A Novel <i>EYA1</i> Splicing Mutation in a Chinese Family With Branchio-Oto Syndrome: A Functional Analysis and Reproductive Intervention

Anhai Chen; Lu Jiang; Zequn Nie; Jian Song; Chufeng He; Lingyun Mei; Yalan Liu

doi:10.21053/ceo.2024.00304

Clinical and Experimental Otorhinolaryngology > Volume 18(4); 2025 > Article

Chen, Jiang, Nie, Song, He, Mei, and Liu: A Novel EYA1 Splicing Mutation in a Chinese Family With Branchio-Oto Syndrome: A Functional Analysis and Reproductive Intervention

Original Article

Clinical and Experimental Otorhinolaryngology 2025; 18(4): 306-317.

Published online: February 3, 2025

DOI: https://doi.org/10.21053/ceo.2024.00304

A Novel EYA1 Splicing Mutation in a Chinese Family With Branchio-Oto Syndrome: A Functional Analysis and Reproductive Intervention

Anhai Chen^1,^2,^3,^4,^*

, Lu Jiang^1,^2,^3,^4,^*

, Zequn Nie^1,^2,^3,^4,^*

, Jian Song^1,^2,^3,⁴

, Chufeng He^1,^2,^3,⁴

, Lingyun Mei^1,^2,^3,⁴

, Yalan Liu^1,^2,^3,⁴

¹Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China

²Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Changsha, China

³Clinical Research Center for Pharyngolaryngeal Diseases and Voice Disorders in Hunan Province, Changsha, China

⁴National Clinical Research Center for Geriatric Disorders (Xiangya Hospital), Changsha, China

Corresponding author: Yalan Liu National Clinical Research Center for Geriatric Disorders (Xiangya Hospital), 87 Xiangya Rd, Changsha 410008, China
Tel: +86-138-7483-5119, Fax: +86-731-85667618 Email: a_lan123@163.com

Co-Corresponding author: Lingyun Mei National Clinical Research Center for Geriatric Disorders (Xiangya Hospital), 87 Xiangya Rd, Changsha 410008, China
Tel: +86-138-7315-6720, Fax: +86-731-85667618 Email: entmly@163.com

^* These authors contributed equally to this study.

Received October 14, 2024 Revised January 20, 2025 Accepted February 2, 2025

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objectives

Branchio-oto syndrome (BOS) is an autosomal dominant disorder characterized by multiple system anomalies, typically sparing the kidneys. BOS exhibits considerable clinical heterogeneity and ethnic variability; most studies have been conducted in European populations rather than in Asian populations, and its prevalence is approximately 1 in 40,000. EYA1 is the most commonly implicated gene in BOS, with mutations ranging from missense to frameshift, splicing, and nonsense variants. Although splicing mutations are important contributors to the disease, previous research has paid less attention to novel mutations that cause aberrant RNA splicing and their pathogenic mechanisms. Furthermore, reproductive interventions aimed at preventing disease transmission have not been reported.

Methods

We collected samples from a three-generation Chinese family affected by BOS. Whole exome sequencing was used to screen for candidate causative genes. A minigene assay was performed to identify aberrant splicing products, and additional molecular biology techniques were employed to analyze the pathogenicity of the resulting mistranslated proteins. Preimplantation genetic testing (PGT), based on single-nucleotide polymorphism analysis, was utilized to prevent hearing loss in this family.

Results

Whole-exome sequencing identified a novel mutation, EYA1:c.1598-2AG>TA, which was classified as pathogenic according to American College of Medical Genetics and Genomics criteria. The minigene assay verified aberrant RNA splicing that is predicted to result in premature termination of EYA1 protein translation. Cytological experiments demonstrated that the truncated EYA1 protein is unstable, exhibits impaired nuclear translocation, and fails to interact properly with SIX1. PGT enabled the proband to give birth to a healthy boy.

Conclusion

We identified a novel splicing variant in the EYA1 gene and elucidated its potential molecular pathogenic mechanism through several functional experiments. Based on these findings, we successfully implemented PGT to ensure the birth of a healthy offspring free from BOS.

Keywords: Branchio-Oto Syndrome; EYA1; Minigene; Hearing Loss; Preimplantation Genetic Testing

INTRODUCTION

Branchio-oto syndrome (BOS; MIM 602588) is an autosomal dominant disorder characterized by hearing loss (HL), preauricular pits, and branchial fistulas. Patients who present with BOSlike symptoms along with renal abnormalities are diagnosed with Branchio-oto-renal syndrome (BORS; MIM 113650) [1]. The clinical phenotypes of BOS and BORS are diverse. In addition to the major symptoms, patients may exhibit additional features, such as facial asymmetry and abnormal taste [2]. The diagnosis of BOS/BORS is based on a well-established clinical framework proposed by Chang et al. [3]. A diagnosis is made when a patient meets at least three major criteria, or two major criteria accompanied by at least two minor criteria, or one major criterion with at least one affected first-degree relative. Ethnic differences have been observed, with the condition reported more frequently in European populations than in Asian populations, and with an estimated prevalence of approximately 1 in 40,000 [4].

As the second leading cause of syndromic deafness, approximately 95% of BOS/BORS patients suffer from various types and degrees of HL [5]. Mixed, conductive, and sensorineural HLs are all observed in BOS/BORS, with incidences of approximately 50%, 30%, and 20%, respectively. The severity of HL can range from mild to profound. BOS/BORS exhibits substantial clinical heterogeneity; our previous findings suggest that sensorineural HL is most frequently observed in patients carrying pathogenic variants in the SIX1 gene [5]. Otological structural abnormalities may occur in various forms and can affect the outer ear (e.g., preauricular pits, cupped ear), the middle ear (e.g., abnormal ossicular chain), and the inner ear (e.g., cochlear dysplasia, enlarged vestibular aqueducts). Additionally, branchial fistulas (observed in approximately 68.5% of cases) and renal anomalies (approximately 38.2%) are also common and problematic for affected individuals.

Three genes have been identified as causative for BOS/BORS: EYA1, SIX1, and SIX5 [6]. The EYA1 gene encodes a conserved phosphatase transactivator that interacts with the SIX1 transcription factor and plays a role in cranial sensory neurogenesis and otic structure differentiation [7,8]. EYA1 variants account for approximately 40% of BOS/BORS cases, with 189 variations recorded in the Human Gene Mutation Database (HGMD) database (http://www.hgmd.cf.ac.uk/). Mutations in EYA1 include frameshift, nonsense, missense, and splicing mutations [9]. Splicing mutations in EYA1 are particularly deleterious. For example, the c.1475+1G>C mutation causes skipping of exon 15 and produces a truncated protein [10]. Similarly, the c.1698+1G>A mutation leads to exon 17 skipping, thereby altering the C-terminal sequence of the EYA domain [11]. Other mutations, such as c.1140+1G>A, c.1598-2G>A, and c.699+5G>A, affect exons 12, 17, and 10 respectively, causing exon skipping [12]. Additionally, the c.639+3A>C mutation results in exon 8 skipping and a premature stop codon [13]. To date, approximately 50 pathogenic mutations affecting EYA1 splicing have been identified. Thus, it is important to focus on aberrant EYA1 splicing and the resulting alterations in EYA domain structure and function in BOS/BORS patients for pathogenicity verification.

Preimplantation genetic testing for monogenic disorders (PGT-M) is a crucial diagnostic technique for preventing the transmission of Mendelian diseases. This approach involves identifying specific causative mutations in embryos through polymerase chain reaction (PCR) and next-generation sequencing and selecting unaffected embryos for implantation. Currently, PGT is widely applied to various forms of hereditary HL [14]. This enables at-risk families to have children free from genetic conditions that lead to hearing impairment, thereby improving quality of life and reducing the burden of these diseases.

In this study, we describe the phenotypic heterogeneity observed in a three-generation Chinese family affected by BOS and identify a novel splicing mutation, EYA1:c.1598-2AG>TA (NM_000503.6), using whole exome sequencing (WES). Aberrant splicing was revealed by in vitro splicing analysis using a minigene assay, which demonstrated a 1 bp deletion at the beginning of exon 17 that likely leads to a premature stop codon. The predicted mutant EYA1 protein exhibited an abnormal structure, suggesting impaired function in its interaction with the SIX1 protein. Molecular biology experiments further confirmed the mislocalization and dysfunction of the mutant protein.

MATERIALS AND METHODS

This study was approved by the Medical Ethics Committee of Xiangya Hospital, Central South University (No. 202106096), and written informed consent was obtained from the patients.

Phenotype characterization

BOS was diagnosed based on a detailed physical examination and medical history of the subjects according to the diagnostic criteria proposed by Chang et al. [3]. The physical examination includes the morphological examination of the external ear, face, and neck region for identification of clinical phenotypes such as preauricular fistulas, microtia, and branchial fistulas. Auditory function tests are done with pure tone audiometry equipment. Morphological analysis of the middle ear (middle ear cavity, auditory ossicles) and inner ear structures (cochlea, vestibule,) using the temporal bone high-resolution computed tomography (HRCT). The function of the urinary system is evaluated through blood tests, and the structure of the urinary system is evaluated through B-ultrasound.

Whole exome sequencing

We had performed the WES on the normal control (I-2) and three patients (I-1, II-1, II-2) within the family. We extracted DNA samples from the subjects’ peripheral blood and used SureSelect (V6 Kit, Agilent) to enrich and capture DNA fragments (100X coverage) containing coding exons and their flanking sequences, and then sequenced them on the Illumina paired-end 150 bp platform. The original sequenced data was converted into raw sequenced reads through base calling analysis using the original image data files obtained by the Illumina sequencing platform. The raw sequenced reads were quality controlled and filtered to obtain the valid sequenced data, which were aligned to the human reference genome (GRCh37/hg19) by Burrows-Wheeler Aligner tools, and then the results were sequenced using Sequence Alignment/Map Tools [15]. The variant calling and function annotation were conducted using Genome Analysis Toolkit (GATK) v.1.6 and ANNOVAR. The detailed WES method and bioinformatics procedures were described previously [16].

Minigene assay

Nested PCR amplification of the EYA1 target region was performed using gDNA from a human control as a template for the wild type minigene. On the other hand, the mutant minigene fragment was obtained by modifying the wild type with site-specific mutations. The PCR product and pcDNA3.1 vector were double digested with Kpn I and Not I respectively and connected to the same enzyme cutting site to generate the recombinant plasmid pcDNA3.1-EYA1-WT/Mut. The pcDNA3.1-EYA1-WT/Mut plasmid was transiently transfected into HeLa cells (CRL3216, ATCC) and 293T cells (CCL-2, ATCC) using NeoFect DNA transfection reagent according to the manufacturer’s instructions and incubated in Dulbecco’s modified eagle medium (DMEM; high glucose) containing 10% fetal calf serum, 37 °C, 5% CO₂. Then, the cellular RNA was extracted, and further reverse transcribed into complementary DNA (cDNA), and the amplification product of the target fragment was obtained by PCR, and analysis of the bands by 1.5% agarose gel electrophoresis.

Three-dimensional model

Utilizing AlphaFold v2.0, we made a three-dimensional prediction for the complete protein structures of EYA1 and SIX1. To simulate the docking of EYA1–SIX1 protein structures, AlphaFold Multimer software was employed, employing the default settings for optimal results [17].

Immunoprecipitation

The overexpression plasmids pLVX-Flag-EYA1-WT/Mut were constructed according to the previous method [18]. We cotransfected the above plasmids with pcDNA3.1-SIX1 (Youbio Biotech Company) separately into 293T cells and cultured for 72 hours. Cells were harvested, an appropriate amount of cell immunoprecipitation (IP) lysis buffer (containing protease inhibitors) was added, and the cells were lysed on ice for 30 minutes, and the supernatant was extracted after centrifugation at 12,000 g for 30 minutes. After protein concentration determination, a small amount of lysate was taken for western blot analysis (Input), and the remaining lysate (6,000 µg) was incubated overnight at 4 °C with slow shaking by adding 30 µL of anti-flag M2 magnetic beads (M8823, Sigma-Aldrich). After the IP reaction, the magnetic beads were given adsorption to the bottom of the tube with the magnetic stand, and carefully remove the supernatant, was washed four times with 200 µL of TBS buffer (50 mM Tris HCL, 150 mM NaCl, pH 7.4), and finally 50 µL of 1×Flag peptide was added and incubated for 30 minutes at room temperature with horizontal shaking for the precipitated proteins.

Nucleoplasm separation experiment

293T cells were transfected with the plasmids pLVX-flag-EYA1-WT/Mut and cultured for 72 hours on 10-cm plate, then harvested and subjected to 200 µL buffer A (10 mM HEPES, 10 mM KCl) containing protease inhibitors and lysed at 4 °C for 15 minutes. The cytoplasmic protein was released by a gentle vortex after adding 10% Nonidet P-40. Subsequently, the cytosolic fraction would be harvested by a 2-minute centrifugation at 12,000 g, 4 °C. The remaining cell fraction was washed three times with buffer A for 2-minute centrifugation at 12,000 g, 4 °C, and then boiled for 10 minutes after adding 50 µL of buffer B. The nuclear protein was obtained by centrifugation for 10 minutes at 12,000 g at room temperature. The success of the separation process was assessed through western blot analysis, utilizing specific markers for nuclear and cytoplasmic proteins.

Cycloheximide treatment

293T cells were seeded in a 6 cm dish and transiently transfected with the plasmids pLVX-flag-EYA1-WT/Mut, then digested and well-distributed to culture in a 6-well plate. Before western blot analysis, cycloheximide (C7698, Sigma-Aldrich) was added to the culture medium at a final concentration of 50 μg/mL to stop protein synthesis at the indicated times.

Western blotting

The concentration of the above-extracted proteins was measured using the bicinchoninic acid assay kit (20201ES76, Yeasen). Protein lysates were then denatured by boiling for 15 minutes with sodium dodecyl sulfate (SDS) loading welling buffer. Protein samples (20 μg) were loaded on an 8% SDS-polyacrylamide gel, then electrophoresed, and transferred to PVDF membranes (Millipore). Primary antibodies used were: Flag (Abmart Cat# M20008), β-actin (Sigma Cat# A5441), GAPDH (Proteintech Cat# 60004-1-Ig), H3 (Proteintech Cat# 17168-1-AP), SIX1 (Proteintech Cat# 10709-1-AP), β-tubulin (Abmart Cat# M20005).

Preimplantation genetic testing

PGT was recommended for the couple, designated as II-1 and II-2, to ensure the birth of a healthy child free from the pathogenic variant c.1598-2AG>TA. Additionally, PGT for aneuploidy was conducted to exclude unrelated sporadic chromosomal abnormalities, thereby reducing the risk of miscarriage. The single-nucleotide polymorphisms (SNPs) identified in the affected individual II-1, his healthy wife II-2, and their son III-1 were utilized for haplotype construction. The PGT process and CNV-Seq analysis was completed by the Reproductive and Genetic Hospital of X. The detailed methods of SNP haplotype construction and genotype analysis were described previously [19]. The healthy embryo was selected for transferring into the uterine cavity following PGT screening.

RESULTS

Identification of the pathogenic gene

As sequencing costs decline and bioinformatics analyses become more standardized and streamlined, targeted capture sequencing and WES of deafness-associated genes have become important methods for the genetic diagnosis of hereditary deafness [20]. We performed WES on peripheral blood DNA from patients I-1, II-1, II-2, and a normal individual (I-2) within the family. Candidate genes—including EYA1, SIX1, and SIX5—were examined, and a novel splicing mutation, EYA1:c.1598-2AG>TA, was identified. Sanger sequencing verified the segregation of this novel variant within the family (Fig. 1). As a canonical splicing variant, EYA1:c.1598-2AG>TA has not been reported in any databases (e.g., gnomAD, ClinVar).

Clinical characteristics

A 31-year-old male patient (II-1) presented to the Department of Otolaryngology and Head and Neck Surgery with bilateral HL, abnormal external ear morphology, and branchial pits. Based on clinical examination and the diagnostic criteria of Chang et al. [3], the proband was diagnosed with BOS. Other family members were also evaluated. Proband II-1 had bilateral preauricular fistulas, bilateral branchial fistulas (with post-excision scarring), and extremely severe mixed HL in the right ear as well as moderately severe mixed HL in the left ear, requiring hearing aids. His renal function tests and urological ultrasound examinations were normal between 2019 and 2022. Otological HRCT revealed malformations in the right middle and inner ears, along with bilateral abnormal enlargement of the Eustachian tubes (Fig. 2B). Patient II-2 exhibited bilateral preauricular fistulas, a unilateral branchial pit on the right side, and severe mixed deafness in both ears, requiring hearing aids. Patient I-1 presented with bilateral preauricular fistulas and branchial pits, along with postoperative scars on his right neck and left preauricular region, and demonstrated moderate to severe HL in the left ear and very severe HL in the right ear. All affected individuals displayed abnormal microtia and facial asymmetry (Fig. 2A). Although the phenotypic severity in this BOS family is comparable to previously reported cases, HL and ear deformities are more pronounced. While renal function was normal in our study, a higher prevalence of renal abnormalities has been reported in other BOS/BORS patients [5,6]. These differences may reflect the clinical and genetic heterogeneity of BOS across different populations.

Discovery of an aberrant splicing product

The minigene assay is an in vitro technique that closely mimics the in vivo splicing process, yielding results nearly identical to those observed in vivo [21]. Accordingly, we successfully constructed both a wild-type (WT) construct and a mutant (Mut) construct containing the targeted mutation (Fig. 3A). We transiently transfected the pcDNA3.1-EYA1-WT/Mut plasmids into HeLa and 293T cells and allowed expression for 48–72 hours. Extracted cDNA was used for targeted PCR and agarose gel electrophoresis, which revealed a band of nearly identical length in both the WT and mutant samples, with no abnormally long or short bands observed (Fig. 3B). Sanger sequencing of the PCR products revealed that the c.1598-2AG>TA mutation leads to aberrant splicing at the mRNA level, specifically resulting in the deletion of one base at position c.1598 in exon 17 (Fig. 3C). This 1 bp deletion causes a frameshift mutation, introducing eight novel amino acids prior to a premature stop codon, which leads to early termination of the EYA1 mutant protein (Fig. 3D).

Three-dimensional protein structure analysis

Due to the premature termination codon, the EYA1 mutant protein is truncated from 592 to 540 amino acids, with the entire truncation located within the EYA domain (Fig. 4A). Based on the aberrant splicing model, we used AlphaFold v2.0 software to predict the crystal structures of both the wild-type and mutant EYA1 proteins. The analysis revealed that the mutant protein lacked several secondary structural elements (marked in green), including β4, β5, α13, α14, and part of the α12 motif (Fig. 4B and C). We simulated the docking of the wild-type EYA1 protein with SIX1 to assess the potential impact of the truncation on the EYA1–SIX1 complex. By marking the protein-protein docking surface in red, we observed that most of the interacting structures reside in the region truncated in the mutant EYA1 protein (marked in green) (Fig. 4E). The molecular docking model illustrates the atomic-level interactions between the wild-type EYA1 protein and SIX1 (Fig. 4F).

Verification of pathogenicity through molecular biology

We transiently transfected 293T cells with the relevant plasmids and conducted nucleoplasm separation analysis, IP, and cycloheximide treatment to extract proteins for verifying pathogenicity at the molecular level. Nucleoplasm separation experiments revealed that, upon co-transfection with a pcDNA3.1-SIX1 plasmid, the wild-type EYA1 protein was localized and expressed in the nucleus, whereas the mutant EYA1 protein failed to localize to the nucleus (Fig. 5A). We then performed an IP experiment to assess whether the mutant EYA1 protein retains the ability to interact with SIX1. In the input group, the expression of the mutant EYA1 protein was noticeably reduced, even with equivalent amounts of protein loaded. To quantify the binding affinity of the mutant protein for SIX1, we normalized the levels of Flag-EYA1 in both the wild-type and mutant samples. The IP results demonstrated that the mutant EYA1 protein was unable to pull down the SIX1 protein (Fig. 5B). Given the observed instability of the mutant protein, we treated transfected 293T cells with cycloheximide and monitored the expression levels of both wild-type and mutant EYA1 proteins over time. Results indicated that the mutant EYA1 protein exhibited significantly reduced stability, with a shortened half-life, and its expression was undetectable after 2 hours of cycloheximide treatment (Fig. 5C).

Preimplantation genetic testing

PGT identifies genetic abnormalities in embryos prior to implantation. Following a complete PGT cycle involving oocyte stimulation, retrieval, and insemination, embryos were obtained for subsequent testing (Fig. 6A). Whole genome amplification was used to amplify biopsy material from a few trophectodermal cells, allowing for comprehensive evaluation of the embryos’ genetic material—including analysis of pathogenic mutations, haplotypes, aneuploidy, and copy number variations. At 18 weeks of gestation, amniocentesis was performed for prenatal genetic diagnosis. Sanger sequencing confirmed that the fetus (patient III-1) did not carry the EYA1:c.1598-2AG>TA mutation (Fig. 1), and chromosomal analysis revealed a normal karyotype (Fig. 6B). CNV-Seq performed on the selected embryos showed no abnormalities in haploid detection or regions of homozygosity (Fig. 6C). The newborn exhibited no typical fistula changes in the external ear and neck regions (Fig. 6D). Furthermore, ear acoustic emission analysis of the newborn demonstrated that all frequencies in both ears, except for 8 kHz, elicited a meaningful distortion product otoacoustic emission with an S/N ratio greater than 6 dB (Fig. 6E). Auditory brainstem response testing at 3 months of age showed bilateral wave V thresholds of 10 dB, acceptable wave differentiation, and normal latencies (Fig. 6F).

Assessment of mutation pathogenicity

Based on American College of Medical Genetics and Genomics criteria [22], the EYA1:c.1598-2AG>TA variant is classified as pathogenic. This classification is supported by the following evidence: PVS1: the variant leads to aberrant RNA splicing and premature termination of EYA1 protein translation; PS3: functional experiments demonstrate that the truncated EYA1 protein is unstable, fails to translocate to the nucleus, and disrupts EYA1–SIX1 interactions; PM2: the variant is absent from major population databases; PP1: the variant co-segregates with HL in affected family members; PP3: multiple computational tools (e.g., PolyPhen-2, MutationTaster, and Sorting Intolerant From Tolerant) predict a deleterious effect on the gene or its product.

DISCUSSION

We analyzed a Chinese family with BOS and identified a novel EYA1 splicing variant, c.1598-2AG>TA. This variant causes aberrant EYA1 pre-mRNA splicing, which alters protein structure and stability and results in defective protein distribution. By investigating its molecular pathogenicity, we have expanded the known mutation spectrum associated with BOS/BORS.

BOS/BORS is an autosomal dominant syndromic form of deafness characterized by HL and various systemic symptoms. Patients often present with otological, branchial, and renal abnormalities, demonstrating both intrafamilial and interfamilial phenotypic variability. In our study, patients exhibited various auricular malformations, sometimes even within the same individual. Cup-shaped auricular deformities were observed in both ears of patient II-2 and in the left ears of patients II-1 and I-1, while helix-shaped malformations were noted in the right ears of patients II-1 and I-1. The pinna develops from six hillocks derived from the first and second branchial arches during the fifth gestational week, and a normal auricular structure is established by the ninth week following proper fusion and development of these hillocks [23]. Incorrect or incomplete fusion can lead to external ear abnormalities such as pinna deformities and preauricular fistulas [24].

External ear malformations are readily apparent on direct observation, whereas middle and inner ear malformations often require temporal bone imaging for accurate diagnosis. These malformations can result in conductive, sensorineural, or mixed HL, with severity ranging from mild to severe. Currently, mouse models deficient in EYA1 and SIX1 display clinical manifestations similar to BOS/BORS patients—including otological structural abnormalities and kidney malformations [25,26]. Otological malformations are among the prominent clinical manifestations of BOS/BORS, which include external, middle, and inner ear malformations. Causative-gene deficiency mouse models present craniofacial anomalies due to the abnormal development of the branchial arches’ morphology, which may explain why the patients developed malformations and functional abnormalities of the ear.

Pathogenic mutations in the EYA1 gene are the primary genetic cause of BOS/BORS, accounting for approximately 40% of cases. These mutations include frameshifts, nonsense mutations, missense mutations, aberrant splicing, deletions, and complex rearrangements. In our study, we identified a novel pathogenic mutation, EYA1:c.1598-2AG>TA, located in the canonical splice region at positions –1 and –2. Although splicing mutations are prevalent among BOS/BORS patients with EYA1 mutations, mutations affecting the two crucial bases (AG) of the 3´ splice acceptor site have not been previously reported. The canonical splice site (GT-AG) is recognized and processed by the U2-type spliceosome [27,28]. Typically, mutations at canonical splice sites occur at the +1 and +2 positions of the 5´ donor splice site or at the –2 and –1 positions of the 3´ acceptor splice site in introns. For example, in the EYA1 gene, mutations such as c.1140+ 1G>A, c.1475+1G>C, c.1598-2G>A, and c.1698+1G>A have been observed [10-12], often resulting in exon skipping. In our case, the nucleotide sequence at the 3´ splice acceptor site was altered from AG to TA. Furthermore, the mutated TA bases were immediately followed by a guanine (G), which may have inadvertently formed a novel 3´ splice acceptor site (AG). This suggests that the EYA1:c.1598-2AG>TA mutation could lead to the production of aberrant pre-mRNA transcripts.

The minigene assay is a valuable tool for identifying aberrant splicing products resulting from mutations and evaluating the effects of abnormal pre-mRNA splicing [8,29]. Misregulated alternative splicing can produce transcripts and proteins with altered or even opposing functions, leading to disease. We performed a minigene assay on both wild-type and EYA1:c.1598-2AG>TA samples using HeLa and 293T cells, followed by Sanger sequencing. Results demonstrated that the mutation causes the skipping of the first base of exon 17, leading to codon mismatches and premature termination of the EYA1 protein. This confirms that the novel mutation disrupts the canonical “GT-AG” splicing rule in intron 16, creating a novel 3´ splice acceptor site and resulting in downstream base skipping with a frameshift.

The minigene experiment revealed that a premature stop codon reduces the EYA1 protein from 592 to 540 amino acids, causing conformational changes and potential functional abnormalities. We used AlphaFold v2.0 to analyze the spatial conformation of the mutant protein. The predicted three-dimensional structure model revealed deletions in multiple secondary structures—including β4, β5, α13, α14, and part of the α12 motif—compared to the wild-type protein. The loss of these secondary structural elements, all located within the key EYA domain, is likely to cause significant functional abnormalities. Previously, Patrick et al. [30] resolved the crystal structures of the EYA2 and SIX1 proteins and elucidated the molecular details of the SIX1-EYA2 complex, providing a valuable model to guide EYA1 protein structure prediction and functional research. We modeled the interaction between the wild-type EYA1 protein and SIX1, analogous to the EYA2-SIX1 complex, to assess potential functional impairments caused by the EYA1 mutant (Fig. 4D). The mutant protein lacks the β4, β5, and α14 motifs (marked in red) at the docking interface, likely disrupting its interaction with SIX1 (Fig. 4E). Key amino acids involved in hydrogen bonding and salt bridge formation are located in the deleted region, undermining the integrity of the interaction. Nonetheless, our analysis revealed mismatched binding at the mutant’s interaction surface (data not shown), suggesting that these structural changes significantly impact the functional integrity of the EYA1–SIX1 complex. The EYA1–SIX1 complex is crucial for organogenesis, particularly for inner ear and kidney development. In the inner ear, it collaborates with SOX2 to activate Atoh1 transcription—promoting hair cell fate specification—and interacts with the SWI/SNF complex to initiate neurodevelopmental programs. In kidney development, EYA1 is essential for renal interstitium formation, and its absence can lead to renal agenesis. EYA1, in conjunction with SIX1 and PAX2, regulates GDNF expression, while SIX1 modulates the BMP4–GREM1 interaction to control ureteric bud morphogenesis. Failure of EYA1 to bind SIX1 can result in developmental defects such as BOR syndrome, which is characterized by ear, kidney, and pharyngeal arch abnormalities, as well as congenital cataracts and cardiac facial syndrome [31,32].

To investigate the functional impact of the mutant EYA1 protein, we conducted several molecular biology experiments. Normally, SIX1 facilitates the nuclear translocation of EYA1 to activate target genes [33]. However, the mutant EYA1 protein exhibited reduced expression and failed to localize to the nucleus, suggesting a diminished capacity for interaction with SIX1. IP experiments confirmed that the mutant protein was unable to bind SIX1. Additionally, the mutant protein displayed poor stability and a shortened half-life, further impairing its function. These findings, together with evidence from Zou et al. [34] demonstrating that BOS/BORS incidence is dose-dependent on EYA1 expression, suggest that the EYA1:c.1598-2AG>TA variant likely contributes to BOS/BORS through haploinsufficiency. Furthermore, Zou et al. [34] showed that Eya1 is essential for the development of sensory epithelia in the inner ear, and that reduced Eya1 expression levels (40% and 21% of normal) in mice result in malformations and complete sensory loss, respectively.

Based on these findings, we successfully applied PGT to enable a BOS-affected proband to give birth to a healthy boy for the first time. This achievement underscores the potential of PGT in preventing the transmission of genetic disorders. However, widespread clinical application of PGT faces several challenges. High costs associated with advanced technical equipment, professional training, and the expenses of detection and analysis limit its accessibility. Geographical disparities and uneven distribution of medical resources result in long wait times and limited access in remote areas. Additionally, patients’ limited understanding of PGT and the complexity of the decision-making process may deter them from utilizing this technology. Technically, PGT has limitations, including the possibility of false results and an inability to detect novel mutations or chromosomal abnormalities arising during embryonic development. Furthermore, ethical concerns—such as the potential for “designer babies” and exacerbation of social inequality—pose significant challenges to the broader application of PGT [14]. Despite these challenges, our successful use of PGT in this case demonstrates its value and potential to improve the lives of families affected by genetic conditions such as BOS.

In conclusion, we identified a novel splicing variant, EYA1: c.1598-2AG>TA, in a Chinese BOS family with three affected individuals. This discovery not only expands the genetic spectrum of EYA1 mutations but also reveals a novel aberrant pre-mRNA splicing event that elucidates the molecular pathogenic mechanism of the resulting truncated protein. These results provide novel insights into the pathogenic mechanism of this mutation, which may have significant implications for the clinical diagnosis, prognosis, and treatment of BOS patients with EYA1 pathogenic mutations.

HIGHLIGHTS

▪ A novel splicing mutation, EYA1:c.1598-2AG>TA, was identified in a Chinese family with branchio-oto syndrome (BOS) and was shown to cause aberrant RNA splicing, leading to premature protein truncation.

▪ Functional experiments revealed that the truncated EYA1 protein was unstable, failed to translocate to the nucleus, and disrupted EYA1–SIX1 interactions, suggesting a molecular pathogenic mechanism.

▪ Based on the discovery of this novel pathogenic mutation and subsequent functional analyses, we successfully applied preimplantation genetic testing for the first time to prevent the transmission of BOS to offspring.

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

ACKNOWLEDGMENTS

This work was supported in part by the Bioinformatics Center, Xiangya Hospital, Central South University. Additional support was provided by the Natural Science Foundation of Hunan Province (Grant No. 2021JJ31084 and 2021JJ41017) and the Foundation of Hunan Provincial Health Commission (Grant No. 202107010047).

We would like to thank the patient and family members who contributed to this study.

AUTHOR CONTRIBUTIONS

Conceptualization: AC, YL, LM. Methodology: AC, LJ, ZN. Formal analysis: AC, LJ, ZN. Data curation: AC, LJ, ZN, JS, CH. Visualization: AC, LJ. Project administration: YL, LM. Funding acquisition: YL, LM. Writing–original draft: AC. Writing–review & editing: AC, LJ, ZN, YL, LM. All authors read and agreed to the published version of the manuscript.

Fig. 1.

Family pedigree and confirmation of the EYA1:c.1598-2AG>TA variant via Sanger sequencing. Co-segregation analysis was performed among family members. Square, male; circle, female; black arrow, proband; quarter-filled symbol, affected individual with distinct phenotypes.

Fig. 2.

Detailed clinical phenotypes of the three individuals with branchio-oto syndrome. (A) Pure tone audiometry (PTA) along with clinical photographs of the external ear and neck structures. Red circles indicate the locations of preauricular and branchial cysts or fistulas; red solid arrows point to auricular malformations. (B) Temporal bone high-resolution computed tomography (HRCT) of proband II-1 demonstrating deformities of the right external and inner ears, along with abnormal bilateral enlargement of the Eustachian tubes. Red hollow arrows indicate the abnormally enlarged Eustachian tubes, and red circle highlights structural abnormalities of the right vestibule, cochlea, and semicircular canals. R, right; L, left.

Fig. 3.

Identification of aberrant RNA splicing via minigene assay. (A) Sanger sequencing confirms the successful construction of wild-type (WT) and mutant (Mut) minigene plasmids. (B) Agarose gel electrophoresis showing complementary DNA (cDNA) bands from WT and Mut constructs. (C) Sanger sequencing reveals a c.1598delG event in the EYA1 pre-mRNA as a result of the novel variant. (D) Aberrant RNA splicing leads to premature truncation of the EYA1 protein.

Fig. 4.

Crystal structure analysis of wild-type and truncated EYA1 proteins. (A) Schematic diagram of the EYA1 exon structure and the coding region of the EYA domain. (B, C) Predicted three-dimensional structures of the EYA domain in wild-type (WT) and mutant (Mut) proteins; the truncated portion in the mutant is highlighted in green. (D) Crystal structure of the EYA1–SIX1 interaction interface, with key regions marked in red. (E) Magnified view of the secondary structure at the protein–protein docking interface. (F) Molecular docking results illustrating the protein–protein interaction interface.

Fig. 5.

Verification of the pathogenicity of the truncated EYA1 protein via molecular biology techniques. (A) Nucleoplasm separation experiments indicate that the mutant EYA1 protein has difficulty entering the nucleus. (B) Immunoprecipitation experiments demonstrate that the truncated EYA1 protein fails to pull down the SIX1 protein. (C) Cycloheximide treatment shows that the truncated protein is rapidly degraded. WT, wild-type; Mut, mutant.

Fig. 6.

Preimplantation genetic testing (PGT) results and clinical evaluation of patient III-1. (A) Patient II-1 delivered a healthy child (III-1) following PGT for monogenic disorders (PGT-M) and PGT for aneuploidy (PGT-A). (B) Chromosomal analysis of III-1 shows a normal karyotype. (C) CNV-Seq of III-1’s embryos reveals no issues with haploidy or runs of homozygosity (ROH). (D) Newborn photographs of III-1 show normal ear and neck appearance without fistulas. (E) Distortion product otoacoustic emissions results for III-1 are normal at all frequencies except 8 kHz of bilateral ears. (F) Bilateral audiory brainstem response testing at 3 months for III-1 demonstrates a 10 dB wave V threshold with normal latency.

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