*4.5. Class 3 Mutations*

The following group of ABCG2 mutations (Class 3) contains those that do not affect protein expression considerably, but impair transport function. At this point, the classification proposed here diverges from the categorization of the CFTR mutations, in which genetic alterations causing impaired gating are classified into Class 3, whereas those leading to diminished channel conductance are categorized into Class 4. This classification cannot be applied to ABCG2 for several reasons. Firstly, the transport mechanism of ABCG2 substantially differs from that of CFTR. Secondly, immense information has been accumulated on the mechanism of action of loss-of-function mutations in CFTR, whereas only limited data on ABCG2 mutations are available in this respect.

The genetic alterations in ABCG2 causing impaired transport activity are exemplified by S248P, P269S, F489L, and A528T [95,114,121–123]. The rare S476P also belongs to this group, although the transport function of ABCG2 bearing this mutation is only slightly reduced [104]. A recent addition to this class is I242T, which has normal protein expression and processing, while its transport activity (urate transport) is completely abolished [125]. A peculiar member of this group is K86M, an artificially generated mutation in the Walker A motif, which leads to a catalytic inactive transporter with close to normal cellular localization [9,126]. By means of these beneficial features, K86M is commonly employed in functional assays as a negative control.

It is worth noting that trafficking (Class 2) mutations cause reduced surface expression, and consequently lead to diminished drug efflux and increased drug sensitivity, to an apparent loss of function. Although generally speaking, the functionality of the transporter is affected in these cases, but the underling mechanism is rather different for Class 2 and Class 3 mutations. When a particular ABCG2 variant is evaluated solely on the basis of functionality in cellular systems, e.g., cell-based cytotoxicity or drug efflux assays, this distinction cannot be drawn. The various assessments to explore the particular impairments in the ABCG2 variants caused by mutations and polymorphisms will be discussed in

Section 5. It is noteworthy that Class 3 mutations can potentially be corrected by so-called potentiators, small molecules that are intended to restore the impaired transport function (see Section 7).
