Additional file 1: of Association of SNPs in LCP1 and CTIF with hearing in 11Â year old children: Findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and the G-EAR consortium

Results for all the SNPs included in the analyses of data from ALSPAC or G-EAR

Additional file 1: of Association of SNPs in LCP1 and CTIF with hearing in 11Â year old children: Findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and the G-EAR consortium

Results for all the SNPs included in the analyses of data from ALSPAC or G-EAR

Immunohistochemistry in the mouse cochlea at P5.

<p>Brown indicates positive staining. A, B, C) Ptprd is localized in hair cells of the organ of Corti (bracket in A, arrowheads in C), in the marginal cells of the stria vascularis (arrow in A, B), in the supporting cells of the Kölliker’s organ (marked by an asterisk in A) and in the spiral ganglions neurons (arrowhead in A); D, E) Cdh13 is expressed in cells of Claudius (red arrowhead in E), outer and inner hair cells (arrowheads in E), Deiters’ cells (bracket in E) and pillar cells (asterisk in E), cells of the Kolliker’s organ (arrows in E) in the organ of Corti. Staining was also noted in interdental cells (arrow in D), stria vascularis (asterisk in D), spiral prominence and external sulcus cells (bracket in D). F, G) Ank2 could be noted in the Hensen’s cells (bracket in G), Deiters’ cells (arrowheads in G)and pillar cells (asterisk in G) in the organ of Corti. Moreover, Ank2 is expressed in the Reissner’s membrane (arrowhead in F) and cells of the Kolliker’s organ (arrow in G). Scale bars: A–C: 20 µm. D, F: 20 µm; E,G: 10 µm. Rectangles label the regions shown in higher magnification.</p

List of candidate genes derived from previously published Meta-Analysis [3] with inclusion criteria satisfied.

<p>Key: Sugg. p-value† = suggestive p-value: p∼10-5,10-6; H.Sugg. p-value‡ = highly suggestive p-value: p∼10-7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Girotto1" target="_blank">[3]</a>.</p

Analysis flow chart.

<p>The diagram illustrates the 4 steps defining our strategy. Relevant details for each step are given: GWAS meta-analysis description, expression studies in the mouse cochlea, replication association study in Silk Road cohort and genotype-phenotype relationship.</p

Results of the gene-based association test for candidate expressed genes.

<p><b>Key:</b> Total N. SNPs = total number of intragenic SNPs for each gene; N. Selected PCs = number of principal components explaining the largest amount of variance and used for the analysis (see Materials and Methods); Variance Explained = Total variance accounted for by the selected principal components.</p

Results of the replication association study for the candidate SNPs of expressed genes.

<p><b>Key</b>: Eff.All = effect allele; *from published work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Girotto1" target="_blank">[3]</a>; Repl. = replication; Maf = minor allele frequency; Str = strand; imp = imputed; gen = genotype; Info Score = imputation quality score form IMPUTE2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Delaneau1" target="_blank">[6]</a>, Conc = concordance for the region encompassing the SNP (chunk dimension = 5 Mb); HWE = Hardy-Weinberg Equilibrium; add = additive; dom = dominant.</p

Further details of shortlisted candidate genes.

<p>Expression in the ear reported in Mouse Genome Informatics (MGI, <a href="http://www.informatics.jax.org/" target="_blank">http://www.informatics.jax.org/</a>); the Morton cDNA human foetal cochlea cDNA library (Morton, <a href="http://brighamandwomens.org/Research/labs/BWH_Hearing/Cochlear_ESTs.aspx" target="_blank">http://brighamandwomens.org/Research/labs/BWH_Hearing/Cochlear_ESTs.aspx</a>); the SHIELD database (SHIELD: Shared Harvard Inner-Ear Laboratory Database, <a href="https://shield.hms.harvard.edu/" target="_blank">https://shield.hms.harvard.edu/</a>); plus localisation within a reported human deafness locus and phenotypic reports from MGI of any mouse mutations of the gene.</p><p>Key: SHIELD* = SHIELD - enriched in hair cells compared to supporting cells; SHIELD** = SHIELD - auditory/vestibular ganglia; DFN = within a human deafness locus?; N/A = data not available.</p

Immunohistochemistry in the mouse cochlea at P5.

<p>Brown indicates positive staining. A, B, C, D) Staining of Grm8 showing expression throughout the cochlea, most notably the root cells (curly brackets in A and B) and root cell processes (asterisks in A and B), the stria vascularis (open arrowhead in A, C), the hair cells (brackets in A, arrowheads in D) and also in the spiral ganglion of the neurons (double asterisks in A); E, F, G, H) Expression of Rimbp2 showing staining in hair cells (bracket in E, arrowheads in F), tectorial membrane (double arrowhead in E), the stria vascularis (open arrowhead in E, G) and in the spiral prominence (arrow in E) and root cell processes (asterisks in E and H). Scale bar indicates 20 µm.</p

Immunohistochemistry in the mouse cochlea at P5 (P4 for Slc16a6 gene).

<p>Brown indicates positive staining. A, B) Expression of Dclk1, showing intense staining in the marginal cells including projections towards the basal cells of the stria vascularis (arrows in A,B); C, D) Expression of Arsg is localized at the top of sensory hair cells in the organ of Corti (bracket in C, arrowheads in D). E, F), Apical hair cells at P4, showing staining in outer hair cells of Slc16a6 of the organ of Corti (bracket in E, arrowheads in F). Note that these samples are from the C3HeB/FeJ strain, which is pigmented. Gabrg3 shows a striking specific expression in the outer and inner hair cells (arrowheads in H). In particular, the inner hair cells have the strongest staining. H, I) Expression of Csmd1 is localized at the top of sensory hair cells in the organ of Corti (arrowheads in H; I) confocal expression shows a strong staining localized in the stereocilia of inner hair cells and weak staining is also present in the stereocilia of outer hair cells. Csmd1 is labelled in green while rhodamine/phalloidin labels actin filaments of stereocilia in red. Merged image (yellow) indicates colocalization of Csmd1 with actin in stereocilia hair bundles. Scale bars: A–H: 20 µm; I: 5 µm.</p