研究背景

综合征性小耳畸形是一类临床表型复杂、病因多样的发育异常，常伴随颅面结构、骨骼及其他器官系统的发育障碍，临床诊断和治疗均具有较大挑战。近年来，随着高通量基因检测技术的广泛应用，部分综合征性小耳畸形的遗传病因逐渐明确，EVEN-PLUS综合征即为典型代表。该病是一种罕见的常染色体隐性遗传疾病，临床以小耳畸形为核心特征，并伴有鼻骨发育不良、骨骼关节畸形等多系统异常表现。目前研究发现，EVEN-PLUS综合征的发生可能与HSPA9基因突变密切相关，但其具体的致病机制及在软骨组织发育过程中的作用仍未完全阐明。HSPA9基因编码的Mortalin蛋白属于线粒体分子伴侣蛋白家族成员，在细胞增殖、分化及组织稳态维持中发挥重要作用，其在软骨细胞中的生物学功能与分子调控机制仍需进一步深入研究。

研究目的

通过临床病例与分子生物学实验，明确一种以小耳畸形为核心表型的综合征——EVEN-PLUS综合征的临床特点及HSPA9基因突变的致病性，进一步从细胞与分子水平深入探讨致病基因HSPA9所编码的Mortalin蛋白在软骨细胞增殖、迁移及分化过程中的功能作用与相关机制，为综合征性小耳畸形的病因诊断及软骨再生治疗提供理论依据。

材料与方法

第一部分：对一名临床表现为双侧小耳畸形合并多系统异常的患者进行详细的临床特征评估和鉴别诊断，运用全外显子测序技术筛查致病基因并确定突变位点，随后借助生物信息学方法评估所鉴定HSPA9基因突变的进化保守性，预测突变对Mortalin蛋白结构与功能可能产生的影响。

第二部分：利用小耳畸形患者残耳软骨组织构建体外软骨细胞培养模型，外源性添加重组Mortalin蛋白，并分别构建HSPA9基因过表达及基因沉默模型。采用CCK-8、划痕实验、ELISA、免疫荧光、qRT-PCR与Western Blot等技术，检测Mortalin对耳软骨细胞增殖、迁移及软骨基质合成的影响，分析相关信号通路。

第三部分：以人脐带血来源间充质干细胞为研究对象，通过软骨诱导分化实验，结合外源Mortalin蛋白干预，运用组织染色、qRT-PCR与Western Blot等技术分析Mortalin蛋白在干细胞软骨分化过程中的作用及分子机制。

研究结果

第一部分：通过临床及影像学检查排除常见综合征性小耳畸形。全外显子测序发现患者HSPA9基因存在两处新型突变（c.389A>G, p.D130G；c.313G>T, p.A105S），生物信息学分析提示突变位于高度保守区域，可能显著影响Mortalin蛋白功能，结合表型和基因型最终明确诊断为EVEN-PLUS综合征。

第二部分：外源Mortalin蛋白能够显著促进耳软骨细胞的增殖活性与迁移能力，并提高软骨特异性基质的表达。对HSPA9基因的过表达与沉默实验，进一步验证了Mortalin对耳软骨细胞增殖和功能维持的调控作用。机制研究显示，Mortalin蛋白可通过激活BMP4与Wnt/β-catenin信号通路关键分子的表达实现上述调控作用。

第三部分：外源Mortalin蛋白有效提高了hMSC向软骨细胞诱导分化的效率，增加了软骨特异性基质表达，并通过选择性调控BMP4、PI3K以及Wnt/β-catenin通路关键分子表达促进干细胞向成熟软骨表型分化。

研究结论

本研究首次报道了EVEN-PLUS综合征HSPA9基因新型致病变异，并从细胞与分子水平系统揭示了Mortalin蛋白在耳软骨细胞增殖及干细胞软骨分化过程中的促进作用及调控机制。Mortalin蛋白通过激活BMP4、Wnt/β-catenin等信号通路，在软骨发育与稳态维持中发挥重要作用。这些结果为综合征性小耳畸形的病因诊断、发病机制研究及软骨再生治疗提供了新的理论依据与潜在靶点。

Background

Syndromic microtia represents a group of developmental abnormalities characterized by highly heterogeneous clinical phenotypes and complex etiologies, often accompanied by craniofacial, skeletal, and multiple organ system involvement, posing significant challenges in clinical diagnosis and treatment. Recently, with advances in high-throughput genetic sequencing, the genetic etiologies of certain syndromic forms of microtia have been progressively clarified, exemplified by EVEN-PLUS syndrome. EVEN-PLUS syndrome is a rare autosomal recessive disorder characterized by microtia, hypoplastic nasal bones, and skeletal-joint abnormalities. Although recent studies suggest a close association between EVEN-PLUS syndrome and mutations in the HSPA9 gene, the exact pathogenic mechanisms and the gene’s role in cartilage development remain unclear. Mortalin, encoded by the HSPA9 gene, is a mitochondrial chaperone protein essential for cellular proliferation, differentiation, and tissue homeostasis. However, its precise biological functions and regulatory mechanisms in chondrocytes require further exploration.

Objective

This study aims to characterize clinically and genetically a syndromic microtia disorder, later identified as EVEN-PLUS syndrome, and to further investigate the role and molecular mechanisms of mortalin, encoded by the disease-associated HSPA9 gene, in the proliferation, migration, and differentiation of chondrocytes. This work provides insights into the pathogenesis of syndromic microtia and offers theoretical evidence for cartilage regeneration strategies.

Materials and Methods

Part I: Detailed clinical evaluations and differential diagnoses were performed for a patient with bilateral microtia accompanied by multi-system abnormalities. Whole-exome sequencing (WES) was conducted to identify candidate pathogenic mutations, followed by bioinformatics analyses to assess evolutionary conservation and predict the potential structural and functional impacts of identified HSPA9 gene mutations on mortalin protein.

Part II: An in vitro chondrocyte culture model was established using auricular cartilage derived from the residual ear tissue of the microtia patient. Cells were treated with exogenous recombinant mortalin protein, and models of HSPA9 gene overexpression and silencing were separately constructed. CCK-8 assays, scratch-wound assays, ELISA, immunofluorescence, qRT-PCR, and Western Blot were used to evaluate the effects of mortalin on chondrocyte proliferation, migration, and cartilage extracellular matrix synthesis, and to analyze relevant signaling pathways.

Part III: Human mesenchymal stem cells derived from umbilical cord blood were induced to differentiate into chondrocytes in the presence or absence of exogenous mortalin protein. Histological staining, qRT-PCR, and Western Blot analyses were performed to determine the functional role and molecular mechanisms by which Mortalin regulates chondrogenic differentiation of stem cells.

Results

Part I: Common syndromic forms of microtia were excluded through detailed clinical and imaging assessments. WES analysis identified two novel mutations in the patient’s HSPA9 gene (c.389A>G, p.D130G; c.313G>T, p.A105S). Bioinformatics analyses indicated these mutations occur in highly conserved regions and are likely to significantly impair mortalin protein function. Integration of genotype and phenotype data established the diagnosis as EVEN-PLUS syndrome.

Part II: Exogenous mortalin protein significantly enhanced the proliferative activity and migration capacity of auricular chondrocytes, as well as upregulated the expression of cartilage-specific extracellular matrix components. Experiments involving overexpression and silencing of the HSPA9 gene further validated the regulatory role of mortalin in auricular chondrocyte proliferation and functional maintenance. Mechanistic studies revealed that mortalin mediates these effects through activation of key molecules within the BMP4 and Wnt/β-catenin signaling pathways.

Part III: Exogenous mortalin protein significantly promoted the efficiency of chondrogenic differentiation in hMSCs, increased cartilage-specific extracellular matrix production, and selectively modulated key molecules within the BMP4, PI3K, and Wnt/β-catenin signaling pathways, facilitating differentiation toward mature chondrocyte phenotypes.

Conclusion

This study reports novel pathogenic variants of the HSPA9 gene in EVEN-PLUS syndrome and systematically elucidates, at the cellular and molecular levels, the promotive roles and underlying regulatory mechanisms of Mortalin protein in chondrocyte proliferation and stem-cell-based cartilage differentiation. Mortalin predominantly functions through activation of BMP4 and Wnt/β-catenin signaling pathways, highlighting its crucial role in cartilage development and homeostasis. These findings provide novel theoretical insights and potential therapeutic targets for diagnosing and understanding syndromic microtia and for developing cartilage regeneration strategies.