Characterization of patient cohort and genotype–phenotype correlation
We evaluated thirteen patients belonging to ten different families, who showed a clinical and neurophysiological phenotype consistent with CMT2A and carried a pathogenic MFN2 variant. Demographic and clinical characteristics of patients enrolled are shown in Tables 1 and 2.
Age of disease onset varied widely, ranging from early childhood to late adulthood (1–69 years, mean 18.5, SD + /− 23.6). Walking difficulties were the most common initial symptoms. Clinical severity also ranged from mild to severe, with most patients presenting a severe phenotype, as already observed by 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-onset CMT2A (1–20 years of age) presented a more severe phenotype comparing with patients with adult-onset CMT2A (> 20 years of age). Interestingly, all the three adult-onset CMT2A patients carried the p.R280H mutation.
Clinical sensory involvement was reported only in 6/13 (46%) patients and did not seem to be associated to specific mutations nor to age of onset. Additional symptoms were described in a total of 8/13 (61%) patients. Among them, optic atrophy and reduced visual acuity occurred in 5/13 (38%) patients, in line with previous reports18.Furthermore, one p.R280H carrier (08-1) showed concomitant alterations in motor and somatosensory evoked potentials (MEP/SSEP). Two patients, one carrying the p.R104W mutation (03-1) and one carrying the p.R280H mutation (06-1), presented a sensorineural hearing defect. Another carrier of the p.R280H mutation (07–1) displayed dysphagia and ptosis.
As already reported, all subjects carrying the p.R104W variant presented 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 ataxic gait (patient 03-2). In addition to that, three patients among them had epileptic manifestations, namely lower limbs myoclonus in two patients from the same family (03-2, 03-3) and epilepsia partialis continua in a third, unrelated patient (02-1). Two patients (02-1, 03-1) displayed punctate white matter alterations at MRI investigation.
Characterization of MFN2 genetic spectrum and identification of one novel mutation
Except for the proband (09-1), all patients carried a known heterozygous missense MFN2 mutation already associated to CMT2A phenotype (p.R94Q5,14,15,18,23,34,35, p.R104W16,22, p.T236M34, p.S249C16, p.R280H5,14,16,17,18,23, p.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). The 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 reported in one family each.
We detected one novel, heterozygous pathogenic variant, namely the c.1069A > G, p.K357E (NM_014874). The mutation, located in MFN2 exon 11, is absent from public databases and is predicted as pathogenic by in silico tools (Supplementary Table 2). MFN2 sequencing in the parents excluded 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), which was found to be necessary for mitochondrial fragmentation and fusion in vitro38. A different amino acid substitution at the same site (c.1071G > C, p.K357N) was previously described by Kijima et al. in 200534. The variant fulfils the ACMG criteria for a likely pathogenic variant33.
The proband carrying the p.K357E mutation presented a severe, early-onset phenotype characterized by severe weakness and muscle atrophy in both proximal and distal segments of lower limbs and in the distal segments of the upper limbs, and mild weakness and atrophy in the proximal segments of the upper limbs. The patient is not able to walk autonomously, uses the wheelchair and needs assistance for daily activities and self-care. She had scoliosis and foot deformities (pes cavus), and a severe restrictive lung disease with respiratory insufficiency requiring nocturnal non-invasive ventilatory support (NIV). She also displayed complete loss of tactile, vibratory and proprioceptive sensation, optic atrophy and bilateral vocal cord paresis determining dysphonia.
The biopsy of the sural nerve was consistent with the clinical severity of the disease showing an almost complete absence of fibers, important connective substitution and several onion bulb formations (Fig. 2), as reported in severe CMT2A cases 22,39,40.