A recurrent de novo mutation in ACTG1 causes isolated ocular coloboma

Abstract Ocular coloboma (OC) is a defect in optic fissure closure and is a common cause of severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole‐exome sequencing (WES) was used to analyze 12 trios (child affected with OC and both unaffected parents). This identified de novo mutations in 10 different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1, and LCP1. Proband‐only WES identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra‐ocular signs of Baraitser–Winter syndrome. We found this mutant protein to be incapable of incorporation into F‐actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin interactions, and no further plausibly causative variants were identified in these genes on resequencing 380 unrelated individuals with OC.

guides were each cloned into SpCas9n-2A-GFP plasmids (Addgene). The repair template includes conservative nucleotide changes to reduce subsequent HDR events at successfully edited loci. These sequences are available on request. RNA was prepared and zygote injections were performed as described previously (McEntagart et al., 2015) (Ran et al., 2013b). Sanger sequencing of embryos collected at E14.5 indicated a specific homozygous p.(Pro70Leu) encoding mutation resulting from HDR of the co-injected repair template at both actg1 alleles. Western blots using an Actg1-specific antibody against cell lysates from primary MEF cultures derived from the collected embryos. Three wild-type, one null mutation arising from a non-specific homozygous editing event, and the Actg1-Pro 70 cell line, were used for analysis.
Signal is absent from the null, but present in all other samples. (b) HEK293 expressing stable single-integrated CDS of either Wt or mutant Actg1 tagged with Nterminal eGFP revealed reduced Leu 70 incorporation into F-actin, in contrast to wild type. (c) The mutant construct did not markedly affect endogenous F-actin or G-actin, as tested by phalloidin or DNase1 fluorophore-conjugates, respectively. (d) Left: In silico disorder predictions indicate that the C-terminal tail region of Twinfilin-1, that encompasses Proline 349 (using the full-length Twf1 isoform NM_001242397.1) is intrinsically unstructured (consensus disorder shaded green). Right: The second ADFhomology domain of Twinfilin-1 solved in complex with G-actin is shown. The folded domain of Twinfilin-1 extends only to Lys 315 , and therefore the location of the disordered tail region with respect to the actin-binding region is unknown. Below: Plasmid constructs expressing FLAG-Actg1 were transiently transfected into stable TET-inducible Twinfilin-1:GFP HEK293 cells and were subjected to coimmunoprecipitation against FLAG epitopes. Twf1-GFP Ser 349 displayed increased binding to FLAG-actg1 compared to WT. (e) Left: the LCP1 side-chain of Asn 608 is exposed on the surface of the protein and therefore is unlikely to greatly destabilize the protein structure. Right: Co-sedimentation assays of TET-inducible Lcp1:GFP HEK293 cells revealed increased Ser 608 compared to WT in the F-actin phase.

UK10K Exome Sequencing
Whole exome sequencing was performed as part of the rare disease component of the UK10K project. This study was approved by the UK Multiregional Ethics Committee (Reference: 06/MRE00/76), and informed consent was obtained from the participating families. Exome sequencing was performed as described (Ramu et al., 2013;McEntagart et al., 2015), briefly aligned using bwa 0.5.9, duplicates marked with Picard 1.43, realignment around indels and base quality scores recalibrated with GATK 1.0.5506, and variants called only with GATK Unified Genotyper. All exome data is available from European Genome-phenome Archive (https://www.ebi.ac.uk/ega) under accession EGAS00001000127. Candidate de novo mutations were identified using DeNovoGear output with a prior probability > 0.1 and were each validated using Sanger sequencing. RNA was not analysed. Nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence. The primers used for validation are available on request.

TET-Inducible Cell Lines
Multiple independent stable tetracycline-inducible Flp-In T-REx 293 (Thermo Scientific) cell lines expressing the wild-type and each variant of Actg1, Twf1 and Lcp1 as full-length N-terminal GFP-fusion proteins were derived from mouse orthologous open reading frames, as previously reported (Rainger et al., 2014).

Actin Filament Co-Sedimentation
Co-sedimentation assays were performed using the G-actin/F-actin Kit (cytoskeleton, Inc) and a TLA-100 Ultracentrifuge (Beckman). Coimmunoprecipitation was performed using the GFP-TRAP_A system (Chromotek), or using FLAG M2 beads (Sigma). The N-terminal FLAG-tagged ACTG1 construct was generated using the pJNK103 DB3.1 vector, a kind gift from Professor Alan Wright, The MRC Human Genetics Unit, Edinburgh, UK.

Mass Spectrometry
Mass spectrometry methods are presented in detail on the Pride EBI (http://www.ebi.ac.uk/pride/). Briefly, reductive dimethylation reactions were performed by adding formaldehyde isotopes CH 2 O or CD 2 O, to generate 'light' and 'heavy' labelled peptides for wild type and mutant samples respectively. Light and heavy labelled peptides were mixed for each sample set. LC-MS/MS was performed by coupling a RSLCnano LC system (Thermo Scientific) to a micrOTOF-II mass spectrometer (Bruker, Germany). Data tables are provided in Additional Table S2.

Western Blotting
For western blots, denatured and reduced protein samples were run on 4-12 % Novex Bis-Tris SDS PAGE gels with NuPAGE MOPS system reagents (Thermo Scientific). Proteins were transferred to nitrocellulose membranes and incubated with 2% BSA -TBS tween-20 (0.1 %) blocking buffer before incubation in primary antibodies overnight at 4 o C. Antibodies used and their dilutions are described in the

Sequence and 3-D structure analysis and in silico mutagenesis
The empirical forcefield FoldX (Guerois et al., 2002;Schymkowitz et al., 2005)  Initiative) was used to assess the impact of Asn608Ser, by FoldX. The crystal structure of rabbit actin bound to mouse twinfilin-1 (PDB ID: 3DAW) (Paavilainen et al., 2007)was initially assessed to locate the position of the Pro349Ser mutation.
Because the C-terminal tail region is located outside the folded domain, a disorder prediction was undertaken on twinfilin-1 isoform 3 with the D 2 P 2 database (Oates et al., 2013), that utilises a range of disorder predictors to highlight potential regions of disorder. Intra-protein residue interactions were determined using the Protein Interactions Calculator (PIC) (Tina et al., 2007). Inter-atomic clashes were analysed using What IF (Vriend, 1990). Surface accessibility was assessed using ASAView (Ahmad et al., 2004). PyMol (The PyMOL Molecular Graphics System, Schrödinger LLC; http://www.pymol.org) was used for 3-D visualisation, analysis and preparation of structure-based figures.