Fig 1: Segregation studies and in silico pathogenicity assessment of the candidate variants identified in CFAP20.a Pedigree of family A with the segregation analysis of CFAP20 mutation (NM_013242; [M] = c.337 C > T; p.Arg113Trp). Whole-genome sequenced individuals are marked with an asterisk. Below, the genotypes of each individual are displayed (left panel). Electropherogram depiction of family A individuals confirming the co-segregation of the variant with the disease (right panel). b Visualization of the T-Coffee alignment of 11 CFAP20 orthologs using the Jalview program. The conservation annotation histogram (below) shows conservation of the physicochemical properties: an asterisk ‘*’ indicates absolutely conserved residues (score 11), a plus symbol ‘+’ marks columns where physicochemical properties are conserved (score 10); less conserved positions are shown in darker colors with decreasing score. Quality of the alignment based on BLOSUM62, and an alignment consensus row are also shown. Positions are colored white to blue according to increasing sequence identity (BLOSUM62 punctuation). c Three-dimensional modeling showing a cartoon view of human CFAP20 protein. The mutated residue (pink) is in a β-strand secondary structure, depicted as an arrow (left panel). A detailed view of wild-type Arg113 vs mutant Trp113 and its interacting amino acids (Ser110, Thr111, and Thr120) (right panel). Hydrogen bonds are shown as blue dashed lines with the donor-acceptor distances depicted in Å.
Fig 2: Analysis of CFAP20 interaction network, and expression profiles.a Protein-protein interaction (PPI) network analysis of CFAP20 showing common interactions between BioGRID (3.5) and IntAct databases. The PPI map was drawn using Cytoscape v3.8.023. Different colors were employed to mark the interactors with a role in the etiopathogenesis of IRDs and other related disorders, using information from different functional databases (OMIM, Uniprot, etc). Each line represents a PPI identified by a different detection method including validated two hybrid, socioaffinity inference, or coimmunoprecipitation. b Relative expression levels of CFAP20 in commercial cDNA derived from five different human tissues. Depicted is the relative amount of mRNA in retina tissue vs. the other tissues, normalized to the expression of the housekeeping gene GAPDH. All the samples were executed in triplicates. Error bars show SD. c Immunohistochemical analysis of CFAP20, using rabbit polyclonal anti-GTL3 antibody (ab225952; alias symbol of CFAP20), on paraffin-embedded sections of human eye of unaffected donors. Magnification: 40x (left) and 60x (right). Scale bars: 50 µm (left) and 20 µm (right). Immunostaining of the tissue sections showed strong positive staining (brown) of CFAP20 in the inner segment of the photoreceptors, followed by the outer plexiform layer, the nucleus of the cells of the inner nuclear layer, and the nucleus of the ganglion cells (arrows). GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, IS inner segment, ONL outer nuclear layer, OPL outer plexiform layer, OS outer segment.
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