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Vascular inflammation contributes to cardiovascular diseases such as aortic aneurysm and dissection. However, the precise inflammatory pathways involved have not been clearly defined. We have shown here that subcutaneous infusion of Ang II, a vasopressor known to promote vascular inflammation, into older C57BL/6J mice induced aortic production of the proinflammatory cytokine IL-6 and the monocyte chemoattractant MCP-1. Production of these factors occurred predominantly in the tunica adventitia, along with macrophage recruitment, adventitial expansion, and development of thoracic and suprarenal aortic dissections. In contrast, a reduced incidence of dissections was observed after Ang II infusion into mice lacking either IL-6 or the MCP-1 receptor CCR2. Further analysis revealed that Ang II induced CCR2+CD14hiCD11bhiF4/80– macrophage accumulation selectively in aortic dissections and not in aortas from Il6–/– mice. Adoptive transfer of Ccr2+/+ monocytes into Ccr2–/– mice resulted in selective monocyte uptake into the ascending and suprarenal aorta in regions of enhanced ROS stress, with restoration of IL-6 secretion and increased incidence of dissection. In vitro, coculture of monocytes and aortic adventitial fibroblasts produced MCP-1– and IL-6–enriched conditioned medium that promoted differentiation of monocytes into macrophages, induced CD14 and CD11b upregulation, and induced MCP-1 and MMP-9 expression. These results suggest that leukocyte-fibroblast interactions in the aortic adventitia potentiate IL-6 production, inducing local monocyte recruitment and activation, thereby promoting MCP-1 secretion, vascular inflammation, ECM remodeling, and aortic destabilization.

Background: Mutations in the transforming growth factor β receptor type I and II genes ( TGFBR1 and TGFBR2 ) cause Loeys–Dietz syndrome (LDS), characterised by thoracic aortic aneurysms and dissections (TAAD), aneurysms and dissections of other arteries, craniosynostosis, cleft palate/bifid uvula, hypertelorism, congenital heart defects, arterial tortuosity, and mental retardation. TGFBR2 mutations can also cause TAAD in the absence of features of LDS in large multigenerational families, yet only sporadic LDS cases or parent–child pairs with TGFBR1 mutations have been reported to date. Methods: The authors identified TGFBR1 missense mutations in multigenerational families with TAAD by DNA sequencing. Clinical features of affected individuals were assessed and compared with clinical features of previously described TGFBR2 families. Results: Statistical analyses of the clinical features of the TGFBR1 cohort (n = 30) were compared with clinical features of TGFBR2 cohort (n = 77). Significant differences were identified in clinical presentation and survival based on gender in TGFBR1 families but not in TGFBR2 families. In families with TGFBR1 mutations, men died younger than women based on Kaplan–Meier survival curves. In addition, men presented with TAAD and women often presented with dissections and aneurysms of arteries other than the ascending thoracic aorta. The data also suggest that individuals with TGFBR2 mutations are more likely to dissect at aortic diameters TGFBR1 mutations. Conclusion: This study is the first to demonstrate clinical differences between patients with TGFBR1 and TGFBR2 mutations. These differences are important for the clinical management and outcome of vascular diseases in these patients.

The vascular smooth muscle cell (SMC)-specific isoform of alpha-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.

Aortic aneurysm and dissection is a common phenotype, accounting for 1% to 2% of all deaths in industrialized countries and ≈50 000 deaths annually in the United States.1 In contrast to abdominal aortic aneurysm, thoracic aortic aneurysm, particularly in the ascending segment, commonly occurs in young individuals in the absence of identifiable environmental risk factors. Marfan syndrome (MFS) is the most common syndromic presentation of ascending aortic aneurysm, but other syndromes such as vascular Ehlers-Danlos syndrome and Loeys-Dietz syndrome (LDS) also have ascending aortic aneurysms and the associated cardiovascular risk of aortic dissection and rupture. Familial segregation of the risk for ascending aortic aneurysm can also occur in the absence of associated systemic findings of a connective tissue abnormality in patients with familial thoracic aortic aneurysm and dissection (FTAAD) or bicuspid aortic valve with ascending aortic aneurysm (BAV/AscAA). The knowledge gained through basic and clinical research focused on MFS has improved and will continue to improve the care of patients with these related conditions. Recent paradigm-shifting discoveries about the molecular pathogenesis of MFS have highlighted the need for a focused research agenda to solidify the gains of the past 30 years and set the stage for future advances in MFS and related conditions. In April 2007, the National Heart, Lung, and Blood Institute (NHLBI) and the National Marfan Foundation convened a working group on research in MFS and related disorders to foster a multidisciplinary discussion. The working group, which included experts in cardiovascular disease, developmental biology, genetics and genomics, and proteomics, was charged with identifying opportunities and barriers to advancing the research agenda and developing recommendations to the NHLBI in the context of the Institute’s strategic plan (http://apps.nhlbi.nih.gov/strategicplan/). MFS is a systemic disorder of connective tissue caused by heterozygous mutations in the gene ( FBN1 ) that encodes the extracellular matrix …

The major function of vascular smooth muscle cells (SMCs) is contraction to regulate blood pressure and flow. SMC contractile force requires cyclic interactions between SMC alpha-actin (encoded by ACTA2) and the beta-myosin heavy chain (encoded by MYH11). Here we show that missense mutations in ACTA2 are responsible for 14% of inherited ascending thoracic aortic aneurysms and dissections (TAAD). Structural analyses and immunofluorescence of actin filaments in SMCs derived from individuals heterozygous for ACTA2 mutations illustrate that these mutations interfere with actin filament assembly and are predicted to decrease SMC contraction. Aortic tissues from affected individuals showed aortic medial degeneration, focal areas of medial SMC hyperplasia and disarray, and stenotic arteries in the vasa vasorum due to medial SMC proliferation. These data, along with the previously reported MYH11 mutations causing familial TAAD, indicate the importance of SMC contraction in maintaining the structural integrity of the ascending aorta.

Background A 24-year-old man presented with previously diagnosed Marfan's syndrome. Since the age of 9 years, he had undergone eight cardiovascular procedures to treat rapidly progressive aneurysms, dissection and tortuous vascular disease involving the aortic root and arch, the thoracoabdominal aorta, and brachiocephalic, vertebral, internal thoracic and superior mesenteric arteries. Throughout this extensive series of cardiovascular surgical repairs, he recovered without stroke, paraplegia or renal impairment. Investigations CT scans, arteriogram, genetic mutation screening of transforming growth factor β receptors 1 and 2. Diagnosis Diffuse and rapidly progressing vascular disease in a patient who met the diagnostic criteria for Marfan's syndrome, but was later rediagnosed with Loeys–Dietz syndrome. Genetic testing also revealed a de novo mutation in transforming growth factor β receptor 2. Management Regular cardiovascular surveillance for aneurysms and dissections, and aggressive surgical treatment of vascular disease.

Background: Children with Marfan syndrome (MFS) treated with angiotensin II receptor blocker (ARB) or beta-blocker (BB) therapy show a reduction in the rate of aortic root (AR) growth, yet substantial variability in effect remains. Adult data suggests that patients with haploinsufficient (HI) mutations may be more responsive to ARBs compared with dominant negative (DN) mutations. This study sought to assess the effect of genotype on the rate of AR growth in children treated with either ARB or BB. Methods: A retrospective review of all individuals with MFS assessed at our institution since 1980 was undertaken; those with ≥2 echocardiograms a minimum of 1 year apart and a documented disease-causing fibrillin-1 (FBN1) mutation were included. One patient with infantile MFS was excluded given extremely AR growth. Genotypes were classified by predicted protein effect as either HI or DN. Multivariable longitudinal linear regression analysis was used to compare average rate of AR growth by genotype and medical therapy using serial echocardiographic data. Results: Overall 41 children were included, including 18 with HI and 23 with DN mutations. Males comprised 58%. There was no difference in median age at first echo (HI 5.4 years, IQR 3.9-9.3 vs. DN 5.2 years, IQR 3.4-10.6 years, p=0.97), follow-up time (HI 5.8 years, IQR 4.6-8.2 vs. DN 4.9 years, IQR 3.3-8.7, p=0.35), AR dimension (HI 26 mm, IQR 23-29 vs DN 26 mm, IQR 22-37, p=0.81), or AR z-score (HI 3.0, IQR 2.3-4.0 vs DN 3.4, IQR 2.4-4.2, p=0.58). AR growth rate was greater in children with DN mutations (z-score annual change HI -0.04 vs DN +0.07, p Conclusions: In our cohort, patients with MFS and DN mutations had slower growth on BB therapy compared to those on ARB therapy. Genetic testing may play an important role in targeted medical therapy in MFS.

The 2014 NIH Physician-Scientist Workforce (PSW) Working Group report identified distressing trends among the small proportion of physicians who consider research to be their primary occupation. If unchecked, these trends will lead to a steep decline in the size of the workforce. They include high rates of attrition among young investigators, failure to maintain a robust and diverse pipeline, and a marked increase in the average age of physician-scientists, as older investigators have chosen to continue working and too few younger investigators have entered the workforce to replace them when they eventually retire. While the policy debates continue, here we propose four actions that can be implemented now. These include applying lessons from the MD-PhD training experience to postgraduate training, shortening the time to independence by at least 5 years, achieving greater diversity and numbers in training programs, and establishing Physician-Scientist Career Development offices at medical centers and universities. Rather than waiting for the federal government to solve our problems, we urge the academic community to address these goals by partnering with the NIH and national clinical specialty and medical organizations.

Abnormalities in the cerebrovascular system play a central role in many neurologic diseases. The on-going expansion of rodent models of human cerebrovascular diseases and the need to use these models to understand disease progression and treatment has amplified the need for reproducible non-invasive imaging methods for high-resolution visualization of the complete cerebral vasculature. In this study, we present methods for in vivo high-resolution (19 μm isotropic) computed tomography imaging of complete mouse brain vasculature. This technique enabled 3D visualization of large cerebrovascular networks, including the Circle of Willis. Blood vessels as small as 40 μm were clearly delineated. ACTA2 mutations in humans cause cerebrovascular defects, including abnormally straightened arteries and a moyamoya-like arteriopathy characterized by bilateral narrowing of the internal carotid artery and stenosis of many large arteries. In vivo imaging studies performed in a mouse model of Acta2 mutations demonstrated the utility of this method for studying vascular morphometric changes that are practically impossible to identify using current histological methods. Specifically, the technique demonstrated changes in the width of the Circle of Willis, straightening of cerebral arteries and arterial stenoses. We believe the use of imaging methods described here will contribute substantially to the study of rodent cerebrovasculature.

Objective: ACTA2 mutations cause 10-14% of familial thoracic aortic aneurysms and dissections. Mice deficient in smooth muscle α-actin (Acta2-/-) develop root and ascending thoracic aortic enlargement associated with thickening of the aortic media and fragmentation and disarray of elastic fibers. We hypothesized that blocking AT1 activation would block the aortic pathology and prevent aortic enlargement in Acta2-/- mice. Methods and Results: Beginning at 4 weeks of age, Acta2-/- mice were treated with losartan or placebo (n≥10) for 6 months and echocardiograms were performed at baseline and every other month. The aortic root in Acta2-/- mice was found to undergo progressive dilatation. After 6 months of treatment, there was no difference in the diameter of the aortic root between wild-type (WT) mice and the losartan treated mice (p=0.44). Histologic analysis of Acta2-/- aortas demonstrated medial thickening and fragmentation of elastic fibers which was normalized by treatment with losartan. Gene expression of matrix metalloproteinase-2 and -9 (Mmp2, Mmp9), along with lumican and decorin, interleukin-6 (Il6) and phosphorylation of RelA (a subunit of nuclear factor κB, NF-κB) was increased in Acta2-/- aortas, and was corrected by treatment with losartan. NADPH oxidase 4 and AT1a mRNA was increased in Acta2-/- aortic smooth muscle cells (SMCs) and aortas. Increase of AT1a was blocked by lowering reactive oxygen species (ROS) with N-actetyl cysteine (NAC). Angiotensin II (AngII) dose response studies suggested Acta2-/- aortic SMCs had increased sensitivity to Ang II. Additionally, Acta2-/- SMCs had increased ROS compared to WT by flow cytometry (P Conclusions: Our results demonstrate that complete loss of α-SMA leads to aortic dilation and pathologic changes by increasing SMC ROS levels, thus increasing sensitivity to AngII, which results in NF-κB activation and increased expression of Mmps and IL6.

We report three families with arterial aneurysms and dissections in which variants predicted to be pathogenic were identified in SMAD2. Moreover, one variant occurred de novo in a proband with unaffected parents. SMAD2 is a strong candidate gene for arterial aneurysms and dissections given its role in the TGF-β signaling pathway. Furthermore, although SMAD2 and SMAD3 probably have functionally distinct roles in cell signaling, they are structurally very similar. Our findings indicate that SMAD2 mutations are associated with arterial aneurysms and dissections and are in accordance with the observation that patients with pathogenic variants in genes encoding proteins involved in the TGF-β signaling pathway exhibit arterial aneurysms and dissections as key features.

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/early/2015/05/11/CIRCRESAHA.117.305108, RATIONALE: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. OBJECTIVE: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. METHODS AND RESULTS: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. CONCLUSIONS: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders, This work was supported by grants from the Ministerio de Economía y Competitividad (RD12/0042/0022 to JMR, RD12/0042/0024 to MS, and RD12/0042/0023 to AGA [FEDER cofounded], and SAF2011-25619 to AGA). MM-A was funded by a MINECO fellowship. SILAC analysis was performed in the Proteomics Laboratory at Vall d'Hebron Institute of Oncology (VHIO), a member of ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. The CNIC is supported by the Ministerio de Economía y Competitividad and the Pro- CNIC Foundation

Bicuspid aortic valve is the most common congenital heart defect and is associated with an aortopathy manifested by dilatation of the ascending thoracic aorta. The clinical consequences of this aortopathy are the need for periodic monitoring of aortic diameters, elective prophylactic surgical aortic repair, and the occurrence of aortic dissection or rupture. This review describes the current knowledge of BAV aortopathy in adults, including incidence, pathophysiologic insights into its etiology, contemporary hypothesis-generating observations into its complications, and recommendations for monitoring and intervention.

Background Cardiovascular disease is the main cause of morbidity and mortality in patients with Marfan syndrome. Many patients with presumed Marfan syndrome do not meet current diagnostic criteria. This study reviews the surgical aspects of aortic disease in 300 patients referred with the diagnosis of Marfan syndrome. Methods During a 16-year period, 300 patients with presumed Marfan syndrome underwent 398 operations on the aorta and branch arteries, including 125 aortic root operations, 59 aortic arch repairs, 31 descending thoracic aortic repairs, and 178 thoracoabdominal aortic repairs. Based on medical record review, patients were classified as confirmed Marfan syndrome if documented features satisfied current diagnostic criteria; patients not meeting these criteria were classified as suspected Marfan syndrome. Results There were 17 operative deaths (4.3%) after the 398 operations. Survival after the initial referral operation was 96.2% ± 1.5% at 1 year, 82.7% ± 2.4% at 5 years, and 74.6% ± 3.1% at 10 years. Presentations, operative details, and outcomes were remarkably similar in the 137 patients (45.7%) with confirmed Marfan syndrome and the 163 patients (54.3%) with suspected Marfan syndrome. Freedom from repair failure, however, was significantly better in patients with confirmed Marfan syndrome (90.3% ± 2.3% at 10 years) than in those with suspected Marfan syndrome (82.0% ± 3.1% at 10 years; p = 0.001). Conclusions Operative treatment of the full spectrum of aortic disease in Marfan patients enables excellent long-term survival. Similarities in surgical aspects of aortic disease suggest that patients with features of Marfan syndrome who do not meet diagnostic criteria should be managed in the same manner as patients with confirmed Marfan syndrome.

Ascending thoracic aortic aneurysms leading to type A dissections can be inherited in an autosomal dominant manner with variable age of onset and decreased penetrance, primarily in women. Three families are described with autosomal dominant inheritance of either ascending aortic aneurysms leading to type A dissections or type B dissections, and a young age of onset of aortic dissections in both men and women. Pedigree analysis suggests that a de novo mutation is responsible for the disease in one family. The discordant age of onset of aortic disease in a monozygotic twin pair in a different family indicates that environmental or stochastic factors may influence the variable expression of disease. Genetic analysis of one family excluded linkage to known loci for TAAD (TAAD1, TAAD2, FAA1, or FBN1) and sequence analysis failed to identify mutations in TGFBR2, the gene encoding transforming growth factor beta receptor type II. Thus, a novel unidentified loci may be responsible for the phenotype in these three families.

Ascending thoracic aortic aneurysms leading to type A dissections (TAAD) can occur in association with a genetic syndrome, such as Marfan syndrome (MFS), or as an autosomal dominant disorder in the absence of syndromic features, termed familial TAAD. Familial TAAD demonstrates genetic heterogeneity, and linkage studies have identified three TAAD loci at 5q13-14 (TAAD1), 11q23 (FAA1), and 3p24-25 (TAAD2). The underlying genetic heterogeneity of TAAD is reflected in the phenotypic variation associated with familial TAAD with respect to age of onset, progression, penetrance, and association with additional cardiac and vascular features. Recently, mutations in the TGFBR2 gene have been identified as the cause of disease linked to the TAAD2 locus, supporting the hypothesis that dysregulation of TGFbeta signaling is a mechanism leading to aneurysms and dissections. The recent identification of the TGFbeta pathway as a key target in the molecular pathogenesis of TAAD has opened new avenues for future genetic and therapeutic research.

Background— A genetic predisposition for progressive enlargement of thoracic aortic aneurysms leading to type A dissection (TAAD) is inherited in an autosomal-dominant manner in up to 19% of patients, and a number of chromosomal loci have been identified for the condition. Having mapped a TAAD locus to 3p24–25, we sequenced the gene for transforming growth factor-β receptor type II ( TGFBR2 ) to determine whether mutations in this gene resulted in familial TAAD. Methods and Results— We sequenced all 8 coding exons of TGFBR2 by using genomic DNA from 80 unrelated familial TAAD cases. We found TGFBR2 mutations in 4 unrelated families with familial TAAD who did not have Marfan syndrome. Affected family members also had descending aortic disease and aneurysms of other arteries. Strikingly, all 4 mutations affected an arginine residue at position 460 in the intracellular domain, suggesting a mutation “hot spot” for familial TAAD. Despite identical mutations in the families, assessment of linked polymorphisms suggested that these families were not distantly related. Structural analysis of the TGFBR2 serine/threonine kinase domain revealed that R460 is strategically located within a highly conserved region of this domain and that the amino acid substitutions resulting from these mutations will interfere with the receptor’s ability to transduce signals. Conclusion— Germline TGFBR2 mutations are responsible for the inherited predisposition to familial TAAD in 5% of these cases. Our results have broad implications for understanding the role of TGF-β signaling in the pathophysiology of TAAD.

The identification of polymorphisms associated with an increase in the risk of developing disease is integral to the development of genetic biomarkers to identify individuals at risk. Based on reports indicating a role for angiotensin-converting enzyme (ACE) in the pathogenesis of intracranial aneurysms (IAs) as well as hypertension, an independent risk factor for IAs, the authors investigated the association between an insertion/deletion (I/D) polymorphism in the ACE gene and IAs in a Caucasian population in the US.The patient population consisted of 162 randomly selected Caucasian patients who underwent surgical repair of an IA at Memorial-Hermann Hospital (Houston, TX) and had no family history of the disease. The ACE I/D polymorphism was typed using polymerase chain reaction amplification of genomic DNA, and allele and genotype frequencies were compared between the patients with IAs and 143 healthy Caucasian volunteers (control group) by performing logistic regression and chi-square tests. The ACE I/D allele frequencies did not differ significantly between the patient and control populations. There were similar allele and genotype frequencies in male and female study participants in both patient and control populations. The authors found no evidence of an association between the allelic or genotypic distribution of the ACE I/D polymorphism and aneurysmal subarachnoid hemorrhage or unruptured IAs.Contrary to findings in two European Caucasian populations (one British and one Polish), this polymorphism did not contribute to the risk of developing IAs in a Caucasian population in the US.

OBJECTIVE: Although previous reports have described patients with both cerebral and aortic aneurysms, any association was believed to be coincidental. In this study, we provide evidence that aortic and cerebral aneurysm formation may share a common genetic predisposition in some families. METHODS: A prospective enrollment of consecutive patients treated for saccular cerebral aneurysm by a single surgeon was constructed. Medical and family histories were obtained. Familial syndromes were identified when two or more first-degree relatives had diagnoses of cerebral or aortic aneurysm. Pedigrees were constructed, and asymptomatic relatives were screened. RESULTS: In 2.5 years, 274 patients were enrolled from 322 eligible patients (85%). A family history of aortic aneurysm was noted in 29 patients (10.5%). These patients were older, included more males, and were overwhelmingly Caucasian. Statistically significant demographic differences were noted when these families were compared with families affected by cerebral aneurysm only. A total of 110 affected family members were identified (average, 3.8 per family; range, 2-12); 59 had cerebral aneurysms, and 51 had aortic aneurysms. In some families, the association may have been coincidental. But in several families, pedigree analyses showed an inheritance pattern likely to represent autosomal dominance with variable penetrance. In addition, both cerebral and aortic phenotypes could be inherited from a parent with an aortic aneurysm, further evidence for a common genetic basis. CONCLUSION: This study, which represents the largest and most complete characterization of families affected by both cerebral and aortic aneurysms, provides evidence that a single gene defect may lead to the development of either lesion.

Contains fulltext : 58851.pdf (Publisher’s version ) (Open Access) A Mexican family with partial congenital nephrogenic diabetes insipidus (NDI) that resulted from a mutation in the aquaporin-2 water channel (AQP2) was characterized, and the source of this rare mutation was traced to the family's town of origin in Mexico. Affected individuals with profound polyuria and polydipsia were homozygous for an autosomal recessive missense V168M mutation in the AQP2 gene. Expression in oocytes revealed that, although retained in the endoplasmic reticulum (ER) to a great extent, a considerable amount of the partially functional AQP2-V168M was expressed at the plasma membrane, and that its ER retention was less than AQP2-T126M, a functional mutant in severe recessive NDI. None of the affected AQP2-V168M individuals had neurologic deficits, which also suggested a milder form of the disease. The homozygous individuals reported subjective improvement in polyuria and polydipsia with the use of dDAVP (1-desamino-8-D-arginine-vasopressin). When clinically tested, infusion of dDAVP at variable doses produced a partial increase in the urinary osmolality in homozygous individuals and decreased their water intake. Heterozygotes were unaffected when compared with controls. Samples were obtained from the population of the Mexican town of origin of the family; 30% of the population was heterozygous for the V168M AQP2 mutation and 1% was homozygous for the mutation. The high frequency of this rare mutation in the town provides evidence for an important health care problem in the village with consequences for future generations.

The major diseases affecting the aorta are aortic aneurysms and dissections, with patients with acute dissections often presenting in the emergency department (ED). Recent studies demonstrate a strong genetic predisposition to thoracic aortic aneurysms and dissections, independent of syndromes traditionally considered to predispose to aortic disease (such as Marfan syndrome). Nonsyndromic familial thoracic aortic aneurysms and dissections are inherited in families as an autosomal dominant disorder and a variable age of onset of the aortic disease. The case reported here illustrates the critical importance of obtaining a family history of thoracic aortic aneurysms and dissections, along with unexplained sudden death, when assessing an individual with chest pain in the ED, regardless of age and in the absence of a known genetic syndrome.

Detection of human DNA polymorphisms using high throughput methods often relies on generating a labeled DNA fraament which is generated by PCR using sequence-specific primers with an end labeled tag to detect a variant. The disadvantage of the synthesis of an end-labeled, sequence-specific primer to assay each DNA variant lies in the costs and time consume. In this report, we have demonstrated a methodology that can generate labeled DNA fragments using a labeled universal primer instead of requiring sequence-specific primers for each DNA variant.

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A(1) and incubation of dermal fibroblasts at 22 degrees C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, alpha-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway.

Enteroaggregative Escherichia coli (EAEC) infection can be identified in 26% of travelers with diarrhea and is associated with fecal interleukin (IL)-8 production. We hypothesized that single-nucleotide polymorphisms (SNPs) in the IL-8 gene are associated with EAEC-related symptoms. Fecal IL-8 production and IL-8 SNPs at 5 loci were identified in 69 US students who remained in Mexico for 5 weeks; 23 subjects had EAEC-associated diarrhea, 7 were asymptomatic EAEC carriers, 22 had nonspecific diarrhea, and 17 were asymptomatic without an enteropathogen. The chances of having EAEC-associated diarrhea were significantly increased among those with the AA genotype at the -251 position (odds ratio [OR], 208.51; 95% confidence interval [CI], 28.5-1525.36) and among those with AT genotype (OR, 14.3; 95% CI, 1.98-105.74), compared with those with the TT genotype at the -251 position. Among subjects with EAEC-associated diarrhea, the AA genotype at the -251 position produced greater concentrations of fecal IL-8 than those with the AT or TT genotype (P=.0053). In the present study, the AA genotype at the -251 position was associated with the occurrence of EAEC-associated diarrhea and increased levels of fecal IL-8.

OBJECTIVE: Although the cause of cerebral aneurysms remains unclear, there is clear evidence that genetic predisposition plays a role. Ten percent of patients report an aneurysm in a first-degree family member. However, studies to date have largely involved Caucasian populations. Our goal was to characterize the familial aggregation of intracranial aneurysms in different ethnic groups. METHODS: We began a prospective, single-center study on patients treated for intracranial aneurysms. Consenting subjects completed a detailed questionnaire regarding the medical history of family members. In familles with two or more affected members, asymptomatic first-degree relatives were screened using computed tomographic or magnetic resonance angiography. RESULTS: In a 2-year period, 292 patients with intracranial aneurysms were treated and 200 were enrolled; these included 124 Caucasians (62%), 34 African-Americans (17%), 38 Hispanics (19%), and 4 Asian-Americans (2%). Forty patients had a famity history (20%). The incidence of family history among the different ethnic groups was similar; it was 19.4% in Caucasians, 20.6% in African-Americans, and 21.6% in Hispanics. One Asian-American patient had a family history. Visual inspection of the pedigrees supported autosomal-dominant inheritance with variable penetrance in all ethnic groups. CONCLUSION: This study examined the incidence of familial cerebral aneurysms in three ethnic groups common to the United States: Caucasian, African-American, and Hispanic. We noted an equivalent rate of familial aneurysms, a finding that has immediate clinical implications. In families that have two or more members with cerebral aneurysms, screening of asymptomatic members should be recommended, regardless of ethnic background.

Background— Familial thoracic aortic aneurysms and dissections (TAAD) occur as part of known syndromes such as Marfan syndrome but can also be inherited in families in an autosomal dominant manner as an isolated condition. Previous studies have mapped genes causing nonsyndromic familial TAAD to 5q13–15 ( TAAD1 ) and 11q23.2-q24 ( FAA1 ). Further genetic heterogeneity for the condition was evident by the presence of TAAD in some families not linked to these known loci. Methods and Results— A 4-generation family with dominant mode of inheritance of TAAD was studied. Affected status was determined by dilation of the ascending aorta, surgical repair of an aneurysm or dissection, or death as the result of aortic dissection. None of the family members evaluated met the diagnostic criteria for Marfan syndrome. After exclusion of known loci for familial TAAD, a genome-wide scan was carried out to map the defective gene causing the disease in the family. A locus was mapped to a 25-cM region on 3p24–25 with a maximum multipoint logarithm of the odds score of 4.28. Conclusions— A third locus for nonsyndromic TAAD was mapped to 3p24–25 and termed the TAAD2 locus. This locus overlaps a previously mapped second locus for Marfan syndrome, termed the MFS2 locus. Future characterization of the TAAD2 gene will determine if TAAD2 is allelic to MFS2 . In addition, identification of the TAAD2 gene will improve the presymptomatic diagnosis of individuals with this life-threatening genetic syndrome and provide information concerning the pathogenesis of the disease.

Small insertions or deletions of nucleotides are common polymorphic variations in the human genome and can result in a predisposition to disease. However, high throughput methods for detecting these variations are limited. This report describes a method to detect this variation based on sequencing the boundaries of nucleotide alterations using the Pyrosequencing technique. This method can optimally detect up to 100 base pair nucleotide insertions and deletions, and also complicated genomic rearrangements.

Objective Fibroblasts from patients with systemic sclerosis (SSc) have an activated phenotype characterized by increased synthesis of extracellular matrix (ECM) components. SPARC (secreted protein, acidic and rich in cysteine) regulates the deposition or assembly of ECM components. The aim of this study was to investigate the role of SPARC in SSc susceptibility by functional and genetic association studies. Methods Complementary DNA (cDNA) microarrays were used to obtain gene expression data on cultured dermal fibroblasts from SSc patients. SPARC protein levels were assessed by Western blotting. Five polymorphic microsatellite markers within 5 cM of the SPARC gene (chromosome 5q31-32) were genotyped in Choctaw Indians, a population previously shown to have a high prevalence of SSc. Discovery of single-nucleotide polymorphisms (SNPs) was accomplished by sequencing the SPARC cDNA. These SNPs were then genotyped in a multi-ethnic cohort of SSc patients to determine potential associations with disease susceptibility in a broader population of SSc patients, as well as with various clinical and immunologic features of SSc. The functional relevance of these SNPs with regard to transcript stability of SPARC was also assessed. Results Microarrays demonstrated increased expression of SPARC, along with other ECM genes, in SSc patients compared with normal controls. SSc fibroblasts also had increased SPARC protein levels. Three of 5 microsatellite markers near SPARC showed significant associations with SSc in the Choctaw SSc patients. Sequencing of SPARC cDNA revealed 3 novel SNPs in the 3′-untranslated region at +998 (CG), +1551 (CG), and +1922 (TG). Homozygosity for the C allele at SNP +998 was significantly increased in SSc patients across ethnic lines. SPARC SNPs +1551 and +1922 demonstrated correlations with Raynaud's phenomenon and pulmonary fibrosis, respectively. Functional studies of SPARC SNP +998 in normal fibroblast cultures suggested a correlation between the SNP +998 C allele polymorphism and an increased messenger RNA half-life. Conclusion This study is the first to show that polymorphisms of the SPARC gene are associated with susceptibility to, and clinical manifestations of, SSc and that they may also be functionally important in influencing SPARC expression in skin fibroblasts.

Experimental and animal studies, as well as genetic mutations in man, have indicated that the development of dentition is under the control of several genes. So far, mutations in MSX1 and PAX9 have been associated with dominantly inherited forms of human tooth agenesis that mainly involve posterior teeth. We identified a large kindred with several individuals affected with molar oligodontia that was transmitted as an isolated autosomal-dominant trait. Two-point linkage analysis using DNA from the family and polymorphic marker D14S288 in chromosome 14q12 produced a maximum lod score of 2.29 at Θ = 0.1. Direct sequencing of exons 2 to 4 of PAX9revealed a cytosine insertion mutation at nucleotide 793, leading to a premature termination of translation at aa 315. Our results support the conclusion that molar oligodontia is due to allelic heterogeneity in PAX9, and these data further corroborate the role of PAX9 as an important regulator of molar development.

Aneurysms and acute dissections are the major diseases affecting the ascending thoracic aorta. These conditions are related; the natural history of an aneurysm involving the ascending aorta is to progressively and asymptomatically enlarge over time, ultimately leading to an acute ascending aortic dissection (type A dissection based on the Stanford classification, type I and II dissections based on the DeBakey classification). Although medical treatments can slow the enlargement of an aneurysm, the mainstay of treatment to prevent dissections is surgical repair of the ascending aortic aneurysm. This is typically recommended when the aneurysm diameter reaches 5.0 to 5.5 cm1; however, studies on patients presenting with acute ascending dissections indicate that up to 60% present with aneurysms 1200 patients presenting with ascending aortic dissection to emergency departments participating in the International Registry of Acute Aortic Dissections (IRAD).4 Article see p 1911 Many of the factors associated with a delay in …

Background Although the risk of thoracic aortic dissection and rupture (TAD) is well-known in bicuspid aortic valve (BAV), the risk of TAD in other congenital heart diseases (CHD), particularly conotruncal lesions like tetralogy of Fallot (TOF), truncus arteriosus, D-transposition of the great arteries (D-TGA), and double outlet right ventricle is currently unknown. The primary purpose of this study was to describe TAD in conotruncal CHD, and the secondary purpose was to explore whether an association exists between TAD and conotruncal CHD. Methods and results Using the Texas Inpatient Public Use Data File, an administrative database of all Texas hospitalizations, including >37.9 million hospitalizations from January 1999 through June 2012, 12,016 cases of TAD and 214 cases of TAD in CHD were identified. The most common lesions were BAV (42%), atrial septal defect (21%), aortic coarctation (7%), ventricular septal defect (6%), and patent ductus arteriosus (4%). Three patients with TOF, 2 with D-TGA, and 1 with truncus arteriosus were admitted with TAD. An exploratory case–control study in patients older than 1year using multilevel logistic regression models to evaluate the association between CHD and TAD that controlled for known TAD risk factors demonstrated a significant association between TAD and BAV (OR 10, 95% CI 8.2–13) but not coarctation of the aorta or any conotruncal lesion. Conclusions TAD in conotruncal CHD is exquisitely rare. In our hospitalized population, there was no increased occurrence of TAD in conotruncal CHD above what would be expected in the rest of the hospitalized population.

To the Editor: In their review on thoracic aortic dilatation associated with bicuspid aortic valve, Verma and Siu (May 15 issue)1 do not mention the increased prevalence of bicuspid aortic valve and its strong association with aortic dilatation and dissection in women with Turner's syndrome.2 This sex-chromosome disorder, which is caused by the loss of all or part of one X chromosome, affects approximately 1 in 2000 live-born females and approximately 80,000 women in the United States. The prevalence of bicuspid aortic valve in Turner's syndrome (approximately 1 in 3) is much greater than that in the general population and . . .

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor β (TGF-β) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P

Genetic studies in patients reveal that mutations to genes that encode contractile proteins in medial smooth muscle cells can cause thoracic aortic aneurysms and dissections. Mouse models of such mutations, including Acta2−/− and Myh11 R247C/R247C, surprisingly do not present with any severe vascular phenotype under normal conditions. This observation raises the question whether these mutations nevertheless render the thoracic aorta increasingly vulnerable to aneurysms or dissections in the presence of additional, epigenetic, factors such as hypertension, a known risk factor for thoracic aortic disease. Accordingly, we compared the structure and biaxial mechanical properties of the ascending and descending thoracic aorta from male wild-type and Myh11 R247C/R247C mice under normotension and induced hypertension. On average, the mutant aortas exhibited near normal biomechanics under normotensive hemodynamics and near normal adaptations to hypertensive hemodynamics, yet the latter led to intramural delaminations or premature deaths in over 20 percent of these mice. Moreover, the delaminated vessels exhibited localized pools of mucoid material, similar to the common histopathologic characteristic observed in aortas from humans affected by thoracic aortic aneurysms and dissections. The present findings suggest, therefore, that mutations to smooth muscle cell contractile proteins may place the thoracic aorta at increased risk to epigenetic factors and that there is a need to focus on focal, not global, changes in aortic structure and properties, including the pooling of glycosaminoglycans / proteoglycans that may lead to thoracic aortic dissection.

Background: Little is known about reliable predictors of thoracic aortic dissection or rupture (TAD) in children and young adults. We sought to determine whether elevated vertebral artery tortuosity is a biomarker of TAD or aortic surgery at an early age. Methods: We identified 208 patients ≤50 years old in the GenTAC Registry who had ≥2.5 cm of either vertebral artery visualized on a computed tomography angiogram (CTA). In a blinded fashion, using a volume-rendered projection, each patient’s vertebral artery tortuosity index (VTI) was calculated using the larger distance factor (% by which actual length exceeds straight-line length) of the two vertebral arteries. We then investigated associations between VTI and freedom from prophylactic or post-TAD surgery. Results: Subjects included 73 with Marfan syndrome (MS), 34 with bicuspid aortic valve, 18 with Loeys-Dietz syndrome (LDS), 16 with familial thoracic aneurysms and dissections, 12 with Ehlers-Danlos syndrome type IV (EDS), 6 with congenital heart disease, and 49 with other aneurysms and dissections. Median age was 38.5 years [interquartile range (IQR) 29.6-44.6]; 23 were ≤18 years. Indication for first surgery was prophylactic in 80 patients and TAD in 49. VTI was highest in LDS (median 50, IQR 21-72), followed by MS (median VTI 27, IQR 16-52), and lowest in EDS (median 7, IQR 4-13). VTI was not associated with age at CTA. When controlling for diagnosis and race/ethnicity, higher VTI was associated with younger age at prophylactic surgery [HR 1.28, 95%CI 1.09-1.50 for every increase in VTI of 20 (VTI20)] and at surgery for TAD (HR 1.38, 95%CI 1.13-1.69 for VTI20). There was no difference in freedom from prophylactic or post-TAD surgery among LDS and MS subgroups (adjusted p=0.95 and p=0.17 respectively). In patients ≤18 years, the association between increased VTI and age at prophylactic surgery was strongest: HR 1.73 (95%CI 1.11-2.69 for VTI20). One patient ≤18 had TAD with VTI 105. Conclusions: Increased vertebral artery tortuosity is associated with earlier thoracic aortic surgery and dissection in patients under 50 years, and may be most predictive in children. VTI can identify patients at high risk for TAD who may benefit from targeted therapies.

Background— ACTA2 mutations are the major cause of familial thoracic aortic aneurysms and dissections. We sought to characterize these aortic diseases in a large case series of individuals with ACTA2 mutations. Methods and Results— Aortic disease, management, and outcome associated with the first aortic event (aortic dissection or aneurysm repair) were abstracted from the medical records of 277 individuals with 41 various ACTA2 mutations. Aortic events occurred in 48% of these individuals, with the vast majority presenting with thoracic aortic dissections (88%) associated with 25% mortality. Type A dissections were more common than type B dissections (54% versus 21%), but the median age of onset of type B dissections was significantly younger than type A dissections (27 years versus 36 years). Only 12% of aortic events were repair of ascending aortic aneurysms, which variably involved the aortic root, ascending aorta, and aortic arch. Overall, cumulative risk of an aortic event at age 85 years was 0.76 (95% confidence interval, 0.64–0.86). After adjustment for intrafamilial correlation, sex and race, mutations disrupting p.R179 and p.R258 were associated with significantly increased risk for aortic events, whereas p.R185Q and p.R118Q mutations showed significantly lower risk of aortic events compared with other mutations. Conclusions— ACTA2 mutations are associated with high risk of presentation with an acute aortic dissection. The lifetime risk for an aortic event is only 76%, suggesting that additional environmental or genetic factors play a role in expression of aortic disease in individuals with ACTA2 mutations.

Vascular Ehlers–Danlos syndrome (vEDS) is an autosomal dominant disease that affects the arteries, bowels, uterus, and skin. Affected individuals can have spontaneous rupture of hollow organs, such as the bowels or gravid uterus, along with arterial dissections and ruptures that lead to premature death. The arterial disease seen in individuals with vEDS is diffuse, involving small to medium arteries and the aorta.1,2 The median lifespan of patients with vEDS is only 48 years with the majority of deaths attributed to vascular complications.3,4 Surgical treatment of vascular disease in patients with vEDS have been limited because of friable and fragile tissues. However, a human treatment trial using celiprolol, a cardioselective β-adenergic blocking agent with β2 agonist properties, was shown to reduce the number of vascular events in patients with vEDS.5 Although there were limitations to the treatment trial, this study provided the first possible medical therapy for vEDS. While much of the morbidity and mortality of this condition is still due to spontaneous rupture of nondilated arteries, progressively enlarging arterial aneurysms have been reported, suggesting that there is a greater role for noninvasive imaging, elective surgery, and medical therapy than previously thought.6,7 Article see p 80 Type III collagen is composed of 3 α1(III) polypeptide chains coiled into a triple helix structure. Each polypeptide chain in the triple helix portion of the protein contains a domain of approximately 330 Gly-X-Y repeats. Disruption of this triple helical structure leads to abnormal protein folding, thereby resulting in pathogenicity.8 Missense mutations altering the glycine codons in the triple helical domain account for the majority of disease-causing mutations and lead to misfolding of type III collagen in the endoplasmic reticulum and retention of seven eighths of misfolded procollagen trimers in the cell.9 This …

Background Development of thoracic aortic aneurysms is the most significant clinical phenotype in patients with Marfan syndrome. An inflammatory response has been described in advanced stages of the disease. Because the hallmark of vascular inflammation is local interleukin‐6 (IL‐6) secretion, we explored the role of this proinflammatory cytokine in the formation of aortic aneurysms and rupture in hypomorphic fibrillin‐deficient mice (mgR/mgR). Methods and Results MgR/mgR mice developed ascending aortic aneurysms with significant dilation of the ascending aorta by 12 weeks (2.7±0.1 and 1.3±0.1 for mgR/mgR versus wild‐type mice, respectively; P P P P Conclusion Activation of IL‐6‐STAT3 signaling contributes to aneurysmal dilation in mgR/mgR mice through increased MMP‐9 activity, aggravating extracellular matrix degradation.

Bicuspid aortic valve (BAV) is the most common adult congenital heart defect and is found in 0.5% to 2.0% of the general population. The term "BAV" refers to a heterogeneous group of disorders characterized by diverse aortic valve malformations with associated aortopathy, congenital heart defects, and genetic syndromes. Even after decades of investigation, the genetic determinants of BAV and its complications remain largely undefined. Just as BAV phenotypes are highly variable, the genetic etiologies of BAV are equally diverse and vary from complex inheritance in families to sporadic cases without any evidence of inheritance. In this paper, the authors discuss current concepts in BAV genetics and propose a roadmap for unraveling unanswered questions about BAV through the integrated analysis of genetic and clinical data. © 2014 by the American College of Cardiology Foundation.

> Everything should be kept as simple as possible, but no simpler. > > —Albert Einstein1 Since its estimated first description >500 years ago by Leonardo da Vinci,2 the bicuspid aortic valve (BAV) has progressively built a reputation; initially, as a curious valvular phenotype with a tendency to develop obstruction and insufficiency. In more contemporary times, however, the BAV is recognized as underlying almost 50% of isolated severe aortic stenosis cases requiring surgery,3 and has been extensively associated with ominous outcomes such as bacterial endocarditis and aortic dissection.4 These associations, coupled with the high prevalence of BAV in humans,5 have prompted investigative efforts into the condition, which although insightful, have generated more questions than answers. This review describes our current knowledge of BAV, but, more importantly, it highlights knowledge gaps and areas where basic and clinical research is warranted. Our review has 2 sections. The first section outlines the multifaceted challenge of BAV, our current understanding of the condition, and barriers that may hamper the advancement of the science. The second section proposes a roadmap to discovery based on current imaging, molecular biology, and genetic tools, recognizing their advantages and limitations. ### A Condition Characterized by Variable Clinical Presentation The clinical presentation and consequences of BAV in humans are exceedingly heterogeneous, with few clinical or molecular markers to predict associated complications.4,6 BAV can be diagnosed at any stage during a lifetime, from newborns7 to the elderly,8 and in the setting of variable clinical circumstances. Some are benign circumstances such as auscultatory abnormalities or incidental echocardiographic findings in otherwise healthy patients8; other circumstances are morbid, such as early severe aortic valve dysfunction, premature congestive heart failure, and thoracic aortic aneurysms (TAAs).8,9 Life-threatening circumstances include bacterial endocarditis and acute aortic dissection.8–11 These complications may present …

Background Turner syndrome is associated with bicuspid aortic valve (BAV), coarctation of the aorta, aortic dilation, and aortic dissection. Vertebral artery tortuosity, as demonstrated by magnetic resonance angiography (MRA), is increased in other disorders associated with thoracic aortic dilation and dissection, including Marfan syndrome and Loeys-Dietz syndrome, and increased tortuosity is associated with earlier adverse cardiovascular outcomes in those groups. We investigated the association between vertebral artery tortuosity and aortic pathology in patients with Turner syndrome.

The pathogenesis of systemic sclerosis (SSc) involves complex interactions between activated fibroblasts eventually leading to fibrosis, and impaired immune tolerance characterized by a variety of circulating SSc-specific autoantibodies. The expression of autoantigens in fibroblasts, a key target tissue in SSc, may play an important role in this process. To obtain a global view of this process, we examined gene expression profiles of SSc dermal fibroblasts using cDNA microarrays. The results show that dermal fibroblasts from SSc patients obtained from either affected or unaffected skin displayed a characteristic pattern of increased SSc autoantigen gene expression compared with that from normal controls. In particular, fibrillarin (p = 0.028), centromeric protein B (p = 0.01), centromeric autoantigen P27 (p = 0.042), and RNA polymerase II (220 kDa; p = 0.02) were significantly overexpressed in SSc fibroblasts. Quantitative RT-PCR confirmed overexpression of these autoantigens and also revealed increased levels of DNA topoisomerase I transcripts in SSc fibroblasts compared with normal control fibroblasts (p = 0.0318). The polymyositis/scleroderma autoantigen gene was overexpressed in some SSc patients (p = 0.09). To examine the specificity of these overexpressed autoantigen genes for SSc and its tissue specificity for fibroblasts, cDNA microarrays of dermal fibroblasts from patients with eosinophilic fasciitis and scleromyxedema were studied as well as PBMC and muscle biopsies from SSc patients. None of these tissues showed significant alterations in gene expression of SSc-specific autoantigens. Therefore, SSc-associated autoantigen genes are selectively overexpressed in SSc dermal fibroblasts, a major tissue involved in disease pathogenesis.

Congenital contractural arachnodactyly (CCA) is an autosomal dominant condition that shares skeletal features with Marfan syndrome (MFS), but does not have the ocular and cardiovascular complications that characterize MFS. CCA and MFS result from mutations in highly similar genes, FBN2 and FBN1, respectively. All the identified CCA mutations in FBN2 cluster in a limited region similar to where severe MFS mutations cluster in FBN1, specifically between exons 23 and 34. We screened exons 22 through 36 of FBN2 for mutations in 13 patients with classic CCA by single stranded conformational polymorphism analysis (SSCP) and then by direct sequencing. We successfully identified 10 novel mutations in this critical region of FBN2 in these patients, indicating a mutation detection rate of 75% in this limited region. Interestingly, none of these identified FBN2 mutations alter amino acids in the calcium binding consensus sequence in the EGF-like domains, whereas many of the FBN1 mutations alter the consensus sequence. Furthermore, analysis of the clinical data of the CCA patients with characterized FBN2 mutation indicate that CCA patients have aortic root dilatation and the vast majority lack evidence of congenital heart disease. These studies have implications for our understanding of the molecular basis of CCA, along with the diagnosis and genetic counseling of CCA patients.

Background —Aortic aneurysms cause significant mortality, and >20% relate to hereditary disorders. Familial aortic aneurysm (FAA) has been described in such conditions as the Marfan and Ehlers-Danlos type IV syndromes, due to defects in the fibrillin-1 and type III procollagen genes, respectively. Other gene defects that cause isolated aneurysms, however, have not thus far been described. Methods and Results —We studied 3 families affected by FAA. No family met the diagnostic criteria for either Marfan or Ehlers-Danlos syndrome. Echocardiography defined involvement of both the thoracic and abdominal aorta. In family ANA, candidate gene analysis excluded linkage to loci associated with aneurysm formation, including fibrillin-1 , fibrillin-2 , and type III procollagen , and chromosome 3p24.2-p25. Genome-wide linkage analysis identified a 2.3-cM FAA locus ( FAA1 ) on chromosome 11q23.3-q24 with a maximum multipoint logarithm of the odds score of 4.4. In family ANB, FAA was linked to fibrillin-1 . In family ANF, however, FAA was not linked to any locus previously associated with aneurysm formation, including fibrillin-1 and FAA1 . Conclusions —FAA disease is genetically heterogeneous. We have identified a novel FAA locus at chromosome 11q23.3-q24, a critical step toward elucidating 1 gene defect responsible for aortic dilatation. Future characterization of the FAA1 gene will enhance our ability to achieve presymptomatic diagnosis of aortic aneurysms and will define molecular mechanisms to target therapeutics.

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained

A large east Texas family with autosomal dominant inheritance of a novel bleeding disorder has been identified. The disorder is characterized clinically by easy bruising, life-threatening bleeding with trauma or surgery, and menorrhagia in affected women. Laboratory studies demonstrated prolongation of the prothrombin time and activated partial thromboplastin time in affected individuals. Paradoxically, assays of known coagulation factors are all within normal limits. To determine the molecular basis of this disease, a candidate gene linkage analysis in this kindred was done. Initially it was hypothesized that the cause of the disease in this family could be an antithrombin III (AT3) mutation that resulted in a constitutively active AT3 in the absence of heparin binding. Linkage studies using DNA from the family and an intragenic polymorphic marker within the AT3 gene showed that the disease mapped to this locus. The coding region and intron/exon junctions of AT3were sequenced using the proband's DNA, but this analysis failed to identify a mutation. Additional family members were recruited for the study, and 16 polymorphic markers around the AT3 gene were analyzed. Using 2 recombinants, the critical interval for the defective gene was narrowed to approximately 1.5 Mb, centromeric toAT3. The factor V (FV) gene was mapped into the disease interval and sequenced; there were no mutations found. Elucidation of the genetic defect causing the bleeding disorder in this family may reveal a novel protein involved in the coagulation cascade.

Mutations in FBN1 cause the autosomal dominant condition, Marfan syndrome. A single-base mutation that results in a skipping of exon 2 of FBN1 was found in a Marfan patient. By sequencing this proband's entire FBN1 gene and comparing the mutated DNA sequence with proband's unaffected family numbers, we confirmed this alteration was the causative mutation. The skipping of exon 2 creates a frameshift and premature termination codon, and forms a truncated fibrillin-1 composed only of 55 amino acids of N-terminus plus 45 nonsense amino acids. The mRNA transcription levels of the mutated FBN1 allele and the deposition of fibrillin-1 into extracellular matrix in fibroblast cells culture were assessed.

Objective To determine if there are abnormalities in fibrillin 1–containing microfibrils in the extracellular matrix (ECM) of primary dermal fibroblasts explanted from patients with systemic sclerosis (SSc). Methods Explanted fibroblasts from unaffected skin of 12 SSc patients were used to examine fibrillin 1–containing microfibrils by immunofluorescence (IF) using a monoclonal antibody (mAb) to fibrillin 1. Metabolic labeling of the fibroblast cultures was used to study the synthesis, secretion, and processing of fibrillin 1, as well as to observe microfibril formation and stability. Microfibrils elaborated by the SSc cells were analyzed by electron microscopy for ultrastructural abnormalities, and the results were confirmed by immunoblotting. Results Control and SSc fibroblasts displayed a prominent meshwork of fibrillin 1–containing microfibrils when visualized by IF using a fibrillin 1 mAb. Paradoxically, metabolic studies indicated a paucity of fibrillin 1 in the ECM in the majority of the SSc fibroblast strains. Subsequent rotary-shadowed electron microscopy revealed reduced amounts of and ultrastructural abnormalities in the microfibrils elaborated by all strains of SSc cells. Immunoblots confirmed the lack of the high molecular weight form of fibrillin 1 in the SSc fibroblasts of Choctaw American Indians. Finally, in vitro studies indicated that the amount of fibrillin 1 in the ECM of SSc cells diminished at a faster rate than the amount of fibrillin 1 in the ECM of control cells with time. Conclusion Although SSc fibroblasts assemble microfibrils, these microfibrils are unstable, suggesting that an inherent defect of fibrillin 1–containing microfibrils may play a role in the pathogenesis of SSc.

Objective Previously, we demonstrated with the use of microsatellite markers that a 2-cM haplotype on chromosome 15q containing the fibrillin 1 gene (FBN1) was strongly associated with systemic sclerosis (SSc) in the Choctaw, a population with high SSc prevalence. In this study, all 69 known FBN1 exons were sequenced to ascertain the presence of changes that might show associations with SSc in the Choctaw and Japanese SSc patients and controls. Methods Screening of FBN1 exons was accomplished by polymerase chain reaction–based fluorescence sequencing of genomic DNA using single-nucleotide polymorphism (SNP) haplotypes, and their frequencies were determined with a new algorithm that recognizes past recombination events between sites. Haplotype phylogenies were inferred using the median-joining network analysis. Results Five SNPs were identified in FBN1. They are located in the 5′-untranslated region (SNP-1), exon 15 (SNP-2), intron 17 (SNP-3), exon 27 (SNP-4), and intron 27 (SNP-5). Only SNP-1 (TC) demonstrated an association with SSc in the Choctaw. Eleven FBN1 SNP haplotypes were ascertained in the Choctaw population, 2 of which (SNPs 5 and 6) were found only in the SSc patients. These same FBN1 SNP haplotypes were associated with SSc in the Japanese. Conclusion A SNP in the 5′-untranslated region of FBN1 (SNP-1, C allele) was strongly associated with SSc in the Choctaw. Furthermore, this polymorphism is present on 2 unique FBN1 haplotypes found only in Choctaw SSc patients. The same 2 haplotypes demonstrate associations with SSc in the Japanese. These data extend the earlier microsatellite studies and are consistent with the hypothesis that FBN1 or a nearby gene on chromosome 15q is involved in SSc susceptibility in the Choctaw and the Japanese.