研究背景：近年来，随着二代测序技术（Next generation sequencing, NGS）突飞猛进的发展，对于各类骨骼畸形疾病的遗传病因的探索愈加深入。然而，目前的研究仍多聚焦于病因学的探索和发病机制的挖掘，对于脊柱畸形这一临床常见的骨骼畸形疾病来说，仅对病因和机制的研究仍不足以弥补临床治疗上的短板。特别是针对先天性脊柱侧凸（Congenital Scoliosis，CS）和特发性脊柱侧凸（Idiopathic scoliosis, IS）等复杂的三维脊柱畸形，尚缺乏从明确遗传病因、建立风险检测手段、评估临床预后风险到指导疾病精准治疗的有效闭环。因此，基于现有的临床表型和遗传数据以及病因学研究手段，探索遗传病因、临床表型和治疗方式与临床预后的潜在关联，对于从病因学方面了解疾病、开展精准治疗、提供风险评估手段及减轻疾病社会和经济负担等方面具有重要意义。

研究目的：基于本课题组前期建立的DISCO研究组（系统解析脊柱侧凸及相关合并症国际协作组，Deciphering Disorders Involving Scoliosis and Comorbidities, DISCO, http://www.discostudy.org/）中纳入的大规模骨骼畸形队列，回顾性分析现有队列中IS患者术后出现围手术期并发症的危险因素，用以明确患者临床风险分层，预估围手术期不良事件的发生；分析现有队列中CS患者信息，全面分析孟德尔遗传病因在CS队列中的贡献，并探索遗传病因与CS临床特征及手术预后的相关性，构建基于分子诊断分型的手术预后风险预测模型；验证软骨形成相关基因突变与CS临床特征的相关性，分析候选基因COMP在骨骼畸形疾病中的致病性。

研究方法：回顾性纳入和分析DISCO队列中146例IS患者的临床及影像资料，对患者围手术期并发症发生的危险因素进行单因素分析和多因素logistic回归分析，研究临床特点和治疗方式对于手术预后的影响；通过对303例CS患者进行全外显子组测序（Whole-exome sequencing, WES）和数据注释及分析，评估和筛选致病的基因突变并进行Sanger验证，根据致病基因所参与的生物学过程进行分子诊断分型，并进一步研究分子诊断分型与临床预后结果的关联性；针对CS队列中经WES明确证实携带软骨形成相关基因变异的患者，采用基因型-表型关联分析的方法探索其基因型与临床表型之间存在的联系，并通过家系对软骨形成相关的候选基因COMP对骨骼畸形中的致病性进行分析。

研究结果：本研究共纳入146例IS患者，其中有21例（14.4%）患者在围手术期出现并发症，多因素回归分析发现围手术期并发症发生的独立危险因素包括Cobb角的变化（Odds ratio [OR] =1.085，95%CI=1.035~1.137，P=0.001）和脊柱截骨术（OR=3.565，95%CI=1.039~12.236，P=0.043）。在纳入的303例CS患者中，经WES及数据分析和解读后，共发现有53（17.5%）例患者具有明确的分子诊断，通过美国国家生物技术信息中心（National Center for Biotechnology Information, NCBI）和基因本体论数据库（Gene Ontology, GO）公共数据明确致病基因所属的生物学过程后，将致病基因及其所致疾病归类为（1）结缔组织缺陷（Connective tissue disorder）；（2）软骨形成缺陷（Chondrogenesis defect）；（3）肌肉形成缺陷（Myogenesis defect）；（4）TBX6基因相关（TBX6-associated congenital scoliosis, TACS）以及（5）其他类别。多因素回归分析发现携带软骨形成相关基因突变（OR=7.73, 95% CI=1.64~42.90, P=0.011）、伴有胸廓/肋骨畸形（OR=2.81, 95% CI=1.19~6.62, P=0.011）以及美国麻醉师协会（American Society of Anesthesiologists, ASA）分级≥3级（OR=14.22, 95% CI=1.18~337.4, P=0.042）的患者更容易发生围手术期及术后并发症。通过总结CS队列中的经WES证实明确携带有软骨形成相关基因突变的8例患者的临床表型特征，并将基因型和表型信息汇总后进行基因型-表型关联分析后发现，从畸形的严重程度上相比，软骨形成相关基因突变的患者与其他患者相比术前最大Cobb角较大。从纳入的家系中，鉴定出一个COMP新的杂合错义突变（c.1153G>T, p. Asp385Tyr），通过明显的家系中共分离（Co-segregation）证据，证明该突变导致了典型的多发性骨骺发育不良（Multiple epiphyseal dysplasia, MED）临床表型，并发现了未曾报道的新的临床表型扁平足。

研究结论：基于146例IS队列，构建了术后不良事件的风险预测模型，为患者术前危险因素分层和治疗方式的选择提供了临床参考；基于303例CS患者的WES队列研究完善了疾病的基因突变谱，证明携带软骨形成相关基因突变可能是导致患者不良预后的重要标志，对CS患者进行术前全面表型评估和WES将有助于精准诊疗的实施；发现COMP基因的c.1153G>T突变导致了经典的MED，并发现新的临床表型，拓展了COMP导致MED的疾病突变谱和表型谱。

Background: In recent years, with the rapid development of Next Generation Sequencing (NGS) technology, the exploration of the genetic etiology of various skeletal deformity diseases has become more and more in-depth. However, the current research still focuses on the exploration of etiology and pathogenesis. For spinal deformity, a common clinical skeletal deformity disease, the research on etiology and pathogenesis is still not enough to make up for the shortcomings of clinical treatment. Especially for complex spinal three-dimensional deformities such as congenital scoliosis (CS) and idiopathic scoliosis (IS), there is still a lack of effective closed-loop from clarifying genetic etiology, establishing risk detection methods, evaluating clinical prognosis risk to guiding accurate treatment of diseases. Therefore, based on the existing clinical phenotype, genetic data and etiological research methods, exploring the potential association between genotype-phenotype and treatment and prognosis is of great significance for understanding the disease, carrying out accurate treatment, providing risk assessment methods and reducing the social burden of the disease.

Objects: Based on the large-scale scoliosis cohort of the DISCO research group (Deciphering Disorders Involving Scoliosis and Comorbidities, http://www.discostudy.org/), we retrospectively analyzed risk factors for postoperative perioperative complications in IS patients who underwent spinal correction surgery. Our aim was to stratify patients for clinical risk and predict the occurrence of perioperative adverse events. We comprehensively analyzed the contribution of Mendelian genetic etiologies in the CS cohort, explored the association between genetic etiologies and CS prognosis, and constructed a genetic risk prediction model based on molecular diagnostic classification. We compared the clinical characteristics of patients with chondrogenesis defect-related gene mutations and patients without chondrogenesis defect-related gene mutations by genotype-phenotype association analysis, with the aim of exploring potential associations between phenotype and genotype, which will assist in the identification of high-risk patients. At the same time, we analyzed the pathogenicity of the candidate gene COMP for skeletal deformities.

Method: The clinical and imaging data of 146 IS patients in the DISCO cohort were collected and retrospectively analyzed. Univariate analysis and multivariate logistic regression analysis were performed on the risk factors of perioperative complications in patients to study the influence of clinical characteristics and treatment methods on clinical prognosis. Based on CS patients from DISCO research group, through whole-exome sequencing (WES) and data annotation and analysis of 303 CS patients, the pathogenic variants were evaluated and screened and Sanger validation was performed. According to the biological process involved in the pathogenic gene, the correlation between molecular diagnostic classification and clinical prognosis was further studied. The genotype-phenotype association analysis was used to study the relationship between chondrogenesis defect-related gene mutations and the clinical manifestations of CS. At the same time, we analyzed the pathogenicity of the candidate gene COMP for skeletal deformities.

Result: Among the 146 IS patients, 21 (14.4%) had perioperative complications. Multivariate analysis found that independent risk factors for perioperative complications included changes in Cobb angle (OR=1.085, 95% CI=1.035-1.137, P=0.001) and spinal osteotomy (OR=3.565, 95% CI=1.039-12.236, P=0.043).

Among the 303 CS patients included, after WES and data analysis and interpretation, a total of 53 (17.5%) patients were found to have a definite molecular diagnosis. By querying the public data of the National Center for Biotechnology Information (NCBI) and the Gene Ontology (GO) database, we identified the biological process to which the disease-causing genes belong. The causative genes were categorized as: 1) connective tissue disorder; 2) chondrogenesis defect; 3) myogenesis defect; 4) TBX6-associated congenital scoliosis (TACS); and 5) others. Multivariate association analysis found that the patients with chondrogenesis-related gene defects (OR=7.73, 95% CI=1.64-42.90, P=0.011), accompanied by thoracic/rib deformities (OR=2.81, 95% CI=1.19-6.62, P=0.011), and the American Society of Anesthesiologists (ASA) grade≥3 (OR=14.22, 95% CI=1.18-337.4, P=0.042) were more likely to develop perioperative and postoperative complications.

By summarizing the clinical characteristics of 8 CS patients with chondrogenesis-related gene mutations in the CS cohort included in this study, a genotype-phenotype association analysis showed that the preoperative maximum Cobb angle was larger in patients with chondrogenesis-related gene mutations.

From the included families, a novel heterozygous missense mutation in COMP (c.1153G>T, p. Asp385Tyr) was identified, and the mutation caused the typical co-segregation of clinical phenotype in the multiple epiphyseal dysplasia (MED) family, and we found an unreported new clinical phenotype of flatfoot.

Conclusion: Based on cohort study of 146 IS patients, a risk prediction model for postoperative adverse events was constructed, which provided a reference for preoperative risk factor stratification and treatment selection.

Based on the cohort study of 303 CS patients, the gene mutation spectrum of the CS has been constructed. Through the integrated analysis of genetic and clinical data, we found that chondrogenesis-related gene mutations may be an important marker leading to poor patient prognosis. In the future, preoperative comprehensive phenotypic assessment and WES for CS patients will facilitate the implementation of precision diagnosis and treatment.

We found that the c.1153G>T mutation of the COMP gene can lead to classic MED disease, and further discovered a new clinical phenotype-flatfoot, which expanded the disease mutation spectrum and phenotype spectrum of COMP causing AD-MED.