Molecular characterisation of heritable thoracic aortic diseases at the Tuscany referral centre for Marfan syndrome and related disorders

Heritable Thoracic Aortic Diseases (HTAD) are broadly classified as syndromic (with systemic manifestations) or non-syndromic (isolated). The main syndromic conditions associated with aortopathy are Marfan syndrome and Loeys-Dietz syndrome. Marfan Syndrome (MFS) is the most common autosomal dominant connective tissue disorder, caused by mutations in the FBN1 gene, encoding fibrillin 1, a component of the extracellular matrix whose altered availability and/or structure may also contribute to a dysregulated Transforming Growth Factor-beta (TGF-β) signalling. MFS is characterized by prominent skeletal, ocular and cardiovascular manifestations, primarily aortic root dilatation. As concerns Loeys-Dietz Syndrome (LDS), it is represented by an autosomal dominant disorder caused by mutations affecting the TGF-β signalling pathway genes (TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 and TGFB3). LDS is clinically characterised by a triad of hypertelorism, bifid uvula/cleft palate, and widespread arterial aneurysms/tortuosity. SMAD3 variants (LDS type 3) cause a syndromic form of HTAD associated with significant osteoarthritis features. Phenotypic studies suggest that LDS patients, particularly those with mutations in TGFBR1, TGFBR2, or SMAD3, are prone to aortic dissection at smaller aortic diameters and younger ages than MFS patients. This study aimed to perform the molecular characterisation of individuals with HTAD by matching genetic information obtained via Next-Generation Sequencing with clinical characteristics. The primary goal was to establish correlations between genotype and phenotype, mainly focusing on vascular manifestations, in patients harbouring pathogenic (P) or likely pathogenic (LP) variants in FBN1 and in LDS genes. A cohort of 275 subjects referred to the Tuscany Referral Centre for aortopathy was analysed. Genomic DNA was manually extracted from whole blood and assessed using Nanodrop One and the Qubit® Fluorometric Method. Library preparation was carried out throught Agilent SureSelect protocol, and sequencing was performed on the Illumina MiSeq platform. Bioinformatics analysis followed GATK guidelines, utilising BWA-MEM for alignment and GATK4 HaplotypeCaller for variant calling. Variants identified were next classified according to ACMG (American College of Medical Genetics) criteria (Richards S et al., Genet Med.2015). Among these patients, n=37 carried Likely Pathogenic (LP)/Pathogenic (P) variants (n=29 in FBN1 gene and n=8 in TGFβ pathway genes), whereas 238 did not show the presence of causative variants in these genes. The patient group with aortopathy but without LP/P variants in FBN1 and TGFβ pathway genes showed a higher prevalence of traditional cardiovascular risk factors, whose burden may contribute to the clinical phenotype, whereas patients with aortopathy and with LP/P variants in FBN1 and TGFβ pathway genes showed a reduced age at the first visit and a higher rate of thoracic aortic surgery. We focused attention on the 37 patients with LP/P variants in the abovementioned genes. Among 29 patients carrying FBN1 variants, we observe that individuals with mutations determining the introduction of a premature stop codon or a defect in the mRNA maturation process potentially associated with haploinsufficiency (HI) have a lower age at first visit and a higher median systemic score compared to those with dominant negative (DN) variants. Among DN variant carriers, a worse phenotype was in particular associated with variants involving conserved Cysteine residues. Data from the present work also showed the contribution of SMAD3 variants in influencing, beyond vascular aortic manifestations widely described in LDS patients, also non-aortic phenotypes. We also compared clinical manifestations between subjects with FBN1 variants and those with TGFβ pathway genes variants, thus confirming a more severe aortic phenotype in LDS patients, also showing a higher dissection rate, and highlighting a higher prevalence of non-aortic manifestations in these patients. Moreover, LDS patients showed a delayed age at diagnosis as well as a delayed age at elective surgery, with respect to FBN1 carriers, so supporting the need of an earlier multidisciplinary diagnostic assessment in order to adequately clinically frame these patients.