Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Abstract

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

Mutations inSPECC1L, encoding sperm antigen with calponin homology and coiled-coil domains 1-like, are found in some cases of autosomal dominant Opitz G/BBB syndrome

“…All three mutant SPECC1L-GFP proteins show a largely punctate expression pattern with poor, non-contiguous association with stabilised microtubules, in contrast to a robust, elaborate network of stabilised microtubules seen with wildtype SPECC1L-GFP. Interestingly, while Gln415Pro and Thr397Pro lay in the second coiled-coil domain, the Gly1083Ser mutation is located in the C-terminal CHD which was also previously shown to be required for SPECC1L stabilisation of microtubules (figure 3M–O) 20. Synthetic mutations outside these domains do not strongly affect microtubule stabilisation (data not shown) 20.…”

“…Interestingly, while Gln415Pro and Thr397Pro lay in the second coiled-coil domain, the Gly1083Ser mutation is located in the C-terminal CHD which was also previously shown to be required for SPECC1L stabilisation of microtubules (figure 3M–O) 20. Synthetic mutations outside these domains do not strongly affect microtubule stabilisation (data not shown) 20. Thus, mutations affecting the second coiled-coil domain or the CHD are likely to affect the same aspect of SPECC1L function and result in similar phenotypes.…”

“…Expression of SPECC1L-GFP results in stabilisation of a subset of microtubules (figure 3A) that appear in a lattice-like pattern and colocalise with acetylated α-tubulin (figure 3B,C), as previously described 20. We used this property of SPECC1L as a functional assay to assess the effect of SPECC1L Thr397Pro and Gly1083Ser mutations.…”

“…Of these, 27 variants (see online supplementary table S2) were shared between the two affected patients (proband and mother), but not in the healthy father (figure 1A). Those variants included a missense variant c.1189A>C (p.Thr397Pro) in SPECC1L (mendelian inheritance in man (MIM) 614140; NM_015330.3), which is required for proper facial morphogenesis 20. The mutation was absent from 1000 Genomes Project, ESP6500SI, or additional exome sequencing data of over 1500 WES samples that we had previously sequenced in our inhouse database.…”

“…The p.Thr397Pro and p.Gly1083Ser mutations were created in full-length in a murine SPECC1L-GFP expression construct, described previously,20 using the Q5 site-directed mutagenesis kit (NEB, Ipswich, Massachusetts, USA) according to manufacturer's protocol. The p.Gln415Pro and C-terminal calponin homology domain truncated (ΔCHD) constructs were created previously 20…”