KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes

Abstract

KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies.

Figure 1

Phenotypes of individuals with KIF7 mutations. (a) Coronal section of the cerebral hemispheres showing lateral ventricles (LV) dilatation in fetus 3 of family 1. (b) Transverse section of the brainstem at the midbrain-hindbrain junction showing deep interpeduncular fossa (arrow), malformed and stretched aqueduct of Sylvius (double arrow) in fetus 3 of family 1. (c,d) Postaxial polydactyly and hallux duplication in fetus 3. (e) Pedigree of family 1. (f,i,l) Sagittal brain MRI section in individuals ACLS-T1, ACLS-T3 and ACLS-T4 showing the corpus callosum agenesis (f,i) or the thin corpus callosum (l), dysplastic superior vermis (arrows) or dilated fourth ventricle (asterisks). (g,h,j,k,m,n) Axial view of the cerebral hemisphere showing corpus callosum agenesis (g,j) or hypoplasia (m). (h,k,n) Axial view of brainstem abnormalities with deep interpeduncular fossa and stretched cerebellar peduncles in three individuals with ACLS. (o) Hallux duplication in ACLS-T1. (p) Hallux duplication in ACLS-N2. (q) Hallux duplication and postaxial polydactyly in ACLS-T4. (r) Schematic representation of KIF7 complementary DNA and protein with the functional domains (kinesin motor, Gli-binding and coiled-coil domains) and localization of KIF7 mutations identified in this study (ACLS and HLS mutations are shown above the scheme and heterozygous variations are shown below the scheme). The mutation found in HLS family 1 is shown in red.

Figure 2

Missense KIF7 variants are pathogenic and may interact genetically with other BBS loci. (a) Representative live lateral views of control (left) or kif7 morphants (right) imaged at 30 h post-fertilization. (b) Magnified view of a. Chevron-shaped somites in control (left) or abnormally shaped somites in kif7 morphant (right) embryos, attributable to ectopic Hedgehog signaling in the zebrafish myotome. The dashed blue line denotes the somite angle measured in the in vivo complementation assay to test KIF7 allele pathogenicity. (c) All eight missense KIF7 alleles identified in cases with ciliopathy were hypomorphic as indicated by the partial ability of mutant human KIF7 mRNA to rescue the somite angle defect induced by kif7 morpholino (MO). The p.Arg1325Gln coding allele (rs73477443; present in the dbSNP database and not in cases of ciliopathy) was not significantly different (NS) from wild-type rescue, providing support for the specificity of the assay. We measured n = 43–71 embryos per injection. The asterisks indicate P < 0.001 for mutant versus wild-type rescue. Error bars, s.e.m. See Supplementary Table 4 for somite measurement data. (d) Co-injection of sub-effective doses of kif7 morpholino and four different bbs gene morpholinos resulted in gastrulation phenotypes in mid-somitic embryos that exceed the sum of affected embryos induced by either individual MO alone. For all co-injections, we observed a marked increase in the proportion of affected embryos with severe phenotypes (Class II; bbs1 and bbs7) or moderately affected embryos (Class I; bbs9 and bbs10), which exceeded the additive effects of kif7 translation-blocking morpholino or bbs morpholino alone. Objective phenotypic criteria have been described previously. We scored n = 71–86 embryos per injection cocktail and repeated masked scoring twice.

Figure 3

Hedgehog signaling in individuals with KIF7 mutations. (a) Heat map representation of microarray expression analysis in the three KIF7 mutated fetuses from family 1 compared to three aged-matched controls (Supplementary Table 4). The heat map shows the upregulation of many of the GLI1, GLI2 and GLI3 direct and/or secondary target genes (upregulated genes in red boxes), confirmed by semiquantitative RT-PCR in fibroblasts for several selected genes relative to GAPDH (b). Error bars, s.d. (c,d) Protein blot analysis for GLI3 on proteins extracted from control and KIF7 mutated fibroblasts. (c) Immunoblot shows the amounts of GLI3-A (GLI3-FL) and GLI3-R (bands are in accordance with a previous study and included a Gli3^−/− mouse extract as a control) in control and case fibroblasts and revealed reduced GLI3-R in KIF7 mutated fibroblasts. (d) Graphical evaluation of the GLI3-FL:GLI3-R ratio in control and case fibroblasts. We used β-actin as the loading control.