Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome
Daniel P. Judge, … , Lynn Y. Sakai, Harry C. Dietz
Daniel P. Judge, … , Lynn Y. Sakai, Harry C. Dietz
Published July 15, 2004
Citation Information: J Clin Invest. 2004;114(2):172-181. https://doi.org/10.1172/JCI20641.
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Article Cardiology

Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome

Abstract

Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome–based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.

Authors

Daniel P. Judge, Nancy J. Biery, Douglas R. Keene, Jessica Geubtner, Loretha Myers, David L. Huso, Lynn Y. Sakai, Harry C. Dietz

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Figure 6

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(A) Cysteine substitution in fibrillin-1 results in impaired microfibril...
(A) Cysteine substitution in fibrillin-1 results in impaired microfibril formation. Cultured dermal fibroblasts from WT, heterozygous (C1039G/+), or homozygous (C1039G/C1039G) mice (for the Fbn1 mutation) were plated at 5 × 105/ml and immunostained for fibrillin-1 using polyclonal anti–fibrillin-1 Ab 9543 after 72 hours of confluence. The quantity and quality of the microfibrils containing the product of the mutant allele appears diminished. (B) Representative aortic wall sections from mice aged 6 months, stained with H&E (left) and Safranin-O (right). Top panels are from a WT control, and bottom panels are from a mouse heterozygous for the C1039G mutation (C1039G/+). Mutant mice have focal areas of medial thickening with disorganization and disruption of elastic lamellae, as well as frequent areas of proteoglycan deposition (arrow). Magnification, ×40 for all sections. Scale bar: 25 μm. (C) Representative aortic wall sections from mice heterozygous for the C1039G mutation and harboring the WT FBN1 transgene, +Tg(WT), or the C1663R-mutant FBN1 transgene, +Tg(mut3). Aortic wall organization is greatly improved by the WT transgene and partly improved by the mutant transgene. Magnification, ×40 for all sections. Scale bar: 25 μm. (D) Average aortic wall thickness (μm) from at least five mice of each genotype at age 6 months. For each mouse, at least six independent sections were examined, and for each section, four measures of the aortic wall thickness were averaged, including the thickest and thinnest, for each section.