Laurence Campens

Novel MYH11 and ACTA2 mutations reveal a role for enhanced TGFβ signaling in FTAAD

BACKGROUND Thoracic aortic aneurysm/dissection (TAAD) is a common phenotype that may occur as an isolated manifestation or within the constellation of a defined syndrome. In contrast to syndromic TAAD, the elucidation of the genetic basis of isolated TAAD has only recently started. To date, defects have been found in genes encoding extracellular matrix proteins (fibrillin-1, FBN1; collagen type III alpha 1, COL3A1), proteins involved in transforming growth factor beta (TGFβ) signaling (TGFβ receptor 1 and 2, TGFBR1/2; and SMAD3) or proteins that build up the contractile apparatus of aortic smooth muscle cells (myosin heavy chain 11, MYH11; smooth muscle actin alpha 2, ACTA2; and MYLK). METHODS AND RESULT In 110 non-syndromic TAAD patients that previously tested negative for FBN1 or TGFBR1/2 mutations, we identified 7 ACTA2 mutations in a cohort of 43 familial TAAD patients, including 2 premature truncating mutations. Sequencing of MYH11 revealed an in frame splice-site alteration in one out of two probands with TAA(D) associated with PDA but none in the series of 22 probands from the cohort of 110 patients with non-syndromic TAAD. Interestingly, immunohistochemical staining of aortic biopsies of a patient and a family member with MYH11 and patients with ACTA2 missense mutations showed upregulation of the TGFβ signaling pathway. CONCLUSIONS MYH11 mutations are rare and typically identified in patients with TAAD associated with PDA. ACTA2 mutations were identified in 16% of a cohort presenting familial TAAD. Different molecular defects in TAAD may account for a different pathogenic mechanism of enhanced TGFβ signaling.

Reference Values for Echocardiographic Assessment of the Diameter of the Aortic Root and Ascending Aorta Spanning All Age Categories

Thoracic aortic dilatation requires accurate and timely detection to prevent progression to thoracic aortic aneurysm and aortic dissection. The detection of thoracic aortic dilatation necessitates the availability of cut-off values for normal aortic diameters. Tools to evaluate aortic dimension above the root are scarce and inconsistent regarding age groups. The aim of this study was to provide reference values for aortic root and ascending aortic diameters on the basis of transthoracic echocardiographic measurements in a large cohort of children and adults. Diameters at the level of the sinuses of Valsalva (SoV) and ascending aorta (AA) were assessed with transthoracic echocardiography in 849 subjects (453 females, age range 1 to 85 years, mean 40.1 ± 21.3 years) and measured according to published guidelines. Linear regression analysis was applied to create nomograms, as well as equations for upper limits of normal and z-scores. SoV and AA diameters were strongly correlated with age, body surface area (BSA), and weight (r = 0.67 to 0.79, p <0.001 for all). Male subjects had significantly larger aortic dimensions at all levels in adulthood, even after BSA correction (p ≤0.004 for all age intervals). Gender-, age-, and BSA-specific upper limits of normal and z-score equations were developed from a multivariate regression model, which strongly predicts SoV and AA diameters (adjusted R(2) for SoV = 0.84 and 0.67 and for AA = 0.82 and 0.74, for male and female subjects, respectively). In conclusion, this study provides widely applicable reference values for thoracic aortic dilatation screening purposes. Age, BSA, and gender must be taken into account when assessing an individual patient.

Gene panel sequencing in heritable thoracic aortic disorders and related entities – results of comprehensive testing in a cohort of 264 patients

BackgroundHeritable Thoracic Aortic Disorders (H-TAD) may present clinically as part of a syndromic entity or as an isolated (nonsyndromic) manifestation. About one dozen genes are now available for clinical molecular testing. Targeted single gene testing is hampered by significant clinical overlap between syndromic H-TAD entities and the absence of discriminating features in isolated cases. Therefore panel testing of multiple genes has now emerged as the preferred approach. So far, no data on mutation detection rate with this technique have been reported.MethodsWe performed Next Generation Sequencing (NGS) based screening of the seven currently most prevalent H-TAD-associated genes (FBN1, TGFBR1/2, TGFB2, SMAD3, ACTA2 and COL3A1) on 264 samples from unrelated probands referred for H-TAD and related entities. Patients fulfilling the criteria for Marfan syndrome (MFS) were only included if targeted FBN1 sequencing and MLPA analysis were negative.ResultsA mutation was identified in 34 patients (13%): 12 FBN1, one TGFBR1, two TGFBR2, three TGFB2, nine SMAD3, four ACTA2 and three COL3A1 mutations. We found mutations in FBN1 (N = 3), TGFBR2 (N = 1) and COL3A1 (N = 2) in patients without characteristic clinical features of syndromal H-TAD. Six TAD patients harboring a mutation in SMAD3 and one TAD patient with a TGFB2 mutation fulfilled the diagnostic criteria for MFS.ConclusionNGS based H-TAD panel testing efficiently reveals a mutation in 13% of patients. Our observations emphasize the clinical overlap between patients harboring mutations in syndromic and nonsyndromic H-TAD related genes as well as within syndromic H-TAD entities, justifying a widespread application of this technique.

Thoracic aortic-aneurysm and dissection in association with significant mitral valve disease caused by mutations in TGFB2

The pathophysiology of thoracic aortic aneurysms and dissections(TAAD) is complex and multifactorial. Classic cardiovascular risk fac-tors play an important role in a majority of patients but genetic fac-tors should always be considered, especially in younger subjectsand/or in the presence of a family history of TAAD. To date, severalgenes have been identified in both syndromic and non-syndromicforms of TAAD, including FBN1 (Marfan syndrome, MFS), TGFBR1/2(Loeys-Dietz syndrome, LDS), SMAD3 (Aneurysm-Osteoarthritissyndrome, AOS), ACTA2 (TAA6, TAAD with livedo reticularis and irisflocculi), MYH11 (TAAD with patent ductus arteriosus), and MYLK(TAAD7) [1–7]. Although clinical features show significant overlap,these entities differ in the extent of vascular involvement and clinicalcourse. As a consequence, molecular studies have become pivotal inthe evaluation, counselling and management of the patient withTAAD. Moreover, the identification of new genes has led to new in-sights into the pathogenesis of aneurysm formation.The crucial role of the transforming-growth-factor β (TGFβ)pathwayin TAAD became evident from both studies on mouse models and theanalysis of components of the TGF β pathway on human aortic tissue ofpatients with these disorders [3,8–11].WepreviouslysequencedTGFB2as a candidate gene for TAAD, but did not identify mutations in 40 pa-tients with isolated aortic root dilatation (Callewaert et al., unpublishedresults). The recent findings by Boileau and colleagues identifying thefirst TGFB2 mutations leading to familial TAAD in association with cere-brovascular disease and mild systemic features reminiscent of Marfansyndrome [16] have urged us toscreenthis geneinapatientgroupasso-ciatingTAADwithawiderphenotypicspectrumincludingcerebrovascu-lar disease, arterial tortuosity, ma rfanoid skeletal features and mitralvalve disease. In total, we assessed the prevalence of TGFB2 mutationsin 146 patients.Using direct sequencing after ampli fication of all exons and flankingintronic sequences of the TGFB2 gene on genomic DNA level (TGFB2NM_001135599.2), we identified 4 heterozygous TGFB2 mutations in 6patients: c.475C>T (p.Arg159X), c.979C>T (p.Arg327Trp), c.980G>A(p.Arg327Gln), and c.1125delT (p.Gly376GlufsX17). We found 2 prema-turetruncatingmutationsandalsoidenti fiedthe firstmissensemutationsin TGFB2. All mutations are expected to result in a loss-of-function. Anoverview of the clinical findings is provided in Table 1 and of the muta-tions in Fig. 1 and Supplemental Table 1.Aortic pathology was universally present at middle-age (z-scores >2.5),butaorticdissectionwasthepresentingfeatureinonlyonepatientat the age of 69 years (patient 1). Importantly, type A dissection oc-curredinanotherpatient5 yearsafterinitialevaluationandatanaorticroot diameter below the classical surgical threshold of 50 mm (patient6). Significant mitral valve prolapse occurred in 4 patients and was thereasonforinitialevaluationintwopatients(2and6)thatbothrequiredsurgical replacement. Patient 4 came to medical attention following aroutine echocardiography showing aortic root dilatation and mitralvalve prolapse without other manifestations. One patient (patient 3)was evaluated following transient ischemic attacks at the age of18 yearsoldwithunderlyingtortuosityofthevertebralarteries.Finally,patient 5 was evaluated for skeletal marfanoid features.Fouroutof6 patientshadskeletaland/orskinmanifestationsrem-iniscent of TGFβ-signalopathies which can be mild; 3 patients hadmyopia of which one had cataract. Importantly, family history wasnegative in 3 patients.Our data indicate a prevalence of TGFB2 mutations in the exam-ined TAAD cohort of around 4% (6/146), which is significantly lowerthan the previously reported prevalence of ACTA2 mutations (16%)[11]. The TGFβ superfamily includes 3 isoforms of TGFβ, TGFβ1, −2,and −3 (for a review [12]). The TGFβs are pleiotropic cytokines, con-trolling a broadrange of biologicalprocesses. The3 TGFβ isoforms ex-hibit both overlapping and divergent properties as illustrated by thephenotype of the respective knockout mouse models. Tgfb2 knockoutmice die perinatally and display a wide range of developmental de-fects, including cardiovascular, pulmonary, skeletal, ocular, inner ear

Characterization of Cardiovascular Involvement in Pseudoxanthoma Elasticum Families

Objective—Pseudoxanthoma elasticum (PXE) is an autosomal recessive connective tissue disorder with involvement of the skin, the retina, and the cardiovascular system. Cardiovascular involvement is mainly characterized by mineralization and fragmentation of elastic fibers of blood vessels and premature atherosclerosis. We conducted an ultrasound study to investigate the cardiovascular phenotype and to propose recommendations for the management of patients with PXE and heterozygous ABCC6 mutation carriers. Approach and Results—Thirty-two patients, 23 carriers, and 28 healthy volunteers underwent cardiac and vascular ultrasound studies. Cardiac imaging revealed left ventricular diastolic dysfunction in patients with PXE with a significantly prolonged deceleration time and lower septal early diastolic velocities of the mitral annulus compared with controls. Carriers also demonstrated significantly prolonged deceleration time. Carotid-to-femoral pulse wave velocity was significantly increased in patients with PXE when compared with carriers and controls. Vascular imaging revealed a high prevalence of peripheral artery disease in both patients and carriers and a significantly higher carotid intima-media thickness compared with controls. Conclusions—The results of this study clearly demonstrate impaired left ventricular diastolic function, impairment of the elastic properties of the aorta, and a high prevalence of peripheral artery disease in patients with PXE. Carriers also seem to exhibit a cardiovascular phenotype with mainly mild diastolic dysfunction and accelerated atherosclerosis. Increased awareness for cardiovascular events in both patients and heterozygous carriers is warranted.

Genes in thoracic aortic aneurysms/dissections : do they matter?

Over the past decade, our knowledge of the genetic background of thoracic aortic aneurysms and dissections (TAAD) has expanded dramatically. This has not only led to a better understanding of the pathogenesis of the disease but also in risk stratification and medical guidance of patients and their families. Strategies for molecular genetic testing have reached a hinge point with the introduction of high throughput techniques based on Next Generation Sequencing (NGS) in routine diagnostics. It is therefore extremely important that clinicians in the field know the indications and limitations of molecular genetic testing. These will be reviewed in this manuscript.

Intrinsic cardiomyopathy in Marfan syndrome: results from in-vivo and ex-vivo studies of the Fbn1C1039G/+ model and longitudinal findings in humans

Background:Mild intrinsic cardiomyopathy in patients with Marfan syndrome (MFS) has consistently been evidenced by independent research groups. So far, little is known about the long-term evolution and pathophysiology of this finding.Methods:To gain more insights into the pathophysiology of MFS-related cardiomyopathy, we performed in-vivo and ex-vivo studies of 11 Fbn1C1039G/+ mice and 9 wild-type (WT) littermates. Serial ultrasound findings obtained in mice were correlated to the human phenotype. We therefore reassessed left ventricular (LV) function parameters over a 6-y follow-up period in 19 previously reported MFS patients, in whom we documented mild LV dysfunction.Results:Fbn1C1039G/+ mice demonstrated LV contractile dysfunction. Subsequent ex-vivo studies of the myocardium of adult mutant mice revealed upregulation of TGFβ-related pathways and consistent abnormalities of the microfibrillar network, implicating a role for microfibrils in the mechanical properties of the myocardium. Echocardiographic parameters did not indicate clinical significant deterioration of LV function during follow-up in our patient cohort.Conclusion:In analogy with what is observed in the majority of MFS patients, the Fbn1C1039G/+ mouse model demonstrates mild intrinsic LV dysfunction. Both extracellular matrix and molecular alterations are implicated in MFS-related cardiomyopathy. This model may now enable us to study therapeutic interventions on the myocardium in MFS.

Marfan Syndrome and Related Heritable Thoracic Aortic Aneurysms and Dissections.

In this overview we aim to address a number of recent insights and developments regarding clinical aspects, etiology, and treatment of Heritable Thoracic Aortic Disease (H-TAD). We will focus on monogenetic disorders related to aortic aneurysms. H-TADs are rare but they provide a unique basis for the study of underlying pathogenetic pathways in the complex disease process of aneurysm formation. The understanding of pathomechanisms may help us to identify medical treatment targets to improve prognosis. Among the monogenetic aneurysm disorders, Marfan syndrome is considered as a paradigm entity and many insights are derived from the study of clinical, genetic and animal models for Marfan syndrome. We will therefore first provide a detailed overview of the various aspects of Marfan syndrome after which we will give an overview of related H-TAD entities.

Absence of cardiovascular manifestations in a haploinsufficient Tgfbr1 mouse model

Loeys-Dietz syndrome (LDS) is an autosomal dominant arterial aneurysm disease belonging to the spectrum of transforming growth factor β (TGFβ)-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 TGFβ receptor 1 and 2 (TGFBR1 and −2), which lead to a paradoxical increase in TGFβ 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.

Genes in Thoracic Aortic Aneurysms and Dissections - Do they Matter?: Translation and Integration of Research and Modern Genetic Techniques into Daily Clinical Practice.

Since the identification of the fibrillin-1 gene as the causal gene for Marfan syndrome, our knowledge of molecular genetics and the applicability of genetic testing in clinical practice have expanded dramatically. Several new syndromes related to thoracic aortic aneurysms and dissections (TAAD) have been described and the list of underlying genes in syndromal and nonsyndromal TAAD already includes more than 10 different genes and is rapidly expanding. Based on this knowledge, our insights into the underlying pathophysiology of TAAD have improved significantly, and new opportunities for targeted treatment have emerged. Clinicians involved in the care of TAAD patients require a basic knowledge of the disease entities and need to be informed on the applicability of genetic testing in their patients and families. Gene-tailored treatment and management is indeed no science fiction anymore and should now be considered as part of good clinical practice. We provide a systematic overview of genetic TAAD entities and practical recommendations for genetic testing and patient management.