Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea

Abstract

The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

Figure 1

Schematic Representation of CHT and Position of the Variants Linked to the Antenatal Form with Arthrogryposis and to the Neonatal Form of CMS (A) The substitutions affecting an amino acid residue already described as critical for CHT activity are in red. All amino acid changes result from mutations that were in the heterozygous state except c.143A>G (p.Asp48Gly), which was homozygous (circle). The variants studied at the functional level are underlined. The changes resulting in DHMN7A (p.Lys499Asnfs^∗13) and linked to ADHD (p.Ile89Val) are also indicated. (B) The alignment of human CHT with the sequences of five model species (rat, mouse, zebrafish, Torpedo mamorata, and Caenorhabditis elegans with the percent of amino acid identities in brackets) demonstrates the high conservation of the substituted residues.

Figure 2

Heterologous Expression and Transporter Activity of CHT in Transfected HEK293T Cells (A) Immunostaining of heterologous CHT using anti-SLC5A7 antibodies showed a membranous staining of the wild-type (WT) and the five variant CHT investigated in HEK293T cells transiently transfected with hSLC5A7 cDNA constructs (c.143A>G [p.Asp48Gly], c.194G>A [p.Gly65Glu], c.313C>T [p.Pro105Ser], c.1082G>A [p.Arg361Gln], and c.1336A>G [p.Arg446Gly]). Nuclei are stained with DAPI (blue). Scale bars represent 10 μm. (B) Evaluation of the choline transporter activity sensitive to HC-3, corresponding to the heterologous expression of CHT activity in transiently transfected HEK293T cells. The y axis bar is interrupted for a better visualization of the residual transporter activity of the variant proteins. (C) Western blot analysis of heterologous CHT in total cell extracts of HEK293 cells transiently transfected with the wild-type or mutant hSLC5A7 constructs performed in denaturating conditions. Three main bands (95 kDa) were observed using a polyclonal antibody directed against human CHT. Anti-GAPDH antibody was used as loading control. No band specific to CHT was observed in extracts from cells transfected with eGFP alone (eGFP), confirming the specificity of the antibody. The size and amount of the bands specific to heterologous CHT were similar between the wild-type (lines WT1 and 2) and the five variants. (D) Evaluation of HC-3-sensitive choline transport when coexpressing the wild-type and the c.313C>T (p.Pro105Ser) and c.194G>A (p.Gly65Glu) mutant constructs in transiently transfected HEK293T cells. The activities recorded in cells coexpressing the indicated combinations of cDNA are compared to the summation of the individual activities obtained when the cells were transfected with the wild-type or the mutant constructs. The results are expressed as mean ± SEM. Statistical significance was calculated by Student’s t test with a level of statistical significance set at p < 0.05 (^∗∗∗p < 0.001).

Figure 3

Immunostaining Analyses of NMJs in Muscle Samples from Individuals 1 and 2 (A) CHT immunostaining (green) and post-synaptic nAChR fluorescent staining (red in b, d, f) on transversal muscle sections showed the presence of CHT at NMJs in individuals 1 (c, d) and 2 (e, f) with a staining intensity similar to the adult control (a, b). (B and C) Representative pictures of muscle biopsies stained for the motor axons with an anti-neurofilament (in green) and for post-synaptic nAChR with α-bungarotoxin (in red). (B) The staining pattern of the two synaptic elements in the young adult individual 1 led us to classify the NMJs in three categories depending upon the innervation status of each NMJ: denervated (b), remodeled (c), or neoformed (d, d′, d′′; d is the merged representation of d′ and d′′). One NMJ from the adult control (a) with well-defined synaptic gutters responsible for the well-circumscribed post-synaptic nAChR fluorescent staining is shown for comparison. (C) Three representative images of immature NMJs observed in the autopsy material of the deceased newborn (individual 2) with no well-differentiated pattern for the postsynaptic apparatus (a, b, c), evidence for accumulation of neurofilament staining in nerve terminal (arrow in b and c), and polyinnervation (arrowhead in c). Scale bars represent 10 μm.