Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures

Abstract

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Figure 1

Family Pedigree and Gene Mapping (A) Pedigree diagram showing all four investigated nuclear families (families 1–4), which interlink into a single extended family. Segregation of the two mutations identified (c.776C>A [p.Ser259^∗] is denoted by X, and c.714_731dup [p.Met241_Gln246dup] is denoted by Dup) is shown (all genotypings were validated by dideoxy sequence analysis). (B) Pictorial representation of the SNP genotype data encompassing the chromosome 19 homozygous (solid box) and compound-heterozygous (dashed box) regions in affected individuals. The locus containing the pathogenic variant is demarcated by SNPs rs2253022 and rs7246244 (2.59 Mb; families 1 and 2).

Figure 2

Kaptin Immunolocalization Studies (A and B) Flag-kaptin colocalized with F-actin-rich foci at the cell body and in growth cones (examples of both are marked by arrow heads in A) in DIV5 rat hippocampal neurons transfected at DIV3. For clarity, the anti-MAP2 immunostaining was omitted from the merged images. Scale bars represent 10 μm. (C and D) Endogenous kaptin immunostained together with the presynaptic marker bassoon at DIV14 (C) and with the postsynaptic marker Shank2 at DIV24 (D). Puncta enriched with anti-kaptin immunoreactivity (marked by arrow heads) were rich in F-actin, as shown by fluorescently labeled phalloidin. (C) Furthermore, they were usually contacted by presynapses. The scale bar represents 5 μm. (D) Postsynapses marked by Shank2 were largely positive for both F-actin and anti-kaptin immunolabeling (arrow heads). For clarity, the anti-F-actin staining was omitted from the merged image in (D). High-magnification images are shown. The scale bar represents 2.5 μm.

Figure 3

Immunolocalization Studies of Altered Kaptin (A–C) Schematic representation of wild-type human kaptin (A), the truncated p.Ser259^∗ (c.776C>A; GFP-kaptin^1–258) (B), and the duplication (GFP-kaptin^{p.Met241_Gln246dup}) (C). On the left is a graphic overview of secondary structures; β sheets are shown as red boxes, and α-helices are shown as blue ellipsoids. On the right are amino acids and secondary-structure elements around amino acid 230. α-helix 4 is predicted to be converted into a β sheet by the insertion of amino acids 241–246, as shown in yellow. (D–F) Localizations of GFP-kaptin, GFP-kaptin^1–258, and GFP-kaptin^{p.Met241_Gln246dup} (green in merges) in COS-7 cells counterstained with phalloidin (red in merged images). Note that whereas wild-type kaptin was distributed in the cytosol and accumulated at F-actin-rich lamellipodia (D), both alterations showed accumulations at perinuclear regions (E and F). (G–I) Transfection of DIV3 rat hippocampal neurons with wild-type and altered kaptin (cells were fixed and imaged at DIV5). Wild-type kaptin was found throughout the cell and showed accumulations at growth cones and F-actin-rich sites of the cell body (G; arrow heads). In contrast, both alterations accumulated in areas of the cell body (H and I; arrows). Merged images show wild-type and altered kaptin in green, MAP2 in red, and phalloidin in blue. Scale bars represent 10 μm.