No abstract

Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by skeletal, cardiovascular and ocular abnormalities. The incidence of the disease is about 1 in 20,000, with life expectancy severely reduced because of cardiovascular complications. As the underlying defect is unknown, MFS diagnosis is based solely on clinical criteria. Certain phenotypic features of MFS are also shared by other conditions, which may be genetically distinct entities although part of a clinical continuum. Immunohistochemical studies have implicated fibrillin, a major component of elastin-associated microfibrils, in MFS aetiology. Genetic linkage analysis with random probes has independently localized the MFS locus to chromosome 15. Here we report that these two experimental approaches converge with the cloning and mapping of the fibrillin gene to chromosome 15q15-21, and with the establishment of linkage to MFS. We also isolated a second fibrillin gene and mapped it to chromosome 5q23-31. We linked this novel gene to a condition, congenital contractural arachnodactyly, that shares some of the features of MFS. Thus, the cosegregation of two related genes with two related syndromes implies that fibrillin mutations are likely to be responsible for different MFS phenotypes.

These results document consistent, relatively specific abnormalities of microfibrillar fibers in the Marfan syndrome. The biomechanical incompetence of these structural elements, due to quantitative or qualitative abnormalities, may account for the pleiotropic clinical manifestations of the disease. Therefore, various defects in the expression, structure, assembly, or degradation of the constituent structural glycoprotein (or glycoproteins) of microfibrils may be implicated in the causation of the Marfan syndrome.

The molecular heterogeneity of collagen is now well established (1-4), and at present, at least four genetically distinct species of collagens (Types I-IV) have been identified in human (3,(5)(6)(7)(8)(9)(10) and other mammalian tissues (11-16). Type I collagen is present in skin, bone, and tendon, and is composed of two identical polypeptide chain subunits, the al Type I chains, and a third structurally different polypeptide subunit, the a2 Type I chain (5,6,12). In current notation, Type I collagen has the subunit structure [al(I)]2a2(I). Type II collagen is present in cartilage, and is composed of three identical a chains different in primary structure from al(I) and a2(I); the subunit structure of Type II collagen is thus all(II)3 (7,14,17). Type III collagen is found in skin, blood -vessels, and fetal membranes and is composed of three identical a chains different than al(I), a2(I), or al(II) chains, and has the structure a l(III)3 (7-10, 15, 18). Type IV collagen is present in basement membranes such as found in glomerulus and lens capsule of the eye, and is composed of three identical al(IV) chains (3,11,16).In addition to the five genetically distinct a chains which compose the four recognized collagen types, other collagen polypeptides have been isolated from bovine (19) (23), and the solubilized collagens were precipitated from the clarified supernate by addition of NaCl to a concentration of 1 M. The precipitate was dissolved in cold 1 M NaCl, 50 mM Tris-HCl at pH 7.5, titrated to pH 8.6 to inactivate residual pepsin, and reprecipitated by exhaustive dialysis against 0.01 M Na2HPO2. The recovered precipitate was redissolved in, and dialyzed against, 0.5 M CH3COOH and lyophilized.For collagen extractability studies, fresh membranes were separated, washed briefly with distilled water, homogenized in water, and lyophilized. Two hundred milligrams (dry weight) of amnion or chorion homogenate was suspended and collected sequentially in 200 ml of the following solutions: H20; 1 M NaCl, 50 mM Tris-HCl at pH 7.5; 4 M guanidine, 50 mM Tris-HC1 at pH 7.5; 4 M guanidine, 50 mM Tris-HCl at pH 7.5, 10 mM dithiothreitol; and 0.5 M CH3COOH. Extractions were performed at 40 for 48 hr with constant stirring. After each extraction, the insoluble residue was harvested, and washed once in 50 ml of the same extracting solvent; the supernatant and wash were pooled and dialyzed for the hydroxyproline assay (24).Differential NaCl Precipitation. Routine differential salt precipitation was performed by dissolving total collagens of amnion, chorion, or both (1 mg/ml) in 1 M NaCl, 50 mM Tris-HCl at pH 7.5, and successively dialyzing versus 1.7 M, 2.6 M, and 4.0 M NaCl containing 50 mM Tris-HCl at pH 7.5. The precipitates obtained at these NaCl concentrations were harvested by centrifugation (5.2 X 105 g-min), dissolved in and dialyzed against 0.5 M CH3COOH, and lyophilized. To further define the salting-out behavior of collagens precipitating between 2.6 and 4.0 M NaCl, several 4.0 M NaCI precipitates were redissolved at...