OBJECTIVES:PIK3CA-Related Overgrowth Spectrum (PROS) is a highly heterogeneous disease. Facial Infiltrating Lipomatosis (FIL) is a rare PROS subset, and its atypical phenotypes, such as macrotia, present diagnostic and therapeutic challenges due to limited data. This study aims to detail the diagnostic and long-term management procedures for an extremely rare case of Macrotia associated with PROS/FIL. METHODS:To confirm the underlying etiology, Whole Exome Sequencing (WES) was performed, complementing routine clinical and pathological examinations. Otoplasty was used as the intervention to reduce ear size. Long-term follow-up was conducted to monitor surgical outcome, stability, and potential recurrence. RESULTS:WES identified a PIK3CA pathogenic variant (p.H1047R). Combined with pathological findings, the patient was definitively diagnosed with PROS manifesting as the FIL phenotype. The reductive otoplasty surgery achieved significant improvement in the affected ear's morphology, reducing the deformity to a minimal, aesthetically pleasing level. Crucially, the 1-year follow-up showed remarkable stability in the near-normal ear contour, without any signs of recurrence or overgrowth. The successful aesthetic restoration significantly alleviated the patient's psychological distress. CONCLUSIONS:This case demonstrates that WES is essential for accurate molecular diagnosis of PROS/FIL in patients presenting with atypical phenotypes like Macrotia. Furthermore, otoplasty is an effective and reliable reconstructive strategy for restoring ear aesthetics in these patients, providing excellent and stable long-term results and improving patient quality of life. LEVEL OF EVIDENCE:Level 4.

Objective:To evaluate the clinical application of ultra-high frequency ultrasonography in the diag-nosis and treatment of recurrent preauricular sinus.Methods:A retrospective analysis was conducted on the clini-cal data of 96 patients(101 ears)with recurrent preauricular sinus who underwent surgical treatment at Eye&ENT Hospital of Fudan University between March 2018 to August 2023.Preoperative 22 MHz ultra-high frequen-cy color Doppler ultrasonography was performed on the lesion site of the affected ears.The surgeries were per-formed under general anesthesia,with the lesions removed from the superficial layer of the temporal fascia under a microscope.The resected tissues were submitted for pathological examination.Results:The concordance rate be-tween preoperative ultrasound findings and intraoperative observations was 97.03％,and the concordance rate with postoperative pathological results was 96.00％.The consistency between the intraoperative observations of the sinus branches and courses and the preoperative ultrasound examination was good(Kappa=0.781 and Kappa=0.790,respectively).There was no recurrence observed during the average follow-up period of 36.6 months.Conclusion:Preoperative ultra-high frequency ultrasonography can effectively diagnose and localize recurrent preauricular sinus,and it provides significant guidance for surgical treatment.

OBJECTIVES:Hemifacial microsomia (HFM) is frequently associated with middle ear anomalies. This study aimed to investigate the association between middle ear anomalies and the severity of mandibular deformity in patients with HFM. METHODS:We retrospectively analyzed patients with HFM treated at the Eye and ENT Hospital of Fudan University between 2017 and 2025. The severity of mandibular deformity was classified using the Pruzansky-Kaban classification system. External ear anomalies were graded according to the Marx classification, and middle ear morphology was evaluated using high-resolution computed tomography with the Jahrsdoerfer scoring system. Audiological outcomes were assessed using pure-tone audiometry. Correlations between mandibular classification and middle ear structures were analyzed using the Kruskal-Wallis H-test, and hearing outcomes were compared using independent samples t-tests. RESULTS:A total of 51 patients were included in this study. External ear deformities and malleus-incus complex abnormalities were prevalent across all HFM subtypes. Incus-stapes articulation differed significantly between type I and type II HFM, and between type I and type III HFM. Significant differences were observed in stapes morphology between type I and type III HFM, as well as between type II and type III HFM. Middle ear cavity development and mastoid pneumatization were significantly reduced in type III HFM compared with type I HFM. Inner ear anomalies were infrequently observed and were limited to a small subset of patients with severe HFM. Patients with more severe mandibular deformities demonstrated larger air-bone gaps and poorer air-conduction thresholds. CONCLUSION:In patients with HFM, the severity of mandibular deformity is significantly associated with middle ear structural abnormalities, including the ossicular chain, middle ear space, and mastoid pneumatization. Assessment of middle ear morphology may provide valuable adjunctive information for disease stratification, hearing outcome prediction, and surgical planning in HFM.

Craniofacial development relies on the migration of cranial neural crest cells (CNCCs) to the first and second pharyngeal arches, followed by their differentiation into various cell types during embryogenesis. Although the CNCC migration has been well-studied, the role of the niche in relation to CNCC remains unclear. Variants in FOXI3 have been implicated in craniofacial microsomia (CFM), yet the molecular mechanisms remain unexplored. FOXI3 is expressed in the ectoderm and auricle epidermis, but not in CNCCs or cartilage. Deletion of Foxi3 in the mouse CNCCs did not disrupt mandible and auricular development, further confirming that FOXI3 does not directly regulate CNCCs. However, Foxi3 deficiency in the ectoderm reduced the production of chondrogenesis-related cytokines derived from ectodermal cells, such as TGF-β1. This impairment affected CNCC proliferation through cell communication, subsequently altering the development of the mandible and auricle. These results emphasize the critical role of FOXI3 in establishing the microenvironment supporting CNCC function. Furthermore, FOXI3 directly regulates target genes associated with translation, thereby orchestrating cytokine production in epidermal cells. The validation using auricle sample from a CFM patient carrying FOXI3 mutation further supports our findings. These insights highlight the function of FOXI3 in creating the niche necessary for CNCC development and provide a basis for understanding the molecular mechanisms driving CFM pathogenesis.

OBJECTIVE:To evaluate whether contralateral EAC skin grafts improve postoperative EAC health in patients with unilateral CAA undergoing atresiaplasty. METHODS:A Zelen-design randomized controlled trial was conducted at a tertiary referral hospital between July 2020 and February 2025. Seventy-four unilateral CAA patients were randomized into case group and control group. Case group: partial reconstruction using contralateral EAC full-thickness skin grafts; remaining defects repaired with 0.3-mm-thick temporal scalp grafts. Control group: entire EAC reconstructed with temporal scalp grafts during atresiaplasty. Pre- and postoperative pure-tone audiometry (PTA), high-resolution temporal bone CT, and postoperative otoscopic evaluation were recorded. Primary outcomes: hearing thresholds, complications (re-atresia or stenosis, tympanic membrane [TM] lateralization). RESULTS:Complete recovery of the donor site from the healthy EAC was achieved in all 42 case-group patients. Fifty patients had follow-up >6 months, 28 patients were case group, and 22 were control group. There were significant air-bone gap (ABG) improvement between 2 groups (case group: 16.2 dB vs. control group: 9.1 dB; P <0.05). Case group demonstrated significantly less TM lateralization compared with control group ( χ 2 =4.711, P <0.05). TM lateralization correlated with poorer hearing outcomes ( P <0.001). CONCLUSIONS:Contralateral EAC grafting techniques improved EAC health compared with conventional scalp grafts. Contralateral EAC grafting resulted in lower rate TM lateralization and better hearing outcomes than noncontralateral grafts.

While bilateral fitting of bone conduction hearing devices (BCHDs) enhances spatial hearing, further improvements are constrained by the unresolved effects of crosstalk - an influential factor that disrupts binaural acoustic cues, such as interaural level difference (ILD) and interaural phase differences (IPD), essential for accurate sound localization. This paper introduces a simplified theoretical model to describe the crosstalk phenomenon and predict the cochlear vibrational responses under bilateral bone conduction (BC) based on principles of wave interference and superposition. The model reveals sound lateralization patterns across different ILD and IPD combinations, different from well-established principles governing air conduction (AC) sound localization, including the precedence effect and intensity rule. These predicted patterns are experimentally validated through cadaveric vibration measurements and are further corroborated in psychoacoustic sound lateralization tests conducted on healthy volunteers. The findings suggest that crosstalk induces wave interference in the skull and leads to the superposition of bilateral signals at the cochleae, resulting in these atypical lateralization patterns. This evidence highlights the inherent challenges of sound localization under BC compared to AC, identifying crosstalk-induced wave interference as a primary obstacle to improved spatial hearing for bilateral BCHD users.

Microtia is one of the most common congenital craniofacial malformations, characterized by the maldevelopment of the external and middle ear. While numerous genes have been implicated in syndromic forms of microtia, the genetic underpinnings of isolated microtia remain poorly understood. In this study, we conducted whole exome sequencing (WES) on 201 pedigrees with isolated microtia to investigate its genetic basis. Bioinformatics analysis identified 1362 deleterious variants corresponding to 332 candidate genes, including 40 previously associated with microtia-related phenotypes. Among these, variants in FOXI3, the most frequently identified pathogenic gene for isolated microtia so far, were detected. Remarkably, the remaining 39 genes, which have been recognized as pathogenic in syndromes with microtia, are also suggested to play a role in isolated microtia. However, the precise molecular mechanisms by which these genes contribute to microtia remain to be elucidated. Furthermore, through protein-protein interaction network analysis, functional annotation, and zebrafish expression profiling, we identified two novel genes, MCM2 and BDNF, as the most promising contributors to the pathogenesis of isolated microtia. Our findings, based on the largest WES study of isolated microtia pedigrees to date, provide new insights into the genetic architecture of isolated microtia and suggest promising avenues for future research.

Objective To explore the characteristics of cellular senescence in human auricular chondrocytes during long-term in vitro culture and to evaluate the effects of anti-senescence treatments on enhancing their chondrogenic function. Methods Auricular chondrocytes exhibited senescence-related characteristics after prolonged expansion in culture. To identify senescence inducers, transcriptome sequencing was performed, with findings corroborated by transmission electron microscopy analyses. Quercetin was employed as an intervention to mitigate cellular senescence progression. The alterations in cellular senescence and mitochondrial function were evaluated. Regenerative cartilage tissue was developed through in vitro chondrogenic induction and in vivo implantation with GelMA hydrogel-loaded cells in nude mice. The impact of quercetin was substantiated through histological examinations. Results Mitochondrial dysfunction was a key characteristic of auricular chondrocytes after long-term expansion culture. Chondrocytes cultured with quercetin showed a lower proportion of senescent cells and reduced mitochondrial dysfunction. The chondrocytes cultured with continuous application of quercetin formed higher quality regenerative cartilage both in vitro and in vivo compared to the control group. Conclusion The results reveal that quercetin attenuates chondrocyte senescence by alleviating mitochondrial dysfunction, thereby preventing the loss of chondrogenic function in chondrocytes subjected to long-term expansion culture.

Tissue engineering technology for cartilage regeneration has increasingly emerged as a preferred method for repairing cartilage defects. However, the loss of chondrocyte-specific phenotypes during in vitro expansion, commonly referred to as dedifferentiation, impedes cartilage regeneration. Current research has yet to fully elucidate this phenomenon, hindering the development of improved cartilage regeneration. Our study employed single-cell sequencing and transposase-accessible chromatin sequencing to identify biomarkers, cell lineages and cellular characteristics within auricular chondrocytes during in vitro expansion. Our results showed that lower passage (P3) chondrocytes exhibited more dedifferentiated phenotypes with increased chromatin accessibility, while higher passage (P6) chondrocytes demonstrated hypertrophic characteristics. Furthermore, we identified that increased calcium influx was closely associated with the early dedifferentiation of chondrocytes, while inhibiting calcium signaling in early dedifferentiated cell could reverse cell phenotypes and promoted cartilage regeneration. In-depth mechanism research revealed that the expression of MYC mRNA was downregulated by increased calcium influx, which subsequently reduced SOX5/SOX6 levels, important transcription factors for chondrocytes, leading to diminished extracellular matrix production and early dedifferentiation. In conclusion, we provide a comprehensive understanding of chondrocyte dedifferentiation and propose new strategies for optimizing cartilage regeneration systems.

Waardenburg syndrome type 2 (WS2) is an autosomal dominant disorder characterized by congenital sensorineural hearing loss, blue iris, and abnormal pigmentation of the hair and skin. WS2 is genetically heterogeneous, often resulting from pathogenic mutations in SOX10 gene. We identified a novel heterozygous frameshift mutation in SOX10 (NM_006941.4: c.22delT, p.S8Rfs*5) in a two-generation Chinese family with WS2 through whole exome sequencing. This mutation was present in both the proband, who exhibited typical features of hearing loss and pigmentation abnormalities, and his father, who showed only mild facial features. Quantitative real-time PCR revealed that the frameshift mutation leads to a reduced expression levels of SOX10 in the peripheral blood of mutation carriers. Our findings expand the spectrum of pathogenic mutations in SOX10 associated with WS2, providing valuable information for prenatal diagnosis and preimplantation screening, and underscore the role of genetic diagnosis in identifying atypical patients.

Treacher Collins syndrome (TCS) is a congenital disorder primarily caused by the mutation in the Treacle Ribosome Biogenesis Factor 1 (TCOF1) gene. However, the significance of many TCOF1 mutations remains uncertain. We report two novel mutations identified in two TCS families and assess their pathogenicity alongside two previously reported mutations. Both novel mutations, c.2115dupG (p.T706DfsTer52) and c.2142+23_2142+52 del (p.A715VfsTer31), result in truncated proteins lacking nuclear location signals (NLSs), which impedes their entry into the nucleus and reduces mRNA expression level. Notably, the mutation c.2142+23_2142+52 del, leading to the retention of a 62 bp intron and disrupting RNA splicing, represents the first documented case of intron retention in TCS patients. Additionally, the previously reported mutation c.136 C> G (p.L46V) hinders protein nuclear location, while mutation c.1719del (p.N574TfsTer22) significantly decreases mRNA levels. Our research expands the spectrum of TCOF1 mutations and provides evidence clarifying their pathogenic nature. These findings are crucial for genetic counseling and prenatal diagnosis for TCS patients.

Congenital ptosis, a genetic disorder involving levator palpebrae muscle dysfunction, is often associated with congenital myopathy. The genetic causes of this condition remain poorly understood. In this study, we identified FOXK2 mutations in five pedigrees with congenital myopathy and ptosis through whole exome sequencing and Sanger sequencing. Zebrafish with foxk2 deficiency exhibited underdeveloped skeletal muscles and reduced mobility, while mice with Foxk2 deletion in skeletal muscle stem cells (MuSCs) showed generalized skeletal muscle abnormalities. Further analysis revealed that FOXK2 deficiency impaired myogenic differentiation in C2C12 cells and disrupted mitochondrial homeostasis in both mouse MuSCs and C2C12 cells. Rescue experiments confirmed the loss-of-function effects of FOXK2 mutation. Coenzyme Q10 treatment improved mitochondrial function and alleviated skeletal muscle development defects in Foxk2 -deficient mice. Preliminary omics analysis suggested FOXK2 directly regulates the expression of mitochondrial function-related genes by modulating chromatin accessibility at its binding sites. Our study identifies FOXK2 as a novel pathogenic gene for congenital myopathy with ptosis and highlights its essential role in skeletal muscle development and mitochondrial homeostasis, offering insights for potential diagnostics and therapies.

Congenital microtia significantly impacts children’s psychological health, yet there has been limited focus on the effect of ear reconstruction surgery on psychological improvements, especially its pathway. This study aims to explore the role of ear appearance in improving psychological health in individuals with congenital unilateral microtia based on social identity theory, highlighting the limited understanding of the specific pathways involved in this relationship, and investigating how social function and benefit mediate the connection between ear appearance and psychological health in patients who underwent autologous cartilage ear reconstruction. A cross-sectional study was conducted, involving 96 patients with congenital unilateral microtia between January and June 2024 at the Eye ENT Hospital of Fudan University. Sociodemographic and clinical data were collected, along with responses to the EAR-Q and Glasgow Children’s Benefit Inventory (GCBI) questionnaires. Data analysis was performed using multiple linear regression and the PROCESS macro in SPSS. Mediation analysis revealed that social function and emotion mediated the relationship between ear appearance and psychological function (total effect = 0.82; direct effect = 0.21; indirect effect = 0.61). Three mediation pathways were identified: ear appearance influenced psychological function through social function, emotional benefit, and a combined effect of both. These findings underscore the critical role of social interaction and emotional health in shaping psychological outcomes for individuals with congenital unilateral microtia following reconstruction. The results offer valuable insights for developing targeted interventions to enhance psychological well-being in this population.

Congenital hearing loss is one of the prevalent birth defects, with approximately 60% of cases attributed to genetic factors. Genetic hearing loss is broadly classified into syndromic and non-syndromic forms, with non-syndromic hearing loss accounting for 70% of cases. MYO15A mutations are known to cause autosomal recessive non-syndromic hearing loss (ARNSHL), while MT-RNR1 mutations follow a maternal inheritance pattern and are linked to aminoglycoside-induced hearing loss. In this study, a family with diverse manifestations of non-syndromic hearing loss was investigated, including aminoglycoside-induced, congenital profound, and post-lingual profound hearing loss. Through whole exome sequencing, distinct genetic etiologies responsible for hearing loss in affected family members were identified. This is the first report to document the co-occurrence ofa compound heterozygous MYO15A mutation alongside an MT-RNR1 mutation within a pedigree. Additionally, it is the first observation of both a homozygous MYO15A c.6956+9C>G mutation and compound heterozygous MYO15A mutations (c.[6956+9C>G] + [4898T>C]) in ARNSHL.These findings broaden the genotype-phenotype spectrum of MYO15A and highlight the critical role of genetic diagnosis in managing hearing loss.

Cartilage defects, whether congenital or acquired, are highly prevalent in clinical practice. Tissue engineering offers a promising strategy for cartilage regeneration; however, the loss of chondrocyte phenotype during in vitro expansion remains a major barrier to the clinical translation of chondrocyte-based engineered cartilage. Emerging evidence has highlighted that alterations in chondrocyte metabolic states can profoundly impact their phenotypic stability. Nonetheless, how metabolic patterns shift during in vitro expansion, and whether metabolic modulation can stabilize the chondrocyte phenotype, remain insufficiently explored.To address these questions, we first utilized single-cell RNA sequencing combined with bulk transcriptomic analysis to profile the metabolic reprogramming of chondrocytes during in vitro expansion. Our findings revealed a distinct shift from glycolytic metabolism toward oxidative phosphorylation dominance. Based on this insight, we engineered a DN (double-net) hydrogel scaffold composed of collagen, PEG (polyethylene glycol), and CNF (nanocellulose). To endow the scaffold with antioxidant functionality, TA (tannic acid) was incorporated by hydrogen bonding to the CNF network, forming an antioxidant DN-TA hydrogel system.To evaluate whether attenuating aerobic metabolism could preserve chondrocyte phenotype, P3 (passage 3) chondrocytes were cultured within the hydrogel scaffold in vitro and then implanted subcutaneously into nude mice. The DN-TA hydrogel effectively preserved the chondrocyte phenotype by activating HIF-1 signaling pathway and reducing ROS (reactive oxygen species).Furthermore, after 8/12 weeks of subcutaneous implantation, the DN-TA scaffold significantly enhanced in vivo cartilage regeneration, as evidenced by increased extracellular matrix deposition and more mature cartilage formation. Collectively, our study demonstrates that reducing aerobic metabolism helps stabilize the chondrocyte phenotype and promotes functional cartilage regeneration. These findings offer novel insights for optimizing cartilage tissue engineering strategies through metabolic modulation.

Recent studies have identified pathogenic variants in the FOXI3 gene associated with craniofacial microsomia pedigrees. In zebrafish, the foxi1 gene is considered a functional homolog of the mouse Foxi3. However, research on foxi3a and foxi3b, which display homologous genes in the naming of FOXI3 in zebrafish, has predominantly focused on their roles in epidermal ionocyte function. Our study reveals that disruption of foxi3a or foxi3b results in a reduced number of cranial neural crest cells (CNCCs) and hypoplastic mandibular cartilage in zebrafish. These findings introduce a new perspective on the functional homologs of FOXI3 and highlight an unrecognized role of foxi3 in zebrafish CNCC and mandibular development.

Efforts in cartilage tissue engineering to repair injuries have seen limited success, primarily due to the inability of scaffold materials to establish a microenvironment conducive to extracellular matrix (ECM) deposition by chondrocytes. Hydrogels, which mimic human tissue, are commonly employed as scaffold materials; however, their constrained network structure and low bioactivity impede chondrocyte ECM deposition, complicating cartilage repair. In this study, we developed dynamic Col-HZ hydrogels featuring adaptive networks by forming hydrazone (HZ) bonds between bioactive natural collagen and synthetic polyethylene glycol (PEG). In contrast to static hydrogels that rely on covalent bonds, Col-HZ dynamic hydrogels facilitate chondrocyte migration and ECM deposition. Additionally, the aldehyde groups on the Col-HZ hydrogel scaffold can engage in dynamic Schiff base bonding with amine groups. Leveraging this non-covalent interaction, we incorporated the bioactivator TD-198946, known to enhance ECM synthesis, into the Col-HZ hydrogel. This significantly boosted ECM deposition and reduced inflammation. Transcriptomic sequencing and bioinformatics analyses indicate that both the dynamic network of the hydrogel and the binding of TD-198946 promote cartilage ECM deposition through modulation of the Wnt/β-catenin signaling pathway. Consequently, the Col-HZ dynamic hydrogel, in combination with TD-198946, creates an improved microenvironment that supports ECM deposition and facilitates cartilage tissue formation.

Craniofacial microsomia(CFM)is a congenital malformation with maxillary and/or mandibular hypoplasia,skin tags,and ear malforma-tions(Luo et al.,2023).Microtia,in its mildest form,can occur alone(Quiat et al.,2023).With a prevalence of 3.8/100,000(Barisic et al.,2014),CFM is the second most common congenital craniofacial ab-normality(Li et al.,2022;Luo et al.,2023).