Mutations in KIF7 implicated in idiopathic scoliosis in humans and axial curvatures in zebrafish

Abstract

Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7^co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7^co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.

Keywords: KIF7; exome sequencing; genetic variants; idiopathic scoliosis; zebrafish.

Figure 1.

a. Pedigree of KIF7 variant in IS family. Arrow indicates proband. Individuals without a GC or GG genotype were not enrolled or did not provide DNA. Individual III:6 possessed the GC genotype and was an obligate carrier for the variant. b. Sanger sequencing showing representative family members with the variant and reference genotypes at NM_198525.3:c.3345C>G.

Figure 2.

kif7^co63 zebrafish display progressive scoliosis that starts at the juvenile period, with an absence of other gross vertebral or other bone abnormalities. A.) Representative anterior-posterior views of juvenile and adult female zebrafish, as shown by micro-CT. Curves on juvenile zebrafish are denoted by white arrows. Pictured adult kif7^co63/co63 zebrafish displays a curve of approximately 68 degrees. B.) Cartilage and bone phenotypes of zebrafish at 33 dpf, as shown by alizarin red/alcian blue staining. White arrows denote separations between vertebrae.

Figure 3.

Characterization of mutation in kif7^co63 in Danio rerio. A.) Genomic DNA sequences of kif7 at exon 3, showing a 5bp deletion/14bp insertion. B.) Kaplan-Meier curve showing survival of wild-type (AB), kif7^co63/+ and kif7^co63/co63 embryos. C.) kif7^co63/co63 adults and embryos show reduced expression of kif7 mRNA.

Figure 4.

kif7^co63/co63 zebrafish show a lack of abnormalities characteristic of ciliopathies. Contrast micro-CT of the brain in wildtype and kif7^co63/co63 zebrafish. Brain does not show hydrocephaly or dilation of the ventricles (arrows).

Figure 5.

Homozygous and heterozygous kif7^co63 zebrafish do not show defects in the formation of central canal structures. Confocal imaging of immunofluorescence of kif7^{co63 /+} (A-A’’, C) and kif7^{co63 /co63} (B-B’’, D) at 5 dpf demonstrating no alterations in cilia (A’, B’) in the central canal (marked by DAPI/nuclei staining (A, B) in kif7^{co63 /co63} mutants (merged in A’’ and B’’) or the Reissner fiber (C, D). Scale bar = 10 microns.

Figure 6.

Gene expression for key hedgehog (Hh) pathway genes within kif7^{co63 /co63} embryos. Smo and Dlg5 appear down- and up-regulated, respectively, while the remaining genes show no differences between WT levels. * indicated p <0.05, ** indicates p < 0.01.