IDENTIFICATION OF 8 NEW MUTATIONS IN BRAZILIAN FAMILIES WITH
MARFAN SYNDROME
Ana B. A. Perez1, Lygia V. Pereira2, Decio Brunoni1, Mayana Zatz2, and Maria
Rita Passos-Bueno1 *
1Centro
de Genética Médica (UNIFESP/EPM), São Paulo, Brazil; 2 Departamento de
Biologia, Instituto de Biociências, University of São Paulo, São Paulo, Brazil.
ADDRESS FOR CORRESPONDENCE:
Dr. Maria Rita Passos-Bueno
Laboratório de Miopatias - IB - USP
Rua do Matão, 277 – São Paulo, SP, Brazil
Telephone = + 55 11 818 75 63
Telefax = + 55 11 818 74 19
E-Mail = passos@usp.br
SHORT TITLE: Marfan Syndrome in Brazilian Patients
Contract grant sponsor: FAPESP; Contract grant sponsor: PADCT
ABSTRACT
Marfan Syndrome (MFS) is a connective tissue disease whose responsible gene,
FBN-1, was mapped to 15q21. Screening for mutations in all the 65 exons of the
FBN-1 gene in each propositus from 34 families were performed to compare the
efficiency of SSCP versus Heteroduplex analysis and to verify if the spectrum of
mutations in Brazilian patients is similar to the one previously reported. Fourteen
different band shifts were detected by SSCP analysis; among these only 6 were also
detected through Heteroduplex analysis, suggesting that SSCP analysis was a more
efficient method. Except for one, the molecular alteration was confirmed in remaining
13 cases by sequencing; five of them were neutral polymorphisms and the eight
others are new pathogenic mutations, as follows: 5 missense, one nonsense and two
deletions leading to a PTC. All of them are located in EGF-like-calcium binding
motifs (EGF-like-cb). Our findings reinforce that cysteine substitutions and PTC
mutations in the region between exons 24-32 are more likely not to be associated
with the neonatal phenotypes and also contribute for a better definition of the critical
regions of the protein.
KEY WORDS: Marfan Syndrome, fibrillin, mutations, SSCP, Heteroduplex,
polymorphism
INTRODUCTION
Marfan Syndrome (MFS) is an autosomal dominant inherited connective
tissue disease, which involves primarily the cardiovascular, ocular, and skeletal
systems. Its incidence is approximately 1/10,000 individuals, and about 15-30% of
them are isolated cases. The phenotype represents a continuum, ranging from a
very severe neonatal form to a very mild one, which sometimes may remain
undiagnosed (Pyeritz, 1996).
The disease is caused by mutations in the fibrillin-1 gene (FBN-1), which is
located at 15q21 (Kainulainen et al., 1990; Tsipouras et al., 1991). FBN-1 is a large
gene with 65 exons spanning 110 Kb of genomic DNA. The gene product is Fibrillin,
that contains many repetitive epidermal growth factor (EGF)-like subunits
interspersed with transforming growth factor-1-binding (TGF-1-BP)-like domains
(Pereira et al., 1993).
FBN-1 mutations have been detected through a great variety of methods,
using both cDNA as well as genomic DNA. However, all of them have shown a very
low detection rate, ranging from 10 to 30% (Pyeritz and Francke, 1993; Dietz and
Pyeritz, 1995). Recently, using primers that allow genomic amplification of the entire
coding sequence of FBN-1 gene, together with Heteroduplex analysis, it was
suggested that nearly 80% of the mutations in MFS patients could be detected
(Nijbroek et al., 1995).
About 100 different FBN-1 mutations have already been detected among MFS
patients, and the great majority are unique to each family. Nearly 70% of the
reported mutations in the FBN-1 gene are missense mutations or small in-frame
deletions or insertions; 15% are premature termination codons (PTC), and 15% are
aberrant splicing events (Dietz and Pyeritz, 1995; Collod-Béroud et al., 1997). The
mutations in FBN-1 gene are spread throughout the gene, usually occurring in exons
that encode EGF-like domains, and involving cysteine residues. No clear genotypephenotype correlation has been found to date, except that mutations in exons 24-32
of FBN-1 gene have been more often associated with the neonatal forms of MFS
(Dietz and Pyeritz, 1995; Putnam et al., 1996).
PATIENTS AND METHODS
Patients
Thirty-four families including 54 patients were evaluated at the Centro de Genética
Médica (UNIFESP-EPM) according to a specific protocol, which was based on the
established signs and diagnostic criteria for the disease (Beighton et al., 1988; De
Paepe et al., 1996). Among the 34 propositus 23 were sporadic and 11 familial
cases.
Genomic DNA Extraction and PCR Amplification
DNA was extracted from peripheral blood leukocytes from all 34 probands and 151
relatives (Miller et al., 1988). Genomic DNA samples were amplified by PCR, using
primers and conditions described elsewhere (Nijbroek et al., 1995).
Single Strand Conformational Polymorphism (SSCP) Analysis
PCR products were analyzed by electrophoresis through the Homogeneous 20 Gel
(Pharmacia) at the Phastsystem (Pharmacia), and silver stained using standard
protocols (Sitnik et al., 1997).
Heteroduplex Analysis
PCR products were analyzed by electrophoresis through MDE gels according to
conditions previously reported (Nijbroek et al., 1995).
DNA Sequencing
PCR products were sequenced in both directions using the “Sequenase Version
2,0 DNA Sequencing Kit” from United States Biochemical (USB) according to
manufacturer’s instructions.
RESULTS
Fourteen different abnormal migrating band patterns were found through
SSCP analysis involving 12 different exons. Of these, only 6 were also detected by
Heteroduplex analysis. The molecular alteration in 13 cases was confirmed by
sequencing, as follows: 10 missense, 1 nonsense and 2 small deletions. In one
individual no mutation was detected and therefore the patient was excluded from
statistical analysis.
In all the cases in which a mutation was detected we were able to test the
DNA changes by SSCP in the relatives of the propositus, which included both
parents and sibs. Six of the changes occurred in isolated cases and they were not
present in neither of their parents, indicating thus that these cases were originated
through new mutations. Two others mutations were found in familial cases, and
segregate only among the affected patients within the family. Therefore, these 8
changes were considered pathogenic (Table 1).
The remaining 5 mutations, which did not cause aminoacid changes, were
also detected in healthy parents and/or sibs. Therefore, they were considered neutral
polymorphisms. The frequency of each of these polymorphisms, based on the
analysis of 68 chromosomes from affected individuals and 100 chromosomes
controls, is described on Table 2.
DISCUSSION
Heteroduplex versus SSCP analysis
In the present report we describe 8 novel FBN-1 pathogenic mutations. The
efficacy of SSCP analysis was estimated as 24% and of Heteroduplex analysis it
decreased to 12%. These values are much lower than the detection rate previously
reported (Nijbroek et al., 1995), although we have used the same conditions as they
did. It is possible that differences between our results and theirs are related to the
sample size, since they reported a detection rate nearly 75% screening only 9
patients.
However, more recently, Hayward et al. (1997), using different Heteroduplex
and SSCP conditions, obtained a detection rate of 28% (17/60), suggesting that their
methods are more sensitive than ours. It is also important to emphasize that 6 out of
the 8 pathogenic changes here detected were found in isolated cases. So, the
detection rate in our sample is higher in isolated cases. These results are apposite to
Hayward et al., (1997), who found a higher detection rate among familial cases.
Pathogenic Mutations
Among the 8 pathogenic changes detected in this study, there are 5
missense, one nonsense and two deletions leading to a PTC. All of them are located
in EGF-like-calcium binding motifs (EGF-like-cb) as expected and are specific to
single families (Tynan et al., 1993; Dietz and Pyeritz, 1995; Nijbroek et al., 1995).
Five of these mutations were associated with the classic phenotype, one with
the classic phenotype without ocular involvement, one with an atypical phenotype,
and one with the neonatal phenotype. Our findings reinforce that cysteine
substitutions and PTC mutations in the region between exons 24-32 are more likely
not to be associated with the neonatal phenotypes.
Based on these results we suggest that the search for mutations in FBN-1
gene may be more efficient and less expensive if one prioritizes exons that
corresponds to EGF-like-cb motifs as for exons 24-32, when the patient has the
neonatal phenotype.
In addition, our findings reinforce the importance of screening for mutations in
the FBN-1 gene in Marfan patients for a better definition of the critical regions of the
fibrillin.
SUMMARY
We describe 8 new mutations in Brazilian families with Marfan Syndrome, all of them
in EGF-like-calcium binding motifs and SSCP seems to be more efficient than
Heteroduplex analysis.
TABLE 1 - NOVEL FBN-1 MUTATIONS AND GENOTYPE/PHENOTYPE CORRELATION
Exon
Exon Typea
Phenotypec
Nucleotide
Amino Acid
Isolated/
Change
Change
14
1836delA
Stop at 624
EGF-cb
F
Classic
28
3545 G/C
C1182S
EGF-cb
I
Neonatal
28
3497 G/A
C1166Y
EGF-cb
I
Classic
32
4011 del T
Stop at 1412
EGF-cb
I
Atypical, pred. sk
36
4490 G/C
C1497S
EGF-cb
I
Classic
44
5467 G/T
E1823X
EGF-cb
I
Classic
44
5453 G/A
C1818Y
EGF-cb
I
Classic (-O)
46
5729 G/T
G1910V
EGF-cb
F
Classic
Familialb
a:EGF-cb= Epidermal Growth Factor calcium binding-like motif
b: pred.= predominantly; sk = skeletal;O = ocular;
c: I = isolated; F=familial
TABLE 2 - FBN-1 POLYMORPHISMS AND FREQUENCIES OF THE ALLELES
Exon
Nucleotide
Amino Acid
MFS proband
Normal control
Change
Change
frequency
frequency
27
3423 G/A
None
0.014
0
29
3675 G/A
None
0.014
0.08
54
6681 A/C
None
0.014
0
15
1875 T/C
None
0.264
0.1
56
6888 G/A
None
0.323
0.17
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