**1. The clinical taxonomy: Malformation vs. disease**

**Aortic valve malformation is a spectrum including Bicuspid Aortic Valve.** Aortic valve malformation has been appreciated since the Renaissance when artists advanced our under‐ standing of anatomy and specifically, Leonardo da Vinci illustrated and described variants of aortic valve morphology [1]. Aortic valve malformation is the most common cardiovascular malformation (CVM), and bicuspid aortic valve (BAV, MIM#109730) is the most common type of aortic valve malformation. BAV is present at birth and is characterized by two rather than three cusps. The incidence of BAV is 1-2% in the general population and affects an estimated 3 million people [2,3]. BAV itself is subclinical and the valve is typically functional, making BAV an endophenotype. Two patterns of BAV morphology are commonly observed: ~70% of isolated cases have fusion of the right and left (RL) coronary cusps with the remainder consisting almost entirely of those with fusion of the right and non (RN) coronary cusps [4,5]. Rarely, cases have shown fusion of the left and non (LN) coronary cusps. In addition to BAV subtypes, there is a spectrum of aortic valve malformation (Figure 1), ranging from various types of unicuspid to quadricuspid aortic valves with the three BAV morphology patterns and a thickened tricommissural aortic valve representing intermediate phenotypes [7]. Presently, it remains unclear to what degree these variations of malformation represent true differences.

**Calcific Aortic Valve Disease is a growing public health problem.** Aortic valve disease is defined by abnormal valve function. Valve disease may manifest as *stenosis*, an obstruction to normal forward blood flow, or *insufficiency*, a defective closure resulting in backward blood flow. Valve disease tends to progress. Ultimately, ventricular function can be compromised. Aortic valve stenosis is the most common manifestation of CAVD and classically presents as angina, syncope and heart failure. The diagnosis can be made clinically and confirmed by echocardiography, which quantifies the severity, and, over time, the progression of disease [8].

© 2013 Hinton; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

pharmacologic treatments for CAVD, the indications for surgical intervention dominate the clinical landscape. Early disease processes and progression remain poorly understood, and

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**Figure 2. Phenotype definition: types of aortic valve disease.** Color Doppler echocardiographic apical four cham‐ ber images demonstrate the two basic types of aortic valve disease. Aortic valve disease is characterized by a dysfunc‐ tional valve and is classified as stenosis (obstruction, A) and/or insufficiency (incompetence, B). Aortic stenosis (AS) and aortic insufficiency (AI) result in hemodynamic perturbations that lead to clinical disease states. Advanced calcific aortic valve disease is typically characterized by stenosis, and histopathology identifies gross calcific nodules in the fi‐ brosa layer of the cusp (asterisks, C), clusters of cartilage like interstitial cells (arrowheads, C), and marked heterogenei‐ ty of extracellular matrix abnormalities (arrows, C). AO aorta; AOV aortic valve; LA left atrium; LV left ventricle.

**Bicuspid Aortic Valve is an independent risk factor for Calcific Aortic Valve Disease.** BAV is an established risk factor for CAVD [3,7,13]. The majority of CAVD cases at any age have an underlying BAV, and longitudinal studies in young adults with BAV have shown that >20% ultimately require surgical intervention [15,22,23]. In addition, those CAVD patients with an underlying BAV tend to develop calcification a decade earlier than those with normal aortic valve morphology [24]. Recently, a National Heart Lung and Blood Institute Executive Statement on CAVD identified a critical need to identify "clinical risk factors for the distinct phases of initiation and progression of AVD" [25], where standard cardiac risk factors including sclerosis have not yet been applicable. There has been avid interest and conflicting reports regarding the potential use of BAV morphology as a specific predictor of CAVD. Fernandes et al identified an association between RN BAV and AVD in a pediatric population, while Tzemos et al found no association in an adult population [5,22]. Exploring AVD in a pediatric population allows for examination of the disease process free from the confounding effects of cardiovascular comorbidities. Risk factors for AVD in children are poorly understood [23], but recently Calloway et al. reported that children with RN BAV and adults with RL BAV were more likely to develop AVD, suggesting BAV morphology may have predictive value for the time course of AVD [26]. It is unclear if AVD in children, which is not characterized by calcification, represents a different genetic type of disease or one end of a spectrum of the same

disease.

there are presently no pharmacologic based treatment options for CAVD.

**Figure 1. Phenotype definition: spectrum of aortic valve malformation.** Aortic valve malformation Parasternal short axis echocardiographic views at the base of the heart showing the aortic valve en face (A-H). Normal tricommis‐ sural aortic valve (TAV) morphology is demonstrated in diastole (A) and systole (B). Distinct morphologies are based on fusion patterns of the commissures (dotted lines, B) as they relate to the right (R), left (L) and non (N) coronary sinuses of Valsalva (A). Aortic valve malformation ranges from unicuspid (UAV) to bicuspid (BAV) to a thickened tricus‐ pid (not shown) to quadricuspid (QAV) morphology. Three normal commissures are demonstrated in panel A, and normal opening of the commissures results in complete cusp separation to the wall of the aorta at the sinotubular junction (yellow arrowheads). UAV manifests as either partial fusion of all three commissures (red arrowheads, C) or complete fusion of both the RN and RL commissures (D). Bicuspid aortic valve (BAV) may manifest as fusion of the RL (E), RN (F), and rarely LN (G) commissures. Rarely, a quadricuspid aortic valve (QAV, H) is identified. Adapted from [6].

Histopathology from diseased valves explanted at the time of surgery from patients with CAVD demonstrates large nodules of overt calcification, in addition to cell-matrix abnormal‐ ities (Figure 2). Research efforts have focused on the valve cusp, and as a result the valve annulus has been largely overlooked [4,7,9]. Human studies investigating valve disease have suggested that the base of the valve cusp and valve annulus regions is the origin of disease processes, including both sclerosis and calcification [10,11]. Greater than 2.5% of the popula‐ tion has AVD, causing more than 25,000 deaths annually in the US [12,13]. The actual direct cost for valve disease in the US alone has been estimated at 1 billion dollars per year [14]. Taken together, the public health impact and burden to society of CAVD is significant and underap‐ preciated. The majority of valve disease at any age has an underlying valve malformation suggesting a genetic basis [15]. Aging is an independent risk factor for CAVD, resulting in a higher prevalence of disease as the population achieves greater longevity [16,17]. Aortic valve sclerosis, a marker of cardiovascular risk, and to a lesser extent valve disease, is present in more than 25% of the aged [18]. Therapy for CAVD remains primarily surgical and is restricted to late stage disease. Aortic valve replacement is the second most frequent cardiovascular surgical procedure [3,9], and the need for re-intervention is common [19]. Bioprosthetic replacement approaches are effective, but not durable [20,21]. Because there is a lack of pharmacologic treatments for CAVD, the indications for surgical intervention dominate the clinical landscape. Early disease processes and progression remain poorly understood, and there are presently no pharmacologic based treatment options for CAVD.

Histopathology from diseased valves explanted at the time of surgery from patients with CAVD demonstrates large nodules of overt calcification, in addition to cell-matrix abnormal‐ ities (Figure 2). Research efforts have focused on the valve cusp, and as a result the valve annulus has been largely overlooked [4,7,9]. Human studies investigating valve disease have suggested that the base of the valve cusp and valve annulus regions is the origin of disease processes, including both sclerosis and calcification [10,11]. Greater than 2.5% of the popula‐ tion has AVD, causing more than 25,000 deaths annually in the US [12,13]. The actual direct cost for valve disease in the US alone has been estimated at 1 billion dollars per year [14]. Taken together, the public health impact and burden to society of CAVD is significant and underap‐ preciated. The majority of valve disease at any age has an underlying valve malformation suggesting a genetic basis [15]. Aging is an independent risk factor for CAVD, resulting in a higher prevalence of disease as the population achieves greater longevity [16,17]. Aortic valve sclerosis, a marker of cardiovascular risk, and to a lesser extent valve disease, is present in more than 25% of the aged [18]. Therapy for CAVD remains primarily surgical and is restricted to late stage disease. Aortic valve replacement is the second most frequent cardiovascular surgical procedure [3,9], and the need for re-intervention is common [19]. Bioprosthetic replacement approaches are effective, but not durable [20,21]. Because there is a lack of

**Figure 1. Phenotype definition: spectrum of aortic valve malformation.** Aortic valve malformation Parasternal short axis echocardiographic views at the base of the heart showing the aortic valve en face (A-H). Normal tricommis‐ sural aortic valve (TAV) morphology is demonstrated in diastole (A) and systole (B). Distinct morphologies are based on fusion patterns of the commissures (dotted lines, B) as they relate to the right (R), left (L) and non (N) coronary sinuses of Valsalva (A). Aortic valve malformation ranges from unicuspid (UAV) to bicuspid (BAV) to a thickened tricus‐ pid (not shown) to quadricuspid (QAV) morphology. Three normal commissures are demonstrated in panel A, and normal opening of the commissures results in complete cusp separation to the wall of the aorta at the sinotubular junction (yellow arrowheads). UAV manifests as either partial fusion of all three commissures (red arrowheads, C) or complete fusion of both the RN and RL commissures (D). Bicuspid aortic valve (BAV) may manifest as fusion of the RL (E), RN (F), and rarely LN (G) commissures. Rarely, a quadricuspid aortic valve (QAV, H) is identified. Adapted from [6].

174 Calcific Aortic Valve Disease

**Bicuspid Aortic Valve is an independent risk factor for Calcific Aortic Valve Disease.** BAV is an established risk factor for CAVD [3,7,13]. The majority of CAVD cases at any age have an underlying BAV, and longitudinal studies in young adults with BAV have shown that >20% ultimately require surgical intervention [15,22,23]. In addition, those CAVD patients with an underlying BAV tend to develop calcification a decade earlier than those with normal aortic valve morphology [24]. Recently, a National Heart Lung and Blood Institute Executive Statement on CAVD identified a critical need to identify "clinical risk factors for the distinct phases of initiation and progression of AVD" [25], where standard cardiac risk factors including sclerosis have not yet been applicable. There has been avid interest and conflicting reports regarding the potential use of BAV morphology as a specific predictor of CAVD. Fernandes et al identified an association between RN BAV and AVD in a pediatric population, while Tzemos et al found no association in an adult population [5,22]. Exploring AVD in a pediatric population allows for examination of the disease process free from the confounding effects of cardiovascular comorbidities. Risk factors for AVD in children are poorly understood [23], but recently Calloway et al. reported that children with RN BAV and adults with RL BAV were more likely to develop AVD, suggesting BAV morphology may have predictive value for the time course of AVD [26]. It is unclear if AVD in children, which is not characterized by calcification, represents a different genetic type of disease or one end of a spectrum of the same disease.

**Careful clinical phenotyping is critical for research, especially genetic discovery.** Phenotype definition and stratification are necessary to advance our understanding of CAVD, especially in the context of genetic discovery. In addition to distinguishing malformation from disease, CAVD phenotyping needs to be detailed and comprehensive using all aspects of the clinical taxonomy, even those currently considered clinically inconsequential. The first step of any human genetic research study is to clearly and precisely define the phenotype. Studies that use too broad or too narrow a phenotype definition may fail to find association with an existing genetic variant or identify a pathologic one. Thus, identification of the phenotype most aligned with the underlying genetic etiology is essential for successful identification of associated genetic variants, a concept recently described as "deep phenotyping" [27]. Cardiovascular risk factors have been established for a variety of cardiovascular diseases, including substantial overlap for CAVD and coronary artery disease (CAD) or atherosclerosis [16,28,29]. While these disease processes often co-occur, as evidenced by the high frequency of concurrent coronary artery bypass grafting and aortic valve replacement surgery, only a small proportion of CAVD patients have CAD [30]. Likewise, there is an increased incidence of CAVD in patients with other cardiovascular disease, including systemic hypertension and chronic kidney disease [31,32]. Substantial investigation has established the adverse effects of common comorbid cardiovascular diseases on the progression of AVD; however, increasing attention on the underlying genetic and developmental processes will identify early mechanisms that incite disease processes. Emerging evidence suggests that both specific genetic factors and clinical cardiac risks may be necessary for disease initiation and progression.

ment of BAV [38]. Pedigree and segregation analyses have consistently identified autosomal dominant inheritance with reduced penetrance and complex inheritance underlying BAV [38-41], acknowledging that BAV is subclinical and therefore may be underestimated. Inter‐ estingly, while BAV is highly heritable, AVD is not, suggesting the phenotypic variability of CAVD is determined largely by non-genetic factors [26]. Consistent with these human observations, an established hamster model of BAV also shows the same characteristics of complex inheritance [42,43]. An additional quantitative measure of familial risk is recurrence risk. The recurrence risk of a disease measures the proportion of relatives who have the disease. BAV recurrence risk in siblings has been estimated to be approximately 9% [44], identifying further evidence of a genetic basis. Linkage analysis determines whether susceptibility variant segregates with disease in families. Previous studies have supported a strong underlying genetic basis for isolated nonsyndromic BAV, including family-based studies that have identified numerous loci [44-46]. Combined, these loci harbor hundreds of genes that may contribute to BAV. Multiple loci identify BAV as a genetically heterogeneous trait. Missense mutations in *NOTCH1* have been identified in a small proportion of nonsyndromic CAVD patients with BAV [47,48]. NOTCH1 is an intriguing biological candidate gene. In animal systems, Notch loss of function recapitulates the AVD phenotype, and actively regulates the maladaptive development of associated calcification, further supporting a mechanistic role [49-51]. In addition, a recent report described copy number variants (CNVs) in 10% of leftsided CVM cases, including BAV and aortic stenosis, potentially identifying new causes and/ or modifiers of CAVD [52]. Association studies have not been used for BAV due to the large number of cases required to perform analyses (typically at least 1000), but combined linkageassociation may be an excellent approach for discovery to leverage the strengths of each method. It is unclear how whole exome sequencing will impact discovery, but combining the various new tools for discovery promises to yield increasing insight into the genetic basis of

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**BAV is a congenital malformation, a defect in cardiac development.** Malformations present at birth often have strong genetic causes, if not monogenic etiology. Primary cardiac develop‐ ment occurs in humans from 2-8 weeks gestation, and semilunar valve (including the aortic valve) formation occurs in the seventh and eighth weeks. The heart is the first organ to form and continued survival of the organism is dependent on the circulation. The primitive heart tube is composed of a myocardial cell layer surrounding an endothelial cell layer. The formation of endocardial cushions is the first event of valve development. Endocardial cushion formation is accomplished by an early epithelial to mesenchymal transition (EMT) that generates a progenitor cell population embedded in a loosely organized extracellular matrix (ECM), followed by a late ECM remodeling stage that results in mature cusp organization (ventricularis, spongiosa, fibrosa) and valve interstitial cells [35-37]. Early defects in this process result in embryonic lethality, but late defects result in viable malformation and disease [53], hypothetically making the mechanisms of late developmental defects more applicable to human disease. It remains unknown why there are uneven frequencies of the different BAV types, but several developmental hypotheses have been proposed including a neural crest contribution that is not necessary but when present results in fusion of the right and left coronary cusps [42]. Further, the relatively rare unicuspid morphology underlies the majority

BAV and CAVD.

**Phenotype definition must expand to include non-clinical paradigms.** Like many diseases, especially cardiovascular diseases, the clinical taxonomy of CAVD is based on anatomy and physiology. Classification schemes are organized with clinical standard of care, particularly surgical intervention, in mind [33,34]. The gold standard for diagnosis of cardiovascular diseases is imaging, such as echocardiography or magnetic resonance, modalities that define anatomy and physiology. While these approaches have been clinically useful, there is sub‐ stantial phenotypic heterogeneity of unclear significance, including for example, the distinc‐ tion between malformation and disease. Expanding phenotype to include an improved understanding of embryologic patterns underlying malformation will provide insight into pathogenesis [35-37]. Increasingly, combinations of phenotypes long held to be independent from a clinical perspective are now understood to be related from an etiologic perspective, challenging classic notions of disease classification. Molecular insights may inform new pharmacologic treatments the same way imaging informs surgical decision-making. Ultimate‐ ly, identifying the genetic causes of disease will require reconciling clinical and molecular taxonomies of disease.
