A number of ESTs and genes were mapped into the region of interest and screened, based upon whether they were expressed in the liver and/or intestine, the organs important in dietary cholesterol retention (K.L. et al.
, manuscript submitted). Three ESTs were expressed in the liver and intestine only, of which one, T99836, was found to encode a ‘half-ABC’ transporter (for ATP binding cassette), and was studied further. A corresponding full-length cDNA was isolated and the gene structure characterized5
. The gene (called ABCG5
) consists of 13 exons, and encodes a 651-residue, 70 kD protein with 6 putative membrane-spanning domains that contains the characteristic ABC signature motifs at its amino-terminal end (in general, ABC proteins consist of 12 membrane spanning domains, hence the term ‘half-transporter’). We screened probands of nine families () for mutations using a combination of SSCP and direct sequencing of PCR products. Based on their haplotype analyses, eight were expected to carry a homozygous mutation and one (family 3300, proband 132) was predicted to be a compound heterozygote (M.-H.L. et al.
, manuscript submitted). Analyses by single strand conformation polymorphism (SSCP) indicated alterations in exons 3, 6, 9 and 13 of the gene (data not shown). Of these, a similar variant in exon 13 was also detected in control samples, suggesting it to be a polymorphic change ().
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.
Frequency of nucleotide changes in unrelated Japanese and North Americans of European descent
As potential sequence variants in exons 3, 6 and 9 were detected only in affected individuals and not in controls, they were directly sequenced. We identified five point mutations: R243X (exon 6, proband 25), R389H (exon 9, probands 46, 113 and 146), R408X (exon 9, proband 140), R419H (exon 9, probands 40 and 132) and R419P (exon 9, proband 157) (). A complex deletion mutation (exon 3) was identified in one proband (). To confirm that the missense nucleotide changes were mutations and not polymorphisms, we used the altered restriction endonuclease recognition sequences as an assay to perform segregation analyses, and to screen normal populations ( and ). Mutations resulting in R243X, R408X, R389H and R419H/P altered cleavage sites of restriction enzymes. R243X mutation segregated with disease in pedigree 500; both parents are carriers and both affected children are homozygous () for the mutation. Similarly, correlations are observed with other mutations (), except R408X, in which the mutation was found in a single individual whose parents were not available for analysis.
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 (more ...)
A homozygous complex deletion and base substitution mutation within exon 3 was identified for proband 63 (). Based upon haplotype analyses (M.-H.L. et al.
, manuscript submitted), we correctly predicted siblings 65, 66 and 67 to be carriers of the mutation and sibling 64, free of it. The predicted effect of this mutation is a frameshift, resulting in an ORF that terminates in exon 5, and the translation of a truncated protein of approximately 20 kD devoid of the transmembrane domains (). It is more probable, however, that such nonsense mutations are likely to lead to rapid mRNA degradation via the nonsense-mediated mRNA decay pathway6
. We screened 145 normal Japanese and 156 US Caucasians for these missense nucleotide changes mutations (); all tested negative.
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, (more ...)
The N-terminal region of ABCG5 is predicted to be cytosolic and contains characteristic ABC motifs; its sequence predicts six transmembrane domains and two potential N-glycosylation (). We propose the use of the name ‘sterolin’ for the protein product of ABCG5
, as this reflects its putative function. Of the two missense mutations of ABCG5
, both are located very close to transmembrane regions. These may lead to disruption of protein stability, as has been previously reported for mutations in DHCR7
leading to the Smith-Lemli-Opitz syndrome7
Selectivity for sterol absorption is a feature of other mammals (such as mice, rats and dogs); cholesterol is absorbed and retained by the body whereas non-cholesterol sterols are not. One might therefore expect the gene whose mutation results in sitosterolemia to be highly conserved among these species. We isolated and sequenced the full-length mouse ABCG5
cDNA and carried out phylogenetic analyses with other known ABC proteins (). Mouse sterolin shows 80% conservation relative to human sterolin at the nucleotide level and 85% identity at the protein level. The nearest non-mammalian neighbor is a yeast putative ABC protein (YOLO75C, Genbank IDs Z74816, Z74817), to which no function has been assigned; its homology with the mouse and human sterolins is confined to its carboxy-terminal region only. And so sterolins would seem to represent a separate ‘family’ of ABC transporters, and are not closely related to either ABC1
, mutated in Tangier disease8–10
, or the multiple drug resistance proteins, MDRs (refs. 11,12
), involved in the transport of phospholipid into bile. However, they are closely related to the ABC8/white
family, the mammalian homologues of which have been shown to be involved in macrophage cholesterol and phospholipid accumulation13
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 (more ...)
Based on the clinical defects in sitosterolemia, the gene is predicted to be expressed in the liver and/or the intestine. Northern-blot analysis showed ABCG5
expression was detected in the liver only5
. However, by RT–PCR analysis, expression is detected in human intestine, and adult and fetal liver (). Consistent with this finding is northern-blot analysis of RNA extracted from mouse tissues: we observed expression in both liver and intestine (). Thus, sterolin is expressed in a tissue-specific manner, consistent with the disease profile.
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 (more ...)
Although we have screened 30 families with sitosterolemia, we have been able to identify mutations in only 9 families. Preliminary analyses, by direct sequencing of all the exons, failed to identify mutations in the remaining probands. This suggests that either more subtle mutations, involving promoter sites or intronic regions may be affected, or that another closely-linked second locus may be involved. Sterolin is probably involved in the selective transport of dietary cholesterol in and out of enterocytes, and in selective sterol excretion by the liver into bile, as evidenced by the consequences when it is deficient. The identification of the specific mechanisms by which these processes come about will be greatly facilitated by the identification of ABCG5 as the gene which, when mutated, causes sitosterolemia.