The importance of genotype-phenotype correlation in the clinical management of Marfan syndrome

This single-centre prospective cohort study included all patients studied in the Hereditary Aortopathy clinic of a tertiary referral hospital who had an initially suspected MFS and presented FBN-1 gene variants between September 2010 and October 2016.

The 532 patients who were studied at the clinic came from 248 non-interrelated families. Patients either presented with aortic complaints that developed at an early age in the absence of known risk factors and/or had family history of aortic events or sudden death, or were a first degree relative of a patient who presented these characteristics.

In the first visit, a thorough physical examination was performed in search of the clinical signs generally associated with syndromic aortopathies, and a transthoracic echocardiogram was also carried out. After this first approach, if the suspicion of hereditary aortopathy (syndromic or not) remained, a blood sample was taken for genetic testing. This was done for 294 patients, in 108 cases directed by clinical suspicion of MFS. All of participants were duly informed about the benefits and risks of having the test, and signed informed consent.

Definite diagnosis of Marfan syndrome was made in accordance with modified Ghent criteria, which state that in the presence of family history, the following findings lead to a definitive diagnosis of MFS: 1) ectopia lentis, or 2) aortic root dilation (Z-score ≥ 2 above 20 years, ≥ 3 below 20 years), or 3) a systemic score ≥ 7 (Table 1). In the absence of a family history, a combination of the following would be necessary: 1) aortic root dilation and ectopia lentis, 2) aortic root dilation and pathogenic FBN-1 mutation, 3) aortic root dilation and systemic score ≥ 7, or 4) ectopia lentis and causal FBN-1 mutation (in this case, as the relationship between the mutation and MFS cannot be established, it is defined as “ectopia lentis syndrome”) [12].

Given the recognised variable clinical presentation of MFS depending on age, with lower expression in younger patients [7, 15], an analysis of baseline characteristics was undertaken for the whole population, above 18 years and below 18 years.

All patients had a transthoracic echocardiogram at each visit, using a GE Vivid T7 (General Electric Company, Connecticut, USA). These studies included M-mode, two dimensional (2D), spectral and color Doppler. In all cases, 2D measurements were taken at the level of the aortic annulus, sinuses of Valsalva, sinotubular junction and ascending aorta, in accordance with the recommendations of current guidelines [16, 17]. Mild aortic dilatation (MAD) was defined as an aortic root or ascending aorta of dimensions higher than the upper limit of the confidence interval at 95% of the distribution of a reference population (Z-score ≥ 2 [18, 19]), but without reaching surgical parameters as defined in current clinical practice guidelines, which is, in any case, 50 mm [17]. Thoracic aorta aneurysm (TA) was defined when the dimensions were higher. The existence of mitral valve prolapse (MVP) was assessed in all patients. The grade of valve regurgitation was assessed and classified as mild, moderate or severe, following American Society of Echocardiography grading [20].

Once the blood samples for genetic analysis had been taken, they were studied in a single reference laboratory, using a massive parallel sequencing library that included 30 genes associated with aneurysm and aortic dissection, of which FBN-1 is one. The FBN-1 variants identified were confirmed by Sanger sequencing in both directions.

The variants were classified as pathogenic if they met the criteria for causal FBN-1 mutations in accordance with modified Ghent criteria (Table 2) [12]. The variants found have been reported following the nomenclature established in the American College of Medical Genetics (ACMG) recommendations [21, 22].

Truncating variants were considered to be those with a nonsense or frameshift effect on the protein.

If findings showed a pathogenic genetic variant, first degree relatives began a cascade screening process consisting of an exhaustive physical examination, a transthoracic echocardiography and genetic testing to screen for the same FBN-1 variant that had been found in the index case.

All patients had a clinical follow-up, at intervals that depended on the clinical presentation and extent of aortic root dilation, in keeping with the recommendations of clinical guidelines. Patients with an aortic diameter larger than recommendations in guidelines (MFS diagnosis and aortic diameter > 50 mm) were referred for aortic surgery [15, 16]. Programmed procedures for TA, without aortic dissection or tear, were classified as prophylactic surgery. In cases of an absence of aortic regurgitation (AR), or in the presence of AR secondary to annular dilation, the options selected were valve-sparing aortic root replacement techniques, either the David procedure (aortic valve reimplanted in a tubular graft and reimplanted coronary ostia) or the Yacoub technique (replacement of the aortic sinuses with or without aortic annuloplasty). In other cases (an emergency situation or significant AR), the chosen procedure was concomitant valve replacement following the classic Bono-Bentall (BB) technique. In our study, the term aortic event included TAs with dimensions within surgical parameters and type A aortic dissections (TAAD). As in previous studies, because there were no patients below 18 years in our event-suffering population, a secondary analysis of the cohort was performed, excluding patients under the age of 18.

Qualitative variables are expressed in percentages and relationship contrasts were analysed using the chi-square (χ [2]) test or, failing that, the Fischer test. Quantitative variables are expressed as a mean ± standard deviation; their distribution was analysed with the Kolmogorov-Smirnov test and intervals were analysed using Student’s t-test for variables that followed a normal distribution, and the Mann-Whitney U for those that did not. All statistical analyses were conducted using SPSS v21 (IBM Corporation, New York, USA).

Of the 532 patients attended at the Hereditary Aortopathy clinic during the study period, genetic studies were requested for clinical indication and suspected MFS for a total 108 patients. Of these, FBN-1 mutations were found in 90 patients (83.33%) corresponding to 58 non-interrelated families. Table 3 contains information about the clinical characteristics of the study population, and a breakdown by age (above or below 18 years). Worthy of note are the low age of the patients studied (31.44 ± 16.92 years) and the higher proportion of male patients (55.6%); 34.4% presented MVP of any grade and 38.9% had a history of EL, which was prevalent in 61.9% of under-18 s. A high proportion of patients are receiving medical treatment with ACE inhibitors/ARBs (48.9%) or beta blockers (58.9%), with lower percentages in children, due either to intolerance or parental refusal. Of the 30% who have undergone surgical intervention, all are adults over the age of 18 who received either prophylactic surgery for TA (18.9%) or emergency surgery for TAAD (7.9%). The reason for intervention (with or without concomitant aortic repair) was MVP with severe mitral regurgitation in 3 patients (3.3%). It is noteworthy that among the prophylactic surgery group of patients, 70.6% were able to benefit from valve sparing aortic root replacement techniques (David or Yacoub). Since 2010, the year in which the Hereditary Aortopathy clinic began, prophylactic surgery has been performed on 11 patients, in 100% of cases using valve-sparing aortic root replacement techniques. Perioperative mortality among these patients was 0%. During a mean follow-up period of 31.43 ± 25.02 months, there were no deaths or emergency procedures among the patients attending consultations at our clinic.

In the 58 families studied, 57 variants of FBN-1 were found. One patient presented with two different FBN-1 variants –c.7754 T > C and c.8176C > T-, and three a priori non-interrelated families that came from the same geographical area presented the same variant, c.6658C > T.

Of the 57 variants in the study, 25 of them (43.9%) had already been published, 23 of which had been identified as associated with MFS, while the remaining 32 (56.1%) are reported for the first time in our study (Table 4).

Of these variants, 11 were de novo (19.3%), that is to say, absence of the same variant was confirmed in both parents. In accordance with the criteria for causal FBN-1 mutations, 46 mutations (80.7%) could be considered pathogenic.

For 84 (93.3%) of the 90 patients with FBN-1 variants, it was possible to give a definite diagnosis of Marfan syndrome in accordance with modified Ghent criteria. The remaining 6 patients were: a 48-year-old woman with systemic characteristics and MVP but no aortopathy, whose variant (p.Met1Lle) has not been previously described and do not fulfill other criteria for causal FBN1 mutation; a 68-year-old male who only presents with TA and a variant of uncertain significance (p.Arg62His); an 8-year-old girl with EL, a systemic score < 7, a normal aortic diameter for her age and a missense variant in exon 2 of FBN-1 (p.Trp71Cys), which could therefore be classified, at least for the time being and pending possibly greater phenotypic expression during her growth, of ectopia lentis syndrome; a 34-year-old woman under study for coronary dissection, with borderline systemic criteria and aortic diameter, who presented a missense type variant in exon 44 (p.Arg1840Cys); a 8-year-old boy with EL and MVP whose variant (p.Arg2335Trp) has not been associated to aortopathy and do not fulfill other criteria for causal FBN1 mutation; and a 30-year-old male with systemic score ≥ 7 and two missense variants - one previously identified as causing MFS, p.Ile2585Thr, and the other also previously published, but with doubts to its causality, p.Arg2726Trp –, but no aortopathy, EL or proven family history.

Among patients with definite diagnosis of MFS, a direct comparison of the two types of variant most frequently found (missense vs. truncating) was performed. Patients with missense variants presented a higher prevalence of EL (56.8 vs. 8.6%, p < 0.001), while those who had truncating variants (nonsense or frameshift) more frequently presented with systemic criteria >7 (54.1 vs. 82.9%, p = 0.006) (Table 5). Of the 44 patients with missense variants, 6 had suffered an aortic event, whereas this figure was 20 of the 35 patients with truncating variants (13.6% vs. 57.1%, p < 0.001). Thus, the majority of the variants observed in the group with aortic events were truncating (71.4%) followed by missense (21.4%) and intronic (7.1%). On the other hand, among the population without aortic events, 67.9% had missense variants (Table 6). In a secondary analysis of the cohort that excluded patients below the age of 18, patients over 18 years without aortic events had a similar frequency of truncating or intronic variants (32.4%) as the patients without events of all ages (32.2%).

Events appeared at slightly earlier ages in patients with truncating variants than in those with missense, although there were no statistically significant differences (41.33 ± 3.77 vs. 37.5 ± 9.62 years, p = 0.162). The median age at time of the aortic event was 40.5, 39.5 and 33.5 years for patients with missense, truncating or intronic variants respectively. The analysis by age for each strata showed 5 patients with aortic events (3 male and 2 female) before the age of 30, of whom 4 (80%) had truncating and 1 (20%) intronic variants. Before the age of 40, 13 patients (8 male, 5 female) had presented an aortic event, in 2 cases with a missense type variant (15.4%), 10 with a truncating variant (76.9%) and 1 with an intronic variant (7.7%). The 2 patients with an aortic event before the age of 40 and a variant that was neither truncating nor intronic were: 1) A male, who underwent surgery at age 38 for TA, with systemic criteria ≥7 and variant p.Gly343Arg in the TGFBP domain of FBN-1, described in previous studies but catalogued by them as “of uncertain significance” [23], who also has a 41 year old brother with TA (aortic diameter of 51 mm) and identification of the same variant; and 2) a female, also operated at 38 years for TA, with systemic criteria and EL, with variant p.Cys1097Tyr in the cb-EGF-like domain of exon 26 of FBN-1 (Additional file 1: Table S1).

In the same way, variant type and the age of event were also compared for patients with prophylactic aortic surgery vs. dissection (Table 6). Patients with dissection (n = 8) less frequently, but not significantly, had missense type variants in comparison with patients undergoing prophylactic aortic surgery (n = 20), (12.5 vs. 25%; p = 0.466). Among the patients with truncating mutation and aortic event, the median age for TAAD was 43 years (n = 7), and for TA was 36 years (n = 13).