Original Contributions Genetic characterization of the Dyscalc locus Veronica V. Colinayo, 1 Jian-Hua Qiao, 2 Peter Demant, 3 Kelly Krass, 1 Aldons J. Lusis, 1,4 Thomas A. Drake 5 1 Department of Microbiology, Immunology and Molecular Genetics University of California Los Angeles, California 90095, USA 2 Department of Pathology, Cedars Sinai Hospital, Los Angeles, California 90048, USA 3 Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands 4 Department of Medicine, University of California, Los Angeles California 90095, USA 5 Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095, USA Received: 27 November 2001 / Accepted: 14 February 2002 Abstract. Calcification occurs frequently in the development of atherosclerotic lesions, and studies in mice have indicated a ge- netic contribution. We now show that one genetic factor contrib- uting to aortic calcification is the Dyscalc locus, previously shown to contribute to myocardial calcification. Thus, the Dyscalc locus, on proximal mouse Chromosome (Chr) 7, segregated with vascular calcification in a large cross between susceptible strain DBA/2J and resistant strain C57BL/6J. Further evidence was observed by analysis of recombinant inbred strains derived from various sus- ceptible and resistant parental strains. Myocardial and vascular calcifications are importantly influenced by multiple modifier loci as well as the Dyscalc gene, making fine mapping of Dyscalc difficult. In order to allow more detailed genetic and biochemical characterization of Dyscalc, we have identified congenic strains containing the Dyscalc locus from resistant strain C57BL/10 on the background of susceptible strain C3H/DiSnA. The congenic strains exhibit little or no myocardial or vascular calcification, unlike the background HcB C3H strain, and the calcification seg- regated as a Mendelian factor, allowing finer mapping of Dyscalc. Cardiac calcification involves pathological deposition of calcium in the form of hydroxyapatite, often in response to injury (Cotran et al. 1994). There has been increased attention focused on calci- fication within vascular media, atherosclerotic lesions, and the myocardium because of the greater appreciation of its pathogenic role and mechanisms of pathogenesis. Calcification of vascular media is a common occurrence in diabetic patients and is a central feature in Mo ¨nckeberg’s arteriosclerosis. Calcification occurring within atherosclerotic lesion is associated with lesion progression and has recently been demonstrated to be a sensitive non- invasively detectable marker for atherosclerosis in asymptomatic patients (Prigent and Steingart 1997). Myocardial calcification is a widespread phenomenon that has been seen in a number of con- ditions including trisomies (Tennstedt et al. 2000), defects in heart structure (Drut et al. 1998), viral infections (Oyer et al. 2000), and responses to cell stress (Drut et al. 1998; Brunnert 1997). A model of spontaneously occurring myocardial calcification exists in mice, termed dystrophic cardiac calcinosis (DCC), in which calcification occurs in the presence of normal calcium and phosphate levels. Previous studies have revealed a genetic component to dystro- phic calcification. For example, different strains of mice placed under the same environmental conditions exhibit varying suscep- tibility to myocardial calcification (Qiao et al. 1994). There have also been mouse models for human genetic diseases, such as fa- milial cardiac myopathy, that have resulted in vascular calcific deposits (Fatkin et al. 1999). Loci contributing to myocardial cal- cification have been identified in mice by using quantitative trait locus (QTL) mapping. A locus on Chr 7, which is inherited as a recessive trait, was identified in a cross between susceptible C3H/ HeJ (C3H) mice and resistant C57BL/6J (B6) mice (Ivandic et al. 1996). Additional evidence for this locus was subsequently ob- served in crosses with B6 and DBA/2 (DBA) mice (van den Broek et al. 1998a; Brunnert et al. 1999). In both crosses, the trait was clearly a complex phenotype involving multiple genetic factors; thus, the location of the gene could not be determined with preci- sion. Although the myocardium is most prominently affected, cal- cification has also been demonstrated in skeletal muscle, kidneys, and tongue (Eaton et al. 1978; Brunnert 1997; and van den Broek et al. 1998a, 1998b). We now report strong evidence that Dyscalc contributes to vascular as well as myocardial calcification. As a step toward identification of the Dyscalc gene, we have identified congenic strains for the locus and have shown that in crosses between con- genic strains and background strain, calcification segregated as a Mendelian element. Materials and methods Mice and diets. BXD RI, BXH RI, B6, and DBA mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). All mice were housed under conditions meeting the guidelines of the Association for Accredita- tion of Laboratory Animal Care. For the BXD and BXH sets, B6 females were mated with DBA or C3H males. For the BXD F 2 population, F 1 progeny were intercrossed. After being weaned at 21 days, female mice were selected and placed on a chow diet (Purina 5001) containing 17.5% protein, 11% fat, 0.8% calcium, and 0.5% phosphorus. The BXD mice were switched to an atherogenic, high-fat, high-cholesterol (HF) diet con- taining 75% chow supplemented with 7.5% cocoa butter, 2.5% dextrose, 1.625% sucrose, 1.625% dextrin, 1.25% cholesterol, and 0.5% sodium cholate (Diet # 90221; Harlan Teklad, Madison, WI) at 12 months of age and maintained on the diet for 16 weeks. The BXH set received the same diet at 4 months of age for 8 weeks as described (Qiao et al. 1994). The mice were given free access to food and had a light/dark cycle of 12 h. The recombinant congenic strains (RCS) mice were developed as pre- viously described (Demant and Hart 1986). Mice from 17 different RCS lines and the parental strains C57BL/10 (B10) and C3H/DiSnA (HcB C3H) were used for the original screening for calcification. These mice were also placed on the HF diet for 16 weeks and sacrificed at 24 weeks of age. Subsequent studies on standard Purina Rodent Chow (Ralston-Purina Company) containing 4% fat revealed that the susceptibility to calcification could be determined by 16 weeks of age in the parental strains and lines HcB 12, HcB 24, and HcB 28. For a closer examination of line HcB 24, the mice were backcrossed to HcB C3H to generate F 1 s, which were brother- sister mated to produce an intercross group (HcB 24 F2s). The mice were housed as described above. Correspondence to: T.A. Drake; E-mail: tdrake@mednet.ucla.edu © Springer-Verlag New York Inc. 2002 Mammalian Genome 13, 283–288 (2002). DOI: 10.1007/s00335-001-2148-1 Incorporating Mouse Genome