MUTANT FIBRILLIN-1 MONOMERS LACKING EGF-LIKE DOMAINS DISRUPT MICROFIBRIL ASSEMBLY AND CAUSE SEVERE MARFAN-SYNDROME

Marfan syndrome (MFS), a heritable connective tissue disorder, is caus ed by mutations in the gene coding for fibrillin-1 (FBN1), an extracel lular matrix protein, One of the three major categories of FBN1 mutati ons involves exon-skipping, To rapidly detect such mutations, we devel oped a long RT-PCR method, Either three segments covering the entire F BN1 coding sequence or a single 8.9 kb FBN1 coding segment were amplif ied from reverse-transcribed total fibroblast RNA, Restriction fragmen t patterns of these RT-PCR products were compared and abnormal fragmen ts were directly sequenced, Six exon-skipping mutations were identifie d in a panel of 60 MFS probands, All skipped exons encode calcium bind ing epidermal growth factor (EGF)-like domains and maintain the readin g 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 norm al levels of synthesis but significantly reduced matrix deposition, Th is dominant-negative effect of the mutant monomers is considered in th e light of current models of fibrillin assembly, Probands with this ty pe of FBN1 mutation include the most severe forms of MFS, such as neon atally lethal presentations.