Autosomal recessive IFT57 hypomorphic mutation cause ciliary transport defect in unclassified oral-facial-digital syndrome with short stature and brachymesophalangia

Abstract

The 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies.

Keywords: IFT57; ciliopathy; exome sequencing; oral-facial-digital syndrome.

Figure 1. Clinical features of the subjects with unclassified OFDS

A) Family tree with the three affected cases; B and C) Pictures and X-Ray (respectively of subjects II-1 and II-2) showing the clinical hallmarks overlapping OFD (median pseudo cleft of upper lip, oral frenulae, pre- and post-polysyndactyly) and EVCS (missing incisors, short stature with mesomelic limb shortening). Skeleton features included additional brachymesophalangia, stocky femoral necks, and vertebral notches. Brain MRI was normal.

Figure 2. Evidence of the pathogenicity of the IFT57 homozygous p.Lys259Lys mutation

A) Homozygosity mapping in Subjects 2 and 3 identifying 2 shared homozygous runs on chromosome 3 and 10, totaling 11 megabases B) Whole exome sequencing for Subject 1, interpretation and variant prioritization focused on variants included in the homozygous regions. C) Sanger sequencing confirmed the familial segregation of the IFT57 variant in the three affected sibs. D) IFT57 cDNA was quantified in cultured fibroblasts, demonstrating a lower quantity of mRNA in the case than in multiple controls.

Figure 3. The synonymous c.777G>A substitution in exon 6 affects IFT57 pre-mRNA splicing

A) Exon 6 is present in the normal transcripts. The G>A substitution identified at the genomic level is detected in all IFT57 normal mRNAs. B) In frame exon 6 skipping. C) In frame skipping of exons 5 and 6. D) Out of frame skipping of exons 4, 5 and 6. Legend: − puro: no puromycin treatment, + puro: puromycine treatment

Figure 4. Functional impact of the mutation on ciliary transport

A) Immunofluorescence showing decreased anterograde ciliary transport in the subject’s fibroblasts. IFT57 staining was low and restricted in most cells to the ciliary bases compared with control cells, which showed base and tip staining. B) Count of ciliary transport showing comparable ciliary number but decreased ciliary tip staining, black bar: healthy control; grey bar: subject C) Decreased GLI1 transcription activation secondary to lowered SHH signaling pathway in the subject’s fibroblasts compared with healthy controls (representative experiment of 4) and in control fibroblasts after siRNA IFT57 delivery (representative experiment of 3).