An Alteration in ELMOD3, an Arl2 GTPase-Activating Protein, Is Associated with Hearing Impairment in Humans

<div><p>Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in <i>ELMOD3</i> at the <i>DFNB88</i> locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.</p></div

The transcripts and expression profiles of the genes that encode ELMOD3 in humans and mice.

<p>(A) Alternative splicing leads to seven isoforms of human <i>ELMOD3</i>. Non-coding segments, sequences encoding ELMO domain and other coding regions of exons are denoted by gray, blue and black boxes, respectively. Also depicted is the mutation that was identified in the DFNB88 family (red arrow). The numbering of the position of mutation c.794T>C (p.Leu265Ser) is based on accession number NM_032213.4. Mice have only three <i>Elmod3</i> alternative transcripts. Primers used for real-time quantitative PCR analyses are represented with black arrows. (B) Real-time quantitative PCR analysis of human and mouse <i>ELMOD3/Elmod3</i> isoforms <i>A/a</i> and <i>B–E/b–c</i>. (C) The leucine residue at amino acid position 265 (accession number NP_115589) is completely conserved across a wide variety of species. Identical residues are boxed in gray. (D) Real-time quantitative RT-PCR analysis of <i>Elmod3</i> isoforms <i>a</i> and <i>b–c</i> in C57BL/6J mouse cochlear and vestibular tissues at three different ages (P0, P10 and P30). C_T indicates the observed threshold number of PCR cycles that was required for the detection of the amplification product; the relative expression level is the calculated difference in C_T between the <i>Elmod3</i> and that of an internal control standard (<i>Gapdh</i>), which was measured in the same sample.</p

ELMOD3 accumulates at actin-based structures.

<p>(A–B) GFP-ELMOD3 localizes on actin structures (elongated microvilli due to co-expression of espin) but the deafness-causing ELMOD3 mutant does not. (A) CL4 epithelial cells were co-transfected with GFP-ELMOD3 and un-tagged Espin expression vectors. Rhodamine phalloidin (red) was used to reveal F-actin and highlight the microvilli on the CL4 cell surface. GFP-ELMOD3 was efficiently targeted to the microvillar actin bundles and the cell membrane. (B) GFP-ELMOD3 harboring the p.Leu265Ser human deafness-associated mutation retained weak microvillar targeting and cell membrane localization; the protein remained in the cytoplasm and accumulated in the nucleus. (C–D) Representative results of the transfection of P2 C57BL/6J mouse inner ear sensory epithelial explants with expression vectors that encoded either GFP-ELMOD3 or p.Leu265Ser ELMOD3. The <i>x–z</i> and <i>y–z</i> plane projections are also presented (lower panels). (C) An inner hair cell (IHC) in the mouse organ of Corti that was transfected with GFP-ELMOD3 reveals localization along the length of stereocilia (arrowhead) and throughout the hair cell body. (D) Mouse organ of Corti hair cell that was transfected with GFP-ELMOD3 (p.Leu265Ser). No concentration and only negligible fluorescence is observed in the stereocilia (arrowhead), and mutated ELMOD3 remains in the cytosol. Scale bar applies to all panels and is 10 µm.</p

ELMOD3 is localized at the plasma membrane.

<p>STORM imaging revealed close proximity between actin filaments and over-expressed GFP-ELMOD3 at the plasma membrane in MDCK cells. (A) A representative two-color STORM image of transfected MDCK cells is shown. Alexa647 phalloidin was used to label F-actin and a Cy3B-conjugated GFP antibody was used to detect GFP-ELMOD3. In most places the GFP-ELMOD3 (green) signal did not co-localize with actin (red) at the plasma membrane. However, at few spots we found partial overlap between ELMOD3 and actin. (B–C) Magnification of the regions inside the boxes in A. (D–E) Zoomed-in views of the regions inside the boxes in B and C, respectively. Scale bars: 5 µm for A, 1 µm for B and C, 200 nm for D and E.</p

Hearing loss segregating in family PKDF468 is associated with a missense ELMOD3 allele.

<p>(A) Pedigree of the family in which the <i>DFNB88</i> locus was mapped. The filled symbols represent affected individuals, and a double horizontal line connecting parents represents a consanguineous marriage. The alleles forming the risk haplotypes are boxed. The short tandem repeat (STR) markers, their relative map positions (Mb) according to UCSC Genome Bioinformatics Build GRCh37 (hg19), and their genetic positions (cM) based on the Marshfield genetic map are shown next to the pedigree. Haplotype analysis revealed a linkage region of 4.3 cM (0.91 Mb) that was delimited by proximal meiotic recombination in individual V:6 (arrowhead) at marker <i>D2S1387</i> (103.16 cM; arrow) and distal recombination at marker <i>D2S2232</i> (107.46 cM; arrow) in individual V:11 (arrowhead). (B) Pure-tone audiograms for family PKDF468 V:2 (25-year-old female) and V:5 (20-year-old male). The symbols ‘o’ and ‘x’ denote air conduction pure-tone thresholds, and the ‘<’ and ‘>’ symbols denote bone conduction thresholds in the right and the left ears, respectively. (C) The linkage interval that defines the <i>DFNB88</i> locus for family PKDF468 partially overlaps with <i>DFNA43</i>. The relative locations and orientations of the genes and mRNAs are indicated with arrows. Individual V:2 DNA sample was used for exome sequencing, while individuals V:5 and V:11 samples were used for Sanger sequencing of coding, non-coding, and flanking sequences of the exon-intron boundaries of the known candidate genes in <i>DFNB88</i> linkage interval.</p

ELMOD3 is linked to the F-actin cytoskeleton.

<p>(A) In transfected MDCK cells, GFP-ELMOD3 (green) localized with F-actin at the cell membrane. Alexa647-phalloidin (blue) was used to stain F-actin, and a ZO1 antibody was used to label the tight junctions (red). (B) When the cells were treated with cytochalasin D (Cyto-D), GFP-ELMOD3 was internalized, and no or negligible localization at the cell membrane was observed. (C) Four hrs post-Cyto-D treatment, GFP-ELMOD3 re-localized with the actin cytoskeleton at the plasma membrane. Scale bar applies to all panels and is 10 µm. (D) Quantification of membranous GFP-ELMOD3 localization. The normalized ratio of GFP-ELMOD3 and ZO1 signal intensities at the cell membrane (n = 30, ** p<0.01) confirmed the internalization of ELMOD3 following Cyto-D treatment.</p

<i>NARS2</i> mutations identified in two unrelated families.

<p>(A) Pedigree of the LS06 family. Filled symbols represent affected individuals and small circles represent carrier individual. The pedigree shows autosomal recessive inheritance of compound heterozygous NARS2 variants [c.969T>A; p.Tyr323*] and [c.1142A>G; p.Asn381Ser]. (B) SDS PAGE and Western blot of control and patient II.1 muscle homogenates (10μg and 20μg of protein), samples were probed for mitochondrial respiratory chain complexes via MitoProfile total OXPHOS human WB antibody cocktail. The result showed significantly decreased amounts of mitochondrial respiratory complex I and IV. (C) SDS PAGE and Western blot of fibroblast lysates from both affected probands (II.1, II.3), their parents (I.1, I.2) and controls using anti-NARS2 antibody and anti-GAPDH antibody as loading control. The expected position of a truncated NARS2 protein product (Δ154aa) stemming from the p.Tyr323* allele is indicated with a black arrow. (D) Pedigree of the PKDF406 family. Filled symbols represent affected individuals, and a double horizontal line represents a consanguineous marriage. Alleles forming the risk haplotypes are boxed. The short tandem repeat (STR) markers, their relative map positions (Mb) according to UCSC Genome Bioinformatics build GRCh37 (hg19), and their genetic positions (cM) based on the Marshfield genetic map are shown next to the pedigree. A haplotype analysis revealed a linkage region delimited by a proximal meiotic recombination at marker D11S911 in individual IV:4 (arrowhead) and distal recombination at marker D11S4082 in individuals IV:8 and IV:9 (arrowhead).</p

NARS2 homodimerization and RNA level: effect of the p.Val213Phe and p.Asn381Ser mutations.

<p>(A) Immunoprecipitates (IP) with anti-GFP antibodies from HEK293T cells transiently transfected with GFP-tagged (arrowhead) and HA-tagged NARS2 (arrow) constructs. Precipitates were immunoblotted with antibodies to the GFP and HA tags. NARS2 homodimerizes, and the p.Val213Phe mutation does not affect the dimerization process. No dimerization was detected with p.Asn381Ser NARS2 construct. (B) Steady state level for mt-tRNA^Asn was assessed by Northern blot and the results were validated by two independent laboratories. 5S-rRNA probe was used as a loading control on the same membrane. In fibroblasts of patient II.1, from LS06 family, the level of mt-tRNA^Asn is decreased compared to his parents and a control sample. Due to high passage number, we could not measure the mt-tRNA^Asn levels in the fibroblast of patient II.3.</p

Summary of the known aminoacyl tRNA-synthetase genes associated with deafness.

<p>Summary of the known aminoacyl tRNA-synthetase genes associated with deafness.</p

Expression of <i>Nars2</i> in mouse inner ear and brain.

<p>(A) RT-PCR analysis of <i>Nars2</i> expression in the C57Bl6/J mouse cochlear (C), vestibular (V) tissues and brain (B) at different developmental stages (P0, P30 and P90). <i>Gapdh</i> expression was used as an internal control. (B-G) Expression of <i>Nars2</i> in the P2 mouse cochlea is shown. Hybridization signals of <i>Nars2</i> antisense (B, D, F) and sense (control) probes (C) in mid-modiolar sections of P2 cochlea are shown. Positive signals were detected in the cochlear epithelium, including the region of the organ of Corti, (D) as indicated by the Myo15a-positive hair cells (E, bracket). Positive signals were also detected in the cells surrounding the cochlear duct (D, asterisks) and neurofilament-positive spiral ganglion (G, SG). B, C, E and G are 12 mm adjacent sections. Abbreviations: cd, cochlear duct, SG, spiral ganglion. The scale bar in C is 100 μm and applies to B and C. The scale bar in G is 100 μm and applies to D-G.</p