探讨Möbius综合征(MBS)患者的临床特征及可能的遗传发病机制。

收集2013年3月至2024年4月首都医科大学附属北京同仁医院眼科中心的14例(28只眼)散发MBS患者的病例资料。其中，男性5例(10只眼)，女性9例(18只眼)；年龄1 ～ 9岁，平均年龄(4.0±0.7)岁。询问或检查并记录患者的性别、年龄、最佳矫正视力、眼球运动、眼前节和眼底、体格发育及颅神经眼眶磁共振成像(MRI)的情况。采集患者及核心家系成员外周静脉血、提取基因组脱氧核糖核酸(DNA)并在illumina平台行全外显子组测序。使用SAMtools和ANNOVAR软件对变异进行识别和注释，对频率数据库中频率低于1%的变异进行过滤。在此基础上，筛选新生突变基因以及≥2个以上患者共有的突变基因。使用R包的maftools和GenVisR软件包分别行共有基因突变特征分析并绘制景观图。年龄、最小分辨角对数(logMAR)视力以及每个样本携带的共有基因突变数量经Shapiro－Wilk检验符合正态分布，以±s表示。性别、单侧或双侧受累、全身发育异常、突变分类、突变类型、突变频谱及共有基因突变频率采用频数和百分比描述。使用R语言ClusterProfiler中的超几何检验进行基因本体(GO)富集分析，使用Benjamini－Hochberg方法进行多重假设检验校正并筛选显著富集的条目。

本研究9例(18只眼)患者的logMAR视力为0.56±0.12。双眼外转受限者有10例(20只眼)，占71.43%(10/14)；单眼外转受限者有4例(4只眼)，占28.57%(4/14)。双侧面瘫者有5例，占35.7%(5/14)；单侧面瘫者有9例，占64.3%(9/14)。伴其他全身发育异常的患者有9例，占64.29%(9/14)。双侧外展神经(CN6)发育不良者有11例，占78.57%(11/14)；单侧CN6发育不良者有3例，占21.43%(3/14)。双侧面神经(CN7)发育不良者有9例，占64.3%(9/14)，单侧CN7发育不良者有5例，占35.71%(5/14)。2例MBS患者发现致病候选基因丛状蛋白D1(PLXND1)新变异2个，分别为杂合错义变异c.C4207T、p.R1403W和剪切位点变异c.2937＋6 G>T。14例患者共筛选出新生突变基因8个，分别为肌联蛋白(TTN)、碳酸酐酶9(CA9)、中间丝相关蛋白(RPTN)、SET结合因子2(SBF2)、丝状肌动蛋白结合蛋白(AFDN)、突触囊泡糖蛋白2C(SV2C)、神经丝蛋白重链(NEFH)和膜金属内肽酶样蛋白1(MMEL1)。≥2个以上患者共有的突变基因总计796个，突变数量为1987个。最常见的突变分类是错义突变，数量为1382个，占69.55%；最多见的突变类型是单碱基替换变异，数量为1534个，占77.20%；最常见的突变频谱是胞嘧啶被替换为胸腺嘧啶，数量为804个，占52.41%。每个样本所携带共有基因突变数量为126～161个，平均(141.93±11.35)个。按照突变频率排名，前10位的共有基因分别为B黑色素瘤抗原家族成员2/3/4/5(BAGE2/3/4/5)、与内体分选复合物Ⅲ相关的钠耐受增加1(IST1)、TTN、高尔基体蛋白A6家族样蛋白2(GOLGA6L2)、NEFH、UBX结构域蛋白11(UBXN11)、二氢硫辛胺支链转酰基酶E2 (DBT)、羰基还原酶4(CBR4)、肌动蛋白相关蛋白3C(ACTR3C)和含V－Set免疫球蛋白域蛋白2(VSIG2)，频率分别为100%、93%、64%、64%、57%、50%、50%、50%、50%和43%。经GO数据库中的生物学过程数据库注释，在本研究全部MBS患者14例(28只眼)中发现的共有突变基因集的基因数量为701个，查询GO数据库获得背景基因集的基因数量为18 722个。比较不同通路的注释基因在此两个基因集中分布的差异，将其概率值按照升序排列，显著富集的通路依次为调控小谷氨酰胺转肽酶介导的信号转导、基于微管的运动、轴突发生、眼形态发生、通过质－膜粘附分子的细胞间粘附、肌动球蛋白结构组装、肌原纤维的组装及微管束的合成，其概率值依次为0.0002、0.004、0.004、0.004、0.004、0.004、0.011及0.011。

MBS患者的临床表型异质性较强，多数患者伴随有除面瘫和眼球外转受限以外的多发先天畸形。基于全外显子组测序的生物信息学分析结果提示参与神经发育和轴突生长过程的多个基因和生物学过程可能与MBS发病相关，进一步揭示了遗传因素在MBS发病中的作用。

To explore the clinical features and possible genetic pathogenesis of patients with Möbius syndrome (MBS).

A total of 14 patients (28 eyes) with sporadic MBs were collected from the Eye Center of Beijing Tongren Hospital affiliated to Capital Medical University from March 2013 to April 2024. There were 5 males (10 eyes) and 9 females (18 eyes) with the average age of (4.0±0.7) years (ranging from 1 to 9 years). The patient′s gender, age, best corrected visual acuity, eye movement, anterior segment and fundus, physical development and cranial nerve orbital magnetic resonance imaging (MRI) were asked or examined and recorded. Peripheral venous blood samples of patients and core family members were collected, genomic DNA was extracted, and full exome sequencing was performed on Illumina platform. SAMtools and ANOVA software were used to identify and annotate the variation, and the variation with frequency less than 1% in the frequency database was filtered. On this basis, newborn mutant genes and mutant genes shared by more than 2 patients were screened. The common gene mutation characteristics were analyzed and the landscape map was drawn using the R package maftools and GenVisR software. Age, logarithm of minimum resolution angle (logMAR) vision and the number of common gene mutations carried by each sample conformed to the normal distribution by Shapiro-Wilk test, which was expressed as ±s. Gender, unilateral or bilateral involvement, systemic dysplasia, mutation classification, mutation type, mutation frequency spectrum and common gene mutation frequency were described by frequency and percentage. The hypergeometric test in the R language ClusterProfiler was used for gene ontology (GO) enrichment analysis, and the Benjamini-Hochberg method was used for multiple hypothesis test correction and screening the items with significant enrichment.

The logMAR visual acuity of 9 patients (18 eyes) was 0.56±0.12. There were 10 cases (20 eyes) with limited binocular exotropia, accounting for 71.43% (10/14). There were 4 cases (4 eyes) with limited monocular exotropia, accounting for 28.57% (4/14); 5 cases with bilateral facial paralysis, accounting for 35.7% (5/14); 9 cases with unilateral facial paralysis, accounting for 64.3% (9/14); 9 patients with other systemic dysplasia, accounting for 64.29% (9/14); 11 cases with bilateral abducens nerve (CN6) dysplasia, accounting for 78.57% (11/14); 3 cases with unilateral cn6 dysplasia, accounting for 21.43% (3/14); 9 cases with bilateral nerve (CN7) dysplasia, accounting for 64.3% (9/14), and 5 cases with unilateral CN7 dysplasia, accounting for 35.71% (5/14). Two novel variants of PLXND1 were found in two MBS patients, which were heterozygous missense variants c. C4207T, p. R1403W and splice site variation c. 2937+ 6 G>T. A total of 8 newly mutated genes were screened out from 14 patients, including myonectin (TTN), carbonic anhydrase 9 (CA9), intermediate filament associated protein (RPTN), set binding factor 2 (SBF2), filamentous actin binding protein (AFDN), synaptic vesicular glycoprotein 2C (SV2C), neurofilament heavy chain (NEFH) and membrane metalloendopeptidase like protein 1 (MMEL1) A total of 796 genes were mutated in more than two patients, and the number of mutations was 1987. The most common mutation classification was missense mutation, with 1382 mutations, accounting for 69.55%. The most common mutation type was single base substitution mutation, the number was 1534, accounting for 77.20%. The most common mutation spectrum was that cytosine was replaced by thymine, accounting for 52.41%. The total number of gene mutations per sample was 126 to 161, with an average of (141.93±11.35). According to the mutation frequency ranking, the top 10 common genes were B melanoma antigen family member 2/3/4/5 (BAGE2/3/4/5), increased sodium tolerance associated with endosome sorting complex Ⅲ 1 (IST1), TTN, Golgi protein A6 family like protein 2 (GOLGA6L2), NEFH, Ubx structure domain protein 11 (UBXN11), dihydrothiooctylamine branched chain transferase E2 (DBT), carbonyl reductase 4 (CBR4), actin associated protein 3C (ACTR3C), and v-set containing immunoglobulin domain protein 2 (VSIG2); and the frequencies of ig2 were 100%, 93%, 64%, 64%, 57%, 50%, 50%, 50%, 50% and 43%. According to the annotation of biological process database in GO database, the number of genes in the common mutation gene set found in 14 patients (28 eyes) with MBS was 701, and the number of genes in the background gene set obtained by querying GO database was 18 722. The differences in the distribution of annotation genes of different pathways in the two genes were compared, and the probability values were arranged in ascending order. The pathways that were significantly enriched were the regulation of small glutamine transpeptidase mediated signal transduction, microtubule based movement, axogenesis, eye morphogenesis, intercellular adhesion through plasma membrane adhesion molecules, actin structural assembly, myofibril assembly and microtubule bundle synthesis, with the probability values of 0.0002, 0.004, 0.004, 0.004, 0.011 and 0.011, respectively.

The clinical phenotypic heterogeneity of MBS patients is strong. Most patients are accompanied by multiple congenital malformations except facial paralysis and limited exophthalmos. Bioinformatics analysis based on full exome sequencing suggested that multiple genes and biological processes involved in nerve development and axon growth may be related to the pathogenesis of MBS, further revealing the role of genetic factors in the pathogenesis of MBS.