Otofaciocervical Syndrome and Its Overlap with Branchio‐Otorenal Spectrum: An Integrated Literature Analysis of EYA1‐Related Disorders, Including a Novel Case with an 8q13.2q13.3 Deletion

1. Introduction

Craniofacial syndromes associated with branchial arch anomalies represent a clinically and genetically heterogeneous group of disorders, often characterized by overlapping features that complicate diagnosis and etiological classification [1,2]. Among these, Otofaciocervical syndrome (OTFCS) is a rare genetic disorder first described by Fara et al. in 1967, with fewer than ten cases reported in the literature [3,4]. It is characterized by peculiar craniofacial traits (e.g., long triangular face, broad forehead, narrow nose and mandible, and high arched palate), ear abnormalities (e.g., Low-set, cup-shaped ears with prominent conchae and a hypoplastic tragus and lobe) often associated with hearing loss, and shoulder girdle anomalies (sloping shoulders, low-set clavicles, winged scapulae, and trapezius hypoplasia). Skeletal anomalies other than girdle anomalies and nasolacrimal duct defects are frequently reported, whereas neurodevelopmental delay and short stature are observed only in some patients [5,6]. OTFCS shares significant phenotypic overlap with branchio-oto-renal spectrum disorders (BORSD) [7,8]. Nonetheless, they have been previously described as clinically distinct entities: phenotypic traits such as facial dysmorphisms and shoulder girdle anomalies were considered specific to OTFCS, whereas BORSD were explicitly characterized by functional and structural renal anomalies (Table 1) [9,10].

Heterozygous variants in EYA1 (eye transcriptional coactivator and phosphatase 1) account for approximately 40-75% of individuals clinically diagnosed with BORSD [11,12], but have also been reported in OTFCS patients [13,14,15]. Other genes in the Pax-Six-Eya-Dach network (PSEDN) are likewise implicated in both phenotypes. Heterozygous mutations in SIX1 (sine oculis homeobox homolog 1) and SIX5 (sine oculis homeobox homolog 5) have been detected in 3.0-45% and 0-3.1% of individuals with BORSD, respectively [16,17,18]. In addition, biallelic PAX1 (paired box 1) variants underlie OTFCS type 2 with T-cell deficiency (OTFCS2) [19,20,21,22], while loss-of-function variants in EYA4 have more recently been reported in a single affected family [4].

Whether OTFCS and BORSD represent distinct nosological entities or instead form part of a broader phenotypic continuum remains unresolved, as the precise genetic basis of OTFCS is not yet fully clarified. Importantly, some individuals with a BORSD diagnosis present with features typical of OTFCS—musculoskeletal and neurodevelopmental involvement—while some OTFCS patients exhibit renal anomalies, suggesting that the two conditions may, at least in part, represent allelic disorders [11,23,24]. This growing body of evidence supports the hypothesis that these disorders may, at least in part, represent allelic conditions.

In this study, we report a novel patient presenting with OTFCS harboring a de novo microdeletion encompassing EYA1, and perform a comprehensive review of all published cases of EYA1-related disorders. By delineating overlaps and distinctions between OTFCS and BORSD, we aim to refine their allelic relationship, improve diagnostic precision, and inform genetic counseling, while contributing to a deeper understanding of the molecular mechanisms underlying these syndromes.

2. Materials and Methods

2.1. Clinical and Molecular Data

Clinical and audiological information was collected for the index patient, including detailed phenotypic characterization with particular attention to branchial, auricular, renal, and neurodevelopmental features. Audiological assessments included the type and degree of hearing loss. Genomic DNA was extracted from peripheral blood samples.

Chromosome microarray analysis (CMA) was performed using the CytoScan XON array (Thermo Fisher Scientific, Waltham, MA, USA).

Multiplex Ligation-dependent Probe Amplification (MLPA) was performed using the SALSA MLPA probemix P153-B1 EYA1 kit (MRC-Holland), and variant analysis was carried out with Coffalyser.Net software (MRC-Holland). The coordinates of detected deletions were mapped to the human genome assembly hg38 (GRCh38). Segregation analysis was performed to determine the inheritance pattern.

2.2. Literature Review and Data Extraction

A systematic literature review was conducted (last search: August 2025) using PubMed, Scopus, Embase, and Google Scholar, with the following keywords: “BORSD”, ‘‘BOR syndrome’’, ‘‘BO syndrome’’, ‘‘OFC syndrome’’, ‘‘OTFC syndrome’’, ‘‘BOF syndrome’’, ‘‘BOU syndrome’’, “branchio-oto-renal”, “branchio-otic”, “Otofaciocervical”, “del”, “deletion”, and ‘‘EYA1’’. Filters applied: English language, human studies, and original clinical/genetic data.

2.3. Inclusion and Exclusion Criteria

Included: case reports, series, or cohorts with (i) EYA1 SNVs/indels or CNVs and (ii) patient-level clinical data covering ≥2 domains (branchial, otologic, renal, craniofacial, musculoskeletal). Excluded: reviews, functional-only/animal studies, or overlapping cohorts (retaining the most complete report).

2.4. Screening and Data Extraction

Two reviewers independently screened titles/abstracts, followed by a full-text review. Extracted data included demographics, clinical features (categorized as BORSD-typical or OTFCS-typical), variant type (missense, truncating, splice, indel, stop-loss, structural/CNV), and deletion coordinates. All variants were described according to HGVS nomenclature using the EYA1 transcript NM_000503.6 and mapped to GRCh38. Duplicates were removed.

3. Results

3.1. Clinical and Molecular Data

A 22-year-old female, born at term, second child of healthy non-consanguineous parents, presented with severe congenital bilateral mixed hearing loss, bilateral preauricular fistulas, hypoplasia of the left shoulder muscles, winged scapula, short stature (<3rd percentile), and a history of speech delay. Chromosome analysis revealed a normal female karyotype (46,XX). The patient previously tested negative for sequence analysis of the EYA1 gene. MLPA analysis identified a heterozygous de novo deletion encompassing the entire coding region of EYA1 at 8q13.3. CMA analysis (Figure 1) confirmed a 2.3 Mb interstitial deletion at 8q13.2q13.3 chromosomal region, which spanned from nucleotides 69,068,130 to 71,362,732 (GRCh38) and involved 12 genes (LINC01592, LINC01603, SULF1, SLCO5A1, PRDM14, NCOA2, LOC101926892, TRAM1, LACTB2-AS1, LACTB2, XKR9, EYA1). The microdeletion occurred de novo because both parents resulted wild-type.

3.2. Literature Review and Data Extraction

The search retrieved more than 200 records in PubMed and additional records in Scopus; after deduplication and eligibility screening, 55 studies and 141 reported SNVs were included. Among these, 54 (38.3%) were frameshift variants (fs), 30 (21.3%) were nonsense variants (ns), 28 (19.9%) were splice-site variants (sp), 26 (18.4%) were missense variants (ms), 2 (1.4%) were stop-loss/stop-like variants (sl), and 1 (0.7%) was annotated as an indel (Figure 2). EYA1 gene SNVs found in the literature in association with OTFC/BORSD spectrum are shown in Table 2, according to the first accession of genotype and/or complete phenotype. OTFCS cases are further characterized in Table 3.

4. Discussion

The present review highlights the complex relationship between BORSD and OTFCS, both associated with EYA1 copy number and sequence variants. BORSD has traditionally been defined by a triad of branchial, otologic, and renal anomalies [7,18]. In contrast, OTFCS has been described as a distinct condition, characterized by musculoskeletal anomalies such as scapular dysplasia and short stature [9,14]. However, our systematic analysis and the present case emphasize that considerable phenotypic overlap exists, and that classical BORSD features may co-occur with OTFCS hallmarks.

The EYA proteins are components of a conserved regulatory network that is often referred to as the PAX–SIX–EYA–DACH developmental network (PSEDN) to reflect better the proteins involved [25]. This network plays a key regulatory role in the early development of multiple organs [26,27]. Notably, all known disease genes implicated in BORSD and OTFCS belong to this network. While OTFCS has also been genetically linked to PAX1 and, in a limited number of patients, EYA4 in [4,14,19], EYA1 remains the major gene implicated in conditions.

Pathogenic EYA1 variants encompass truncating, missense, splice-site, stop-loss, and copy-number alterations, and have been documented in association with BORSD, OTFCS, and intermediate phenotypes [6,28]. The variant class alone is insufficient to predict the clinical presentation. We observed that large EYA1 deletions are enriched among BORSD cases, accounting for about 20% of cases in the literature [29,30], and two-thirds of reported EYA1 SNVs were predicted to be loss-of-function (LoF), consistent with the notion that haploinsufficiency is the main disease mechanism. Conversely, OTFCS – which has been hypothesized as a contiguous gene deletion syndrome [14]– has also been observed with missense and splice variants [13,15]. Complex rearrangements, inversions, and insertions further contribute to the mutational spectrum [31,32].

A particularly noteworthy finding from our review is that all published patients with OTFCS due to EYA1 defects presented with renal anomalies. Since renal involvement has been traditionally associated with BORSD, this observation undermines the concept of a strict clinical separation between the two syndromes. Instead, it suggests that musculoskeletal involvement in OTFCS and renal anomalies in BORSD are not mutually exclusive, but somewhat variable manifestations of the same allelic defect.

The wide spectrum of presentations of EYA1-related disorders suggests that modifying factors, such as genetic background, environmental influences, or stochastic events during development, may critically modulate the expressivity of EYA1 variants [33]. Analogous patterns are well recognized in other genetic conditions such as COL2A1-related skeletal dysplasias and TBX6-related segmentation defects, where allelic heterogeneity and modifiers account for wide phenotypic variability [34,35,36,37]. Rather than being distinct syndromes, BORSD and OTFCS may represent different clinical expressions of EYA1 dysfunction within the context of the broader PSEDN. Reports of identical or highly similar EYA1 anomalies resulting in divergent phenotypes in different families further support this model [29,38,39].

From a clinical standpoint, acknowledging OTFCS and BORSD as allelic disorders has significant implications. It underscores the need to systematically evaluate musculoskeletal and developmental features in patients diagnosed with BORSD, and conversely, to ensure comprehensive renal and auditory assessments in patients with OTFCS. Grouping both under the umbrella of EYA1-related disorders would enhance and streamline variant interpretation, strengthen genetic counseling, and support the development of follow-up protocols that integrate renal, otologic, and skeletal surveillance.

Future studies should pursue three main directions: (i) large-scale genotype–phenotype analyses integrating both BORSD and OTFCS cases; (ii) functional studies to elucidate the molecular impact of different EYA1 variants; and (iii) investigation of potential second-site modifiers within the PSEDN network that might influence phenotypic outcome.

5. Conclusions

Our findings consolidate the model of BORSD and OTFCS as allelic disorders within a unified EYA1-related spectrum. This reclassification is critical for clinical practice: it improves diagnostic accuracy, mandates comprehensive phenotyping—most notably, systematic renal screening in all OTFCS patients—and refines prognostic and genetic counseling. Future research integrating deep phenotyping, genomic data, and functional studies will be essential to elucidate the mechanisms underlying the striking phenotypic variability within this spectrum.