Charcot-Marie-Tooth (CMT) disease is the most common inherited neuropathy with an estimated prevalence of 1 in 2,500 (1). It is a clinically and genetically heterogeneous group of disorders that is characterized by progressive weakness and atrophy of the extremities and loss of sensory function (2). Neurophysiological findings in median nerve differentiate CMT into two groups: demyelinating CMT (CMT1) with slow motor nerve conduction velocity (MNCV) (<38 m/s) and axonal CMT (CMT2) with normal or a slight reduction of MNCV (≥ 38 m/s) (2). It is generally accepted that CMT is a monogenic condition. Up till now, more than 100 causative genes have been reported as causal for CMT (http://neuromuscular.wustl.edu/time/hmsn.html), among which PMP22 and MFN2 are the most common causative genes for CMT1 and CMT2, respectively (3). Of note, patients with PMP22 duplication or MFN2 mutation, such as p.I126S and p.R94W, have a heterogeneous clinical presentation in terms of age at onset, disease severity, and clinical progression (4, 5). Thus, a single mutation may not completely explain the intrafamilial heterogeneity of CMT. Next-generation sequencing (NGS) affords opportunities to detect the co-occurrence of variants in dual or multiple genes, providing insights into the wide phenotypic variability of the disease.

In this study, we screened 189 Chinese CMT families by using multiplex ligation probe amplification (MLPA) combined with NGS technologies. We are further reporting families with significant intrafamilial phenotypic heterogeneity in the presence of concomitant heterozygous MFN2 and GDAP1 variants and a PMP22 duplication combined with an MPZ variant.

In this study, the patient with CMT1 harboring a de novo PMP22 duplication and an inherited MPZ variant presented with an earlier disease onset and a severer phenotype than her father harboring only the MPZ variant (Figure 1A). Each variant was sufficient to cause disease, suggesting a cumulative “double trouble” effect of these two concomitant variants (Figure 2A). MPZ is the major structural protein of peripheral myelin expressed by Schwann cells (more than 50%) (13). Mutation in MPZ was associated with unfolded protein response activation and protein aggregates, leading to the apoptosis and demyelination of the Schwann cell and altered axonal interaction (13, 14). PMP22 is also highly expressed in myelinating Schwann cell (2–5%) and directly contribute to myelin organization (15). PMP22 duplication disrupts the development and maintenance of normal myelin in the Schwann cell (16). Thus, simultaneous variants in these two myelin-related genes might exert double deleterious effects in Schwann cells, which finally leads to more severe defects in the myelination of axons. Of interest, the co-occurrence of PMP22 duplication and variant in another CMT gene (GJB1, LITAF) had also been reported (17, 18). Considering PMP22 duplication as the most common cause of CMT1 patients (approximately 70%) (3, 19, 20), one of the changes observed in cases with CMT1 with variants in two genes is usually the PMP22 duplication.

Two patients with concomitant variants in MFN2 and GDAP1 were severer affected than their parents harboring a single MFN2 variant (Figures 1B,C). Heterozygous MFN2 variants were the primary mutations that could establish the diagnosis. The variant c.3G>T in GDAP1 changed the initiation codon, which has been reported in previous literature (21–23). The GDAP1 c.713G>T missense change occurred at the same position (p.W238) as another pathogenic variant observed (21, 24). The bioinformatic analysis supported the deleterious effect of both variants. Of note, WES ruled out other candidate genetic variants that could be involved in phenotypic expression in these families. Taken together, the heterozygous state of GADP1 variants could increase the disease severity in association with the inherited MFN2 variant, indicating their role of “genetic modifier” (Figure 2B). Another patient with CMT2 with de novo MFN2 variant and inherited heterozygous GDAP1 variant (Figure 1D) presented with early disease onset and moderate phenotype. The variant c.767A>G in GDAP1 is widely recognized as causative of autosomal recessive (AR) CMT (25–27) and might also modulate phenotypic expression in this case. MFN2 and GDAP1 serve as a protein partners in regulating mitochondrial dynamics (MFN2 for fusion, GDAP1 for fission) and are involved in the same mitochondrial function such as energy coupling (28, 29). Mutant GDAP1 exerts a loss of function mechanism and aggravate mitochondrial damage (dynamics dysfunction and energy deficit) caused by MFN2 mutation, which could explain the exacerbation of the CMT2A phenotype. The deficits in mitochondrial function might be their underlying mechanism which still needs to be further studied. MFN2 and GDAP1 are among the more frequent causative genes of CMT2, and this might be the reason why concomitant variants in these two genes have been repeatedly encountered in patients with variants in two CMT2 genes. Of interest, the frameshift p.V205Sfs^*26 variant in MFN2 associated with subclinical neuropathy in our study suggested that MFN2 is not sensitive to haploinsufficiency. Previous reports have described patients with heterozygous state of MFN2 variants (p.E308X, p.V160fs^*26 and del ex7-8) developing a subclinical phenotype or asymptomatic presentation (30, 31), indicating that null variants in MFN2 might be associated with a minimal phenotype.

This study identified a 2.1% (4/189) prevalence of concomitant variants in our cohort. The concomitant variants in families with CMT have been documented in several studies. Recently, a Japanese cohort study identified 5 out of 1,005 families with CMT harboring coinherited variants, accounting for 0.5% (5/1,005) of the total, in which the “double trouble” effect of concomitant variants were the underlying causes (32). Compared with the “genetic modifier” effect of GDAP1 variants observed in this study, the coexistence of homozygous AR-CMT2K (p.Q163X) or heterozygous autosomal dominant-CMT2K (p.H123R, p.E222K, and p.R120W) variant in GDAP1 and heterozygous variant in MFN2 had been reported associating with a more severe phenotype, suggesting the “double trouble” effect of concomitant MFN2 and GDAP1 variants (28, 30, 33, 34). Moreover, concomitant variants were also related to true digenic inheritance (DI) that has been defined as the coinheritance of two nonallelic mutations, both are indispensable to establish the diagnosis (35). The existence of true DI was reported in a family with CMT with heterozygous MFN2 p.L741V and GDAP1 p.Q163^* variants (36). These results suggest that the occurrence of concomitant variants was not uncommon in CMT.

There was an important limitation to this study. Because we conducted a retrospective analysis of target NGS data, genetic variants in non-CMT-causing genes could not be detected. WES (or whole-genome sequencing [WGS]) would detect all possible genetic modifiers. The application of WES (WGS) in all the families with phenotypic variabilities could provide further insights into the clinical and genetic heterogeneity of CMT.

In summary, the coexistence of variants in PMP22/MPZ and MFN2/GDAP1 related to more severe phenotypes accounted for 2.1% of patients with CMT in our cohort. Cumulative deficits on myelination or mitochondrial function might be their underlying mechanism. The possibility of the coexistence of variants in distinct causative genes should be taken into consideration when significant clinical heterogeneity is observed.

The data presented in this study were deposited in Figshare open access repository doi: 10.6084/m9.figshare.17192927. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Third Xiangya Hospital of Central South University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

RZ designed and conceptualized study. YX, ZL, XL, LL, BW, WC, JG, LS, and RZ contributed patient material and clinical data. SH and HZ contributed acquisition of neurophysiological data. ZL and ZH interpreted the genetic data. YX provide the first draft of the manuscript. RZ and BT revised the manuscript. All authors contributed to the article and approved the submitted version.

This study is funded by the National Natural Science Foundation of China (81771366 and 82001338) and the China International Medical Foundation (CIMF-Z-2016-20-1801).

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.