbers of viable cells were determined using absorbance at 560 nm.
Western blotting
Protein synthesis in NSC34 cells was determined using standard Western blotting with anti-myc Ab (Abcam, Cambridge, UK), anti-actin Ab, anti-mouse secondary Ab, and anti-rabbit secondary Ab (Sigma, St. Louis, MO, USA). An OXPHOS detection cocktail (Abcam) was used based on standard Western blotting. ECL plus Western blotting substrate (Thermo Scientific, Rockford, IL, USA) were used for detection of proteins.