NAV3 genetic variants cause intellectual disability, microcephaly, speech, and development delay in humans.a Pedigrees of seven families from diverse ethnicities with affected individuals due to genetic variants in NAV3 with multiple inheritance patterns. Filled symbols represent affected individuals, while a double horizontal line connecting parents shows a consanguineous marriage. Affected individuals of PKMR471, PK1, PE30, and F01 families are homozygous for the NAV3 variants. In contrast, family FM1 has a dominant inheritance pattern, and affected individuals of families PED4263 and MI01 have de novo variants. b Representative images of facial features of PKMR471 affected individual (IV:3) with severe ID showing dentofacial dysmorphism, microcephaly, hypertelorism, and strabismus, while affected individual of family PE30 (II:4) with moderate ID has microcephaly and skin pigmentation problems. c Bar graph representing frequency salient phenotypic features observed in the individuals that harbor genetic variants in NAV3. ID, speech, and developmental delay, and cephalic are commonly shared features. d MRI of the control and an affected individual of family PK1. Similar to control, the overall brain morphology of the affected individual was also unremarkable. However, the affected individual had mild delayed myelination (marked by red arrows) represents mild delayed myelination. Two minor developmental venous anomalies through the right side of the midbrain and cerebellum.

In-silico analysis and 3D protein modeling support deleterious impact of NAV3 variants. a Schematic representation of human NAV3 gene (top) and encoded protein (bottom) structures, along with the variants identified in this study. NAV3 in humans is comprised of 39 coding exons, while encodes Neuron Navigator 3 (NAV3) protein with multiple functional domains, including the Calponin homology region (CH), microtubule-binding domain (MTBD), ATPases (AAA), and three coiled-coil regions (green ovals). b The Missense Tolerance Ratio (MTR) graph and Intolerance landscape visualization of NAV3 via MetaDome are presented with relative positions of the variants identified in our cohort. All of the identified variants are located in relatively intolerant regions of the NAV3. c High conservation of the residues, replaced due to the missense variants found in this study, was observed during evolution. d Three-dimensional (3D) protein modeling illustrating an overview of NAV3 protein. The left panels represent wild-type residues at given positions, while their alternate mutated residues are in the right panels. Color code: green element; targeted amino acid; interacting amino acids: mustard, pink, purple, red, badge, rose, and burgundy, while hydrogen bonding between the residues is shown with yellow dotted lines along with the distances in Å. For p.Ser1326Asn substitution, a loss of hydrogen bond with p.Ser1128 (purple) was predicted. Similarly, for the p.Ser1681Arg variant, loss of hydrogen bond with p.Glu1734 (badge) was predicted. The p.Arg2261Cys variant is predicted to abolish four hydrogen bonds: two with p.Arg2264 (pink) and two with p.Glu2320 (mustard).

NAV3 interacts with microtubules in COS7 cells. GFP-tagged WT and mutated NAV3 proteins (green) showed an overlap expression (yellow) with microtubules, decorated with acetylated tubulin antibodies (red). Granular expression of NAV3 was observed at dendrite-like structure formations and tip ends of microtubules. White arrow heads show the granule type expression at +Tip ends of microtubules. Similar to WT protein, the p.Arg2261Cys, p.Ser1326Asn, and p.Ser1681Arg variants harboring NAV3 also showed overlapping expression with stable microtubule structures. However, the protein-truncating variants, except p. Asp1208Argfs*58, did not show +Tip granule formations. Among the variants analyzed, NAV3 with p.Thr1617Tyrfs*9 and p.Trp332* had reduced or no bundling and show centralized expression in nucleus. Scale bar: 20 um.

ID-associated variants impact NAV3 ability to stabilize microtubule in HEK293T cells. a Representative confocal images of GFP-NAV3 wild type (WT) and mutated proteins (green) overexpressing HEK293T cells treated with 10 μM nocodazole for 1 h, and immuno-labeled with microtubule acetylated tubulin (orange) and tyrosinated tubulin (red) markers. Compared to cells over-expressing WT GFP-NAV3 with a stable microtubular network despite the nocodazole treatment, all the ID-associated variants impact the microtubule-stabilization function of NAV3. Dotted white lines show zoomed in areas in insets. Scale bar: 20 μm. b Bar graph quantification ratio of stable to dynamic microtubules for all respected treated groups. At least 10 transfected cells per construct were imaged and quantified. All the variants expressing cells results were compared to WT using the paired student t-test. NAV3 proteins harboring ID-associated variants showed significantly reduced microtubules stability (**** p < 0.0001; *p < 0.0350; **p < 0.00220). Error bars represent standard error of the mean. c Single-cell RNA (sc-RNA) expression profile of mouse cell clusters at early organogenesis (top panel) generated from transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation (data available from UCSC cell browser). The transcriptome data (bottom panel) shows the highest expression of Nav3 in the neural tube and postmitotic neurons, while expression also observed in inhibitory and inter neurons. Dispersed expression in neuronal progenitor cells were also observed.

Zebrafish nav3 morphants show severe development abnormalities. a Schematic representation of zebrafish nav3 gene along with position and sequence of ATG translational blocker (ATG_MO) and splice site (SS_MO) morpholinos. b Representative images of control (Control_MO), nav3 ATG_MO (10 ng), and SS_MO (12 ng) morphants. Based on their developmental morphology, nav3 morphants were grouped into severe, moderate, mild, and normal classes. Severe class morphants had underdeveloped heads and eyes, small body length, missing swim bladders, yolk and heart edema, and curved tails, while moderate class morphants had small head size, yolk, and heart edema with curved tail in morphants. The mild class morphants had small head sizes, yolk, and heart edema, while normal class morphants had no apparent morphological difference from WT or control injected larvae. c RT-PCR followed by gel electrophoresis revealed smaller PCR product in SS_MO injected morphants (top). Sanger sequencing of the PCR products confirmed aberrant splicing (exon 2 skipping, deletion of 118 bp; schematically represented in bottom panel) due to the SS_MO injection in zebrafish larvae. d, e Phenotype assessment-based bar graphs for control, nav3 ATG_MO, SS_MOs. d With increase in ATG_MO concentration, the phenotypic abnormalities among injected larvae also escalated. e Compared to control, significantly reduced (**p < 0.0048 and ****p < 0.000123) number of morphologically normal larvae observed in ATG_ and SS_MOs injected morphants. f Head-to-length ratio bar graph in uninjected, control_MO, and nav3 ATG_MO morphants. Compared to controls, nav3 morphants have statistically significant reduced head sizes (**p = 0.006931 and ****p = 0.000027, respectively), suggesting microcephaly. g Confocal images of zebrafish brain from the neuro-d transgenic line, injected with control (top) or nav3 (bottom) morpholinos. Control_MO injected larvae show normal morphology of optic tectum (OT), torus longitudinalis (TL), habenula (Hb), and corpus cerebelli (Cc) regions. In contrast, nav3 morphants had significantly reduced (****p < 0.000001) OT (highlighted with white dashed lines), quantified in (h). Scale bar: 100 um. i Violin plot showing statistically significant (****p < 0.000001) decreased movement in nav3 morphants as compared to controls, when subjected to light stimulus. Error bars in (d, e, f, h, I), represent standard error of the mean.

NAV3-WT mRNA injections rescued the phenotype in morphants as compared to variants harboring mRNAs. a Bar graph representation of morphant morphological phenotypes after injection of four different concentrations (250 pg, 500 pg, 750 pg, and 1000 pg) along with nav3 ATG_MO. The 500 pg concentration of NAV3-WT mRNA injection revealed the most significance rescue of normal class larvae. NS not-significant; ***p < 0.000250; and ****p < 0.00001. b Rescue experiments performed via co-injecting human NAV3WT or variants harboring mRNAs (500 pg) along with ATG_MO (8 ng) revealed significant rescue of normal developmental class in WT mRNA injected morphants compared to ATG_MO alone. The normal phenotype class in morphants from NAV3WT and the variants mRNAs co-injected were compared for statistical analysis (***p < 0.0001; ****p < 0.00001). Importantly, the ID-associated variant harboring NAV3 mRNAs could not rescue the normal developmental class of zebrafish, supporting their pathogenic nature. Error bars in (a, b) represent standard error of the mean.

NAV3 variants cause a spectrum of Neurodevelopmental disorders (NDD) in humans. Bar diagram of NAV3 gene and protein structure showing mutations identified in this study at the top and mutations reported previously for Autism Spectrum Disorder (ASD) with other cooccurring NDDs at the bottom. Overall, among the 30 known variants, 10 are located in the MTBD domain, making it a suspectable hotspot region.