The multiple drug resistance 3 (MDR3) protein is a canalicular phospholipid 
translocator involved in the bile secretion and encoded by the ABCB4 gene. Its 
deficiency is related to a large spectrum of liver diseases. Taking into account 
the increased evidence about the involvement of synonymous variants in inherited 
diseases, this study aims to explore the putative effects of silent genetic 
variants on the ABCB4 expression. We performed an exhaustive computational 
approach using ESE finder, RegRNA 2.0, MFOLD, SNPfold, and %MinMax software 
added to the measurement of the Relative Synonymous Codon Usage. This analysis 
included 216 synonymous variants distributed throughout the ABCB4 gene. Results 
have shown that 11 synonymous coding SNPs decrease the ESE activity, while 8 of 
them change the codon frequency. Besides, the c.24C>T variation, located 21 
nucleotides downstream the start A (Adenine) U (Uracil) G (Glutamine) AUG causes 
an increase in the local stability. Moreover, the computational analysis of the 
3'UTR region showed that six of the eight variants located in this region 
affected the Wild Type (WT) pattern of the miRNA targets sites and/or their 
proper display. The 26 sSNPs retained as putatively functional possessed a very 
low allele frequency, supporting their pathogenicity. In conclusion, the 
obtained results suggest that some synonymous SNPs in the ABCB4 gene, considered 
up to now as neutral, may be involved in the MDR3 deficiency.