Mutations in the PLEKHG5 gene is relevant with autosomal recessive intermediate Charcot-Marie-Tooth disease

DNA was purified from blood using a QIAamp blood DNA purification kit (Qiagen, Hilden, Germany). Patient samples were prescreened for 17p12 duplication, which is a major genetic cause of demyelinating CMT, by using hexaplex microsatellite PCR [21]. Sequencing was also determined for the coding exons of major CMT-relevant genes, including MPZ, PMP22, GJB1, and MFN2.

Exome sequencing was performed for the proband of FC307 family according to Lee et al. [22]. Functionally significant variants (missense, nonsense, exonicindel and splicing site variants) were first selected from WES data, and then novel or uncommon variants (MAF ≤0.01) registered in the dbSNP137 [23] and 1000 Genomes database [24] were chosen. Homozygous or compound heterozygous mutations were finally selected. Candidate variants were confirmed by Sanger’s sequencing method using an ABI3130XL automatic genetic analyzer (Applied Biosystems, Foster City, CA). The underlying cause was determined by the presence of mutation only in the patient within the family and the absence in 300 controls. Conservation analysis of protein sequences was performed using MEGA5 version 5.05 software [25].

The proband (Figure 1A, III-1), her parents (Figure 1A, II-3, and −7), and grandparents (Figure 1A, I-1, -2, -3, and −4) were examined for motor and sensory impairments, deep tendon reflexes, and muscle atrophy. The strength of flexor and extensor muscles was assessed manually using the medical research council (MRC) scale. Physical disabilities were assessed using a CMT neuropathy score (CMTNS) [26]. Sensory impairments were assessed in terms of the level and severity of pain, temperature, vibration, and position. Age at onset was determined by asking patients for the age when symptoms, i.e., distal muscle weakness, foot deformity, or sensory change, first appeared.

Nerve conduction studies were performed with a surface electrode. Motor nerve conduction velocities (MNCVs) of the median and ulnar nerves were determined by providing stimulation 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 manner, the MNCVs and CMAPs of the peroneal and tibial nerves were determined. Sensory nerve conduction velocities (SNCVs) and action potentials (SNAPs) were obtained from the median, ulnar, and sural nerves. Needle electromyography (EMG) was performed for bilateral proximal and distal limb muscles. The patient and her parents underwent tests for visually evoked potentials and brainstem auditory evoked potentials.

The proband was studied by examining MRIs of the brain, hip, thigh, and lower leg, using a 1.5-T system (Siemens, Erlangen, Germany). Lower limb imaging was 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). The following protocol was used: T1-weighted spin-echo (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).

The distal sural nerve of patient (III-1) was biopsied at 19 years of age. The density of myelinated fibers (MFs), axonal diameter, and myelin thickness were determined from the semi-thin transverse sections using a computer-assisted image analyzer (AnalySIS; Soft Imaging System, Münster, Germany). Ultrathin cut samples (60–65 nm) were contrasted with uranyl acetate and lead citrate for electron microscopy (H-7650, Hitachi, Japan). An immunohistochemical study was performed with anti-PLEKHG5 antibody (N-15: sc-130100, 1:50 dilution, Santa Cruz Biotech, Santa Cruz, CA) and the findings were compared with a control case (female, 39 years old).

PLEKHG5 (BC015231), which is fused with a c-myc epitope, was obtained from Capital Biosciences (Rockville, MD). Site-directed mutagenesis was performed to generate mutant PLEKHG5 (p.Thr663Met and p.Gly820Arg) using a QuickChange Site-directed mutagenesis kit (Stratagene, La Jolla, CA). DNA transfections into HEK293 cells were performed using each DNA and Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Protein expression was analyzed by immunocytochemistry using anti-Myc antibody (Abcam, Cambridge, UK) after transfection of each vector. Reporter assay for NF-κB was achieved using a reporter vector, pNF-κB-Luc-reporter (from Dr. Kim HT, Sungkyunkwan University, Korea), and a luciferase assay system (Promega, Madison, WI).

To identify the disease-associated genetic defect of our patient, we performed exome sequencing of the affected individual (Table 1). We identified novel compound heterozygous mutations, c.1988C > T (p.Thr663Met, paternal origin) and c.2458G > C (p.Gly820Arg, maternal origin) in the PLEKHG5 gene in a Korean CMT family (family ID: FC307) (Figure 1A and 1B). The mutations of her parents were inherited from their mothers, respectively (Figure 1A). Neither mutation has been reported in dbSNP137 or the 1000 Genomes database. In the 300 Korean controls, the p.Thr663Met mutation was not found, while the p.Gly820Arg was found in three control samples. Both mutation sites were well conserved across species (Figure 1C). In silico analysis using PolyPhen 2 software [27] predicted that both mutations may affect protein function with scores of 0.993 (p.Thr663Met) and 1.000 (p.Gly820Arg). In addition to the compound heterozygous mutations in PLEKHG5, several other variants were identified in the more than 50 CMT-associated genes (Table 2). However, no variant was considered to be causative, because all the variants were polymorphic; moreover, they did not fit the recessive inheritance characteristic.

The clinical manifestations of the present patient and the previously described patients with PLEKHG5 mutations are shown in Table 3. The proband, a 19-year-old woman, was the first child of healthy nonconsanguineous Korean parents. On neurological examination and after electrophysiological studies, the parents did not show any abnormalities. The proband was born at full term and the perinatal history was unremarkable. Early motor milestones were not delayed, and one year after her birth, she was able to walk. At age 8 years, she experienced frequent falling and noticed muscle weakness of the distal lower limbs. She started to walk with short leg braces at 16 years of age. Neurological examination at 19 years of age revealed muscle weakness and atrophy of bilateral distal muscles, predominantly at lower limbs rather than upper limbs. Bilateral pes cavus, steppage gait, and atrophic changes of intrinsic foot and calf muscles were noted, but scoliosis was not observed. Sensitivity to pinprick, touch, position, and vibration decreased. Vibration and position senses were more severely disturbed than pain and touch senses. Knee and ankle jerks were absent. No pyramidal or cerebellar signs were detected. CMTNS was 15. Elevated serum creatine kinase levels were revealed (246 μmol/L, reference value: <185 μmol/L).

Data from the nerve conduction study are summarized in Table 4. Median MNCVs ranged from 24.7 m/s to 29.3 m/s. The results also revealed prolonged motor latencies, and no motor responses were elicited by stimulation of peroneal and tibial nerves at 19 years of age. SNAPs on bilateral sural nerves were absent on all electrophysiological studies (at 14, 15, 16 and 19 years old). In the upper limb, SNCVs and SNAPs were decreased on bilateral median and ulnar nerves. Needle EMG showed a neurogenic pattern of muscle degeneration. Visually evoked potentials and brainstem auditory-evoked potentials were normal.

Brain and hip MRIs were normal. However, thigh and lower leg MRIs of the patient demonstrated hyperintense signal abnormalities. T1-weighted images showed severe muscle atrophy and fatty replacement in the lower leg muscles compared with the thigh, which is compatible with a hypothesis of length-dependent axonal degeneration (Figure 2A and 2B). At the thigh level, fatty replacement of the vastus lateralis and semimembranous muscles was observed, but more muscle remained than fat. Sartorius, gracilis, biceps femoris, and rectus femoris muscles contained some fatty streaks (Figure 2C). We could observe a distinct pattern of muscle involvement; lower leg MRI showed selective and predominant involvement of anterior and lateral compartment muscles; however, superficial and deep posterior compartment muscles revealed mild manifestations (Figure 2D and 2E).

Semi-thin transverse sections showed loss of large- and medium-sized MFs, with small MFs remaining (Figure 3A). The number of MFs was reduced (297/mm²) compared with a control (20-year-old female, 9,800/mm²) (Figure 3B). The range (1.2-4.6 μm) and average (2.4 μm) MF diameter were also reduced compared with the control (2–12.5 μm and 4.5 μm, respectively). The distribution pattern of MF diameter was unimodal and composed of 75% of <3 μm MFs. In addition, MF% area and g-ratio (axonal diameter/MF diameter) were also reduced. The MF% area in this case was 0.2% (normal distal sural nerve in 20-year-old female: 25.2%). The range and average of g-ratio (axonal diameter/MF diameter) were 0.38-0.73 and 0.56 ± 0.09, respectively (mean g-ratio at age 21–50 years old: 0.66). The g-ratio >0.7 (abnormally thin myelin sheath) consisted of 10% of MFs and g-ratio <0.4 (abnormally thick myelin sheath) consisted of 1.7%. Electron microscopic examination revealed that the remaining small MFs showed occasional focal folding of myelin with very rare evidence of regeneration (clusters of regenerating fibers) (Figure 3C and 3D). The unmyelinated axons showed suggestive clustering (reinnervated Büngner bands with unmyelinated fibers) and atrophy. Endoneurial fibroblast proliferation and large amounts of collagen deposition were well documented.

The immunohistochemical study with anti-PLEKHG5 antibody showed a focal positive reaction in Schwann cell nuclei in this patient (Figure 3E) compared with a diffuse positive reaction in axons and Schwann cell nuclei in the control case (Figure 3F). To further investigate the impact of the mutant proteins in cellular function and localization, we generated mutants using a BC015231 clone as a template. Cellular localization of the proteins was analyzed after transfection of the genes into HEK293 cells. Overall, the proteins were localized in the cytoplasm and there was no distinctive aggregation of any of the three proteins (Figure 4).

The proteins were examined for ability to activate the NF-κB pathway using a luciferase assay. Transfection of wild-type PLEKHG5 significantly induced luciferase activity. However, elevated levels of the activity were reduced in mutant protein-expressing cells (Figure 5).