Paired box (PAX) genes encode a family of nine transcription factors that function as master regulators of embryogenesis, organogenesis, and lineage specification. Their tightly regulated spatial and temporal expression is essential for the development of multiple organ systems, including the central nervous system, eyes, kidneys, immune system, musculoskeletal system, and endocrine organs. Germline mutations of PAX genes result in a broad and often pleiotropic spectrum of human disease, reflecting the developmental programs governed by each family member. Pathogenic variants in PAX genes underlie diverse congenital disorders such as aniridia (PAX6), renal coloboma syndrome (PAX2), otofaciocervical syndrome with immunodeficiency (PAX1), Waardenburg syndrome (PAX3), maturity-onset diabetes of the young (PAX4), and tooth agenesis (PAX9). These conditions frequently demonstrate variable expressivity, incomplete penetrance, and overlapping phenotypes, which make it challenging to be clinically recognized. Beyond embryogenesis and embryologic development, emerging evidence indicates that several PAX proteins remain active in postnatal tissue maintenance, adult stem cell regulation, immune function, and regenerative responses (particularly PAX7 in skeletal muscle satellite cells and PAX5 in B-cell homeostasis), further expanding their clinical relevance. This review provides a synopsis of the major, clinically relevant, germline PAX gene mutations, emphasizing genotype-phenotype correlations, developmental mechanisms, and disease classification across the organ systems. By integrating molecular genetics with human pathology, we highlight the diagnostic implications of PAX genes as central determinants of congenital disease and provide a framework for understanding how alterations in the developmental transcriptional networks translate into human pathology.

Otofaciocervical syndrome (OTFCS) is a rare disorder characterized by facial, auditory, and shoulder girdle anomalies. Its significant phenotypic overlap with branchiootorenal spectrum disorders (BORSD)-both linked to EYA1 (EYA transcriptional coactivator and phosphatase 1) gene defects-has raised questions about whether they are distinct entities or part of a single clinical spectrum. We report a novel OTFCS patient with a de novo microdeletion spanning EYA1 and review all published cases of EYA1-related disorders. Our analysis reveals that all EYA1 variant types (truncating, missense, CNV, etc.) can cause BORSD, OTFCS, or hybrid phenotypes, firmly supporting their status as allelic disorders. Crucially, all reported OTFCS patients with EYA1 variants had renal anomalies, a feature previously considered a hallmark of BORSD. We conclude that BORSD and OTFCS constitute a single EYA1-related diagnostic continuum. This reclassification mandates the development of follow-up protocols that integrate renal, otologic, and skeletal surveillance in EYA1-related disorders, including OTFCS, and refines prognostic and genetic counseling.

Objective: To determine the diagnosis of microtia-associated syndrome through genetic testing. Methods: Peripheral venous blood samples were collected from members of a two-generation family with a syndrome associated with ear malformations (3 patients and 1 normal control). Pathogenic mutations were identified using whole exome sequencing analysis, Sanger sequencing validation, and bioinformatics analysis. Based on the genetic diagnosis and a review of the literatures, the patients' clinical phenotypes were thoroughly evaluated to confirm the clinical diagnosis. Results: All three patients carried a novel heterozygous insertion mutation (c.1171_1172insGGCC: p.Pro391fs) in the paired box 1 (PAX1) gene. This mutation showed genotype-phenotype co-segregation within the family and was predicted to be pathogenic. Consequently, the family was diagnosed with autosomal dominant otofaciocervical syndrome 2. The clinical phenotypes of the patients included not only ear malformations and conductive hearing loss but also branchial cleft fistula, preauricular fistula, bilateral facial asymmetry, spinal deformities, and short stature, which were major symptoms of otofaciocervical syndrome 2. Imaging also revealed previously unreported phenotypes, including parotid gland malformation and facial nerve dysplasia. Conclusion: The heterozygous insertion in PAX1 (c.1171_1172insGGCC: p.Pro391fs) found in this family causes otomandibular-cervical syndrome type 2 in an autosomal dominant manner, leading to congenital anomalies affecting external and middle ear, craniofacial region, and spine. 目的： 通过遗传学检测明确一个耳畸形相关综合征家系的诊断。 方法： 采集一个两代耳畸形相关综合征家系成员中3例患者及1例正常对照者的外周静脉血，利用全外显子组测序分析、Sanger测序验证和生物信息学分析鉴定致病性变异。根据基因诊断结果结合文献回顾详细评估患者临床表型，明确临床诊断。 结果： 3例患者均携带配对盒1（paired box 1，PAX1）基因新的杂合插入变异（c.1171_1172insGGCC：p.Pro391fs）。该突变在家系内呈现基因型-表型共分离，预测具有致病性，该家系患者被诊断为常染色显性遗传模式的耳-面-颈综合征2型。患者的临床表型除耳畸形和传导性听力损失外，还包括鳃裂瘘管、耳前瘘管，双侧面部不对称、脊柱畸形以及身材矮小等耳-面-颈综合征的主要症状。通过影像学检查，还发现患者有既往未被报道的表型，即腮腺畸形以及面神经发育不良。 结论： 本家系中PAX1基因杂合插入变异（c.1171_1172insGGCC：p.Pro391fs）可导致常染色显性遗传耳-面-颈综合征2型，引起外中耳、颅面、脊柱等处先天发育异常。.

Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Otofaciocervical syndrome (OTFCS) is a rare genetic disorder of both autosomal recessive and autosomal dominant patterns of inheritance. It is caused by biallelic or monoallelic mutations in PAX1 or EYA1 genes, respectively. Here, we report an OTFCS2 female patient of 1st consanguineous healthy parents. She manifested facial dysmorphism, hearing loss, intellectual disability (ID), and delayed language development (DLD) as the main clinical phenotype. The novel homozygous variant c.1212dup (p.Gly405Argfs*51) in the PAX1 gene was identified by whole exome sequencing (WES), and family segregation confirmed the heterozygous status of the mutation in the parents using the Sanger sequencing. The study recorded a novel PAX1 variant representing the sixth report of OTFCS2 worldwide and the first Egyptian study expanding the geographic area where the disorder was confined.