Male EBP disorder with neurologic defects (MEND syndrome) is an extremely rare disorder with a prevalence of less than 1/1,000,000 individuals worldwide. It is inherited as an X-linked recessive disorder caused by impaired sterol biosynthesis due to nonmosaic hypomorphic EBP variants. MEND syndrome is characterized by variable clinical manifestations including intellectual disability, short stature, scoliosis, digital abnormalities, cataracts, and dermatologic abnormalities. The goal of this study was to investigate the disease-causing variants in a family of two patients affected with MEND syndrome. The genomic DNA of the two patients with MEND syndrome was subjected to whole exome sequencing to identify disease-causing variants. Segregation of the identified variant was tested through Sanger sequencing. Several in-silico tools were used to evaluate the pathogenicity of the variant. Protein's 3D structure analysis systems were used to predict the impact of the identified variant on the binding and function of the mutated EBP protein including AlphaFold, PyMOL, AutoDock, ChimeraX and Discovery Studio. A novel pathogenic missense EBP variant NM_006579.3:c.556T > C (Trp186Arg) was found segregating in the affected family. In-silico analysis and molecular docking results supported the pathogenicity of the identified variant. Our study expands the mutation spectrum of EBP and adds to the restricted reports of MEND patients. It strengthens the body of evidence that supports the role of EBP in the MEND syndrome phenotype. To our knowledge, this is the first report of this disorder from Pakistan.

Male EBP disorder with neurologic defects (MEND) syndrome is an X-linked disease caused by hypomorphic mutations in the EBP (emopamil-binding protein) gene. Modifier genes may explain the clinical variability among individuals who share a primary mutation. We studied four males (Patient 1 to Patient 4) exhibiting a descending degree of phenotypic severity from a family with MEND syndrome. To identify candidate modifier genes that explain the phenotypic variability, variants of homeostasis cholesterol genes identified by whole-exome sequencing (WES) were ranked according to the predicted magnitude of their effect through an in-house scoring system. Twenty-seven from 105 missense variants found in 45 genes of the four exomes were considered significant (-5 to -9 scores). We found a direct genotype-phenotype association based on the differential accumulation of potentially functional gene variants among males. Patient 1 exhibited 17 variants, both Patients 2 and 3 exhibited nine variants, and Patient 4 exhibited only five variants. We conclude that APOA5 (rs3135506), ABCA1 (rs9282541), and APOB (rs679899 and rs12714225) are the most relevant candidate modifier genes in this family. Relative accumulation of the deficiencies associated with variants of these genes along with other lesser deficiencies in other genes appears to explain the variable expressivity in MEND syndrome.