PMC9947397_01
Phenotype of index patient with compound heterozygous NUP85 mutation. (A) Pedigree. (B) Pictures of affected individual (II.2). (C) MRI images of the head (sagittal) T2 images of II.2 shows reduced brain volume. (D) Representation of identified compound heterozygous variants by whole genome sequencing in the NUP85 cDNA c.454C > A in exon 6 (maternally inherited) and c.487C > A in exon 7 (paternally inherited) and NUP85 wild-type protein (p.L152I, p.L163I). (E) Mutations lie in the highly conserved region of the NUP85 protein across species. (F) Unchanged levels of total NUP85 protein in II.2-derived fibroblasts compared to controls [NUP85 (75kDa), actin (43kDa) (loading control)] (n = 3, one-way ANOVA, Tukey's multiple comparison test, p = 0.2973). (H) Cell viability in II.2-patient derived fibroblasts is significantly reduced compared to controls (n=8, one-way ANOVA, Tukey's multiple comparison test, ****p < 0.0001). (I) Structural overlay of human NUP85 based on PDBid 7R5K (purple) and the L152I/L163I double mutant (wheat). Top panel: Overlay of the overall structures; bottom panel: magnification of the hinge region (indicated by red line), the structures have been overlaid using the central region of the molecules. The mutations are located in this hinge region and are indicated in ball and stick mode. (J) Significant change of the interaction pattern due to mutations. Magnification of the interaction patterns as evaluated using the program Arpeggio under standard settings. Top panel wild-type, bottom panel-double mutant. The mutated residues are indicted in the colors as in (I).
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F1
The index patient (II.2) was a 3.6-year-old boy born at term as a second child to non-consanguineous parents without complications (Figures 1A, B). Primary microcephaly was already severe at birth with the occipitofrontal head circumference (OFC) of 31 cm [-3 standard deviations (SD), <3rd centile] (Supplementary Figure S1, Supplementary material) (Table 1). At the age of 3 years, the OFC was 46 cm (<-3 SD), while the weight (11 kg, -2 SD) and height (86 cm, -1 SD) were normal (Supplementary Figure S1). The boy displayed facial dysmorphism (almond-shaped eyes, simplified ears, short philtrum) and Pes adductus (Figure 1B). Cranial MRI at 1.4 years of age revealed reduced global brain volume and delayed myelination (Figure 1C). To identify the underlying genetic cause of the disease phenotype, we performed the whole genome sequencing (WGS) in the index family and identified compound heterozygous missense mutations in the NUP85 gene (NM_024844.5) in the index patient: c.454C > A, g.73211897C > A (inherited from mother) and c.487C > A, g.73214291C > A (inherited from father) (Figure 1D). NUP85 protein leading to an exchange of leucine to isoleucine (p.L152I and p.L163I) (NP_079120.1) (Figure 1E). No other variants were identified that met the filtration criteria in the WGS analysis. Western blot analysis on patient-derived fibroblasts revealed the unaltered levels of NUP85 protein between controls and patient samples, indicating the presence of mutant protein (Figures 1F, G) (n = 3, one-way ANOVA). Cell viability was significantly reduced in the patient-derived fibroblasts compared to controls (Figure 1H, n = 8, one-way ANOVA). Since both the mutations L152I and L163I are located at the end and the beginning of two helices with the linker in between as part of the MHD, it is predicted to interfere with the helix arrangement in the MHD region, which in turn alters its orientation and might affect the interaction of NUP85 with NUP214 complex on the cytoplasmic side and NUP205 on the nuclear side of the complex (Figures 1I, J).