Characterization of the patient cohort and genotype-phenotype correlation
We evaluated thirteen patients from ten different families who presented a clinical and neurophysiological phenotype consistent with CMT2A and carried a pathogen MFN2 Variant. Demographic and clinical characteristics of the enrolled patients are presented in Tables 1 and 2.
The age of onset varied greatly, ranging from early childhood to late adulthood (1–69 years, mean 18.5, SD +/- 23.6). Difficulty walking was the most common initial symptom. Clinical severity also ranged from mild to severe, with most patients presenting with a severe phenotype, as observed in previous studies (Table 1, Supplementary Table 1). 15:16. CMTESv2 in adult patients ranged from 3 to 19 points (mean 12.7, SD +/- 5), while Rasch-modified CMTESv2 ranged from 3 to 26 points (mean 16.5, SD +/- 6.9). Overall, patients with childhood CMT2A (aged 1-20 years) displayed a more severe phenotype compared to patients with adult CMT2A (>20 years). Interestingly, all three adult-onset CMT2A patients carried the p.R280H mutation.
Clinical sensory involvement was only reported in 6/13 (46%) patients and did not appear to be related to specific mutations or age of onset. Additional symptoms were reported in a total of 8/13 (61%) patients. Optic atrophy and decreased visual acuity occurred in 5 of 13 (38%) patients, consistent with previous reports18. Furthermore, one p.R280H carrier (08-1) showed simultaneous changes in motor and somatosensory evoked potentials (MEP/SSEP). Two patients, one with the p.R104W mutation (03-1) and one with the p.R280H mutation (06-1), had sensorineural hearing loss. Another carrier of the p.R280H mutation (07-1) showed dysphagia and ptosis.
As previously reported, all subjects carrying the p.R104W variant demonstrated central nervous system involvement with intellectual disability and developmental delay, in some cases with dysarthric speech (patients 03-2 and 03-3), spastic paraparesis (patient 03- 1) and atactic gait (patient 03-2). In addition, three patients among them had epileptic manifestations, namely lower extremity myoclonus in two patients from the same family (03-2, 03-3) and epilepsy partialis continua in a third unrelated patient (02-1). Two patients (02-1, 03-1) showed punctate white matter changes on MRI.
Characterization of the MFN2 genetic spectrum and identification of a novel mutation
With the exception of subject (09-1), all patients carried a known heterozygous missense MFN2 Mutation already associated with the CMT2A phenotype (p.R94Q5,14,15,18,23,34,35p.R104W16:22S.T236M34S.S249C16p.R280H5,14,16,17,18,23S.A383V16,36,37). Most of them (p.R94Q, p.R104W, p.T236M, p.S249C and p.R280H) are located within the highly conserved GTPase domain (Fig. 1A). Affected members of three separate kindreds carried the p.R280H mutation. The p.R104W mutation was observed in four patients from two families. The other mutations (p.R94Q, p.T236M, p.S249C, p.A383V) were each reported in one family.
We discovered a new, heterozygous pathogenic variant, namely c.1069A > G, p.K357E (NM_014874). The mutation is in MFN2 Exon 11, is absent from public databases and predicted to be pathogenic by in silico tools (Supplementary Table 2). MFN2 sequencing in the parents ruled out the presence of the variant, thus confirming its de novo occurrence (Fig. 1C). The p.K357E replaces a highly conserved lysine (Fig. 1B) in the R3 region (Fig. 1A) that has been found to be necessary for mitochondrial fragmentation and fusion in vitro38. Another amino acid substitution at the same site (c.1071G > C, p.K357N) was previously reported by Kijima et al. described. in the year 200534. The variant meets the ACMG criteria for a probable pathogenic variant33.
The subject carrying the p.K357E mutation had a severe, early-onset phenotype characterized by severe weakness and muscle atrophy in both the proximal and distal segments of the lower limbs and in the distal segments of the upper limbs, as well as by Slight weakness and atrophy in the proximal segments was characterized upper extremity segments. The patient is unable to walk independently, uses a wheelchair and requires assistance with daily activities and self-care. She had scoliosis and foot deformities (hollow foot), and severe restrictive pulmonary disease with respiratory failure requiring nocturnal noninvasive ventilation (NIV) support. She also showed complete loss of tactile, vibratory, and proprioceptive sensation, optic atrophy, and bilateral vocal cord paresis, which determine dysphonia.
Biopsy of the sural nerve was consistent with the clinical severity of the disease, showing an almost complete absence of fibers, important connective tissue replacement, and multiple onion bulb formations (Fig. 2) as reported in severe CMT2A cases 22,39,40.