Branchiootorenal syndrome (BOR1, MIM# 113650) is an autosomal dominant disorder characterized by branchiogenic malformations, various degrees of hearing loss, and renal involvement (Zheng et al., 2021). Branchiootic syndrome (BOS1, MIM# 602588) is distinguished by normal renal anatomy and function. The incidence of this disorder is approximately 1 in 40,000 live births, and accounts for about 2% of children with severe deafness (Li et al., 2018).

Molecular studies have been performed in patients with BOR/BOS, which revealed the most known causative genes were EYA1 (BOR1, MIM# 113650, BOS1, MIM# 602588), SIX1 (BOS3, MIM# 608389), and SIX5 (BOR2, MIM# 610896). Of these genes, EYA1 variants—including frameshifts, and partial duplications—account for the majority of diagnosed patients (Dantas et al., 2015; Wang et al., 2018). EYA1 is a transcription factor important in developing several tissues, including the branchial arches, kidney, eyes, and ear. However, no significant relationship has been shown between the nature of the mutations and the clinical features associated with BOR/BOS. Whole-exome sequencing (WES) is an efficient strategy to identify the coding sequences of a genome, in which 85% of pathogenic mutations are located, and has shown significant advantages in diagnosing genetic disorders (Rabbani et al., 2014; Yang et al., 2015).

Herein, we report a Chinese child with BOS with a novel pathogenic mutation in the EYA1 gene and secretory otitis media (SOM) and vestibular hypofunction (VH), which has not been previously reported. WES and Sanger sequencing was used to analyze the proband’s DNA sample. We conducted this study to expand both the mutational spectrum and clinical characteristics of BOS.

BOS is a rare autosomal dominant disorder with high genotype heterogeneity (Chen et al., 2019). It is expressed differently in various individuals, and currently, there is no evidence of genotype-phenotype correlations in patient with BOS (Unzaki et al., 2018). In 2004, Chang et al. (2004) proposed a widely accepted diagnostic criteria for BOR/BOS using the major and minor criteria. The major criteria include branchial arch anomalies, hearing loss, preauricular pits, and renal anomalies; minor criteria include external, middle, and inner ear anomalies, preauricular tags, facial asymmetry, and palatal abnormalities (Table 1). Patients without a family history are diagnosed with BOR if they meet three or more of the above major criteria or two major and at least two minor criteria (Chang et al., 2004). A previous study reported the frequency of major criteria in BOR as follows: hearing loss 95.4%, preauricular pits 87%, branchial arch anomalies 86.5%, and renal anomalies 58.3% (Li et al., 2020). From a clinical standpoint, hearing loss is the most common and constant feature of the EYA1 gene mutations and is detected in more than 90% of affected individuals (Chang et al., 2004). Treatment of BOS is mainly symptomatic, including the surgical removal of branchial arch fistulae and cochlear implants (Men et al., 2020).

The proband in this study had known BOS clinical presentations such as bilateral preauricular pits, bilateral cervical branchial arch fistulae, hearing loss, right external ear, and bilateral inner ear anomalies. In addition to the above manifestations, the patient had SOM and bilateral VH, phenotypes not previously reported in other studies. While SOM in an 8-year-old child is not rare, SOM in the proband occurred on the same side as the external ear anomaly. This indicates that BOS may affect the Eustachian tube and ear ventilation, which can lead to inflammation. The proband also has VH, which has not previously been associated with BOS. The most common etiology of bilateral VH is gentamicin ototoxicity, followed by other rare entities, such as autoimmune inner ear disease, meningitis, Meniere’s disease and vestibular neuritis; these features were not present in the proband (Hain et al., 2018). HRCT indicated lateral semicircular canal-vestibule dysplasia in the proband’s left ear (Figures 2B, E), which may have caused VH. This finding warrants more attention to the vestibular functions in BOS patients in the future.

Nowadays, WES can sensitively detect sequence variants. In this study, we identified a novel EYA1 frameshift mutation c.1697_1698delinsT [p.(Lys566IlefsTer73)] using read-depth analysis of WES data in this family with BOS and verified by Sanger sequencing in the proband but not in unaffected family members. The EYA1 gene, located on chromosome 8q13.3, is the human homolog of the Drosophila EYA1 gene, which is essential for eye development in this species (Orten et al., 2008). The EYA1 protein is also important for the ear, branchial arches, and kidney development. Animal model studies have shown the EYA1 homozygous-deficient mice lack ears and kidneys, and the EYA1 heterozygous-deficient mice present phenotypes resembling BOR (Xu et al., 1999; Xu et al., 2002). The known pathogenic genes of BOR/BOS include EYA1, SIX1, and SIX5. The EYA1 gene mutations account for approximately 40% of BOR cases, while SIX5 and SIX1 genes account for 5% and less than 1%, respectively (Shah et al., 2020). To date, more than 240 pathological mutations in the EYA1 gene have been reported. In China, EYA1 c.466C>T [p.(Gln156Ter)], and EYA1 c.1735delG [p.(Asp579 fs)] were the first variants of EYA1 identified in 2012 (Wang et al., 2012). With the development of next-generation sequencing technology, an increasing number of novel EYA1 mutations have been reported in Chinese patients with BOS/BOR (Table 2). Han et al. (2021) recruited 7 members of a Chinese family, 4 of whom affected with BOS. EYA1 c.1627C>T [p.(Gln543Ter)] was identified as a novel mutation and pathogenic cause of BOS. Similarly, Xing et al. (2020) also collected blood samples from 6 members of a Chinese family, 4 of whom affected by BOS, and found a novel frameshift variant, c.1075_1077delinsAT [p.(Gly359 fs)] in the EYA1 gene. These results suggest that the BOR/BOS criteria are potential indications for molecular studies to diagnose EYA1-associated syndromes (Chen et al., 2019; Li et al., 2020).

EYA1 is also affected by gene dose effects, and clinical phenotypic heterogeneity may be related to insufficient EYA1 gene dosage. However, gene activity can be recognized only when the number of encoded proteins exceeds a certain threshold. In addition, gene regulation of specific molecules that encode different amounts of gene products explains the wide phenotypic variation in BOS patients between or within families (Wang et al., 2018).

Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the following three criteria were applied to interpret this EYA1 c.1697_1698delinsT as a pathogenic variant with an important role in EYA1-assoicated conditions. First, it is a frameshift mutation in the EYA1 and causes gene loss-of-function; this is a known disease mechanism (very strong pathogenic criterion; PVS1). The absence of this variation in the proband’s parents suggests that this is a de novo mutation (strong pathogenic criterion; PS2). Finally, since this variant was not found in the Genome Aggregation Database (gnomAD), the rarity of the mutation was recognized (moderate pathogenic criterion; PM2).

This de novo mutation in our proband contributes to an autosomal dominant disorder. According to the law of autosomal dominant inheritance, it is estimated that the offsprings of the proband have a 50% increased risk of inheriting EYA1 pathogenic variants. Therefore, detecting the pathogenic variants for this disease is of great importance, and patients suspected of BOS should be monitored accordingly for associated otologic and second branchial arch anomalies. With the emergence of new sequencing technologies in recent years, many novel pathogenic mutations have been reported, but many unknown pathogenic variants still need to be discovered. The novel EYA1 frameshift mutation discovered in this study can be equip BOS genetic testing in the future for early diagnose of diseases and to assist with the personalized prenatal screening.

We identified a novel heterozygous de novo pathogenic frameshift mutation in the EYA1 gene in a proband with BOS. Previously unreported clinical manifestations of BOS, including bilateral vestibular hypofunction and unilateral secretory otitis media, were observed. Our results expand both the mutational and phenotypic spectra of BOS, suggesting that the mutation of a key amino acid is an etiological factor. Considering the high phenotypic heterogeneity of BOS in patients whose diagnosis is difficult based on clinical manifestations alone, genetic testing for candidate pathogenic gene mutations can successfully complement clinical diagnosis.