Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation

Abstract

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox-Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor.

Figure 1

Subject IS10-017a1 frontal (a) and side view (b) at age 8 years and subject IS10-017a2 frontal (c) and side view (d) at age 7 years demonstrating ocular hypertelorism and anteverted nares. Both subjects had normal head circumference, and were non-ambulatory with notable persistent hypotonia.

Figure 2

Representative EEG tracings of subjects with KPNA7 mutations showing evolution from infantile spasms to Lennox–Gastaut syndrome. (a) Subject IS10-017a1 at age 7 months showing ictal electrodecrement with background delta activity. (b) At age 24 months she had multifocal epileptiform discharges with high amplitude slowing. (c) Subject IS10-017a2 at 4 months with ictal electrodecrement time-locked with an epileptic spasm. (d) At 4 years and 11 months, her EEG showed slow spike and wave discharges followed by a period of abrupt attenuation with superimposed fast frequency activity consistent with tonic seizures. Measurement bar: vertical axis=100 μV; horizontal axis=1 s.

Figure 3

Brain MRI of subject IS10-017a1 showing thinning of the splenium of the corpus callosum (arrowhead) and cerebellar vermis hypoplasia (arrow) on T1 sagittal (a). The loculated nature of the posterior fossa cysts can be seen on T1 axial (b and c; arrows). The pillars of the fornix were asymmetric, as seen on T1 axial (d; arrow) and T1 coronal (e; arrow) views. Her sister, subject IS10-017a2, had similar findings of partial agenesis of the corpus callosum (arrowhead) and cerebellar vermis hypoplasia on T1 sagittal (f). The posterior fossa cysts were similarly loculated on T2 axial views (g and h; arrows). The pillars of the fornix appeared normal on T2 axial (i; arrow) and T1 coronal (j; arrow) views.

Figure 4

Compound heterozygous mutations c.1015C>G and c.1030G>C in KPNA7 identified by WES of subject IS10-017a1 were confirmed by Sanger sequencing in both of the affected subjects and parents. The c.1015C>G (p.(Pro339Ala)) mutation (a) and the c.1030G>C (p.(Glu344Gln)) mutation (b) were present in both affected subjects in a compound heterozygous manner. The c.1015C>G (p.(Pro339Ala)) mutation was carried by the mother and the c.1030G>C (p.(Glu344Gln)) mutation was carried by the father. Neither mutation was found in the unaffected siblings.

Figure 5

Amino-acid substitutions in the nuclear import receptor KPNA7 map to a helix implicated in NLS binding. (a) Ribbon structure of KPNA2 (green) with the bipartite NLS from Rb (red) generated using PDB 1PJM. The KPNA7 model (purple) was produced by threading the KPNA7 sequence onto IPJM. The amino-acid substitutions identified in the affected subjects are indicated on both models. (b) Sequence alignment from helix 3 of ARM 7 from human KPNA proteins. Positions of amino-acid identity across 5 or more family members are shown in bold, and the KPNA7 substitutions are marked with arrows. (c) Binding assays performed with the bipartite NLS of Rb immobilized on beads and WT and Glu354Gln (E354Q) forms of KPNA2. The full colour version of this figure is available at European Journal of Human Genetics online.