Identification and functional characterization of novel GDAP1 variants in Chinese patients with Charcot–Marie–Tooth disease

Abstract

Objective: To identify and characterize the pathogenicity of novel variants in Chinese patients with Charcot-Marie-Tooth disease. Methods: Multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES) were performed in 30 unrelated CMT patients. Minigene assay was used to verify the effect of a novel splicing variant (c.694+1G>A) on pre-mRNA. Primary fibroblast cell lines were established from skin biopsies to characterize the biological effects of the novel variants p.L26R and p.S169fs. The mitochondrial structure was observed by an electron microscope. The expression level of protein was analyzed by Western Blotting. Mitochondrial dynamics and mitochondrial membrane potential (MMP, Dwm) were analyzed via immunofluorescence study. Mitochondrial ATP levels were analyzed via bioluminescence assay. The rate of oxygen consumption was measured with a Seahorse Bioscience XF-96 extracellular flux analyzer. Results: We identified 10 pathogenic variants in three known CMT related genes, including three novel variants (p.L26R, p.S169fs, c.694+1G>A) and one known pathogenic variant (p.R120W) in GDAP1. Further, we described the clinical features of patients carrying pathogenic variants in GDAP1 and found that almost all Chinese CMT patients with GDAP1 variants present axonal type. The effect of c.694+1G>A on pre-mRNA was verified via minigene splice assay. Cellular biological effects showed ultrastructure damage of mitochondrial, reduced protein levels, different patterns of mitochondrial dynamics, decreased mitochondrial membrane potential (Dwm), ATP content, and defects in respiratory capacity in the patient carrying p.L26R and p.S169fs in GDAP1. Interpretation: Our results broaden the genetic spectrum of GDAP1 and provided functional evidence for mitochondrial pathways in the pathogenesis of GDAP1 variants.

GDAP1 Involvement in Mitochondrial Function and Oxidative Stress, Investigated in a Charcot-Marie-Tooth Model of hiPSCs-Derived Motor Neurons

“…However, the weak GDAP1 expression detected in fibroblasts does not exclude a GDAP1 role in this cell type, and the possibility of conducting functional studies on them [16][17][18]. In our case, in the examination of electron transport chain's (ETC) activity, any big difference emerged between controls and patient, neither in fibroblasts, nor in MNs.…”

“…The disruption of cristae structure has already been associated to other pathological conditions induced by mutations or lack of proteins involved in mitochondrial dynamics, such as optic atrophy 1 (OPA1) protein [42], or mitofusin 2 (Mfn2) protein [43]. Cristae abnormalities were also reported in nerves' axonal mitochondria of a CMT2 patient, carrying the c.174_176delGCCinsTGTG (p.Pro59Valfs*4) mutation in GDAP1 [44], but also, more recently, in muscular tissue of a patient carrying the c.77T>G (p.Leu26Arg) and the c.505_511del (p.Ser169*) GDAP1 mutations [18]. Interestingly, some additional findings of our cell culture need to be pointed out.…”

“…These cell types cannot be obtained from humans, and used in vitro as cellular models. Given the inability to culture human neural cells, the only model available in these conditions was represented by human fibroblasts [16][17][18]22], which, unfortunately, poorly express GDAP1 [22]. The aim of this study was to go beyond limits imposed by existing animal and cellular models, developing a new solid model of human motor neurons, carrying the homozygous p.Ser194* mutation in GDAP1, to investigate GDAP1 functions and GDAP1-associated mechanisms in CMT disease.…”

“…Three main model organisms have been developed to elucidate GDAP1 role in cellular physiology, through its up-or down-regulation, in Drosophila melanogaster [10,11], its depletion, in mice [12,13], or its mutation in yeasts [14]. Concerning the cellular models, given the inaccessibility of human neurons, all functional studies employed a large amount of alternative strategies, such as primary cultures of murine neurons [9,15], rat Schwann cells [7], and human fibroblasts [16][17][18], but also transfected, or non-transfected, cell lines, such as Cos7, HeLa, SH-SY5Y, N1E-115, HT22 [7][8][9]15,[19][20][21][22][23][24]. These existing models have been fundamental to highlight some GDAP1 functions.…”

Genetic approaches and pathogenic pathways in the clinical management of Charcot-Marie-Tooth disease

“…Among the most commonly involved genes in the pathogenesis of CMT, we can find GDAP1. This gene encodes ganglioside-induced differentiation-associated protein 1 (GDAP1), which is an atypical glutathione S-transferase (GST) [54] with glutathione-conjugating and membrane-remodeling functions [55] . This protein can be found in the outer mitochondrial membrane (OMM) and the mitochondria-associated membranes (MAMs), and it is mainly expressed in neurons [56][57][58] .…”