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Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe marfan syndrome
1Howard Hughes Medical Institute, Stanford University Medical Center, CA 94305, USA.
Abstract:
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.
Insights
Researchers developed a long RT-PCR method to detect exon-skipping mutations in the fibrillin-1 (FBN1) gene, identifying six such mutations in Marfan syndrome patients. These mutations lead to reduced matrix deposition, correlating with severe Marfan syndrome forms.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Marfan syndrome (MFS) is a heritable connective tissue disorder.
- Mutations in the fibrillin-1 (FBN1) gene are a primary cause of MFS.
- Exon-skipping represents a significant category of FBN1 mutations.
Purpose of the Study:
- To develop a rapid method for detecting exon-skipping mutations in the FBN1 gene.
- To identify and characterize FBN1 exon-skipping mutations in Marfan syndrome patients.
Main Methods:
- Development of a long reverse transcription-polymerase chain reaction (RT-PCR) assay.
- Amplification of full-length or large segments of FBN1 cDNA from fibroblast RNA.
- Analysis of RT-PCR products using restriction fragment patterns and direct sequencing.
- Pulse-chase analysis to assess fibrillin protein synthesis and matrix deposition.
Main Results:
- Six distinct exon-skipping mutations in FBN1 were identified in 60 MFS probands.
- All identified skipped exons encode calcium-binding epidermal growth factor (EGF)-like domains and maintain the reading frame.
- Five mutations resulted from splice site point mutations, and one from a donor splice site deletion.
- Pulse-chase experiments showed normal fibrillin synthesis but significantly reduced matrix deposition, indicating a dominant-negative effect.
Conclusions:
- The developed long RT-PCR method is effective for detecting FBN1 exon-skipping mutations.
- Exon-skipping mutations in FBN1 can lead to severe Marfan syndrome phenotypes, including neonatally lethal forms.
- The identified mutations impair fibrillin matrix deposition, contributing to the pathophysiology of Marfan syndrome.