[Characterization of copy number variations in concomitant exotropia using single nucleotide polymorphism microarray]

Objective: To investigate the genetic characteristics of copy number variations (CNVs) in families with concomitant exotropia. Methods: A family-based molecular genetic study. The study included 25 unrelated concomitant exotropia families who visited Tianjin Eye Hospital between August 2019 and August 2021. Patients and healthy individuals from these families were included as subjects. Clinical data were collected through detailed medical history interviews, family history documentation, and comprehensive ophthalmic and systemic examinations to confirm the diagnosis of probands and affected family members. Peripheral blood samples were obtained for genomic DNA extraction, followed by single nucleotide polymorphism (SNP) genotyping using single nucleotide polymorphism array (SNP array). CNV detection was performed using the PennCNV tool. Gene enrichment analysis and protein-protein interaction (PPI) network analysis were conducted to explore the biological functions of candidate genes and their potential pathogenic mechanisms. The Benjamini-Hochberg method was used for multiple comparison correction, and node permutation randomization test was used for significance assessment of network analysis. Results: A total of 74 subjects from 25 families with concomitant exotropia were enrolled in the study, including 55 patients with concomitant exotropia. Among all subjects, 33 were male (44.6%) and 41 were female (55.4%). Among the 55 patients with concomitant exotropia, 27 were male (49.1%) and 28 were female (50.9%). The age at onset was 6.0 (3.3, 10.0) years. Using the prism and alternate cover test, the deviation angle was -30^Δ (-39^Δ, -25^Δ) at distance (6 m) and -35^Δ (-40^Δ, -30^Δ) at near (33 cm). Based on the SNP genotyping technique, this analysis identified seven novel concomitant exotropia-associated CNVs, located at chromosomal regions 2p25.1, 20q13.33, 15q22.2, 7q34, 13q14.2, 8p21.1, and 3q25.33. Analysis of familial distribution showed that the CNV at 2p25.1 had the highest detection rate (28%), being present in seven families. Co-segregation analysis revealed that CNVs at 15q22.2, 7q34, and 13q14.2 exhibited complete co-segregation with the disease phenotype (100% penetrance), whereas regions at 2p25.1, 20q13.33, 8p21.1, and 3q25.33 showed incomplete penetrance of 73.3%, 92.3%, 83.3%, and 80%, respectively. These seven CNVs covered the exonic regions of nine protein-coding genes, including ROCK2, SYCP2, VPS13C, RB1, BRAF, CCDC25, ESCO2, PBK and IFT80. Gene enrichment analysis revealed that these genes were enriched in pathways associated with muscular movement regulation (q<0.001). Gene intersection analysis identified two genes (ESCO2 and BRAF) that overlap with genes related to oculomotor neural development, and four genes (ESCO2, BRAF, RB1 and IFT80) that overlap with genes related to extraocular muscle development. PPI analysis revealed that ROCK2, BRAF, RB1, PBK, and ESCO2 proteins formed a significant functional cluster with a higher number of edges than random expectation(μ=0.09±0.42,P=0.002,Z=9.31). Conclusions: This study reports seven CNVs associated with concomitant exotropia. These CNVs exhibit genetic characteristics of scattered chromosomal distribution, variable penetrance and functional clustering of target genes.