Exome Sequencing and Molecular Modeling Reveal Novel Loci in Familial Multiple Sclerosis: The Importance of BTNL3 and BTNL8 in Disease Pathogenesis.

Multiple sclerosis (MS) is a chronic autoimmune disorder with a complex interplay of genetic and environmental factors. The familial aggregation of MS cases, especially within genetically related families, suggests a strong genetic component with high penetrance. This study explores the genetic factors contributing to MS in two multiclient MS families. Whole exome sequencing (WES) was performed on affected and unaffected members of the two multi-incident MS families with a history of genetic homogeneity. Selected variants were validated using appropriate molecular methods and linkage analysis under an autosomal recessive model. In silico analyses including protein modeling with AlphaFold3 and molecular docking using HADDOCK2.4, were conducted to evaluate the functional impact of the identified variants. Our study revealed two co-segregating copy number variants (CNVs) in BTNL3 and BTNL8 genes in one family. In silico modeling showed that the BTNL8*3 fusion protein, resulting from the identified CNVs, exhibited reduced binding affinity with the Vγ4 T-cell receptor (TCR). Comparison of the binding affinity between the BTNL8-BTNL3 heterodimer and BTNL8*3 fusion protein with Vγ4 TCRs revealed HADDOCK scores of -23.8 ± 4.8 and 8.8 ± 9.2, respectively, suggesting altered T-cell activation and a potential role in MS pathogenesis. A rare MBL2 variant (p.Pro101Leu) was also found in the second family, though its incomplete segregation with the MS phenotype suggests it may act as a genetic modifier. This study underscores the importance of both single-nucleotide variants and copy number in familial MS. The segregation pattern and characteristics of the identified variants in BTNL3 and BTNL8 support their potential association with disease risk. The BTNL8*BTNL3 fusion may influence γδ T cell selection and MS pathogenesis, warranting further functional studies.