A New Mouse Model for Marfan Syndrome Presents Phenotypic Variability Associated with the Genetic Background and Overall Levels of Fbn1 Expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19–24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression

Targetting of <i>Fbn1</i>.

<p>(A) Scheme of <i>Fbn1</i> targeting. From top to bottom, fibrillin1 protein with the internally deleted region (residues 770–1042); schematic representation of the corresponding <i>Fbn1</i> gene region with the probes (I and III) used in the Southern-blot analysis, and the sizes of BamHI (b) and HindIII (h) fragments; and targeted <i>Fbn1</i>^mgΔloxPneo allele with the neoR flanked by lox-P sequences (triangles). (B) Southern blot of genomic DNA from correctly targeted ES clone digested with BamHI, and hybridized to probe I. (C) sequence of the cDNA from the <i>Fbn1</i>^mgΔloxPneo allele showing the junction of exons 18 and 25; (D) Heredogram of founder heterozygotes in a mixed 129/Sv/CD-1 background. Hetreozygotes were phenotypically normal until the F3, where one severely affected heterozygote was found (arrow), whose X-ray is shown. (E) Relative quantification of total <i>Fbn1</i> mRNA levels in wild-type (WT), heterozygous (HE) and homozygous (HO) fetal fibroblasts by Real Time RT-PCR analysis (P<0.05).</p

Phenotypic variability within the 129/Sv strain.

<p>(A) Comparison of histological slides and radiography of 129/Sv heterozygotes at 3 months of age show phenotypic variation in this strain. Wild-type (+/+), asymptomatic (AS) and severely affected (SE) heterozygous mice. (B) Average mean linear intercept at 3 and 6 months of age (*P<0.42, **P<0.0006, ***P<0.006). (C) Average thickness of thoracic aortic media at 3 and 6 months of age (*P<0.33, **P<0.04, †P<0.8, ††P<0.01). (D) Average KR at 3 and 6 months (*P<0.03, **P<0.04). (MI) Mildly affected animals. The number of animals analyzed in each group (n) and the ages (3 m and 6 m represent 3 months and 6 months, respectively) are shown.</p

Correlation between <i>Fbn1</i> expression and phenotype severity in 129/Sv heterozygotes.

<p>(A) Real-time RT-PCR analysis of wild-type (WT – left panel) and mutant (right panel) <i>Fbn1</i> alleles in heterozygous 129/Sv animals. Individual animals are represented by different colored bars. For each animal three technical replicas were performed. Note that, despite the expression variability among different animals, the ratio between mutant and normal <i>Fbn1</i> transcripts is constant. (B to D) Correlation between level of expression of total <i>Fbn1</i> (Y axis) and the severity of (B) lung, (C) vascular (AWT: aortic wall thickness), and (D) skeletal phenotypes in heterozygous 129/Sv animals. Diamonds correspond to the animals represented in (A). Pearson's correlation coefficients (R) and the statistical significance (P) are shown. Note strong correlation (|R|≥0.75) between levels of total <i>Fbn1</i> expression and severity of the phenotype in the three affected systems.</p

Immunofluorescence of cultured fetal fibroblasts.

<p>(A) Wild-type, (B) heterozygous and (C) homozygous fibroblasts immune-stained for fibrillin-1 (scale bars 50 µm). In contrast to the elaborate network of immunoreactive fibrillin-1 seen in control lines, heterozygous and homozygous cell cultures show a diffuse pattern of immunoreactive material. (D) Representative field at higher magnification to show intracellular deposition of mutant protein (scale bar 25 µm).</p