Recurrent mutations in known autosomal recessive non-syndromic hearing loss (ARNSHL) genes in 6 Pakistani families.

<p>Acc. No., accession number of reference sequence; Chr, chromosome; Ex, exon; EVS, exome variant server; In, intron; SNPs, single nucleotide polymorphisms; Ref, references.</p

Effect of <i>TMC1</i> intronic mutation c.362+18A>G using a minigene approach.

<p>Electropherogram of the partial cDNA sequence of RNA derived from cells transfected with the pCI-NEO with either the mutant or wildtype <i>TMC1</i> exon 8. The mutation leads to the insertion of 17bp at the 3′end of exon 8, which can be predicted to result in a premature stop codon in exon 9 (p.Glu122Tyrfs*10).</p

Predicted effect of mutation c.726C>G (p.Cys242Trp) on the three dimensional structure of TMPRSS3.

<p>A) Wild type protein structure with an intact disulphide bridge showing position of the mutated residue (magenta). B) Close-up view of the structure showing the wild type residue cysteine (green) and the mutant residue tryptophan (red). In case of the mutant residue there will be no disulphide bridge at this position.</p

Spectrum of recurrent <i>GJB2</i> mutations in Pakistani families with autosomal recessive non-syndromic hearing loss (ARNSHL).

<p>As reference sequence NM_004004.5 was employed. EVS, exome variant server;</p>#<p>The pathogenicity of this mutation is controversial.</p

Novel mutations identified in known genes for autosomal recessive non-syndromic hearing loss (ARNSHL) in the current study.

<p>Acc. No., accession number of reference sequence; Chr, chromosome; Ex, exon; EVS, exome variant server; hg19, human genome assembly 19; In, intron; NA, not applicable; ND, not determined; SNPs, single nucleotide polymorphisms; PhyloP, phylogenetic P-values; Polyphen, polymorphism phenotyping; SIFT, sorting intolerance from tolerance.</p

Pedigrees and the segregation of novel mutations in known deafness genes.

<p>Unfilled circles indicate unaffected females, unfilled squares indicate unaffected males, filled circles indicate affected females, filled squares indicate affected males, double lines represent consanguineous marriages, slashed line across the symbols indicate deceased individual, + indicates wild type allele, M indicates mutant allele.</p

Effect of MYO15A c. 9948G>A using a minigene approach.

<p>An agarose gel containing RT-PCR products detected from HEK293T cells transfected with the wildtype and mutant minigene construct and a schematic representation of the identified splicing products. The RT-PCR products were verified by sequence analysis. The c.9948G>A mutation leads to skipping of exon 61.</p

NINL interactome screen identifies MICAL3.

<p>(<b>a</b>) Strep-SILAC and TAP (tandem affinity purification) experiments show that NINL interacts specifically with MICAL3 (Yellow). The solid line between NINL and MICAL3 symbolizes a direct interaction, whereas the dashed lines indicate interactions determined by IP. (<b>b</b>) Co-immunoprecipitation of eGFP-MICAL3 with FLAG-NINL^isoB, but not with FLAG-STRAD. The immunoblot (IB) in the top panel shows that eGFP-tagged MICAL3 co-immunoprecipitated with FLAG -tagged NINL (lane 2), whereas FLAG-tagged STRAD used as a negative control (lane 3) did not. The anti-GFP immunoprecipitates are shown in the middle panel; protein input is shown in the bottom panel. Reciprocal IP experiments using anti-FLAG antibodies confirmed the co-immunoprecipitation of eGFP-tagged MICAL3 with FLAG-tagged NINL^isoB (lane 2) and not with STRAD (lane 3) shown in the top panel. The anti-FLAG immunoprecipitations are shown in the middle panel; protein input is shown in the bottom panel. A co-immunoprecipitation experiment using untagged eGFP as a negative control (right panel) showed that eGFP-tagged MICAL3 immunoprecipitates with FLAG-tagged NINL^isoB but not with untagged eGFP.</p

Genetic interaction between <i>ninl</i> and <i>cc2d2a</i>.

<p>(<b>a-d</b>) Partial <i>ninl</i> knockdown enhances the cystic kidney phenotype of <i>cc2d2a</i> mutants. (<b>a-c</b>) Glomerulus and proximal pronephric tubules highlighted in the transgenic line Tg(wt1b-EGFP). (<b>a</b>) Injection of a low dose of <i>ninl</i> atgMO (0.75 ng/nl) causes no cysts in wild-type larvae. (<b>b</b>) <i>cc2d2a</i>-/- larvae display small dilatations of the proximal tubules (arrow) in ~40% of cases. (<b>c</b>) Injection of this low dose of <i>ninl</i> atgMO in the <i>cc2d2a</i>-/- background leads to large dilatations of the proximal tubules and glomerular space (arrow) in 89% of mutants. <b><i>g</i></b> glomerulus, <b><i>p</i></b> pancreas. (<b>d</b>) Quantification of the glomerular + proximal tubular area displayed as a scatter plot, demonstrating a significant increase in proximal pronephric area in <i>cc2d2a</i>-/- larvae injected with low-dose <i>ninl</i> atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint is an individual fish. (<b>e-g’</b>) Immunohistochemistry with anti-opsin antibody (4D2, green) on retinal cryosections of 4dpf <i>cc2d2a</i>-/- uninjected larvae (<b>f-f”</b>) and <i>cc2d2a</i>-/- larvae injected with subphenotypic doses of <i>ninl</i> MO (<b>g’g”’</b>), that cause no mislocalization in wild-type fish (<b>e-e’</b>), demonstrates that partial <i>ninl</i> knockdown increases the mislocalization of opsins (<b>e’-g’</b>). (<b>h</b>) Quantification of the mean intracellular fluorescence displayed as a scatter plot shows significant increase in intracellular fluorescence in <i>cc2d2a</i>-/- larvae injected with low dose of <i>ninl</i> atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint represents the mean intracellular fluorescence from 10 photoreceptors in one individual fish. Cell membrane and outer segments are stained with bodipy (red in <b>e-</b>g). Nuclei are counterstained with DAPI. Scale bars are 100 μm in (a-c) and 4 μm in (e-g’). (<b>i</b>) Pedigree of a consanguineous family with one affected boy (UW48-3) and 4 unaffected siblings. UW48-3 carried a homozygous missense <i>CC2D2A</i> mutation as well as a frameshift mutation in <i>NINL</i> leading to premature truncation. (<b>j</b>) Pedigree of a family where the affected individual (UW36-3) carries the same homozygous <i>CC2D2A</i> mutation as in (<b>i</b>) but no additional rare deleterious variants. (<b>k</b>) Pedigree of a family where the affected individual (UW07-3) carries compound heterozygous <i>C5ORF42</i> frameshift mutations and a nonsense mutation in <i>NINL</i>. (<b>l</b>) Pedigree of a family where the affected individual (UW57-3) carries compound heterozygous <i>TMEM67</i> mutations and a missense <i>NINL</i> mutation. The phenotype of the affected individuals is detailed in <i>italic</i> on each pedigree under the corresponding mutations. <i>MTS</i> Molar Tooth Sign, <i>DD</i> Developmental Delay, <i>ESRF</i> End-Stage Renal Failure.</p

CC2D2A and NINL co-localize at the ciliary base in hTERT-RPE1 cells and in zebrafish retina.

<p>(<b>a, a’</b> and inset) When expressed alone, eCFP-tagged CC2D2A (green signal) localizes to the ciliary base (basal body, accessory centriole). The cilium is marked by anti-polyglutamylated tubulin (red signal, <b>a’</b> and inset). eCFP-tagged CC2D2A (green signal; <b>b</b>) also (partly) localizes to the ciliary transition zone, which was visualized using anti-RPGRIP1L as a marker (red signal; <b>b</b>). (<b>c, c’</b> and inset) mRFP-tagged NINL isoform B was localized at the ciliary base (cilium in green, <b>c’</b> and inset). (<b>d</b> and inset) mRFP-tagged NINL isoform B (red signal) localizes adjacent to the ciliary transition zone (anti-RPGRIP1L; green signal). (<b>e-e”</b> and inset) Co-expression of mRFP-tagged NINL isoform B (red signal) and eCFP-tagged CC2D2A showed co-localization of both proteins around the ciliary base (yellow signal). (<b>f</b>) In wild-type larval zebrafish retina (4 dpf), Cc2d2a marked by anti-Cc2d2a antibodies (red signal) is localized apically to the photoreceptor basal body (marked by anti-centrin antibodies, green signal). (<b>g</b>) Ninl, stained with anti-Ninl antibodies, (red signal) is localized at the zebrafish photoreceptor ciliary base, partially overlapping with and apical to the green centrin signal. (<b>h</b>) Cc2d2a localization is unaffected by <i>ninl</i> knockdown and (<b>i</b>) Ninl localization is normal in <i>cc2d2a</i>^<b>-/-</b> larvae. (<b>j</b>) Schematic representation of the localization of Ninl and Cc2d2a in zebrafish photoreceptor cells. (<b>f-i</b>) are immunostainings on cryosections from 4 dpf larvae. Nuclei were stained with DAPI (blue signal) in all panels. Scale bars are 10 μm in a-e, and 4 μm in f-i.</p