The Proprotein Convertase Furin as a Novel Aneurysm Predisposition Gene Impairing TGF-β Signalling

Genetics Cardiovascular System Life Sciences Medicine
aortic aneurysm Furin TGF-β signalling proprotein convertase genetic predisposition

Academic Background

Aortic aneurysm (AA) is a condition characterized by abnormal dilation of the aorta, commonly occurring in the abdominal and thoracic aorta. Aortic aneurysms are more prevalent in individuals over the age of 65, and if not diagnosed and treated promptly, they can lead to fatal ruptures. Although age, smoking, hypertension, and male gender are considered significant risk factors, these factors are neither necessary nor sufficient, indicating that genetic susceptibility plays a crucial role in the disease’s development. Currently, in the majority of aortic aneurysm cases, including familial aortic aneurysms, no known pathogenic genetic variants have been identified. Only about 2% of unselected abdominal aortic aneurysm patients and 5% of unselected thoracic aortic aneurysm patients carry (likely) pathogenic variants in known aortic aneurysm genes. This suggests that most aortic aneurysms have a complex genetic background, potentially involving interactions among multiple genes.

The transforming growth factor-beta (TGF-β) signaling pathway plays a significant role in the development of aortic aneurysms. Furin, a proprotein convertase, is involved in the maturation of the TGF-β precursor. Therefore, Furin is considered a potential susceptibility gene for aortic aneurysms. This study aims to explore the genetic susceptibility of Furin gene variants in aortic aneurysms and their impact on the TGF-β signaling pathway through whole-exome sequencing and functional experiments.

Source of the Paper

This study was conducted by a team of researchers from KU Leuven in Belgium and Erasmus MC in the Netherlands. The primary authors include Zongsheng He, Arne S. Ijpma, and Danielle Majoor-Krakauer, among others. The paper was published online on April 18, 2024, in the journal Cardiovascular Research, titled “The proprotein convertase furin is a novel aneurysm predisposition gene impairing TGF-β signalling.”

Research Process

1. Patient Cohort and Whole-Exome Sequencing

The study included 781 aortic aneurysm patients and their affected relatives who were consecutively diagnosed at Erasmus MC between January 2009 and July 2019. Rare Furin gene variants were detected in these patients through whole-exome sequencing (WES). Sequencing data were aligned to the reference genome (hg19) using the BWA software, variant calling was performed using the GATK software, and variant annotation was carried out using the ANNOVAR tool.

2. Functional Validation of Furin Gene Variants

In vitro experiments, researchers constructed expression vectors for 13 Furin gene variants (e.g., R81C, P169T, V210A, etc.) and performed transient transfection in HEK-293T cells. The effects of these variants on Furin protein maturation, secretion, and enzymatic activity were analyzed using immunoblotting. Additionally, the protease activity of these variants was assessed using a fluorogenic substrate (Pyr-RTKR-AMC).

3. Patient-Derived Fibroblast Experiments

Skin fibroblasts were obtained from seven patients carrying Furin gene variants. The processing of the TGF-β precursor, phosphorylation levels of downstream effectors SMAD2 and ERK1/2, and mRNA expression levels of the TGF-β-responsive gene ACTA2 were analyzed in these cells. Furthermore, the impact of Furin on the TGF-β signaling pathway was validated by knocking down Furin gene expression.

4. Histological and Immunohistochemical Analysis of Aortic Tissue

Histological and immunohistochemical analyses were performed on the ascending aortic aneurysm tissue of a patient carrying the P169T variant. The expression of Furin, collagen, fibrillin, TGF-β, and ACTA2 was compared with healthy control tissue.

Main Results

1. Discovery of Furin Gene Variants

Among the 781 aortic aneurysm patients, 13 rare Furin gene variants were identified in 3.7% (29) of unrelated patients, with 72% of these patients having multiple aneurysms or dissections. These variants showed reduced Furin protein maturation and enzymatic activity in vitro.

2. Impact of Furin Variants on the TGF-β Signaling Pathway

Patient-derived fibroblasts exhibited impaired processing of the TGF-β precursor, reduced phosphorylation levels of SMAD2 and ERK1/2, and decreased mRNA expression of the ACTA2 gene. Knockdown of Furin gene expression further confirmed its regulatory role in the TGF-β signaling pathway.

3. Pathological Changes in Aortic Tissue

The aortic tissue of the patient carrying the P169T variant showed moderate to severe aneurysm characteristics, including elastin fragmentation, smooth muscle cell disorganization, and reduced collagen and fibrillin. Immunohistochemical analysis revealed significantly decreased expression of Furin, TGF-β, and ACTA2.

4. Clinical Characteristics

Among the 29 patients carrying Furin gene variants, 72% had multiple aneurysms, 41% had thoracic aortic aneurysms or dissections, and 58% had medium-sized artery aneurysms. Additionally, 41% of the patients exhibited features of connective tissue disorders, such as Marfanoid habitus, scoliosis, and joint hypermobility.

Conclusion

This study is the first to confirm that the Furin gene is a genetic susceptibility factor for aortic aneurysms. Its variants contribute to the development of aortic aneurysms by affecting the TGF-β signaling pathway. Furin gene variants are relatively common in patients with abdominal aortic aneurysms, thoracic aortic aneurysms, and medium-sized artery aneurysms, and their effects are modulated by individual genetic backgrounds. The study recommends including the Furin gene in the diagnostic gene panel for aortic aneurysms to better identify high-risk patients and family members.

Research Highlights

  1. Newly Discovered Genetic Susceptibility Gene: The Furin gene is the first to be confirmed as associated with aortic aneurysms.
  2. Regulatory Mechanism of the TGF-β Signaling Pathway: The study reveals the role of the Furin gene in aortic aneurysm development through the regulation of the TGF-β signaling pathway.
  3. Multidisciplinary Research Approach: The study combines whole-exome sequencing, in vitro functional experiments, patient-derived cell experiments, and histological analysis to comprehensively validate the function of the Furin gene.
  4. Clinical Significance: The findings provide new insights for the genetic diagnosis and family screening of aortic aneurysms, aiding in the early identification of high-risk individuals and intervention.

Research Value

This study not only reveals the genetic susceptibility of the Furin gene in aortic aneurysms but also provides new perspectives on the role of the TGF-β signaling pathway in the development of aortic aneurysms. The results have significant clinical implications, contributing to the improvement of diagnostic and therapeutic strategies for aortic aneurysms, particularly in the early screening and prevention of familial aortic aneurysms.