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

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.

PTEN/MMAC1 mutations identified in small cell, but not in non-small cell lung cancers

“…To determine if these exons were homozygously deleted, we co-ampli®ed each of these exons in the presence of control primers for FBN1 exon 36 (Liu et al, 1996) using semi-quantitative PCR conditions, as described in Materials and methods. All the PCR reactions showed clear bands of the control FBN1 exon 36, but some of these reactions clearly showed no PCR ampli®cation of PTEN/MMAC1 exons.…”

“…PCR products were examined for heteroduplexes by subjecting 4 ± 7 ml of each sample, containing approximately 100 ng of PTEN/MMAC1 exon sequence, to high performance liquid chromatography under conditions of partial denaturation (Oefner and Underhill, 1995;Liu et al, 1996). The DHPLC method can separate DNA heteroduplexes from homoduplexes, so that we can determine which PCR products had sequence variations.…”

“…Every PTEN/MMAC1 exon was coampli®ed with control primer pairs speci®c for exon 36 of the ®brillin-1 (FBN1) gene (Liu et al, 1996) as an internal control. Standards were made by mixing normal DNA and PC-3 cell line DNA which is homoxygously deleted for the PTEN/ MMAC1 gene to estimate PTEN/MMAC1 gene losses of 0%, 25%, 50%, 75% and 100%, respectively (data not shown).…”

Fibrillin Assembly Requires Fibronectin

“…Although many publications in recent years have addressed the spatial organization of fibrillins in microfibrils Downing et al, 1996;Liu et al, 1996;Reinhardt et al, 1996;Qian and Glanville, 1997;Baldock et al, 2001;Lee et al, 2004;Baldock et al, 2006;Kuo et al, 2007), relatively little information is available about mechanisms and components involved in microfibril formation. Revealing these mechanisms is ultimately critical for the understanding of the pathobiology of microfibrillopathies associated with mutations in fibrillins.…”

“…Extracted microfibrils from cell culture or tissues display a typical bead-on-a-string ultrastructure, having a 50 -55-nm periodicity when analyzed by electron microscopy after rotary shadowing Kielty et al, 1991). Although many publications over recent years have addressed the spatial organization of fibrillins as it relates to the bead-on-a-string microfibrillar structure Reinhardt et al, 1996;Downing et al, 1996;Liu et al, 1996;Qian and Glanville, 1997;Baldock et al, 2001Baldock et al, , 2006Lee et al, 2004;Kuo et al, 2007), little information is available about the dynamic processes and components involved in microfibril formation. The pathogenetic relevance of this is highlighted by the fact that microfibril assembly is frequently disturbed in patients with fibrillinopathies (Tiedemann et al, 2004).…”