Preferential pre‐mRNA utilisation of an upstream cryptic 5′ splice site created by a single base deletion mutation in exon 37 of the FBN‐1 gene

Abstract

A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin-1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5′ splice site in exon 37 which was utilised in preference to the normal 5′ splice site during pre-mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48-bp deletion from the 3′ end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16-amino-acid sequence from a central eight-cysteine repeat motif of the fibrillin-1 molecule. Interestingly, the cryptic 5′ splice site in exon 37 and the normal 5′ splice site had equally strong consensuses for splice-site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre-mRNA splicing. Analysis by reverse-transcription PCR indicated that, in the patients skin fibroblasts, the steady-state level of the mis-spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse-chase biosynthetic labelling indicated that close to normal amounts of fibrillin-1 were being synthesised and secreted by the cells. However, in contrast to control cells cultured from an unaffected individual, little fibrillin-1 was detected, either biosynthetically or by immunofluorescence, in the extracellular matrix produced by the proband's fibroblasts. Thus, the slightly shorter mutant fibrillin-1 molecules appeared to be exerting a powerful dominant-negative effect on the incorporation of normal fibrillin-1 molecules into microfibrils in this culture system. This severe inhibition of microfibril synthesis in cell culture contrasts with the 'classic' phenotype of the proband, suggesting that factors influencing microfibril formation may differ greatly between in vivo and in vitro environments.Keywords : cryptic splicing ; exon mutation; fibrillin; Marfan syndrome. FBN-1, the gene for fibrillin-1, a 350-kDa glycoprotein component of extracellular microfibrils [1Ϫ3], have been causally linked to the congenital disorder, Marfan syndrome and related phenotypes [4Ϫ6]. Marfan syndrome is a common (about 1 in 10 000 live births) autosomal dominant condition of connective tissue which affects the cardiovascular, skeletal and ocular systems. The syndrome is characterised by a range of abnormalities including overgrowth of the ribs and long bones, distension of the ascending aorta, heart valve defects, arachnodactyly, and lens dislocation. Although penetrance is complete, the severity and range of clinical features are variable, often within the same family. Even individuals with mild skeletal abnormalities can die prematurely, usually from dissecting aneurysm of the aorta [4]. The rate of detection of fibrillin-1 gene (FBN-1) mutations in Marfan patients is currently about 20% of those screened, although higher detection rates have been reported using heteroduplex analysis of...

Qualitative and quantitative analysis of FBN1 mRNA from 16 patients with Marfan Syndrome

“…This finding is explained by introduction of a CSS, which may cause splicing at a position of a transcript where it is usually not spliced [ 35 ]. Introduction of CSS has previously been associated with human diseases, including MFS [ 36 , 37 ].…”

Microfibril-associated Glycoprotein-2 Specifically Interacts with a Range of Bovine and Human Cell Types via αVβ3 Integrin

“…Endothelial cells were obtained from the umbilical arteries of 210-day-old bovine fetuses using the method of Wall et al (34). Human skin fibroblasts obtained from normal adult subjects have been described previously (35). Human osteoblastic cells, grown from trabecular bone explants of normal adults, were a kind gift from Dr. D. Haynes (Department of Pathology, University of Adelaide).…”