Biallelic mutations in neurofascin cause neurodevelopmental impairment and peripheral demyelination

Abstract

Axon pathfinding and synapse formation are essential processes for nervous system development and function. The assembly of myelinated fibres and nodes of Ranvier is mediated by a number of cell adhesion molecules of the immunoglobulin superfamily including neurofascin, encoded by the NFASC gene, and its alternative isoforms Nfasc186 and Nfasc140 (located in the axonal membrane at the node of Ranvier) and Nfasc155 (a glial component of the paranodal axoglial junction). We identified 10 individuals from six unrelated families, exhibiting a neurodevelopmental disorder characterized with a spectrum of central (intellectual disability, developmental delay, motor impairment, speech difficulties) and peripheral (early onset demyelinating neuropathy) neurological involvement, who were found by exome or genome sequencing to carry one frameshift and four different homozygous non-synonymous variants in NFASC. Expression studies using immunostaining-based techniques identified absent expression of the Nfasc155 isoform as a consequence of the frameshift variant and a significant reduction of expression was also observed in association with two non-synonymous variants affecting the fibronectin type III domain. Cell aggregation studies revealed a severely impaired Nfasc155-CNTN1/CASPR1 complex interaction as a result of the identified variants. Immunofluorescence staining of myelinated fibres from two affected individuals showed a severe loss of myelinated fibres and abnormalities in the paranodal junction morphology. Our results establish that recessive variants affecting the Nfasc155 isoform can affect the formation of paranodal axoglial junctions at the nodes of Ranvier. The genetic disease caused by biallelic NFASC variants includes neurodevelopmental impairment and a spectrum of central and peripheral demyelination as part of its core clinical phenotype. Our findings support possible overlapping molecular mechanisms of paranodal damage at peripheral nerves in both the immune-mediated and the genetic disease, but the observation of prominent central neurological involvement in NFASC biallelic variant carriers highlights the importance of this gene in human brain development and function.

Keywords: neurodevelopment; neurofascin; peripheral demyelination.

Figure 1

Schematic diagram showing the different domains of a myelinated axon. The axonal region around the node of Ranvier is expanded to show the different axonal domains: the node of Ranvier where voltage-gated Na⁺ channels (Na_v1.6 and Na_v1.1) are expressed, the paranode where the myelin is attached to the axon, and the juxtaparanode where most voltage-gated K⁺ channels (KCNQ2/3 and K_v1) are located. Each of these domains is characterized by the expression of specific cell adhesion molecules; at the nodes Nfasc186 binds gliomedin (GLDN) and NrCAM, which are secreted by Schwann cells in the nodal gap lumen, at the paranode Nfasc155 forms a complex with CNTN1/CASPR1 to form the septate-like junctions, and at the juxtaparanode the CNTN2/CASPR2 complex enables the sequestration of K_v1 channels. Adapted and modified from Arancibia-Carcamo and Attwell (2014).

Figure 2

Pedigrees, Sanger sequencing, multiple-sequence alignment of NFASC and clinico-radiological natural history of patients. (A) Pedigrees of the six families carrying biallelic NFASC mutations and Sanger sequencing electropherograms confirming the mutations. (B) Facial pictures of Patients 1–3; note the facial hypomimia present in all cases, the muscle weakness and inability to hold his neck in Patient 1 carrying the p.P694T variant, and the tongue protrusion in Patients 2 and 3 carrying the frameshift mutation p.P939Ter. (C) MRIs of Patients 1–3 and 10 showing loss of cerebral white matter and atrophic changes of corpus callosum and brainstem: T₂-weighted coronal MRI showing enlarged lateral ventricles in Patient 1 indicative of cortical volume loss (top left). T₁-weighted sagittal MRI-sequence of Proband 2 displaying thin corpus callosum as well as cortical volume loss (top right). Axial T₂-weighted sequence in Patient 3 showing progressive loss of cerebral white and grey matter (bottom left). T₁-weighted sagittal MRI-sequence of Proband 10 displaying thin corpus callosum as well as cortical volume loss (bottom right). (D) In silico modelling of the 3D structure of the p.N130D and p.R359P variants. In the top left image [wild-type (WT) protein], a hydrogen bonding interaction with the side chain of glutamine 46 is visible. The residue of lysine 49 is distant from the point of mutation and forms a salt bridge with the carbonyl moiety of residue tyrosine 416. In addition, a weaker hydrophobic (cation-π) interaction forms between the side chains of lysine 49 and tyrosine 416 (red dashes, connecting the charged nitrogen atom with the centre of the phenyl ring). The top right image shows the different hydrogen bonding network formed in the mutant: the mutated aspartate residue forms three hydrogen bonding interactions, two involving its side chain and the side chain of residue lysine 49, and the third between its amino moiety and the side chain of residue glutamine 46. No interactions are visible between residues lysine 46 and tyrosine 416 that are further apart (∼6 Å) and with an unsuitable geometry. In the bottom left image (wild-type protein), a strong hydrogen bonding network connects residues aspartate 352, arginine 370 and aspartate 402 positioned on three different β-strands. The absence of the arginine residue in the mutant (bottom right) impedes the formation of the hydrogen bonding network, distancing the three β-strands. (E) A schematic representation of Nfasc155 and Nfasc186 showing the position of all NFASC variants. (F) Inter-species alignment performed with Clustal Omega shows the complete conservation down to invertebrates of the amino acid residues affected by the substitutions.

Figure 3

Membrane targeting of Nfasc155 variants. The homozygous Nfasc155 variants associated with a severe pathology diminish Nfasc155 protein level. The variants p.P705T and p.P939Ter inhibit the association of CASPR1/CNTN1 with Nfasc155. (A–E) HEK cells were transfected with Myc-tagged Nfasc155 variants and surface Nfasc155 was monitored by incubating the live cells with anti-Nfasc155 IgG (red) prior to fixation and permeabilization. Nfasc155 was then revealed using an anti-Myc antibody (green). Wild-type Nfasc155, but also the variants p.N124D, p.R370P, p.P705T and p.S831P were readily targeted at the cell surface, and did not show signs of intracellular retention. (F and G) HEK cells were co-transfected with GFP (green) and either wild-type Nfasc155 or p.P939Ter variant, then cells were immunostained for Myc (red). In contrast to wild-type Nfasc155, p.P939Ter was not detectable, indicating that this mutation strongly affects Nfasc155 expression. Nuclei are stained DAPI (blue). Scale bar = 10 µm. (H) Western blot analysis of HEK cells transfected with Nfasc155 variants and revealed with anti-Myc antibodies or anti-a-tubulin antibodies as loading control. (I) Protein expression levels were analysed by normalizing the signals to the corresponding α-tubulin signal, then to wild-type Nfasc155 in four independent experiments. The expression levels of p.P705T, p.S831P and p.P939Ter variants were significantly decreased compared to wild-type Nfasc155 (Mann-Whitney test). *P < 0.01; **P < 0.005; ***P < 0.001; by unpaired two-tailed Student’s t-tests for two samples of equal variance and by one-way ANOVA followed by Bonferroni’s post hoc tests. Bars represent mean and SEM. Molecular weight markers are shown on the left (in kDa). N.S. = not significant. (J–O) N2A cells were transfected with GFP (green) in combination with CNTN1 and CASPR1 and were incubated for 2 h with cells transfected with tandem tomato (magenta) and wild-type Nfasc155 (J), p.P705T (M) or pP939Ter (O). As negative control, N2A cells transfected with Nfasc155 (magenta) were incubated with cells transfected with GFP. (P) The numbers of green and magenta cell aggregates (dashed line circles in J–O) per visualization field were quantified in each condition (n = 4 experiments for each condition). p.R370P, p.P705T, p.S831P and p.P939Ter mutations significantly decreased aggregate formation. ***P < 0.001 by unpaired two-tailed Student’s t-tests and by one-way ANOVA followed by Bonferroni’s post hoc tests. Bars represent mean and SEM. Scale bar = 20 µm.

Figure 4

Severe involvement of peripheral myelinated fibres and partial disruption of Nfasc155 at the paranode in Patient 1. Confocal images of cutaneous innervation from hairy skin (leg and arm) of the patient compared to a control showing well preserved unmyelinated fibres in the epidermis and dermis (B and B1 compared to A), severe loss of myelinated fibres around a hair follicle and the few remaining ones presenting a very weak MBP (red) immunoreactivity (D, D1 arrowheads compared to C), in a nerve bundle with few remaining myelinated fibres presenting a very faint staining with MBP antibody presence of nodes marked with Nfasc186 (green) (F and F1 compared to E), paranodes marked with panNeurofascin (green) (H, H1 arrowheads compared to G) and paranodes marked with CASPR (green) (J and J1 arrowheads compared to I). Scale bar = 100 µm in A, B, B1; 50 µm in C, D, D1; 30 µm in F–J1.

Figure 5

Severe involvement of peripheral myelinated axons in Patient 3 with complete lack of Nfasc155 expression at paranodes. Confocal images of cutaneous innervation from hairy and glabrous skin (thigh and fingertip) of the patient compared to a control showing quite preserved unmyelinated fibres in the epidermis (A and A1) and around autonomic annexes (see arrector pili muscle, arrowheads in C); severe loss of myelinated fibres with evidence in a nerve fascicle of few segments with severe myelin abnormalities in the distal site (leg) (B and B1); more preserved myelinated fibres with evidence around a hair follicle of several myelinated fibres showing loss of myelin in their distal segments in the more proximal site (thigh) (C, arrowheads in C1); loss of mechanoreceptors and myelinated fibres (compared to the control in E and E1) and morphological abnormalities of myelinated fibres with tracts of demyelination in the fingertip (D, arrowheads in D1) (compared to the control in E and E1). Scale bar = 100 µm in A, A1, C, C1, D, D1, E, E1 and 30 µm in B and B1.