4th International Symposium on the Marfan Syndrome

Eur J Pediatr (1996) 155 : 725-750 9 Springer-Verlag 1996

MARFA

CENTENNIAL

EVENT

1896-1996

4th International Symposium on the Marfan Syndrome

SYNDROME

1stInternational Workshopfor People withMarfanSyndrome Davos, Switzerland August 11-14, 1996

Chairs:

Beat Steinmann, Division of Metabolic and Molecular Diseases, University Children's Hospital, CH-8032 Ziirich Michael Raghunath, Institute of Physiological Chemist17 and Pathobiochemistry, D-48149 MOnster

Scientific board:

P. Byers (Seattle), R. Devereux (New York), H. Dietz (Baltimore), B. Gloor (Ziirich), L. P eltonen (Helsinki ), R. Pyeritz (Pittsburgh), L. Sakai (Portland), B. Sykes (Oxford)

Honorary member:"

V. A. McKusick (Baltimore)

Coordinated by:

Malfan Stiftung (Schweiz) in cooperation with the International Federation of Marfan Syndrome Organizations (IFMSO)

Co-sponsered by:

Worm Health Organization (WHO)

INVITATION R. Dreifuss Federal Councillor Federal Department of Home Affairs, Bundeshaus West, CH-3003 Berne, Switzerland It is a great pleasure to welcome you in D a r e s to the 4th International Symposium on the Marfan Syndrome and the 1st international Workshop for People with Marfan Syndrome. This international meeting is especially important for two reasons. The first is that a hundred years have passed since Dr Antoine B. Marfan described for the first time the syndrome to which his name was given. Since then substantial progress has been made in medicine. Early diagnosis is now possible, together with appropriate methods of treatment. In spite of this, however, not all those who suffer from Marfan syndrome (MFS) have the good fortune to receive treatment. More information is still necessary to increase public awareness. This meeting will make an important contribution towards this goal. The second important feature is that this is the first time those involved in research are being joined by people suffering from MFS. I am particularly glad about their inclusion and bid them a very special welcome to Davos. During the workshop they will be able to share their personal experiences, observations and conceres. This will be of inestimable value in developing partnership and full understanding of where behavioural and treatment responsibility lies. Cooperation between people involved in research, care and treatment on the one hand and those afflicted with MFS on the other is the best possible way towards coping with the complex

consequences of MFS in a spirit of mutual respect. Only in this way can doctors, insurers and employers become truly aware of the need of those with MFS. Let me thank the members of the Organizing Committee for preparing the meeting with so much personal commitment. I wish you all a fruitful exchange of views.

A. M A R F A N : HIS L I F E A N D T I M E S B. Rfittimann 1, B. Steinmann 2 1Institute and Museum of the History of Medicine and 2Division of Metabolic and Molecular Diseases, Department of Pediatrics, University of Zurich, CH-8006 and CH-8032 Zurich, Switzerland Antonin-Bemard-Jean Marfan (23 Jun 1858-11 Feb 1942) was a pioneer of paediatrics in France. He gained an international reputation as a clinician, investigator and teacher, especially in the Mediterranean parts of Europe and the Middle East, in Canada and Latin America, but he received also an honorary felloship of the Royal Society of Medicine of Great Britain in 1934. Marfan was born at Castelnaudary near Carcassonne where his father was a local general practitioner who tried to dissuade him from medicine, in vain. He trained in medicine at Toulouse and Paris and started a straight, conventional and yet a most successful career; he became an externe, an interne, a MD, chef de clinique, mddecin des h@itaux and agrdgd, and in 1910, professor of therapeutics. Four years later Marfan was appointed to the new post especially created for him - of professor of infantile hygiene and

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clinic at the University of ParAs. Since 1901 he worked at the H@ital des Enfants-Malades. In 1920 his chair was transferred to the Hospice des Enfants-Assistds where he also assumed the directorship of the Inst#ut de Pudriculture. He retired in 1928 but continued his scientific and social work. The main topics of Marfan's research and teaching were diphtheria, rickets, infant feeding, and especially pulmonary tuberculosis; his observations gave rise to the concept known as Marfan's law which was seminal in the development of the BCG vaccine. He published extensively on paediatric subjects and was, together with Grancher and Comby, co-editor and co-author of an important treatise of children's diseases in five volumes (1897/8). He founded and for many years edited the journal Le Nourrisson. Few could have suspected that his name and interest in him would persist because of a short case presentation. At the Socidtd Mddicale des H@itaux de Paris on 28 Feb 1896, he described a 5year-old girl, Gabrielle P., who had disproportionally long limbs and digits (pattes d'araign~e, spider legs, arachnodactyly) and an asthenic physique, a condition for which he suggested the term dolichostdnorndlie (Bull M6m Soc Mdd H6p Paris 13:220-226, 1896). In 1929, A. Carrau proposed, with good reasons, that the incomplete and unsatisfactory terms such as arachnodactyly, acromacria, hyperchondroplasia be replaced by the eponyme Marfan syndrome (Maladie de Matfan) (Le Nourrisson 17 : 82-92, 1929). Marfan's second and last (!) contribution to dolichostenomelia was 42 years later when he reviewed the 150 similar cases reported since (Ann Mdd 44 : 5-29, 1938). Marfan's case report fell into a time period recalled as the Belle Epoque and Fin de siOcle. There was an unbroken and unquestioned belief in the advances of science and in universal progress. For medicine it had brought the development of microbiology, of hygiene, of anti- and asepsis, and at the end of 1895 the discovery of X-rays. These great steps radically changed the appearance of the whole medical world, and the traditional physician was to become a 'technician', an artisan moderne as illustrated by ToulouseLautrec. Since then the Marfan syndrome has been extensively studied, its defect elucidated in 1991, and has become a paradigm in molecular medicine.

MARFAN SYNDROME: A PERSONAL A C C O U N T V. A. McKusick Johns Hopkins University, Baltimore, Maryland, USA It is 100 years since Prof. Marfan's report (1896), 45 years since my first encounter with a particularly instructive patient that set off my lifelong interest in the Marfan syndrome, and 40 years since the first publication of Heritable Disorders of Connective Tissue (1956) which defined disorders with a generalized genetic defect in one or another element of connective tissue. My studies of Marfan syndrome in the 1950s established the natural history of the cardiovascular, especially aortic, complications and demonstrated that multiple pleiotropic features can be related to a unitary connective tissue defect. Knowledge of the nature of that unitary defect had to wait for map-based gene discovery 35 years later: assignment of both the Marfan syndrome and the fibrillin gene to chromosome 15 and the clinching of a connection by demonstration of point mutations in FBN1 in 1991. Much was learned about the diagnosis and management of Marfan syndrome in the interval: the simulating condition homocystinuria, qualitative and quantitative variation in the same and different families, paternal age effect in new mutations, actuarial information on survivorship from cardiac complications, unusual clinical features such as meningocele. The importance of three aspects of the improved management of Marfan syndrome cannot be overstated. Beginning about 1970, echocardiography came in for

noninvasive monitoring of the aortic root, use of beta-adrenergic blockade and related agents was started for protection of the ascending aorta, and composite graft surgery for replacement of the aortic valve and ascending aorta was invented. Finally, the genetic support groups that started about 20 years ago have been a source of great satisfaction to all of us who have been privileged to work with patients and families with Marfan syndrome.

THE C O N T I N U U M OF M I C R O F I B R I L L A R DISORDERS: WHAT IS MARFAN SYNDROME? R. E. Pyeritz Medical College of Pennsylvania and Hahnemann University, Pittsburgh, Pennsylvania, USA In 1986, the diagnosis of the Marfan syndrome was established by the Berlin nosology. Over the past decade, two factors have created problems for clinicians and investigators alike. First, the nosology has been found wanting in many individual cases. This was perhaps not unexpected, given the empiric nature of clinical criteria. Second, molecular information about the basic defect in the Marfan syndrome has proven useful in relatively few instances. When coupled with the recognition that phenotypically related conditions also share defect in components of the microfibril, and in fibrillin-1 itself, this second factor clearly forced the need to revisit the nosology. Revised criteria have been proposed and will be reviewed (DePaepe et al.: A m J Med Genet 6 2 : 4 1 7 4 2 6 , 1996). Notable departures from the Berlin nosology include: broadening of the skeletal manifestations to warrant identification of a "major criterion" based on 4 of 8 signs being present; restricting the criteria for diagnosis in relatives of a clearly affected individual; and identifying the role of DNA-based testing in establishing the diagnosis. The continued efforts to refine diagnostic criteria only serve to emphasize persistent shortcomings. The phenotype of the Marfan syndrome remains incompletely defined. Most manifestations are age-dependent, so younger people perforce have fewer criteria than older ones. Most manifestations are difficult to quantify. The diagnostic specificity and sensitivity of each manifestion are poorly understood, and our designations of "major" and "minor" criteria are based more on accumulated experience than critical analysis. Of course, all of these problems also exist for the conditions most commonly confused with the Marfan syndrome, so the amount of careful research needing to be done is substantial indeed.

REVISED DIAGNOSTIC CRITERIA FOR THE MARFAN S Y N D R O M E A. De Paepe L, R. B. Devereux 2, H. C. Dietz 3, R. C. M. Hennekam 4, R. E. Pyeritz 5 i Dpt Med Genet, Gent, Belgium; 2Div Cardiol, Cornell Med Centre, New York, USA; 3 Dpt Paediat, John Hopkins Univ, Baltimore, USA; 4 Dpt Paediat & Hum Genet, Univ Amsterdam, The Netherlands; 5 Dpt Hum Genet, Medicine & Paediat, Pittsburgh, USA Until recently, the diagnosis of the Marfan syndrome (MFS) has relied on a set of clinical criteria, known as the Berlin Nosology (Beighton et al, 1988). Although these proved very useful, some shortcomings have emerged, in particular with the advent of molecular testing. One major concern was the fact that the criterion of a

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positive family history could produce a bias in favour of overdiagnosis. We propose a revision of the diagnostic criteria, reached after consensus with other professionals in the field. These new criteria are still based on a combination of major and minor clinical manifestations in different organ systems. The major differences with the previous version are: 1) more stringent requirements for diagnosis in relatives of an unequivocally affected individual (a major criterion in the family history and one major criterion in an organ system and involvement of a 2nd organ system); 2) skeletal involvement as a major criterion if at least 4 of 8 typical skeletal manifestations are present (other major criteria being: dilatation/dissection of ascending aorta, ectopia lentis, dural ectasia); 3) potential contribution of molecular analysis to the diagnosis (presence of causal FBN1 mutation or diseaseassociated FBN1 haplotype). In addition, initial criteria for diagnosis of conditions partially overlapping with MFS are presented. It is hoped that these revised criteria can serve as an international standard for clinical diagnosis of MFS, for comparing results of clinical and molecular studies and for investigations of genetic heterogeneity and genotype-phenotype correlations (Am J Med Genet 62:417-426, 1996).

LIFE EXPECTANCY AND E X P E C T A T I O N S F O R PATIENTS WITH THE MARFAN SYNDROME D. M. Milewicz I, D. Johnston 2, E. S. Crawford 3, J. Coselli 3, R. Finkbohner 1 1The University of Texas-Houston Medical School, Houston, Texas; 2University of Texas, MD, Anderson Cancer Center, Houston, Texas; 3Baylor College of Medicine, Houston, Texas Development of surgical therapy for aortic aneurysms and dissections has lead to treatment of the life-threatening cardiovascular complications associated with Marfan syndrome. The present study determines the effect of surgical therapy on the life expectancy of patients with Marfan syndrome and the clinical course of these patients after aortic aneurysm repair. Medical records were reviewed on 192 patients with Marfan syndrome who underwent aortic aneurysm repair during the past 26 years; 103 patients were interviewed, and complete preoperative and postoperative medical information was obtained. Survival curves were generated and data were analyzed. The median cumulative probabiliy of survival was 61 years, significantly increased compared with the median survival of 47 years for patients with Marfan syndrome determined 30 years ago (P < .006). The majority of patients (53%) had second surgeries to repair subsequent aneurysms or dissections at other sites, the vast majority of which involved the aorta. The most common pattern of aneurysm repair was proximal ascending aortic aneurysm repair, followed by descending thoracic aneurysm surgery. The following variables predicted patients requiring second vascular surgeries: presence of acute or chronic dissection at the time of the first surgery, hypertension after the first surgery, and a history of smoking. Review of all medical problems of the patient group revealed possible unidentified pleiotropic manifestations of the Marfan syndrome, which may be related to aging of this population. These medical problems included the onset of arthritis at a young age, varicose veins, ruptured or herniated discs, and prolapse of the uterus or bladder in women. In summary, the life expectancy of patients with Marfan syndrome undergoing surgical repair of aortic aneurysms has improved and is consistent with increased survival. Patients who have a dissection at the time of the first aortic surgery are more likely to require subsequent aortic surgery than are patients who undergo prophylactic composite graft repair of an aortic aneurysm.

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LIFE EXPECTANCY IN BRITISH M A R F A N SYNDROME

POPULATIONS

J. R. Gray, A. B. Bridges, R, West, L. McLeish, A. G. Stuart, J. Dean, M. Porteus, M. Boxer, S. J. Davies Instit Med Genet, University Hospital of Wales, Cardiff CF4 4XW, UK There is generalised awareness of the significant morbidity and mortality associated with Marfan syndrome since the original study by Murdoch in 1972. The past two decades have seen significant improvements in both the surgical and medical management, alongside improved ascertainment of the disorder. The precise improvement in well ascertained British populations has not been precisely addressed. The study population consisted of 205 patients with a definite diagnosis of Marfan syndrome ascertained primarily through genetics clinics in Wales and Scotland reviewed during the period 1970-1995. Information was ascertained from patients/family members, medical records and death certificates. In 174 individuals data was complete on sex, cardiovascular severity and familial status. Survivals were compared one variable at a time (univariate analysis) by the logrank test and plotted using Kaplan Meier estimate. Cox proportional hazard modelling was employed to evaluate survival taking account of more than one variable at a time (multivariate analysis). In total there were 45 deaths representing 22% of the cohort. Mean age at death was 45.3 + / - 16.5 years. Both the familial status and severity were significant variables on univariate analysis whilst multivariate analysis confirmed severity as the best independent indicator of survival. These findings and survival curves generated will help in the counselling of families and individuals with Marfan syndrome.

THE DEVELOPMENT OF THE ELASTIC

EXTRACELLULAR

MATRIX

DURING THE EMBRYOGENESIS OF THE CARDIOVASCUALR

SYSTEM

J. M. Hurle Departamento de Anatomia y Biologia Celular, Unversidad de Cantabria, Santander, Spain The elastic matrix constitutes a specialized component of the extracellular matrix which confers resiliency to tissues and organs subjected to repeated deformations. In this study we report the distribution of elastin, fibrillin, emilin, and collagen type VI during the development of the avian heart. Fibrillin and emilin appear in the cardiac jelly in the stages of tubular heart. Elastin appears in the stages of heart septation. Initial positivity for elastin was restricted to the developing outflow tract of the heart. Fibrillin and emilin are also present in this region. In the course of heart maturation elastin, fibrillin, emilin in association with collagen type VI are progressively deposited in the developing valvular apparatus, subendocardium and epicardium. The myocardial interstitium exhibits intense positivity for fibrillin, emilin and collagen type VI but lacks elastin. Possible roles for the elastic matrix during cardiogenesis are discussed.

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FACTORS AFFECTING THE MATERIAL PROPERTIES OF THE AORTA W. J. Rogers Jr. Medical College of Pennsylvania and Hahnemann University, Pittsburgh, Pennsylvania, USA The material properties of the aorta result from the combined effects of its structure and geometry. Three concentric regions make up the structural organization of the aorta. A single layer of endothelial cells attached to an underlying lamina comprises the intima. The next outward layer (media) contains vascular smooth muscle, elastin and collagen. Finally the adventitia provides a primarily connective tissue girdle. These layers define the change in vessel cross sectional area at a given intravascular pressure or the overall stress strain relationship. The proportion of these components varies significantly from the proximal aorta with 15% collagen, 47% elastin, and 35% smooth muscle to 35%, 15 and 50% respectively in the descending aorta. This is in part responsible for the increased distensibility in the ascending aorta. In Marfan patients, while the proportion of elastic fibers is normal their structure is disordered and fragmented resulting in a less compliant wall more susceptible to dilation and dissection. For a given wall composition and intravascular pressure increasing the vessel radius without a proportional increase in wall thickness will result in greater circumferential wall stress. At lower intravascular pressure compliant elastic fibers accommodate changes in pressure by stretching and increasing aortic diameter. At higher pressure elastic fibers are maximally stretched and the pressure-area relationship is dominated by stiff connective tissue resulting in more stress on the aortic wall. In that it is not possible to directly determine the aortas' structure, invasive and non-invasive indirect methods are used to measure mechanical properties. The change in aortic area for a given change in pressure (distensibility) can be determined invasively using a manometertipped catheter to continuously record intravascular pressure, or estimated using applanation tonometry of the carotid artery. Area may be measured using echocardiography or magnetic resonance imaging. Distensibility in dilated ascending Marfan aortas is 50% less than that in age matched normals. The descending aorta also shows reduced distensibility in the absence of any dilatation. The goal of pharmacologic intervention is to either directly alter the mechanical properties of the aorta, reduce blood pressure and thereby operate on a different portion of the wall stress-strain curve or reduce blood ejection velocity and reduce the hemodynamic forces on the aortic wall. p-blockade in relatively high doses, has been shown to reduce progressive dilation presumably by its negative inotropic effect and attendant reduction in ejection velocity. Vasodilators such as nitroprusside increase compliance by reduction of blood pressure but may also impact stiffness through vasodilation of the surrounding smooth muscle. Thus, pharrnacologic therapy may compensate for altered mechanical properties on either a structural or hemodynamic level.

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DETECTION OF ABNORMAL AORTIC ARCHITECTURE IN MARFAN SYNDROME WITH ULTRASONIC TISSUE CHARACTERIZATION M. Bosner D. Recchia, N. Kouchoukos, S. Wickline Washington University School of Medicine, St. Louis, Missouri, USA Aneurysmal dilation and dissection of the aorta are frequent complications and the primary cause of death in Marfan syndrorae. To

determine whether abnormal vessel architecture of the Marfan aorta can be delineated with ultrasonic tissue characterization, nondissected aortic tissue samples were obtained from patients with Marfan syndrome (n = 11) who underwent thoracic aortic aneurysm or dissection surgery. Human aortic tissue (n = 8) were obtained at post-mortem from subjects without evidence of aortic disease. Integrated backscatter (IB) was measured at more than 400 independent arterial sites from both tissue types with a 50 Mhz acoustic microscope. The directional dependence of backscatter, or anisotropy (variation in IB as a function of the plane of analysis, parallel vs. perpendicular), was also determined for each tissue sample. The IB from Marfan aortae was significantly lower than that from normal aortic tissue (-40.9 + 2.9 vs -32.6 + 2.2 Db, > 11 fold, P < .0001). Marfan aortae manifested substanitally less ultrasonic anisotropy than did normal aortae (12.4 _+ 3.3 Db vs 24.1 + 3.7 Db, for Marfan vs normal; 14 fold, P < .0001). Histologic analyis of tissues stained with yon Gieson's stain revealed a marked overall decrease, disorganization, and fragmentation of elastin fibers in each Marfan specimen as compared with normal specimens. The collagen content of the Marfan aortae was not significantly greater than normal aortae. Thus, quantitative ultrasonic tissue characterizatin sensitively detects abnormalities in aortic wall organization in Marfan syndrome and may provide a useful tool for diagnosis and monitoring evolution of this disease.

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NONINVASIVE IMAGING AND PROGNOSIS OF AORTIC DISEASE IN MARFAN SYNDROME COMPARED TO ARTERIAL HYPERTENSION Y. v. Kodolitsch ~, O. Simic 2, K. Langes 1, R. Spielmann 3, C. A. Nienaber 1 University Hospital Eppendorf, Dept. of Cardiology, Hamburg, Martinistrage 52, D-20246 Hamburg, Germany; 2 St. Georg Hospital, Dept. of Cardiac Surgery, Hamburg, Germany; 3 Dept. of Radiology, University Kiel, Germany In an attempt to identify specific anatomic and prognostic features of ascending aortic pathology associated with the Marfan syndrome [MFS], 35 consecutive pts. with MFS [16 W, 19 M; 35 + 12 years] were compared to a control group of 85 consecutive pts [24 W, 61 M; 59 _+ 11 years] with ascending aortic pathology associated with long-standing arterial hypertension [HTN]. All pts. with MFS were imaged by transthoracic [TTE; n = 26] and transesophageal echocardiography [TEE; n = 11 ], contrast enhanced computed tomography [XCT; n = 16] and/or magnetic resonance imaging [MRI; n = 14]; diagnostic findings were validated against intraoperative [n = 30] or angiographic findings [n = 2 t ] and compared to diagnostic results in the control group of pts. with HTN. All pts. were followed for 5 + 4 years. With overt aortic dissection being found in 20/35 [57%], intramural hemorrhage in 3/35 [8%] and aortic root or ascending aortic aneurysm in 12/35 [34%], the profile of aortic pathology was similar in MFS as compared to HTN. Although with 91% compared to 57%, aortic regurgitation was more frequent in MFS than in HTN [P < .05], and with 6 i % compared to 40%, aortic pathology in MFS was more frequently confined to the ascending aortic segment [P < .05], no specific anatomic features of aortic pathology were identified with MFS. Sidebranch occlusion with renal, mesenteric or coronary malperfusion, pericardial effusion and severe hypotension were more frequently seen HTN [P < .05]. Sensitivities and specificities of TEE, XCT and MRI were high for the detection of the entire spectrum of aortic pathology irrespective underlying disease. However, with a sensitivity of 53%, TTE was not adequate

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for the detection of dissecting lesions [P < .05 vs TEE, XCT and MRI]. 30-day [95% vs 71%; P < .05] and 1-year survival [90% vs 64%; n.s.] of pts with surgically treated aortic dissection was higher with MFS than HTN. However, with 17% compared to 6%, respectively, the frequency of reoperation was higher in MFS than in HTN [n.s.]. Conclusion: 1. There is no "Marfan-specific" aortic pathology 2. Aortic pathology in MFS should be evaluated noninvasively using TEE, CXT or MRI 3. TTE is not adequate for aortic surveillance 4. Early postsurgical survival is higher in MS than HTN 5. Follow-up using TEE, XCT or MRI might be crucial to improve long-term survival.

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CARDIOVASCULAR EXAMINATION IN MARFAN PATIENTS: THE SWISS CONSENSUS T. Carrel I, H. Saner, R. Jenni, K. Ammann, A. Hoffmann, I. Oberhfinsli, M. Rothlin 1Clinic for Thoracic and Cardiovascular Surgery, University Hospital Berne, CH-3010 Berne, Switzerland, and the Working Group W A T C H of the Swiss Society of Cardiology The diagnosis, management and follow-up of non-operated and operated Marfan patients requires a multidisciplinary approach. In Switzerland, the pediatrician, the family doctor or the cardiologist usually acts as the coordinator for management of the multiple aspects of this disease. More recently, a Working group for Adult and Teenager Congenital Heart Disease (WATCH-group) was established within the Swiss Society for Cardiology with the aim of optimizing the management of grown-up patients with congenital heart disease. The main recommendations concerning the cardiovascular examinations, management and follow-up of Marfan patients are briefly presented here: Thefirst examination includes a full patient's interview, a clinical examination, chest X-ray as well as Doppler echocardiography. If there is any suspicion of pathologic findings or if any cardiac and vascular structure cannot be visualized adequately, trans-

Anulus ESD parastema[ tong axis (-3.0 cm) Sinus of Valsalva ESD parastemal long axis (-3.7 cm)

1- . . . . . . . . . . . . . . . . . . . . . I I rI I

I

1

Ascending Aorta parastemal long axis (-3.7 crn)

[/ / /

9 Aortic arch suprastemal (-3.1 cm)

proximal Descending Aorta suprastomal (-2.8 cm)

I L . . . . . . . . . . . . . . . . . . . .

Abdominal Aorta at diaphragm subxyphoidal view (-2,8 cm)

Fig. 1 Recommended evaluation of the thoracic aorta and upper limits in the adult

esophageal echocardiography and/or magnetic resonance imaging is performed. Follow-up examinations take place at different intervals (from 6 months to 3 years), depending on the original cardiovascular pathology and the previous observations of the thoracic aorta9 In adult patients, the indication for surgical repair of the thoracic aorta should be discussed at a diameter of 50 mm; in children, surgery should be considered when the diameter of the aortic root/ascending aorta exceeds by 2 • the diameter of the aortic arch. In presence of aortic and/or mitral regurgitation, indications for surgery are similar to those of non-Marfan patients. Follow-up after surgery is recommended at yearly intervals. Postoperative evaluation should include echocardiography and computed tomography or magnetic resonance imaging of the operated and non-operated aortic segments. A close radiographic follow-up allows the detection of potential late complications and proper planning of elective reoperations. In addition, several points should also be discussed in this population: prophylaxis against endocarditis, ability to practice sport, problems related to pregnancy and insurance. Further, the patient and his family should be informed about the available assistance and goals of the Swiss Marfan Foundation.

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INTERNATIONAL NORMALIZED RATIO (INR)HOME MONITORING IMPROVES LONG TERM ANTICOAGULATION

AND QUALITY

OF LIFE AFTER BENTALL'S PROCEDURE ON PATIENTS WITH MARFAN SYNDROM H. Mair, C. Detter, P. G6hring, M. Lindenmeir, T. Fischlein, A. Welz, E. Kreuzer, D. Seidel, B. Reichart Department of Cardiac Surgery and Institute of Clinical Chemistry, Klinikum Groghadern, University of Munich, D-81377 Munich, Germany After cardiovascular surgery on patients with Marfan Syndrom (MFS), such as Bentall's operation, a stable long term anticoagulation is required to avoid thromboembolic (TE) or bleeding complications9 On average 14 month after Bentall's procedure 6 MFS patients (mean age: 35 years, m : w = 5:1) were trained in a 2 days course to measure their INR out of 25 gl capillary blood from the fingertip and the portable anticoagulation monitor CoaguChek plus (Boehringer Mannheim). Therapeutical range was INR 2,54,5. After receiving the INR-result within 1 minute, patients adjusted their own Marcumar | dosage9 We asked the patients every 6 months to send us their documentation of the selfdetermined INRvalues9 Five patients prefered the home monitoring, especially because of the convenient testing and independence from medical institutions. They measured their INR once a week. The follow up after the training was on average 18 months (median: 15 months)9 82% of the self-controlled values were within the therapeutical range. Before selfmonitoring, GPs measured the INR once every 2-7 weeks9 Only 58% of these values were within the therapeutical range. The INR-values of the CoaguChek plus had a good correlation to our laboratory (r = 0.85). So far, we observed no major bleedings or TE complications with permanent disability in these patients. In conclusion, INR home monitoring is well accepted by patients. They feel an improvement in quality of life. Frequent selftesting at home increases compliance and leads to a better anticoagulation status and therefore reduces life-threatening complications.

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META-ANALYSIS OF I f - B L O C K A D E R. Devereux New York Hospital, Cornell Medical Center, New York, NY 10021, USA

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A NOVEL MULTIDISCIPLINARY APPROACH TO THE DELIVERY OF HEALTH CARE FOR MARFAN SYNDROME AND RELATED DISEASES M. S. Bosner, A. C. Braverman, P. Turnbough, N. T. Kouchoukos Barnes-Jewish Hospital North, and Washington University School of Medicine, St. Louis, Missouri, USA Marfan syndrome is a genetic disorder which results from a defect in the structure and/or function of fibrillin, an integral connective tissue protein. The clinical manifestations primarily include defects in the musculoskeletal, ophthalmologic, and cardiovascular systems and morbidity and mortality results primarily from cardiovascular abnormalities including complications of aortic aneurysm including rupture/dissection, aortic and mitral valve insufficiency and resultant LV dilation and congestive heart failure. We have designed and implemented a multidisciplinary approach to the evaluation and assessment of individuals with definite and/or suspected Marfan syndrome or related diseases as well as individuals and families screened for suspected inherited disorder of connective tissue. We are organized to see these subjects on a single day, when they are screened by the geneticists, ophthalmologists and cardiovascular specialists (cardiologists and surgeons). All testing is done on campus and results are immediately available for a thorough and rapid assessment. In addition various other consultants (pulmonary, ob-gyn, orthopedics, physical medicine, genetic counselors et al.) are readily available during scheduled clinic time. We feel this is of great benefit to patients and families who do not have to make multiple physician/hospital visits. In addition, it is cost effective since clinic charges are negotiated with the health care carriers and our hospital system on a group basis, and thus are significantly lower than if individual physician and technical charges were placed.

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COMPUTERIZED SERIAL AORTIC ROOT DIAMETERS AID SURGICAL DECISIONS RE OPTIMAL TIMING OF AORTIC ROOT REPLACEMENT IN MARFAN SYNDROME T. Treasure I, A. Child 1, O. Valencia t, C. Reynolds 2, S. Gallivan 2 St. George's Hospital Medical School, London, UK; 2Clinical Operational Research Unit, University College, London, UK At present, optimal timing of aortic root replacement to prevent dissection is based largely on serial echocardiographic assessment of aortic root diameter. To aid decision making, a prototype computer system has been developed which displays serial aortic root measurements, standardized for height and age. These data are

available on a lap-top computer in the clinic. The diameter of the aortic root is measured at 3 different levels on each occasion, annulus (A), sinuses of Valsalva at tip of open cusp (V), and sinotubular junction (T). The internationally accepted level of high risk of spontaneous dissection occurs at 5.0 cm(V), when elective surgery should be considered. A retrospective study has been carried out using records of 81 classically affected adult Marfan syndrome patients, using only the first available echo assessment for the present analysis. Multiple linear regression has been utilized to examine the potential influence of patients' height, age and gender on aortic root diamter. The surgeon can elect to replace the aortic root when (V) reaches 95% prediction interval. We will now test these data as a method of prediction of risk of aortic dissection.

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ELECTROCARDIOGRAPHIC (ECG) ABNORMALITIES IN CHILDREN WITH MARFAN SYNDROME D. G. Wilson l, S. Davies 2, V. Booker l, A. G. Stuart 1 Congenital Heart Disease Centre, and 2Institute of Medical Genetics, University Hospital of Wales, Cardiff CF4 4XW, UK Cardiac rhythm disturbance is a significant cause of morbidity and mortality in Marfan syndrome (MFS), but the incidence in childhood is unknown. We have observed a spectrum of ECG abnormalities in children with MFS under serial follow-up in specialist clinic. Patients with suspected MFS were assessed using clinical examination, ECG and echocardiography. In 3 years, 88 patients were referred (age 14 d-19 yr, median 8.9 yr, 46 males) of whom 43 fulfilled criteria for MFS; 6 additional patients had probable MFS. At initial assessment, of the 49 patients with MFS/probable MFS, only 1 complained of palpitations. 12-Lead ECG abnormalities were observed in 18 patients and included interventricular conduction delay (ICD; 13), nodal beats (2), prominent Q/S waves (3), left ventricular (LV) hypertrophy (1), premature ventricular complexes (PVCs; 1), and ventricular tachycardia (> 2 beats; 1). Echocardiographic features included aortic root dilatation (40), aortic regurgitation (13), mitral regurgitation (14) and prolapse (MVP; 10) and LV dilatation (2). Follow-up data are available on 39 cases. Arrhythmia-related symptoms were noted in 6 patients (chest pain; 4, palpitations; 2). New ECG findings were seen in 9 (ICD; 6 and in 4 this was intermittent, PVCs- 2, premature atrial beats; 1). 24 hour Holter recordings were performed in 4 patients; 3 were abnormal (frequent PVCs/ventricular bigeminy; 2, supraventricular tachycardia; 1). Although numbers were small, there was a poor correlation between mitral valve prolapse, symptoms of palpitation and ECG evidence of arrhythmias. In this study ECG abnormalities were observed in 55% of children with MFS or probable MFS. Symptoms due to arrhythmias were uncommon. The reported ECG findings may reflect the electrophysiological consequences of fibrillin abnormalities within the myocardium and their long-term significance is unclear.

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18 S U R G E R Y FOR MARFAN C A R D I O V A S C U L A R DISEASE: A MULTI-CENTER ASSESSMENT AT THE BEGINNING OF THE SECOND CENTURY V. L. Gott Johns Hopkins Hospital, Baltimore, MD 21287, USA Ten Marfan centers in Europe, Canada and America are being surveyed for their overall data on surgery of Marfan cardiovascular disease. The following centers will be submitting their surgical data to the senior author by June 1, 1996 (the senior Marfan surgeon's name appears in parentheses): Hannover Medical Center (Hans G. Borst), Hospitalier Piti&Salp&ribre (Christian E. A. Cabrol), University of Zurich (Marko Turina), University of Toronto Hospital (Tirone E. David), Texas Heart Institute (Denton A. Cooley), Baylor Medical Center (Joseph S. Coselli), Mount Sinai Hospital, New York City (M. Arisan Ergin), Barnes-Jewish Hospital, St. Louis (Nicholas T. Kouchoukos), Stanford Medical Center (D. Craig Miller), and Johns Hopkins Hospital (Vincent L. Gott). After collating all of the Marfan surgical data from these ten centers, we will analyze, among other things, the actuarial survival for all of the patients and by univariate and multivariate analysis determine the significant independent predictors of early or late mortality. Various morbidity factors will be analyzed with determination of the actuarial freedom from late endocarditis, thromboembolism and problems of the unoperated aorta. The ten centers will also be surveyed regarding the use of aortic valve-sparing procedures for Marfan patients undergoing surgery. At the present time, we anticipate that data on more than 1,000 Marfan surgical patients will be submitted by these ten centers.

19 EARLY AND LATE RESULTS OF T H E C O M P O S I T E - G R A F T OPERATION IN MARFAN PATIENTS: ARE THERE VALID SURGICAL ALTERNATIVES? T. Carrel, J. Gysi, P. Mohacsi, B. Meyer, U. Althaus Clinic for Thoracic and Cardiovascular Surgery, Department of Cardiology, University Hospital Berne, CH-3010 Berne, Switzerland

Objective. The most significant cardiovascular pathology encountered in Marfan patients is dilatation of the aortic root and of the proximal ascending aorta, associated with or without substantial aortic regurgitation. Different methods of managing patients with Marfan syndrome and ascending aortic aneurysm, aortic regurgitation and/or aortic dissection have been reported in the past. The composite graft operation remains the procedure of choice which has lead to considerable improvment of early and late results in these particular patients. However, most recently there have been attractive developments in the field of aortic surgery, which may be considered as interesting alternatives for well selected subjects, in whom anticoagulation with Cumadine | should be avoided or is refused. Own experience. We reviewed our experience in the treatment of 43 consecutive Marfan patients who underwent elective (n = 31) or emergency (n = 12) surgery on the aortic root and/or ascending aorta; these patients represent 6.5% of the overall number of patients with surgery of the ascending aorta treated in our institution during the same period (1975-1995). The following pathology was encountered: aneurysm of the ascending aorta without (n = 10) or

with (n = 18) aortic regurgitation, acute dissection of the ascending aorta in 12 patients, chronic dissection in 3 patients. Composite graft replacement was performed in 30 patients, aortic valve replacement and supracoronary graft in 8, and isolated graft replacement of the ascending aorta in 5. Concomitant mitral valve surgery was performed in 4 patients and coronary artery bypass in 2. In one patient abdominal fenestration immediatly after the throacic aortic repair was necessary. Results. Hospital mortality was 16.6% (2/12) after emergency operation and 9.6% (3/31) after elective procedure. Main causes of perioperative mortality were hemorrhage in 3 patients and myocardial failure in 2. A total of 6 patients underwent a reoperation on the aortic root and/or on the distal ascending aorta/aortic arch after a mean follow-up of 6.5 + 2.7 years. Reasons for redo-operation were aortic root aneurysm in 2, pseudoaneurysm at a suture line in 2 and aneurysm of the distal ascending aorta and/or arotic arch in 2 patients. Actuarial survival is 82 + 4.5% at 5 and 60 + 5.6% at 10 years. Causes of late death include rupture of unoperated aortic segment, recurrent distal dissection and cardiac failure. Alternatives. Most recently, several surgical alternatives have been introduced in selected adult patients who refused peroral anticoagulation as well as in infants and adolescents: the confection of a composite graft with a biologic prosthesis, the insertion of a tube-valve homograft in the so called "extended miniroot" technique, the aortic root replacement with preservation of the native aortic valve and the most demanding Ross procedure, consisting in the transfer of the main pulmonary artery and the pulmonary valve in the aortic position, with subsequent replacement of the excised structures with a homograft. Since only limited experience is currently available, each method will briefly be presented. Conclusions. Composite graft replacement gives very satisfactory early and late results in Marfan patients: preventive surgery (as soon as diameter > 50-55 mm) gives better results than emergency treatment. Long-term follow-up should be clearly defined for early detection of late complications and proper planing of redo-operations. The advantages and disadvantages of each alternative must be outweighted against the perioperative risk and long-term results of the well-established treatment of annulo-aortic ectasia, that is eradication of the pathological tissue (e.g. aortic wall and aortic valve) and replacement with a composite graft. Moreover each new option should compare favourably with this classical operation, which fulfills the goal of a final procedure during the first operation.

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MARFAN SYNDROME: S U R G I C A L T E C H N I Q U E AND PRELIMINARY RESULTS OF TAILORING AORTOPLASTY OF CHRONIC EXPANDING AORTIC DISSECTION H. Schumacher, H. H. Eckstein, F, Kallinowski, J. R. Allenberg Department of Surgery, Division of Vascular Surgery, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany

Purpose. Tailoring aortoplasty has been reported to effectively prevent ischemia-reperfusion injury of the viscera and spinal cord and to restore more safely reperfusion to all critical aortic branches. We review our early experience with tailoring aortoplasty of chronic expanding aortic dissection in Marfan syndrome. Methods. Tailoring aortoplasty of the aortic segment IV was combined with standard graft replacement of the noncritical portions (segment III and V) of the aorta in patients with postdissection aneurysm ( 0 6 cm). Aortic tailoring consists of extended longitudinal aortotomy and removal of all intraaortic dissection mem-

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branes from the descending thoracic aorta to below the renal arteries. The enlarged aorta is closed and plicated creating a single normal-sized lumen ( 0 2.5 to 3.5 cm) containing the origins of the important arteries to the spinal cord, viscera, and kidneys. Results. 6 patients (28 to 68 years; mean 42 years) have successfully undergone the combined procedure in the last 3 years (1993-1995). All survived without paraplegia or significant visceral ischemia. Mean follow-up was 15 months (range, 4 to 40 months) and did not reveal any radiologic evidence of dilatation of the tailored segment. Conclusions. Tailored aortoplasty of chronic expanding aortic dissection seems to be a safe technique with favorable early results obtained in a smaII number of patients. Reduction in diameter and thus wall tension of the tailored aortic segment, growth of neointima and active collagen deposition support the success of this procedure.

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DEVELOPMENT OF A COMPUTER BASED TRAINING PROGRAM FOR VASCULAR SURGERY OF THE MARFAN SYNDROME A. Mehrabi, H. Schumacher, O. Sierp, Ch. Gltickstein, C. Herfarth, J. R. Allenberg, F. Kallinowski Department of Surgery, University of Heidelberg, D-69120 Heidelberg, Germany The Marfan syndrome (MFS) is a heritable disorder of the connective tissue. The wealth of informations about the MFS requires the use of modern informational technology for its teaching. Developing computer based training (CBT) systems is a possible solution. CBT programs contain material of a specific field and offer interactivity using multimedia components such as text, graphics, animation, sound, digital slide shows, and videos as well as quizzes. The aim of this project was the development of a CBT program on vascular aspect of the MFS. Using SuperCard, a teaching module for MFS is developed. This CBT program contains detailed clinical information regarding pathogenesis, diagnostic processes, radiography, video demonstration of surgical techniques, and questions. Video clips and vivid animations combine theoretical knowledge with practical experience. From prospective testing with a structured questionnaire, CBT is expected to gain in all evaluated criterias (distinctness, detailed description, presentation of materials, structure, motivation of learning, time saved learning and memory retention) 15%-20% better scores than a lecture on the same topic. Based on these evaluations, CBT modules are an appropriate teaching and learning future system that is well accepted. In conclusion, CBT programs should be integrated into medical education as a valuable supplement Furthermore, CBT can serve to inform special interest groups. In diseases with small patient numbers, medical knowledge is usually unevenly distributed with leading experts far apart. CBT programs can serve as an information container from which to draw knowlege necessary to counsel patients and relatives or to allocate centers for appropriate treatment.

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TWO-STAGE REPLACEMENT OF THE THORACO-ABDOMINAL AORTA USING THE "ELEPHANT TRUNK" TECHNIQUE T. Canel Clinic for Thoracic and Cardiovascular Surgery, University Hospital Berne, CH-3010 Berne, Switzerland In complex aortic surgery, the elephant trunk technique was developed to facilitate staged aortic replacement on the downstream or upstream aorta using additional intravascular graft length. The trunk can be placed in the proximal descending aorta as an extension of a total aortic arch graft or in the more distal aorta as an extension of a descending thoracic aortic graft. We demonstrate the case of a 63 year-old patient who presented with complex aortic pathology, including chronic type B dissection with a rapidly increasing diameter of the descending aorta. Preoperative assessment revealed a severe aortic regurgitation, an annulo-aortic ectasia and a true aneurysm of the aortic arch. In this video, we will present the two stages of the surgical procedure, using the elephant trunk technique for reconstruction of the aortic arch and subsequent replacement of the descending aorta. At a core temperature of 18 ~ C, cardiopulmonary bypass is discontinued and the arch replacement performed in deep hypothermic circulatory arrest. The dissecting membrane is generously resected to allow unproblematic placement of the prosthesis into the descending aorta. The supraaortic vessels are excised with a generous patch of surrounding aortic wall. The doubled-up graft is inserted into the descending aorta, and the distal anastomosis is created between the fold in the prosthesis and the aorta, immediatly distally to the left subclavian artery. The subsequent replacement of the thoraco-abdominal aorta is scheduled 4 to 8 weeks after initial surgery, depending on the patient's recovery and the urgency by which the remaining native aorta has to be replaced. Surgical approach is performed through a thoraco-phrenico-laparotomy. Medial visceral rotation allows a strictly retroperitoneal exposure of the abdominal aorta. Femorofemoral or atrio-femoral bypass is instituted in moderate hypothermia and the operation performed with the heart beating. Clamping of the descending aorta is performed well away from adhesions caused by previous surgery, the elephant trunk is pulled out and connected to a new graft in accomodation to the extent of distal aortic repair. W e consider this method suitable for the treatment of complex aortic disease - especially in Marfan patients -- including true aneurysms involving the whole thoracic aorta, acute and chronic dissection combined with true aneurysm of an additional aortic segment and in type B dissection with retrograde extension into the ascending aorta.

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TWO FRIBRILLINS AND ONE OR MANY MICROFIBRILS? L. Pereira, R. Ramirez Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York, USA The discovery of two structurally related fibriltins and their causal association to related disorders has raised new issues in matrix biology. Do morphologically similar microfibrils contain different combinations of fibrillin molecules, and do these have different

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biomechanical and physiological properties? Do the fibrillin molecules contribute to microfibril assembly and function in the same way or do they have distinct roles? These questions are relevant to the understanding of matrix physiology and etiopathogenesis of Marfan syndrome and related disorders. As a first step toward this goal, we previously established the spatiotemporal pattern of expression of the two fibrillin genes during mouse development. Based on these data, we proposed that fibrillins 1 and 2 have respectively homeostatic and regulatory functions. To test this hypothesis, we have recently targeted the fibrillin genes by homologous recombination in mouse embryonic stem cells. The results of the fibrillin 1 gene (Fbn]) targeting will be presented. Targeting of F b n l abrogated production of normal fibrillin-1; heterozygous mice appeared to be normal, whereas homozygotes died just after birth, and were indistinguishable from wild type littermates. Histopathologic examination revealed abnormal formation of elastic tissue in elastic vessels. In some mice, there was indication of atrial dissection without overt extracardiac hemorrhage; in others, there was dilatation of the abdominal aorta with onset of dissecting aneurysm. In both sets of animals, elastic fibers throughout appeared in some vessels to be clumped or to contain more amorphous content which enlarged the diameter of the elastic lamina multifocally. Overall, the analysis indicated an abnormality in the amount of elastic tissue present. More importantly, the study inferred a predominant role of fibrillin-i in tissue homeostasis.

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FIBRILLIN: AN EXTRACELLULAR MATRIX PROTEIN CONSERVED FROM JELLYFISH TO HUMANS S. Reber-Mfiller l, T. Spissinger 2, J. Spring I, V. Schmid ~ University of Basel, Institute of Zoology, Rheinsprung 9, CH-4051 Basel, Switzerland; 2 University of Basel, Biocenter, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Extracellular matrix (ECM)-molecules are mosaic proteins build from modular units. They are found either in animals or in plants but not in both, nor in prokaryotes. This suggests that E C M molecules arose at a time when plants and animals diverged. Therefore, the study of ECM-molecules of a phylum that has split very early from the main line of animal evolution like the cnidarians will help to understand the evolution of metazoan ECM. One of the monoclonal antibodies (mAb 44) generated in our laboratory against the E C M of the jellyfish Podocoryne carnea stains a fibrillar component in immunohistochemical preparations of this ECM. The antibody was used to isolate a cDNA clone from a Podocoryne carnea expression library. The deduced amino acid sequence of this cDNA fragment reveals > 40% identity to human fibrillins. From the partial cDNA sequence it can be concluded that the fibrillin modules and their arrangements are highly conserved from cnidarians to humans. Additionally, fibrillin-like microfibrils can be visualized by electron microscopy after rotary shadowing in E C M extracts of Podococw~e carnea. The dimensions of the beaded fibrils compare very well with mammalian fibrillin but show a less defined ultrastructure. The presence and distribution of the m A B 44 antigen was surveyed in the different stages of the life cycle of'Podocoryne carnea and also in several other cnidarian species by immunohistochemical methods. The results reveal a species and life-stage specific expression pattern of fibrillin, probably reflecting the biomechanical requirements of the animals.

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AFTER ALL, I T ' S A PROTEIN: POSTTRANSLATIONAL MODIFICATIONS OF PROFIBRILLIN M. Raghunath Institute of Physiological Chemistry & Pathobiochemistry, University of MOnster, D-48149 Mtinster, Germany The initial fascination about the knowledge of the full sequence of the cDNA of both fibrillins and the multitude of individual mutations segregating with the MFS or CCA phenotype has given way to the recognition that the biological properties of a protein cannot be solely deduced from its amino acid sequence. Although computer-assisted analyses of the genes allowed predictions about Nglycosylation, promotion of cell adhesion via RGD motifs, or calcium binding to EGF-like motifs, it is now left to cell biology to verify these deductions on the protein level, to characterise posttranslational modifications which are not predictable, and to explore their biological meaning. A typical example for such an unpredictable modification is the occurrence of xylose in O-linked glycans of fibriltin (Glanville et al.: J BioI Chem 2 6 9 : 2 6 6 3 0 26634, 1995). The role of glycans in fibrillin remains to be defined yet; most certainly they are not just a decoration. Furthermore, there is recent evidence that profibrillin (the existence of which was a matter of dispute for many years) is cleaved extracellularly by a calcium-dependent proprotein convertase of the PACE (paired basic amino acid cleaving enzyme)-class. This adds the first extracellular matrix protein to a list of known substrates such as proinsulin, proNGF, provWF, proopiomelanocorticotropin. It is conceivable that the trimming of profibrillin is a prerequisite for microfibril assembly. If this was correct, this would designate a proteolytic enzyme as a potential modulator of the MFS phenotype, since reduced conversion of profibrillin was found to be associated with dolichostenomelia (Milewicz et al.: J Clin Invest 9 5 : 2 3 7 3 - 2 3 7 8 , 1995). Another enzyme - t i s s t l e transglutaminase - creates covalent y-glutamyl-~-lysine cross-links in microfibrils. Fibrillin appears to be a substrate for this enzyme in both cell culture and dermis. It is conceivable that mutations disrupting transglutaminase cross-linking sites in fibrillin directly impair microfibrillar stability and may lead to a MFS phenotype. Based on these data from the basic research side, it will not be uninteresting to find out whether purified or recombinant tissue transglutaminase can be applied as a novel biological glue in the repair and stabilization of fragile tissues in the Marfan syndrome.

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FIBRILLIN MICROFIBRIL ORGANISATION AND C O M P O S I T I O N C. M. Kielty School of Biological Sciences, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK Our abiIity to interpret genotype- phenotype relationships in Marfan syndrome and related diseases is limited by lack of molecular definition of microfibril organisation. Consequently, we have developed and applied new experimental approaches to elucidate microfibril structure and composition. Using scanning transmission electron microscopy (STEM), we have measured microfibrillar mass of preparations of intact native fibrillin-containing microfibrils from normal and MFS tissues and cell layers. These studies, in conjunction with detailed theoretical

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modelling based on fibrillin-1 primary structure and molecular dimensions, have highlighted marked differences in mass and its distribution in these microfibrils. Our studies have highlighted tissuespecific differences in microfibril composition and organisation, and patient-specific differences in the response of microfibrils from some MFS patients to calcium chelation and addition. We have also investigated the potential association of proteoglycans with intact fibrillin-containing microfibrils from foetal bovine elastic tissues and with newly-synthesised fibrillin in human and bovine cell cultures. A combination of ultrastructural (rotary shadowing following chondroitinase treatment, STEM, positive staining, and cuprolinic blue staining) and biochemical (cell labelling, digestion, immunoprecipitation and SDS-PAGE) analyses indicates that chondroitin sulphate proteoglycans associate with fibrillin and contribute to microfibril assembly. This association has major implications for microfibril function in health and disease. Indeed, preliminary studies have highlighted differences between MFS cell lines in the capacity of fibrillin monomers to interact with chondroitin sulphate in vitro. Grants. Medical Research Council, UK

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FIBRILLIN: EVIDENCE THAT CHONDROITIN SULPHATE PROTEOGLYCANS ARE COMPONENTS OF MICROFIBRILS AND ASSOCIATE WITH NEWLY-SYNTHESISED MONOMERS C. M. Kielty, S. P. Whittaker, C. A. Shuttleworth School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Manchester M13 9PT, UK We have investigated the potential association of proteoglycans with intact fibrillin-containing microfibrils from foetal bovine elastic tissues End with newly-synthesised fibrillin in human and bovine cell cultures. Microfibril integrity was disrupted by chondroitinase ABC lyase and chondroitinase AC lyase, but not by keratinase or hyaluronidase. Following chondroitinase treatment, beads were disrupted but the underlying fibrillar scaffold appeared intact. Cuprolinic blue was prominently associated with beaded domains at a critical electrolyte concentration. Electron-dense rods were often associated with cuprolinic blue-treated microfibrils isolated from fixed tissues. Positive staining revealed charged foci at the beads. Newly-synthesised fibrillin could be labelled with ~5S TranSlabel, 3H-glucosamine or 35S-SO4 but its electrophoretic mobility was not influenced by treatment with chondroitinase ABC or AC lyase. A diffuse 35S-SO4 -labelled chondroitinase-sensitive component with a resistant band (Mr 35,000) coimmunoprecipitated with fibrillin. These experiments indicate that chondroitin sulphate proteoglycans associate with fibrillin and contribute to microfibril assembly. This association has major implications for microfibril function in health and disease.

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FIBRILLIN EXPRESSION AND DEPOSITION BY VASCULAR ENDOTHELIAL CELLS: IMPLICATIONS FOR MARFAN SYNDROME C. M. Kielty l, D. G. Wilson 2, G. Stuart 2, C. J. P. Jones 3, S. Davies 4, C. A. Shuttleworth 1 l School of Biological Sciences, and 3Department of Pathology, 2.205 Stopford Building, University of Manchester M13 9PT, UK 2Paediatric Cardiology, and 4 Institute of Medical Genetics, University Hospital of Wales, Cardiff CF4 4XW, UK We have shown at protein and mRNA levels that freshly isolated and low passage (passage 1-2) porcine aortic endothelial cells and human endothelial cells (HUVEC) express fibrillin. The cells were labelled with 35S- TranSlabel for 16 hours prior to immunoprecipitation from medium and cell layer compartments with anti-fibrillin antiserum. SDS-PAGE and fluorography revealed the presence of monomeric and aggregated forms of fibrillin in medium and cell layers. Analysis of immunoprecipitated counts indicated that the aortic endothelial cells synthesised markedly more fibrillin than HUVECs. RT-PCR and Northern analyses using total endothelial cell RNA confirmed the presence of mRNA encoding fibrillin-1 in both cell types. We also demonstrated the presence of assembled fibrillin-containing microfibril in cell layers following extraction and rotary shadowing EM. Detailed transmission EM analysis of fixed tissues sections of porcine aorta and non-Marfan human adult aortae revealed the presence of abundant microfibrils immediately subjacent to the endothelial cells and aligned in the direction of blood flow. Concurrent analysis of aortic sections removed from two Marfan patients during aortic root repair revealed extreme endothelial fragility and marked subendothelial microfibrillar disorganisation. In situ hybridisation of formalin-fixed sections confirmed the expression of fibrillin-1 by normal and Marfan aortic endothelial cells. These data suggest that fibrillin-l-containing microfibrils are deposited by vascular EC and act as a flexible anchorage for endothelial cells in juveniles and adults, thus playing a central role in normal aortic function. We postulate that vascular dysfunction in Marfan patients may reflect abnormlities not only of the medial microfibrils associated with elastic fibres but also the subendothelial microfibril population.

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FIBRILLIN-CONTAINING MICROFIBRILS: STEM ANALYSIS REVEALS CALCIUM-DEPENDENT ALTERATIONS IN MASS DISTRIBUTION AND PERIODICITY T H A T S U G G E S T A MOLECULAR BASIS FOR STRUCTURAL FLEXIBILITY M. J. Sherratt, D. F. Holmes, C. A. Shuttleworth, C. M. Kielty School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Manchester M I 3 9PT, UK We have carried out scanning transmission electron microscopy (STEM) on intact fibrillin-containing microfibrils isolated from foetal bovine aorta to derive new insights into microfibril organisation. By this method, intact unstained, unshadowed fibrillin-containing microfibrils revealed an average periodicity of 54.8 nm and a mass per unit length of 33.7 kDa/nm. STEM analysis revealed that chelation or addition of calcium induces profound, reversible

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and inverse variations in both parameters. Thus, periodicity was reduced and mass per unit length increased upon removal of calcium, but when microfibrils were saturated with calcium, increased periodicity and decreased mass per unit length were apparent. The reversibility of these changes was also confirmed. Equilibration of microfibrils in 4M GuC12 resulted in reduced periodicitiy and increased mass per unit length, and a morphology resembling that of calcium-free preparations. Peaks of high mass corresponding to beaded domains interspersed with troughs of low mass were the major features of the axial distribution of microfibrillar mass, and these were conserved between all preparations. Asymmetric peaks of mass were apparent in calcium-saturating conditions. These experiments clearly demonstrate that bound calcium directly and reversibly influences microfibrillar periodicity and organisation. Similar structural changes occurring in vivo may represent a physiological mechanism for modulating microfibrillar dimensions and mechanical properties. We have also carried out a comprehensive survey of theoretical microfibrillar mass distributions analysis based on fibrillin-1 primary structure and dimensions. We have demonstrated that fibrillin-1 as sole structural component can generate a periodicity of 50-55 nm in certain alignments, but the mass of fibrillin-1 alone is always insufficient to account for the peaks mad throughs of mass characteristic of isolated microfibrils. These data can be reconciled by the periodic presence of other microfibriIlar component(s).

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PROFIBRILLIN IS TRIMMED EXTRACELLULARLY TO FIBRILLIN AT A TETRABASIC CLEAVAGE SITE BY A PACE-LIKE CONVERTASE M. Raghunath ~, T. Ritty 2, D. Hamstra 3, A. Rehemtulla 3, D. Milewicz 2 LInstitute of Physiological Chemistry & Pathobiochemistry, University of MOnster, D-48149 MOnster, Germany; 2Dept. of Internal Medicine, University of Texas Medical School, Houston, Texas, USA; 3Dept. of Radiation Oncology, The University of Michigan, Ann Arbor, Michigan, USA The conversion of profibrillin-1 to fibrillin-1 was studied in cell culture and by expression of a miniprofibrillin consisting of the aminoterminus, exons 23 through 44 and the carboxyterminus. The carboxyterminus contains a stretch of basic amino acids harbouring a tetrabasic motif (RGRKRR,I, ST) that is recognised by proprotein convertases belonging to the PACE/furin family. Both wild type profibrillin and the in vitro transcribed/translated miniprofibrillin were converted to their corresponding lower molecular weight forms fibrillin and minifibrillin (approx. 20 kDa smaller) in the presence of furin/PACE, PACE4, PC 1/3, PC2 and fibroblast cultures. An alternative construct lacking the carboxy terminal domain and therefore the putative cleavage site was not converted by either enzyme. Remarkably, cleavage of wild type profibrillin-1 occurs strictly extra-pericellularly. Interestingly, PACE/turin - a trans Golgi endoprotease - accepts profibrillin as substrate only in vitro but not in the intact cell. Therefore, the intracellular cleavage of proFib to Fib by PACE/furin seems to be inhibited by a yet unknown mechanism. Also, native mutant profibrillin with a substitution of the -p6 arginine of the cleavage site which is not converted in fibroblast cultures was rapidly trimmed by PACE added to the conditioned culture medium. This rules furingFPACE out as the cleaving enzyme but sheds light on a -p6 arginine as a specific substrate requirement for the enzyme in question. Nevertheless, we could efficiently inhibit conversion in cell culture by the addition

of a fnrin inhibitor (Dec-RVKR-chloromethylketone), either by crossinhibition of the genuine cleaving enzyme or by inhibition of other proprotein convertases which in turn activate profibrillin convertase. We have herewith identified the carboxyterminal cleavage site of profibrillin and the candidate family of enzymes responsible for it. Profibrillin appears to be the first extracellular matrix protein to be shown to be a substrate for PACE-like enzymes.

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FIBRILLIN, THE MAIN COMPONENT OF ELASTIC MICROFIBRILS, IS MODIFIED BY TRANSGLUTAMINASE IN CULTURED DERMAL FIBROBLASTS AND HUMAN DERMIS M. Raghunath I, T. B~ichi2, D. Aeschlimann 3, B. Steinmann 4 i Institute of Physiological Chemistry and Pathobiochemistry, University of MOnster, D-48149 MOnster, Germany; 2Electron Microscopic Central Laboratory and 4 Division of Metabolic and Molecular Diseases, Dept. of Paediatrics, University of Z0rich, Switzerland; 3Dept. of Medicine, University of Wisconsin, Madison, Wisconsin, USA We investigated the role of transglutaminases in the post-translational modification of fibrillin through T-glutamyl-~-lysine crosslinks during microfibril formation in cell culture and in tissue. Transglutaminase-mediated cross-linking was demonstrated by the incorporation of monodansylcadaverine (MDC), a potent amine donor substrate for the enzyme, in dermal fibroblast cultures. Confocal laser scanning microscopy revealed that microfibrils rather than the pericellular fibronectin assembly are the target for transamidation in cell culture. We confirmed transglutaminase activity in tissue and found incorporation of MDC into microfibrils of human dermis in a periodic manner as revealed by immuno electron microscopy. In fibroblast cultures, [3H]putrescine was incorporated into fibrillin, fibronectin and high molecular mass complexes by endogenous transglutaminase. A similar pattern of labeled proteins was obtained after addition of purified tissue transglutaminase to conditioned fibroblast culture medium demonstrating that fibrillin is a glutaminyl substrate for tissue transglutaminase. Rotary shadowing electron microscopy of microfibrils isolated from fibroblast cultures that had been treated with MDC to compete with transglutaminase cross-linking revealed a broad spectrum of structural alterations. Our findings suggest that fibrillin containing microfibrils are cross-linked by tissue transglutaminase in vitro and in vivo. We speculate that 7-glntamyl-e-lysine crosslinks play a major role in microfibril formation and stabilization and thus might be involved in the pathophysiology of heritable microfibrillar disorders such as the Marfan syndrome.

32 THE STRUCTURAL

CONSEQUENCES

OF MUTATIONS IN FIBRILLIN-1 EGF-LIKE DOMAINS P. A. Handford Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK We are interested in determining the structural and functional roie of fibrillin domains as a first step towards understanding the mole-

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cular basis of the Marfan syndrome. We have recently determined the solution structure of a covalently linked pair of calcium binding (cb) epidermal growth factor (EGF)-like domains from human fibrillin-1. The two domains are in a rigid, rod-like arrangement, stabilised by interdomain Ca 2+ binding and hydrophobic interactions. Since the amino acids which are involved in the key packing interactions are highly conserved in fibrillin cbEGF pairs, we suggest that the relative orientation of all tandem cbEGF domains in fibrillin is similar to that seen in this structure. The implications of the structure for fibrillin cbEGF organisation within the microfibril will be discussed. Almost all reported point mutations in fibrillin-1 cbEGF domains can be classified into three distinct groups by their effects at the protein level on 1) disulphide bond formation, 2) calcium binding, 3) intra-/intermolecular interactions. The mutations in this last group are among the most interesting since some produce amino acid changes which would not obviously perturb structure, and therefore there is no clear explanation for the disease phenotype. These may provide the first clues to the specificity of fibrillin cbEGF interactions within the microfibril.

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INTERACTION OF THE FIBRILLINS WITH LAMININ [i2 IDENTIFIED BY YEAST TWO-HYBRID ANALYSIS P. A. Rupp, C. L. Maslen Oregon Health Sciences University, Portland, Oregon, USA Although much is now known about the fibrillins, little is known about their assembly into the complex, multi-protein microfibril or how the fibrillins associate with other proteins in the ECM. Each fibrillin has a unique proline-rich region. The proline-rich region of fibrillin-1 (Prol) encompasses amino acid residues 383-448. When fibrillin-1 and fibrillin-2 are aligned according to their maximum similarity this position is replaced by a glycine-rich region in fibrillin-2. However, fibrillin-2 has a different proline-rich region (Pro2) which is much smaller and covers amino acid residues 27-46. Although the overall similarity between Prol and Pro2 is limited, each contains a short sequence closely related to the consensus sequence for the Src-homology-3 (SH3) binding domain proteins and a more extensive sequence that is predicted to form a polyproline type II motif (PPII). The established function of all SH3 binding and many PPII domains is the mediation of proteinprotein interaction. We hypothesized that the proline-rich regions of the fibrillins are important sites of protein-protein interaction and that the sequence differences between Pro1 and Pro2 are indicative of functional differences between the two proteins. Initially, a yeast-based Two-Hybrid system (Clontech, Palo Alto, CA, USA) was employed to screen a human placenta cDNA library for proteins that interact with Prol. After dual selection using the HIS a n d ~-gal reporter genes, 11 positive clones were identified. Ten of the clones encoded unknown proteins and one encoded domain I of the laminin 92 (Lam~2) chain. Since laminins are found in basement membranes and fibrillin-containing elastic microfibrils insert into basement membranes this became a top candidate for interactions with the fibrillins. Consequently, we have further characterized the interaction using a protein-based affinity assay, which confirms the Two-Hybrid interaction results. Subsequent experiments demonstrated that the Pro2-Lam~2 interaction is of higher affinity than Prol-Lam[32, indicating a difference in specificity. Additional experiments are underway to determine the specific sites of interaction, to quantitate the relative affinities for Pro 1 and Pro2 and to determine the physiologic relevance of these interactions.

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AN INVESTIGATION I N T O T H E P O T E N T I A L ADHESIVE BEHAVIOUR OF FIBRILLIN-1 USING RECOMBINANT FRAGMENTS H. Dobson, M. Boxer Department of Pathology, Ninewells Hospital, Dundee, DD1 95Y, Scotland, U K Fibrillin-1 is a large (M r = 350 kDa) extracellular matrix glycoprorein important in the microfibrillar network of many connective tissues. Like many such proteins, fibrillin consists of a series of repeat structures. Repeats do not necessarily have a universal function: they appear to have precise structural constraints that result in the fomration of a 'scaffold' on which specific active groups such as recognition sequences for adhesion - can be presented. In the absence of such groups, repeats can function as spacers, facilitating precise spatial orientation of active sequences located on other groups. Within fibrillin, two commonly repeated structures are the EGFlike domains (both calcium- and non-calcium-binding) and the TGF-~31 binding protein-like motif. One of these latter motifs (module 34) of fibrillin-1 contains the adhesive sequence Gly-ArgAsp (RGD), The family of cell adhesion receptors known to recognise such sequences is the integrin family. In this work, we have used a P-GEX expression system to produce fusion proteins of a number of the fibriltin modules. These modules include: module 34, a doublet of module 34 and 35 (an EGF-cb-like motif), and module 21 (a TGF-~I binding protein-like motif). We have used a 72-well plate adhesion assay to investigate the adhesion of a number of different cell types to these modules and to various proteins including fibronectin and collagen, as well as several small RGD-containing peptides. The strategy for selection of cell types to study involved the consideration of those likely to be affected in the fibrillin disorder Marfan syndrome, and included fibroblasts, endothelial cells, smooth muscle cells, lung epithelium (normal and carcinoma) and a chondrocyte cell line. Sections of intact tissue were also obtained and stained with anti-fibrillin antibodies to determine whether any in vitro observations were likely to be important physiologically. Having established an adhesion assay, we then used a variety of antibodies, including anti-integrin antibodies, as well one against module 34, to reveal which integrins are important in the binding of these cell types and also show that while the RGD site in module 34 is active in cell adhesion, there is also possibly a synergistic site that facilitates spreading located in the adjacent module 35.

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FIBRILLIN-1 IMMUNOSTAINING IN 270 INDIVIDUALS WITH SOME PHENOTYPIC FEATURES OF THE MARFAN SYNDROME M. Godfrey., J. Cisler, S. De Bie, L. J. Kugler, A. De Paepe University of Nebraska Medical Center, Omaha, NE 68198, USA, and University Hospital, B-9000 Gent, Belgium Abnormal fibrillin immunofluorescence (Fib IF) was the first clue that defects in fibrillin-1 cause the Marfan syndrome (MFS). Since those early reports, we have received numerous requests for Fib IF in patients who, at the time of request did not fulfill the accepted clinical criteria of MFS. W e have analyzed skin sections and/or cultured fibroblasts from some 270 individuals. Clinical evaluation for all patients included echocardiography and ophthalmologic examination. The following clinical features were assessed: Skeletal-

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dolichostenomelia, pectus deformity, scoliosis, arachnodactyly, high arched palate, joint laxity and/or dislocation, pes planus; Ocular-ectopia lentis, myopia; Cardiovascular-aortic dissection/dilatation, mitral valve prolapse, aortic and/or mitral insufficiency; Other-striae, pneumothorax. Statistical analyses were performed using a two-tailed Fisher's exact test. Patients with skeletal abnormalities were divided into three groups (0 or 1; 2 to 4; or 5 or more; skeletal features). There is a statistically significant association between having 5 or more skeletal abnormalities and abnormal Fib IF in fibroblast cultures (P < 0.01). This is true even if patients with ectopia lentis are excluded. Similar analyses with ectopia tentis showed a significant statistical association with fibroblast cultures (P < 0.01) and skin sections (P < 0.05). The only association between Fib IF and cardiac features was between skin sections and aortic dissection/dilatation. Of individuals in whom Fib IF analyses were available for both skin and fibroblast cultures, 63% were concordant. Significantly, only 11% of patients with abnormal Fib IF in skin sections have normal fibroblast IF; whereas, 55% of those with abnormal fibroblast IF have normal skin IF (P < 0.001). Molecular studies will be needed to ascertain the specificity of the Fib IF findings in patients with skeletal abnormalities. The utility of Fib IF remains as a means to exclude the possibility of MFS in isolated cases and in families where linkage is not possible or not informative.

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DERMAL FIBROBLAST CULTURE AS A M O D E L S Y S T E M TO S T U D Y N O R M A L FIBRILLIN-1 A S S E M B L Y A N D D E F E C T S IN D I S T I N C T G R O U P S OF I N D I V I D U A L S W I T H MARFAN SYNDROME T. Brenn t, T. Aoyama l, U. Francke 2, H. Furthmayr l Laboratory of Experimental Oncology, Department of Pathology, Howard Hughes Medical Institute and 2 Departments of Genetics and Pediatrics, Stanford University, Stanford, CA 94305-5324, U S A At the Center for Marfan Syndrome and Connective Tissue Diseases at Stanford University we have recruited individuals with Marfan syndrome (MFS), with equivocal symptoms (not meeting clinical criteria for MFS) and with single organ involvement to participate in a study searching for mutations in the fibrillin-1 (FBN1) gene and evaluating FBN1 biosynthesis in dermal fibroblasts. The results of the biosynthetic studies are presented here. A quantitative pulse chase assay was used in the initial phase of the work and abnormalities in FBN1 production and deposition of newly synthesized and radiolabeled FBN1 molecules allowed to distinguish 5 groups as defined by Aoyama et al. (J. Clin. Invest. 94: 130-137, 1994). Groups I and II showed defects in production and deposition, groups III and IV showed defects in deposition only, and group V was indistinguishable from normal control fibroblasts. The group distribution (I-V) for the initial study population of 80 was as follows: 13.7%, 26.2%, 23.8%, 23.8%, and 12.5% indicating that - 50% are in the most severely affected groups II and IV. While > 95% of MFS showed defects, surprisingly 75% of non-MFS also showed abnormal FBN1 biosynthesis albeit with a large shift to groups I, III and V. The study was repeated on a second population of 90 individuals and a largely similar group distribution was found in spite of the fact that the clinical diagnosis have not been completely decoded: 8.2%, 24.7%, 31.5%, 35.6% and 16.0%. Immunofluorescence analysis of fibroblasts from different groups showed clear defects in amount and pattern of microfibrillar structures for the most severely affected groups, with more subtle abnormalities in group III and normal mi-

crofibrillar patterns in group I. Immunoelectron microscopic analysis of microfibrils deposited by fibroblasts grown in nylon mesh for several weeks indicated differences in complexity, thickness and arrangement of microfibrillar bundles. We postulate that the distinction into these groups is due to shared pathogenetic mechanisms. Our work further suggests that these methods have considerable potential to define homogeneous groups of individuals, to refine diagnostic criteria, and to assess prognosis before the onset of life-threatening complications.

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S T R U C T U R E A N D F U N C T I O N OF CA 2+ B I N D I N G E G F - L I K E D O M A I N S IN H U M A N F I B R I L L I N - 1 C. M. Cardy, S. Kettle, P. Handford Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK Mutations in the gene encoding fibrillin-1, a 350 kDa glycoprotein localised to the 10-12 nm microfibrils of the extracellular matrix, cause the Marfan syndrome. The predominant motif of fibritlin-1 is a Ca 2+ binding epidermal growth factor (EGF)-like domain, of which there are 43. The nuclear magnetic resonance (NMR) structure of a pair of covalently-linked, contiguous Ca 2+ binding EGFlike domains from fibrillin-1 has recently been determined. The two domains are in a rigid, rod-like arrangement, stabilized by interdomain Ca 2+ binding and hydrophobic interactions. We have devised a recombinant system to study the effects of defined mutations in Ca 2+ binding EGF-like domains on microfibril structure. A human fibroblast-derived cell line has been identified which produces correctly assembled microfibrils and exhibits a high transfection efficiency. A full-length human fibrillin cDNA has been constructed using RT-PCR and mutations introduced to "knock out" Ca 2+ binding to individual EGF-like domains. Both wild-type and mutant cDNAs have been transfected into this fibroblast line and R N A analysis performed to determine the level of mutant transcript. W e are currently examining the effect of such mutations on microfibril structure by rotary shadowing electron microscopy. These studies, in conjunction with the structural data provided by N M R analysis and an investigation into the properties of the microfibrils elaborated by the fibroblast line, will help to define the arrangement of fibrillin monomers within the microfibril.

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PATIENTS A N D P A T I E N C E IN T H E S T U D Y OF T H E P A H T O G E N E S I S OF F I B R I L L I N - 1 G E N E MUTATIONS H. Dietz, Z. Eldadah, S. Sood, E. Bull, R. Montgomery Johns Hopkins University School of Medicine, Baltimore, Maryland, USA The phenotypic spectrum associated with mutations in the gene encoding fibrillin-1 is wide and expanding. An ongoing goal is to better understand the pathogenesis of disease, and hence the origins of intrafamilial and interfamilial clinical variability, through phenotype-to-genotype correlations. An early observation was that mutations causing Marfan syndrome (MFS) are most frequently found in EGF-like domains and cluster at residues that dictate domain structure or calcium binding. Moreover, it has been observed that mutations causing the most severe form of disease cluster in a

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critical region flanked by exons 24-32. We have now obseawed that mutations within this region that substitute cysteine residues or create premature termination codons are associated with typical disease while more conservative changes or splicing defects are more generally associated with the severe phenotype. Selected substitutions of residues within this region with no known significance for domain structure or function have been identified in families segregating extremely mild phenotypes, characterized by skeletal features, myopia, mitral valve prolapse, and striae distensae (MASS phenotype). Thirty three probands and families segregating isolated or predominant aortic disease have been studied. Linkage analysis allows exclusion of the genes encoding the fibrillins, elastin, MFAP 1 and MFAP2 in about 25 % of familial cases. Direct mutation analysis suggests that defects in fibrillin- 1 are rare among individuals with isolated aortic disease. Locus heterogeneity has been considered an unlikely basis for interfamilial variability in the MFS. Indeed, our analyses have demonstrated conclusive evidence of linkage to FBN1 in the vast majority of cases. Recently we have observed a number of obligate recombinants between intragenic markers and the phenotype in a large family with classic ocular, skeletal, and cardiovascular manifestations of MFS. The possibilities of 1) intragenic recombination, 2) independent segregation of multiple predisposing alleles, and 3) linkage to other genes encoding microfibriilar eiements are being investigated. FBN1 mutations have recently been identified in individuals with the Shprintzen-Goldberg syndrome, a rare disorder that associates features of MFS with a distinct and recurrent pattern of neuropsychiatric and structural defects. These data suggest that fibrillin- 1 may influence early patterning of the cranium and the central nervous system. Our recent observation that FBN1 transcripts are expressed as early as the 8-cell stage of human development is consistent with this hypothesis. In addition, a patient with overlap features of MFS and Williams syndrome who is haploinsufficient for the elastin locus has been identified, perhaps manifesting a convergence of 2 pathogenetic mechanisms involving the elastinmicrofibril system.

39 Pl148A IN FIBRILLIN-I: DISEASE CAUSING MUTATION, DISEASE MODIFIER, OR NORMAL POLYMORPHISM? M. Wang, K. Mathews, M. Godfrey University of Nebraska Medical Center, Omaha, NE 68198, USA Phenotypic variability is a hallmark of most heritable connective tissue disorders. The fibrillin- 1 (FBN 1) microfibrillopathies, Marfan syndrome (MFS) (both classic and neonatal), familial and sporadic ectopia lentis, Marfan-like skeletal deformities, late onset aortic aneurysms, and most recently Shprintzen-Goldberg syndrome (SGS), are no exception. Few recurring FBN1 mutations have been described since, like many other heritable disorders of connective tissue, most families have private mutations. Two previous reports (Tynan, K. et al. Hum. Mol. Genet. 2: 1813, 1993 and Sood, S. et al. Nat. Genet. 12:209, 1996) have described P1148A substitutions in fibrillin-1. In the former paper, P1148A was found in 3 MFS patients, 2 patients with aortic aneurysms, and one of 44 controls. In the latter paper, P1148A was found in 3 of 60 affected families, one classic MFS, one with late onset aortic aneurysm, and one with SGS. They did not find this substitution in 163 controls. We have indentified the P1148A substitution in 4 affected individuals, but not in 120 controls. One patient had neonatal MFS, one had an aortic dissection and aneurysm, one with familial ectopia lentis, and one had classic MFS. We have identified

an additional change in the patient with classic MFS. It is of interest that five of the six individuals harboring P1148A in the Tynan study were of Asian descent, including the control. Two of our four patients with the P1148A substitution were Japanese. The ethnicity of the patients in the Sood paper were not specified. Additional studies on Asian populations will be important in helping to determine the pathogenicity of the P1148A substitution in fibrillin.

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A SOFTWARE AND A DATABASE FOR THE ANALYSIS MUTATIONS IN THE HUMAN FBN1 GENE G. Collod ~, C. Beroud I M. Coulon', M. Lackmy-Port-Lys ~, M. Guillotel 1, T. Soussi 2, C. Junien l, 3, C. Boilean 1, 3 11NSERM U383, H6pital Necker-Enfants Malades, 149-161, rue de Sbvres, F-75015 Paris, France; 2INSERM U301, 27, rue Juliette Dodu, F-75010 Paris, France; 3Laboratoire Central de Biochimie, d'Hormonologie et de G6n6tique Mol6culaire, H6pital Ambroise Par~, 9, avenue Charles de Gaulle, F-92104 Boulogne, France Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were described first in the heritable connective tissue disorder, Marfan syndrome (MFS). More recently, FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS. Seventy six mutations in the FBN1 gene have been reported. These mutations are private, essentially missense, non recurrent and widely distributed throughout the gene. To date, no clear genotype/phenotype relationship has been observed. This is not surprising when considering past experience with other disease genes such as the type I procollagen genes for which many mutations were accumulated before genotype/phenotype relationships emerged, [n an effort to standardize the information regarding FBN1 mutations, we have developed a computerized data base that currently contains information about the published mutations of the fibrillin gene FBN1 and some only reported in meeting proceedings. The current version of the database contains 76 entries. Subsequently, the software will be expanded as the database grows and according to the requirements of its users. New functions could be implemented. The current database and subsequent updated versions are (will be) available on request to G.C. or C.B6. on floppy disc using Apple format and Microsoft Excel | The software package is available on a collaborative basis. Notification of omissions and errors in the current version as welt as a specific data would be gratefully received by the authors.

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LONG RT-PCR ANALYSIS OF FBN1 TRANSCRIPT FOR RAPID DETECTION OF EXON SKIPPING MUTATIONS IN MARFAN SYNDROME W. Liu I, C. Qian 1, T. Brenn 4, H. Furthmayr 4, U. Francke 1, 2, 3 ~Howard Hughes Medical Institute, Departments of ; Genetics, 3 Pediatrics and 4 Pathology, Stanford University Medical Center, Stanford, CA 94305, USA Marfan syndrome (MFS), a heritable connective tissue disorder, is caused by mutations in the gene coding for fibrillin-l (FBN1), an

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extracellular matrix protein. One of the three major categories of FBN1 mutations involves exon skipping. To rapidly detect such mutations, we developed a Long RT-PCR method. Either three segments covering the entire FBN1 coding sequence or a single 8.9 kb FBN1 coding segment were amplified from reverse-transcribed total fibroblast RNA. Restriction fragment patterns of these RT-PCR products were compared and abnormal fragments were directly sequenced. Six exon-skipping mutations were identified in a panel of 60 MFS probands. All skipped exons encode epidermal growth factor (EGF)-like calcium binding domains and maintain the reading frame. In five patients, exon-skipping was due to point mutations in splice site sequences, and one had a 6 bp deletion in a donor splice site. Pulse-chase analysis of fibrillin protein revealed normal levels of synthesis but significantly reduced matrix deposition. Two multiple exon deletion mutations were also identified by this method. Both were due to genomic deletions. Patients with either type of FBN1 mutation include the most severe presentations of MFS such as one neonatally lethal case with skipping of exon 31 and another with deletion of exon 44-46. Thus, this method provides a powerful tool for rapid detection of exon skipping mutations that are correlated with a severe or classic MFS phenotype.

42P A N O V E L FBN1 GENE MUTATION IN AN EGF-LIKE M O T I F I N A PATIENT WITH E C T O P I A L E N T I S P. Booms, U. Vetter, P. N. Robinson Forschungslabor ftir P~idiatrische Molekularbiologie, Abteilung fiir Allgemeine P~idiatrie, Zentrnm ftir Kinderund Jugendmedizin der Humboldt-Universit~it, D-10098 Berlin, Germany Mutations in the fibrillin gene located on chromosome 15 (FBN1) may cause not only Marfan syndrome (MFS), but also autosomal dominant ectopia lentis, a disorder with or without mild skeletal changes but lacking the aortic lesions of MFS. W e screened a total of 18 dermal fibroblast cell lines from unrelated patients with Marfan syndrome (n = 16) or ectopia lentis (n = 2) for mutations in exons 1-27 of the fibrillin-1 gene using temperature gradient gel electrophoresis (TGGE) analysis of FBN1 cDNA. Using this system abnormal bands were observed for a cDNA-amplicon spanning exons 14-16 in one patient. Direct sequencing of amplified cDNA revealed a G-to-A transversion in one FBN1 allele at nucleotide 1762 which predicts the substitution by a tyrosine residue (Y) of a highly conserved cysteine residue (C) at codon 587 (C587Y) in the calcium-binding EGF-like domain coded by exon 14. G1762A was confirmed by sequencing from genomic DNA. Allele-specific oligonucleotide (ASO) hybridization analysis of a panel of MFS patients and normal controls showed the mutation to be specific for the patient. The 15-year old female patient has bilateral ectopia lentis as well as moderate scoliosis and arachnodactyly. Echocardiography showed normal findings for the heart and ascending aorta, with an aortic root diameter of 3.17 cm and no mitral valve prolaps. No other family members showed evidence of ectopia lentis or manifestations of MFS. It remains unknown why certain mutations result only in ectopia lentis without associated cardiovascular findings. To our knowledge, only one other FBN1 mutation has been identified in a patient with ectopia lentis, a substituion of a lysine for glutamic acid (E2447K) in the calcium binding EGF-like motif coded by exon 59. Interestingly, the mutation found in our study affects the third cysteine residue of an EGF-like motif coded by exon 14. Thus, mutations affecting different EGF-like motifs as well as different residues within these motifs may cause ectopia lentis.

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MOLECULAR BASIS OF THE MARFAN SYNDROME: OF MOLECULAR DEFECTS IN THE FIBRILLIN 1 GENE (FBN 1) BY SSCP AND DIRECT PCR-SEQUENCE ANALYSIS IDENTIFICATION

U. Grau, H. Mair, C. Detter, D. Seidel, B. Reichart, H.-G. Klein Institute of Clinical Chemistry and Department of Cardiovascular Surgery, Klinikum Groghadern, University of Munich, Germany Cardiovascular complications, such as aortic aneurysm and dissection drastically reduce life expectancy of individuals with Marfan Syndrome (MFS), whereas preventive surgery substantially improves prognosis in these patients. The aims of this study are 1) to substantiate a clinical diagnosis of MFS with molecular analysis and subsequently to identify allele carriers in affected families and 2) to investigate the correlation between the cardiovascular phenotype of MFS and defects in specific regions of the F B N 1 gene. W e studied the F B N 1 gene in a pilot study group of 15 patients (age 34 + 9 years) with cardiovascular phenotype of MFS, who underwent cardiovascular surgery in our hospital. 65 exons including the adjacent intron regions were amplified by PCR and screened for single stranded conformation polymorphisms (SSCP) using commercially available polyacrylamide gels. Band shifts were observed in 8 unrelated patients. PCR amplicons with an ambivalent electrophoretic pattern were analyzed by automated DNA sequencing on an ABI system. Direct DNA sequence analysis of both, sense and anti-sense strands identified novel point mutations in 2 unrelated patients. A G---~A transition, located 21 bases downstream of the splice donor site of intron 12, and a G--+A transition at codon 984 (exon 24), converting Val to Ile. Both substitutions were confirmed by restriction analysis. A 3-year old daughter of the latter patient, thus far phenotypically not affected, did not exhibit this defect. Conclusions: 1) The domain encoded by exon 24 has been suggested to play a critical role for calcium binding which may be disturbed by the amino acid substitution. 2) Intron 12 contains several cryptic splice sites, which may be activated by the mutation and lead to alternative splicing. 3) Defects in other genes than FBN 1 are likely to be also associated with the cardiovascular phenotype of MFS.

44P NOVEL FIBRILLIN GENE MUTATIONS I N V O L V E D IN MARFAN SYNDROME J. Shabbeer, S. Rooker. P. Johnson, M. Yacoub, A. H. Child Heart Science Centre, Harefield Hospital, Middlesex, U K The involvement of fibrillin 1 (FBN 1) in Marfan Syndrome (MFS) has been well established. The heteroduplex analysis method of Nijbroek et al (1995) is being used to characterise FBN1 gene mutations in our patients diagnosed with MFS. All of the patients display classic three system involvement, with aortic and mitral valve complications. We have identified several previously unreported mutations in these patients. The majority of these are missense, and are predicted to cause amino acid substitutions in epidermal growth factor (EGF)-like domains with a consensus for calcium binding. One new mutations causing neonatal presentation of MFS was found in exon 25 of the previously described neonatal cluster. As with approximately one third of all known FBN1 gene mutations it is predicted to result in the substitution of a cysteine residue. One other 'adult' mutation in an EGF-like domain affects

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a site involved in ~-hydroxylation of Asp/Asn residues. We have also identfied a nonsense mutation early in FBN 1 that is associated with a severe adult disease phenotype. Having confirmed the mutation in the proband this information was used in prenatal diagnosis by screening the DNA sample obtained from chorionic villus cells, correctly identifying an unaffected female. We are investigating the effect of these and other mutations on fibritlin m R N A and protein expression.

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WHY IS THE 'NEONATAL REGION' OF THE FIBRILLIN GENE SO IMPORTANT? L. Peltonen Department of Molecular Genetics, University of Helsinki and National Public Health Institute, Finland Distinct majority of fibrillin (FBN1) mutations resulting in exceptionally severe clinical phenotype (neonatal form) of Marfan syndrome (MFS) are clustered in the limited region of this gene, in exons 24-32 encoding the longest continuous stretch of EGF-motifs, characteristic for this polypeptide. This 'neonatal region' most probably plays a key role for the multimerization of fibrillin polypeptides to fibers or for the stability of these multimers. A total of 10 different mutations in neonatal patients are so far identified in this region. Only mutations resulting in premature STOP codon and low transcript levels of the mutant allele seem to result in milder form of the MFS. The same seems to hold for five reported mutations changing Cys to another amino acid in this neonatal region. They all result in the classical MFS, most probably due to severe folding defect of the polypeptide preventing the incorportation of the mutated fibrillin into microfibrils. To analyze the significance of this region and to dissect the specific consequences of various mutations, we have produced the neonatal cDNA construct encoding the polypeptide consisting of 8-Cys motifs on both sides of the stretch of 12 EGF-motifs. The multimerization of secreted polypeptides into minifibers has been demonstrated by rotary shadowing EM evidencing for proper folding of this region in vitro. A spectrum of mutations mimicking MFS-mutations identified on this region in MFS-patients has been introduced into this neonatal construct and the synthesized fibrillin polypeptides as welt as formed short "minifibers" have been analyzed in transient and stable expression systems. Differences in processing, post-transIational modification and incorporation of mutant polypeptides into ECM have been observed proving the usefulness of this in vitro model in monitoring the consequences of neonatal mutations including interferences of mutant polypeptides with fiber formation. These data should increase our understanding on the moleculm pathogenesis of the most severe form of MFS. Grants. The Academy of Finland, The National Marfan Foundation, US

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NEONATAL MARFAN SYNDROME AND SEVERE LETHAL CONGENITAL CONTRACTURAL ARACHNODACTYLY: CLINICAL AND MOLECULAR COMPARISONS M. Godfrey, M. Wang, K. Mathews, M. Wahl, S. D. Cederbaum, J. M. Graham, C. L. Clericuzio University of Nebraska Medical Center, Omaha, NE 68198, USA; University of New Mexico, School of Medicine, Albuquerque, NM 87131, USA; UCLA School of Medicine and Cedars-Sinai Medical Center, Los Angeles, CA 90048 USA The identification of specific mutations can provide independent evidence to help classify overlapping clinical phenotypes. The gamut of clinical phenotypes caused by mutations in the gene encoding fibrillin-1 (FBN1), on chromosome 15q, is such an example. At the most severe end of this spectrum is neonatal Marfan syndrome (nMFS), characterized by arachnodactyly, joint contractures, dysmorphic facies, and cardiovascular abnormalities including aortic root dilatation, and mitral and tricuspid valve involvement. These patients often die of congestive heart failure in the first year of life. Congenital contractural arachnodactyly (CCA) is a less common heritable connective tissue disorder characterized by a marfanoid habitus, flexion contractures, scoliosis, and abnormal pinnae. Recently, mutations in the gene encoding flbrillin-2 (FBN2), on chromosome 5q, have been found in CCA. Several reports in the literature have described a severe lethal form of CCA, whose additional clinical features included structural visceral anomalies: interrupted aortic arch, gastrointestinal abnormalities, and cardiac septal defects. However, its distinction from nMFS had remained in doubt. We have identified eight FBN1 mutations in nMFS, including two outside the "neonatal region" (between exons 24 and 34) and three FBN2 mutations in patients with severe lethal CCA. Molecular analyses can help to confirm the clinical distinction between nMFS and severe lethal CCA, which is suggested by differing patterns of cardiovascular and other visceral anomalies.

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AN EXTRA N-GLYCOSYLATION SITE IN FIBRILLIN-1 RESULTS IN NEONATAL MARFAN SYNDROME L. L0nnqvist z, L. Karttuneu ~, T. Rantam~iki 1, C. Kielty 2, M. Raghunath 3, L. Peltonen 1 t Dept Hum Mol Genet, Natl Publ Health Inst, Helsinki, Finland; 2 School of Biological Sciences, Univ of Manchester, UK; 3 Inst of Physiological Chemistry and Pathobiochemistry, University of Mtinster, D-48149 Mtinster, Germany Mutations leading to creation of an extra N-glycosylation site in the polypeptide chain have only very rarely been encountered in human diseases. We detected a point mutation T3143C in the exon 25 of the fibrillin- 1 (FBN1) gene in a patient with neonatally lethal Marfan syndrome (nMFS). This point mutation leads to the substitution of threonine for isoleucine at position 1048. A new potential N-glycosylation site is formed at Asn-1046, the local amino acid sequence being changed from Asn-Thr-Ile to Asn-Thr-Thr which

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is in agreement with the consensus sequence for N-linked glycosylation Asn-Xaa-Thr/Ser. Several lines of evidence indicate that this extra N-glycosylation site, created by the mutation, is utilized. A population of profibrillin-1 molecules migrating more slowly on SDS-PAGE than the control's sample was observed in the patient's sample. Immunohistochemical and ultrastructural analyses revealed that the microfibril formation was severely affected in the patient's fibroblast culture. In the presence of tunicamycin, an inhibitor of N-glycosylation, the patient's cell culture was capable of producing a better organized microfibril network. The creation of a neonatal cDNA construct consisting of exons 24-37 of the FBN1 gene was also proven to be a powerful tool in the analyses of the consequences of this mutation. The polypeptide translated from the minigene construct carrying the analogous I1048T mutation migrated more slowly on SDS-PAGE than the corresponding wild type polypeptide. Treatment with either tunicamycin, endoglycosidase H or Nglycosidase F abolished the migration difference indicating that the difference was originally related to the over-N-glycosylation of the mutant polypeptide. We conclude that excessive N-glycosylation due to a newly formed N-glycosylation site represents an interesting novel pathogenic mechanism for Marfan syndrome and should stimulate further studies. Grants. The National Marfan Foundation, US

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NEONATAL MARFAN S Y N D R O M E AND RESPIRATORY DISEASE J. C. S. Dean 1, D. J. Lloyd 2, G. F. Cole ? Department of Medical Genetics; 2Neonatal Unit; 3 Department of Medical Paediatrics, Aberdeen Royal Hospitals NHS Trust, Foresterhill, Aberdeen, Scotland, UK Neonatal Marfan syndrome is a severe form of the disease usually associated with cardiac valvular regurgitation and aortic dilatation resulting in death in the first year of life. In addition to the usual skeletal and ocular features, flexion contractures, crumpled ears, redundant skin and a progeroid facial appearence are not uncommon. The disease may be associated with mutations in exons 23-32 of the fibrillin gene and with deficient decorin production. We describe a patient with neonatal Marfan syndrome presenting as a newborn with arachnodactyly, joint laxity, abdominal wall laxity, sunken eyes giving a progeroid facial appearance and blue sclerae. Iridodonesis was noted at 2 months of age. There were initial feeding difficulties and later concerns about poor muscle tone. At 6 months of age, she developed a respiratory infection complicated by recurrent pneumothorax. Emphysematous bullae were seen on chest X-ray. The aortic root was dilated. She died from respiratory failure 7 days from the onset of symptoms. In neonatal Marfan syndrome, attention is usually focused on the cardiovascular abnormalities which often lead to death. However, in one literature series, 7/22 patients had respiratory disease. Review of the Aberdeen Marfan Clinic revealed a point prevalence of Marfan syndrome in North East of Scotland (population 0.5 million) of 1/14000, suggesting reasonably complete ascertainment. 10 patients were defined as "childhood" cases, being diagnosed at under 18 years of age, 2 of 3 sporadic cases in this group (including the subject of this report) suffered recurrent pneumothorax with emphysema but none of the 7 familial cases were so affected. 3/33 adult patients have suffered pneumothorax and a further adult has emphysema and pulmonary fibrosis without pneumothorax none are sporadic cases. Serious lung disease such as pneumothorax and emphysema appears to be more common in sporadic childhood-diagnosed Marfan syndrome (including neonatal Marfan

syndrome), than in familial adult cases. This may reflect biased ascertainment of severe cases in the sporadic childhood group, but could also reflect altered interactions between certain mutated fibrillins and other matrix proteins affecting lung integrity. In view of the morbidity and potential mortality from lung disease in Marfan patients, this area warrants further study.

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IN VITRO EXPRESSION OF THE NEONATAL MARFAN MUTATIONS T. Rantam~iki 1, L. Karttunen I, C. M. Kielty 2, L. Peltonen ~ Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland; 2 School of Biological Sciences, University of Manchester, UK Neonatal Marfan syndrome (nMFS) represents the most severe, neonatally lethal form of different Marfan syndrome (MFS) phenotypes. Several mutations in nMFS have been detected in the fibrillin-1 gene (FBN1). These mutations appear to have clustered in a distinct region of FBN1, exons 24-32. These exons code for a part of the longest stretch of consecutive EGF-like (epidermal growth factor like) motifs in fibrillin polypeptide. We have constructed a FBN1 minigene to study the consequences of different nMFS mutations by in vitro expression. This construct contains exons 24-32 of FBN1 cDNA inserted into a SV-Poly expression vector together with a signal sequence derived from a lysosomal enzyme, aspartylglucosaminidase. Several nMFS as well as a couple of classical MFS mutations were introduced into this minigene using an in vitro mutagenesis kit. For transient transfection COS-1 cells are transfected with different minigenes, then pulse-labeled, and medium, cells and E C M are harvested at different time-points. Polypeptides are immunoprecipitated with a polyclonal antibody and then analyzed on SDS-PAGE and fluorography. So far, all the minigens have been shown to be expressed and the polypeptides secreted into the medium. Some variation in the processing of different polypeptides is seen. In some cases the polypeptides have also been detected in ECM. We have also set up a stable cell line in CHO cells that expresses the wild type minigene. By rotary shadowing electron microscopy we could demonstrate that these cells produce fibrillin 'minifibers' that are seen as short linear fibrillar structures in cell layers. Grants. The National Marfan Foundation, US

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NEONATAL MARFAN SYNDROME: A CASE REPORT S. de Knecht ~, J. L. Yntema 2, B. HameP, J. R. M. Cruysberg 4 1Paediatric Heart Center and Paediatric Pulmonology, Dept. of Paediatrics; 3 Dept. of Human Genetics; 4 Dept. of Ophthalmology, University of Nijmegen, The Netherlands Aim of the case report: The Marfan syndrome has a wide variability in expression. Symptoms in adults and older children are well known but may differ from the neonatal Marfan syndrome in which serious problems lead to ealry disability and death, especially cardiac valve insufficiency and puhnonary emphysema. A full-term newborn girl of a mother with classical Marfan syndrome and a father with skeletal findings of Marfan syndrome, was admitted one day after birth because of a large diaphragmatic

742

hernia in the right thorax. She had the typical marfanoid habitus; mitral valve insufficiency (MI); mitral- and tricuspid valve prolapse and aortic diameter > P95. Bilateral blepharophimosis of the eyelids was an unusual finding. After lateral canthotomy normal eyes were seen. The hernia was operated on. At the age of two months a second admission was necessary because of severe dyspnea, possibly due to severe MI. However, no sings of pulmonary hypertension at heart catheterization, no high pCO2 level and no pulmonary engorgement on X-ray were found. On echo/Doppler cardiography a Iarge left atrium with severe MI was seen. Aftefload reduction did not change the clinical condition and at last mitral valve replacement by a St. Jude prosthesis was performed. At the time of surgery diffuse pulmonary emphysema was noted. A short period after surgery the patient's condition improved, but after a respiratory syncytial viral infection she died at the age of four months. Post m o r t e m investiations confirmed severe heart disease and pulmonary emphysema. Conclusions: be aware of fatal pulmonary emphysema in the neonatal Marfan syndrome.

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MICROFIBRILLAR PROTEINS: THE LONG AND THE SHORT OF IT J. Rosenbloom Department of Anatomy and Histology, University of Pennsylvania School of Dental Medicine, Philadelphia, P A 19104, USA Microfibrils having a diameter of 10-12 nm are widely distributed in many tissues of the body, often but not always in association with elastin. Characterization of the microfibrils remains incomplete, but recent findings have suggested that the proteins composing the microfibrils can be grouped into two classes: (1) large ones > 150 kDa and (2) small ones < 50 kDa. The large class contains two closely related gene families, the fibrillins (FBNs) and latent TGF-~-binding proteins (LTBPs), which share structural domains, including epidermal growth factor and 8-cysteine motifs. Presently, two distinct FBNs and three LTBPs are known. Phylogenetic analysis suggests that these two gene families have evolved from a common ancestral gene. The small class of proteins is more heterogeneous and as a group they have been designated microfibril associated proteins (MFAPs). Sequence analysis has not revealed any homology among them. MFAP1 is a 439 amino acid, highly acidic protein whose human gene is located near the FBN1 locus, 15q15q21. MFAP2, previously designated MAGP, is a 183 amino acid protein with a wide tissue distribution, whose human gene locus is lp36.1-p35. MFAP3 is a 362 amino acid protein whose human geue is near the FBN2 locus, 5q21-q31. It is not known whether the linkage of these two MFAPs near FBN loci has any functional significance or is merely coincidental. Several other glycoproteins including emilin and a 36 kDa protein have been localized to the microfibrils. While it is likely that the FBNs provide the basic scaffolding of the microfibrils, the function of the other proteins is unclear. They may stabilize the microfibril structure, interact with other matrix components as has been demonstrated for MFAP2 and elastin and thereby act as nucleation sites in fiber formation, or serve as cytokine storage depots as has been suggested for the LTBPs. Grants. NIH grants AR20553 & AR41474.

52

FBN2 MUTATIONS IN PATIENTS WITH CONGENITAL C O N T R A C T U R A L ARACHNODACTYLY

AND RELATED PHENOTYPES D. M. Milewicz l, E. S. Park l, C. M. Aalfs 2, R. C. M. Hennekam 2, H. Zhang 3, F. Ramirez 3, E. A. Putnam J ~Univ. of Texas Houston Medial School; aUniv, of Amsterdam; 3Mount Sinai School of Medicine New York, New York, USA Congenital contractural arachnodactyly (CCA) or Beals syndrome is an autosomal dominant condition phenotypically related to the Marfan syndrome (MFS). We have recently established that CCA results from mutations in FBN2, a gene that encodes fibrillin-2 found in 10-12 nm microfibrils. The previously characterized mutations in unrelated patients were C1252Y in exon 29 and C1433S in exon 33. We have subsequently characterized novel FBN2 mutations in three unrelated CCA patients. We identified an exon 29 splicing error in two affected siblings with unaffected parents. The splicing defect is due to an A to G transition at the - 1 5 position in the 3' splice site of intron 28. The affected siblings were heterozygous for the mutation. Analysis of the parents' DNA revealed that the father was a somatic mosaic with the mutation present in his hair bulb and buccal cells, but not in white blood cells; presumably, the father was also a germline mosaic. Another unrelated CCA patient was heterozygous for a mutation resulting in I1092T in exon 25. This mutation is not predicted to alter the secondary structure of the EGF-like domain encoded by exon 25, but does introduce a novel glycosylation site into the domain, suggesting a unique pathogenesis due to this particular mutation. A third putative mutation in exon 24 (G1056D) was identified in a CCA family. We have also identified a family with features of CCA, as well as a characteristic facies with hypertelorism, a broad forehead and flat facial profile. The phenotype segregates with FBN2 (LOD score > 3). These results indicate that FBN2 mutations producing CCA are private. The predicted effects of many of the FBN2 mutations on fibrillin-2 are similar to those of FBN 1 mutations. All the currently characterized FBN2 mutations occur in a region of the gene equivalent to the location of FBN1 mutations that produce the severe, neonatal MFS phenotype. Finally, our data suggest that FBN2 mutations result in conditions related to CCA.

53

ABERRANT SPLICING OF FIBRILLIN-2 IN A FAMILY WITH CONGENITAL CONTRACTURAL

ARACHNODACTYLY

C. L. Maslen t, D. Babcock s, M. Raghunath 2, B. Steinmann 3 Oregon Health Sciences University, Portland, Oregon, USA; 2University of Mtinster, D-48149 Mtinster, Germany; 3 University Children's Hospital, CH-8032 Ziirich, Switzerland Congenital Contractural Arachnodactyly (CCA) is an autosomal dominant disorder that is phenotypically similar to, but genetically distinct from Marfan syndrome. Genetic linkage analysis implicated the fibrillin-2 gene (FBN2) as the CCA locus. Mutation analysis of single CCA patients indicate that defects in FBN2 may be responsible for that disorder. However, co-segregation of a mutant allele with the disease phenotype has not been established. We have investigated the primary cause of CCA in a large, well characterized kindred with four generations comprising 18 affected individuals. Previous studies showed linkage of the CCA phenotype

743

to FBN2. Mutation analysis of the proband's cDNA using NonIsotopic Rnase Cleavage Assay (NIRCA, A m b i o n Corp., Austin Texas, USA) identified the presence of a skipped exon that was subsequently identified as exon 31. D N A sequence analysis of genomic D N A identified the splice site alteration responsible for the exon-skipping event. The occurrence of exon skipping was confirmed in the cDNA of an affected sibling. Genomic D N A from 29 additional available family members, both affected and unaffected, was then analyzed for the splice site mutation. The results clearly demonstrate co-segregation of the abberant splice site with the CCA phenotype. This unequivocally establishes, for the first time, that mutations in FBN2 are responsible for the CCA phenotype.

54 A SINGLE

MUTATION

THAT

RESULTS

IN AN ASP-->HIS S U B S T I T U T I O N AND PARTIAL E X O N S K I P P I N G WITH

CONGENITAL

I N A PATIENT CONTRACTURAL

ARACHNODACTYLY C. Maslen ~, D. Babcock ~, C. Gasner 2, U. Francke 2 Oregon Health Sciences University, Portland, Oregon, USA; 2 Stanford University Medical Center, Stanford, California, U S A Recent investigations of the molecular basis of congenital contractural arachnodactyly (CCA) indicate that mutations in the fibrillin2 gene (FBN2) cause CCA. In order to determine the range and nature of FBN2 mutations associated with CCA, we are examining cRNA from a series of patients using Non-Isotopic Rnase Cleavage Assay (NIRCA; Ambion Inc., Austin, Texas, USA). A NIRCA positive result indicated the presence of a mutation in cRNA prepared from cultured fibroblasts derived from CCA patient, FB904. The patient has the classic features of CCA; multiple contractures, arachnodactyly, and crumpled ears, with no apparent heart or eye manifestations. This mutation had previously gone undetected when cDNA from this individual was analyzed by chemical mismatch cleavage analysis, indicating that NIRCA may be a more robust technique for detecting fibrillin mutations. D N A sequence analysis of the NIRCA positive region detected a G to C transversion at nucleotide 3340 (G3340C), which predicts the substitution of histidine for asparagine at amino acid residue 1114. This asparagine residue acts as a calcium-binding ligand in the 12th calcium-binding pEGF-like domain. In addition, the G3340C mutation alters the last nucleotide of exon 25; position - t of the 5' donor splice site, which is a highly conserved G (77%). Subsequent RT-PCR and D N A sequence analyses demonstrate that this missense mutation also acts as a splice site error, resulting in partial skipping of that exon. Consequently, the complex manifestation of this genotype may result in two different populations of mutant fibrillin-2 molecules as components of elastic microfibrils in one individual. However, the phenotype of the affected individual indicates that if both mutant fibrillin-2 forms are present in the matrix, they are not appreciably less well tolerated than is a single defective molecule.

55

IDENTIFICATION OF FIBULIN-2 AS A BINDING LIGAND FOR FIBRILLIN-1 D. P. Reinhardt 1, T. Sasaki 2, B. J. Dzamba l, D. R. Keene l, M.-L. Chu 3, W. G6hring 2, R. Timpl 2, L. Y. Sakai 1 1Shriners Hospital for Crippled Children, Portland, Oregon, USA; aMax-Planck-Institut ftir Biochemie, Martinsried, Germany; 3 Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania Fibrillins are major components of microfibrils 10-12 nm in diameter, which are found in both elastic and non-elastic tissues. Only very little is known about the molecular structure of microfibrils. Several protein components have been suggested, but their interaction with fibrillins are obscure. To identify possible binding ligands for fibrillin-1, we tested binding of fibronectin, laminin-1, B M40 (SPARC), fibulin-1C and -D, fibulin-2 and collagens type I, II, III, IV, V, and XI to recombinant fibrillin-1 peptides in solid phase assays. Only fibulin-2 showed significant binding to rF11, comprising the N-terminal half of fibrillin-1. By overlapping recombinant peptides the fibulin-2 binding site was narrowed down to the N-terminus of fibrillin-1 (amino acid position 4 5 4 5 0 ) . Surface plasmon resonance demonstrated a calcium-dependent binding with a KD = 56 nM. Ultrastructural localization in skin showed immunogold labeling of fibulin-2 on microfibrils. Immunohistochemistry demonstrated colocalization of fibrillin-1 and fibulin-2 in skin, periochondrium, blood vessels, and in some basement membrane zones (kidney glomerulus) but not others (lung alveoli). The ciliary zonule does not appear to contain fibulin-2. These results suggest a tissue-specific function for the fibulin-2/fibrillin-1 interaction.

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INTERACTIVE EXPRESSION OF FIBULIN-2, FIBRILLIN AND FIBRONECTIN (FN) IN NORMAL AND REGENERATING SKIN M. Raghunath I, M. Tsch6drich-Rotter a, R. Peters 2, R. Timpl 3 1Institutes of Physiological Chemistry & Pathobiochemistry and 2Medical Physics & Biophysics, University of Mtinster, Germany; 3Max-Planck Institute for Biochemistry, Martinsried, Germany We studied the temporo-spatial expression of fibulin-2 in skin regenerating from autologous keratinocyte grafts in burned children. Biopsies taken between 5 d to 2 yrs post grafting (pg) were analysed by confocal laser scanning microscopy. Normal skin: complete colocalisation of fibulin-2/fibrillin on elastic fibrils of the reticular dermis and blood vessels. Despite extensive colocalisation on the transpapillary part of the microfibrillar apparatus, fibulin-2 was barely present on the fine elastin-free microfibrils radiating into the basement membrane. Regenerating skin: both fibrillin and fibulin-2 were abundant 7 d pg (far earlier than elastin expression) but in a strikingly discordant distribution. Fibrillin formed a distinct layer of numerous fusiform fibrils along the basement membrane, whereas fibulin-2 was present separately below. Both proteins formed independent fibrillar systems also in the reticular dermis without significant colocalisation. However, over time both fibrillar systems became congruent: 4 mo pg there was extensive colocalisation of fibulin-2/fibrillin in the reticular dermis, after 17 mo and 2 yrs pg there was substantial colocalisation also in the papillary microfibrillar apparatus. Fibulin-2/FN double labelling revealed an inverse pattern: complete colocalisation at 7 dp and

744

discordant distribution 17 to 2 yrs pg. In particular, the fibrillar brilliant anti-FN pattern at early time points pg changed into a faint granular distribution throughout the dermis and along the subbasement membrane region as in normal skin. Our findings imply, that fibulin-2 is a genuine component of the cutaneous microfibrillar apparatus, but has an independent morphogenesis as fibrillar system, probably mediated by fibronectin. Analyses of fibroblast cultures show separate distribution of fibrillin/FN but partial colocalization of fibulin-2 with both proteins. This suggests the interaction of fibulin-2 with both FN fibrils and fibrillin-microfibrils.

57 EXCLUSION OF THE FIBULIN-2 GENE AT 3p24.2-p25 AS THE SECOND LOCUS FOR MARFAN SYNDROME G. Collod 1, M.-L. Chu 2, M. Coulon ~, R. TimpP, T. Sasaki 3, G. Jondeau 4, J.-P. Bourdarias 4, C. Junien t, 4, C. Boileau ~,4 INSERM U383, H6pital Necker-Enfants Malades, Paris, France; 2Department of Biochemistry & Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; 3Max-Planck-Institute, Martinsried, Germany; 4 CHU Ambroise Pard, Boulogne, France Marfan syndrome (MFS) is an autosomal dominant connective-tissue disorder. It involves predominantly three systems (skeletal, ocular, and cardiovascular systems) and is characterized by highly variable expressivity. By excluding the disease locus fibrillin 1 (FBN1) in a large French family with typical cardiovascular and skeletal anomalies, we raised the tissue of genetic heterogeneity in MFS and the implication of a second locus (MFS2). Linkage analyses, perfumed in this family with dispersed anonymous DNA markers, have localized MFS2 at 3p24.2-p25. In this chromosomal area, the fibulin-2 gene (FBLN2) has been mapped. This gene was considered as an interesting positional and functional candidate since it encodes an extracellular matrix protein. Linkage analyses were performed using the candidate gene approach. Family members were genotyped for two intragenic markers. Several recombinants were identified and linkage was excluded between MFS2 and FBLN2. The exclusion data were in agreement with cellular data that showed normal synthesis, secretion and accumulation in cultured fibroblasts from affected family members. Our results show that the MFS2 locus is not FBLN2.

58P CYSTEINE MUTATIONS IN FIBRILLIN-2 CAUSE TYPICAL CONGENITAL CONTRACTURAL ARACHNODACTYLY

(FBN2)

M. Wang l, S. Belleh 3, V. M. Der Kaloustian 2, V. Proud 3, M. Godfrey 1 l University of Nebraska Medical Center, Omaha, NE 68198 USA; McGill University, Montrdal, Qudbec H3H 1P3, Canada; 3 University of Alabama, Birmingham, AL 35294, USA Congenital contractural arachnodactyly (CCA) is an autosomal dominant connective tissue disorder that has some phenotypic skeletal overlap with the Marfan syndrome (MFS). The typical clinical features of CCA include joint contractures, arachnodactyly, and crumpled ears. In contrast to MFS, CCA does not typically affect the aorta and eyes. Genetic linkage studies have linked FBN2 to CCA

and recently two cysteine mutations have been described in CCA patients. We have performed FBN2 mutation screening in patients with CCA using MDE heteroduplex analysis of 45 overlapping fragments of RT-PCR products that span the entire cDNA of FBN2. Direct sequencing using an automatic sequencer was carried out for the samples in which heteroduplex bands had been detected. By means of this strategy, two new mutations in patients with CCA have been identified. A T to G transversion at position 6211 was detected in a 13 year old boy with progressive contractures of his extremities and mild scoliosis. This mutation causes a Cys to Gly change. The second mutation was found in an 8 year old girl with crumpled ears, contractures, and arachnodyctyly. A G to T transversion was found at position 3422 of FBN2 cDNA which resulted in Cys to Phe change. The mutations have been confirmed in genomic DNA. Mutations in these and the previously described patients suggest that, like FBN1 mutations in MFS, most mutations in typical CCA will be cysteine substitutions.

59P

LINKAGE ANALYSES IN FINNISH FAMILIES WITH ANNULO-AORTIC ECTASIA L. Karttunen l, T. Savunen 2, L. Peltonen I 1 National Public Health Institute, Dept. of Hum. Mol. Genet., Helsinki, Finland; 2Turku University Central Hospital, Dept. of Surgery, Turku, Finland Annulo-aortic ectasia (AAE) is an anatomical entity consisting of an insufficiency of the aortic valve and aneurysm of the ascending aorta. AAE can occur secondary to atherosclerosis, trauma, or syphilis, but there are also familial cases, where AAE is either found as an autosomal, dominantly inherited disease or where the pattern of inheritance is not yet solved. AAE patients have an inherent structural defect in the media of the aortic wall, which is indistinguishable to that found in Marfan syndrome (MFS), but the patients with familial AAE lack the characteristic musculoskeletal and ocular symptoms of MFS. The aortic wall is composed of strenght providing collagens and elastin and elastin-associated microfibrils. Defects in genes encoding these components have recently been identified in different syndromes involving the cardiovascular system. For example, MFS is caused by mutations in the fibrillin-1 gene, dominantly inherited supravalvular aortic stenosis results from large deletions in the elastin gene, and in patients with Ehlers-Danlos syndrome type IV, mutations in collagen type III chain have been identified. Although no specific biochemical marker has thus far been demonstrated in AAE families, the primary defect in AAE individuals is likely to involve some of the components of connective tissue specifically expressed in the aortic root. In an attempt to identify the gene defect in AAE, we have used linkage analyses in seven Finnish families and followed the segregation of intragenic polymorphic and flanking markers of genes encoding components of extracellular matrix to test if any of these would be linked to the disease. Genes encoding fibfillin-1 and -2, elastin, collagen type III chain, three collagen type VI chains, lysyl oxidase, lysyl oxidase-like protein, microfibril-associated glycoprotein-1, microfibril-associated protein-1 and -3 have all so far been clearly excluded as disease causing genes.

745

60P

P R E F E R E N T I A L S Y N T H E S I S OF FIBRILLIN-2 BY C U L T U R E D H U M A N K E R A T I N O C Y T E S M. Raghunath ~, E. A. Putnam 2, A. P. Withers 3, M. Boxer 3, L. Brnckner-Tuderman 4, M. Duvic 5, B. Steinmann 6, D. M. Milewicz 2 Institute of Physiological Chemistry and Pathobiochemistry and 4 Dept. of Dermatology, University of Mtinster, Germany; 2 Depts of Internal Medicine and 5 Dermatology, University of Texas Medical School, Houston, Texas, USA; 3 Dept. of Pathology, Ninewells Hospital, Dundee, Scotland, UK; 6 Division for Metabolic and Molecular Disorders, University Children's Hospital, CH-8032 Ztirich, Switzerland 10-12 nm fibrillin containing microfibrils represent a characteristic anchoring system of the dermo-epidermal junction of the human skin. Dermal fibroblasts can lay down a matrix of fibrillin-microfibrils, whereas cultured h u m a n keratinocytes appear to do not. However, immunocytochemistry indicated intracellular presence of fibrillin in keratinocytes which was increased in the presence of TGF~32. Metabolic labelling experiments with I2 different human keratinocyte cell lines showed synthesis and secretion of fibrillin, which was markedly increased upon stimulation with TGF[32. The identity of fibrillin was determined by radioimmunoprecipitation with the novel polyclonal antibody ERP3 recognising the carboxyterminus of both fibrillin-1 and fibrillin-2. Differentiation between fibritlin-1 and 2 was accomplished with another set of antibodies and revealed that keratinocytes produced significantly more fibrillin-2 than fibrillin-1, in contrast to the predominant fibrillin-1 production by cultured dermal fibroblasts. RT-PCR using primers distinguishing between the highly homologous fibrillin-1 and fibrillin-2 demonstrated m R N A for both fibrillins in both fibroblasts and keratinocytes. However, Northern analyses using probes to unique regions of the FBN1 and FBN2 cDNAs showed FBN1 expression in fibroblasts, but not in keratinocyte RNA. We conclude, that fibrillin production is not restricted to mesenchymal cells. Furthermore, we have identified a cell culture source producing predominantly fibrillin-2 which should facilitate studies of fibrillin-2 at the protein level and the formation of fibrillin-2 containing microfibrils.

61

F I B R I L L I N IN C A R T I L A G E : W H E R E IS T H E O V E R G R O W T H ? L. Y. Sakai, D. P. Reinhardt, N. L. Charbonneau, D. R. Keene Shriners Hospital for Crippled Children, Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201, USA Fibrillins must be important components of developing cartilage, since mutations in FBN1 and FBN2 both result in abnormalities affecting long bone growth. However, we do not yet understand why mutations in the fibrillin genes cause skeletal abnormalities. And, we do not know what special functions might be performed by fibrillins in cartilage. As a first step toward investigating these questions, we have examined the distribution of fibrillins in developing and maturing h u m a n cartilage. For these studies, we have utilized monoclonal antibodies which recognize fibrillin-1 and do not crossreact with fibrillin-2. During fetal development, fibrillin1 is abundant in a broad zone around all cartilages. Around mature cartilage and bone, this boundary zone is equivalent to the perichondrium and periosteum. Fibrillin-1 is also present within the

cartilage matrix. Electron microscopy reveals single microfibrils within the matrix of fetal cartilage. In teenagers and adults, fibrillin-1 containing microfibrils aggregate laterally to form special banded fibers within the cartilage matrix. These banded fibers represent unique aggregates of microfibrils, which are not found in other tissues like skin and cornea of comparable age and are distinguishable from amianthoid fibers. Since these banded fibers can be isolated from cartilage and analyzed ultrastructurally, they appear to be covalently crosslinked. Fibrillin-1 banded fibers are found primarily surrounding the chondrocyte. Fibrillin-1 microfibrils appear not to be present in the rest of the growth plate. Therefore, the structural and functional contributions made to cartilage by fibrillin- 1 are restricted to the perichondrium and to the pericellular matrix.

62

PREGNANCY AND THE MARFAN SYNDROME A DUTCH STUDY J. Lind l, H. C. S. Wallenburg 2 Department of Obstetrics and Gynecology, Westeinde Hospital, Den Haag, The Netherlands; 2Department of Obstetrics and Gynecology, Erasmus University Medical School, Rotterdam, The Netherlands

Objective. To collect relevant data on the course and outcome of pregnancies in women with the Marfan syndrome in order to provide rational counseling to women contemplating pregnancy. Design and metods. With the help of the Dutch Foundation of Marfan Patients 116 pregnancies of Marfan patients were found and analyzed including the condition of the newborn. The diagnosis had been confirmed in each case. Results. A mutation was found in 20,6% of the pregnant women analyzed. Early abortion occurred in 14,8% of cases and the preterm delivery rate was 3,8%. Preeclampsia occurred in 7,6% of cases. Two women suffered cerebral vascular accidents. Dissections of the aorta occurred in five women (4,3%); in all of these cases the aorta diameter was 40 m m or more. An abnormal fetal presentation during labor was found in 8,4% of cases; the Cesarean section rate was 6,4%. Post partum bleeding occurred in 6,9% of the mothers. Maternal death did not occur. A floppy infant syndrome was observed in 6,4% of the new borns. The perinatal mortality rate was 0,8%. Conclusion. Pregnancy in the Marfan syndrome is generally well tolerated if no serious cardiovascular complications are present. In this study, the incidence of aortic dissection was lower than usually suggested in published case reports and reviews. Pregnancy is preferably to be avoided when the aortic diameter is 40 m m or more.

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S E R I A L E C H O C A R D I O G R A P H Y IS OF L I M I T E D VALUE IN P R E D I C T I N G A O R T I C D I S S E C T I O N IN P R E G N A N T M A R F A N PATIENTS G. Brice, T. Treasure, C. Pumphrey, G. Leech, A. Child St. George's Hospital Medical School, London SW17 ORE, UK Pregnancy in women with the Marfan syndrome poses two problems: the unquantified risk of aortic dissection and the 50% risk of having a child who will inherit the syndrome. Serial echocardiog-

746

raphy has been the mainstay of preventive management for the pregnant Marfan patient, with studies recommended pre-conception, each trimester and six months post-partum. As part of an international collaborative study to determine the efficacy of this regime, six Marfan patients who had been followed at our centre during pregnancy were ascertained and serial echo results re-examined. Of the six women 1 suffered aortic dissection 4 weeks post-parturn requiring aortic root replacement, 1 dissected and died at 38 weeks gestation with peri-mortem delivery of a live infant. A third woman suffered a type B dissection at 35 weeks gestation during her 5th pregnancy, with a live infant being delivered. Conservative treatment of the aneurysm was employed. All three patients dissected with an aortic root dimension of less than 5 cm, the point at which surgery is normally recommended, and without notable dilatation from the pre-conception dimension. Conversely, another of the 6 patients had aortic root dimensions of 5.4 cm throughout pregnancy and did not undergo aortic root replacement for another 4 years at which time the aorta measured 6.3 cm. It is therefore proposed that, whilst being a useful tool in the risk stratification of the non-pregnant Marfan patient, other prognostic indicators should be used alongside trans-thoracic echocardiography in order to better counsel Marfan patients wishing to embark on pregnancy.

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THE OPERATIVE TREATMENT OF S P I N A L D E F O R M I T Y IN PATIENTS WITH MARFAN SYNDROME W. Morgenstern, P. Metz-Stavenhagen, H_ J. V61pel Center of Spine Surgery, Wuner-Wicker-Klinik, D-34537 Bad Wildungen, Germany The incidence of spinal deformity in patients with Marfan syndrome is between 40 and 75%. The most common type of deformity is combined scoliosis with or without kyphotic component. In comparison to idiopathic scoliosis the onset of deformity is at the age of six years and is progressive through adolescence. Between 1986 and 1993 we operated 18 patients between 10 and 26 years (13 female and 5 male). The average follow-up was 23,7 months. 15 patients had posterior fusion with implants, three had anterior fusion. The average correction was 43,3% in the thoracic and 50,1% in the lumbar spine. Kyphosis was corrected with an average of 47,4%. In 70% correction of the spine balance was achieved, 6 of 8 patients had improvement of the sagittal alignment. 11 patients had significant correction of lumbar lordosis. The average correction of the tilt of the lumbar end vertebra was 50%. There were no neurological complications and no pseudarthroses. One female patient required prolonged intensive care because of recurring pneumothorax. One patient showed a Crankshaft phenomenon due to persisting anterior growth after posterior fusion. In comparison with other authors the complication rate was low. Consideration of the sagittal alignment in planning the correction is of major importance. The time for surgery should not be too early to avoid bending of the fusion mass during growth and not too late, since older patients have a higher complication rate and more pain.

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CRANIOFACIAL PROBLEMS IN T H E M A R F A N S Y N D R O M E L. Westling, A. Bresin, B. Mohlin, S. Kopp Dep. of Orthodontics, Faculty of Odontology, University of G6teborg, G6teborg, Sweden; Dep. of Oral Physiology, Faculty of Odontology, Karolinska Institutet, Huddinge, Sweden Very little is published about the prevalence and severity of the craniofacial signs and symptoms in the Marfan syndrome (MFS). Noteworthy for the dentist are a high palate, dental crowding and temporomandibular joint (TMJ) hypermobility and dysfunction. Material. 29 males and 27 females with the diagnosis of MFS (0.5-53 yrs, median 24 yrs). Reference group. 1009 healthy subjects referred for orthodontic treatment. Methods. Dental casts for measurements of the palatal vaults were available in 34 MFS subjects and all orthodontic patients. Profile X-rays were analysed in 34 MFS patients and 79 orthodontic patients selected because of high palatal index 1 (P.I.; palatal height/palatal width). Five females (18-52 yrs, mean 38 yrs) with MFS and severe orofacial pain had MRI or X-ray tomograms of the TMJs. Results. Malocclusion was found in 87% of the MFS subjects, 65% _> 11 years had been referred for orthodontic treatment. 43% of the MFS subjects and 5% of the orthodontic reference group had high palatal vaults (> 2 S.D.2). High palatal index (P.I. _> 2 S.D. 2) was found in 64% of the MFS group and in 9% of the orthodontic patients. Crowded teeth were recorded in 78% of the MFS subjects, insignificantly more common in the mandible than in the maxilla. Cephalometric analysis of the MFS patients showed enlarged frontal sinus and large intermaxillary and obtuse gonial angles (steep mandible) indicating a marked vertical facial growth. In comparison with the reference group, selected on the presence of high palatal vaults, the anterior and posterior facial height as welt as the distance between the cephalometric points nasion and sella (anterior cranial base) of the MFS subjects were significantly greater. 54% of the MFS subjects _> 16 years reported orofacial pain and jaw dysfunction symptoms. Pain in the face and the jaws, difficulties in opening the mouth, TMJ sounds, and unilateral headache were prevalent. 31% had craniomandibular dysfunction problems severe enough to require treatment compared to a population mean < 5%. All patients subjected to TMJ imaging had bony structural changes. Conclusion. The study shows that MFS patients have a great need of orthodontic and/or craniomandibular specialist treatment and suggests that TMJ dysfunction ought to be considered as a musculoskeletal manifestation of the MFS. References. JWestling et al. (1993) A m J Med Genet 47 : 159; 2Redman et al. (1966) J Dent Res 45 : 266

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P S Y C H O S O C I A L I M P A C T OF T H E M A R F A N S Y N D R O M E IN ADOLESCENTS AND YOUNG ADULTS A. Van Tongm-loo, A. De Paepe Department of Medical Genetics, University Hospital Gent, Belgium We started a pilot study aiming to evaluate the psychological effects and consequences of the Marfan syndrome in patients be-

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tween 16 and 30 years of age. Through a semi-structured interview, we evaluated the impact of the disease on the daily life of the patients, and on specific items such as education, occupation, relationships and family planning. A second part of the investigation consisted in a battery of standardized psychological tests, aiming to evaluate the patients' perception on the quality of life, their anxiety and depression level, and their personality. The following psychological tests were used: Sickness Impact Profile (SIP), State and Trate Anxiety Inventory (STAI), Beck Depression Inventory (BDI), Multiphasic Minnesota Personality Inventory (MMPI). The results of these tests were compared to those of a control group of healthy individuals, friends of the patients, who live in the same environment and belong to the same social class. Although the study is still ongoing, preliminary results reveal that the disorder represents a serious burden on the physical activities of the patients. All report that their way of life is significantly influenced by the disease and dominated by concerns of their health. Looking "different" than their peers has negative implications for their self-image. As a consequence, they are more introvert. All remember a time in life when they were teased by peers. In the female patients, the problem of child bearing represents a major concern. All patients emphasized the need for professional guidance, by means of accurate information in the disease and psychological support. Nevertheless, most patients come to terms with their disease.

67P

MARFANOID FEATURES AND CRANIOSYNOSTOSIS,

THE S H P R I N T Z E N - G O L D B E R G S Y N D R O M E : A UNIQUE ENTITY? C. StoW, S. Gilgenkrantz 2 1Service de G6n6tique M6dicale, CHU, F-67000 Strasbourg, France; 2Laboratoire de Cytogdndtique, CRTSH, F-54500 Vandoeuvre, France The association of craniosynostosis and marfanoid features was first described by Shprintzen and Goldberg (1982) in two sporadic cases. Since then several other patients with and without mental retardation have been reported. Recently, a similar syndrome was published in a family with autosomal dominant inheritance. Here we report on two new cases and discuss the origin of this rare syndrome. One case, a boy was followed up during 17 years. The other, also a boy, was seen at 5 years of age. Both had the following features: craniofacial anomlies (craniosynostosis, downslanting palpebral fissures, ptosis, ocular proptosis, strabismus, micrognathia, highly arched palate, cleft palate, low-set ears), skeletal anomalies (arachnodactyly, camptodactyly, talipes equinovarus, hypermobile joints, long feet, scoliosis, pectus deformity - excavatum or carinatum - , and functional disorders (developmental delay, mental retardation, obstructive apnea). Since there is another syndrome quoted in McKusick as Shprintzen-Goldberg syndrome (MIM 182210), the suggestion of Lacombe and Battin to name the described syndrome "ShprintzenGoldberg marfanoid syndrome" should be taken into account. Nine cases were reported until now establishing the features of this syndrome, characterized by craniosynostosis most often of the sagittal suture, mental retardation or developmental delay, anomalies of the hand and feet with arachnodactyly and camptodactyly. We do not think, that patients with mental retardation or without mental retardation represent different entities. They may be the ends of a continuous spectrum. Most cases of the Shprintzen Goldberg syn-

drome were sporadic. In a recent paper however the same phenotypic features were observed in a mother and her two children.

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O R T H O P E D I C A S P E C T S IN M A R F A N SYNDROME - RADIOLOGIC FEATURES L. Lavisse ~, S. Chagnon ~, A. Foudali 1, J. M. Le Parc 2, G. Jondeau 2, B. Chevallier 4, D. Montagnon 5 Departments of I Radiology, 2 Rheumatology, 3 Cardiology, 4pediatric, 5 Genetic, Ambroise Par6 Hospital, Boulogne-Billancourt, France

Purpose. To analyse skeletal features in Marfan syndrome in order to review the skeletal diagnostic criteria, to evaluate their incidence and to outline the management of the most common orthopedic problems. Materials and methods. 60 patients with Marfan syndrome (according to internationally accepted criteria) were evaluated by a multidisciplinary medical team. Roentgenograms were obtained for all of them (standing anteroposterior and lateral roentgenograms of the spine, pelvis and sometimes hands and feet). Results. Scoliosis was the most frequently skeletal manifestation (70%). There are parallels with adolescent idiopathic scoliosis, but with longer multiple and more progressive curves. 5 sagittal profiles of spine were described (based on thoracic kyphosis' aspect and on how transition between kyphosis and lordosis occurred). Kyphosis associated to scoliosis were more often symptomatic with arthrosis lesions observed. Spondylolisthesis was present (18%) with a mean slip. Posterior vertebral scalloping was a main sign for dural ectasia which had no morbidity. Acetabular protrusion occurred in 40% (grade 2 or 3), usually with few symptoms except when capital femoral deformities were present. Foot deformities were variable and common, and seldom needed orthopedic treatment.

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CHEMICAL COMPOSITION OF THE EYE THE Z O N U L A R A P P A R A T U S AS A MODEL

SYSTEM

TO STUDY

THE S T R U C T U R E OF M I C R O F I B R I L S R. Mayne Dept. of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA During the development of the eye, the lens arises from an invagination of the ectoderm to form the lens placode. At the same time, the retina is formed from forebrain nervous tissue which develops into the optic cup and eventually almost completely surrounds the developing lens vesicle. As the lens differentiates, it becomes completely surrounded by a thick basement. Attached to this basement membrane are a series of fine filaments (called the zonular apparatus) which are also attached to basement membrane-like material located at the ciliary body close to the retina. Accommodation or focusing of the eye, is achieved by the ciliary muscles altering the force on the zonular apparatus and thereby changing the shape of the lens. It is the failure of these filaments which results in lens dislocation as commonly observed in Marfan syndrome. Initial analyses of the zonular apparatus by transmission electron microscopy revealed a series of microfibrits (called zonular

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fibrils) of about 10 nm in diameter but with little further discernable structure. However, rotary shadowing observations revealed that the fibrils consist of a series of beads interconnected by 6-8 filaments. Further support for this structure was obtained from experiments in which the zonular fibers were stretched by exerting force on the lens and examination of the fibrils by rotary shadowing. The structure of the zonular fibrils appears identical to the elastin microfibrils observed in a variety of connective tissues. Zonular fibrils were dissected from bovine eyes and used to prepare monoclonal antibodies. All antibodies obtained were against fibrillin and reacted with microfibrils present in a variety of connective tissues including gingiva, dermis, blood vessels, and tendon. In addition, analyses of the zonular fibrils after preparation of CNBr peptides and subsequent separation by SDS P A G E gave Nterminal amino acid sequences that were located in fibrillin 1. Zonular fibrils are closely related to the 10 nm microfibrils found in a wide variety of other connective tissues. In the eye, the major structural component of these microfibrils is apparently fibrillin 1. Grants. NIH EY 09908

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MANAGEMENT OF THE EYE IN THE MARFAN SYNDROME INCLUDING THE USE OF INTRAOCULAR LENSES

lens dislocation in 13 patients with Marfan syndrome. Eight eyes had moderate or major grades of myopia. Retinal tears or a retinal detachment (RD) had been treated before lens extraction in 4 eyes. No visual improvement (visual acuity < 0.2) was achieved in 3 out of 19 eyes. In these eyes, visual loss was caused by pre- or postoperative retinal detachment and/or secondary glaucoma. Group l: Three eyes had intracapsutar cataract extraction ([CCE) without pars plana vitrectomy (PPV): 2 of these eyes went blind due to retinal detachment (RD) and glaucoma, one eye achieved a visual acuity (VA) of 0.8. Group 2." ICCE was combined with PPV in 7 eyes. Five eyes remained aphakic; final V A was 0.8 to 1.0 in 3 eyes, and 0.4 and 0.7 in 2 eyes. In 2 eyes, an anterior chamber lens (ACL) was implanted: V A was reduced to 0.2 and 0.8 due to secondary keratopathy; the ACL had to be removed from one eye. Group 3: The dislocated lens was removed via pars plana in 5 eyes. Post-operative complications were not observed. VA did not improve in one eye with pre-operative, intractable RD. In 3 eyes, final V A was 0.8 to 1.0; in 1 eye, V A was reduced to 0.3 due to high myopia. Group 4: In 4 eyes, ICCE and PPV was combined with sulcus fixation of a posterior chamber lens (PCL). Post-operative V A ranged from 0.3 to 1.0. Apart from an early post-operative ocular hypotony, no serious complications were observed in this group. In conclusion, dislocated lenses in Marfan syndrome may be safely extracted in combination with a vitrectomy via pars plana. In non-myopic eyes, sulcus fixation of an artificial PCL offers a proven alternative for a satisfactory visual rehabilitation.

I. H. Maumenee Johns Hopkins Hospital, Baltimore, MD 21287, USA All parts of the eye may be affected in the Marfan syndrome: orbit, extraocular muscles, cornea, iris, anterior chamber angle, sclera, lenses, retina and to a lesser degree the vitreous. The resulting abnormalities are: enophthalmos, antimongoloid slanting, strabismus, megalocomea and astigmatism, glaucoma, microcornea, simplified iris structure, retinal detachments. We have learned to remove dislocated or cataractous lenses and to insert posterior chamber lenses, fixating them in the ciliary sulcus. This surgical approach is only appropriate for those patients whose axial length of the bulbus is not excessive. Intercalary staphylomas are seen in patients who had iutraocular surgery in the face of a significant increase in axial length as a sign of scleral thinning. Eyes are still lost due to retinal detachments. At present we are directing a major effort towards understanding of the pathogenesis of the retinal detachment seen in patients with the Marfan syndrome.

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THE CHALLENGE OF IMPLANT FIXATION IN THE SURGERY OF A SUBLUXATED CRYSTALLINE LENS A. Galand C.H.U., B-4000 Liege, Belgium

There are several possibilities of implant fixation in the eye offering a total absence of posterior capsule: anterior chamber lens, iris supported lens ("Lobster Claw"), iris or ciliary sutured lens. Each of those systems has important drawbacks. W e try to optimize an implant which could be fixated in the ciliary body without suture. W h e n a part of the capsular bag remains, an implant with haptic in the ciliary sulcus can be a solution.

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ADVANCES IN THE MANAGEMENT OF DISLOCATED LENSES IN MARFAN SYNDROME H. U. Bachmann M. Boehnke, F. Koemer Department of Ophthalmology, University of Berne, Inselspital, Freiburgstrasse, CH-3010 Berne, Switzerland During the past 3 decades, surgical techniques for subluxated or dislocated lenses have been improved. Major advance was achieved with the invention of vitreoretinal surgery and of various techniques for the implantation of artificial lenses. In Marfan syndrome, the management of dislocation of the lens is complicated by an increased risk of retinal detachment. In a retrospective survey, we analyzed various surgical approaches and the post-operative outcome of 19 eyes operated for

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INTRAOCULAR LENS (IOL) IMPLANTATION IN DISLOCATION OF THE LENS V. De Molfetta, R. Maggi Eye Department, S. Gerardo Hospital, Monza, Italy Lensectomy is commonly performed in Marfan cases. Although satisfactory results have been reported in several statistics, secondary implantation of an intraocular lens (IOL) in the posterior chamber (PC) is seldom considered in such cases. The authors report on the use of a PC IOL for scleral fixation in 3 points of the pars plana, suitable for implantation in total absence of a posterior capsule and zonule. As shown in the video, after pars plana lensec-

749

tomy and vitrectomy a limbal section is performed and the lens implanted as a primary procedure. Vitrectomy is considered quite safe in eye surgery. Nevertheless, in eyes with an increased risk of retinal detachment, as is the case in Marfan children, it could be desirable to resort to more conventional lens surgery. A video shows open-sky surgery performed in a 3-year-old Marfan child, and in a 9-year-old boy with traumatic dislocation of the lens. However, lens removal is performed, implantation of the present lens with an almost closed-eye technique would be possible if desired.

74P SURGICAL TREATMENT OF ECTOPIA LENTIS IN PATIENTS WITH MARFAN SYNDROME IN DENMARK J. Fuchs, T. Rosenberg The National Eye Clinic for the Visually Impaired, Hellerup, Denmark Surgical treatment of dislocated lenses has been associated with a high frequency of complications and a poor visual outcome. In or-

der to evaluate the outcome of lensectomy in Danish Marfan patients a retrospective study was conducted collecting information on a national scale concerning patients with ectopia lentis and/or Marfan Syndrome. Information about 79 Marfan patients undergoing surgical treatment of dislocated lenses in 1932-93 was obtained. One hundred and sixteen eyes were operated. The most frequent indication for operation was optical distorsion leading to an unacceptable visual accuity (52%). Information about pre- and postoperative visual accuity (VA) was available in 99 of 116 eyes. In 86% of these VA was improved by operation. In 3.5% VA was unaltered, and in 10.5% VA deteriorated due to early or late postoperative complications. The most important complication was retinal detachment (RD) which occurred in 20 eyes (17.2%). All of these were operated before 1983. In 46 eyes operated after 1983 no RDs had occurred after a mean follow-up time of 2 years. W e conclude that surgical treatment of dislocated lenses in Marfan patients have become safer during the last decade with few serious complications and a good visual outcome.