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And Suggest Distinct Pathogenetic Mechanisms Takeshi Aoyama,* Uta Francke,4911 Harry C Quantitative Differences in Biosynthesis and Extracellular Deposition of Fibrillin in Cultured Fibroblasts Distinguish Five Groups of Marfan Syndrome Patients and Suggest Distinct Pathogenetic Mechanisms Takeshi Aoyama,* Uta Francke,4911 Harry C. Dietz,l and Heinz Furthmayr* *Departments of Pathology, tGenetics, §Pediatrics, I1Howard Hughes Medical Institute, Stanford University, Stanford, California 94305; and IDepartment of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Abstract musculoskeletal, and cardiovascular systems ( 1). Several years ago, Sakai et al. (2) isolated fibrillin from the culture medium Pulse-chase studies of [3S]cysteine-labeled fibrillin were of dermal fibroblasts and provided substantial evidence for this performed on fibroblast strains from 55 patients with Mar- protein to be the major component of 10-nm microfibrils. Mi- fan syndrome (MFS), including 13 with identified mutations crofibrils are abundant in elastic and non-elastic tissues in or- in the fibrillin-1 gene and 10 controls. Quantitation of the gans that are significantly affected in patients with the Marfan soluble intracellular and insoluble extraceliular fibrillin al- syndrome. A dramatic decrease in the amount of such microfi- lowed discrimination of five groups. Groups I (n = 8) and brils has previously been demonstrated in the skin and in cul- H (n = 19) synthesize reduced amounts of normal-sized tured fibroblasts from such patients (3). fibrillin, while synthesis is normal in groups m (n = 6), Genetic linkage studies with random probes mapped the IV (n = 18), and V (n = 4). When extracellular fibrillin MFS locus to chromosome 15 (4). The fibrillin cDNA was deposition is measured, groups I and III deposit between 35 cloned (5) and mapped to the same site on human chromosome and 70% of control values, groups II and IV < 35%, and 15 by in situ hybridization (6). Subsequently, genetic linkage group V > 70%. was established between the Marfan syndrome and the fibrillin A deletion mutant with a low transcript level from the gene (FBNI) on chromosome 15 by using intragenic markers mutant allele and seven additional patients have the group (7). Several unique and independent missense, nonsense, and I protein phenotype. Disease in these patients is caused by deletion mutations in FBNJ have been reported, which make a a reduction in microfibrils associated with either a null al- strong argument for the primary involvement of this large gene lele, an unstable transcript, or an altered fibrillin product in the causation of MFS (8-14). The basis for considerable synthesized in low amounts. In 68% of the MFS individuals intra- and interfamilial phenotypic variability remains unknown. (groups II and IV), a dominant negative effect is invoked Unique beaded microfibrils (2, 3, 15) are the presumptive func- as the main pathogenetic mechanism. Products made by the tional units that are affected. Even though the beaded microfibril mutant allele in these fibroblasts are proposed to interfere presumably contains other proteins in addition to fibrillin ( 16), with microfibril formation. Insertion, deletion, and exon all linkage studies and mutational analyses to date point to the skipping mutations, resulting in smaller fibrillin products, FBN1 gene as the major locus responsible for the phenotype. exhibit the group II phenotype. A truncated form of fibrillin MFS is an autosomal dominant disorder in patients hetero- of 60 kD was identified with specific fibrillin antibodies in zygous for mutations at the FBNJ locus and, therefore, the one of the group II cell culture media. Seven of the nine normal allele is expected to produce fibrillin mRNA of normal known missense mutations, giving rise to abnormal, but nor- size and amount. It is not known whether null mutations exist mal-sized fibrillin molecules, are in group IV. (J. Clin. In- and cause MFS that do not produce mutant fibrillin and that vest. 1994. 94:130-137.) Key words: fibrillin biosynthesis e allow unimpeded matrix deposition of wild-type fibrillin made dominant negative mutation * FBNJ * extracellular matrix from the normal allele. In contrast, a mutant allele that gives * elastin-associated microfibril rise to defective fibrillin molecules could interfere with normal fibrillogenesis and affect the integrity of the microfibrils by a Introduction dominant negative mechanism (17), similar to that postulated of No direct The Marfan syndrome (MFS)' is a systemic disorder of connec- for certain types osteogenesis imperfecta (18). tive tissue and its clinical features involve primarily the ocular, evidence has been provided as yet, however, to prove that this mechanism plays a role in the pathogenesis of MFS. McGookey Milewicz et al. (19) have previously reported variable abnor- T. Aoyama's present address is Third Division, Department of Internal malities in cultured fibroblasts from patients with MFS with Medicine, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. respect to the rate of synthesis, secretion and the amount of Address correspondence to H. Furthmayr, Department of Pathology, fibrillin in the extracellular matrix. Implied pathogenetic mecha- Stanford University, 300 Pasteur Drive, Stanford, CA 94305-5324. nisms were not correlated with genotype. In a recent study we Received for publication 14 January 1994 and in revised form 23 have determined that missense mutations involving cysteine March 1994. residues in the FBNJ gene result in delayed fibrillin secretion, but this may not be the most important effect of such muta- 1. Abbreviation used in this paper: MFS, Marfan syndrome. tions (20). J. Clin. Invest. Here we report a comprehensive study of 55 fibroblast © The American Society for Clinical Investigation, Inc. strains from clinically well characterized MFS patients by quan- 0021-9738/94/07/0130/08 $2.00 titative pulse-chase methods. Based on the amounts of newly Volume 94, July 1994, 130-137 synthesized cellular fibrillin and the insoluble extracellular frac- 130 Aoyama et al. tion deposited during the 20-h chase period, five cellular pheno- A B C types were distinguished. Correlation of genotype to cellular and clinical phenotype suggests that FBNJ mutations cause dis- woo ease by different pathogenetic mechanisms. WO..1.1~~~~~~~.... Methods Studypopulation. Patients (n = 55), who satisfied the diagnostic criteria T(h) 04 8 20 8 20 4 8 20 established by Beighton et al. (21), and 10 normal controls were se- Cell ECM Medium lected for from the Marfan Clinic of the study Syndrome Stanford 1. the Northern California of the Figure Synthesis, secretion, and incorporation of fibrillin into the University Hospital, Chapter National extracellular matrix cultured control Marfan Association and the Johns Medical Center by fibroblasts. In A and B., cells Hopkins (Baltimore, were labeled with These include nine fibroblast with [355]cysteine and fibrillin was isolated from the cul- MD). strains missense mutations ture medium (A, left lane) and the cell lysate (B, left lane) by (20) and four with various other mutations as described purifica- recently (11, tion using a monoclonal antibody immunoaffinity column (A and B, 13, 14). right lanes) and samples were analyzed SDS-PAGE and Cell culture and Skin were by autoradiog- pulse-chase labeling. biopsy samples raphy. In C, cell lysate (Cell), NP-40 insoluble matrix components obtained under an from and controls either approved protocol patients (ECM) and tissue culture medium of control fibroblasts are at cardiovascular or visits. Dermal fibroblast (Medium) surgery during outpatient shown that were for 30 min and chased for or 20 h cultures were established in Dulbecco's modified medium pulse-labeled 4, 8, Eagle's (T[h]). Most of the newly synthesized and labeled fibrillin (F), col- (DME) with 15% fetal calf serum (FCS). The procedures used for pulse-chase lected after the 30-min pulse at time 0, is secreted from the cells during labeling, analysis by SDS-polyacrylamide gelectrophoresis the 4-8-h chase period. It accumulates in the medium that time and were done as described during (SDS-PAGE) quantitation recently (20). and a fraction of the labeled molecules is incorporated into the extracel- column. Monoclonal antibodies to fibrillin Immunoaffinity (mAb69 lular matrix within 8-20 h. was and donated Dr. L. Health Science Univer- Fibrillin quantitated in 30-min cell 201, kindly by Sakai, Oregon lysates (synthesis) and 20 h ECM (deposition) and not in the were to CNBr-activated media, sity, Portland, OR) coupled Sepharose (Phar- because the latter showed too much variability (not shown). macia Fine Chemicals, Piscataway, NJ) as described previously (2). Cell culture medium was incubated with antibodies coupled to Sepha- rose for 3 h at room temperature. The gel was then washed extensively with 50 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.05% Tween value±2 SD, Table I; see data presented below in Figs. 2 and 20. Bound material was eluted from the column with 1 M acetic acid 3 A). Likewise, "decreased deposition" was defined as the and immediately neutralized with 4 M Tris, followed by dialysis against amount of fibrillin incorporated into the extracellular matrix phosphate buffered saline (PBS). The eluates were analyzed by SDS- being < 70% of controls (control value±2 SD, Table I; com- PAGE as described (20). pare also Figs. 2 and 3 B). In control fibroblasts, the amount of intracellular fibrillin after a 4-h chase was 18±4.8% com- Results pared with that at time 0. When > 30% of fibrillin
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