Mutation screening by conventional Sanger sequencing on specific regions of the RYR1 gene was performed in all family members for whom DNA was available (Figure 1). In detail, primers were designed using Primer3 software (http://frodo.wi.mit.edu/primer3) to amplify all RYR1 exons (11). Genomic DNA was extracted from peripheral blood leucocytes by standard procedures (31, 32). Amplified DNA fragments were directly sequenced on an ABI3500 Genetic Analyzer (Applied Biosystems), using the Big-Dye Terminator v3.1 kit and analyzed with Sequencher 5.0 software (Gene Codes Corporation). DNA mutation numbering was based on cDNA reference sequence (NM_000540.2), taking nucleotide +1 as the A of the ATG translation initiation codon. The mutation nomenclature used follows that described at http://www.hgvs.org./mutnomen/.

Stimulators and electrodes used in this study derives from devices developed in the EU project RISE (21, 28). The FES training procedure was developed by Dr. H. Kern and colleagues and previously applied on SCI patients with complete lesion of the conus cauda to promote recovery of muscle mass, structure and function in the lower limb (27–29, 33). The patient was provided with stimulators and electrodes at home, and after appropriate training and instructions, was able to perform stimulation at home. Two pairs of large electrodes, each having an area of 200 cm², were strapped to the anterior surface of the thighs in proximal and distal positions (see Figure S2, for additional detail). Twitch contractions were elicited by biphasic rectangular current pulses lasting 100 to 150 ms and up to ±200 mA amplitude, representing an impulse energy up to ~2 Joules, to recruit fibers throughout the Quadriceps Femoris muscles. The long duration of the impulses needed for stimulation precluded the use of frequencies that would elicit tetanic contractions: training was initiated at 2 Hz, delivered for 15 min/day (series of 4 s on, 2 s off), 3 days/week. During the first/second month, muscle excitability progressively increased allowing an increase of 2 Hz stimulation, 2 × 10 min stimulation (2 min rest). As training proceeded, excitability of the muscle continued to increase, and from the third month onwards, training time stimulation were increased to 3 × 10 min (2 min rest).

Muscle structure and ultra-structure was assessed by Magnetic Resonance Imaging (MRI), histology, and Electron Microscopy (EM). Muscle function was assessed by measuring maximum isometric force (MIF) and stabilometric test (ST), and using a complete set of functional tests to assess mobility in activities of daily living (ADL): Timed Up and Go Test (TUGT), 10 m Self Paced Walk Test (SPWT), and Short Physical Performance Battery (SPPB) as in Kern et al. (30). The effect of FES on muscle structure and function was assessed by doing all the tests mentioned above before the beginning of the 26 months of FES stimulation (T0), and at 3 different time points: T1, 5 months; T2, 14 months; T3, 26 months. See Figure 2 for additional detail.

The 55-year-old Caucasian woman enrolled in the study was diagnosed with CCD (CCD; OMIM# 117000) at the age of 44 (Center for Neuromuscular Diseases; Ospedale Clinicizzato SS. Annunziata, I-66100 Chieti, Italy). CCD was confirmed by histological examination of muscle biopsies at the time of the initial diagnosis (not shown). Presence of areas of internal disarray in skeletal muscle fibers was also confirmed in the present study (please see Figure S1, for more detail). The patient reported that her disease had been aggravated by orthopedic complications (scoliosis diagnosed at the age of 17 years) that limited her daily activities to a point in which she retired from working activities at the age of 54. At the time of enrolment in the present study a medical check-up was performed (anthropometric data, medical aptitude, and anamnesis): her vital signs were all within normal limits, except for a moderately high body mass index (Table 1). The score of muscle strength test by the Medical Research Council scale was 3/5 in both upper and lower limbs (42). Patient had a waddling gait and difficulty in climbing stairs.

Sequence analysis of the RYR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in the substitution of the Arg in position 2452 with a Trp (p.R2452W) and a duplication (c.12853_12864dup12, p.Thr4285_Thr4288dup) in exon 91, involving 4 amino acid residues (Thr, Ala, Ala, Thr, p.Thr4285_Thr4288dup) in exon 91 was also observed. To test how these two mutations segregate in the family, DNA samples from relatives were obtained and analyzed by direct sequencing (Figure 1). This analysis indicated that mutations are independently transmitted in the family and that the proband inherited the c.12853_12864dup12 duplication from the father and the c.7354C>T mutation from the mother. Analysis of the daughter and the son of the proband, indicated that both inherited only the duplication from the father. From a clinical point, neither the parents nor the sons of the proband presented signs of myopathy or CCD.

Stimulators and electrodes used in this study derives from devices developed in the EU project RISE (21, 28). The patient was provided with stimulators and electrodes, and after appropriate training and instructions, was able to perform FES at home (please see Figure S2, for more detail). The effect of FES on muscle function and structure was assessed by measurement of maximum isometric force (MIF) of leg extensor muscles, magnetic resonance imaging of lower extremities (MRI), a complete set of functional tests to assess mobility in activities of daily living (ADL) and, finally, histological and electron microscopy (EM) analysis of muscle biopsies. Functional tests and structural studies were performed before the beginning of the 26 weeks of FES stimulation (T0), and at 3 different time points during FES training: T1 (5 months), T2 (14 months), and T3 (26 months). See section Materials and Methods and Figure 2 for additional detail about stimulation protocols and adjustment of parameters during the different phases.

Figures 3A–C shows representative color MRI of thigh muscles after 5, 14, and 26 months of FES training (T1, T2, and T3, respectively). Unfortunately, data at T0 were not collected. Data collected from measurements of MRI scan images are plotted in Figures 3D–F. The average cross sectional area (CSA) of the quadriceps (brown in Figures 3A–C) increased of about 15% from T1 to T2 and of about 18% at T3 (Figure 3D). The size of quadriceps muscle increased similarly in the right and left leg, while no changes were detected in the hamstrings muscles, i.e., the posterior muscles of the thigh (green in Figures 3A–C). Note that, during stimulation, the surface electrodes were placed on the front part of the thigh, in correspondence to quadriceps, and, as a consequence, hamstrings were not directly stimulated by FES. Figure 3F shows increased percentage of free fat mass at T3. These results indicate that FES treatment increased muscle mass at T2 and T3 and reduced fat fraction area at T3. See Table S1 for additional detail.

To investigate the beneficial effect of FES treatment on muscle strength and balance of the patient, maximum isometric force (MIF) and orthostatic balance during a stabilometric test (ST) were tested.

We monitored whether improved muscle force and balance while standing (Figure 4) was associated with an effective improvement of the patient's Activities of Daily Living (ADL). A complete set of tests to assess mobility of the patient was employed as in Kern et al. (30) to evaluate the effect of FES at the different time points of the study: Timed-Up-And-Go-Test (TUGT), 10 m Self Paced Walk Test (SPWT), and Short Physical Performance Battery (SPPB) (Figure 5). The results of these tests were slightly contradictory, as at T1 and T2 the results collected point to a worsening of the patient's performance, while a possible improvement was detected at T3: (a) shorter total times in TUGT test (−3.2%; Figure 5A); (b) an improvement in 10 m walking performance, time needed to cover 10 m (speed) and number of steps during SPWT decreased (−5.1% and −4.2%, respectively; Figure 5B); (c) higher score obtained in the SPPB (6 vs. 8 points; Figure 5C). Taken together, these data suggest a slight improvement of the patient's ADL at T3. The reason for the partially negative outcome at T1 and T2 will be further discussed below (see section Discussion).

Muscle biopsies of Vastus Lateralis (VL) muscle were analyzed by EM (Figure 6) and by histology for quantitative analysis (Figures 6, 7; an image in cross section at lower magnification is provided in Figure S1) before (T0) and after 14 months of FES training (T2).

At the EM analysis (Figure 6) fibers presented with different appearances: (a) apparently normal fibers, which displayed a normal cross striation pattern of the skeletal muscle fibers (Figure 6A); (b) fibers with unstructured cores (Figure 6B), extended areas in which myofibrils were disrupted and replaced by amorphous cytoplasmic material; and (c) fibers with contracture cores (Figure 6C), regions with laterally-packed and/or over-contracted myofibrils. In unstructured cores, mitochondria were often damaged (Figure 6B), while in contracture cores both mitochondria and SR were scarce or completely missing (Figure 6C). Figure 7 shows two representative histological images from biopsies before (Figure 7A) and after the 14 months of FES training (Figure 7B): we used this approach to evaluate whether FES improved fiber structure and/or reduced the number of cores. Before FES training (T0), only 18% of skeletal fibers analyzed displayed the normal cross striation pattern, with the remaining 82% presenting structural abnormalities, i.e., either unstructured or contracture cores (see Figure 6). Following FES training, while recovery was definitely not complete, the percentage of fibers containing cores was significantly reduced from 82 to 53% (Figure 6, columns B and C), accompanied by a parallel increase (from 18 to 47%) in the percentage of fibers with a fairly normal appearance (Figure 6, column A).

CCD is a disorder usually associated with a mild to moderate degree of disability. Weakness is usually slowly progressive, but orthopedic complications often limit the ability of CCD adult patients to perform physical exercise (2). While the use of anti-oxidants is being considered for the treatment of patients (17, 18, 20), CCD has currently no cure, and the development of strategies aiming to maintain muscle mass and strength could be of help. FES has been previously used to rescue or maintain muscle functions in individuals with no, or limited, capability to perform physical exercise: FES was indeed beneficial to improve muscle function in patients with spinal cord injury (27–29) and in elderly individuals (30).

Here we tested the possibility of using FES to improve muscle function in a 55-year-old female patient diagnosed with CCD at the age of 44 by providing stimulators and electrodes to the patient at home (see Figure S2). Sequence analysis of the RYR1 gene in the patient identified a missense mutation (c.7354C>T) in exon 46, which had been previously reported in an individual with MHS (43) and a duplication (c.12853_12864dup12) in exon 91 (Figure 1). This duplication has not been reported in the literature, but occurs in a region of the RYR1 gene where other duplications associated with MHS have been reported (44, 45). Actually, the duplication reported here partly overlaps with the one we report in Levano et al. (45). The patients inherited the c.12853_12864dup12 from the father, while the c.7354C>T mutations was inherited from the mother. Absence of clinical evidence of myopathy in the family members carrying only one of the two mutation in the RYR1 gene and evidence of central core in the proband carrying two mutations agrees with a recessive form of CCD (19, 20, 46, 47).

The results of tests and experiments aiming to evaluate the effects of FES on muscle function and structure were reasonably encouraging. According to MRI analysis, FES treatment resulted in a selective increase in muscle mass (at T2 and T3) in stimulated quadriceps (Figure 3), while no improvement was exerted on the hamstring due to an unfavorable position of the electrodes used to stimulate tight muscles. A reduction in fat mass area was observed at T3. In addition, functional tests reported in Figure 4 also led to some positive outcomes:

Maximum isometric force (MIF) of leg extensors improved in both legs, already significantly at the fifth month of stimulation (8% at T1), and continued to increase steadily until the end of the treatment to achieve an improvement of 11% at 26 months (T3) (Figure 4). The increase in force may result from the combined effect of an increase in muscle mass (Figure 3; discussed above) and of the improvement in ultrastructure of skeletal fibers (Figures 6, 7). The qualitative analysis was supported also by quantitative data revealing a significant decrease in the percentage of fibers presenting structural cores from T0 to T2: 82 vs. 53% (Figure 6).

The results of the battery of ADL tests were less straightforward than the ones collected with other approaches, as at T1 and T2 there were no evidence of improvement, but rather a general worsening of performance in the various tasks, while at T3 scores were improved compared to T0 (Figure 5). The reason for this partial negative outcome is unclear. However, one of the possible explanations is the low back-pain lamented by the patient for several months during the trial, a problem that had temporally caused a significant reduction in her daily walking activities. However, it is important to underline that despite the low back pain, at the end of the FES treatment (T3) all ADL tests showed a slight improvement in the patient's performance. The result of ADL tests (Figure 5) are considered in literature as a parameter to score the degree of independence (27, 51, 52) and an indirect index of muscle strength (52).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.