Bardet-Biedl syndrome is an autosomal recessive disease characterized by rod-cone dystrophy, postaxial polydactyly, kidney defects, obesity, mental retardation and hypogonadism. Here, we report different genotypes in two Bardet-Biedl syndrome affected sisters with a different clinical phenotype regarding severity. The proband of the family was examined by Next Generation Sequencing (NGS) using clinical exome and filtering by syndromic and non-syndromic genes associated with retinal dystrophies. Targeted NGS revealed two novel variants in the MKKS and CEP290 genes in homozygosis state in the proband. Segregation analysis revealed the presence of the same MKKS homozygous variant in her younger affected sister but not the CEP290 variant. Both sisters presented different clinical manifestation, at different ages, with a more severe renal and retinal defect in the case of the sister carrying mutations in both genes. Another unaffected sister showed only homozygosity for the CEP290 variant, thus supporting the non-pathogenic role of this mutation in BBS phenotype. In this study, NGS proved to be a powerful and efficient sequencing method to identify causal variants in different genes. However, it remarks the importance of the segregation analysis and clinical information to establish the pathogenicity of new variants. The two affected sisters present different genotypes and clinical manifestation, suggesting that the novel CEP290 variant could be acting as a modifier, making the phenotype more severe in the sister homozygote for MKKS and CEP290 genes. On the other hand, the difference in the age of both sisters highlight the important role of monitoring disease progression also to confirm the modifier role of genetic variants. • An Iranian family in which two sisters were diagnosed with Bardet-Biedl syndrome was genetically studied. • The older affected sister was examined by Next Generation Sequencing revealing two novel variants in the MKKS and CEP290 genes in homozygosis state in the proband. • Segregation analysis revealed the presence of the same MKKS homozygous variant in her younger affected sister but not the CEP290 variant. • Both affected sisters presented different clinical manifestation, suggesting that the novel CEP290 variant could be acting as a modifier, making the phenotype more severe in the sister homozygote for MKKS and CEP290 genes. • On the other hand, the different age of the sisters highlights the important role of disease progression monitoring also to confirm the modifying role of genetic variants. [ABSTRACT FROM AUTHOR]