Since 2008, when angiotensin II type I receptor blockade with losartan was introduced in the prevention of cardiovascular manifestation of Marfan syndrome (MFS), a specific treatment to address the cardiovascular lesions became available. The present study aimed to compare the response of such in an unselected cohort of patients with genotyped MFS. At a tertiary university children’s hospital, 20 pediatric and adolescent patients aged 1.7 to 21.6 years with genetically proven MFS were enrolled in a prospective treatment study of losartan for evaluation of the aortic dimensions and elasticity indexes. The mean follow-up period was 33 ± 11 months. A significant reduction in the normalized aortic dimensions with losartan was observed in the valve, root, sinotubular junction, and ascending aortic segments (p = 0.008, p <0.001, p = 0.012, and p = 0.001, respectively). No correlation between elasticity behavior and the decrease in the aortic dimension with losartan therapy was detectable. A significant correlation between stronger improvement and younger age at onset (r = 0.643, p = 0.002) and a longer therapy duration (r = −0.532, p = 0.016) was verifiable. However, no correlation between improvement with therapy and the type of mutation or presentation of clinical forms was remarkable. Elasticity also seemed to improve but not significantly. In conclusion, in our cohort of young patients with MFS, a significant improvement with losartan monotherapy was proved in all affected proximal aortic segments, with a better response to therapy when started at an earlier age and with a longer therapy duration.
Marfan syndrome (MFS) is an autosomal dominantly inherited connective tissue disorder caused by mutations of the fibrillin-1 gene (FBN1), located on chromosome 15q21. Fibrillin-1 is a component of elastic fibers, leading to weaker connective tissue formation, predominantly in the musculoskeletal, ocular, and cardiovascular systems in the case of mutation. Furthermore, it sequesters the large latent complex of transforming growth factor-β binding protein (TGF-β) and regulates its bioavailability for activation. In 2003, Neptune et al demonstrated that dysregulation of TGF-β activation is an important pathogenetic factor contributing to the genesis of MFS, opening new avenues for potential therapeutic strategies. Angiotensin’s activity can influence TGF-β signaling by stimulating TGF-β mRNA and protein expression, probably through its induction of thrombospondin. Angiotensin II and angiotensin II type I (AT1) receptor expression is increased in the MFS aortic tissue and is associated with cystic media necrosis, leading to aneurysm formation. Therefore, AT1-receptor blockade, for example using losartan, induces a decrease in TGF-β signaling by a reduction in the free TGF-β levels. In a mouse model, it was shown that losartan not only prevented, but also reversed, aortic root dilation and elastic fiber degeneration in FBN1-deficient mice. Subsequent to these findings, losartan was tested in 18 pediatric patients with MFS and aggressive aortic disease, with a dramatic stabilization of the aortic root diameters. This prompted us to adopt our current medical strategy in our pediatric population with MFS. We report the results of our prospective, nonrandomized losartan monotherapy study in 20 pediatric patients with MFS on their aortic root dimensions and tissue elasticity.
Methods
The patients were prospectively selected from the Pediatric and Young Adult Marfan Outpatient Clinic (Vienna, Austria). All had been classified according to the newly established Ghent criteria. The inclusion criteria were a clinical diagnosis of MFS according to the current Ghent criteria, the presence of an FBN1-mutation, age 1 to 22 years at the start of therapy, and a minimum of 12 months of follow-up with a minimum of 2 follow-up visits. The exclusion criteria were Marfan-like syndromes, intolerance to AT1-receptor blockers, renal insufficiency, an aortic root diameter of ≥50 mm, and previous aortic root replacement therapy. The institutional review board of the Vienna University Hospital committee on human research and the Austrian Competent Authority of Security and Health approved the study protocol (approval reference number 462/2009). The study was registered in the European Union Drug Regulating Authorities Clinical Trials database (EudraCT No. 2009-016139-36). All patients and/or their guardians provided written informed consent before inclusion in the present study.
A total of 20 patients with genetically confirmed MFS (13 males) fulfilling the study criteria were eligible for evaluation of the effectiveness of losartan treatment, independent of aortic root dilation. A total of 40 matching healthy children and adolescents with normal functioning tricuspid aortic valves, referred during the study period for routine checkups, were prospectively recruited as a cross-section sample control cohort.
Genomic DNA was extracted from peripheral whole blood using the salt extraction method. All coding exons and adjacent introns were amplified by polymerase chain reaction using our own designed primers (primer sequences available on request). The polymerase chain reaction products were subsequently purified and sequenced using an ABI BigDye Terminator Cycle Sequencing kit, version 3.1 (ABI Applied Biosystems, Foster City, California), on a 3130XL sequencer using the manufacturer’s procedures.
Of the 20 patients with MFS, 2 had just recently been diagnosed and the other 18 had been followed up at the Marfan Clinic before initiation of the present study. Of these 18, 9 had been receiving β-blocker therapy and 1 had been receiving dual therapy with a β blocker and an angiotensin-converting enzyme inhibitor before the study initiation. At the beginning of the study, 10 patients started losartan monotherapy as their first medication, 8 of 9 switched from β-blocker to losartan monotherapy, and 1 patient, and the patient with previous β-blocker and angiotensin-converting enzyme inhibitor therapy, continued with β blockers as a new dual therapy with losartan owing to their progressive aortic root dilation. During therapy adjustment, the β blockers were gradually reduced during the first 4 weeks of losartan initiation. In all patients, losartan therapy was started with 0.2 mg/kg twice daily, with the dosage doubled every 2 weeks to the greatest tolerable dosage without arterial hypotension (maximum 1.4 mg/kg/day or 100 mg/day for patients with a body weight of ≥70 kg). A therapeutic level at a minimum of 1 mg/kg should be reached at 3 months at the latest. Home monitoring of blood pressure with weekly contact using electronic mail during medication adjustment was performed.
The baseline examination started after clinical and genetic confirmation of MFS. The patients diagnosed before 2010 were reclassified according to the revised Ghent criteria. Medication was started after the baseline examination. The clinical follow-up examinations were at months 3, 6, and 12 and every 6 months thereafter. During the follow-up period, anamnestic evaluations were performed, covering adverse events, side effects, signs of inadequate low blood pressure, and the development of orthopedic or ocular symptoms, which were checked routinely. The cardiac evaluation was achieved using detailed transthoracic echocardiography and electrocardiography at every follow-up visit.
All echocardiographic measurements were made by the same investigator. Within the echocardiographic assessment—performed with a Vivid E9 scanner (GE Medical, Horton, Norway)—the diameters of the aortic valve, aortic root, sinotubular junction, and ascending aorta were measured perpendicular to the aortic flow in the parasternal long-axis using the inner-edge to inner-edge method. All values were normalized to the current body surface area of the patient. The data are given with the z-score values compared with the normal values; z-scores of ≥2 were considered to indicate dilatation. The elastic properties of the aortic root were measured by M-mode from the parasternal long-axis view. Offline calculations of the mean systolic and diastolic diameters were performed for calculation of the stiffness and distensibility, as previously described. The blood pressure was measured simultaneously at the right forearm using a Philips SureSigns VS2 (Andover, Massachusetts). The stiffness z-score values were calculated using the reference data obtained from the control group.
Statistical analysis was performed with PASW Statistics, version 18.0 (SPSS, Chicago, Illinois). For continuous variables, Student’ t test, for nonparametric parameters, the Kruskal-Wallis test, and for the follow-up data equation, paired t tests were used. Correlation structures were tested using the Pearson parametric correlation coefficient. The data are presented as 2-sided p values, considered statistically significant at p <0.05.
Results
In the patients with MFS, 17 different FBN1 mutations, including 10 novel mutations, were detected; 2 and 3 patients had the same mutation because they belonged to the same families. Of the 17 mutations, 3 (18%) were splice mutations, 6 (35%) caused premature termination codons (frameshift mutations or nonsense mutations), and 8 (47%) were missense mutations (5 involving and 3 not involving a cysteine residue).
The baseline characteristics of the patients with MFS before the start of losartan treatment and their 40 matching controls are listed in Table 1 . The patients with MFS had highly significant greater aortic root dimensions and reduced elasticity values. The characteristic features concerning the MFS symptoms are listed in Table 2 . Because of the known malignant outcome in neonatal patients with MFS, 2 patients presenting with this type of MFS (patients 1 and 5) were excluded from additional analysis. Subdividing the remaining patients and the control cohort into age subgroups of <10, 10 to 15, and >15 years of age showed significant differences in the aortic dimensions and z-scores for all MFS subgroups compared with the control subgroups. A significant increase in stiffness and decrease in distensibility occurred in the patients with MFS who were >10 years of age, with still close to normal levels in the youngest MFS subgroup.
| Variable | MFS Group (n = 20) | Control Group (n = 40) | p Value |
|---|---|---|---|
| Age (yrs) | 0.745 | ||
| Mean ± SD | 11.3 ± 6.3 | 10.8 ± 5.3 | |
| Range | 1.7–21.6 | 0.8–17.7 | |
| Male gender | 13 (65) | 22 (55) | 0.464 |
| Weight (kg) | 41.6 ± 24.3 | 43.1 ± 22.4 | 0.815 |
| Weight z-score | −0.19 ± 1.80 | 0.31 ± 1.16 | 0.272 |
| Height (cm) | 154.1 ± 36.9 | 142.6 ± 35.1 | 0.255 |
| Height z-score | 2.23 ± 1.42 | 0.47 ± 1.38 | <0.001 ∗ |
| BSA (m 2 ) | 1.30 ± 0.53 | 1.29 ± 0.51 | 0.972 |
| Body mass index (kg/m 2 ) | 15.7 ± 3.4 | 18.9 ± 4.0 | 0.002 ∗ |
| Systolic blood pressure (mm Hg) | 100 ± 12 | 104 ± 13 | 0.178 |
| Diastolic blood pressure (mm Hg) | 59 ± 7 | 59 ± 7 | 0.958 |
| Aortic valve diameter (mm) | 23.2 ± 4.9 | 18.0 ± 4.1 | <0.001 ∗ |
| Aortic valve diameter/BSA (mm/m 2 ) | 20.0 ± 6.3 | 15.7 ± 5.1 | 0.011 ∗ |
| Aortic valve z-score | 3.11 ± 1.05 | 0.70 ± 0.95 | <0.001 ∗ |
| Aortic root diameter (mm) | 32.5 ± 7.9 | 22.8 ± 5.3 | <0.001 ∗ |
| Aortic root diameter/BSA (mm/m 2 ) | 27.6 ± 8.9 | 19.9 ± 6.7 | 0.002 ∗ |
| Aortic root z-score | 3.48 ± 1.41 | 0.20 ± 1.04 | <0.001 ∗ |
| Sinotubular junction diameter (mm) | 24.9 ± 5.8 | 18.4 ± 4.6 | <0.001 ∗ |
| Sinotubular junction diameter/BSA (mm/m 2 ) | 21.5 ± 7.7 | 16.0 ± 5.1 | 0.007 ∗ |
| Sinotubular junction z-score | 2.76 ± 1.50 | 0.06 ± 1.02 | <0.001 ∗ |
| Ascending aortic diameter (mm) | 23.0 ± 5.1 | 18.5 ± 4.2 | 0.003 ∗ |
| Ascending aortic diameter/BSA (mm/m 2 ) | 19.7 ± 6.4 | 16.0 ± 5.6 | 0.046 ∗ |
| Ascending aorta z-score | 1.50 ± 1.57 | −0.77 ± −1.07 | <0.001 ∗ |
| Stiffness | 5.98 ± 2.81 | 3.31 ± 0.87 | 0.003 ∗ |
| Stiffness z-score | 3.05 ± 2.95 | 0.01 ± 1.00 | <0.001 ∗ |
| Distensibility (cm 2 × dynes −1 × 10 −6 ) | 5.36 ± 3.06 | 8.19 ± 2.24 | 0.007 ∗ |
| Distensibility (kPa −1 × 10 −3 ) | 43.3 ± 23.8 | 67.2 ± 19.5 | 0.002 ∗ |
| Pt. No. | Birth Year | Gender | Family History | Aortic Root Diameter (mm) | Aortic Root Diameter/BSA (mm/m 2 ) | Aortic Root z-Score | Aortic Stiffness | Aortic Stiffness z-Score | Ectopic Lentis | Systemic Score |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 ∗ | 2008 | F | No | 21.6 | 44.1 | 3.64 | 12.35 | 10.39 | Yes | 7 |
| 2 | 2006 | F | Yes | 19.7 | 28.6 | 1.64 | 3.78 | 0.54 | No | 4 |
| 3 | 2005 | M | No | 25.7 | 31.4 | 3.31 | 2.21 | −1.26 | Yes | 6 |
| 4 | 2005 | M | Yes | 22.2 | 28.0 | 1.77 | 3.81 | 0.57 | No | 1 |
| 5 ∗ | 2004 | M | No | 35.5 | 55.4 | 7.35 | 8.78 | 6.29 | Yes | 16 |
| 6 | 2004 | M | No | 20.8 | 28.5 | 1.51 | 5.20 | 2.17 | Yes | 1 |
| 7 | 2004 | M | No | 24.5 | 33.6 | 3.02 | 5.00 | 1.94 | Yes | 8 |
| 8 | 2000 | M | No | 24.9 | 23.3 | 1.63 | 5.44 | 2.03 | Yes | 6 |
| 9 † | 1998 | M | Yes | 32.9 | 31.9 | 4.52 | 2.92 | −0.44 | No | 7 |
| 10 | 1998 | M | No | 39.7 | 22.6 | 4.03 | 4.05 | 0.85 | No | 10 |
| 11 | 1997 | F | No | 41.8 | 23.1 | 4.41 | 8.13 | 5.54 | No | 6 |
| 12 | 1996 | M | Yes | 41.7 | 26.9 | 5.07 | 6.26 | 3.39 | No | 5 |
| 13 ‡ | 1995 | M | Yes | 37.6 | 17.8 | 2.46 | 10.56 | 8.33 | Yes | 3 |
| 14 † | 1994 | M | Yes | 34.1 | 24.3 | 3.54 | 8.46 | 5.92 | No | 7 |
| 15 ‡ | 1993 | M | Yes | 44.5 | 22.7 | 4.78 | 5.79 | 2.85 | Yes | 3 |
| 16 | 1991 | F | No | 18.7 | 23.1 | 3.71 | 4.14 | 0.95 | Yes | 8 |
| 17 | 1991 | M | Yes | 3.2 | 19.3 | 3.27 | 6.07 | 3.17 | No | 6 |
| 18 | 1991 | F | No | 39.3 | 23.7 | 4.17 | 6.38 | 3.53 | Yes | 5 |
| 19 | 1990 | F | No | 32.3 | 21.8 | 2.74 | 5.08 | 2.04 | Yes | 9 |
| 20 ‡ | 1990 | F | Yes | 40.0 | 20.0 | 3.10 | 4.77 | 1.68 | No | 5 |
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