Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption

Abstract

The molecular mechanisms regulating the amount of dietary cholesterol retained in the body, as well as the body's ability to exclude selectively other dietary sterols, are poorly understood. An average western diet will contain about 250-500 mg of dietary cholesterol and about 200-400 mg of non-cholesterol sterols. About 50-60% of the dietary cholesterol is absorbed and retained by the normal human body, but less than 1% of the non-cholesterol sterols are retained. Thus, there exists a subtle mechanism that allows the body to distinguish between cholesterol and non-cholesterol sterols. In sitosterolemia, a rare autosomal recessive disorder, affected individuals hyperabsorb not only cholesterol but also all other sterols, including plant and shellfish sterols from the intestine. The major plant sterol species is sitosterol; hence the name of the disorder. Consequently, patients with this disease have very high levels of plant sterols in the plasma and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. We previously mapped the STSL locus to human chromosome 2p21 and further localized it to a region of less than 2 cM bounded by markers D2S2294 and D2S2291 (M.-H.L. et al., manuscript submitted). We now report that a new member of the ABC transporter family, ABCG5, is mutant in nine unrelated sitosterolemia patients.

Fig. 1

Pedigrees with sitosterolemia. The parents are depicted as obligate carriers, and the affected individuals are depicted as filled symbols. None of these pedigrees are consanguineous.

Fig. 2

Mutational analyses in ABCG5 in sitosterolemia probands. a, The top panels show the sequence electropherograms; the middle panels, the sizes and restriction enzyme fragments; and the bottom panels, the gel electrophoresis of digested PCR fragments. Mutation R243X (left-hand panels) results in a gain of the AlwnI site. Using this as an assay, this mutation segregated within pedigree 500. Similarly, mutation R419P causes a loss of the second BstUI site and can be shown to segregate appropriately in pedigree 4000 (right-hand panels). BstUI can also be used to screen for R419H (data not shown). Additionally, the first BstUI site is altered by mutation R389H (middle right panels). Thus, digestion of the exon 9 PCR product with BstUI allows screening of all three mutations. Mutation R408X alters AvaI site (middle left panels). Control samples are indicated by the letters C1 and C2, with + or – indicating digested and undigested samples. Marker tracks (M) are as indicated. b, Following the detection of an abnormal SSCP for exon 3, direct sequencing of the PCR product revealed complex deletion and base substitutions, as depicted by the CLUSTALW alignment of the mutant product with the wild-type sequence. After re-designing the primer-pairs (exon3F and exon3R) to better detect the 20-bp size difference, the mutant alleles could be shown to segregate within pedigree 2100. Lane 2 contains a mixture of wild-type and mutant PCR products that were mixed, denatured and annealed before loading. The slower migrating band is therefore a hybrid between a wild-type strand and a mutant strand. Its presence is detected in heterozygotes only (lanes 5, 6 and 7). Based upon haplotype analyses, siblings 65, 66 and 67 are predicted to be carriers and sibling 64 is homozygous wild type, in good agreement with the segregation of the deletion. Maternal non-paternity was detected in this family and her sample was excluded from analyses (data not shown).

Fig. 3

Predicted sterolin structure and position of the mutations. The polypeptide is predicted to have a very large N termini and both the N and C termini are predicted to be cytosolic. The transmembrane domains are contained within the second half of the polypeptide, encoded by exons 9–13. Two potential N-glycosylation sites are as indicated on a loop that faces the lumenal or cell surface. The polymorphic variant, Q604E (open circle) is also within this loop. The mutations (dark filled circles) are as indicated, with stop codon mutations indicated by the crosses.

Fig. 4

Phylogenetic analysis of human, mouse and yeast ABC proteins. Phylogenetic analyses of sterolin and ABC proteins were carried out as described in the text. Each ABC family member was included on the basis of identification by its ATP-binding domain and the inclusion of approximately 10 aa upstream and 120 aa downstream of this site. Some ABC proteins contain two ATP-binding sites. In these cases, each site was analyzed independently; the first site is depicted in by the continuous lines, and the second site by the broken lines. Sterolin forms a distinct family (bold solid lines), with its nearest neighbor being the White gene family, the mammalian homologues of which include ABC8 family members.

Fig. 5

Analysis of Abcg5 expression. a, PCR products were obtained from cDNA synthesized from RNA extracted from different human tissues. A correctly spliced 250-bp fragment is expected for detection of expression (indicated by arrow), compared with a genomic fragment of 838 bp (compare cloned cDNA fragment, track 4, with genomic DNA fragment, track 3). A number of other PCR products were also consistently obtained, but did not contain ABCG5 sequence. b, Tissue expression of mouse Abcg5 was analyzed by northern-blot analysis. An expected 2.5-kb message was detected, with a fainter band of about 3.5 kb, in liver and small intestine only (tracks 4 and 8).