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 5

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(A) Drawing depicting cDNA resulting from proper homologous recombinatio...
(A) Drawing depicting cDNA resulting from proper homologous recombination of the endogenous Fbn1 allele with the targeting vector for the C1039G mutation. Above is a representation of the genomic segment prior to Cre-mediated recombination to remove the NeoR flanked by loxP sequences. (B) Representative Southern blot demonstrating correct targeting before and after Cre-mediated loxP recombination. Left lane is from a WT, untargeted mouse, with an undigested 21-kb KpnI fragment; middle lane is from a C1039G heterozygous mutant mouse without Cre-recombinase, resulting in an untargeted band (21 kb) and a targeted band (5.4 kb); right lane is from a C1039G heterozygous mutant mouse after Cre-mediated recombination, resulting in an untargeted band (21 kb) and a targeted band (3.2 kb). (C) Northern blot demonstrating that the level of expression of Fbn1 in mice heterozygous or homozygous for the C1039G allele is unchanged in comparison with untargeted (WT) mice. Expression is normalized to expression of the gene encoding β-actin (βACT). The level of Fbn1 expression in heterozygous and homozygous targeted mice, relative to that seen in WT, is indicated.