**1. Discovery to Therapy**

In 1974, Bhattacharyya and Conner described a new lipid storage disorder in two sisters who presented with tendon and tuberous xanthomas and elevated plasma levels of phytosterols, sitosterol, campesterol, and stigmasterol [1]. Absorption of radiolabeled β-sitosterol was reported to be thirty-five times greater than that of normal subjects. They named their new lipid disorder β-sitosterolemia (hereon referred to as sitosterolemia), but it would be another 26 years before the discovery of *ABCG5 ABCG8* as the causative gene defect. Subsequent case reports established recessive genetics of the disease and greatly expanded its potential clinical presentation, which may include elevated low density lipoprotein (LDL) cholesterol, premature coronary artery disease and death, hemolytic anemia, macrothrombocytopenia, splenomegaly, adrenal dysfunction, elevated liver function tests, and cirrhosis [2–13]. Clinical studies in individuals with sitosterolemia revealed reductions in cholesterol synthesis, biliary cholesterol secretion, plasma clearance, and fecal elimination of neutral sterols [8,9,14,15]. Despite the absorptive phenotype and metabolism of phytosterols to bile acids, the clinical management of these patients with low sterol diets and bile acid binding resins resulted in modest and inconsistent reductions in plasma phytosterols [5,16].

Following the elimination of key genes in the esterification, absorbance, biosynthesis, and regulation of cholesterol metabolism, the sitosterolemia locus was mapped to a 0.5 centimorgan region on chromosome 2p21 [17,18]. The breakthrough would come two years later when investigators were studying agonists for liver X receptors (LXR-α NR1H3, LXR-β NR1H2), sensors of excess cholesterol that promote cholesterol mobilization from

**Citation:** Williams, K.; Segard, A.; Graf, G.A. Sitosterolemia: Twenty Years of Discovery of the Function of *ABCG5 ABCG8*. *Int. J. Mol. Sci.* **2021**, *22*, 2641. https://doi.org/10.3390/ ijms22052641

Academic Editor: Thomas Falguières

Received: 7 January 2021 Accepted: 26 February 2021 Published: 5 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

macrophages, metabolism to primary bile acids, and the excretion of neutral and acidic sterols [19,20]. A microarray screen of liver and intestinal transcripts from mice treated with an LXR agonist (T0901317) revealed a modest induction (2.5-fold) of the mouse brown gene, so named as it is the ortholog of the ABC transporter that determines brown eye color in *Drosophila* [21,22]. The human ortholog to mouse brown mapped to 2p21, *STSL* (OMIM: *STSL1*: 210250/*STSL2*:618666). At virtually the same time, independent groups identified individuals in multiple kindreds harboring nonsense mutations in either *ABCG5* or *ABCG8* with sitosterolemia, but not their unaffected family members [22,23]. *STSL1* and *STSL2* encode a pair of ATP binding cassette (ABC) half transporters in the G-subfamily. They reside on opposite strands of the DNA with initiation codons separated by a mere 374 base pairs. In these early reports, transcripts were restricted to the liver and intestine, the abundance of which increased in response to dietary cholesterol, suggesting the function of the transporters was to oppose intestinal absorption and promote biliary secretion of neutral sterols.

The discovery of ezetimibe as the inhibitor of Neiman–Pick C-1-Like 1 (NPC1L1), and cholesterol absorption was a breakthrough in the clinical management of sitosterolemia [24,25]. Ezetimibe (10 mg/day) was tested as a cholesterol-lowering agent in healthy subjects with moderate hypercholesterolemia. A detailed analysis of plasma sterols revealed that in addition to cholesterol, plasma phytosterols were also reduced, indicating that phytosterols and cholesterol shared a common, ezetimibe-sensitive pathway for absorption, thus suggesting the drug might be effective in the treatment of sitosterolemia (Figure 1 (1)(2)). Ezetimibe was subsequently shown to reduce plasma phytosterols in sitosterolemic subjects by >20% after eight weeks [26]. In a two-year follow-up study, plasma sitosterol and campesterol levels were reduced by 44% and 51%, respectively [27]. It should be noted that phytosterol levels in these subjects remained well above normal. However, the reductions observed with ezetimibe as a single agent or as an adjunctive therapy resolves many of the clinical manifestations of sitosterolemia (reviewed in [28]).

**Figure 1.** Enterohepatic sterol flux and regulation of *ABCG5 ABCG8*. (1) Bile acid micelles facilitate the solubilization of dietary and endogenous sterols in the proximal small intestine. Phospholipids not depicted. (2) NPC1L1 facilitates uptake of cholesterol and phytosterols into intestinal enterocytes (3) Cholesterol is incorporated into chylomicrons, delivered to the plasma compartment through the lymphatic system, and cleared by the liver. ABCG5 ABCG8 also promotes cholesterol secretion into the intestinal lumen. (4) Phytosterols are poorly absorbed and largely returned to the intestinal lumen by ABCG5 ABCG8. (5) Bile acids are reabsorbed in the distal small intestine, stimulate FXR-

dependent expression of FGF15/19, and are returned to the liver through the portal system. (6) In the liver, bile acids stimulate ABCG5 ABCG8 catalytic activity and promote the formation of bile acid micelles that serve as acceptors for ABCG5 ABCG8 mediated biliary cholesterol secretion. (7) Cholesterol metabolites (oxysterols), through LXR, and bile acids, through FXR and in cooperation with FGF15/19, activate ABCG5 ABCG8. The half transporters heterodimerize, traffic to the canalicular surface, and promote biliary phytosterol and cholesterol secretion. (8) Excess cholesterol, phytosterols and bile acids that are not absorbed/reabsorbed are eliminated from the body.
