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Human Molecular Genetics Pages 1581-1588
Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe Marfan syndrome
Introduction
Results
   Identification of exon-skipping mutations in the FBN1 gene
   Genomic analyses and population studies
   Pulse-chase studies of fibrillin protein with EGF-like domain deletion
Discussion
   Effects of exon-skipping mutations on protein and clinical phenotypes
Materials And Methods
   Subjects and nucleic acid preparation
   Long RT-PCR
   Mutation detection and sequencing
   Pulse-chase studies of fibrillin protein
Acknowledgements
References

Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe Marfan syndrome

Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe Marfan syndrome Wanguo Liu1, Chiping Qian1, Kimberly Comeau1, Thomas Brenn2, Heinz Furthmayr2 and Uta Francke1,3,*

1Howard Hughes Medical Institute, 2Department of Pathology and 3Department of Genetics, Stanford University Medical Center, Stanford, CA 94305, USA

Received April 24, 1996; Revised and Accepted July 22, 1996

Marfan syndrome (MFS), a heritable connective tissue disorder, is caused by mutations in the gene coding for fibrillin-1 (FBN1), an 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 calcium binding epidermal growth factor (EGF)- like domains and maintain the reading frame. In five probands, 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 labelled fibrillin protein revealed normal levels of synthesis but significantly reduced matrix deposition. This dominant-negative effect of the mutant monomers is considered in the light of current models of fibrillin assembly. Probands with this type of FBN1 mutation include the most severe forms of MFS, such as neonatally lethal presentations.

INTRODUCTION

The Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with an incidence of ~1 in 10 000 live births, with no ethnic bias (1 ). The disease is characterized by cardiovascular, ocular, skeletal and other connective tissue manifestations with great phenotypic variability (2 ,3 ). The MFS gene was linked to the chromosome 15q21 region (4 ,5 ) and the gene involved, fibrillin-1 (FBN1), was identified by a candidate gene cloning approach (6 -11 ). Detection of mutations in the 9.6 kb FBN1 cDNA, encoded by 65 exons that are distributed over 110 kb, has proved to be a challenge, not only because current technologies for mutation detection have limited sensitivities, but also because almost all of the FBN1 mutations identified are unique (12 ). Several strategies and approaches have been described for FBN1 mutation screening, such as multiple sets of primers to amplify overlapping fragments from coding sequence (13 ,14 ), and exon-by-exon screening of genomic DNA that not only detects mutations in coding but also in splice site sequences (15 ,16 ), combined with single strand conformation analyses (SSCA) or heteroduplex analysis (13 -16 ). To date, 5 years after the FBN1 gene was cloned, a total 61 FBN1 mutations have been reported, that account for only ~10-15% of all MFS samples screened (12 ,17 ). Systematic screening of all exons, amplified individually by heteroduplex analysis, promises to result in a higher rate of mutations detection, although the reported optimistic number of 78% was based on only nine probands studied (16 ). New screening strategies or mutation detection methods are clearly desirable for a better understanding of the role of the FBN1 gene in the causation of MFS.

Three categories of FBN1 mutations have been described in MFS probands: (i) missense mutations including cysteine substitutions in epidermal growth factor (EGF)-like domains as well as small in-frame deletions or insertions; (ii) mutations causing premature termination of translation; and (iii) mutations causing exon-skipping or genomic exon deletions that maintain the reading frame. Correlation studies between FBN1 gene defects and the MFS phenotype have revealed that probands with severe, early-onset forms of MFS often have mutations in the middle region of FBN1 between exons 24 and 32 (17 -19 ), while probands with reduction in the steady-state level of mutant allele transcript due to premature termination mutations have a milder phenotype (14 ,20 ). The correlation between phenotype and mutations leading to exon-skipping, however, has not yet been systematically studied in individuals with MFS.

We have developed a simple and highly efficient long RT-PCR approach for rapid detection of exon-skipping mutations in FBN1 that has allowed us to detect six different exon-skipping mutations: five novel and one recurring. The effects of these mutations on fibrillin synthesis and deposition have been studied in cultured cells and are correlated with the clinical phenotypes.

RESULTS

Identification of exon-skipping mutations in the FBN1 gene

Sixty unrelated MFS probands were examined for exon-skipping mutations in the coding region of the FBN1 cDNA. Total RNA was extracted from cultured fibroblasts and, after reverse transcription, the entire FBN1 cDNA was amplified in three overlapping fragments, 3-4 kb each, by using three sets of primers (Table 1 ). These PCR products were cleaved with restriction enzymes chosen to generate individually identifiable subfragments with known exon content, based on the FBN1 coding sequence. Comparison of the restriction patterns of long RT-PCR products amplified from normal and patient samples allowed the detection of one mutation in the 4 kb fragment including exons 1-31 of the cDNA (Fig. 1 a), two mutations in fragments from exons 28 to 52 (Fig. 1 b) and three mutations in fragments spanning exons 47-65 (Fig. 1 c).


Figure 1. Comparison of the restriction fragment patterns of long RT-PCR products digested with restriction enzymes (a) DdeI, (b) TaqI and (c) FokI. Sample N is a control and the others are proband samples. Lane 1 in each gel is a 100 bp DNA fragment ladder. Arrows point to altered-size fragments in patient lanes that were sequenced.


Table 1 Primers used for long RT-PCR in FBN1 mutation analysis

DNA from the altered bands was either gel-purified or re-amplified with one of the 22 primer pairs described earlier (14 ) and directly sequenced. Analysis of the cDNA sequence data revealed the deletion of a single exon (exons 31, 46, 49, 56 and 58) in each altered fragment in five proband samples (Fig. 2 a-e) and a partial (51 bp only) deletion of exon 18, due to usage of a cryptic splice site, in one sample (Fig. 2 f). Further investigation by RT-PCR with a sense primer in exon 16 and an antisense primer that spans the FBN1 exon 17/19 boundary revealed FBN1 transcript heterogeneity in this proband, with products skipping the entire exon 18 also present (data not shown). In all six cases, the mutant transcripts maintain the reading frame and delete a single calcium-binding EGF-like domain. All of the skipped exons are located in the D region of the FBN1 cDNA that contains 41 of the 43 EGF-like calcium binding domains.


Figure 2. Direct sequencing analysis of the DNA isolated from the altered-size restriction fragments reveals deletions of exons 31, 46, 49, 56 and 58 (a-e) and a 51 bp deletion in exon 18 (f).

In an alternative approach, a single fragment covering 8.9 kb of coding region from exon 1-65 was amplified with a single set of primers (Table 1 ) using the Expand PCR System (Boehringer Mannheim). The 8881 bp RT-PCR products and their restriction fragment patterns, derived from one normal individual and two MFS probands with exon-skipping mutations, are illustrated in Figure 3 . Successful amplification was obtained for 9 of 14 samples tested and was found to be dependent on the quality of the mRNA.


Table 2 . Mutations leading to skipping of EGF-like domain exons
*Synthesis and deposition of fibrillin protein are expressed as % of control; for definition of groups see reference 21.

Genomic analyses and population studies

To identify the mutations that cause the exons to be skipped during transcription, we amplified the skipped exons from genomic DNA with intron primers (16 ). The PCR products were then directly sequenced, and the mutations identified are summarized in Table 2 .

Skipping of exon 46, due to a G -> A transversion at the +5 position of the consensus 5' splice site, was previous reported (16 ) and recurred in our patient FB815. This is the fourth case reported and makes this the most frequent recurring FBN1 mutation in unrelated MFS patients. The mutation occurs at a CpG hotspot on the non-coding strand and generates a new NlaIII site. The presence of this mutation was then searched for in another 120 unrelated MFS and 90 normal control DNA samples by NlaIII digestion of PCR products, with negative results. Searches for the presence of the other five mutations in 45 normal and 90 patient DNA samples by either restriction analyses or SSCA were all negative indicating that each of these mutations is unique (data not shown). Family studies revealed that one case is familial and four are de novo mutations.

Pulse-chase studies of fibrillin protein with EGF-like domain deletion

Quantitative pulse-chase studies with labelled cysteine revealed levels of fibrillin synthesis and secretion in the normal range (Table 2 ) in all six patient fibroblast samples. This is consistent with the preserved stability of the mutant mRNA that was specifically tested and confirmed in three of the samples by allele-specific mRNA analyses (data not shown). Although the mutant and normal fibrillin monomers cannot be distinguished on the polyacrylamide gel, the pulse-chase data indicate that the mutant fibrillin monomers are synthesized and secreted normally, since the total level is in the normal range. Deposition of newly synthesized fibrillin into the extracellular matrix was very much reduced, however, with four samples falling into group IV (deposition <35% of normal control) and two into the lower part of group III (35-70% deposition) (Table 2 ) (21 ). As we have recently shown, the levels of matrix deposition of newly-synthesized fibrillin monomers depend critically on the density of the pre-existing microfibrillar network (22 ) in cell culture. These results suggest that the mutant fibrillin monomers, shortened by one EGF-like domain, interfere significantly with microfibril formation.

DISCUSSION

Effects of exon-skipping mutations on mutant mRNA stability and detection.

A long RT-PCR method has been efficiently used for the detection of exon-skipping and exon deletion mutations in the FBN1 gene of patients with MFS. This method has several advantages compared with previously reported RT-PCR methods (14 ,15 ). First, with only three sets of primers, overlapping fragments covering the entire coding region of FBN1 gene are amplified. Second, the products are 3-4 kb in length with at least four exons overlapping, so that no alleles with deletions up to three exons will be missed. Third, there is no need for SSCA or heteroduplex analyses that both have limited sensitivities for mutation detection (23 -26 ). Alternatively, we show that a single fragment covering the 8.9 kb coding sequence can also be amplified by using the Expand PCR System. Our success of amplifying analyzable products, however, was limited to 9 of 14 mRNA samples tested and appeared to be related to the RNA quality, with standard extraction methods (27 ) giving better results than commercial RNA isolation kits.


Figure 3. (a) Long RT-PCR amplification products of the 8881 bp FBN1 coding sequence from total RNA of a normal control (N) and two MFS probands. (b) Restriction patterns of these amplicons digested with AlwI and KpnI. Arrows indicate altered bands. Marker lanes (left) contain (i) HindIII [lambda] DNA fragments and (ii) a 100 bp ladder.

Splice site mutations that may result in exon-skipping have also been detected by a genomic exon-by-exon screening approach combined with heteroduplex or SSC analysis (18 ,19 ), but multi-exon deletions would be missed by this approach. The advantages of this method are that only genomic DNA is needed and that its sensitivity is not influenced by mutant RNA instability that can be caused by various kinds of mutations (28 -30 ). But not all splice site mutations, detected by genomic screening, will result in exon-skipping. Therefore, the analysis will have to be extended to RNA studies for understanding the molecular basis of a particular phenotype. Since the FBN1 gene contains 65 exons (31 ), however, to screen all exons is very time-consuming. Furthermore, the efficiency of mutation detection by heteroduplex or single-strand conformation analysis, on which this approach depends, is quite variable. Indeed, three of the exon-skipping mutations that we identified by the long RT-PCR method (in FB815, FB835 and FB1070) had been previously missed by short RT-PCR/SSCA screening (14 ), although others have reported the detection of exon-skipping by short RT-PCR analysis (20 ,32 ). In two of these three cases, we had also missed the mutations with the genomic exon amplification/MDE gel approach, carried out as described in reference 16 .

The main disadvantage of any RT-PCR approach is that the sensitivity is affected by the stability of the mutant mRNA. Our studies of fibrillin protein levels in fibroblasts from patients with mutations causing the skipping of a calcium-binding EGF-like domain indicate, however, that fibrillin synthesis and secretion were in the normal range (>70% of control) (Table 2 ). This result is consistent with RNA analyses revealing normal levels of mutant mRNAs that have spliced out exons but maintain the reading frame in 3/3 of our cases (data not shown) and others reported in the literature (16 ,19 ). In two other reported cases, mutant mRNA levels were reduced to 16 or 25% respectively (20 ,32 ). In these cases, however, the exon-skipping either caused a frameshift resulting in a stop codon downstream (20 ), or the mutation changed a codon within the skipped exon to a premature termination codon that apparently was responsible for the skipping (32 ). In both cases, one can invoke the well-established phenomenon of mRNA instability associated with stop codons. To address the issue whether the long RT-PCR approach would be able to detect mutant transcripts that are present in reduced amounts, we carried out mixing experiments and were able to detect altered-size restriction fragments derived from mutant mRNA at the 16 and 25% levels (data not shown). At any rate, reduced levels of mutant mRNA due to instability are not associated with exon-skipping mutations that maintain the reading frame. Because of the repetitive nature of the FBN1 gene, with most exons consisting of multiples of 3 nt and encoding complete structural domains, splice-site mutations that lead to skipping of 60 of the 63 internal exons will fall into this category.

The high sensitivity of the long RT-PCR method allowed us not only to detect the six exon skipping mutations reported here but also other exon-skipping or multiple exon deletion mutations caused by different mutational mechanisms (data not shown). Prior to this study, only six of the 61 FBN1 mutations reported (10%) caused exon-skipping (19 ,20 ,32 ,33 ). Considering the total number of MFS samples screened previously by other methods, however, the detection rate for exon-skipping mutations only amounts to 1-2%, much lower than the 10% that we found here. Our results with the long RT-PCR method suggest that at least 10% of MFS patients have exon-skipping or multiple exon deletion mutations that could rapidly be detected with the methods described here.

Effects of exon-skipping mutations on protein and clinical phenotypes

Genotype/phenotype comparisons may be useful to define the sub-group of MFS patients who are likely candidates for this kind of mutation. The clinical spectrum of MFS includes a range of severity in cardiovascular, ocular and skeletal systems, from equivocal, classic, severe to neonatal lethal MFS. Including the six probands reported in this paper, FBN1 mutations have been identified in a total of 70 unrelated MFS families (12 ,17 ), of which 15 had exon-skipping mutations (21%). With the exception of the three cases with recurrent exon 46 skipping mutations who had a classic MFS phenotype (16 ) and our proband FB835 who has transcript heterogeneity with preponderance of a mutant mRNA with a 51 bp exon 18 deletion, the other 11 cases of exon-skipping mutations had a severe MFS phenotype including four with neonatal MFS which is at the most severe end of the phenotypic spectrum of MFS (Table 3 ). Five of our six patients with exon-skipping mutations have dislocated lenses, severe scoliosis and aortic involvement early in life. One of them had a neonatally lethal disorder to be described in detail elsewhere (Spinazzola et al., manuscript submitted). These data indicate that MFS patients with in-frame skipping of an exon in their FBN1 gene tend to have a severe phenotype. The correlation holds with respect to the fibrillin protein phenotype, with all of them in groups III or IV, characterized by normal production and severe-to-less severe reduction in extracellular matrix deposition of fibrillin (21 ). The strong dominant negative effect of this type of mutation seen in the pulse-chase assay could be modulated, however, by low levels of mutant mRNA and/or protein.


Table 3 Exon skipping mutations in FBN1 gene and resulting phenotype

We suggest that the chain of pathogenetic events is as follows. The mutant mRNA missing an in-frame exon is stable and is translated efficiently into mutant fibrillin monomers that are slightly shorter and not distinguishable by SDS-PAGE from wild-type monomers. Both forms of fibrillin are secreted normally and presumably are present in the same amount in the extracellular matrix, where the structurally abnormal fibrillin molecules are predicted to participate in the formation of microfibrils. The currently favored model of fibrillin assembly is based on immunolocalization of region-specific monoclonal antibodies in relationship to the beaded microfibrils seen by electron microscopy (34 ). Strong evidence supports this model of head-to-tail organization of multiple parallel aligned fibrillin monomers, with the beads covering the interacting N- and C-terminal ends (Fig. 4 a). In this model, there would be one bead per unit length of the fibrillin molecule. The measured interbead distances in tissue microfibrils, however, and recent NMR structure determinations of two covalently linked calcium-binding EGF-like fibrillin-1 domains (35 ) are more consistent with a model in which monomers span two interbead regions (Fig. 4 b). Because of the internal repeat of the FBN1 domain structure, a perfect lateral alignment of the, presumed globular, 8-cysteine domains appears to be maintained in this model of staggered monomers. In contrast with the previous model (Fig. 4 a), a portion of the long region of uninterrupted calcium-binding EGF-like domains (encoded by exons 25-36) is predicted to participate in the bead formation. While in both models exact phasing of monomers is required and deletion of any exon would perturb their precise lateral alignment, it may be significant that most exon-skipping mutations causing radical disruption of microfibril formation and severe neonatal lethal presentation involve exons 31 (this report) and 32 (19 ). In the staggered model, these domains would be part of the bead structure and could be important for alignment with the interacting amino- and carboxy-termini of the staggered set of monomers. The other skipped exons we identified (marked with arrows on Fig. 4 b) fall into predicted interbead regions. Uncertainties about the precise interbead distances of individual microfibrils, that can be extended in vitro, in relation to measurements of the length of isolated fibrillin monomers, and of the amount of fibrillin structure packed into the beads, however, make it difficult to choose between these two models at this time.


Figure 4. Alternative models of fibrillin assembly. The domain structure of the fibrillin monomer is deduced from the cDNA sequence (39) and ovals symbolize the location of the globular beads seen by electron microscopy of isolated microfibrils (34). (a) Parallel alignment of head-to-tail interacting monomers with one bead per monomer. (b) Staggered alignment with 50% overlap of monomers leading to two beads per monomer in the microfibril structure. This model is favored by recent NMR solution structure analysis of a pair of calcium-binding EGF-like fibrillin domains (35). The EGF-like domains deleted by the exon-skipping mutations reported here are indicated by arrows and the numbers refer to the exons skipped.

MATERIALS AND METHODS

Subjects and nucleic acid preparation

Study subjects were recruited from the Stanford University Marfan Clinic, the Northern California Chapter of the National Marfan Foundation and the Cardiovascular Surgery Department of Stanford University Hospital. Fifty-five of the 60 subjects in this study met the established diagnostic criteria for MFS (36 ). The remaining five had equivocal MFS-like features. Normal control samples were from unrelated persons with no features of MFS. Skin biopsies were obtained under an IRB-approved protocol and primary fibroblast cell strains were established. Genomic DNA and total RNA were extracted from fibroblast cultures by standard methods (27 ).

Long RT-PCR

Reverse transcription was performed using Superscript II (Gibco-BRL) following the manufacturer's protocol (Perkin Elmer Cetus). Amplification of three overlapping fragments covering the entire FBN1 cDNA sequence was obtained using three pairs of PCR primers (nos 1-3 in Table 1 ) and the following conditions: 94oC for 30 s; 57oC for 30 s (primers no. 1), 55oC for 45 s (primers no. 2) or 56oC for 30 s (primers no. 3); 72oC for 60 s (primers no. 1) or 45 s (primers nos 2 and 3) for 35 cycles, followed by a final extension step at 72oC for 10 min. For amplification of the 8.9 kb coding sequence in a single segment we used a pair of 30mer PCR primers (no. 4 in Table 1 ) and the Expand Long Template PCR System (Boehringer Mannheim). The PCR was carried out in thin wall tubes with a thermal profile including denaturation for 10 s at 94oC, followed by 35 cycles of denaturation (10 s at 94oC), annealing (30 s at 61oC) and extension (8 min at 68oC), followed by a final extension step of 7 min at 68oC.

Mutation detection and sequencing

Exon-skipping was predicted by comparison of the restriction patterns between the long RT-PCR products amplified from normal control of study subject samples. Altered RT-PCR fragments detected in agarose gel were either purified with the QIAquick Gel Extraction Kit (QIAGEN) or reamplified with one of the 22 pairs of primers previously reported (14 ) and directly sequenced with fluorescent terminators on an ABI Prism 377 DNA sequencer. The genomic DNA amplification of the deleted exons was performed using intron primers flanking these exons (16 ), kindly provided by H. Dietz (Marfan Consortium). These amplicons were sequenced manually with the Sequenase PCR Product Sequencing Kit (70170 USB, Amersham Life Science).

Pulse-chase studies of fibrillin protein

Primary fibroblast cultures of the six probands and normal controls were metabolically labelled with [35S]cysteine. Harvesting of cell and matrix fractions and quantitation of fibrillin were performed as described by Aoyama et al. (37 ) except that, for some samples, a phosphorimager rather than densitometry of autoradiograms was used for quantitation of fibrillin signals on gels. The classification scheme for patients, based on quantitation of synthesis and matrix deposition values, has been described (21 ) and clinical correlations were delineated previously (38 ).

ACKNOWLEDGEMENTS

We thank E. Valero for expert technical assistance, H. Dietz for intron primers and clinical information, T. Aoyama for pulse-chase data, and C. Gasner, C. Miller and M. Bialer for clinical samples. The work in the laboratory of U.F. was supported by the HHMI and in the laboratory of H.F. by the National Marfan Foundation. T.B. is a recipient of a fellowship from the Deutsche Forschungsgemeinschaft.

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34 Reinhardt,D.P., Keene,D.R., Corson,G.M., Pöschl,E., Bächinger,H.P., Gambee,J.E. and Sakai,L.Y. (1996) Fibrillin-1: Organization in microfibrils and structural properties. J. Mol. Biol., 258, 104-116. MEDLINE Abstract

35 Downing,A.K., Knott,V., Werner,J.M., Cardy,C.M., Campbell,I.D. and Handford,P.A. (1996) Solution structure of a pair of calcium-binding epidermal growth factor-like domains: Implications for the Marfan syndrome and other genetic disorders. Cell, 85, 597-605. MEDLINE Abstract

36 Beighton,P., De Paepe,A., Danks,D., Finidori,G., Gedde-Dahl,T., Goodman,R., Hall,J.G., Hollister,D.W., Horton,W., McKusick,V.A., Opitz,J.M., Pope,F.M., Pyeriitz,R.E., Rimoin,D.L., Sillence,D., Spranger,J.W., Thompson,E., Tsipouras,P., Viljoen,D., Winship,I. and Young,I. (1988) International nosology of heritable disorders of connective tissue. Am. J. Med. Genet., 29, 581-594. MEDLINE Abstract

37 Aoyama,T., Tynan,K., Dietz,H.C., Francke,U. and Furthmayr, H. (1993) Missense mutations impair intracellular processing of fibrillin and microfibril assembly in Marfan syndrome. Hum. Mol. Genet., 2, 2135-2140. MEDLINE Abstract

38 Aoyama,T., Francke,U., Gasner,C. and Furthmayr,H. (1995) Fibrillin abnormalities and prognosis in Marfan syndrome and related disorders. Am.J. Med. Genet., 58, 169-176.

39 Corson,G.M., Chalberg,S.C., Dietz,H.C., Charbonneau,N.L. and Sakai,L.Y. (1993) Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 5' end.Genomics, 17, 476-484. MEDLINE Abstract

*To whom correspondence should be addressed at: Howard Hughes Medical Institute, Beckman Center, Room B205, Stanford University Medical Center, Stanford, CA 94305-5428, USA

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