Combining ES, cosegregation analysis and RNA splicing experiments, we identified a novel splice site altering variant (c.5383 + 6T > A) in the TECTA gene segregating in a large Asian-Chinese pedigree with early onset ADNSHL and determined the genetic cause of moderate to severe mid-frequency sensorineural hearing loss in this family.
In this study, the heterozygous c.5383 + 6T > A variant in TECTA we identified segregating with the NSHL in this large family is novel (absent from all population databases), highly conserved, and predicted to eliminate the donor splice site of exon 16 by in silico analysis. We confirmed aberrant splicing of exon 16 due to c.5383 + 6T > A by using in vitro minigene splicing assay and in vivo RNA analysis (Fig. 2 and Fig. 3). Interestingly, the c.5383 + 6T > A variant in minigene seems to affect the intron15 intermediate retention of 67bp and the exon 16 skipping, while in vivo RNA analysis showed a consequential exon 16 skipping. We revealed that the variant c.5383 + 6T > A lead to exon 16 skipping in vitro using in vivo RNA analysis at the same time, the results of which are consistent with each other. In previous studies, because of the low levels expressed in TECTA in the blood, total RNA of affected individuals and ethnically matched control individuals were isolated from EBV-transformed lymphoblasts[10, 11], which is costly and time-consuming, and the operation is relatively difficult as well. In our study, we used the PAXgene Blood RNA tube to collect RNA of whole blood for protection of RNA stability[12] and undertook further in vivo RNA analysis successfully, which was time-efficient and low-cost and our research protocols can probably be used as reference by other researchers.
The α-tectorin encoded by TECTA is one of the main non-collagenous proteins of the tectorial membrane (TM), which enables the motion of the basilar membrane to optimally drive the inner hair cells at their best frequency[13], and are strips of extracellular matrix lies over the stereocilia of the hair cells that are essential for the mechanical transmission and amplification of sound[14]. In mice, targeted deletion of the TECTA gene results in complete detachment of the TM from the organ of Corti[15]. In humans, nearly all recessive DFNB21 mutations in TECTA result in premature stop codons that may result in either truncated α-tectorin protein products or nonsense-mediated degradation of the TECTA mRNA, and are considered loss-of-function mutations[16–18]. The DFNA8/12 mutations in TECTA, which cause dominant hearing loss, all substitute highly conserved amino acids. The various missense mutations in TECTA that cause DFNA8/12 can be subdivided into classes with a clear genotype/phenotype correlation.
In our study, carriers of this mutation displays clinical pure mid-frequency hearing loss with an age span from three months to seventy-two years. Therefore, the progressive deterioration in high frequency hearing observed in III-4 and III-5 and III-11 (all of them over fifty years old) may be attributed to the old age. As we know, autosomal dominant hearing loss is highly heterogeneous in the phenotypes. For instance, the 5383 + 5delGTGA mutation in the ZA-ZP inter-domain, which could be contributing to in high frequency hearing loss phenotype in a Brazilian family, but c.5331G > A (p.L1777L) was found to segregate with the mid-frequency hearing loss in an Dutch family[10]. The established genotype-phenotypic correlations suggest that missense mutations in the ZP region cause mid-frequency hearing loss, while missense mutations in the ZA region lead to high-frequency hearing loss[19]. The 37 amino acids predicted to be lacking are located N-terminally from the Zona Pellucida domain (Fig. 6) and include two consensus N-glycosylation sites, which is important for the alpha-tectorin folding and cell-extracellular matrix at tachment. It may affect the proteolytic processing, structure, and/or function of this domain, resulting in a clinical phenotype similar to that of ZP domain missense mutations[10]. It is observed in mice that the ZA domain is vital for the normal development of the marginal band, covernet and Hensen’s stripe. In contrast, the ZP domain is more critical for ensuring these peripheral features are correctly placed or attached to the TM[20]. However, the impact of the ZA-ZP interdomain on the structure and function of the alpha-tectorin protein remains to be elucidated. The research mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane suggests that: in humans with the Y1870C missense mutation in TECTA causes a 50 to 80dB hearing loss. In transgenic mice with the Y1870C mutation in TECTA, the tectorial membrane's matrix structure is disrupted, and its adhesion zone is reduced in thickness. These abnormalities do not seriously influence the tectorial membrane's known role in ensuring that cochlear feedback is optimal, because the sensitivity and frequency tuning of the mechanical responses of the cochlea are little changed[15, 20]. The phenotype in the heterozygous mouse suggests haploinsufficiency is the pathogenic mechanism.
The individuals with the audiograms evaluated for over 10 years showed non progressive hearing loss. It was previously reported that the word recognition score(WRSs) in patients with TECTA mutations are better than those individuals with age-related hearing deterioration presenting with the same levels of hearing loss[21]. This phenomenon was a result of the fact that thresholds are maintained at high frequencies [17], and the lack of primary damage to the function of hair cells and cochlear nerves in TECTA patients may also be pertinent. Patients with cysteine-replacing mutations in TECTA demonstrate progressive hearing loss, while those with other mutations have non-progressive clinical manifestations [22]. The mutation in our study were not cysteine-replacing, and none of the patients showed progressive hearing loss; therefore, our data are consistent with the findings of previous studies.
Up to now, over 80 different variations in TECTA have been identified for ADSNHL (Fig. 6). At present, the TECTA gene mutations reported in the literature mostly occur in Iraq Lang family and Japanese family, with only four variation reported in Chinese family, including c.257_262delinsGCT[23], c.990C > A[24], c.5945C > A[25], and c.1893C > T[26]. Due to a lack of genetic screening, the mutation rate of TECTA in Chinese population still remains unknown. In the present study, we expand the mutation spectrum of TECTA, which is associated with ADSNHL.
TECTA are most frequently reported to cause mid frequency sensorineural hearing loss (MFSNHL), while other three genes, COL11A2 (DFNA13, DFNB53)[27, 28], EYA4 (DFNA10)[29], and CCDC50 (DFNA44) [30, 31], are also reported to cause MFSNHL. The MFSNHL phenotypes that are allelic to these traits also show midfrequency-like types of audiograms, but usually at substantially lower thresholds: DFNB37 (COL11A2)[32], COL11A1 was solely associated with Marshall and Stickler syndromes[33], but the recent reports expands its phenotypic spectrum to include nonsyndromic deafness (DFNA13), suggesting that variation in COL11A1 exert their effect through a dominant-negative gain of function mechanism[34]. In previous reports, the audiometric configuration for EYA4-associated hearing loss was a gradual high-frequency hearing loss or a fat-type hearing loss, and the rate of hearing loss progression caused by EYA4 variants was considered to be 0.63 dB/year [35]. The variation in CCDC50 were reported to cause DFNA44, and the DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system[31]. In mice, CCDC50 presumed loss-of-function mouse mutants which showed normal hearing thresholds up to 6 months old, thus indicating that haploinsufficiency is unlikely to be the pathogenic mechanism. Moreover, in vivo and in vitro results obtained indicate that the variation in CCDC50 exert their effect through dominant-negative gain of function mechanism[30].
In conclusion, we presented a novel splice-site altering variant in TECTA that segregates in a large family with postlingual deafness, and a healthy male child was born by PGT, thus expanding the phenotypic spectrum of pathogenic variants in TECTA. The implications of this discovery are valuable in the clinical diagnosis, prognosis, and treatment of patients with TECTA pathogenic variants.