Identification of fibrillin 1 gene mutations in patients with bicuspid aortic valve (BAV) without Marfan syndrome

Bicuspid aortic valve (BAV) is the most common congenital heart disease [1, 2]. BAV and Marfan syndrome (MFS) share some clinical features such as the increased prevalence of thoracic aortic aneurysm (TAA) and dissection (TAD), as well as overlapping histopathological features [3]. MFS is associated with mutations in the fibrillin 1 (FBN1) gene in more than 90% of patients, and less commonly in the transforming growth factor beta receptor 2 and 1 (TGFBR2, and TGFBR1) genes [4]. While the genetic background of MFS is well described at present, that of BAV is poorly defined.

BAV has been associated with NOTCH1 gene mutations in a few cases [5], and with actin alpha 2 smooth muscle aorta (ACTA2) gene mutations (12%) in a subgroup of patients with TAA, livedo reticularis on the upper and lower limbs, and iris flocculi [6]. Other genes are suspected to be associated, and more chromosomal loci associated to BAV have been reported [7, 8].

We have recently demonstrated a 4-fold increase in the prevalence of BAV in a large cohort of unrelated MFS patients (12 BAV in 257 MFS, 4.7%) with respect to the general population screened by echocardiography including primary school students in Italy (0.5%), detecting FBN1 mutations in 2 out of 3 subjects who consented to undergo DNA mutation analysis [9]. These findings are consistent with data showing decreased FBN1 mRNA or protein content in a subgroup of BAV patients [10], which suggest that FBN1 may be one of the genes associated with BAV.

To date, however, the demonstration of FBN1 mutations in patients with BAV is lacking. Thus, we screened for FBN1 mutations in the selected patients with BAV and thoracic aortic dilation not fulfilling the clinical criteria for MFS.

All patients [8 men and 2 women, age range 24–42 years] were Italian. Two of them (P8 and P10) (Table 1) had MASS syndrome (an acronym for myopia, mitral valve prolapse, aortic dilatation, skeleton features, skin features); most displayed systemic features such as pectus excavatum, scoliosis, pes planus, cutaneous striae, mitral valve prolapse and myopia (Table 1). All had fusion of the right and left coronary aortic valve cusps, and 8 of 10 patients had maximum aortic enlargement at the level of the aortic root and not at the ascending aorta.

To the best of our knowledge, this is the first study reporting pathogenetic fibrillin 1 mutations in patients with BAV and aortic dilatation/aneurysm in whom MFS and other more severe type 1 fibrillinopathies were clinically excluded according to the updated Ghent criteria [4].

The mutations detected in two unrelated patients are all arginine substitutions. The Arg529Gln mutation detected in P1 is reported at the UMD-FBN1 database (http://​www.​umd.​be/​FBN1/​4DACTION/​WV/​2451) in a male proband of France geographic origin with an incomplete MFS phenotype. Unfortunately, no further information on BAV occurrence in this patient is available. Moreover, a single nucleotide substitution in the same codon causing a preterminal stop codon was previously described in a Norwegian patient displaying a classic Marfan phenotype with ectopia lentis, thoracic aorta dilatation and systemic features [15]. According to Polyphen-2 and MuPro, the Arg529Gln mutation is probably damaging and contributes to decreased protein stability. P2 carried two mutations, one of which (Arg2726Trp) has been previously associated with variable clinical phenotypes, including mitral valve prolapse and myopia [14], isolated skeletal features [16], combined skeletal and ocular manifestations [17], mild skeletal abnormalities [18]; and a family in which the mutation appeared incompletely penetrant [19]. The Arg2726Trp mutation was reported in one chromosome in 1000Genomes and NHLBI Exome Variant Server databases as rs61746008 (http://​www.​1000genomes.​org/​ and http://​evs.​gs.​washington.​edu/​EVS/​). The other mutation (Arg636Gly) identified in P2 has never been reported in the literature, although another single nucleotide substitution responsible for a different aminoacid change at the same codon (Arg636Ile) in a MFS patient with aortic root dilatation, ectopia lentis and minor involvement of skeleton was previously described [17]. According to SIFT, both the Arg2726Trp and Arg636Gly mutations are classified as damaging, with decreased protein stability as evaluated in silico by MuPro.

It is unknown at present if the two mutations at the FBN1 locus identified in our patient are in cis, on the same chromosome, or in trans. The infrequent presence of double mutations has been reported in many human gene-causing diseases such as hypertrophic cardiomyopathy [20, 21] and arrhythmias associated with the lamin A/C (LMNA) gene [22].

The detection of FBN1 point mutations in patients with BAV with aortic dilatation/aneurysm but without MFS adds to the striking clinical heterogeneity of type I fibrillinopathies to include a small number of patients bearing the most common congenital heart disease. These data also provide further evidence of the heterogeneity of the BAV syndrome [12], with demonstration that aortic dilatation/aneurysm develops in a subgroup of patients as a manifestation of an inherited connective tissue disorder, including FBN1 mutations in a minority of patients. It is noteworthy that the two patients carrying the mutations displayed a family history of TAA in one and MVP in the other, without systemic features which were otherwise prevalent in the remaining subjects. Moreover, both had aortic aneurysm size attaining the threshold for surgery notwithstanding the young age [23, 24], with the largest diameter localized at the level of the sinuses of Valsalva, which is the less prevalent phenotype of aortic dilatation in BAV individuals [1, 25]. Della Corte, et al., first named this pattern as “root phenotype” [25] and have subsequently demonstrated that it may be a marker of more severe aortopathy warranting closer surveillance [26]. Interestingly, 8 out of our 10 patients displayed this phenotype, in association with a certain degree of systemic characteristics suggestive of a connective tissue disorder. Finally, the 2 patients bearing FBN1 mutations had significant aortic regurgitation, which is a powerful predictor of loss of aortic medial elastic fibers in patients with ascending aortic aneurysms and aortic valve disease [27]. These findings call for greater focus on the BAV-related cardiovascular abnormalities rather than on the MFS-like systemic features, which may well coexist and warrant investigation in BAV patients in general, but are not associated with the FBN1 mutations identified in the present study. On the other hand, these FBN1 mutations do not completely fulfill the definition of the major criterion for MFS according to the revised Ghent criteria because they have never been detected in Marfan patients with TAA [4]. Therefore our two BAV/TAA patients did not achieve the diagnosis of MFS.

The FBN1 gene has been previously associated with various conditions, including MFS, neonatal MFS, Shprintzen-Goldberg syndrome, marfanoid neonatal progeroid syndrome, familial arachnodactyly, ectopia lentis, isolated ascending aortic aneurysm and dissection, aortic root dilatation without dissection, skeletal and skin abnormalities (MASS phenotype), Marfan-like syndromes, autosomal dominant Weill-Marchesani syndrome (WMS), mitral valve prolapse, and sclerodermia. Recently, mutations in the FBN1 gene were reported in two other syndromes [28, 29]. The interfamilial clinical heterogeneity at the FBN1 locus is further characterized by a striking intrafamilial variability (OMIM*134797).

Contrasting data have been reported regarding the genetic background of BAV-related aortopathy. A decrease in FBN1 mRNA and protein has been demonstrated in some BAV patients suggesting a possible involvement of FBN1 with BAV [10]. Moreover, single nucleotide polymorphisms (SNPs) spread in the area of the FBN1 gene, which predispose to TAA, have been reported [30]. On the other hand, other investigators have screened BAV patients for mutations in FBN1, TGFBR2, and TGFBR1 genes and failed to detect any mutation, concluding that the FBN1 gene is not, or only rarely, associated with BAV [31]. More recently, a mutation in the TGFBR2 gene was reported in a patient classified as aortic dilatation/aneurysm but otherwise not well defined clinically [32]. Another recent study, comparing gene expression in subjects with BAV and tricuspid aortic valves, reported an increase of FBN1 mRNA only in the subjects with tricuspid aortic valves [33]. Thus, it is conceivable that BAV represents the phenotypic manifestation of many distinct clinical outliers underlined by genetic, molecular, and structural anomalies that do not follow a common path [7].

Our findings may have relevant clinical implications in the future, if confirmed by larger studies. Although multiple similarities have been shown between MFS and BAV patients, recent improvements in knowledge regarding the natural history of the aortopathy in BAV [34‐36] have raised concerns regarding the direct application of surgical criteria adopted in MFS patients to those with BAV and aortic dilatation/aneurysm [37, 38]. On the other hand, cardiovascular events are considerable in patients with FBN1 mutations and remain so throughout life, with men appearing to be at higher risk for an aortic event than women [39]. While we acknowledge that the size of the present study and its retrospective nature do not allow conclusions regarding the indications for surgery, our findings support the need of future studies aimed to characterize BAV patients with aortic dilatation/aneurysm on the basis of their aortic phenotype and other clinical stigmata of connective tissue disorders, eventually performing genetic testing when appropriate. Whether such an approach would result in a different outcome, thus affecting therapeutic choices in patients with BAV and aortic dilatation/aneurysm, should be a pivotal aim of such future research.

The novel implication of the present findings is the need of a multidisciplinary approach (including internal medicine, medical genetics, cardiology, ophthalmology, cardiovascular surgery, orthopaedic, and molecular biology experts) in the global assessment and management of patients with BAV. Due to the high prevalence of this disorder and its multispecialty requirements, our findings suggest a tailored diagnostic and therapeutic approach, which should be addressed in future prospective studies.

In conclusion, this current findings expand the concept that BAV is a heterogenous disorder with a wide spectrum of clinical manifestations. Beyond the common phenotype of isolated BAV and the previously reported association of BAV in MFS patients [9], we described BAV patients in whom the clinical diagnosis of MFS has been excluded, carrying mutations in FBN1 gene. These data also expands the clinical spectrum of the type 1 fibrillinopathies to include BAV. These results underscore the importance of accurate clinical characterization of BAV aortopathy, including investigation of systemic connective tissue manifestations and genetic testing.