*4.6. Class 4 Mutations*

As mentioned above, Class 4 of ABCG2 mutations differs from that of CFTR variants. According to our proposed classification, this group collects the mutations resulting in a transporter with altered substrate specificity. These mutations are represented by the rare F431L (rs750568956, MAF < 0.0001). ABCG2-F431L is normally expressed in cells, localized to the plasma membrane, mediates porphyrin transports, and confers resistance to mitoxantrone [95,113], but these cells exhibit decreased resistance to methotrexate and various tyrosine kinase inhibitors [95,138]. Conflicting results on altered resistance to camptothecin analog SN-38 (the active metabolite of irinotecan) have been published in connection with this mutant [95,128].

Other members of this class are the R482G and R482T missense mutations, which have been identified from drug-selected cell lines, when ABCG2 was originally cloned [4,5]. In the beginning, the actual sequence of the wild type ABCG2 was ambiguous; causing some controversies in the initial characterization of the transporter, but later it was made clear that there is an arginine at position 482 in the wt ABCG2. Amino acid alterations at R482 strongly influence the substrate profile of the transporter, which is exemplified by methotrexate or uric acid being a substrate of the wt ABCG2 but not of the R482G variant; and inversely, doxorubicin or daunorubicin is exported by ABCG2-R482G, but not by the wild type [3,86,129–132].
