Nonsyndromic hearing loss (NSHL, OMIM: # 610572) is a common congenital condition. Genetic factors are the major cause of NSHL, accounting for more than 70% of the cases (Wang et al., 2018; Smith et al., 2005). To date, at least 127 genes and 170 loci have been implicated in the pathogenesis of NSHL (Morgan et al., 2019; An et al., 2019). MARVELD2, located at the DFNB49 locus on chromosome 5q13.2, is one of the genes associated with NSHL (Sadeghi et al., 2020). Mutations in MARVELD2 can result in bilateral, moderate to severe NSHL (Zheng et al., 2019). MARVELD2 contains seven exons and encodes a 558 amino acid protein called tricellulin. Tricellulin is a tight junction protein that is expressed in the inner ear epithelial cells and plays a crucial role in maintaining the barrier and fence functions of the tight junctions. Tricellulin regulates the transport of water, ions, and large and small molecules using the paracellular pathway and restricts the free flow of cell membrane lipids and proteins (Gao et al., 2023; Mariano et al., 2011). It controls lateral ion diffusion that is essential for normal hearing. Therefore, MARVELD2 mutations may cause varying degrees of hearing loss (Taghipour-Sheshdeh et al., 2019; Kitajiri and Katsuno, 2016; Kitano et al., 2019; Kamitani et al., 2015).

In this case, we evaluated a newborn male child who failed the screening test for hearing; but was found to have normal hearing upon further evaluation. However, both his parents had hearing impairments. Genetic testing of the child revealed the presence of the MARVELD2 c.663G>A and the MT-RNR1 1555A>G mitochondrial gene mutations. Family tracing analysis revealed that the father of the child was the proband, with a homozygous MARVELD2 c.663G>A mutation. The child’s MT-RNR1 1555A>G mitochondrial gene, encoding the 12S rRNA mutation, was inherited from the maternal lineage via an extranuclear (mitochondrial) inheritance pattern, whereas the heterozygous MARVELD2 c.663G>A mutation was inherited from the homozygous father.

This study aimed to analyze the molecular, epidemiological, and clinical phenotypes of the members of a family with deafness carrying the MARVELD2 c.663G>A nonsense mutation. We aimed to provide a basis for future deafness gene-related genetic screening and identification, genetic counseling and prevention of NSHL, and to provide valuable guidance for the diagnosis and research of deafness genes. Our study has enriched the mutation spectrum of MARVELD2 and improved our understanding of its role in NSHL, which has significant implications for clinical diagnosis and management.

A 1-year-old boy failed the postnatal hearing screening test at the Hainan Provincial Women and Children’s Medical Center. After consultation, researchers found that both parents of the child were deaf, and there was no consanguineous marriage in the family. Further hearing evaluation revealed that the boy’s hearing was normal. Genetic testing revealed that he harbored a compound heterozygous mutation MARVELD2 c.663G>A and MT-RNR1 1555A>G. Gene tracking of the family revealed that the father was a proband of the MARVELD2 c.663G>A mutation. The heterozygous 12S rRNA gene mutation encoded by the MT-RNR1 1555A>G mitochondrial gene carried by the child was inherited from the maternal lineage, and the heterozygous MARVELD2 c.663G>A gene mutation was inherited from the homozygous father. This study was approved by the Ethics Committee of the Hainan Women and Children’s Medical Center. All participants provided informed consent for genetic testing and release of their case details.

The proband (Figure 1 II6) was homozygous for the MARVELD2 c.663G>A mutation and showed profound congenital deafness and language impairment. In this family, the grandparents had normal hearing, but the grandfather (Figure 1 I1) who had passed away, the grandmother, and two brothers (Figure 1 I2, II2, II7) were heterozygous carriers of the MARVELD2 c.663G>A mutation although their hearing is normal.

We performed family pedigree analysis and next-generation sequencing (NGS) to confirm that the father (Figure 1 II6) was the proband who carried the pathogenic mutation MARVELD2 c.663G>A. This mutation caused autosomal recessive NSHL with severe bilateral hearing loss (Figure 2B). The other four individuals (Figure 1 I2, II2, II7, III3) in the family were heterozygous carriers of the MARVELD2 c.663G>A mutation and had normal hearing.

NGS analysis revealed the novel mutation, c.663G>A, in the MARVELD2 gene. The Sanger verification electropherograms validated that the father was homozygous for the pathogenic mutation MARVELD2 c.663G>A, which resulted in a nucleotide substitution of G by A at position c.663. The mother had a wild-type MARVELD2 gene. Their son was heterozygous for the pathogenic mutation MARVELD2 c.663G>A (Figure 2A). The raw sequence reads have been uploaded to the National Center for Biotechnology Information database (NCBI; GenBank accession no. PRJNA976897). Up to now, 12 mutations of MARVELD2 causing NSHL have been reported, as shown in Table 1. However, the MARVELD2 mutation identified in this study was not found in any of the previously reported studies.

As mentioned in Section 1, MARVELD2 c.663G>A introduces a nonsense mutation, that leads to truncated transcripts of the protein (Figure 2C) due to the change to a premature stop codon (p.Trp221*). We used three-dimensional structure model prediction (I-TASSER, S731348, and S730824), bioinformatic analysis, and database annotation to assess the pathogenicity of this mutation. We found that the location of the nonsense mutation results in the loss of essential domains of the tricellulin protein, such as the occludin-ELL domain, which is critical for the protein’s function (Mariano et al., 2011; Li et al., 2005). This caused the proband to present with severe NSHL (Figures 2B, E, F). These domains are involved in protein-protein interactions and tight junction formation, which are important for normal hearing.

NSHL is a common sensorineural disorder with an increasing incidence. NSHL exhibits genetic heterogeneity, with most cases showing autosomal recessive inheritance (Safka Brozkova et al., 2012). MARVELD2 encodes a four-transmembrane protein consisting of seven exons and 558 amino acids (Riazuddin et al., 2006; Nayak et al., 2013). Mutations in human MARVELD2 which is located at the DFNB49 locus have been reported to cause NSHL in different studies (Nayak et al., 2015). In this study, we used NGS and Sanger sequencing to evaluate a family with NSHL, identifying a rare mutation (c.663G>A) in MARVELD2. The mutation caused the tricellulin protein to become truncated at glutamic acid position 221, resulting in a 60.4% amino acid loss compared to the wild-type protein. To the best of our knowledge, this is the first report of the identification of the mutation.

Tricellulin is ubiquitously expressed in epithelial junctions of tissues and organs throughout the body. However, the only obvious phenotype of the MARVELD2 mutant alleles is deafness. Tricellulin encoded by MARVELD2 contains two conserved domains with important functions, four-transmembrane domain and an Occludin-ELL domain (Pfam accession number PF07303) located at the C-terminus. The tricellulin C-terminus interacts with the cytosolic adapter protein ZONA occludens-1 (ZO-1) and is thereby linked to the actin cytoskeleton (Riazuddin et al., 2006; Ikenouchi et al., 2005). Mutations that cause structural damage to the C-terminus in patients with NSHL result in restricted binding to ZO-1. This observation emphasizes the importance of the C-terminal domain for the physiological function of tricellulin. For example, an affected NSHL family member in a Pakistani family was homozygous for a transition mutation (c.1498C>T) that creates a nonsense codon p.R500X in exon five of MARVELD2, which results in a truncation within the occludin-ELL domain of the protein’s C-terminus (Riazuddin et al., 2006). In another four cases in Pakistani family (c.1183-1G>A, c.1331 + 1G>A, c.1331 + 2T>C, c.1331+2deITGAG), mutations occurred at the splice donor where exon 4 and intron 4 intersect, changing the original splicing position of hnRNA and leading to premature termination of protein translation (Riazuddin et al., 2006; Chishti et al., 2008; Nayak et al., 2015). In addition, two variants c.1555delinsAA and c.1543delA occurred in Iranian families, resulting in frameshift mutations that caused premature stop codons (Babanejad et al., 2012; Sarmadi et al., 2020). These variants all resulted in the lack of complete domains of the terminal occludin-ELL encoded by exons 5, 6, and 7. All reported variants that affect the integrity of the occludin-ELL domain have led to the occurrence of NSHL. The new truncation mutation identified in our study results in direct deletion of the C-terminus, causing severe hearing impairment.

The proband was homozygous for the MARVELD2 mutation due to its recessive inheritance pattern, and there was no consanguineous marriage within the family, which originated from Hainan Province, China. Because the mutation shows recessive inheritance, it indicates that the mutated gene has a certain carrying scale in Hainan Province. No large-scale epidemiological surveys have been conducted to estimate the prevalence of this mutation. Further research is required to determine its incidence and carrier frequency. Our findings suggest that MARVELD2 mutations must be considered in patients with NSHL in Hainan Province. Further, our study expands the spectrum of clinical manifestations associated with gain-of-function mutations in the tricellulin protein, and contributes to the identification of additional MARVELD2 gene variants.

NGS and Sanger sequencing, combined with database annotation, confirmed that the nonsense mutation c.663G>A in MARVELD2 is pathogenic and causes NSHL, and shows an autosomal recessive inheritance pattern.