A truncating mutation in SERPINB6 is associated with autosomal-recessive nonsyndromic sensorineural hearing loss

Abstract

More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DFNB91) to chromosome 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DFNB91 and encodes for an intracellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss.

Figure 1

The DFNB91 Locus, Audiograms, and Molecular Studies in Family 728 (A) Haplotypes created with SNP markers show complete cosegregation of a locus at 6p25 (DFNB91) in family 728. (B) Audiograms of seven members of family 728. 301, 102, and 103 have heterozygous, 201, 101, 105, and 106 have homozygous p.E245X mutation in SERPINB6. (C) Electropherograms showing the p.E245X (c.733G>T) mutation in SERPINB6. (D) Relative quantity values of SERPINB6 cDNA obtained from peripheral blood leukocytes of homozygotes and heterozygotes in family 728. Difference between homozygotes and heterozygotes is statistically significant (p = 0.034). (E) The western blotting results of SERPINB6 in peripheral blood leukocytes in homozygous and heterozygous members of family 728. The 42 kDa protein is clearly visible in NIH/3T3, HeLa cells, and a healthy person with wild-type SERPINB6. A reduced SERPINB6 band is visible in a heterozygote. The SERPINB6 protein band is completely absent in homozygotes.

Figure 2

Detection of Serpinb6 in the Developing Mouse Inner Ear by Immunohistochemistry and In Situ Hybridization (A–D) Weak Serpinb6 was detected in E13.5 utricular sensory epithelium. However, Serpinb6 was not in utricular hair cells, which were labeled with myo7a (B). Lines show examples of hair cells that are devoid of Serpinb6 (A). (E–H) Distinct Serpinb6 was detected in E16.5 crista hair cells (lines). (I–L) Weak Serpinb6 was detected in E16.5 cochlear hair cells. Multiple inner and outer hair cells were shown due to a section angle (J). (M–P) Serpinb6 was upregulated in P6 inner and outer hair cells (IHC, OHC), with mainly cytoplasm distribution. Ptprq labeled hair bundles (N)⁴⁰. In addition, Serpinb6 was detected in the GER at this stage. (Q–T) In situ hybridization showed very weak Serpinb6a expression in E18.5 cochlear hair cells and GER (Q), whereas the expression was significantly increased in P6 cochlear hair cells and GER (S). Control sense probe did not produce any signal (R, T). Abbreviations: Coch, cochlea; Cr, crista; Ut, utricle; anti-sen, antisense probe; sen, sense probe. Magnification: 40×.

Figure 3

The Effects of Transient Overexpression of SERPINB6 on Lysosomes in HeLa Cells (A–C) Distribution of LysoTracker Red 99-DND Florescence in HeLa cells transfected with different GFP constructs. Distribution of the intensity range and frequency of LysoTracker-red was different in SERPIN-B-6-WT cells from both mutant and control cells after water treatment. Histograms show that most lysosomes lost their integrity in mutant and control cells, while some lysosomes remain less disrupted in SERPINB6-WT cells. (D–G) Representative examples of images of wild-type and mutant SERPINB6-transfected HeLa cells. Green signal shows the overexpressed wild-type or mutant SERPINB6, which is localized in the cytoplasm. Red signal shows intact lysosomes. After osmotic stress, cells became round and lysosomes became indistinguishable with a general pinkish hue in the cytosol. However, this effect was less pronounced in cells where wild-type SERPINB6 is overexpressed. (H) Averaged LysoTracker Intensities with standard deviation with PBS and water. Averaged LysoTracker intensity difference in WT SERPINB6-overexpressed cells is less than those in mutant SERPINB6-overexpressed and control cell lines (p = 0.003 and 0.024, respectively, for the mutant and control cells).