Bi-allelic Loss-of-Function Variants in NUP188 Cause a Recognizable Syndrome Characterized by Neurologic, Ocular, and Cardiac Abnormalities

Abstract

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.

Keywords: Ashkenazi Jewish; NUP188; autosomal recessive; congenital cataracts; congenital heart defects; dendritic arborization; microcephaly; negative geotaxis; nuclear pore complex; nucleoporin.

Figure 1

Facial and Digital Features of Affected Individuals Note the high anterior hair line, broad forehead, small and downslanting palpebral fissures, wide nasal bridge with bulbous tip, and down-turned corners of the mouth in the facial photographs. The feet are notable for broad halluces, overlapping toes, and camptodactyly. The hand photographs show long gracile fingers and camptodactyly, as well as preaxial polydactyly in individual 1.

Figure 2

NUP188 p.Ile302Valfs^∗7 and p.Tyr1048^∗ Result in Loss of Full-Length NUP188 (A) Western blot of nuclei isolated from individual 1 and control fibroblasts stained with an antibody against NUP188 reveals full-length NUP188 (~188 kDa) is undetectable in individual 1. LAMB1 is used as a loading control. Individual 1 has compound heterozygous truncating variants NUP188 p.Ile302Valfs^∗7 and p.Tyr1048^∗. Experiment was performed in triplicate, and representative images are shown. (B) Immunofluorescent staining of NUP188 in fibroblasts from individual 1 and a control individual. NUP188 (green) localizes to the nucleus in control fibroblasts but is undetectable in fibroblasts from individual 1. The experiment was performed in triplicate (n = 20 cells each), and representative images are shown. The scale bar represents 20 μm. Immunoblot and immunofluorescence data for individual 2 corroborate staining patterns seen in individual 1 and can be found in in Figure S1.

Figure 3

Impaired SV40 NLS-GFP and hnRNPA1 NLS-GFP Import into the Nucleus in Affected Individuals Representative immunofluorescent images showing less GFP (green) transport into the nuclei of affected individuals’ fibroblasts compared to the GFP transport into the nuclei of control fibroblasts. Disruption of NUP188 in affected individuals inhibited the “classic” importinα-importinβ1 nuclear import pathway (SV40 NLS) and the transportin-1 pathway (hnRNPA1 NLS) but not the rate of diffusion (no NLS). DAPI (blue) was used to define the boundaries of the nucleus, and wheat-germ agglutinin (not shown) was used to define the cell boundaries for quantification of the ratio of nuclear GFP fluorescent intensity to cytoplasmic GFP fluorescent intensity. Error bars represent SEM. The scale bar represents 20 μm. The experiment was performed in triplicate with a combined n = 47–70 per condition, representative images shown. ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 by two-tailed unpaired Student’s t test.

Figure 4

Nup188 Deficiency Results in Impaired Sensory Dendrite Tiling Representative images of the interface between C4da neurons ddaC and v’ada. C4da neurons are genetically labeled with ppk-CD4tdGFP. The WT image on the left shows extensive dendrite tiling; a minimal vacant area between the dendrite arbors is outlined in purple. On the right is the Nup188^10B mutant, and a larger vacant area is outlined in red. The scale bar represents 20 μm. The vacant area at the interface not covered by dendrites (the area inside the outline) is quantified. Error bars, SEM. n = 10 neurons from four larvae. ^∗∗∗p < 0.001 by two-tailed unpaired Student’s t test.

Figure 5

Nup188 Deficiency Leads to Decreased Negative Geotaxis and Seizures (A) Schematic depiction of the negative geotaxis apparatus. Vials containing flies are secured in the red container, which is then tapped downward so that flies are knocked to the bottom. The response of flies is video recorded. (B) The average height climbed up from the bottom of the vial by the flies was measured three seconds after tapping. Males (M) and females (F) in each group were tested separately. WT flies climbed significantly farther up the vial than Nup188^10B flies. (C) The average percent of flies showing seizure activity in a 10 s period after tapping. WT males = 9 vials, 210 flies total; Nup188^10B males = 9 vials, 205 flies total; WT females = 6 vials, 149 flies total; Nup188^10B females = 6 vials, 119 flies total. Error bars, SEM. ^∗∗p < 0.01, ^∗∗∗p < 0.001 by one-way ANOVA followed by Tukey’s test.