A nonsense variant in FBN1 caused autosomal dominant Marfan syndrome in a Chinese family: a case report.

BACKGROUND: Marfan syndrome (MFS) is a common autosomal dominant inherited disease, and the occurrence rate is around 0.1-0.2‰. The causative variant of FNB1 gene accounts for approximately 70-80% of all MFS cases. In this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene. This finding extended the variant spectrum of the FBN1 gene and will provide a solution for patients to bear healthy offspring by preimplantation genetic testing or prenatal diagnosis. CASE PRESENTATION: The patient was treated due to tachycardia during excitement in a hospital. Echocardiography showed dilatation of the ascending aorta and main pulmonary artery, mitral regurgitation (mild), tricuspid regurgitation (mild), and abnormal left ventricular filling. Electrocardiograph showed sinus rhythm. In addition, flutters of shadows in front of his eyes and vitreous opacity were present in the patient. Genomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated controls. Potential variants were screened out by next-generation sequencing and confirmed by MLPA & Sanger sequencing. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the relative mRNA quantitation in the patient. A heterozygous nonsense variant c.3217G > T of the FBN1 gene, which resulted in p. Glu1073Term, was identified in both patients. Only wild type bases were found in the cDNA sequence of the patient. Real-time fluorogenic quantitative PCR results showed that the relative expression level of FBN1 cDNA in the patient was only about 21% compared to that of normal individuals. This variant c.3217G > T of the FBN1 gene introduces a Stop codon in the cb-EGF12 domain. We speculated that a premature translational-termination codon (PTC) was located in the mRNA and the target mRNA was disintegrated through a process known as nonsense-mediated mRNA decay (NMD), which led to a significant decrease of the fibrillin-1 protein, eventually causing clinical symptoms in the patient. CONCLUSIONS: In this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene, which eventually led to Marfan syndrome in a Chinese family.

Assessment of Bones Deficient in Fibrillin-1 Microfibrils Reveals Pronounced Sex Differences.

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFß) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFß signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches.

Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-ß antibody, angiotensin II (AngII), anti-TGF-ß antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-ß dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-ß dependent.

An integrative systems approach identifies novel candidates in Marfan syndrome-related pathophysiology.

Marfan syndrome (MFS) is an autosomal dominant genetic disorder caused by mutations in the FBN1 gene. Although many peripheral tissues are affected, aortic complications, such as dilation, dissection and rupture, are the leading causes of MFS-related mortality. Aberrant TGF-beta signalling plays a major role in the pathophysiology of MFS. However, the contributing mechanisms are still poorly understood. Here, we aimed at identifying novel aorta-specific pathways involved in the pathophysiology of MFS. For this purpose, we employed the Fbn1 under-expressing mgR/mgR mouse model of MFS. We performed RNA-sequencing of aortic tissues of 9-week-old mgR/mgR mice compared with wild-type (WT) mice. With a false discovery rate <5%, our analysis revealed 248 genes to be differentially regulated including 20 genes previously unrelated with MFS-related pathology. Among these, we identified Igfbp2, Ccl8, Spp1, Mylk2, Mfap4, Dsp and H19. We confirmed the expression of regulated genes by quantitative real-time PCR. Pathway classification revealed transcript signatures involved in chemokine signalling, cardiac muscle contraction, dilated and hypertrophic cardiomyopathy. Furthermore, our immunoblot analysis of aortic tissues revealed altered regulation of pSmad2 signalling, Perk1/2, Igfbp2, Mfap4, Ccl8 and Mylk2 protein levels in mgR/mgR vs WT mice. Together, our integrative systems approach identified several novel factors associated with MFS-aortic-specific pathophysiology that might offer potential novel therapeutic targets for MFS.

Everolimus depletes plaque macrophages, abolishes intraplaque neovascularization and improves survival in mice with advanced atherosclerosis.

BACKGROUND AND AIMS: Inhibition of the mechanistic target of rapamycin (mTOR) is a promising approach to halt atherogenesis in different animal models. This study evaluated whether the mTOR inhibitor everolimus can stabilize pre-existing plaques, prevent cardiovascular complications and improve survival in a mouse model of advanced atherosclerosis. METHODS: ApoE-/-Fbn1C1039G+/- mice (n = 24) were fed a Western diet (WD) for 12 weeks. Subsequently, mice were treated with everolimus (1.5 mg/kg daily) or vehicle for another 12 weeks while the WD continued. RESULTS: Despite hypercholesterolemia, everolimus treatment was associated with a reduction in circulating Ly6Chigh monocytes (15 vs. 28% of total leukocytes, p = 0.046), a depletion of plaque macrophages (2.1 vs. 4.1%, p = 0.040) and an abolishment of intraplaque neovascularization, which are all indicative of a more stable plaque phenotype. Moreover, everolimus reduced hypoxic brain damage and improved cardiac function, which led to increased survival (100 vs. 67% of animals, p = 0.038). CONCLUSIONS: Everolimus enhances features of plaque stability and counters cardiovascular complications in ApoE-/-Fbn1C1039G+/- mice, even when administered at a later stage of the disease.

Axitinib attenuates intraplaque angiogenesis, haemorrhages and plaque destabilization in mice.

AIM: An increased density of intraplaque (IP) microvessels in ruptured versus nonruptured human plaques suggests that IP neovascularization has a major causative effect on plaque development and instability. Possibly, vascular endothelial growth factor (VEGF) or other angiogenic factors mediate IP microvessel growth and plaque destabilization. Because apolipoprotein deficient mice with a heterozygous mutation (C1039G+/-) in the fibrillin-1 gene (ApoE-/-Fbn1C1039G+/-) manifest substantial IP neovascularization, they represent a unique tool to further investigate angiogenesis and its role in atherosclerosis. Here, we examined whether administration of axitinib (inhibitor of VEGF receptor-1,-2 and -3) inhibits IP neovascularization and stabilizes atherosclerotic plaques. METHODS: ApoE-/-Fbn1C1039G+/- mice were fed a western diet (WD) for 20weeks. After 14weeks WD, mice received axitinib (35µg/g) or solvent i.p. 4×/week for 6weeks. Cardiac function was monitored to evaluate the effect of axitinib on atherosclerosis-driven complications such as myocardial infarction. RESULTS: Axitinib significantly reduced IP neovascularization, with subsequent less prevalence of IP haemorrhages. The smooth muscle cell content doubled, whereas the amount of macrophages decreased. Overall cardiac function was improved in axitinib-treated animals. Moreover, the number of animals with myocardial infarction was decreased by 40%. Coronary plaque formation was observed in almost all control animals whereas treated animals showed a 30% reduction in the occurrence of coronary plaques. CONCLUSIONS: Inhibition of VEGF receptor signalling by axitinib attenuates intraplaque angiogenesis and plaque destabilization in mice.

Gross deletions in FBN1 results in variable phenotypes of Marfan syndrome.

BACKGROUND: A mutation in FBN1 is primarily attributed to Marfan syndrome (MFS). So far, >1800 unique FBN1 mutations have been identified, with the vast majority being single-nucleotide substitutions, small deletions, and insertions. The rearrangement of large fragments of FBN1 accounts for only 1.7% of all variants. The aim of this study was to investigate the characteristics of large genomic rearrangements in FBN1 among MFS patients and to evaluate the correlations between genotype and phenotype. METHODS: Systematic sequencing of the disease-related genes FBN1, TGFBR1, and TGFBR2, was carried out previously for 26 unrelated patients with MFS. No small mutations were found. Subsequently, multiplex ligation-dependent probe amplification was performed for the detection of copy number variations in these patients. The breakpoints were determined by gap PCR and sequencing. Transcription level analysis was conducted in patients whose RNA sample was available. RESULTS: Four gross deletions were identified in FBN1. Three deletions (exons 6, 48-53, and 49-50) were predicted to be in-frame deletions; the remaining deletion (exons 1-36) was expected to induce the loss of one copy of the FBN1 gene. The breakpoints of these four deletions were cloned, and revealed deletion sizes of 16,551, 10,346, 4563, and 187,047bp, respectively. Patients with in-frame deletions of exons 48-53 and 49-50 showed severe clinical phenotypes; Patient with an exon 6 deletion showed mild potential MFS phenotypes. And the patient had classic MFS with a deletion of exons 1-36. CONCLUSIONS: We characterized four large genomic rearrangements in FBN1. FBN1 haploinsufficiency correlated with a classic MFS phenotype, while in-frame deletions between exons 24-53 of FBN1 tended to cause severe clinical phenotypes.