**8. Conclusions and Future Directions**

The last twenty years have revealed a great deal about the role of *ABCG5 ABCG8* in cholesterol metabolism and in the defense against the accumulation of dietary xenosterols. The master regulators of neutral and acidic sterol metabolism modulate G5G8 abundance and activity as a component of the integrated machinery that maintains sterol homeostasis. Much, however, remains unknown about the hormonal and intracellular signals that promote G5G8 translocation to the biliary surface and G5G8-mediated cholesterol secretion. Beyond G5G8, the hepatocyte orchestrates the clearance of excess cellular cholesterol by metabolism to bile acid or incorporation into very low density and high-density lipoproteins, either on the surface of the particles or in the hydrophobic core following esterification. What regulates and under what conditions does the intracellular flux of cholesterol favor G5G8-dependent biliary secretion? Investigations of intestinal G5G8 regulation and activity have been more limited than in the liver. In what ways does the regulation of G5G8 in the enterocyte differ, if any, from the hepatocyte? Targeting G5G8 to promote TICE is conceptually attractive to promote RCT, but studies have yet to reveal a route to a potential therapeutic and the molecular mechanisms that mediate TICE remain elusive.

Sequencing of large numbers of genomes and exomes reveals that disease-causing mutants in *ABCG5 ABCG8* are significantly more common that previously appreciated. The combination of the required instrumentation, expertise, and cost for routine clinical laboratory analysis of plasma xenosterols is presently impractical. Conversely, genetic screening has become substantially less costly and increasingly common across healthcare systems. Genetic testing may offer a more practical means to identify and diagnose sitosterolemics. Such an approach requires the cataloguing and inclusion of *ABCG5 ABCG8* mutants among the various genetic testing platforms in use. A major limitation for such an approach is the lack of validation of suspected and likely pathogenic variants that may disrupt G5G8 function. Frameshift mutations in exon 13 of both *ABCG5* and *ABCG8* cause sitosterolemia, indicating little tolerance for truncation of the protein. Thus, deletions or frameshifts are expected to be Class I mutants. A significant number of splice donor and acceptor variants have also been identified and are likely pathogenic, but have yet to be formally analyzed. Of the 37 known missense variants, only 13 have been analyzed for maturation. Although most failed to form mature complexes and have been designated as Class II, three retained at least some degree of G5G8 maturation (G5:E146Q, G8:R543S, G8:G574R). Future investigations of the structural and functional impact of these and other missense mutants and variants will advance our understanding of sterol transport, the molecular dynamics of the transporter, and potential therapeutics for sitosterolemia as well as gall bladder and cardiovascular risk reduction.

**Author Contributions:** A.S. and K.W. crafted the initial draft. G.A.G. revised and edited the final draft. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institutes of Health, grant numbers 1R01DK113625, 1P20GM130456, 5P30GM127211.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
