Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and have been associated with a wide range of overlapping phenotypes. Clinical care is complicated by variable age at onset and the wide range of severity of aortic features. The factors that modulate phenotypical severity, both among and within families, remain to be determined. The availability of international FBN1 mutation Universal Mutation Database (UMD-FBN1) has allowed us to perform the largest collaborative study ever reported, to investigate the correlation between the FBN1 genotype and the nature and severity of the clinical phenotype. A range of qualitative and quantitative clinical parameters (skeletal, cardiovascular, ophthalmologic, skin, pulmonary, and dural) was compared for different classes of mutation (types and locations) in 1,013 probands with a pathogenic FBN1 mutation. A higher probability of ectopia lentis was found for patients with a missense mutation substituting or producing a cysteine, when compared with other missense mutations. Patients with an FBN1 premature termination codon had a more severe skeletal and skin phenotype than did patients with an inframe mutation. Mutations in exons 24–32 were associated with a more severe and complete phenotype, including younger age at diagnosis of type I fibrillinopathy and higher probability of developing ectopia lentis, ascending aortic dilatation, aortic surgery, mitral valve abnormalities, scoliosis, and shorter survival; the majority of these results were replicated even when cases of neonatal MFS were excluded. These correlations, found between different mutation types and clinical manifestations, might be explained by different underlying genetic mechanisms (dominant negative versus haploinsufficiency) and by consideration of the two main physiological functions of fibrillin-1 (structural versus mediator of TGFβ signalling). Exon 24–32 mutations define a high-risk group for cardiac manifestations associated with severe prognosis at all ages. Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and have been associated with a wide range of overlapping phenotypes. Clinical care is complicated by variable age at onset and the wide range of severity of aortic features. The factors that modulate phenotypical severity, both among and within families, remain to be determined. The availability of international FBN1 mutation Universal Mutation Database (UMD-FBN1) has allowed us to perform the largest collaborative study ever reported, to investigate the correlation between the FBN1 genotype and the nature and severity of the clinical phenotype. A range of qualitative and quantitative clinical parameters (skeletal, cardiovascular, ophthalmologic, skin, pulmonary, and dural) was compared for different classes of mutation (types and locations) in 1,013 probands with a pathogenic FBN1 mutation. A higher probability of ectopia lentis was found for patients with a missense mutation substituting or producing a cysteine, when compared with other missense mutations. Patients with an FBN1 premature termination codon had a more severe skeletal and skin phenotype than did patients with an inframe mutation. Mutations in exons 24–32 were associated with a more severe and complete phenotype, including younger age at diagnosis of type I fibrillinopathy and higher probability of developing ectopia lentis, ascending aortic dilatation, aortic surgery, mitral valve abnormalities, scoliosis, and shorter survival; the majority of these results were replicated even when cases of neonatal MFS were excluded. These correlations, found between different mutation types and clinical manifestations, might be explained by different underlying genetic mechanisms (dominant negative versus haploinsufficiency) and by consideration of the two main physiological functions of fibrillin-1 (structural versus mediator of TGFβ signalling). Exon 24–32 mutations define a high-risk group for cardiac manifestations associated with severe prognosis at all ages. Marfan syndrome (MFS [MIM 154700]) is a connective-tissue disorder, with autosomal dominant inheritance and a prevalence of 1 in 5,000–10,000 individuals.1Pyeritz RE Marfan syndrome: current and future clinical and genetic management of cardiovascular manifestations.Semin Thorac Cardiovasc Surg. 1993; 5: 11-16PubMed Google Scholar The cardinal features of MFS involve the ocular, cardiovascular, and skeletal systems.2Judge DP Dietz HC Marfan’s syndrome.Lancet. 2005; 366: 1965-1976Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar The skin, lung, and dura may also be involved. MFS is notable for its variability in age at onset, tissue distribution, and severity of clinical manifestations, both among and within affected families. Because of the high population frequency and the nonspecific nature of many of the clinical findings for MFS, clinical diagnostic criteria for this disorder have been established,3Beighton P de Paepe A Danks D Finidori G Gedde-Dahl T Goodman R Hall JG Hollister DW Horton W McKusick VA et al.International nosology of heritable disorders of connective tissue, Berlin, 1986.Am J Med Genet. 1988; 29: 581-594Crossref PubMed Scopus (561) Google Scholar–4De Paepe A Devereux RB Dietz HC Hennekam RC Pyeritz RE Revised diagnostic criteria for the Marfan syndrome.Am J Med Genet. 1996; 62: 417-426Crossref PubMed Scopus (1287) Google Scholar the latest being the Ghent criteria, which superseded the previous so-called Berlin criteria. Study of the molecular determinants of phenotypical variations in MFS has been possible only since the identification of the causative fibrillin-1 (FBN1) gene5Dietz HC Cutting GR Pyeritz RE Maslen CL Sakai LY Corson GM Puffenberger EG Hamosh A Nanthakumar EJ Curristin SM et al.Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene.Nature. 1991; 352: 337-339Crossref PubMed Scopus (1480) Google Scholar (MIM 134797). Fibrillin-1 has a modular structure, with 47 repeats of six-cysteine epidermal-growth-factor (EGF)–like motifs, 43 of which are of the calcium-binding (cb) type (cb-EGF).6Handford PA Fibrillin-1, a calcium binding protein of extracellular matrix.Biochim Biophys Acta. 2000; 1498: 84-90Crossref PubMed Scopus (66) Google Scholar Fibrillin-1 monomers associate to form complex extracellular macroaggregates, termed “microfibrils,” which are important for the integrity and homeostasis of both elastic and nonelastic tissues.7Sakai LY Keene DR Engvall E Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils.J Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (866) Google Scholar–8Pereira L Andrikopoulos K Tian J Lee SY Keene DR Ono R Reinhardt DP Sakai LY Biery NJ Bunton T et al.Targeting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome.Nat Genet. 1997; 17: 218-222Crossref PubMed Scopus (296) Google Scholar The protein also contains seven eight-cysteine motifs, which bear homology to motifs found in the latent transforming-growth-factor beta–binding proteins (TGFβ-BPs), and a proline-rich region. The relationship between FBN1 and TGFβ signaling has been underscored by the identification of mutations in TGFBR2 (MIM 190182) in patients with MFS and Marfan-like conditions9Mizuguchi T Collod-Beroud G Akiyama T Abifadel M Harada N Morisaki T Allard D Varret M Claustres M Morisaki H et al.Heterozygous TGFBR2 mutations in Marfan syndrome.Nat Genet. 2004; 36: 855-860Crossref PubMed Scopus (497) Google Scholar and in pathological studies in knockin and knockout mice.10Neptune ER Frischmeyer PA Arking DE Myers L Bunton TE Gayraud B Ramirez F Sakai LY Dietz HC Dysregulation of TGF-β activation contributes to pathogenesis in Marfan syndrome.Nat Genet. 2003; 33: 407-411Crossref PubMed Scopus (1067) Google Scholar Indeed, fibrillins and TGFβ-BPs constitute a family of structurally related and interacting proteins. In MFS mouse models, deficiency of fibrillin-1 alters matrix sequestration of the large latent complex of TGFβ, rendering the cytokine more prone for activation.10Neptune ER Frischmeyer PA Arking DE Myers L Bunton TE Gayraud B Ramirez F Sakai LY Dietz HC Dysregulation of TGF-β activation contributes to pathogenesis in Marfan syndrome.Nat Genet. 2003; 33: 407-411Crossref PubMed Scopus (1067) Google Scholar Recently, a specific fibrillin-1 sequence encoded by exons 44–49 has been shown to regulate the bioavailability of endogenous TGFβ1.11Chaudhry SS Cain SA Morgan A Dallas SL Shuttleworth CA Kielty CM Fibrillin-1 regulates the bioavailability of TGFβ1.J Cell Biol. 2007; 176: 355-367Crossref PubMed Scopus (184) Google Scholar Two major mutation categories—premature termination codons (PTCs) and inframe mutations—have been reported in the FBN1 gene.12Collod-Beroud G Le Bourdelles S Ades L Ala-Kokko L Booms P Boxer M Child A Comeglio P De Paepe A Hyland JC et al.Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database.Hum Mutat. 2003; 22: 199-208Crossref PubMed Scopus (232) Google Scholar A total of 559 pathogenic mutations were reported in the last update of the Universal Marfan Database (UMD)–FBN1,12Collod-Beroud G Le Bourdelles S Ades L Ala-Kokko L Booms P Boxer M Child A Comeglio P De Paepe A Hyland JC et al.Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database.Hum Mutat. 2003; 22: 199-208Crossref PubMed Scopus (232) Google Scholar and most of these are unique to individual families. Two-thirds are missense mutations, the majority of which are cysteine substitutions. Besides classic MFS, mutations in FBN1 have been associated with a broad spectrum of phenotypes, including neonatal MFS,13Booms P Cisler J Mathews KR Godfrey M Tiecke F Kaufmann UC Vetter U Hagemeier C Robinson PN Novel exon skipping mutation in the fibrillin-1 gene: two “hot spots” for the neonatal Marfan syndrome.Clin Genet. 1999; 55: 110-117Crossref PubMed Scopus (79) Google Scholar isolated ectopia lentis14Lonnqvist L Child A Kainulainen K Davidson R Puhakka L Peltonen L A novel mutation of the fibrillin gene causing ectopia lentis.Genomics. 1994; 19: 573-576Crossref PubMed Scopus (76) Google Scholar (MIM 129600), isolated ascending aortic aneurysm and dissection,15Milewicz DM Michael K Fisher N Coselli JS Markello T Biddinger A Fibrillin-1 (FBN1) mutations in patients with thoracic aortic aneurysms.Circulation. 1996; 94: 2708-2711Crossref PubMed Scopus (166) Google Scholar isolated skeletal features,16Hayward C Porteous ME Brock DJ A novel mutation in the fibrillin gene (FBN1) in familial arachnodactyly.Mol Cell Probes. 1994; 8: 325-327Crossref PubMed Scopus (37) Google Scholar, 17Milewicz DM Grossfield J Cao SN Kielty C Covitz W Jewett T A mutation in FBN1 disrupts profibrillin processing and results in isolated skeletal features of the Marfan syndrome.J Clin Invest. 1995; 95: 2373-2378Crossref PubMed Scopus (146) Google Scholar and Weill-Marchesani syndrome18Faivre L Gorlin RJ Wirtz MK Godfrey M Dagoneau N Samples JR Le Merrer M Collod-Beroud G Boileau C Munnich A et al.In frame fibrillin-1 gene deletion in autosomal dominant Weill-Marchesani syndrome.J Med Genet. 2003; 40: 34-36Crossref PubMed Scopus (197) Google Scholar (MIM 608328). So far, genotype-phenotype correlations in FBN1 have been weak except for the cluster of mutations in exons 24–32 reported in neonatal MFS.13Booms P Cisler J Mathews KR Godfrey M Tiecke F Kaufmann UC Vetter U Hagemeier C Robinson PN Novel exon skipping mutation in the fibrillin-1 gene: two “hot spots” for the neonatal Marfan syndrome.Clin Genet. 1999; 55: 110-117Crossref PubMed Scopus (79) Google Scholar, 19Liu W Qian C Comeau K Brenn T Furthmayr H Francke U Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe Marfan syndrome.Hum Molec Genet. 1996; 5: 1581-1587Crossref PubMed Scopus (76) Google Scholar, 20Putnam EA Cho M Zinn AB Towbin JA Byers PH Milewicz DM Delineation of the Marfan phenotype associated with mutations in exons 23-32 of the FBN1 gene.Am J Med Genet. 1996; 62: 233-242Crossref PubMed Scopus (110) Google Scholar, 21Dietz HC Pyeritz RE Mutations in the human gene for fibrillin-1 (FBN1) in the Marfan syndrome and related disorders.Hum Mol Genet. 1995; 4: 1799-1809Crossref PubMed Scopus (413) Google Scholar, 22Robinson PN Booms P Katzke S Ladewig M Neumann L Palz M Pregla R Tiecke F Rosenberg T Mutations of FBN1 and genotype-phenotype correlations in Marfan syndrome and related fibrillinopathies.Hum Mutat. 2002; 20: 153-161Crossref PubMed Scopus (153) Google Scholar Indeed, previous reported studies investigating genotype-phenotype correlations were performed with a maximum of 101 patients.23Schrijver I Liu W Odom R Brenn T Oefner P Furthmayr H Francke U Premature termination mutations in FBN1: distinct effects on differential allelic expression and on protein and clinical phenotypes.Am J Hum Genet. 2002; 71: 223-237Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 24Loeys B De Backer J Van Acker P Wettinck K Pals G Nuytinck L Coucke P De Paepe A Comprehensive molecular screening of the FBN1 gene favors locus homogeneity of classical Marfan syndrome.Hum Mutat. 2004; 24: 140-146Crossref PubMed Scopus (171) Google Scholar, 25Biggin A Holman K Brett M Bennetts B Adès L Detection of thirty novel FBN1 mutations in patients with Marfan syndrome or a related fibrillinopathy.Hum Mutat. 2004; 23: 99Crossref PubMed Scopus (59) Google Scholar, 26Arbustini E Grasso M Ansaldi S Malattia C Pilotto A Porcu E Disabella E Marziliano N Pisani A Lanzarini L et al.Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies.Hum Mutat. 2005; 26: 494Crossref PubMed Scopus (74) Google Scholar, 27Rommel K Karck M Haverich A von Kodolitsch Y Rybczynski M Muller G Singh KK Schmidtke J Arslan-Kirchner M Identification of 29 novel and nine recurrent fibrillin-1 (FBN1) mutations and genotype-phenotype correlations in 76 patients with Marfan syndrome.Hum Mutat. 2005; 26: 529-539Crossref PubMed Scopus (80) Google Scholar Those authors compared patients with mutations leading to a PTC versus patients with missense mutations, as well as subjects with missense mutations involving a cysteine versus individuals with other missense mutations. Moreover, they focused on major cardiac, ocular, and skeletal involvement. Differences between the groups, with regard to patients’ age at follow-up, were not taken into account. Large sets of both clinical and molecular data are needed to study (1) the association between FBN1 mutation type and severity of the disease and (2) the specificity of organ involvement in relation to a mutation type. The international UMD-FBN1, set up in 1995, provides these data for 1,013 probands with known FBN1 mutations who were recruited from specialized MFS clinics all over the world. We report the results of the statistical analysis of these data. These results provide possible clues into pathophysiological processes. A total of 1,191 probands who had received a diagnosis of MFS or another type I fibrillinopathy were identified between 1995 and 2005 via the framework of the UMD-FBN112Collod-Beroud G Le Bourdelles S Ades L Ala-Kokko L Booms P Boxer M Child A Comeglio P De Paepe A Hyland JC et al.Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database.Hum Mutat. 2003; 22: 199-208Crossref PubMed Scopus (232) Google Scholar and participating centers. The inclusion criteria in our study were (1) the presence of a heterozygous pathogenic FBN1 gene mutation and (2) the availability of clinical information. Overall, 178 patients (15%) had to be excluded from the study (no clinical data available for 129; insufficient data about cardiac, ocular, or skeletal involvement for 44; two different mutations on the same allele for 4; and compound heterozygosity for FBN1 mutations for 1). The 1,013 patients included in our study originated from 38 countries on five continents. The majority (72%) were white Europeans or were of European ancestry, 14% were from North and South America, 8% were from Oceania, 4% were from Asia, and 2% were from Africa. Table 1 summarizes the participation of the different laboratories in the study. Patient age at inclusion ranged from birth to 72 years. The clinical data were collected mainly from standardized questionnaires sent to the referring physician, and a minority were from previous publications that reported sufficient clinical data. The clinical information included a range of qualitative and quantitative clinical parameters, including age at diagnosis of MFS or another type I fibrillinopathy and the presence or absence of clinical features including cardiac, ophthalmologic, skeletal, dermal, pulmonary, and dural manifestations. The age at diagnosis and at surgery for aortic dilatation, ectopia lentis, and scoliosis was also noted. All questionnaires were collected by one individual (L.F.) to rule out duplication of patients in the study. To avoid bias as a result of familial clustering, affected family members of a proband were not included in the analysis.Table 1Number of Patients Recruited to the Study and Their Laboratory of OriginOriginNo. of Patients (Laboratories)Belgium167 (1)United Kingdom166 (7)Germany156 (4)France154 (3)United States146 (8)Italy89 (2)Australia80 (1)Asia22 (6)Other European countries22 (3)Other North and South American countries11 (2) Total1,013 (37) Open table in a new tab The pathogenic nature of a putative mutation was assessed using recognized criteria. In brief, all nonsense mutations, all deletions or insertions (in or out of frame) were considered pathogenic; for all splice mutations, the wild-type and mutant strength values of the splice sites were compared using genetic algorithms,28Shapiro MB Senapathy P RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression.Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (1915) Google Scholar, 29Senapathy P Shapiro MB Harris NL Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project.Methods Enzymol. 1990; 183: 252-278Crossref PubMed Scopus (614) Google Scholar, 30Beroud C Hamroun D Collod-Beroud G Boileau C Soussi T Claustres M UMD (Universal Mutation Database): 2005 update.Hum Mutat. 2005; 26: 184-191Crossref PubMed Scopus (86) Google Scholar and only mutations displaying significant deviation from the norm were included. Missense mutations were considered pathogenic when at least one of the following features was found: (1) de novo missense mutation, (2) missense mutation substituting or creating a cysteine, (3) missense mutation involving a consensus cb residue,21Dietz HC Pyeritz RE Mutations in the human gene for fibrillin-1 (FBN1) in the Marfan syndrome and related disorders.Hum Mol Genet. 1995; 4: 1799-1809Crossref PubMed Scopus (413) Google Scholar (4) substitution of glycines implicated in correct domain-domain packing,31Downing A Knott V Werner J Cardy C Campbell ID Handford PA Solution structure of a pair of calcium-binding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders.Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar and (5) intrafamilial segregation of a missense mutation involving a conserved amino acid. For 38 mutations not displaying one of the above features, additional data provided by SIFT,32Ng PC Henikoff S Predicting deleterious amino acid substitutions.Genome Res. 2001; 11: 863-874Crossref PubMed Scopus (1763) Google Scholar, 33Ng PC Henikoff S SIFT: Predicting amino acid changes that affect protein function.Nucleic Acids Res. 2003; 31: 3812-3814Crossref PubMed Scopus (3483) Google Scholar BLOSUM-62,34Henikoff S Henikoff JG Amino acid substitution matrices from protein blocks.Proc Natl Acad Sci USA. 1992; 89: 10915-10919Crossref PubMed Scopus (4056) Google Scholar and biochemical values (Kristine Yu's Web site) were gathered and analyzed using a new UMD tool (M. Frédéric, C. Boileau, D. Hamroun, M. Lalande, M. Claustres, C. Béroud, G. Collod-Béroud, unpublished data). The proportion of each specific clinical feature or system involved was compared in the different groups of mutation types or locations. The following were each considered as a system: the skeleton, the eye, the heart, the skin, and the dura. The clinical features of each system are listed in table 2. No attempts were made to incorporate dilatation of the pulmonary artery, calcification of the mitral valve annulus, apical blebs, flat cornea and iris, or ciliary muscle hypoplasia in the analyses, since those phenotypes were rarely evaluated. Similarly, the axial globe length had rarely been measured, and the definition of myopia varied widely from center to center. For this reason, myopia of any degree was included. In consequence, the presence or absence of minor ophthalmological involvement could not be assessed. The ages at diagnosis and at surgery were collected for scoliosis, ectopia lentis, and aortic dilatation or dissection. The probability of surgery for ectopia lentis was studied only in the group of patients with ectopia lentis. Similarly, the probability of aortic dissection and surgery for aortic dilatation or dissection was studied only in the group of patients with aortic dilatation. The number of systems involved was also assessed according to the Ghent nosology.4De Paepe A Devereux RB Dietz HC Hennekam RC Pyeritz RE Revised diagnostic criteria for the Marfan syndrome.Am J Med Genet. 1996; 62: 417-426Crossref PubMed Scopus (1287) Google Scholar Patients were classified as having MFS if the diagnostic Ghent nosology criteria were met, including the presence of an FBN1 mutation as a major feature and, in a second step, excluding the presence of an FBN1 mutation as a major feature. All other patients were considered as displaying a type I fibrillinopathy. For the purpose of this study and in the absence of consensus diagnostic features, patients were classified as having neonatal MFS when severe features of MFS, including severe valvular insufficiencies, were present before age 4 wk.Table 2Frequency of Clinical Features in the Different Systems Involved in MFS and Type I Fibrillinopathies (N=1,013)System and Clinical Feature(s)No. of EventsNo. of Available DataPercentageSkeletal: Arachnodactyly751969aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.78 Dolichostenomelia522947aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.55 Joint laxity600956aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.63 Scoliosis508965aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.53 Pectus deformitybIncludes pectus excavatum, 246 (26%) of 962; pectus carinatum, 288 (30%) of 962; and undefined pectus malformation.570962aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.59 Limited elbow extension153974aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.16 Protrusio acetabulae69298aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.23 Facial dysmorphism443913aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.49 High-arched palate639932aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.69 Dental malocclusion372843aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.44 Pes planus402864aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.47 Orthopedic surgery113983aNineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.12 Major skeletal involvement3271,01332 Minor skeletal involvement5641,01356Ocular: Ectopia lentis5421,01354 Myopia45386552 Cataract399834 Retinal detachment659807 Glaucoma199052 Surgery for ectopia lentis12291013 Other eye surgeries439055 Major eye involvement5421,01354Cardiac: Dilatation of the ascending aorta7751,01377 Dissection of the ascending aorta1451,01314 Dilatation or dissection of the descending or abdominal aorta before age 50 years661,0137 Mitral valve prolapse53398354 Mitral regurgitation31395933 Aortic insufficiency20597521 Aortic surgery282101128 Isolated valvular surgery4510044 Major cardiac involvement7761,01377 Minor cardiac involvement1081,01311Skin: Striae44494547 Herniae9698810 Minor skin involvement4801,01347Lung: Pneumothorax731,0027 Minor lung involvement731,0137CNS: Dural ectasia15429253 Major CNS involvement1541,01315a Nineteen patients were classified as having minor, major, or neither minor nor major skeletal features, but details about their skeletal manifestations were not available.b Includes pectus excavatum, 246 (26%) of 962; pectus carinatum, 288 (30%) of 962; and undefined pectus malformation. Open table in a new tab Mutation screening, with the consent of the patient or a guardian, was performed in the 38 different laboratories by use of SSCP analysis, denaturing high-performance liquid chromatography, heteroduplex analysis, long-range RT-PCR, or direct sequencing of RNA extracted from cell lines or of genomic DNA from peripheral-blood samples. PTC mutations were classified as those that would be likely to produce no or a truncated FBN1 protein (frameshifts, stop codons, and out-of-frame splice mutations), whereas inframe mutations were classified as missense mutations, inframe deletions/duplications, or inframe splice mutations. Twenty-nine splice mutations could not be classified and were therefore excluded from the analyses that compared types of mutations. The frequency of many features of MFS increases with age. Since the patients had different lengths of follow-up, χ2 tests are not appropriate for comparing clinical features between groups. Thus, we used a time-to-event analysis technique to estimate a reliable cumulative probability of observing the different manifestations of MFS. This technique could be applied for the following events: diagnosis of type I fibrillinopathy and diagnosis of scoliosis, ectopia lentis, and/or aortic dilatation or dissection, as well as surgery for these different manifestations, for which the ages at diagnosis were systematically collected. In all time-to-event analyses, the baseline date (time zero) was the date of birth. The time-to-event diagnosis was defined as the interval between the baseline date and the date of first observation of the event. Subjects who did not manifest the studied event during the follow-up course were censored at their last follow-up. Subjects for whom the age at diagnosis of a specific manifestation was not available were excluded from these analyses (a maximum of 4% of patients). The number of observations for each clinical feature is indicated in table 2. The Kaplan-Meier method35Kaplan E Meier P Nonparametric estimation from incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (46320) Google Scholar was used to estimate the cumulative probabilities of clinical manifestations of the disease at ages 10, 25, and 40 years, to describe the diagnosis of clinical features over time. Clinical differences among the different mutation groups (different locations or types of mutation) were tested using the nonparametric log-rank test. Overall survival was also described and compared, using the same method, according to the type/location of the mutation. To underline the importance of taking into account the time to diagnosis of clinical features, we compared the results obtained for aortic dilatation using a χ2 test and the time-to-event technique. For the other features (skeletal features other than scoliosis, skin, lung, and dural involvement) for which the age at diagnosis was not collected, age at last follow-up was the only information available about the time of observation of clinical features. To indirectly take into account the patient’s length of follow-up even in this situation, we adjusted all comparisons of MFS manifestation proportions for the age at last follow-up, categorized into 10-year age groups. These adjusted comparisons were performed using the Mantel-Haenszel (MH) test.36Mantel N Haenszel W Statistical aspects of the analysis of data from retrospective studies of disease.J Natl Cancer Inst. 1959; 22: 719-748PubMed Google Scholar Since this test is appropriate only if the relationship between the mutation type and the clinical manifestation is similar in the different strata o