Absence of cardiovascular manifestations in a haploinsufficient Tgfbr1 mouse model.
ABSTRACT: Loeys-Dietz syndrome (LDS) is an autosomal dominant arterial aneurysm disease belonging to the spectrum of transforming growth factor ? (TGF?)-associated vasculopathies. In its most typical form it is characterized by the presence of hypertelorism, bifid uvula/cleft palate and aortic aneurysm and/or arterial tortuosity. LDS is caused by heterozygous loss of function mutations in the genes encoding TGF? receptor 1 and 2 (TGFBR1 and -2), which lead to a paradoxical increase in TGF? signaling. To address this apparent paradox and to gain more insight into the pathophysiology of aneurysmal disease, we characterized a new Tgfbr1 mouse model carrying a p.Y378* nonsense mutation. Study of the natural history in this model showed that homozygous mutant mice die during embryonic development due to defective vascularization. Heterozygous mutant mice aged 6 and 12 months were morphologically and (immuno)histochemically indistinguishable from wild-type mice. We show that the mutant allele is degraded by nonsense mediated mRNA decay, expected to result in haploinsufficiency of the mutant allele. Since this haploinsufficiency model does not result in cardiovascular malformations, it does not allow further study of the process of aneurysm formation. In addition to providing a comprehensive method for cardiovascular phenotyping in mice, the results of this study confirm that haploinsuffciency is not the underlying genetic mechanism in human LDS.
Project description:Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-? receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-? signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-? signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-? in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-? signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-? target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-?1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-?1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-? signaling contributes to postnatal aneurysm progression in LDS.
Project description:Loeys-Dietz syndrome (LDS) associates with a tissue signature for high transforming growth factor (TGF)-? signaling but is often caused by heterozygous mutations in genes encoding positive effectors of TGF-? signaling, including either subunit of the TGF-? receptor or SMAD3, thereby engendering controversy regarding the mechanism of disease. Here, we report heterozygous mutations or deletions in the gene encoding the TGF-?2 ligand for a phenotype within the LDS spectrum and show upregulation of TGF-? signaling in aortic tissue from affected individuals. Furthermore, haploinsufficient Tgfb2(+/-) mice have aortic root aneurysm and biochemical evidence of increased canonical and noncanonical TGF-? signaling. Mice that harbor both a mutant Marfan syndrome (MFS) allele (Fbn1(C1039G/+)) and Tgfb2 haploinsufficiency show increased TGF-? signaling and phenotypic worsening in association with normalization of TGF-?2 expression and high expression of TGF-?1. Taken together, these data support the hypothesis that compensatory autocrine and/or paracrine events contribute to the pathogenesis of TGF-?-mediated vasculopathies.
Project description:BACKGROUND: The Loeys-Dietz syndrome (LDS) is a rare autosomal dominant disorder characterized by thoracic aortic aneurysm and dissection and widespread systemic connective tissue involvement. LDS type 1 to 4 are caused by mutations in genes of the TGF-? signaling pathway: TGFBR1 and TGFBR2 encoding the TGF-? receptor (LDS1 and LDS2), SMAD3 encoding the TGF-? receptor cytoplasmic effector (LDS3), and TGFB2 encoding the TGF-?2 ligand (LDS4). LDS4 represents the mildest end of the LDS spectrum, since aneurysms are usually observed in fourth decade and the progression of the disease is slower than in the other forms. CASE PRESENTATION: We report the clinical and molecular findings of an LDS4 Italian family. Genetic testing included TGFBR1, TGFBR2, SMAD3, and TGFB2 analysis by Sanger sequencing. In order to verify the effect of the identified splice mutation, RT-PCR analysis was performed.The proband, a 57-year-old woman, showed high palate, hypoplasic uvula, easy bruising, joint hypermobility, chronic pain, scoliosis, multiple relapsing hernias, dural ectasia, and mitral valve prolapse. Magnetic resonance angiography revealed tortuosity and ectasia of carotid, vertebral, cerebral, and segmental pulmonary arteries. Arterial aneurysm and dissection never occurred. Her 39- and 34-year-old daughters presented with a variable degree of musculoskeletal involvement. Molecular analysis disclosed the novel c.839-1G>A splice site mutation in the TGFB2 gene. This mutation activates a cryptic splice acceptor site in exon 6 leading to frameshift, premature termination codon and haploinsufficiency (p.Gly280Aspfs*41). CONCLUSIONS: Our data confirm that loss-of-function mutations in TGFB2 gene do not always lead to aggressive vascular phenotypes and that articular and skeletal signs are prevalent, therefore suggesting that LDS4 must be considered in patients with sparse signs of LDS and related disorders also in the absence of vascular events.
Project description:A number of autosomal dominantly inherited disorders, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), are associated with predisposition to thoracic aortic aneurysms and dissections (TAADs). In the majority of cases, mutations in genes encoding components of the transforming growth factor-? (TGF-?) signaling pathway, such as FBN1, TGFBR1, TGFBR2 and SMAD3, underlie the disease. Recently, a familial syndromic form of TAAD with other clinical features that overlap the MFS-LDS spectrum has been described to be caused by heterozygous loss-of-function mutations in TGFB2, encoding the TGF-?2 ligand of TGF-? serine/threonine kinase receptors (TGFBRs). We analyzed the TGFB2 gene by sequencing in a cohort of 88 individuals with a Marfan-like phenotype and/or TAAD, who did not have mutations in known genes causing thoracic aortic disease. We identified the novel heterozygous c.1165dupA mutation in exon 7 of TGFB2 in three members of a family, a 51-year-old male, his brother and nephew with aortic aneurysms, cervical arterial tortuosity and/or skeletal abnormalities as well as craniofacial dysmorphisms. The 1-bp duplication causes a frameshift leading to a stable transcript with a premature stop codon after seven TGF-?2-unrelated amino acids (p.Ser389Lysfs*8). As the resulting protein is unlikely functional and by considering data from the literature, we support the notion that functional haploinsufficiency for TGF-?2 predisposes to thoracic aortic disease. Taken together, TGFB2 is a rarely mutated gene in patients with syndromic TAAD, and the clinical features of our TGFB2 mutation-positive individuals fit in the scheme of LDS, rather than MFS-related disorders.
Project description:BACKGROUND: Loeys-Dietz syndrome (LDS) is a rare autosomal dominant disorder showing the involvement of cutaneous, cardiovascular, craniofacial, and skeletal systems. In particular, LDS patients show arterial tortuosity with widespread vascular aneurysm and dissection, and have a high risk of aortic dissection or rupture at an early age and at aortic diameters that ordinarily are not predictive of these events. Recently, LDS has been subdivided in LDS type I (LDSI) and type II (LDSII) on the basis of the presence or the absence of cranio-facial involvement, respectively. Furthermore, LDSII patients display at least two of the major signs of vascular Ehlers-Danlos syndrome. LDS is caused by mutations in the transforming growth factor (TGF) beta-receptor I (TGFBR1) and II (TGFBR2) genes. The aim of this study was the clinical and molecular characterization of two LDS patients. METHODS: The exons and intronic flanking regions of TGFBR1 and TGFBR2 genes were amplified and sequence analysis was performed. RESULTS: Patient 1 was a boy showing dysmorphic signs, blue sclerae, high-arched palate, bifid uvula; skeletal system involvement, joint hypermobility, velvety and translucent skin, aortic root dilatation, tortuosity and elongation of the carotid arteries. These signs are consistent with an LDSI phenotype. The sequencing analysis disclosed the novel TGFBR1 p.Asp351Gly de novo mutation falling in the kinase domain of the receptor. Patient 2 was an adult woman showing ascending aorta aneurysm, with vascular complications following surgery intervention. Velvety and translucent skin, venous varicosities and wrist dislocation were present. These signs are consistent with an LDSII phenotype. In this patient and in her daughter, TGFBR2 genotyping disclosed in the kinase domain of the protein the novel p.Ile510Ser missense mutation. CONCLUSION: We report two novel mutations in the TGFBR1 and TGFBR2 genes in two patients affected with LDS and showing marked phenotypic variability. Due to the difficulties in the clinical approach to a TGFBR-related disease, among patients with vascular involvement, with or without aortic root dilatation and LDS cardinal features, genotyping is mandatory to clarify the diagnosis, and to assess the management, prognosis, and counselling issues.
Project description:Loeys-Dietz syndrome (LDS) is a connective tissue disorder first described in 2005 featuring aortic/arterial aneurysms, dissections, and tortuosity associated with craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations. Heterozygous mutations in 6 genes (TGFBR1/2, TGFB2/3, SMAD2/3), encoding components of the TGF-? pathway, cause LDS. Such genetic heterogeneity mirrors broad phenotypic variability with significant differences, especially in terms of the age of onset, penetrance, and severity of life-threatening vascular manifestations and multiorgan involvement, indicating the need to obtain genotype-to-phenotype correlations for personalized management and counseling. Herein, we report on a cohort of 34 LDS patients from 24 families all receiving a molecular diagnosis. Fifteen variants were novel, affecting the TGFBR1 (6), TGFBR2 (6), SMAD3 (2), and TGFB2 (1) genes. Clinical features were scored for each distinct gene and matched with literature data to strengthen genotype-phenotype correlations such as more severe vascular manifestations in TGFBR1/2-related LDS. Additional features included spontaneous pneumothorax in SMAD3-related LDS and cervical spine instability in TGFB2-related LDS. Our study broadens the clinical and molecular spectrum of LDS and indicates that a phenotypic continuum emerges as more patients are described, although genotype-phenotype correlations may still contribute to clinical management.
Project description:The Loeys-Dietz syndrome (LDS) is a connective tissue disorder affecting the cardiovascular, skeletal, and ocular system. Most typically, LDS patients present with aortic aneurysms and arterial tortuosity, hypertelorism, and bifid/broad uvula or cleft palate. Initially, mutations in transforming growth factor-β (TGF-β) receptors (TGFBR1 and TGFBR2) were described to cause LDS, hereby leading to impaired TGF-β signaling. More recently, TGF-β ligands, TGFB2 and TGFB3, as well as intracellular downstream effectors of the TGF-β pathway, SMAD2 and SMAD3, were shown to be involved in LDS. This emphasizes the role of disturbed TGF-β signaling in LDS pathogenesis. Since most literature so far has focused on TGFBR1/2, we provide a comprehensive review on the known and some novel TGFB2/3 and SMAD2/3 mutations. For TGFB2 and SMAD3, the clinical manifestations, both of the patients previously described in the literature and our newly reported patients, are summarized in detail. This clearly indicates that LDS concerns a disorder with a broad phenotypical spectrum that is still emerging as more patients will be identified. All mutations described here are present in the corresponding Leiden Open Variant Database.
Project description:Loeys-Dietz syndrome (LDS) is a rare connective tissue genetic disorder that is caused by a pathogenic variant in genes of transforming growth factor (TGF) beta receptor 1 (TGFBR1), TGFBR2, mothers against decapentaplegic homolog 2 (SMAD2), SMAD3, TGFB2, or TGFB3. It is characterized by aggressive vascular pathology, aneurysms, arterial tortuosity, bifid uvula, hypertelorism, and cleft palate. Here we present a 42-year-old female patient with LDS. The patient underwent rapidly progressing artery aneurysms and life-threatening aortic dissection. Spontaneous fracture of the first metatarsal bone was noted in her medical record. Physical examination revealed a delayed wound healing on her left abdomen. Considering these clinical manifestations, we speculated that there was a genetic defect in the connective tissue, which provides strength and flexibility to structures such as bones, skins, ligaments, and blood vessels. Thus, whole exome sequencing (WES) was performed on the proband and revealed a heterozygous missense pathogenic variant (c.1613T > C/p.Val538Ala) in TGFBR2, which was a de novo variant in the proband as confirmed by the segregation analysis in parental samples. Although this variant was discovered and associated with the phenotype of LDS previously, the pathogenicity of the variant had not been confirmed by cellular functional assay yet. To further validate the effects of the variant in vitro, we assessed the canonical TGF-? signaling pathway in mutant cells. Our results showed that the p.Val538Ala variant significantly decreased TGF-?-induced gene transcription and the phosphorylation of Smad2, which were consistent with other pathogenic variants of TGFBR2. In conclusion, this study demonstrates that the p.Val538Ala pathogenic variant in TGFBR2 leads to aberrant TGF-? signaling and LDS in this patient.
Project description:Patients with mutations in either TGF-? receptor type I (TGFBR1) or TGF-? receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-? signaling. Similarly, mutations in TGF-? receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-? ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-?2 and TGF-? receptor type III (T?RIII); activation of a T?RI/T?RIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted T?RI/T?RIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-? signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-? signaling may be beneficial for the prevention of congenital craniofacial birth defects.
Project description:Transforming growth factor beta (TGF-?) is a potent inhibitor of cell growth. TGFBR1 6A is a polymorphism consisting of a 9-base pair in-frame deletion within exon 1 of the type I TGF-? receptor (TGFBR1), which results in a receptor with decreased TGF-? signaling capability. The discovery of an association between TGFBR1*6A and cancer susceptibility led to the hypothesis that hypomorphic variants of the TGF-? signaling pathway may predispose to the development of cancer. This hypothesis was tested in vivo with the development of a mouse model of Tgfbr1 haploinsufficiency. Tgfbr1 (+/-) mice developed twice as many intestinal tumors as Tgfbr1 (+/+). Tgfbr1 haploinsufficiency was also associated with early onset adenocarcinoma and increased tumor cell proliferation. A case control study identified two haplotypes associated with constitutively decreased TGFBR1 and substantially increased colorectal cancer risk indicating that TGFBR1 may act as a potent modifier of cancer risk.