Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm

Abstract

Loeys-Dietz syndrome (LDS) associates with a tissue signature for high transforming growth factor (TGF)-β signaling but is often caused by heterozygous mutations in genes encoding positive effectors of TGF-β signaling, including either subunit of the TGF-β receptor or SMAD3, thereby engendering controversy regarding the mechanism of disease. Here, we report heterozygous mutations or deletions in the gene encoding the TGF-β2 ligand for a phenotype within the LDS spectrum and show upregulation of TGF-β signaling in aortic tissue from affected individuals. Furthermore, haploinsufficient Tgfb2(+/-) mice have aortic root aneurysm and biochemical evidence of increased canonical and noncanonical TGF-β signaling. Mice that harbor both a mutant Marfan syndrome (MFS) allele (Fbn1(C1039G/+)) and Tgfb2 haploinsufficiency show increased TGF-β signaling and phenotypic worsening in association with normalization of TGF-β2 expression and high expression of TGF-β1. Taken together, these data support the hypothesis that compensatory autocrine and/or paracrine events contribute to the pathogenesis of TGF-β-mediated vasculopathies.

Figure 1. Phenotypic characteristics of patients with TGFB2 mutation

Significant clinical features of individuals with TGFB2 mutations include mild hypertelorism (widely spaced eyes; 1-II:1, 3-III:1 and 7-III:1), malar hypoplasia (flat cheek bones;1-II:1,3-III:1, 4-II:1 and 7-III:1), retrognathia (receding chin; 1-II:1, 3-III:1, 4-II:1 and 7-III:1), arachnodactyly (long fingers; 1-II:1 and 4-II:1), pectus excavatum (7-III:1), pes planus (flat feet; 1-II:1 and 3-III:1) and hammer toes (1-II:1). Permission to publish photographs was obtained from the affected individuals or their parents.

Figure 2. Mutational Analysis of TGFB2 in aneurysm patients

(A.) Schematic representation of the microdeletions on chromosome 1q41. The TGFB2 gene is indicated in red. Pedigrees for two patients with de novo chromosomal microdeleletions completely overlapping TGFB2 (1-II:1 and 2-II:1) are shown. (+) Indicates presence of the described mutation in an associated individual while (−) indicates lack of mutation. (B-C.) TGFB2 mutations and pedigrees for families 3–8. Pedigrees document an autosomal dominant pattern of inheritance. Mutations are annotated at the nucleotide (c.) and protein level (p.; three letter code for amino acids is used; reference transcript: Ensembl ENST00000366929 or NCBI NM_001135599.2). Circle, female; square, male; open symbol, unaffected; shaded symbol, affected; diagonal line, deceased. The location of mutations in relation to the exons (numbers) of TGFB2 and the domain organization is shown (LAP; latency associated peptide; RKKRA potential furin cleavage site). Evolutionary conservation of the mutated residues in TGFB2 and related human cytokines (TGFB1/3) is shown.

Figure 3. Cardiovascular Pathology in Human Subjects with TGFB2 mutations

(A.) Multidetector computed tomography (MDCT) with 3 dimensional reconstruction of head and neck vessels demonstrating tortuosity of the distal cervical internal carotid arteries bilaterally (arrows, center panel) as well as the V1 segment of the left vertebral artery (arrows, left panel). MDCT imaging in modified sagittal view of dilated aorta at sinuses of Valsalva (arrows, right panel), Bars= 2 cm. (B.) Movat’s pentachrome staining of human aortic samples demonstrating an increase in proteoglycan deposition (Blue staining in Movat’s pentachrome) and elastic fragmentation (Black in Movat’s pentachrome) in MFS, LDS, and patient with TGFB2 mutation (7:III-1) versus control, Bar= 200µM, Enlargement Bar= 80µM. (C.) Masson’s Trichrome staining of human aortic samples with increased collagen deposition (Blue in Masson’s Trichrome) in MFS, LDS, and patient with TGFB2 mutation (7:III-1) versus control, Bar= 200µM, Enlargement Bar= 80µM. (D.) Immunocytochemical staining of the aortic media for phosphorylated Smad2 protein, CTGF, TGFB1, and TGFB2. Panels show control aorta (Control) and patient aorta with TGFB2 mutation (7:III-1). Quantification of fraction of pSmad2 positive nuclei (pSMAD2) or staining (CTGF, TGFB1, TGFB2) represents staining of three control aortas (Co) versus patients 7:III-1 and 5:II-2 (Pts), Error bars equal 2 SEM, (*p<0.05). Bar= 80µM.

Figure 4. Haploinsufficiency for Tgfb2 causes aortic root aneurysm in mice

(A.) Parasternal long axis echocardiographic systolic images of the aortic root of 8 month old wild type (n=10), Tgfb2^+/− (n=6), and Fbn1^+/C1039G mice (n=9). Arrows denote root dimension. Bar= 0.75 mm (B.) Echocardiographic quantification of dimensions at the aortic valve (AoV), aortic root (AoR), sinotubular junction (STJ), and ascending aorta (AscAo) in wild type, Tgfb2^+/− and Fbn1^+/C1039G mice at 8 months of age. (†p<0.005, †† p<0.001). There was no significant difference in aortic dimension between Tgfb2^+/− and Fbn1^+/C1039G mice at this age. (C.) Western blot analysis of murine ascending aortas demonstrating increased phosphorylation of Smad2, Smad3, and ERK proteins in 8 month old Tgfb2^+/− and Fbn1^+/C1039G mice. Graphs representing phosphoprotein western blot quantification standardized to GAPDH expression, Error bars equal 2 SEM, (*p<0.05, **p<0.01, ††p<0.001).

Figure 5. Synergistic pathology in Tgfb2^+/−:Fbn1^+/C1039G double heterozygous mice

(A.) Photomicrographs and echocardiographic aortic root quantification of WT (n=8), Tgfb2^+/− (n=8), Fbn1^+/C1039G (n=7), and Tgfb2^+/−: Fbn1^+/C1039G (n=11) mice. Orange arrow demonstrates a large sinus of Valsalva aneurysm. (**p<0.01, †p<0.005, ††p<0.001) Bar = 1.5 mm. (B.) Worsened aortic phenotype from Tgfb2^+/−: Fbn1^+/C1039G double heterozygous mice. Panels of VVG (upper row) and Masson’s Trichrome (bottom row) stained aortas from 4 month old mice demonstrating elastin fragmentation and increased collagen deposition in Tgfb2^+/−: Fbn1^+/C1039G mice. Bar= 20 µm. (C.) Immunohistochemistry of phosphorylated Smad2 in aortas from 4 month old WT, Tgfb2^+/−, Fbn1^+/C1039G, and Tgfb2^+/−: Fbn1^+/C1039G mice. Bar= 20 µm (D.) Transcript analysis of ascending and descending aortas of two month old WT (n=3), Tgfb2^+/− (n=3), Fbn1^+/C1039G (n=3), and Tgfb2^+/−: Fbn1^+/C1039G (n=3) mice normalized to GAPDH expression, Error bars equal 2 SEM, (*p<0.05).