Clinical and experimental evidence suggest a link between KIF7 and C5orf42-related ciliopathies through Sonic Hedgehog signaling

Abstract

Acrocallosal syndrome (ACLS) is an autosomal recessive neurodevelopmental disorder caused by KIF7 defects and belongs to the heterogeneous group of ciliopathies related to Joubert syndrome (JBTS). While ACLS is characterized by macrocephaly, prominent forehead, depressed nasal bridge, and hypertelorism, facial dysmorphism has not been emphasized in JBTS cohorts with molecular diagnosis. To evaluate the specificity and etiology of ACLS craniofacial features, we performed whole exome or targeted Sanger sequencing in patients with the aforementioned overlapping craniofacial appearance but variable additional ciliopathy features followed by functional studies. We found (likely) pathogenic variants of KIF7 in 5 out of 9 families, including the original ACLS patients, and delineated 1000 to 4000-year-old Swiss founder alleles. Three of the remaining families had (likely) pathogenic variants in the JBTS gene C5orf42, and one patient had a novel de novo frameshift variant in SHH known to cause autosomal dominant holoprosencephaly. In accordance with the patients' craniofacial anomalies, we showed facial midline widening after silencing of C5orf42 in chicken embryos. We further supported the link between KIF7, SHH, and C5orf42 by demonstrating abnormal primary cilia and diminished response to a SHH agonist in fibroblasts of C5orf42-mutated patients, as well as axonal pathfinding errors in C5orf42-silenced chicken embryos similar to those observed after perturbation of Shh signaling. Our findings, therefore, suggest that beside the neurodevelopmental features, macrocephaly and facial widening are likely more general signs of disturbed SHH signaling. Nevertheless, long-term follow-up revealed that C5orf42-mutated patients showed catch-up development and fainting of facial features contrary to KIF7-mutated patients.

Fig. 1

Craniofacial features of patients with biallelic KIF7 or de novo SHH variants. a–d Deceased female patient 1 at 2 years and 2 months a–c and in adulthood d whose mother was shown to be a heterozygous carrier of a KIF7 variant; e–h male patient 2 with pathogenic KIF7 biallelic Swiss founder variants at 3 months (e), 3 years (f) and 33 years (g, h) of age; i–l the previously reported male patient (case 4 of Putoux et al. 2012) with the same biallelic variants as patient 2 at the age of 3 months (i, j) and 26 years (k, l); m, n female patient 3 with KIF7 homozygous Swiss splice site pathogenic variants at 5 months (m) and 21 years of age (n); o–p male patient 13 with a de novo SHH variant at 29 years. Note the shared facial features of broad and high forehead, hypertelorism, flat nasal root, thin upper and everted lower lips, and retracted but relatively large chin. Patient 3 (m, n) shows the mildest dysmorphism corresponding with the milder intellectual disability, likely due to the leakiness of the splice site variant

Fig. 2

Clinical features of patients with C5orf42 biallelic variants. a–d male patient 9 at 1 year 3 months (a–c) and 9.5 years (d); e–i female patient 10 at 2 years 8 months (e), about 6.5 years (f) and about 14 years (h, i); j–k female patient 11 at about 2.5 (j) and 7.5 (k) years of age; l–m patient 12 (brother of patient 11) at 8 months (l) and 6 years (m) of age. Note the hallux duplication in patients 9 (c) and 10 (g) and ACLS-like craniofacial features in all patients being more evident in early childhood but becoming milder with time

Fig. 3

Evaluation of the primary cilia and response to the SHH agonist SAG in fibroblasts of patients 11 and 12 with compound heterozygous (likely) pathogenic variants in C5orf42 compared to the controls. a, b Representative fibroblast images from a control and patient 12 immunostained for acetylated-α-tubulin (green). c, d Fibroblast cultures of the patients showed significantly fewer and shorter primary cilia compared to those of the controls. Results are expressed as mean values ± SD (*P < 0.05, **P < 0.001, Mann–Whitney). e, f qPCR analysis of GLI1 and PTCH1 expression in control and patient fibroblasts following 48 h serum starvation and subsequent 48 h treatment with SAG. After treatment, patient fibroblasts showed significantly lower level increase in the expression of GLI1 and PTCH1 transcripts compared to the controls. Results are expressed as mean values ± SEM (*P < 0.05, **P < 0.001, t test)

Fig. 4

Silencing C5orf42 in the developing neural tube of chicken embryos resulted in pathfinding errors of commissural axons at the midline. The pathfinding behavior of dI1 commissural axons at the floor plate was analyzed in open-book preparations of the spinal cord (a; see Methods section for details). Axonal trajectories (red) are visualized by the injection of DiI into the area of commissural neuron cell bodies. In untreated control embryos, commissural axons grew ventrally, entered the floor plate to cross the midline and turned rostrally at the floor-plate exit site (arrows, b). No difference in commissural axon pathfinding was observed in control-treated embryos (c). In contrast, after silencing C5orf42 in the neural tube, dI1 commissural axons failed to cross the midline, stalled in the floor plate (arrow heads) and also failed to turn rostrally along the contralateral floor-plate border (open arrow, d). The floor plate is indicated by dashed lines. Bar: 50 µm. Quantification of phenotypes as average percentage of DiI injection sites per embryo with the given phenotypes (e, shown with SEM; *P < 0.05). Green bars for untreated control embryos, blue bars for control-treated embryos-expressing GFP, red bars for embryos after RNAi-mediated silencing of C5orf42 (LOF, loss-of-function). In addition, silencing C5orf42 in cranial neural crest cells of developing chicken embryos resulted in facial dysmorphism. In comparison with the control-treated embryos (f), embryos lacking C5orf42 in cranial neural crest cells developed aberrant facial features (g). The nasal structure (arrowhead) was much wider than in age-matched control heads stained with Alcian Blue. Similarly, the jaw was broader in experimental compared to control embryos (indicated by black line) and eye distance was increased