Siddharth K. Prakash

TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome

A predisposition for thoracic aortic aneurysms leading to acute aortic dissections can be inherited in families in an autosomal dominant manner. Genome-wide linkage analysis of two large unrelated families with thoracic aortic disease followed by whole-exome sequencing of affected relatives identified causative mutations in TGFB2. These mutations—a frameshift mutation in exon 6 and a nonsense mutation in exon 4—segregated with disease with a combined logarithm of odds (LOD) score of 7.7. Sanger sequencing of 276 probands from families with inherited thoracic aortic disease identified 2 additional TGFB2 mutations. TGFB2 encodes transforming growth factor (TGF)-β2, and the mutations are predicted to cause haploinsufficiency for TGFB2; however, aortic tissue from cases paradoxically shows increased TGF-β2 expression and immunostaining. Thus, haploinsufficiency for TGFB2 predisposes to thoracic aortic disease, suggesting that the initial pathway driving disease is decreased cellular TGF-β2 levels leading to a secondary increase in TGF-β2 production in the diseased aorta.

Bicuspid Aortic Valve Identifying Knowledge Gaps and Rising to the Challenge From the International Bicuspid Aortic Valve Consortium (BAVCon)

> 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 …

Rare Copy Number Variants Disrupt Genes Regulating Vascular Smooth Muscle Cell Adhesion and Contractility in Sporadic Thoracic Aortic Aneurysms and Dissections

Thoracic aortic aneurysms and dissections (TAAD) cause significant morbidity and mortality, but the genetic origins of TAAD remain largely unknown. In a genome-wide analysis of 418 sporadic TAAD cases, we identified 47 copy number variant (CNV) regions that were enriched in or unique to TAAD patients compared to population controls. Gene ontology, expression profiling, and network analysis showed that genes within TAAD CNVs regulate smooth muscle cell adhesion or contractility and interact with the smooth muscle-specific isoforms of α-actin and β-myosin, which are known to cause familial TAAD when altered. Enrichment of these gene functions in rare CNVs was replicated in independent cohorts with sporadic TAAD (STAAD, n = 387) and inherited TAAD (FTAAD, n = 88). The overall prevalence of rare CNVs (23%) was significantly increased in FTAAD compared with STAAD patients (Fishers exact test, p = 0.03). Our findings suggest that rare CNVs disrupting smooth muscle adhesion or contraction contribute to both sporadic and familial disease.

Recurrent chromosome 16p13.1 duplications are a risk factor for aortic dissections.

Chromosomal deletions or reciprocal duplications of the 16p13.1 region have been implicated in a variety of neuropsychiatric disorders such as autism, schizophrenia, epilepsies, and attention-deficit hyperactivity disorder (ADHD). In this study, we investigated the association of recurrent genomic copy number variants (CNVs) with thoracic aortic aneurysms and dissections (TAAD). By using SNP arrays to screen and comparative genomic hybridization microarrays to validate, we identified 16p13.1 duplications in 8 out of 765 patients of European descent with adult-onset TAAD compared with 4 of 4,569 controls matched for ethnicity (P = 5.0×10−5, OR = 12.2). The findings were replicated in an independent cohort of 467 patients of European descent with TAAD (P = 0.005, OR = 14.7). Patients with 16p13.1 duplications were more likely to harbor a second rare CNV (P = 0.012) and to present with aortic dissections (P = 0.010) than patients without duplications. Duplications of 16p13.1 were identified in 2 of 130 patients with familial TAAD, but the duplications did not segregate with TAAD in the families. MYH11, a gene known to predispose to TAAD, lies in the duplicated region of 16p13.1, and increased MYH11 expression was found in aortic tissues from TAAD patients with 16p13.1 duplications compared with control aortas. These data suggest chromosome 16p13.1 duplications confer a risk for TAAD in addition to the established risk for neuropsychiatric disorders. It also indicates that recurrent CNVs may predispose to disorders involving more than one organ system, an observation critical to the understanding of the role of recurrent CNVs in human disease and a finding that may be common to other recurrent CNVs involving multiple genes.

Rare, Nonsynonymous Variant in the Smooth Muscle-Specific Isoform of Myosin Heavy Chain, MYH11, R247C, Alters Force Generation in the Aorta and Phenotype of Smooth Muscle Cells

Rationale: Mutations in myosin heavy chain (MYH11) cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, nonsynonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. Objective: To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). Methods and Results: The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11R247C/R247C mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11R247C/R247C mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11R247C/R247C mice were dedifferentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11R247C/R247C SMCs to a Rho activator rescued the dedifferentiated phenotype of the SMCs. Conclusions: These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury.

Copy number gain at Xp22.31 includes complex duplication rearrangements and recurrent triplications

Genomic instability is a feature of the human Xp22.31 region wherein deletions are associated with X-linked ichthyosis, mental retardation and attention deficit hyperactivity disorder. A putative homologous recombination hotspot motif is enriched in low copy repeats that mediate recurrent deletion at this locus. To date, few efforts have focused on copy number gain at Xp22.31. However, clinical testing revealed a high incidence of duplication of Xp22.31 in subjects ascertained and referred with neurobehavioral phenotypes. We systematically studied 61 unrelated subjects with rearrangements revealing gain in copy number, using multiple molecular assays. We detected not only the anticipated recurrent and simple nonrecurrent duplications, but also unexpectedly identified recurrent triplications and other complex rearrangements. Breakpoint analyses enabled us to surmise the mechanisms for many of these rearrangements. The clinical significance of the recurrent duplications and triplications were assessed using different approaches. We cannot find any evidence to support pathogenicity of the Xp22.31 duplication. However, our data suggest that the Xp22.31 duplication may serve as a risk factor for abnormal phenotypes. Our findings highlight the need for more robust Xp22.31 triplication detection in that such further gain may be more penetrant than the duplications. Our findings reveal the distribution of different mechanisms for genomic duplication rearrangements at a given locus, and provide insights into aspects of strand exchange events between paralogous sequences in the human genome.

MAT2A Mutations Predispose Individuals to Thoracic Aortic Aneurysms

Up to 20% of individuals who have thoracic aortic aneurysms or acute aortic dissections but who do not have syndromic features have a family history of thoracic aortic disease. Significant genetic heterogeneity is established for this familial condition. Whole-genome linkage analysis and exome sequencing of distant relatives from a large family with autosomal-dominant inheritance of thoracic aortic aneurysms variably associated with the bicuspid aortic valve was used for identification of additional genes predisposing individuals to this condition. A rare variant, c.1031A>C (p.Glu344Ala), was identified in MAT2A, which encodes methionine adenosyltransferase II alpha (MAT IIα). This variant segregated with disease in the family, and Sanger sequencing of DNA from affected probands from unrelated families with thoracic aortic disease identified another MAT2A rare variant, c.1067G>A (p.Arg356His). Evidence that these variants predispose individuals to thoracic aortic aneurysms and dissections includes the following: there is a paucity of rare variants in MAT2A in the population; amino acids Glu344 and Arg356 are conserved from humans to zebrafish; and substitutions of these amino acids in MAT Iα are found in individuals with hypermethioninemia. Structural analysis suggested that p.Glu344Ala and p.Arg356His disrupt MAT IIα enzyme function. Knockdown of mat2aa in zebrafish via morpholino oligomers disrupted cardiovascular development. Co-transfected wild-type human MAT2A mRNA rescued defects of zebrafish cardiovascular development at significantly higher levels than mRNA edited to express either the Glu344 or Arg356 mutants, providing further evidence that the p.Glu344Ala and p.Arg356His substitutions impair MAT IIα function. The data presented here support the conclusion that rare genetic variants in MAT2A predispose individuals to thoracic aortic disease.

Aortic Disease Presentation and Outcome Associated with ACTA2 Mutations

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.

Diabetes and Reduced Risk for Thoracic Aortic Aneurysms and Dissections: A Nationwide Case-Control Study

Background Vascular diseases are the principal causes of death and disability in people with diabetes. At the same time, studies suggest a protective role of diabetes in the development of abdominal aortic aneurysms. We sought to determine whether diabetes is associated with decreased hospitalization due to thoracic aortic aneurysms and dissections (TAAD). Methods and Results We used the 2006 and 2007 Nationwide Inpatient Sample (NIS) to determine TAAD discharge rates. Control subjects were randomly selected to achieve three controls per case. Predictor variables in multilevel logistic regression included age, race, median income, diabetes, and hypertension. We estimated that the average rate of hospital discharge for TAAD among individuals diagnosed with diabetes was 9.7 per 10 000, compared to 15.6 per 10 000 among all discharges. The prevalence of diabetes was substantially lower in TAAD (13%) than in control (22%) records. After adjustment for demographic characteristics, the negative association between diabetes and TAAD remained highly significant in both NIS datasets. Compared to discharges without diabetes, those with chronic complications of diabetes were least likely to be diagnosed with TAAD (OR [odds ratio] 0.17, 95% CI, 0.12–0.23). A significant association remained between uncomplicated diabetes and TAAD. We replicated these findings in an independent group of patients who were hospitalized with acute thoracic aortic dissections. Conclusions The principal implication of our findings is that diabetes is independently associated with a decreased rate of hospitalization due to TAAD in proportion to the severity of diabetic complications. Future studies should consider diabetes in predictive models of aneurysm expansion or dissection. (J Am Heart Assoc. 2012;1:jah3-e000323 doi: 10.1161/JAHA.111.000323.)

Bicuspid aortic valve aortopathy in adults: Incidence, etiology, and clinical significance

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.