Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec 22;8(24):e172665.
doi: 10.1172/jci.insight.172665.

TMTC4 is a hair cell-specific human deafness gene

Jiang Li  1   2 , Byung Yoon Choi  3 , Yasmin Eltawil  4 , Noura Ismail Mohamad  4 , Yesai Park  4 , Ian R Matthews  4 , Jin Hee Han  3   5 , Bong Jik Kim  5 , Elliott H Sherr  1   2 , Dylan K Chan  4
Affiliations

TMTC4 is a hair cell-specific human deafness gene

Jiang Li et al. JCI Insight. .

Abstract

Transmembrane and tetratricopeptide repeat 4 (Tmtc4) is a deafness gene in mice. Tmtc4-KO mice have rapidly progressive postnatal hearing loss due to overactivation of the unfolded protein response (UPR); however, the cellular basis and human relevance of Tmtc4-associated hearing loss in the cochlea was not heretofore appreciated. We created a hair cell-specific conditional KO mouse that phenocopies the constitutive KO with postnatal onset deafness, demonstrating that Tmtc4 is a hair cell-specific deafness gene. Furthermore, we identified a human family in which Tmtc4 variants segregate with adult-onset progressive hearing loss. Lymphoblastoid cells derived from multiple affected and unaffected family members, as well as human embryonic kidney cells engineered to harbor each of the variants, demonstrated that the human Tmtc4 variants confer hypersensitivity of the UPR toward apoptosis. These findings provide evidence that TMTC4 is a deafness gene in humans and further implicate the UPR in progressive hearing loss.

Keywords: Cell stress; Genetic diseases; Mouse models; Otology.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Floxed Tmtc4 transgenic mouse (Tmtc4fl/fl).
(A) LoxP sequences were inserted in the introns up- and downstream of Tmtc4 exon 3 and confirmed by Sanger sequencing. (B) Tmtc4 expression is significantly decreased in the brain and cochlea of Rosa26CreER/Tmtc4fl/fl mice as compared with Cre–/Tmtc4fl/fl mice. ***P < 0.001, ****P < 0.0001 by 1-way ANOVA.
Figure 2
Figure 2. Tmtc4 expression in Myo15Cre/Tmtc4fl/fl mice.
(A) Myo15Cre/TdTomato reporter demonstrates Myo15Cre-driven TdTomato expression only in hair cells (P5 cochlear explant). (B) P5 cochlear explants were stained with anti-Myo7a antibody (red) to label hair cells and were subjected to RNAScope to detect Tmtc4 RNA (green). They were also stained with DAPI to identify nuclei (blue). Myo15Cre/Tmtc4fl/+ mouse (top) exhibited Tmtc4 expression in hair cells (colocalized with Myo7a) as well as outside of hair cells. In Myo15Cre/Tmtc4fl/fl cKO mice (bottom), Tmtc4 expression was specifically lost in hair cells. Representative images from 3 experiments for each condition are shown.
Figure 3
Figure 3. Auditory function in Tmtc4-cKO mice.
(A and B) ABR (A) and DPOAE (B) thresholds are elevated in adult (>P30) cKO mice in which Tmtc4 is subject to recombination by Cre driven by the hair cell–specific Myo15Cre promoter (Myo15Cre+/Tmtc4fl/fl), compared with Cre– littermate controls (Myo15Cre–/Tmtc4fl/fl). (C) Progression of hearing loss is seen in Myo15Cre/Tmtc4fl/fl cKO mice from onset of hearing (P15) through P28. (D) ABR thresholds in adult (>P30) Prox1CreER+/Tmtc4fl/fl mice, in which Tmtc4 is specifically knocked out in supporting cells, are not elevated compared with Cre– controls (Prox1CreER–/Tmtc4fl/fl). *P < 0.05; **P < 0.001, 2-tailed unpaired Student’s t test between genotypes.
Figure 4
Figure 4. Hair cell loss in Myo15Cre/Tmtc4-cKO mice.
(A) Whole-mount IHC against Myo7a to label hair cells was performed at the apical, middle, and basal turns of P30 and P45 Myo15Cre/Tmtc4-cKO mice of the indicated genotypes. Scale bar: 50 μm. (B) Hair cell loss in Myo15Cre/Tmtc4-cKO mice. Inner and outer hair cell (IHC, left; OHC, right) counts were made in the apical, middle, and basal turns of cochleae from P45 Myo15Cre– (n = 4) and Cre+/Tmtc4fl/fl (n = 4) mice. *P < 0.05; **P < 0.001 by 2-tailed unpaired Student’s t test between genotypes.
Figure 5
Figure 5. Identification of human TMTC4 variants.
Variants from exome sequencing data (n = 92,080) underwent filtering steps. After each filtering step, candidate variants were narrowed down to 8 candidates: 2 variants in 4 genes (TMTC4, ATAD3A, MCM3AP, MDGA1). After Sanger sequencing confirmation and segregation study, 2 variants of TMTC4 remained as potential causative variants.
Figure 6
Figure 6. TMTC4 variants in human hearing loss.
TMTC4 is the likely cause of nonsyndromic progressive sensorineural hearing loss. (A) Three-generation pedigree consistent with autosomal recessive inheritance pattern. Black: hearing loss; white: normal hearing; cross: deceased. Genotyping at c.547 and c.575 loci of TMTC4 demonstrates cosegregation of compound heterozygous rare variants c.547 G>A and c.575 C>T with hearing loss in patients 788 and 595 (pathogenic variants in red). (B) Audiograms for patients 788 and 595 show noise-induced notch at 2 kHz and high-frequency–predominant sensorineural hearing loss. (C) Lymphoblastoid cell lines were established from 4 human family members. mRNA levels of CHOP and S-XBP1 (opposing proapoptotic and prohomeostatic effectors of the UPR, respectively) were measured by qPCR, and the CHOP/S-XBP1 ratio, which reflects the proapoptotic balance of the UPR, was calculated. n = 4 for each condition. One-way ANOVA followed by Tukey’s multiple-comparison test was performed, with pairwise comparisons relative to patient 789 (as a normal-hearing control) performed. At baseline (left), only patient 595 had significant elevation of the CHOP/S-XBP1 ratio. After treatment with 1 mM thapsigargin (TG) for 6 hours, cells from the 2 patients with hearing loss (788 and 595) had significant elevation of the CHOP/S-XBP1 ratio. **P < 0.001. (D) CHOP/S-XBP1 ratio for the 4 patients was correlated with hearing level (bilateral pure-tone average to 0.5–8 kHz tones). The correlation was not significant for baseline CHOP/S-XBP1 ratio (black line) but was statistically significant for thapsigargin-induced levels (red line; linear regression R2 = 0.94, P < 0.05). (E) HEK cells were generated harboring homozygous E183K or homozygous A192V mutations, and CHOP and S-XBP1 mRNA levels were measured as in C. E183K and A192K mutant cells exhibited elevated CHOP/S-XBP1 ratios at baseline (left). Upon UPR induction with 1 μm TG (right), E183K cells had a greater CHOP/S-XBP1 ratio, whereas A192V cells had a lower CHOP/S-XBP1 ratio, compared with WT cells. n = 4 for each condition. *P < 0.01; **P < 0.001 by 2-tailed unpaired Student’s t test relative to WT.
See this image and copyright information in PMC

References

    1. Li J, et al. Deletion of Tmtc4 activates the unfolded protein response and causes postnatal hearing loss. J Clin Invest. 2018;128(11):5150–5162. doi: 10.1172/JCI97498. - DOI - PMC - PubMed
    1. Larsen ISB, et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci U S A. 2017;114(42):11163–11168. doi: 10.1073/pnas.1708319114. - DOI - PMC - PubMed
    1. Wang W, et al. Impaired unfolded protein response in the degeneration of cochlea cells in a mouse model of age-related hearing loss. Exp Gerontol. 2015;70:61–70. doi: 10.1016/j.exger.2015.07.003. - DOI - PubMed
    1. Chan DK, Rouse SL. Sound-induced intracellular Ca2+ dynamics in the adult hearing cochlea. PLoS One. 2016;11(12):e0167850. doi: 10.1371/journal.pone.0167850. - DOI - PMC - PubMed
    1. Rouse SL, et al. Integrated stress response inhibition provides sex-dependent protection against noise-induced cochlear synaptopathy. Sci Rep. 2020;10(1):18063. doi: 10.1038/s41598-020-75058-w. - DOI - PMC - PubMed

Publication types