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HUMAN MUTATION 24:140^146 (2004)

RESEARCH ARTICLE

Comprehensive Molecular Screening of the FBN1


Gene Favors Locus Homogeneity of Classical
Marfan Syndrome
B. Loeys,1 J. De Backer,1 P. Van Acker,1 K. Wettinck,1 G. Pals,2 L. Nuytinck,1 P. Coucke,1
and A. De Paepe1n
1
Ghent University Hospital, Center for Medical Genetics, Ghent, Belgium; 2Vrije Universiteit Medisch Centrum, Amsterdam, The Netherlands

Communicated by David Rimoin


In order to estimate the contribution of mutations at the fibrillin-1 locus (FBN1) to classical Marfan syndrome
(MFS) and to study possible phenotypic differences between patients with an FBN1 mutation vs. without, a
comprehensive molecular study of the FBN1 gene in a cohort of 93 MFS patients fulfilling the clinical diagnosis
of MFS according to the Ghent nosology was performed. The initial mutation screening by CSGE/SSCP allowed
identification of an FBN1-mutation in 73 patients. Next, sequencing of all FBN1-exons was performed in 11
mutation-negative patients, while in nine others, DHPLC was used. This allowed identification of seven and five
additional mutations, respectively. Southern blot analysis revealed an abnormal hybridization pattern in one
more patient. A total of 23 out of the 85 mutations identified here are reported for the first time. Phenotypic
comparison of MFS patients with cysteine-involving mutations vs. premature termination mutations revealed
significant differences in ocular and skeletal involvement. The phenotype of the eight patients without proven
FBN1 mutation did not differ from the others with respect to the presence of major cardiac, ocular, and skeletal
manifestations or positive familial history. Most likely, a portion of FBN1-mutations remains undetected
because of technical limitations. In conclusion, the involvement of the FBN1-gene could be demonstrated in at
least 91% of all MFS patients (85/93), which strongly suggests that this gene is the predominant, if not the sole,
locus for MFS. Hum Mutat 24:140–146, 2004. r 2004 Wiley-Liss, Inc.

KEY WORDS: Marfan syndrome; MFS; fibrillin-1; FBN1; mutation analysis


DATABASES:
FBN1 – OMIM: 134797, 154700 (MFS); GenBank: NM_000138.2

INTRODUCTION et al., 1995]. Identification of a locus on chromosome 3


was based on the analysis of a single kindred that
Marfan syndrome (MFS; MIM# 154700) is a segregates a phenotype similar to, but not identical to
connective tissue disorder with autosomal dominant MFS [Collod et al., 1994].
inheritance and a prevalence of 4–6 per 100,000 The purpose of this study was twofold: first, to
individuals [Pyeritz and McKusick, 1979]. The cardinal estimate the contribution of mutations at the FBN1
features involve the ocular, skeletal, and cardiovascular locus to classical MFS by performing a comprehensive
system. The clinical diagnosis of MFS is based on a set of molecular study of the FBN1 gene in a large cohort of
major and minor clinical criteria, known as the ‘‘Ghent
nosology’’ [De Paepe et al., 1996]. MFS is caused by
mutations in the fibrillin-1 gene (FBN1; MIM# 134797) The Supplementary Material referred to in this article can be ac-
[Dietz et al., 1991]. The gene is 200-kb in length, and cessed at www.interscience.wiley.com/jpages/1059 -7794/suppmat
the more than 550 different FBN1-mutations [Robinson Received 24 November 2003; accepted revised manuscript 5 April
et al., 2002; Le Bourdelles et al., 2002] detected so far 2004.
n
are widespread throughout the gene and largely unique Correspondence to: Anne De Paepe, MD, PhD, Ghent University
Hospital, Center for Medical Genetics, De Pintelaan 185, B-9000,
to each family. Because of variation in patient popula-
Ghent, Belgium. E-mail: Anne.Depaepe@UGent.be
tions and screening techniques, it is difficult to L. Nuytinck’s present a⁄liation is Innogenetics NV, Ghent, Belgium.
accurately estimate the sensitivity of FBN1 mutation Grant sponsor: Fund for Scienti¢c Research-Flanders; Grant
detection. number: G.0290.02.
Due to incomplete mutation uptake and the sugges- DOI 10.1002/humu.20070
tion of a second locus on chromosome 3p, the issue of Published online in Wiley InterScience (www.interscience.wiley.
locus heterogeneity in MFS remains under debate [Dietz com).
r2004 WILEY-LISS, INC.
COMPREHENSIVE FBN1 SCREENING IN CLASSIC MFS 141

MFS patients; second, to identify possible phenotypic NM_000138.2 was used, with the A of the ATG translation
differences between patients with an FBN1 mutation vs. initiation start site as nucleotide +1. Intron–exon boundaries are
as previously defined [Pereira et al., 1993].
without, and within different subgroups of FBN1
mutations. Sequencing. Aberrantly migrating PCR fragments, detected
by SSCP or CSGE, were analyzed on an ALF automated DNA
MATERIALS AND METHODS sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden).
Patient Samples Direct sequencing analysis of those samples that remained
mutation-negative after SSCP or CSGE screening, was carried out
This study includes a cohort of 93 consecutive patients in whom using the Big Dye Terminator kit (Applied Biosystems, Warring-
the clinical diagnosis of classical MFS was made according to the ton, Cheshire, UK) and the samples were run on an ABI 377
Ghent nosology, either after personal examination by one of the Genetic Analyzer (Applied Biosystems, Foster City, CA).
investigators (B.L., J.D.B., and A.D.P.) or by another clinical DHPLC. DHPLC analysis was carried out using the WAVE
geneticist familiar with MFS based on a detailed checklist. The DNA Fragment Analysis System (Transgenomic, Cheshire, UK).
patients included 46 male and 47 female subjects, with a mean age The primers, temperatures, and conditions as optimized in Matyas
of 25 years. A total of 61 patients were adults (418 years of age) et al. [2002] were used.
and 32 patients were children (r18 years of age). We accepted
Southern blot analysis. Genomic DNA was digested with
the clinical diagnosis of MFS in a sporadic patient if major
various restriction enzymes (TaqI, EcoRI, and HindIII) according
manifestations in at least two organ systems were present and if at
to the manufacturer’s (New England Biolabs, Beverly, MA)
least one other organ system was involved. In familial cases, the
recommendations. After electrophoretic separation, the restriction
diagnosis in a relative of a MFS patient was established in the
fragments were transferred to Hybond-N+membranes (Amer-
presence of one major criterion in at least one organ system and
sham Pharmacia Biotech, Uppsala, Sweden). All hybridizations
involvement of a second organ system. From each patient, we
were carried out using standard procedures [Sambroock et al.,
obtained either a blood sample or a skin biopsy specimen for
1989], with four cDNA probes covering overlapping portions of
fibroblast culture, or both.
the FBN1 gene (cDNA primers probe sets 1F-1396R, 1012F-
Genomic DNA (gDNA) was extracted from peripheral blood
2815R, 2699F-5824R, and 5722F-8696R).
leukocytes by the QIAamp DNA blood mini kit (Qiagen,
Valencia, CA) or from skin fibroblasts by the EasyDNA kit
(Invitrogen, Carlsbad, CA). Total RNA was prepared from skin RESULTS
fibroblasts with TRIzol Reagent (Life Technologies, Rockville, Stepwise Analysis of 93 Patients Ful¢lling MFS
MD), and first-strand complementary DNA (cDNA) was Diagnostic Criteria
synthesized by Moloney murine leukemia virus reverse transcrip-
tase (MMLV-RT) (Life Technologies, Rockville, MD). Initial mutation analysis by CSGE and/or SSCP
allowed identification an FBN1-mutation in 73 of the
Mutation Analysis 93 patients (Table 1). Of these 73 mutations, 62 have
been previously reported by us (n=58) [Loeys et al.,
Strategy of themolecularFBN1analysis. For the molecular 2001] or others (n=4) [Nijbroek et al., 1995; Matsukawa
analysis of the FBN1 gene, a three-step strategy was developed. In et al., 2001; Katzke et al., 2002; Halliday et al., 2002],
a first step, all patients were subjected to single strand and 11 mutations are reported here for the first time. Of
conformation polymorphism (SSCP) analysis or conformation-
sensitive gel electrophoresis (CSGE) on gDNA (n=62) or, if these 73 mutations, 21 were identified on cDNA,
available, on cDNA (n=31). If initial screening of cDNA or whereas 52 were identified on gDNA. Of the 21 positive
gDNA was negative by either one of these techniques, the analysis cDNA samples, 16 were picked up after initial cDNA
was repeated (n=18) on gDNA or cDNA, respectively, if possible. screening, whereas five mutations were detected in
In a second step, mutation negative samples (n=20) were analyzed patients in whom prior gDNA analysis was negative.
either with exon by exon sequencing (n=11) or by denaturing Remarkably, all five mutations were splicing errors (n=3)
high-performance liquid chromatography (DHPLC) analysis
or frameshift mutations (n=2). Eight of the 52 mutations
(n=9) of the FBN1 genomic DNA. In a third step, Southern
blot analysis was done on six mutation-negative samples. identified by gDNA analysis were identified in patients in
The causal nature of the missense mutations was assumed on whom initial cDNA screening was negative. The nature
the basis of the familial segregation of the mutation, its absence in of these eight mutations was variable: four missense,
50 controls, and/or the occurrence of the same mutation in an three nonsense, and one frameshift mutation. In
unrelated patient with MFS. addition, seven new polymorphisms were found after
CSGE/SSCP. Initial mutation screening was performed by gDNA analysis (Table 2).
CSGE and SSCP, as described by Ganguly et al. [1993] and Orita The mutation analysis with CSGE and/or SSCP
et al. [1989]. Primers for screening were initially provided by the
failed to detect an FBN1-mutation in 20 out of the 93
international Marfan consortium (coordinated by H. Dietz, Johns
Hopkins University, Baltimore, Maryland). Mutation screening of patients. In order to determine whether this was due to
FBN1 genomic fragments was performed by amplification of 65 technical reasons only or possibly also to locus hetero-
fragments, each covering one exon, with flanking intron sequences geneity, we repeated the mutation analysis in the
and an average size of 260 bp. Primer sequences for cDNA mutation-negative patients either with direct sequencing
screening were initially obtained from D. Milewicz and E. Putman of each FBN1-exon (in 11 samples) or with DHPLC
(The University of Texas, Houston, Texas). The complete cDNA analysis (in nine other patients). In this way, seven and
analysis comprised amplification of 24 overlapping PCR fragments
with an average size of 450 bp. Lists with currently used gDNA
five additional mutations were identified, respectively
and cDNA FBN1 primers are available in supplementary material (Fig. 1). Remarkably, none of these mutations had been
online (Supplementary Tables S1 and S2, available online at reported previously. One mutation, c.3388delC
www.interscience.wiley.com/jpages/1059-7794/suppmat). FBN1 (p.His1130fsX31), was found in three unrelated index
cDNA reference sequence with GenBank accession No. patients.
TABLE 1. List of the Clinical and Molecular Data in 93 Patients Ful¢lling the Clinical Diagnostic Criteria for MFSd
142

Cardio-
Skeletal Ocular vascular Family Exon FBN1 Nucleotide
No. Age system system system Lung Skin Dura history Methoda Exon type change Consequenceb Referencec
1 45 M M M 0 m 2 M g /DHPLC þ 1 NH2-terminal c.31delC p.Leu11fsX6 n
2 19 M 0 M 0 m M 0 g /c þ 1 NH2-terminal c.124delG p.Ala42fsX65 1
3 52 m 0 M 0 m 2 M g /c þ 2 NH2-terminal c.247 þ1G4A p.His56fsX72 1
4 22 m M M 0 0 2 M g þ 3 EGF-like #01 c.248-3C4G cDNA na 1
LOEYS ET AL.

5 28 M 0 M 0 m 2 0 g þ 3 EGF-like #01 c.266G4T p.Cys89Phe 1


6 34 m M 0 0 m M M g þ 4 EGF-like #02 c.364C4T p.Arg122Cys 1
7 37 M 0 M 0 m 2 0 g þ 6 Hybrid motif #01 c.643C4T p.Arg215X 2
8 31 M M M 0 0 0 0 g þ 6 Hybrid motif #01 c.718C4T p.Arg240Cys 1
9 49 m 0 M 0 m 0 M c /g þ 8 cb EGF-like #02 c.959dupA p.Tyr320X 1
10 30 M 0 M 0 m 2 M c /seq þ 9 8-cys #01 c.9891G4C p.Asp330 _Thr382del n
11 29 M M M 0 m 2 0 g þ 9 8-cys #01 c.1076G4A p.Cys359Arg n
12 14 m M M 0 m M M g þ 9 8-cys #01 c.1098G4T p.Trp366Cys 1
13 46 m 0 M 0 0 2 M g þ 9 8-cys #01 c.1147G4A p.Gln383Tyr and r.spl? 1
14 34 m M M 0 m M M g /seq þ 11 EGF-like #03 c.1463G4T p.Cys488Phe n
15 21 m 2 M 2 m 2 M g þ 12 cb EGF-like #03 c.1556A4G p.Tyr519Cys n
16 19 m M M 0 m 2 M g þ 13 cb EGF-like #04 c.1633C4T p.Arg545Cys 1
17 3 m M M 0 0 2 M g þ 13 cb EGF-like #04 c.1678G4A p.Gly560Ser 1
18 36 M M 0 0 0 2 M g þ 13 cb EGF-like #04 c.1709G4A p.Cys570Tyr 1
19 41 m M 0 0 m M M g þ 14 cb EGF-like #05 c.1794C4G p.Cys598Trp 1
20 31 m M M 0 0 0 0 c þ 14 cb EGF-like #05 c.1837 þ1G4A p.Ser614fsX25 n
21 36 m M M 0 0 0 M c þ /g þ 15 cb EGF-like #05 c.18381G4A r.spl? n
22 5 M M M 0 0 2 0 c þ 15 cb EGF-like #06 c.1843_1845delAAC p.615delAsn 1
23 3 M M M 0 0 2 0 c þ 16 8-cys #02 c.2055C4G p.Cys685Trp 3
24 4 M M M 0 0 2 M g /c þ 17 8-cys #02 c.2114-1G4C p.Ala705 _Ser722del 1
25 9 M M M 0 m 2 0 c /g þ 19 cb EGF-like #08 c.2327G4A p.Cys776Tyr 1
26 24 m M M 0 0 2 0 c þ 19 cb EGF-like #08 c.2341T4C p.Cys781Arg 1
27 7 m M M 0 0 0 0 g þ 19 cb EGF-like #08 c.2341T4C p.Cys781Arg 1
28 27 m M M 0 m 2 M g /seq þ 20 cb EGF-like #09 c.2433C4G p.Cys811Trp n
29 37 m 0 M 0 0 M M g /c /seq þ 21 Hybrid motif #02 c.2562G4A p.Trp854X n
30 39 M 0 M m m M M g þ 21 Hybrid motif #02 c.2586dupT p.Cys863fsX1 1
31 24 m M M 0 0 2 0 g þ 21 Hybrid motif #02 c.2646C4T p.Ala882Val n
32 16 M m M 0 0 0 M c þ 21 Hybrid motif #02 c.2677G4A p.Ser893fsX18 1
33 32 0 M M m 0 M M g þ 23 cb EGF-like #10 c.2738A4G p.Glu913Gly 1
34 18 M 0 M 0 0 2 M g þ 24 8-cys #03 c.2953G4A p.Gly985Arg 1
35 16 m M M 0 m 0 0 c þ 25 cb EGF-like #11 c.3164G4A p.Cys1055Tyr 1
36 2 m M M 0 0 2 0 c /g þ 26 cb EGF-like #12 c.3217G4A p.Glu1073Lys 4
37 28 m M M 0 m 2 0 c /g þ 26 cb EGF-like #12 c.3299G4T p.Gly1100Val n
38 17 m M M 0 0 2 0 g þ 26 cb EGF-like #12 c.3302A4G p.Tyr1101Cys 1
39 24 m M m m m 0 M g /DHPLC þ 27 cb EGF-like #13 c.3388delC p.His1130fsX31 n
40 27 M 0 m 0 m 0 M g /DHPLC þ 27 cb EGF-like #13 c.3388delC p.His1130fsX31 n
41 17 M 0 M 0 m 2 M c /seq þ 27 cb EGF-like #13 c.3388delC p.His1130fsX31 n
42 8 M M M 0 0 2 0 c þ 28 cb EGF-like #14 c.3559 _3560insT p.His1187fsX5 n
43 3 m 0 M 0 0 2 M g þ 29 cb EGF-like #15 c.3623delG p.Cys1208fsX21 1
44 33 m M M 0 m 2 0 c þ 32 cb EGF-like #18 c.4016G4A p.Cys1339Tyr 1
45 24 m M M 0 m 2 M g þ 34 cb EGF-like #20 c.4285T4A p.Cys1429Ser 1
46 14 M M M 0 m 2 0 c þ 36 cb EGF-like #22 c.4460-8G4A p.Thr1486 _Asp1487insValLeu 1
47 24 M m M 0 m 2 M g /DHPLC þ 36 cb EGF-like #12 c.4473delC p.Cys1491fsX28 n
48 25 M M M 0 m M M c /g þ 37 8-cys #04 c.4615C4T p.Arg1539X 1
49 41 M M 0 0 0 2 M g þ 37 8-cys #04 c.4621C4T p.Arg1541X 1
50 35 M 0 M 0 m 2 0 g þ 37 8-cys #04 c.4621C4T p.Arg1541X 5
51 26 m m M 0 m 0 M g þ 37 8-cys #04 c.4699_4721dup p.Trp1574fsX14 1
52 15 M 0 M m m 2 M c þ 38 8-cys #04 c.4750G4T p.Glu1584X 1
53 29 m M M 0 m M M c /g þ 38 8-cys #04 c.4786C4T p.Arg1596X 1
54 26 M 0 M 0 m 2 0 g þ 39 cb EGF-like #23 c.4930C4T p.Arg1644X 1
55 30 M M M 0 m M M c þ 43 cb EGF-like #25 c.5369G4C p.Arg1790Pro 1
56 38 m 0 M 0 m 2 M c /g þ 43 cb EGF-like #25 c.5372G4A p.Cys1791Tyr 1
57 41 m m M 0 m 2 M g þ 44 cb EGF-like #26 c.5470 _5484dup cDNA na 1
58 49 m 0 M 0 0 2 M g þ 44 cb EGF-like #26 c.5499dupT p.Cys1833fsX1 1
59 29 m M M m 0 M M c þ 44 cb EGF-like #26 c.5504G4A p.Cys1835Tyr 1
60 7 M M M 0 0 2 0 g þ 46 cb EGF-like #28 c.5726T4C p.Ile1909Thr 1
61 10 M M M 0 0 2 0 g þ 46 cb EGF-like #28 c.5788 þ5G4A cDNA na 1
62 8 M m M M 0 2 0 g /seq þ 47 cb EGF-like #29 c.5809G4T p.Gly1937X n
63 10 M 0 m 0 0 2 M g þ 47 cb EGF-like #29 c.5898delA p.Pro1966fsX13 1
64 1 m M M 0 0 2 0 g þ 48 cb EGF-like #30 c.5930G4A p.Cys1977Tyr 1
65 45 M M m 0 m 2 M g þ 48 cb EGF-like #30 c.6037 þ1G4C cDNA na n
66 26 M M M 0 0 M M c /g þ 49 cb EGF-like #31 c.6160C4T p.Gln2054X 1
67 40 M M m 0 m 2 0 g /c þ 52 cb EGF-like #32 Unknown p.Asp2127 _Val2165del 1
68 40 M M M 0 0 M M g /c þ 52 cb EGF-like #32 c.6379 þ2T4G p.Asp2127 _Val2165del 1
69 12 m M M 0 m 2 M g þ 53 cb EGF-like #33 c.6583G4T p.Gly2195X 1
70 15 M 0 M 0 0 2 M g þ 54 cb EGF-like #34 c.6667A4C p.Asn2223His 1
71 4 m 0 M 0 0 2 M g þ 55 cb EGF-like #35 c.6844C4T p.Arg2282Trp 1
72 27 M M M 0 m 2 0 g þ 55 cb EGF-like #35 c.6844C4T p.Arg2282Trp 1
73 17 M 0 0 0 m M 0 g /DHPLC þ 56 cb EGF-like #36 c.6881A4C p.Glu2294Ala n
74 14 M 0 m 0 0 2 M c þ 58 cb EGF-like #37 c.7217G4A p.Cys2406Tyr 1
75 4 M 0 M 0 0 2 M g þ 59 cb EGF-like #38 c.7398T4C p.Tyr2466X 1
76 35 m M M 0 m 2 M g þ 60 cb EGF-like #39 c.7465T4G p.Cys2489Gly n
77 50 m 0 M 0 0 M 0 g þ 61 cb EGF-like #40 c.7577delA p.Asn2526fsX155 1
78 35 m 0 M 0 0 2 M g þ 62 cb EGF-like #41 c.7742G4T p.Cys2581Phe 1
79 21 M M M 0 m 2 0 c þ 62 cb EGF-like #41 c.7754T4C p.Ile2585Thr 1
80 32 M 0 M 0 m M 0 c /seq þ 62 cb EGF-like #41 c.[7809delC; p.Asn2603fsX5 n
c.7807 _7808dupAA]
81 34 M M M 0 m 2 0 c þ 63 cb EGF-like #42 c.7864T4A p.Cys2622Ser n
82 23 m 0 M 0 m 2 M g þ 63 cb EGF-like #42 c.7872C4G p.Asn2624Lys 1
83 30 0 0 M 0 m 0 M g þ 63 cb EGF-like #42 c.8002G4T p.Gly2668Cys 1
84 10 m M M 0 0 0 0 g þ 64 cb EGF-like #43 c.8149G4T p.Glu2717X 1
85 5 m M M 0 0 2 0 g þ 65 COOH-terminal c.8512dupA p.Lys2838fsX5 n
86 45 M 0 M 0 0 2 M g /DHPLC 
87 34 M 0 M 0 m 2 0 g /DHPLC 
88 14 m M m 0 0 2 M g /DHPLC 
89 28 M M M 0 m M 0 c /g /DHPLC 
90 19 m M M m 0 2 0 c /g /seq 
91 15 m M M 0 0 2 0 c /g /seq 
92 5 M 2 M 0 m 2 0 c /g /seq 
93 21 M M M 0 m 2 0 g /seq 
a
g, mutation screening of genomic FBN1 DNA; c, mutation screening of copy FBN1 DNA; , no mutation identi¢ed; þ , mutation identi¢ed.
b
cDNA na, copy DNA not available.
c
Novel mutations are marked with an asterisk ( n) and printed in bold type; previously reported mutations with references: 1Loeys et al. [2001]; 2 Matsukawa et al. [2001]; 3Katzke et al. [2002]; 4Nijbroek et al. [1995];
5
Halliday et al. [2002].
d
Intron^exon boundaries are as previously de¢ned [Pereira et al.,1993].
COMPREHENSIVE FBN1 SCREENING IN CLASSIC MFS

FBN1 cDNA reference sequence with GenBank accession No NM _000138.2 was used with the A of the ATG translation initiation start site as nucleotide þ1.
M, major involvement; m, minor involvement; seq, complete FBN1 sequencing; DHPLC, denaturing high performance liquid chromatography.
143
144 LOEYS ET AL.

TABLE 2. Novel FBN1 Polymorphisms Identi¢ed inThis Study n comprised three major categories (Table 1): 1) 41
Position Exon/intron AA codon a¡ected missense mutations (48%); 2) 13 nonsense mutations
(16%) and 20 frameshift mutations (22%) leading to
c.442 þ61 _442 þ67del Intron 4 premature termination codons; and 3) 11 mutations
c.783T4C Exon 7 p.Asn261
c.1715-5T4C Intron 13 (14%) affecting or predicted to affect splicing or causing
c.2855-37G4A Intron 23 in frame deletions.
c.3780G4A Exon 30 p.Glu1260 The distribution of the major manifestations in the
c.5789-64G4T Intron 46
c.8082A4C Exon 64 p.Arg2694
total cohort of MFS patients (n=93) was as follows:
ectopia lentis in 52 patients (56%), significant aortic
FBN1 cDNA reference sequence with GenBank accession no.
NM _000138.2 was used with the A of the ATG translation initiation start
dilatation or dissection in 81 patients (87%), and major
site as nucleotide þ1. Intron-exon boundaries are as previously de¢ned skeletal involvement in 47 patients (50%). Overall, 53
[Pereira et al.,1993]. out of the 93 patients had a positive family history
(57%), whereas 40 were sporadic.
The phenotype of the eight patients without proven
93 classical MFS patients:
FBN1 mutation did not differ from the others with
73 FBN1 mutations identified with SSCP/CSGE
respect to the distribution of major cardiac (7/8;
P=0.47), ocular (4/8; P=0.72), and skeletal manifesta-
20 patients without FBN1 mutation
tions (5/8; P=0.48), or positive familial history (2/8;
P=0.06) (w2 analysis with one degree of freedom [df]).
We found a significant difference in the incidence of
11 complete FBN1 sequencing 9 FBN1 DHPLC screening ectopia lentis in the MFS patients with cysteine
substitutions (20/27) vs. those with premature termina-
tion codons (11/33) (Po0.01). On the other hand,
7 additional FBN1 mutations: 5 additional FBN1 mutations: major skeletal involvement was more frequent in the
c.1463G>T, c.2433C>G c.31delC latter group (20/33 vs. 8/27; Po0.025). The difference
c.2562G>A, c.5809G>T c.3388delC (2x) was not significant for the presence of major cardiovas-
c.989-1G>C, c.3388delC c.6881A>C cular manifestations (23/27 vs. 29/33) or positive family
c.[7809delC;7807-7808dupAA] c.4473delC history (14/27 vs. 21/33). In contrast to Schrijver et al.
[2002], we did not find aortic dissections to be more
common in patients harboring premature termination
Overall mutation detection rate in 93 MFS patients with mutations (4/33 vs. 3/27) (w2 analysis with 1 df).
SSCP/CSGE followed by sequencing or DHPLC:
91% (85/93) FBN1 mutations identified
DISCUSSION
This study describes a two-line approach, using
Southern blot analysis in 6/8 MFS patients without mutation: DHPLC and gene sequencing as second line techniques
1 patient showed aberrant pattern for the analysis of samples from patients fulfilling the
diagnostic criteria for MFS and in which no FBN1
mutation had been detected using SSCP and/or CSGE.
Previous studies used either CSGE [Körkko et al., 2002]
8% (7/93): involvement of FBN1 not confirmed
or DHPLC [Halliday et al., 2002] alone as screening
methods or have compared different techniques [Yuan
FIGURE 1. Stepwise analysis of 93 patients ful¢lling MFS diag-
nostic criteria. Corresponding protein changes of mutations re-
et al., 1999; Matyas et al., 2002] for an identical panel of
presented in this ¢gure are shown inTable 1. mutations. Liu et al. [2001] reported that no major
deletions were found by Southern blot analysis, but long-
As a last step, the presence of total gene or large range RT-PCR detected two large genomic deletions out
intragenic deletions was investigated by Southern blot of 105 unique disease-causing mutations.
analysis in 6 out of 8 patients (not in Patients 86 and 93, The overall detection rate of FBN1 mutations in this
because of lack of DNA). This analysis revealed an study group was 85 out of 93 (91%). The pick-up rate of
abnormal hybridization pattern (extra band on TaqI- 78% (73/93) for the initial SSCP/CSGE screening is
digestion) for patient 92 after hybridization with probe grossly comparable with the better detection rates in
1F-1396R, suggesting the presence of an intragenic previous reports in patients fulfilling the Ghent criteria
deletion in the FBN1 gene. for MFS [Körkko et al., 2002]. The fact that 12 of the
initial 73 mutations were identified after second screen-
ing on gDNA or cDNA, respectively, illustrates com-
Genotype and Phenotype Study
plementarities of both targets.
Overall, 85 FBN1 mutations were identified in the 93 In our series, mutational analysis of samples negative
MFS patients. A total of 27 mutations created or after SSCP and/or CSGE screening, using either direct
substituted a crucial cysteine residue. The type of sequencing or DHPLC allowed us to detect 7 out of 11
FBN1 mutation identified was heterogeneous and (64%) and 5 out of 9 (55%) additional mutations,
COMPREHENSIVE FBN1 SCREENING IN CLASSIC MFS 145

respectively. These figures are remarkably high if one robustness of selection criteria is the most important
takes into account that all the samples in our study had determinant of the outcome of mutational studies
already been screened by SSCP and/or CSGE. Matyas [Hayward et al., 1997; Katzke et al., 2002].
et al. [2002] reported a high detection rate (89–99%) Within the mutation-positive group, mutation-type–
using DHPLC, but in this study the investigators largely specific clinical subgroups seem to emerge. We did not
analyzed samples with known mutations, which might identify differences in cardiovascular symptoms between
have influenced the outcome. both groups but, as described by Schrijver et al. [2002],
Our data confirm the findings by Körkko et al. [2002], the PTC mutations appear to have more severe skeletal
who found that CSGE is a sensitive and specific findings, whereas the cysteine substitution group had a
technique for detecting FBN1 mutations. However, in significantly greater incidence of ectopia lentis.
this study we were able to detect five mutations with In conclusion, the involvement of the FBN1 gene
DHPLC in previous SSCP/CSGE–negative samples. could be demonstrated in at least 91% of all MFS
When we reanalyzed the SSCP and CSGE gels of the patients, which strongly suggests that this gene is the
mutation-negative samples in which a mutation was predominant, if not the sole, locus for MFS. Additional
subsequently identified by sequencing or DHPLC, we study of the FBN1 promoter region or other regulatory
found that one mutation, c.[7809delC; 7807_7808du- sequences may be required to definitively resolve the
pAA] (p.Asn2603fsX5), was post hoc visible on the question of locus homogeneity in MFS. Based on the
CSGE gel, although it was very subtle, and therefore it results of this study and after reviewing the available
was initially overlooked. One mutation. c.989– literature, we conclude that mutational analysis of cDNA
1G>C(p.Asp330_Thr382del), was definitely missed using DHPLC or CSGE as first screening techniques is
because the screening was only performed on cDNA. the preferred approach in clinical implementation of
The other mutations were not detectable by SSCP or FBN1 analysis. Detection rates of up to 90% in molecular
CSGE. Interestingly, only 3 of the 12 mutations detected FBN1 screening studies are currently achievable. How-
by DHPLC or sequencing were missense mutations, ever the number of DHPLC-based mutational studies of
whereas the other nine mutations were nonsense the FBN1 gene is still limited. Moreover, with respect to
mutations (n=2) or mutations affecting the splicing or the cost and manpower involved, DHPLC may also be
reading frame (n=7). The latter might be explained by the preferred technique because of its suitability for
the fact that some of the initial genomic exon primers automation. Direct sequencing and gDNA SSCP analysis
used in SSCP and CSGE screening were in too close can be used as alternative secondary techniques.
proximity to the exon–intron boundaries. Although cDNA analysis requires a skin biopsy, which
One explanation for the incomplete FBN1 mutation is less patient friendly and less suitable for routine use, it
uptake in classical MFS might be the failure to detect requires a significantly lower workload than genomic
gross deletions. Up to now, only three multiexon DNA analysis. In addition, the availability of cDNA is of
deletions have been identified [Kainulainen et al., tremendous value in the study of splicing errors. Also,
1992; Liu et al., 2001]. This can be explained in part the need for prenatal diagnosis may justify cDNA studies,
by the screening methods that have been used in the especially in small families for which linkage is not
past. For this reason, we analyzed six mutation-negative feasible [Loeys et al., 2002].
samples with Southern blotting. In one patient, an
abnormal hybridization pattern was detected and is ACKNOWLEDGMENTS
under further investigation. This finding, together with
the data of Liu et al. [2001], suggests that a small This work was supported by grant G.0290.02 from the
percentage (1–2%) of Marfan patients carry multiexon Fund for Scientific Research-Flanders to ADP. B. Loeys
deletions. Southern blot analysis might not be the most and J. De Backer are research fellows of the Fund for
sensitive technique to detect gross genomic deletions. It Scientific Research-Flanders (FWO) and Bijzonder
is, however, unlikely that gross deletions are the Onderzoeksfonds (BOF) of the Ghent University,
explanation for the remaining 9% of patients in which respectively.
no FBN1 mutation was identified in the study. Linkage
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