gbb845-sup-0001-AppendixS1.docxWord 2007 document10K Data S1: Additional analysis using absolute hearing levels instead of MHL.
gbb845-sup-0002-FigureS1.docWord document834K Figure S1: Average ABR thresholds as a function of time and sound frequency for all tested strains. For some strains, no data are available at either 2 or 3 weeks of age, because data collection was more sparse at these early ages. Additional information about the ABR measurements in the different strains is provided in Table S1.
gbb845-sup-0003-FigureS2.docWord document37K Figure S2: Highly suggestive locus at chromosome 10. (a) Suggestive QTL at chromosome 10, highest peak at 4 kHz (LRS = 17.1; additive allele effect = 6.8). Frequencies 8 and 16 kHz show peaks with decreasing magnitude (LRS = 16.3 and 14.0, respectively). Approximate suggestive (*; LRS = 10.7) and significant (**; LRS = 18.8) values are indicated by horizontal, dashed lines. Presence of the D2 allele increases trait values. This locus has a higher correlation with MHL values at 4 kHz (r = 0.62) than the known mutation in the fascin-2 gene (r = 0.44). Calculation of the partial correlation showed that the locus at chromosome 10 could explain 28.4% in the variation of the MHL at 4 kHz after accounting for the effects of fascin-2. (b) Zoomed-in view of the genes present at the locus on chromosome 10.
gbb845-sup-0004-FigureS3.docWord document567K Figure S3: Gene expression analysis for genes of the suggestive chromosome 10 locus. (a) Relative gene expression levels are shown. The bars represent different BXD strains in the sequence (from left to right): BXD13, BXD11, B6, BXD73, BXD32 and D2. Shading indicates allele inheritance from either B6 (dark gray) or D2 (light gray). Significant differences (anova) between strains (hatched line) or for the presence of the B6 or D2 allele (black lines; values without parental strains) are indicated (see Table S3). *P < 0.01; **P < 0.001; ***P < 0.0001. (b) Spearman rank correlations (r) of MHL at 4 kHz with gene expression of Zdhhc17 and Osbpl8 (P < 0.1; see Table S3). The parental origin of the allele is indicated for each sample.
gbb845-sup-0005-tS1.xlsExcel spreadsheet44K Table S1: Extended overview of ABR threshold data. The number of mice used in our study, including distribution of sexes, is given for each strain. Second, the average best hearing threshold and hearing threshold at the endpoint (12 weeks) are provided per frequency. The difference between these two gives the MHL used in our analysis.
gbb845-sup-0006-tS2.xlsExcel spreadsheet104K Table S2: Analysis of SNPs within the significant and suggestive QTLs on chromosome 18 and 10, respectively. Genes on ahl9 (chromosome 18) and chromosome 10 were analyzed for SNPs in exons (left, green) and introns (right, blue). Indicated is the location (Mb) of the SNP, the sequence of the SNP (D2) and the reference population (B6), the type of SNP (SC, synonymous coding; NSC, non-synonymous coding; 5′ UTR, exon: 5′ UTR; 3′ UTR, exon: 3′ UTR; NA, sequence not annotated in Genome browser; Nonsplice Site, intron not at a splice site) and whether the SNP was also present in the normal hearing strain, CBA/J, DBA/1J and DBA/2HaSmnJ. Note that for chromosome 10 for all genes, except Nav3, the sequence of B6 and good hearing strains was mostly similar and therefore only the total number of SNPs is indicated.
gbb845-sup-0007-tS3.docWord document55K Table S3: Gene expression analysis of genes at the suggestive QTL on chromosome 10. Displayed are genes (see Table 1) used in the qPCR analysis across six strains with divergent hearing loss, their gene symbol, the average expression level, the anova results (α = 0.001) across strains and the presence of the B6 or the D2 allele (*without parental lines; α = 0.01), the mean of ranks when strain averages were used for the analysis of having the B6 or D2 allele, the Spearman rank and Pearson product–moment correlation (and P-value) with MHL at 4 kHz and the LRS score of this trait. XH, Relative expression level (log 2) across strains (Expression) ≥ 10; H, 10 > Expression ≥ 8; M, 8 > Expression ≥ 5; L, 5 > Expression. Significant (bold; anova, P < 0.001; correlation, P < 0.05) and trends for (italics; correlation, 0.05 < P < 0.1) differences are highlighted. Note that no significant results were obtained in any analysis for Cdh23, a gene on chromosome 10 outside the suggestive QTL for MHL.
gbb845-sup-0008-tS4.docWord document62K Table S4: Primer sequences used for expression analysis of genes at ahl9 (chromosome 18) and chromosome 10. Genes outside the 1-LOD interval are shown in gray.
gbb845-sup-0009-tS5.xlsExcel spreadsheet151K Table S5: Genome-wide Pearson correlation of MHL at 4 kHz with total brain gene expression. Using, the dataset of MHL at 4 kHz was uploaded and correlated using a Pearson product–moment correlation with expression of two total brain databases: UCHSC BXD Whole Brain M430 2.0 (Nov06) RMA and INIA Brain mRNA M430 (Jun06) RMA. Genes present at ahl9 are given in bold and dark blue, those nearby in light blue. Note that only the correlation of Dym is significant and in the corresponding direction as in the cochlear samples (see Fig. 5).

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