Fibrillin-1 is a major component of the extracellular microfibrils, where it interacts with other extracellular matrix proteins to provide elasticity to connective tissues, and regulates the bioavailability of TGFβ family members. A peptide consisting of the C-terminal 140 amino acids of fibrillin-1 has recently been identified as a glucogenic hormone, secreted from adipose tissue during fasting and targeting the liver to release glucose. This fragment, called asprosin, also signals in the hypothalamus to stimulate appetite. Asprosin levels are correlated with many of the pathologies indicative of metabolic syndrome, including insulin resistance and obesity. Previous studies and reviews have addressed the therapeutic potential of asprosin as a target in obesity, diabetes and related conditions without considering mechanisms underlying the relationship between generation of asprosin and expression of the much larger fibrillin-1 protein. Profibrillin-1 undergoes obligatory cleavage at the cell surface as part of its assembly into microfibrils, producing the asprosin peptide as well as mature fibrillin-1. Patterns of FBN1 mRNA expression are inconsistent with the necessity for regulated release of asprosin. The asprosin peptide may be protected from degradation in adipose tissue. We present evidence for an alternative possibility, that asprosin mRNA is generated independently from an internal promoter within the 3' end of the FBN1 gene, which would allow for regulation independent of fibrillin-synthesis and is more economical of cellular resources. The discovery of asprosin opened exciting possibilities for treatment of metabolic syndrome related conditions, but there is much to be understood before such therapies could be introduced into the clinic.

Marfan syndrome (MFS) is an autosomal dominantly inherited connective tissue disorder. Aortic dilatation/dissection and ectopia lentis are the most severe features, which affect physical functioning and psychological well-being. In Aboriginal Australians, there is little psychosocial research on genetic conditions. This study explored the physical, psychological, and practical impacts of MFS on Aboriginal Australians. Eighteen (8 affected and 10 unaffected) members of a large Aboriginal Australian family with MFS participated in an ethically approved study. Semi-structured qualitative interviews were conducted, transcribed verbatim, and analyzed thematically. All individuals reported challenges from MFS, negatively affecting day-to-day living. Severe vision impairment was perceived as the greatest challenge, contributing to feelings of stigma and exclusion. With aging, concerns shifted toward cardiac complications. The unpredictability of lens dislocation and aortic dissection was reported to be psychologically challenging. Participants described MFS-related barriers to obtaining and retaining employment, especially following cardiac surgery; with consequential psychological and financial hardships. Participants articulated that their cultural drive to support the ill and respectfully mourn the deceased, regardless of distance, resulted in a significant financial burden. Additionally, when hospitalization and/or funerals occurred, financially solvent individuals were expected to share resources, without any expectation of repayment or reciprocity (i.e., 'demand sharing', common in Aboriginal Australian culture). This study documents the nature and pervasiveness of uncertainty for both affected and unaffected members of an MFS family. Many reported challenges are consistent with other MFS cohorts (including stigma, social exclusion, and unemployment). However, our findings suggest that cultural values may exacerbate the financial costs of MFS for Aboriginal Australians.

Basal cell nevus syndrome (also known as Gorlin Syndrome; MIM109400) is an autosomal dominant disorder characterized by recurrent pathological features such as basal cell carcinomas and odontogenic keratocysts as well as skeletal abnormalities. Most affected individuals have point mutations or small insertions or deletions within the PTCH1 gene on human chromosome 9, but there are some cases with more extensive deletion of the region, usually including the neighboring FANCC and/or ERCC6L2 genes. We report a 16-year-old patient with a deletion of approximately 400,000 bases which removes only PTCH1 and some non-coding RNA genes but leaves FANCC and ERCC6L2 intact. In spite of the small amount of DNA for which he is haploid, his phenotype is more extreme than many individuals with longer deletions in the region. This includes early presentation with a large number of basal cell nevi and other skin lesions, multiple jaw keratocysts, and macrosomia. We found that the deletion was in the paternal chromosome, in common with other macrosomia cases. Using public databases, we have examined possible interactions between sequences within and outside the deletion and speculate that a regulatory relationship exists with flanking genes, which is unbalanced by the deletion, resulting in abnormal activation or repression of the target genes and hence the severity of the phenotype.

Dermatology Research Centre, The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia Prince Charles Hospital Clinical Unit, The University of Queensland, School of Clinical Medicine, Brisbane, Queensland, Australia Department of Metabolic Medicine, Queensland Children's Hospital, Brisbane, Queensland, Australia Translational Genomics Group, Institute of Health and Biomedical Innovation, Queensland University of Technology at Translational Research Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia Department of Endocrinology, James Mayne Building, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia

Causal attributions are important determinants of how health threats are processed and affect health-related behaviors. To date, there has been no research on causal attributions in genetic conditions in Aboriginal Australians. Forty members of a large Aboriginal Australian family with Marfan syndrome (MFS) were invited to participate in an ethically approved study exploring causal attributions, including perceived causes of phenotypic variability within the family. Eighteen individuals consented to conduct semi-structured qualitative interviews, which were recorded, transcribed verbatim and analyzed thematically. Most participants knew that MFS was genetic, but there were diverse theories about inheritance, including beliefs that it skipped generations, was affected by birth order and/or gender, and that it co-occurred with inheritance of blue eyes within this family. The mutation was thought to have been inherited from British settlers and initially triggered by disease or diet. Factors believed to modify disease severity included other genes and lifestyle factors, particularly alcohol and substance abuse and stress. Generally, this family did not endorse “blaming” chance or a higher power for phenotypic variability, though some felt that the spirits or a deity may have played a role. In conclusion, although participants knew MFS was a genetic condition, many speculated about the role of non-genetic causes in initiating the original mutation; and the gene-environment interaction was thought to affect severity. This study demonstrates a successful approach for exploring causal attributions in other genetic conditions in First Australians.

BACKGROUND:Marfan syndrome (MFS) is a dominant monogenic disorder caused by mutations in fibrillin 1 (FBN1). Rarely, compound heterozygosity for FBN1 mutations has been described. METHODS:A large kindred with MFS was assessed clinically over decades, and genetically using exome and/or Sanger sequencing. RESULTS:A previously identified FBN1 missense variant (p.Tyr754Cys) was confirmed in all subjects with MFS. An additional variant (p.Met2273Thr), previously associated with incomplete MFS, was identified in three siblings. These three compound heterozygous individuals had aortic dilatation at early age (all <30 years): one also had cerebral and ocular aneurysms; and one, who had undergone surgical repair aged 18 years, died from aortic dissection at 31 years. In contrast, their heterozygous father (p.Tyr754Cys) with MFS died at 57 years (myocardial infarction) without requiring surgical intervention and one heterozygous (p.Tyr754Cys) sibling has aortic dilatation presenting >40 years but not requiring surgical intervention. Another heterozygous (p.Tyr754Cys) sibling did require aortic root repair (28 years). The heterozygous (p.Met2273Thr) mother had aortic dilatation diagnosed at age 68 years but has not required surgical repair. CONCLUSION:Although compound heterozygosity or homozygosity is rare in MFS, it should be considered when there is an unusually severe phenotype in a subset of family members.

Thoracic aortic aneurysm (TAA) develops in about 35% of those with bicuspid aortic valve (BAV) but the cause is unkown. Some studies have identified Notch1 gene mutations in BAV while expression of osteopontin ( OPN ) gene has been reported to be significantly elevated in BAV associated TAA. Furthermore OPN gene expression has been shown to regulate apoptosis and through a CD44 antigen dependent mechanism that regulates autophagy. Apolipoprotein E ( ApoE ) gene mutations have been correlated with atherosclerosis and with BAV stenosis however an association with development of TAA in BAV is not clear. We have previously shown using electron microscopy a significant increase of VSMC apoptosis and autophagy in BAV TAA. To determine the role of Notch1, OPN and ApoE gene function in BAV associated TAA we measured mRNA expression using RT PCR methods. Immunohistochemistry was used to analyse aortic wall degeneration and VSMC loss (OPN, CD44, apoptosis and autophagy). TAA tissue was obtained from 9 subjects (7M, 2F; 57±19yr) with BAV at the time of surgery. Samples of control aorta were obtained from 5 organ donor subjects (TAC; 2M, 3F; 54±13yr). There was significant increase in mRNA expression of OPN (1.42±0.69 vs 0.24±0.09; mean±SD, P≤0.05) and ApoE (1.38±0.55 vs 0.25±0.57; P≤0.05) but not Notch1 (1.24±0.16 vs 0.91±0.23) in BAV VSMC compared to controls. BAV VSMC apoptosis (≤ 30% of total VSMCs; caspase-3+) and autophagy (≥50% of total VSMCs; LC3+) was observed but not in controls. Notch1 protein staining was negligible but there was strong OPN expression in the subendothelial layer co-localised with inner elastic lamina degeneration. Focal OPN expression in medial layer was co-localised with VSMC apoptosis and some autophagy. CD44 expression in BAV TAA was minimal and no co-localisation was found with VSMC autophagy. In summary up-regulation of OPN and ApoE but not Notch1 mRNA was found in BAV TAA. This was associated with OPN protein up-regulation that co-localised with areas of VSMC apoptosis and autophagy in the absence of CD44. The findings suggest a potentially important role for OPN and ApoE gene function in VSMC death independent of CD44 in BAV associated TAA

Background: The expression of transforming growth factor beta (TGF-beta) and Smad3 regulates extracellular matrix homeostasis and inflammation in aortic aneurysms. The expression of Smad3 depends on signaling by angiotensin II (AngII) receptor pathways through TGF-beta receptor-dependent and - independent pathways.Methods: To determine the expression of AngII type 1 (AT(1)R) and type 2 receptors (AT(2)R), TGF-beta, and Smad3 in thoracic aortic aneurysms, we performed immunohistochemistry testing on tissue and cultured cells derived from subjects with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) malformation and from normal aortas of subjects who were organ donors.Results: MFS and BAV aneurysm tissue showed enhanced accumulation of TGF-beta and Smad3 in vascular smooth muscle cells (VSMCs) and in inflammatory cells in the subintimal layer and tunica media. The normal aortic wall exhibited minimal TGF-beta and Smad3 staining. Cultured VSMCs from MFS and BAV samples showed nuclear Smad3 and strong cytoplasmic TGF-beta expression in the cytoplasmic vesicles. In control cells, Smad3 was located mainly in the cytoplasm, and weak cytoplasmic TGF-beta was distributed with a pattern similar to that of the aneurysm-derived cells. Compared to normal aorta cells, AT(1)R and AT(2)R expression was increased in both aneurysm types. Treatment of cultured VSMCs with the AT(1)R antagonist losartan caused both reduced TGF-beta vesicle localization and nuclear expression of Smad3.Conclusions: Increased TGF-beta and Smad3 expression in aneurysm tissue and cultured VSMCs is consistent with aberrant TGF-beta expression and the activation of Smad3 signaling. Losartan-mediated reduction in TGF-beta expression and the cytoplasmic localization of Smad3 support a role for AT(1)R antagonism in the inhibition of aneurysm progression.

Osteogenesis imperfecta (OI) and Marfan syndrome (MFS) are common Mendelian disorders. Both conditions are usually diagnosed clinically, as genetic testing is expensive due to the size and number of potentially causative genes and mutations. However, genetic testing may benefit patients, at-risk family members and individuals with borderline phenotypes, as well as improving genetic counseling and allowing critical differential diagnoses. We assessed whether whole exome sequencing (WES) is a sensitive method for mutation detection in OI and MFS. WES was performed on genomic DNA from 13 participants with OI and 10 participants with MFS who had known mutations, with exome capture followed by massive parallel sequencing of multiplexed samples. Single nucleotide polymorphisms (SNPs) and small indels were called using Genome Analysis Toolkit (GATK) and annotated with ANNOVAR. CREST, exomeCopy and exomeDepth were used for large deletion detection. Results were compared with the previous data. Specificity was calculated by screening WES data from a control population of 487 individuals for mutations in COL1A1, COL1A2 and FBN1. The target capture of five exome capture platforms was compared. All 13 mutations in the OI cohort and 9/10 in the MFS cohort were detected (sensitivity=95.6%) including non-synonymous SNPs, small indels (<10 bp), and a large UTR5/exon 1 deletion. One mutation was not detected by GATK due to strand bias. Specificity was 99.5%. Capture platforms and analysis programs differed considerably in their ability to detect mutations. Consumable costs for WES were low. WES is an efficient, sensitive, specific and cost-effective method for mutation detection in patients with OI and MFS. Careful selection of platform and analysis programs is necessary to maximize success.

Thoracic aortic aneurysm (TAA) associated with congenital bicuspid aortic valve, Marfan syndrome, Loeys-Dietz syndrome and familial aortic aneurysm is characterised by abnormal expression of TGF-beta (TGFB) and matrix metalloproteinase (MMP)-2 and MMP-9. We examined the effect of the angiotensin II receptor antagonist, losartan, and doxycycline, an MMP inhibitor, on MMP expression in cultured endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) derived from subjects with TAA. VSMCs and ECs (104/mL, passage 2–4) from subjects with thoracic aneurysm (n = 8; age: 31–72; 6F, 2M; MFS = 3, BAV = 1, LDS = 1, FAA = 3) and organ donor control subjects (n = 3; age: 37–59; 2F; 1M) were cultured for 7 d. They were then incubated in either losartan (1 μg/mL) or doxycycline (2 μg/mL) for 24 h. Monocyte conditioned media (MCM) was also used to simulate the effects of inflammation. MMPs secreted into the conditioned media were measured using gelatine zymography. Cells containing nuclear Smad3 (nSmad3) staining, a marker of downstream TGFB receptor activation, were counted. In ECs and VSMCs there was increased 62 kDa MMP-2 (1.7 and 1.8 fold respectively, p < 0.05) and 82 kDa MMP-9 (5.9 and 2.2 fold, p < 0.05) compared to controls. MCM treatment resulted in a further increase in MMPs compared to cells in the absence of MCM. Expression of MMP-2 was reduced with losartan (EC = 0.8 fold; VSMC = 0.7 fold, p < 0.05) and doxycycline (EC = 0.3 fold; VSMC = 0.4 fold, p < 0.05) compared to non treated cells. There were similar findings for MMP-9. Reduced nSmad3 number was found in losartan (EC = 83 ± 24%; VSMC = 92 ± 10%) and doxycycline (EC = 59 ± 32%; VSMC = 76 ± 21%) treated cells compared to control cells. MCM treatment reduced nSmad3 in VSMCs (73 ± 18%) but had no effect in ECs (96 ± 6%). TAA ECs and VMSCs secrete higher levels of MMP-2 and MMP-9 compared to controls. Losartan and doxycycline inhibit MMP-2 and MMP-9 expression and reduce nSmad3 count suggesting inhibition of downstream effects of TGFB receptor activation. The results support a rationale for the use of these agents in subjects with thoracic aortic aneurysm disease.

Marfan syndrome is a multisystem disorder of connective tissue that is inherited in an autosomal dominant fashion, and results from mutation of the FBN1 gene on human chromosome 15. There are a number of conditions of the connective tissue with a similar phenotype that can be confused with Marfan syndrome. Modifications of the diagnostic criteria have recently been published, facilitating the differentiation of Marfan syndrome from these conditions. It is still difficult to use modern genetic testing for diagnosis because Marfan syndrome can be caused by many different mutations in FBN1, a large gene with 65 coding segments, while mutations in other genes can cause overlapping phenotypes. Several clinical trials of drug therapy, including the antihypertensive drug losartan, are in progress.

Objectives: Tissue from Marfan syndrome (MFS) or bicuspid aortic valve (BAV) aneurysm is characterized by vascular smooth muscle cell (VSMC) loss, cystic medial necrosis and elastic tissue destruction. We examined morphological changes in aneurysm tissue using light microscopy (LM) and transmission electron microscopy (TEM). Conclusions: The findings suggest that morphological abnormalities in VSMC precede recognizable alterations of EL, consistent with the hypothesis that the primary defect in the pathogenesis of MFS and BAV aneurysm arises within VSMCs.

Increased expression of transforming growth factor-beta (TGFβ) and Smad3 is associated with fibrosis and inflammatory cell infiltration in abdominal aortic aneurysm. In Smad3-null mice there is reduced extracellular matrix (ECM) deposition but enhanced neointimal hyperplasia in response to vascular injury suggesting a TGFβ/Smad3 role in ECM regulation and cell proliferation. In vitro studies show that exogenous TGFβ administration leads to phosphorylation and nuclear translocation of Smad3 while angiotensin II (AngII) induces fibrosis through TGFβ and Smad3 pathways. We investigated TGFβ/Smad3 signaling using immunohistochemical and cell culture studies in thoracic aortic aneurysm tissue derived from subjects with Marfan syndrome (MFS; 3M, 2F, 46±24yr, mean +/−SD) and bicuspid aortic valve malformation (BAV; (3M, 2F, 65±13yr) as well as normal aorta from organ donor subjects (3M, 2F 40±11yr). MFS and BAV tissue showed co-localisation of TGFβ1–3 and Smad3 in myofibroblasts, vascular smooth muscle cells (VSMCs) and chronic inflammatory cells in the subintimal layer and tunica media and in fibroblasts in tunica adventitia. In normal aortic wall there was minimal TGFβ and Smad3 staining. Cultured VSMCs (passage 1–5) from MFS and BAV showed nuclear Smad3 and strong cytoplasmic TGFβ1–3 expression in numerous vesicles and in areas of exocytosis and extracellular localisation. In control cells there was much weaker TGFβ1–3 staining compared to aneurysm derived cells. Smad3 staining in normal cells was located in the cytoplasm alone. Compared to normal tissue and cells AngII receptor type1 and 2 (ATR1 and 2) expression was increased in both aneurysm tissue and in cultured VSMCs derived from aneurysm. Cultured VSMCs were treated for 48h with the ATR1 antagonist losartan (10μM). This caused disappearance of TGFβ1–3 vesicle localisation and nuclear expression of Smad3. The findings of increased TGFβ1–3 and Smad3 expression in aneurysm tissue and cultured cells are consistent with aberrant TGFβ signalling and with activation of the Smad3 signalling pathway. Furthermore reduction in extracellular TGFβ and de-activation of Smad3 expression associated with losartan treatment supports a role for ATR1 antagonism in inhibition of aneurysm progression. Figure 1: Risk of IMH Conversion to Typical Dissection

We hypothesised that in human thoracic aortic aneurysm associated with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) abnormal expression of angiotensin II (AngII) receptors leads to changes in disposition of circulating vascular progenitor cells (CPCs) and adverse effects on tissue remodelling. Expression of AngII type 1 and type 2 receptors (AT1R, AT2R) and disposition of CPCs were compared in MFS and BAV aneurysm and in an ApoE−/− mouse aneurysm model. Tissue from subjects with MFS (three males, two females; 28–67 years) and BAV (three males, two females; 53–73 years) and normal thoracic aorta from organ donor subjects (two males, three females; 27–72 years) were collected. Female 5-month-old ApoE−/− mice transplanted with green fluorescent protein (GFP)+/+ ApoE−/− bone marrow were infused with AngII for 2 weeks. In MFS and BAV abnormal AT1R and AT2R expression and CD34+ CPCs were detected in areas of intimal thickening. There was expression of AT1R, AT2R and occasional CD34+ cells in media and adventitia. In the animal model AngII resulted in either thoracic aortic dissection (29%) or significant intimal thickening (71%). GFP+ CPCs were found in cells forming intimal thickening in thoracic and abdominal aorta. Increased accumulation of GFP+ cells was found in adventitia corresponding to areas of intimal thickening. Aortic dissection was present in regions of the aorta without intimal thickening and without adventitial GFP+ cells. The study suggests altered AT1R and AT2R regulation affects CPC disposition and vascular wall remodelling. Absence of intimal and adventitial CPCs was a precursor for aneurysm development and dissection.

Altered expression of angiotensin II type-1 and type-2 receptors (AT1, AT2) has been implicated in the development of aortic aneurysm. We used immunohistochemistry to examine the expression of AT1 and AT2 in thoracic and abdominal aortic aneurysm. Thoracic and abdominal aortic tissue samples were collected from organ donor subjects (normal controls; 4M, 9F; age 40±12yr, mean ± SD), and subjects with Marfan syndrome (MFS; 6M, 3F; 42±24yr), bicuspid aortic valve (BAV; 10M, 4F; 62±17yr) or abdominal aortic aneurysm (AAA; 8M, 2F; 72±10). In each type of aneurysm there was mild expression of AT1 and moderate expression of AT2 associated with intimal thickening and the tunica adventitia compared to mild expression of AT1 and negligible expression of AT2 in control aorta. In aneurysm tissue AT1 and AT2 expression, transforming growth factor (TGF)-beta1 expression and progenitor cell (CD34+) localization were also present in areas of tissue remodeling associated with focal medial degeneration, angiogenesis and areas of inflammation. In cell culture experiments aortic fibroblasts (AF) derived from adventitial aneurysm tissue showed moderate expression of AT1 and strong expression of AT2 while control AF showed mild expression of AT1 and negligible expression of AT2. Negligible to mild expression of active TGF-beta1 was observed in all cultured AF. Interestingly adventitial aneurysm cultured in endothelial growth media resulted in the appearance of spindle shaped cells with CD31+ endothelial colonies sprouting among them. Moderate active TGF-beta1 and AT1 and strong AT2 expression were observed in these CD31+ cells. Cultured control adventitia only had spindle cell outgrowth and negligible active TGF-beta1, AT1 and AT2 expression. In summary aneurysm tissue and cultured AF and CD31+ cells of aneurysm origin showed increased expression of AT2 and an absence of increased AT1 expression. Increased AT2 expression in areas of pathological remodeling suggests an active role for AT2 in aneurysm development. Co-localization of increased AT2 with active TGF-beta1 expression and CD34+ cells suggests a role for AT2 in endothelial differentiation and angiogenesis.

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34).Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls.Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.