THOC1 deficiency leads to late-onset nonsyndromic hearing loss through p53-mediated hair cell apoptosis

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.


Introduction
Age-related hearing loss affects over 40% of the population older than 65 years [1]. Apoptosis of the inner ear sensory hair cells, which are nonregenerative in mammals, is a key step towards this process [2,3]. Over the past two decades, more than 40 genes have been implicated in the genetic causes of late-onset, progressive hearing loss (Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org/). Dysfunction of those genes have been shown to hinder the repair or stability of critical auditory components such as cytoskeleton structure, intercellular junction, fluid homeostasis and synaptic transmission [4]. While such findings provided invaluable resources to gain insights into the molecular basis of the age-related hearing loss, the mechanism underlying the direct cause of the progressive hair cell loss remains elusive. To date, only TJP2 and DIABLO, two genes associated with dominant, late-onset, progressive hearing loss DFNA51 and DFNA64, respectively, have been reported to participate in the apoptosis pathway [5,6]. Overexpression of the tight junction protein TJP2 due to a genomic duplication was shown to result in decreased phosphorylation of GSK-3β and altered expression of the apoptosis-related genes, increasing the susceptibility of inner ear cells to apoptosis [6]. A missense p.Ser126Leu mutation in the proapoptotic gene DIABLO retained its proapoptotic function but renders the mitochondria susceptible to calcium-induced loss of the membrane potential [5].
THOC1 is an essential member of the conserved THO/TREX ribonucleoprotein (RNP) complex that functions in cotranscriptional recruitment of mRNA export proteins to the nascent transcript [7]. Along with the assembly of other THO/TREX proteins, it has been shown to regulate the coordinated gene expression required for cancer development as well as self-renewal and differentiation of embryonic stem cells [8]. Mice homozygous for the Thoc1 null allele is embryonic lethal, while those with the Thoc1 hypomorphic allele exhibited a dwarf phenotype with severely compromised gametogenesis [7,9,10]. So far, no THOC1 mutations have been reported in humans and the function of THOC1 in inner ear has not been explored. In this study, we identified a missense mutation in THOC1 as the probable cause of late-onset, progressive, non-syndromic hearing loss in a large, dominant family.
Deficiency of Thoc1 was shown to lead to hair cell apoptosis through the p53-mediated pathway.

Clinical characteristics of Family SH
Family SH had at least 15 members affected by adulthood-onset hearing loss within 4 generations (Figure 1a). The nonsyndromic, bilateral hearing loss was most predominant in the high frequencies, beginning mildly during the third decade and gradually progressed to severe-to-profound in the fifth and sixth decades (Supplementary Figure S1). All affected subjects reported tinnitus, while vestibular dysfunction or other clinical abnormalities were not observed. No inner ear malformation was detected by temporal bone computed-tomography (CT) scanning (data not shown).

Identification of the p.L183V mutation in THOC1
Because the onset of the hearing loss was during the third decade for Family SH, only unaffected family members over 40 years old were included in the linkage analysis in this study. Multipoint genome-wide linkage analysis of the 9 affected and 12 unaffected family members (marked with asterisks in Figure 1a Table S1).
Whole-exome sequencing of four affected (III-4, III-9, III-10 and III-18) and two unaffected (III-16 and IV-6) family members (marked with triangles in Figure 1a) identified a total of three candidate variants (Supplementary Table S2 Figure   S2). The p.L183V variant was predicted to be pathogenic by computational programs Mutation Taster, PROVEAN and SIFT and was not seen in 1000 Chinese Han normal hearing controls.

The expression of thoc1 in mouse and zebrafish hair cells
To further elucidate the role of THOC1 in hearing, we investigated the expression of THOC1 in mouse and zebrafish. Cross-section and whole mount immunostaining of P0 mouse inner ear showed that THOC1 was specifically expressed in inner and outer hair cells (Figure 2a These results suggest that Thoc1 conservatively contribute to the formation of hair cells in vertebrates.

Thoc1 deficiency caused hair cell developmental defects in zebrafish
In order to examine whether thoc1 was required for the formation of hair cell, the CRISPR/Cas9 system was utilized to generate a series of thoc1 mutants in Tg(pou4f3:gap43-GFP) transgenic zebrafish line, in which the membrane of hair cell is labeled with GFP [11]. To obtain viable but hypomorphic alleles, we chose the target sites at the exon 20, the second last exon of zebrafish thoc1 (Supplementary Figure   S4a). The selected gRNA-Cas9 system efficiently induced a series of frameshifting indels in the targeting sites that were predicted to truncating a small portion of the C-terminal protein (Supplementary Figure S4b In addition, we examined the fast escape reflex, the C-shaped startle response. It was found that the probability of the C-startle response in thoc1 deficient zebrafish was significantly lower than that in control zebrafish embryo and adults, suggesting thoc1 mutants have hearing problems (Supplementary Figure S6, S7). To verify these phenotypes in thoc1 mutants were indeed caused by loss of function of thoc1, we examined whether thoc1 knockdown could phenocopy the mutants. A splicing-blocking mophorlino (MO) was validated to efficiently interfere the thoc1 pre-mRNA splicing, caused the exon3 (61 bp) deletion and lead to the reading frame shift (Figure 4a, b). Sanger sequencing analysis confirmed this result (data not shown).
It was demonstrated that thoc1 morphants had the same phenotypes with those in thoc1 mutants (Figure 4c). In addition, both human THOC1 mRNA and zebrafish thoc1 mRNA significantly rescued the hair cell defects in thoc1 morphants (Figure 4c, d). These results substantiate that hair cell defects were specifically caused by inactivation of thoc1.

The p. L183V mutation in THOC1 impaired its function in hair cell formation
To testify whether the c.C547G/p.Leu183Val mutation in THOC1 impairs its function in hair cell formation, we explored the effect of this mutation during hair cell development. We setup 6 groups for microinjection and did confocal imaging analysis of hair cells at 4 days post fertilization (dpf): wild-type control, standard MO control, MO, MO + zthoc1 mRNA, MO + hTHOC1 mRNA, MO + hTHOC1 (C547G) mRNA.
The mutation of THOC1 significantly reduced the ratio of embryos been rescued by microinjection of hTHOC1 (C547G) mRNAs into thoc1 morphants (Figure 4c, d).

Inactivation of thoc1 induced apoptosis in neuromasts
To understand the cellular mechanism by which the hair cells were dramatically decreased, we did the confocal imaging analysis of the residual cells. In thoc1 mutants, we found that around 83% of these hair cells had an abnormal morphology, such as distorted shape, quite smaller, and even became cell fractions (Figure 5a). These results suggest a number of hair cells underwent apoptosis. To test this hypothesis, we carried out Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis in thoc1 mutants. It was revealed that thoc1 deficiency resulted in dramatic hair cell and supporting cell apoptosis in neuromasts (Figure 5b).
In addition, we performed the transcriptome sequencing analysis of the thoc1 mutated embryos at 48 hpf. We found that 455 genes were significantly up-regulated, while 1312 genes were significantly down-regulated in thoc1 mutants compared to the sibling control (Figure 6a

Discussion
As a key component of the TREX protein complex, THOC1 is evolutionarily conserved (supplementary Figure S2) and plays an essential role for coordinated gene expression during early and postnatal development [7,9,10]. In humans, non-synonymous variants in THOC1 are extremely rare, as no such variants have minor allele frequencies higher than 0.002 in the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/). In this study, we presented to our knowledge the first THOC1 mutation associated with human mendelian disorders. Even though THOC1 regulates expression of a wide range of genes required for crucial biological processes such as embryogenesis, organogenesis and cellular differentiation [8], mice with hypomorphic alleles in Thoc1 were viable [10] and are associated with tissue-specific defects such as compromised fertility [9]. In our study, the affected members of Family SH showed no abnormalities other than the hearing loss, further demonstrating that systematic reduced level of functional Thoc1 is generally tolerated and may lead to certain tissue-restricted disorders.
To elucidate the role of THOC1 in the inner ear, we generated a series of Thoc1 Surprisingly, though hair cell apoptosis is a key step towards progressive and age-related hearing loss, THOC1 represents only one of a very few deafness-causing genes directly involved in this process. Given THOC1 regulates distinct and specific sets of downstream genes in different tissues and development stages, the Thoc1 deficiency zebrafish generated in our study presented a good model to study the coordinated control of hair cell apoptosis at the molecular level. Indeed, by transcriptome sequencing, we identified a list of genes that were differentially expressed in the Thoc1 mutant zebrafish ( Figure 6). Among them, genes in pathways associated with apoptosis such as p53 signaling, DNA replication and cell cycle were enriched.
Previous studies have suggested dual roles of Thoc1 in relation to cell proliferation and apoptosis depending on the context of different tissues and cell types.
In embryonic development of Rb1 null mice, Thoc1 is required to for increasing expression of E2f and other apoptotic regulatory genes [12]. On the other hand, Thoc1 is overexpressed in a variety of cancers [13,14], and depletion of Thoc1 in those cancer cells, but not in normal cells, induce apoptotic cell death [15]. In mouse models, hypomorphic Thoc1 allele lead to germ cell apoptosis that in testes correlates with the decreased number of primary spermatocytes [9]. In consistence with the latter findings, our study demonstrated that Thoc1 deficiency may induce hair cell apoptosis in zebrafish, which likely underlies the pathogenic mechanism of the late-onset, progressive hearing loss in Family SH. Accompanied with the Thoc1 deficiency was the increased expression of a number of downstream apoptotic genes such as P53, Bax, Casp3 and Casp9 ( Figure 6). The p53 signaling pathway has been implicated in the hair cell apoptosis during age-related hearing loss [16]. In our study, we showed that inhibition of P53 by Pifithrin-α significantly alleviated the hair cell apoptosis in the Thoc1 MO-knockdown zebrafish, presenting a potential new strategy for preventing of the age-related hearing loss.
In conclusion, our study identified THOC1 as a new causative gene for late-onset, progressive hearing loss in humans. Functional studies showed that Thoc1 deficiency unleashes expression of proapoptotic genes in the p53 signaling pathway and results in hair cell apoptosis in zebrafish. These findings may provide important insight into the molecular basis of age-related hearing loss.

Subjects and clinical evaluation
Members of Family SH was recruited through the Affiliated Hospital of Nantong University in Nantong, Jiangsu Province, China. A total of 9 affected and 18 unaffected members participated in the present study (Figure 1a, asteriskes). All subjects received comprehensive auditory evaluation including pure tone audiometry (PTA), otoscopic examination and temporal bone high-resolution CT scanning.
Family history and general physical examination were performed to exclude the possible syndromic hearing loss. Informed consent was obtained from all participating subjects and this study was approved by the ethics committee of Affiliated Hospital of Nantong University.

Genome-wide linkage analysis
Genome-wide multipoint linkage analysis was performed using the

Immunostaining of THOC1 in mouse inner ear
Immunofluorescence staining of THOC1 was performed in cross-section and whole mount samples of mouse inner ear as previously described [17].

Zebrafish Line and Startle Response Test
The zebrafish embryos and adults were maintained in zebrafish Center of Nantong University under standard conditions in accordance with our previous procedures [18,19]. The transgenic zebrafish lines Tg(pou4f3:gap43-GFP) and Tg(cldnb:lynGFP) were used as previously described [20]. Sound-evoked C-shaped startle response was tested at larval and adult stage in a well-plate and a plastic tank and recorded with a high-speed camera.

RNA Isolation, Reverse Transcription (RT) and Realtime PCR
Total RNA was extracted from zebrafish embryos by TRIzol reagent according to the manufacturer's instructions (Thermo Fisher Scientific, USA). Genomic contaminations were removed by DNaseI, and then 2 µg total RNA was reversely transcribed using a reversed first strand cDNA synthesis kit (Fermentas, USA) and stored at −20 • C. The sequences of PCR primers, used for validating the Splice-blocking Morpholino, were: 5'-TCCGTCTCACTTCGACTTCA-3' and 5'-TCCCAGCAGAGTAAAATGTGT-3'. The Realtime PCR analysis was performed according to the procedure described in our previous work [18]. The primers were used for the bax:

Whole Mount in situ Hybridization
A 498 bp cDNA fragment of Thoc1 was amplified from the cDNA library that established from wild type (WT) AB embryos using the specific primers of thoc1 F1 controlling the false discovery rate, P-value of genes were set <0.05 to detect differential expressed ones.

Microscopy and Statistical Analysis
Zebrafish embryos were anesthetized with E3/0.16 mg/mL tricaine/1% 1-phenyl-2-thiourea (Sigma, USA) and embedded in 0.8% low melt agarose, and then were examined with a Leica TCS-SP5 LSM confocal imaging system. For the results of in situ hybridization, Photographs were taken using an Olympus stereomicroscope MVX10. All images of THOC1 immunostaining in mouse were imaged using the Leica SP5 confocal microscope. Statistical analysis was performed using GraphPad Prism software. T-test and one-way analysis of variance (ANOVA) were used, and P < 0.05 were considered statistically significant.