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.