Mutations in the netrin-1 gene cause congenital mirror movements
Aurélie Méneret, … , Emmanuel Roze, David Markie
Aurélie Méneret, … , Emmanuel Roze, David Markie
Published September 25, 2017
Citation Information: J Clin Invest. 2017;127(11):3923-3936. https://doi.org/10.1172/JCI95442.
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Research Article Development Neuroscience

Mutations in the netrin-1 gene cause congenital mirror movements

Abstract

Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.

Authors

Aurélie Méneret, Elizabeth A. Franz, Oriane Trouillard, Thomas C. Oliver, Yvrick Zagar, Stephen P. Robertson, Quentin Welniarz, R.J. MacKinlay Gardner, Cécile Gallea, Myriam Srour, Christel Depienne, Christine L. Jasoni, Caroline Dubacq, Florence Riant, Jean-Charles Lamy, Marie-Pierre Morel, Raphael Guérois, Jessica Andreani, Coralie Fouquet, Mohamed Doulazmi, Marie Vidailhet, Guy A. Rouleau, Alexis Brice, Alain Chédotal, Isabelle Dusart, Emmanuel Roze, David Markie

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Figure 5

Expression of the WT and mutated netrin-1–AP and netrin-1 constructs.

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Expression of the WT and mutated netrin-1–AP and netrin-1 constructs. (A...
(A–C) HEK293 cells were transfected with mouse and human WT and mutated netrin-1–AP plasmids and grown for 48 hours. Western blot showed the presence of the WT and mutated proteins in total lysates at the expected molecular weight (A) but no detection of the mutated proteins in the supernatant, contrary to that seen with WT (B). AP assay of the supernatants (C) showed no difference in the initial rate of reaction between nontransfected cells (X) and cells transfected with mutated netrin-1–AP plasmids, indicating that mutated netrin-1–AP levels in the supernatant were under the detection level. The experiments were replicated 3 times. The antibodies used were anti–netrin-1 (A, B, and D–F), anti-actin (A), and anti–α-tubulin (D). Flp-In TRex tetracycline transactivator HeLa cells were transfected with the WT or 3 netrin-1 constructs harboring the NTN1 mutations (D–G). Stable cell lines were grown for 24 hours in the presence or absence of doxycycline. Western blot showed the presence of WT and mutated proteins in total lysates at the expected molecular weight in the presence of doxycycline (D) but no detection of the mutated proteins in the supernatant, contrary to that seen with WT (E). A small amount of mutated proteins could be detected in the supernatant after concentration (F). In all cases, no netrin-1 protein could be detected in the absence of doxycycline (D–F). (G) The ratio of netrin-1/α-tubulin was significantly reduced only for the mutated I518del and C601R forms compared with WT (1-way ANOVA [F (3,12) = 6.44, P = 0.008] followed by Bonferroni’s post-hoc test, *P < 0.05 and **P < 0.01). h, human, m, mouse; Net1, netrin-1. The experiments were replicated 3 times.