Genomic Duplication and Overexpression of TJP2/ZO-2 Leads to Altered Expression of Apoptosis Genes in Progressive Nonsyndromic Hearing Loss DFNA51 Tom.

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Genomic Duplication and Overexpression of TJP2/ZO-2 Leads to Altered Expression of Apoptosis Genes in Progressive Nonsyndromic Hearing Loss DFNA51 Tom Walsh, Sarah B. Pierce, Danielle R. Lenz, Zippora Brownstein, Orit Dagan-Rosenfeld, Hashem Shahin, Wendy Roeb, Shane McCarthy, Alex S. Nord, Carlos R. Gordon, Ziva Ben-Neriah, Jonathan Sebat, Moien Kanaan, Ming K. Lee, Moshe Frydman, Mary-Claire King, Karen B. Avraham The American Journal of Human Genetics Volume 87, Issue 1, Pages 101-109 (July 2010) DOI: 10.1016/j.ajhg.2010.05.011 Copyright © 2010 The American Society of Human Genetics Terms and Conditions

Figure 1 Linkage Analysis and Identification of the Inverted Genomic Duplication in Family T (A) Family T with progressive hearing loss indicated by filled symbols. (B) Audiogram of hearing loss by age. (C) Chromosome 9 region of linkage to the hearing loss phenotype. Recombinant individuals are indicated in the notation of (A). (D) arrayCGH scan indicating the family-specific duplication of ∼270 kb in genomic DNA of II-7. arrayCGH also detected homozygosity for a polymorphic deletion of 8760 bp within the larger novel duplicated region. The study was approved by the Helsinki Committees of Tel Aviv University and the Israel Ministry of Health and by the Human Subjects Division of the University of Washington. The American Journal of Human Genetics 2010 87, 101-109DOI: (10.1016/j.ajhg.2010.05.011) Copyright © 2010 The American Society of Human Genetics Terms and Conditions

Figure 2 Characterization of the Inverted Duplication at TJP2 (A) Sketch of the inverted duplication. (B) PCR products, ∼7 kb in size, amplified with primers specific to the inverted duplication, from genomic DNA of affected (A) and unaffected (Un) relatives of family T. The American Journal of Human Genetics 2010 87, 101-109DOI: (10.1016/j.ajhg.2010.05.011) Copyright © 2010 The American Society of Human Genetics Terms and Conditions

Figure 3 Expression of Tjp2 in the Mouse Inner Ear (A) Expression of Tjp2 transcript at ages E16.5, P0, 1 week, 1 month, 3 months, and 9 months, determined by real-time qPCR, reveals decline with age until after hearing maturation at P30, then a stable level during adulthood. Values represent mean ± SD of samples run in triplicate. The experiment was repeated twice; a representative experiment is shown. (B) Schematic representation of the P0 cochlea (top) and saccule (bottom). The inner and outer hair cells of the cochlea make up the sensory epithelium; nonsensory supporting cells include Deiters', pillar, and Hensen cells. The saccule contains sensory hair cells and nonsensory supporting cells. (C and D) Immunofluorescence confocal images of the cochlea (C) at E16.5 and P0 and of the saccule (D) at E16.5. Tjp2 expression (red) is seen mainly at cell junctions (arrowheads) and also in the cytoplasm and nuclei (arrows) of hair cells and adjacent supporting cells. The vestibular pattern of expression was similar in the utricle and cristae at these ages (data not shown). Negative control staining of P0 cochlea without primary antibody against Tjp2 demonstrates the low background of the secondary antibody. Hair cell cytoplasm and nuclei are marked by myosin VI (MyoVI, green) and DAPI (blue), respectively. Paraffin-embedded sections were stained with the primary antibodies goat anti-myosin VI (Santa Cruz Biotechnology), rabbit anti-ZO-2 (Cell Signaling), and mouse anti-ZO-1 (Zymed) and the fluorescence-conjugated secondary antibodies donkey anti-goat 488, donkey anti-rabbit 594, and donkey anti-mouse rhodamine (Molecular Probes). Imaging was done with the LSM 510 confocal microscope (Zeiss). All procedures involving animals met NIH guidelines and were approved by the Animal Care and Use Committees of Tel Aviv University and the University of Washington. Scale bar represents 10 μm. The American Journal of Human Genetics 2010 87, 101-109DOI: (10.1016/j.ajhg.2010.05.011) Copyright © 2010 The American Society of Human Genetics Terms and Conditions

Figure 4 Overexpression of TJP2 and Reduced Phosphorylation of GSK-3β Associated with the Genomic Duplication (A) Expression of TJP2 transcript in lymphoblasts of family T relatives with (DUP) and without (WT) the genomic duplication, determined by real-time qPCR. Values represent mean ± SEM of data from three independent experiments, which included cells from four individuals without the duplication and six with the duplication. (B–E) Lymphoblast cell lysates of family T relatives were analyzed by immunoblotting with primary antibodies rabbit anti-TJP2 (Santa Cruz Biotechnology), mouse anti-GSK-3β (BD Transduction Laboratories), rabbit anti-phospho(Ser9)-GSK-3β (p-GSK-3β) (Cell Signaling Technology), and mouse anti-β-actin (Sigma) and IRDye-conjugated secondary antibodies (LI-COR Biosciences). Levels of TJP2 relative to β-actin (B) and levels of p-GSK-3β relative to GSK-3β (D) were quantified from western blots (C, E). Blue bars represent results from individuals without the duplication; red bars represent results for individuals with the duplication. Individuals are labeled as in Figure 1. Values in (B) and (D) represent mean ± SEM of data from three independent experiments; representative western blots are shown. The American Journal of Human Genetics 2010 87, 101-109DOI: (10.1016/j.ajhg.2010.05.011) Copyright © 2010 The American Society of Human Genetics Terms and Conditions

Figure 5 Effects of the TJP2 Duplication on Expression of Apoptosis-Related Genes Real-time qPCR analyses of RNA from lymphoblasts of family T relatives (blue and red bars and individual samples as in Figure 4). (A) Expression of genes identified as differentially expressed in the screen of apoptosis-related genes. (B) Expression of BCL2 family genes. Gene expression was normalized to the geometric mean of the expression of the endogenous control genes HPRT, TBP, and UBC.41 Values represent mean ± SEM of data from three independent experiments. The result for BCL2L11 is indicated in both (A) and (B). The American Journal of Human Genetics 2010 87, 101-109DOI: (10.1016/j.ajhg.2010.05.011) Copyright © 2010 The American Society of Human Genetics Terms and Conditions