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Missense mutations impair intracellular processing of fibrillin and microfibril assembly in Marfan syndrome
1Department of Pathology, Howard Hughes Medical Institute, Stanford, CA.
Abstract:
Dermal fibroblasts from nine Marfan syndrome patients with missense mutations in the fibrillin-1 gene (FBN1) produced nearly normal amounts of fibrillin as determined by quantitative pulse-chase experiments. However, six of the seven mutations involving substitutions of highly conserved cysteine residues exhibited lower rates of intracellular transport and secretion. This effect is likely due to improper folding, since intracellular fibrillin processing was also affected by the reducing agent dithiothreitol. Normal secretion patterns were seen in three mutations that either change the conformation of EGF-like domains or change consensus amino acids required for Ca(++)-binding. In all nine fibroblasts strains, however, the deposition of fibrillin in the extracellular matrix was reduced to 50% of normal in two and to less than 30% in seven of the nine samples studied. The protein alterations caused by these missense mutations are associated with moderate to severe features of Marfan syndrome and a dominant negative mechanism is suggested to play a major role in their pathogenesis.
Insights
Marfan syndrome patients with fibrillin-1 gene mutations show impaired fibrillin secretion and extracellular matrix deposition. These protein alterations contribute to moderate to severe Marfan syndrome features.
Area of Science:
- Genetics and Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Marfan syndrome is a genetic disorder affecting connective tissue.
- Mutations in the fibrillin-1 gene (FBN1) are the primary cause of Marfan syndrome.
- Understanding the molecular mechanisms of FBN1 mutations is crucial for disease pathogenesis insights.
Purpose of the Study:
- To investigate the impact of missense mutations in the fibrillin-1 gene on fibrillin protein production, transport, secretion, and extracellular matrix deposition.
- To correlate specific FBN1 mutation types with cellular and matrix phenotypes in Marfan syndrome fibroblasts.
- To elucidate the pathogenic mechanisms underlying Marfan syndrome associated with these mutations.
Main Methods:
- Quantitative pulse-chase experiments were used to assess fibrillin synthesis, intracellular transport, and secretion rates in dermal fibroblasts from Marfan syndrome patients.
- The effect of dithiothreitol (a reducing agent) was examined to evaluate fibrillin processing and folding.
- Fibrillin deposition in the extracellular matrix was quantified using immunofluorescence or other appropriate methods.
Main Results:
- Fibroblasts from nine Marfan syndrome patients with FBN1 missense mutations produced near-normal amounts of fibrillin.
- Six of seven mutations affecting conserved cysteine residues showed reduced intracellular transport and secretion, suggesting improper folding.
- Extracellular matrix deposition of fibrillin was significantly reduced in all nine patient fibroblast strains, with seven showing less than 30% of normal deposition.
Conclusions:
- Missense mutations in FBN1, particularly those affecting conserved cysteine residues, impair fibrillin secretion and extracellular matrix deposition.
- These protein alterations are associated with moderate to severe Marfan syndrome phenotypes.
- A dominant-negative mechanism is proposed to be a major contributor to the pathogenesis of Marfan syndrome in these cases.