Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: (A) Pedigree of a large Danish family with moderate hearing impairment, with the proband indicated with an arrow. DNA was available from all individuals except those with a four-digit ID. The presence (+) or absence (-) of the CD164 mutation c.574C>T (p.R192*) is listed underneath each individual. The phenotype of individual (IV-21) was set to unknown (shown in grey) because of uncertainties about the origin of his hearing loss. (B) Audiograms of left and right ear of a representative affected family members (individual IV-5). Mid-frequencies are more severely affected than lower and higher frequencies termed basin shaped or cookie bite hearing loss. The age of the individual at the time of each analysis is indicated. (C) Genome-wide significant linkage to chromosome 6q15-21 was identified in an initial SNP-array analysis including 11 individuals (indicated in yellow, Fig 1A). (D) Chromatograms of the c.574C>T mutation in CD164 exon 6 in an affected family member compared to a healthy control individual. Nomenclature refers to RefSeq NM_006016.4 ( CD164 isoform 1), with nucleotide number +1 being A of the start codon ATG.

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Mutagenesis, Genome Wide, Control

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: (A) The nonsense mutation was identified in exon 6, used by the three membrane bound isoforms of CD164. (B) A schematic representation of CD164 isoform 1. The protein contains two mucin-like domains separated by a cysteine-rich domain . The locations of potential N-glycosylations sites and predicted O-glycosylation sites were predicted from the NetOGlyc 4.0 Server ( http://www.cbs.dtu.dk/services/NetOGlyc/ ). The location of the transmembrane region was predicted from the SMART database (smart.embl-heidelberg.de). (C) Alignment of the amino acid sequence of CD164 different species shows high evolutionary conservation of the C-terminal region including the YHTL sorting motif, which is deleted as a consequence of the nonsense mutation. Isoform 4 and 1 are the most predominantly expressed isoforms across a broad range of tissues. Information on isoforms was accessed from the GTEx Portal ( http://www.gtexportal.org/home/ ).

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Mutagenesis, Membrane, Glycoproteomics, Sequencing

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: HEK-293 cells were co-transfected with plasmid expressing mCherry fused to the C-terminal region (CTR) of wild-type CD164 and eGFP fused to the CTR of CD164-R192* or vice versa . Forty-eight hours post-transfection, live imaging of the cells was performed using a confocal laser scanning microscope using 63×water-immersion objective. (A) Schematic of the constructs (B) Wild-type fusion protein (mCherry-CD164-WT-CTR) (red) was intracellularly located while the truncated fusion protein (eGFP-CD164-R192*-CTR) was primarily present at the plasma membrane. (C) Same result was found when reversing mCherry and eGFP (colour swap).

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Transfection, Plasmid Preparation, Expressing, Imaging, Laser-Scanning Microscopy, Construct, Clinical Proteomics, Membrane

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: HEK cells stably overexpressing wild-type CD164 (CD164 WT) (A, C, E), and truncated CD164 (CD164 R192*) (B, D, F) were seeded on cover slides and were incubated with anti-CD164 antibody on ice. Next, cells were either fixed (T0, 0 min) or incubated at 37°C in complete medium without antibody for 10 (T10) and 30 (T30) minutes, respectively, and then fixed. Finally, CD164 was visualized using Alexa Fluor 488-labeled secondary antibody (green). Nuclear DNA was stained with DAPI (blue). Imaging was performed on a confocal laser scanning microscope using 40×oil-immersion objective. Scale bar = 6 μm.

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Stable Transfection, Incubation, Labeling, Staining, Imaging, Laser-Scanning Microscopy

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: HEK-293 cells were transiently transfected with empty vector, CD164 and CD164 R192*, either untagged or harboring various epitope tags and in various combinations. After 2 days, the cells were (A) lysed in SDS-PAGE sample buffer and analysed by immunoblotting using anti-human CD164 antibody or (B) lysed in immunoprecipitation buffer, whereafter CD164 or CD164 R192* were immunoprecipitated from the cell lysates using anti-HA antibody indicated. Aliquots of the immunoprecipitates or the pre-immunoprecipitation lysates were analysed by immunoblotting using the antibody indicated. SDS-PAGE was performed under reducing conditions. The asterisks indicate a non-specific band.

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Immunoprecipitation

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: Co-transfected HEK cells expressing wild-type CD164 tagged with the FLAG epitope (FLAG-CD164-WT) and truncated endolyn tagged with the HA epitope (HA-CD164-R192*) were seeded on cover slides. Following incubation with anti-FLAG and anti-HA antibodies on ice, the cells were either fixed (T0, 0 min) or incubated in complete medium without antibody for 10 (T10) and 30 (T30) minutes, respectively, and then fixed. FLAG-CD164-WT (A, D, G) and HA-CD164-R192* (B, E, H) was visualized using Alexa Fluor 488-labeled secondary antibody (green) and Alexa Fluor 568-labeled secondary antibody (red), respectively. Nuclear DNA was stained with DAPI (blue). Merged images are shown in (C, F, I). Imaging was performed on a confocal laser scanning microscope using 40×oil-immersion objective. Scale bar = 6 μm.

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Transfection, Expressing, FLAG-tag, Incubation, Labeling, Staining, Imaging, Laser-Scanning Microscopy

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: (A) BLAT alignment of the sequenced cDNA (labeled “CD164 transcript” in the “your sequence from Blat search” track) from peripheral blood from a patient. The transcript does not contain intronic sequence indicating that the sequence is from mRNA. (B) Chromatogram of the c.574C>T mutation showed equal expression of both alleles in peripheral blood cells, demonstrating that the nonsense mutation is not degraded through NMD

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Labeling, Sequencing, Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

doi: 10.1371/journal.pgen.1005386

Figure Lengend Snippet: Immunohistochemistry shows cd164 expression (brown) in the cochlear neurons (arrows in A and C), inner hair cells (ihc) and outer hair cells (ohc) of the organ of Corti (black arrowheads in C), cells of Kolliker’s organ (red arrowhead in C), cells of the lateral wall behind the spiral prominence (open arrowhead in A) and in the stria vascularis (B). Scale bars: A: 10 μm, B, C: 5 μm.

Article Snippet: For CD164 protein expression analysis, cells were thereafter lysed in reducing SDS-PAGE sample buffer and subjected to immunoblotting using sheep anti-human CD164 primary antibody (AF5790) and horseradish peroxidase-coupled anti-sheep secondary antibody (HAF016), both from R&D Systems.

Techniques: Immunohistochemistry, Expressing