Two genes that map to the STSL locus cause sitosterolemia: genomic structure and spectrum of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively

Abstract

Sitosterolemia is a rare autosomal recessive disorder characterized by (a) intestinal hyperabsorption of all sterols, including cholesterol and plant and shellfish sterols, and (b) impaired ability to excrete sterols into bile. Patients with this disease have expanded body pools of cholesterol and very elevated plasma plant-sterol species and frequently develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. In previous studies, we have mapped the STSL locus to human chromosome 2p21. Recently, we reported that a novel member of the ABC-transporter family, named "sterolin-1" and encoded by ABCG5, is mutated in 9 unrelated families with sitosterolemia; in the remaining 25 families, no mutations in sterolin-1 could be identified. We identified another ABC transporter, located <400 bp upstream of sterolin-1, in the opposite orientation. Mutational analyses revealed that this highly homologous protein, termed "sterolin-2" and encoded by ABCG8, is mutated in the remaining pedigrees. Thus, two highly homologous genes, located in a head-to-head configuration on chromosome 2p21, are involved as causes of sitosterolemia. These studies indicate that both sterolin-1 and sterolin-2 are indispensable for the regulation of sterol absorption and excretion. Identification of sterolin-1 and sterolin-2 as critical players in the regulation of dietary-sterol absorption and excretion identifies a new pathway of sterol transport.

Figure 1

Pedigrees with sitosterolemia. The parents, who are depicted as obligate carriers (half-black symbols), and the affected individuals (black symbols) are indicated. Only one of these pedigrees, 3400, is consanguineous. The boxed pedigrees are those that are mutant for sterolin-1 (see the Results section).

Figure 2

A, Structure and organization of ABCG5 and ABCG8. The intronic sizes (see table 1) were estimated on the basis of either long-range PCR products or, when available, genomic sequence. B, Promoter-region DNA sequence between the two first exons of each gene. The minimum start-transcription sites were estimated on the basis of both the presence of particular sequences in cDNA and the ability of an oligonucleotide located in such sequences to amplify spliced cDNA sequences. We estimate that the maximum distance between the two genes' start-transcription sites is ⩽140 bp. No canonical TATA sequence can be identified in this minimal region.

Figure 3

Alternative splicing of ABCG8 mRNA transcripts. The genomic sequence of exon 7–exon 8 region is shown (A), with the splice-acceptor site for exon 8, which contains a CAG repeat (underlined) that results in two different processed transcripts being made. The majority of clones are spliced by the first AG (B, left), but ∼10% of transcripts are spliced by the second AG (A, downward-facing arrowhead).

Figure 4

Homology of human, mouse, and rat ABC proteins. A, Phylogenetic analyses of sterolins and other ABC proteins, performed as described in the text. Each ABC-family member was included on the basis of identification based on its ATP-binding domain and the inclusion of ∼10 amino acids upstream and 120 amino acid downstream of this site. For ABC proteins that contain two ATP-binding sites, each site was analyzed independently; the first site is represented by the thinner unbroken lines, the second site by broken lines. Reassuringly, all such proteins clustered, indicating that such “full” ABC proteins are closely related and probably evolved after being organized as full ABC proteins. Sterolin, which forms a distinct family, is represented by the thicker unbroken lines; its nearest neighbor is the WHITE gene family, the mammalian homologues of which include ABC8-family members. B, Human, mouse, and rat sterolin proteins, aligned by CLUSTALW. The Walker A (i.e., ATP-binding domain) and Walker C motifs are indicated by the series of downward-pointing black triangles and the series upward-pointing black triangles, respectively; the Walker B motif is indicated by the sequence of alternating upward- and downward-pointing black triangles. The thick horizontal lines indicate the predicted transmembrane domains. Residues with the highest homology are denoted by the black portions of the boxes, those with moderate homology by the unblackened boxes and portions of boxes. Missense mutational changes are indicated by black ovals and diamonds, and polymorphic coding changes are indicated by the unblackened ovals and diamonds; changes pertaining to sterolin-1 (ABCG5) are indicated, above the alignments, by diamonds, and those pertaining to sterolin-2 (ABCG8) are indicated below the alignments, by ovals. In sterolin-2, serine 376, which is deleted by alternative splicing, is indicated by the solitary upward-pointing triangle; phenylalanine 570, which is deleted by a 3-bp deletion, is indicated by the arrow (see the text).

Figure 4

Homology of human, mouse, and rat ABC proteins. A, Phylogenetic analyses of sterolins and other ABC proteins, performed as described in the text. Each ABC-family member was included on the basis of identification based on its ATP-binding domain and the inclusion of ∼10 amino acids upstream and 120 amino acid downstream of this site. For ABC proteins that contain two ATP-binding sites, each site was analyzed independently; the first site is represented by the thinner unbroken lines, the second site by broken lines. Reassuringly, all such proteins clustered, indicating that such “full” ABC proteins are closely related and probably evolved after being organized as full ABC proteins. Sterolin, which forms a distinct family, is represented by the thicker unbroken lines; its nearest neighbor is the WHITE gene family, the mammalian homologues of which include ABC8-family members. B, Human, mouse, and rat sterolin proteins, aligned by CLUSTALW. The Walker A (i.e., ATP-binding domain) and Walker C motifs are indicated by the series of downward-pointing black triangles and the series upward-pointing black triangles, respectively; the Walker B motif is indicated by the sequence of alternating upward- and downward-pointing black triangles. The thick horizontal lines indicate the predicted transmembrane domains. Residues with the highest homology are denoted by the black portions of the boxes, those with moderate homology by the unblackened boxes and portions of boxes. Missense mutational changes are indicated by black ovals and diamonds, and polymorphic coding changes are indicated by the unblackened ovals and diamonds; changes pertaining to sterolin-1 (ABCG5) are indicated, above the alignments, by diamonds, and those pertaining to sterolin-2 (ABCG8) are indicated below the alignments, by ovals. In sterolin-2, serine 376, which is deleted by alternative splicing, is indicated by the solitary upward-pointing triangle; phenylalanine 570, which is deleted by a 3-bp deletion, is indicated by the arrow (see the text).

Figure 5

Northern analyses of human sterolin-2 expression. A, Tissue expression of sterolin, analyzed by hybridization to poly (A)+ RNA from different human tissues. The upper panel shows that significant hybridization was detected for liver RNA only (lane 8) and not in small-intestine RNA (lane 9); the bottom panel shows hybridization to β-actin after the membrane has been stripped. B, Results of RT-PCR analyses of cDNAs from different human tissue types, suggesting that sterolin-2 transcripts can be detected in a number of different tissue types, including the small intestine (top panel). The PCR products are derived from amplification across exons. Amplification of a control transcript, β-actin, also is shown (β). NC = water-only control.

Figure 6

Effect of mutations, in either ABCG5 or ABCG8, on plasma sitosterol levels. Plasma sitosterol levels were grouped on the basis of whether affected individuals had mutant sterolin-1 (ABCG5) or mutant sterolin-2 (ABCG8) and were compared with those in their parents (obligate heterozygotes), in their unaffected siblings, and in controls. Plasma sitosterol levels in individuals with mutant sterolin-1 were not different from those in individuals with mutant sterol-2.