The Mouse Mutation “Thrombocytopenia and Cardiomyopathy” (trac) Is within the ABCG5 Gene: A Single Gene Model for Human Mediterranean Macrothrombocytopenia.

Homozygosity for a new spontaneous mouse mutation named “thrombocytopenia and cardiomyopathy” (trac) results in thrombocytopenia, dilated cardiomyopathy, and infertility. A/J-trac/trac mice show a precipitous drop in platelet numbers and increases in platelet volume by 4 weeks of age. By 2–3 months of age, trac/trac mice have a 20-fold decrease in platelet number, a 3-fold increase in platelet volume, and a greatly increased bleeding time. Blood smears showed abnormally large platelets and megakaryocytoid cells. Homozygotes (trac/trac) also developed mild microcytic anemia accompanied by the presence of stomatocytes, a doubling of reticulocyte numbers, and a two-fold decrease in WBC counts. Increased numbers of megakaryocytes were present in bone marrow, spleen, and lungs. Ultrastructural studies of trac/trac megakaryocytes showed a poorly developed demarcation system and a failure to form platelet territories. The trac/trac platelets were enlarged, spherical, and contained numerous small alpha granules. The thrombocytopenia was not associated with defects intrinsic to bone marrow progenitor cells. Although thrombopoietin (TPO) levels were decreased, TPO treatment failed to reverse the thrombocytopenia. To identify the responsible gene, we produced a fine structure genetic map of a 5 megabase interval containing the trac locus on mouse chromosome 17. The human syntenic region is Chr 2p21–p22. Analyses of 1100 F2 progeny from intercross matings of (A/J x C57BL/6) F1 +/trac mice narrowed the interval to 0.3 Mb containing 4 genes. Sequencing of these genes revealed a G to A mutation at base 1435 (refseq nm031884) of Abcg5 (ATP-binding cassette sub-family G, member 5). This G>A base change results in a tryptophan codon (UGG) at amino acid position 463(uniprot) being changed to a premature stop codon (UAG). The transmembrane helices prediction program, TMHMM, predicts that the premature stop codon would truncate the last four of the six transmembrane domains of the ABCG5 protein. No DNA alterations were found in any of the other candidate genes. Genetic crosses of +/trac mice with mice doubly transgenic for the closely linked human ABCG5 and ABCG8 genes (stock B6SJL-Tg(ABCG5/ABCG8)14-2Hobb/J) showed that the transgenes normalized platelet counts and volumes in trac/trac mice. ABCG5 (sterolin-1) functions as part of a heterodimer, with ABCG8, that regulates plant sterol uptake. The trac/trac mutant mice have greatly elevated plasma levels of plant sterols. When placed on a phytosterol-free diet, the thrombocytopenia is reversed. Recent studies have shown that Mediterranean Macrothrombocytopenia is caused by mutations in ABCG5 or ABCG8. Identification of the molecular basis of the mouse Abcg5trac mutation provides a new model for studying the role of phytosterols in pathogenic changes in the hematopoietic, cardiovascular, and reproductive systems.