Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

Cardiovascular surgery in Marfan syndrome: implications of new molecular concepts in thoracic aortic disease

Acute dissection and rupture of aortic aneurysms comprise for 1-2% of all deaths in industrialized countries. Dilation of the aorta is caused by a multitude of mechanisms including inherited connective tissue disorders such as Marfan syndrome (MFS). MFS is one of the most common inherited connective tissue disorders affecting 1 in 5000 individuals. Although the phenotype of MFS can be quite variable, aneurysmal dilation of the aortic root and consecutive acute aortic dissection is the leading cause of death in this patient population. Over the past years it has been shown that a comprehensive understanding of this disorder provides greater understanding of vascular wall biology and identifies pathways relevant to aortic aneurysms and dissection in general. The current review discusses the surgical management of patients with MFS with a special emphasis on indications for surgery in this complex group of patients.

Should aortic arch replacement be performed during initial surgery for aortic root aneurysm in patients with Marfan syndrome?

OBJECTIVES: The aim of this study was to investigate whether total arch replacement (TAR) during initial surgery for root aneurysm should be routinely performed in patients with Marfan syndrome (MFS). METHODS: Retrospective analysis of 94 consecutive MFS patients fulfilling Ghent criteria who underwent 148 aortic surgeries and were followed at this institution during the past 16 years. RESULTS: The mean follow-up interval was 8.8 ± 7 years. Initial presentation was acute aortic dissection (AAD) in 35% of patients (76% Type A and 24% Type B) and aneurismal disease in 65%. TAR was performed in 8% of patients during initial surgery for AAD (otherwise a hemi-arch replacement was performed) and 1.6% in elective root repair. Secondary TAR had to be performed in only 3% of patients without, but in 33% following AAD (33% Type A and 33% Type B; P = 0.0001). Thirty-day, 6-month, 1-year and overall mortalities were 3.2, 5.3, 6.4 and 11.7%, respectively. Operative and 30-day mortalities in secondary aortic arch replacement were zero. Secondary TAR after AAD did not increase the need for the replacement of the entire thoracoabdominal aorta during follow-up compared with patients without secondary TAR (37 vs 40%, P = 1.0). CONCLUSIONS: MFS patients undergoing elective root repair have small risk of reinterventions on the aortic arch, and primary prophylactic replacement does not seem to be justified. In patients with AAD, the need for reinterventions is precipitated by the dissection itself and not by limiting the procedure to the hemi-arch replacement in the emergency setting. Limiting surgery to the aortic root, ascending aorta and proximal aortic arch is associated with low mortality in MFS patients presenting with AAD.

Identification and in silico analyses of novel TGFBR1 and TGFBR2 mutations in Marfan syndrome-related disorders

Very recently, heterozygous mutations in the genes encoding transforming growth factor beta receptors I (TGFBR1) and II (TGFBR2) have been reported in Loeys-Dietz aortic aneurysm syndrome (LDS). In addition, dominant TGFBR2 mutations have been identified in Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). In the past, mutations of these genes were associated with atherosclerosis and several human cancers. Here, we report a total of nine novel and one known heterozygous sequence variants in the TGFBR1 and TGFBR2 genes in nine of 70 unrelated individuals with MFS-like phenotypes who previously tested negative for mutations in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1). To assess the pathogenic impact of these sequence variants, in silico analyses were performed by the PolyPhen, SIFT, and Fold-X algorithms and by means of a 3D homology model of the TGFBR2 kinase domain. Our results showed that in all but one of the patients the pathogenic effect of at least one sequence variant is highly probable (c.722C > T, c.799A > C, and c.1460G > A in TGFBR1 and c.773T > G, c.1106G > T, c.1159G > A, c.1181G > A, and c.1561T > C in TGFBR2). These deleterious alleles occurred de novo or segregated with the disease in the families, indicating a causative association between the sequence variants and clinical phenotypes. Since TGFBR2 mutations found in patients with MFS-related disorders cannot be distinguished from heterozygous TGFBR2 mutations reported in tumor samples, we emphasize the importance of segregation analysis in affected families. In order to be able to find the mutation that is indeed responsible for a MFS-related phenotype, we also propose that genetic testing for sequence alterations in TGFBR1 and TGFBR2 should be complemented by mutation screening of the FBN1 gene.

Large genomic fibrillin-1 (FBN1) gene deletions provide evidence for true haploinsufficiency in Marfan syndrome

Mutations in the FBN1 gene are the major cause of Marfan syndrome (MFS), an autosomal dominant connective tissue disorder, which displays variable manifestations in the cardiovascular, ocular, and skeletal systems. Current molecular genetic testing of FBN1 may miss mutations in the promoter region or in other noncoding sequences as well as partial or complete gene deletions and duplications. In this study, we tested for copy number variations by successively applying multiplex ligation-dependent probe amplification (MLPA) and the Affymetrix Human Mapping 500 K Array Set, which contains probes for approximately 500,000 single-nucleotide polymorphisms (SNPs) across the genome. By analyzing genomic DNA of 101 unrelated individuals with MFS or related phenotypes in whom standard genetic testing detected no mutation, we identified FBN1 deletions in two patients with MFS. Our high-resolution approach narrowed down the deletion breakpoints. Subsequent sequencing of the junctional fragments revealed the deletion sizes of 26,887 and 302,580 bp, respectively. Surprisingly, both deletions affect the putative regulatory and promoter region of the FBN1 gene, strongly indicating that they abolish transcription of the deleted allele. This expectation of complete loss of function of one allele, i.e. true haploinsufficiency, was confirmed by transcript analyses. Our findings not only emphasize the importance of screening for large genomic rearrangements in comprehensive genetic testing of FBN1 but, importantly, also extend the molecular etiology of MFS by providing hitherto unreported evidence that true haploinsufficiency is sufficient to cause MFS.

Proteomic analysis in aortic media of patients with Marfan syndrome reveals increased activity of calpain 2 in aortic aneurysms

BACKGROUND: Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation. METHODS AND RESULTS: Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin. CONCLUSIONS: Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

Circulating transforming growth factor-beta in Marfan syndrome

BACKGROUND: Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-beta (TGF-beta). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-beta activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-beta might be mirrored in circulating TGF-beta concentrations. METHODS AND RESULTS: Serum obtained from MFS mutant mice (Fbn1(C1039G/+)) treated with losartan was analyzed for circulating TGF-beta1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-beta1 concentrations increased with age and were elevated in older untreated Fbn1(C1039G/+) mice compared with wild-type mice (P=0.01; n=16; mean+/-SEM, 115+/-8 ng/mL versus n=17; mean+/-SEM, 92+/-4 ng/mL). Losartan-treated Fbn1(C1039G/+) mice had lower total TGF-beta1 concentrations compared with age-matched Fbn1(C1039G/+) mice treated with placebo (P=0.01; n=18; 90+/-5 ng/mL), and circulating total TGF-beta1 levels were indistinguishable from those of age-matched wild-type mice (P=0.8). Correlation was observed between circulating TGF-beta1 levels and aortic root diameters in Fbn1(C1039G/+) and wild-type mice (P=0.002). In humans, circulating total TGF-beta1 concentrations were elevated in patients with MFS compared with control individuals (P<0.0001; n=53; 15+/-1.7 ng/mL versus n=74; 2.5+/-0.4 ng/mL). MFS patients treated with losartan (n=55) or beta-blocker (n=80) showed significantly lower total TGF-beta1 concentrations compared with untreated MFS patients (P< or =0.05). CONCLUSIONS: Circulating TGF-beta1 concentrations are elevated in MFS and decrease after administration of losartan, beta-blocker therapy, or both and therefore might serve as a prognostic and therapeutic marker in MFS.

Suburothelial myofibroblasts in the human overactive bladder and the effect of botulinum neurotoxin type A treatment

BACKGROUND: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). OBJECTIVE: To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic DO (NDO) or idiopathic DO (IDO) and whether this is affected by successful treatment of DO with botulinum neurotoxin type A (BoNTA). DESIGN, SETTING, AND PARTICIPANTS: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre, open-label study of intradetrusor BoNTA injections. Control tissue was obtained from 10 patients undergoing pelvic-floor repair procedures who had no overactive bladder (OAB) symptoms. This study is registered with ClinicalTrials.gov, number NCT00662064. INTERVENTIONS: Bladder biopsies performed with flexible cystoscopes were obtained from control subjects and from NDO and IDO patients before BoNTA treatment and at 4 wk and 16 wk after treatment. They were studied with quantitative immunofluorescence using antibodies to connexin 43 (Cx43), vimentin, and c-kit. MEASUREMENTS: Differences in Cx43, vimentin, and c-kit immunoreactivity between control subjects and NDO or IDO patients (primary outcomes). Changes in NDO or IDO, Cx43 immunoreactivity, and c-kit immunoreactivity after BoNTA treatment (secondary outcomes). RESULTS AND LIMITATIONS: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNTA treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNTA treatment. CONCLUSIONS: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.

Petrology and geochronology of "muscovite age standard" B4M

Muscovite B4M, distributed in 1961 as an age standard, was ground under ethanol. Five grain size fractions were obtained and characterized by X-ray diffraction. They display a mixing trend between a phengitic (enriched in the fraction <0.2 µm) and a muscovitic component (predominant in the fraction >20 µm). High-pressure phengite is preserved as a relict in retrograde muscovite. Electron microprobe analyses of the distributed mineral separate reveal at least four white mica populations based on Si, Al, Mg, Na, Fe and F. Rb/K ratios vary by one order of magnitude. Rb–Sr analyses link the mineralogical heterogeneity to variable Rb/Sr and 87Sr/86Sr ratios. The grain size fractions define no internal isochron. Relict fine-grained phengite gives older ages than coarse-grained retrograde greenschist facies muscovite. The inverse grain size–age relationship also characterizes 39Ar/40Ar analyses. Cl/K anticorrelates with step ages: Cl-rich coarse muscovite is younger than Cl-poor fine relict phengite. Sr and Ar preserve a similar isotopic inheritance despite peak metamorphism reaching 635±20 °C. A suitable mineral standard requires that its petrological equilibrium first be demonstrated. Relicts and retrograde reaction textures are a guarantee of isotopic disequilibrium and heterogeneous ages within single crystal at the micrometre scale.

Quantitative high-resolution warm season rainfall recorded in varved sediments of Lake Oeschinen, northern Swiss Alps: calibration and validation AD 1901–2008

High-resolution, well-calibrated records of lake sediments are critically important for quantitative climate reconstructions, but they remain a methodological and analytical challenge. While several comprehensive paleotemperature reconstructions have been developed across Europe, only a few quantitative high-resolution studies exist for precipitation. Here we present a calibration and verification study of lithoclastic sediment proxies from proglacial Lake Oeschinen (46°30′N, 7°44′E, 1,580 m a.s.l., north–west Swiss Alps) that are sensitive to rainfall for the period AD 1901–2008. We collected two sediment cores, one in 2007 and another in 2011. The sediments are characterized by two facies: (A) mm-laminated clastic varves and (B) turbidites. The annual character of the laminae couplets was confirmed by radiometric dating (210Pb, 137Cs) and independent flood-layer chronomarkers. Individual varves consist of a dark sand-size spring-summer layer enriched in siliciclastic minerals and a lighter clay-size calcite-rich winter layer. Three subtypes of varves are distinguished: Type I with a 1–1.5 mm fining upward sequence; Type II with a distinct fine-sand base up to 3 mm thick; and Type III containing multiple internal microlaminae caused by individual summer rainstorm deposits. Delta-fan surface samples and sediment trap data fingerprint different sediment source areas and transport processes from the watershed and confirm the instant response of sediment flux to rainfall and erosion. Based on a highly accurate, precise and reproducible chronology, we demonstrate that sediment accumulation (varve thickness) is a quantitative predictor for cumulative boreal alpine spring (May–June) and spring/summer (May–August) rainfall (rMJ = 0.71, rMJJA = 0.60, p < 0.01). Bootstrap-based verification of the calibration model reveals a root mean squared error of prediction (RMSEPMJ = 32.7 mm, RMSEPMJJA = 57.8 mm) which is on the order of 10–13 % of mean MJ and MJJA cumulative precipitation, respectively. These results highlight the potential of the Lake Oeschinen sediments for high-resolution reconstructions of past rainfall conditions in the northern Swiss Alps, central and eastern France and south-west Germany.

Prospective risk stratification of sudden cardiac death in Marfan's syndrome.

BACKGROUND Marfan syndrome (MFS) is a variable, autosomal-dominant disorder of the connective tissue. In MFS serious ventricular arrhythmias and sudden cardiac death (SCD) can occur. The aim of this prospective study was to reveal underlying risk factors and to prospectively investigate the association between MFS and SCD in a long-term follow-up. METHODS 77 patients with MFS were included. At baseline serum N-terminal pro-brain natriuretic peptide (NT-proBNP), transthoracic echocardiogram, 12-lead resting ECG, signal-averaged ECG (SAECG) and a 24-h Holter ECG with time- and frequency domain analyses were performed. The primary composite endpoint was defined as SCD, ventricular tachycardia (VT), ventricular fibrillation (VF) or arrhythmogenic syncope. RESULTS The median follow-up (FU) time was 868 days. Among all risk stratification parameters, NT-proBNP remained the exclusive predictor (hazard ratio [HR]: 2.34, 95% confidence interval [CI]: 1.1 to 4.62, p=0.01) for the composite endpoint. With an optimal cut-off point at 214.3 pg/ml NT-proBNP predicted the composite primary endpoint accurately (AUC 0.936, p=0.00046, sensitivity 100%, specificity 79.0%). During FU, seven patients of Group 2 (NT-proBNP ≥ 214.3 pg/ml) reached the composite endpoint and 2 of these patients died due to SCD. In five patients, sustained VT was documented. All patients with a NT-proBNP<214.3 pg/ml (Group 1) experienced no events. Group 2 patients had a significantly higher risk of experiencing the composite endpoint (logrank-test, p<0.001). CONCLUSIONS In contrast to non-invasive electrocardiographic parameter, NT-proBNP independently predicts adverse arrhythmogenic events in patients with MFS.