**2. The genetic basis of BAV and CAVD**

**BAV has a strong genetic basis, but the precise causes remain unknown.** Heritability estimates the proportion of a disease attributable to genetics. BAV heritability estimates are high, ranging from 75 to 89%, indicating that major genetic factors contribute to the develop‐

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 BAV and CAVD.

**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.

taxonomies of disease.

176 Calcific Aortic Valve Disease

**2. The genetic basis of 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

**BAV has a strong genetic basis, but the precise causes remain unknown.** Heritability estimates the proportion of a disease attributable to genetics. BAV heritability estimates are high, ranging from 75 to 89%, indicating that major genetic factors contribute to the develop‐

**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 of cases of critical aortic stenosis in the newborn and is associated with hypoplastic left heart syndrome (HLHS), suggesting genetic ("severe" malformation) and environmental (flow perturbations) factors combine to result in disease manifestation [15,54,55]. Elucidation of the genetic basis of both BAV and CAVD will result in a reconciled classification system that integrates the molecular basis of cardiac development with the pathologic basis of disease in a clinically meaningful manner.

**Genetic factors contributing to CAVD are numerous and relatively small.** Common complex traits are generally the result of numerous factors, each with a small additive effect and none necessary or sufficient to cause disease [56]. Coronary artery disease (CAD) and systemic hypertension (HTN) are well-described examples of this type of trait. While there is unequiv‐ ocal evidence that BAV with CAVD is a complex trait, it is not nearly as common as CAD or HTN, and is more strongly linked to developmental processes, therefore it is likely that BAV/ CAVD is an "intermediate" phenotype between the "rare single-gene" and "common com‐ plex" diseases. Importantly, this suggests that it is more likely to discover clinically useful patterns of variants associated with CAVD. Clinically, CAVD, CAD and HTN are considered discrete disease states, but there is a preponderance of epidemiologic and molecular evidence suggesting some pathogenesis is shared. Therefore, variants that have been identified studying individuals with CAD and HTN may inform risk assessment in patients with CAVD. Just as some clinical cardiovascular risk factors are common to all cardiovascular disease states, some genetic variants may pertain to predisposition of any cardiovascular disease depending on the aggregate risk (Figure 3). For example, the 10q24 locus has been identified in probands from BAV, CAD, HTN, thoracic aortic aneurysm (TAA) and intracranial aneurysm families [44, 57-60], suggesting the gene(s) in this region plays a role in each of these related cardiovascular phenotypes and therefore may be a general cardiovascular risk variant. It remains unclear whether a specified number of general cardiovascular risk variants are sufficient to cause any one disease, or more intuitively both *specific* and *general* disease variants are necessary.

may initiate additional disease processes that incite CAVD (e.g. endothelial dysfunction). Taken together, a nonspecific cardiovascular insult in the context of a specific genetic predis‐ position for BAV may be necessary and sufficient for the manifestation of CAVD. As the genetic and developmental basis of valve malformation and disease is elucidated, opportunities for novel medical therapies will emerge and potentially preclude or delay the need for surgery. Defining regulation of valve tissue maintenance and homeostasis will provide exciting

**Figure 3. Shared predisposing genetic risk variants in common cardiovascular diseases.** Cardiovascular diseases characterized by complex inheritance may have genetic variants specific to the clinical disease state, e.g., CAVD, CAD, HTN (yellow), as well as nonspecific genetic variants that may contribute to two (green) or three (blue) different cardi‐

Genetics of Bicuspid Aortic Valve and Calcific Aortic Valve Disease

http://dx.doi.org/10.5772/55506

179

**Complex inheritance is characterized by a liability threshold.** Polygenic conditions are characterized by a fixed number of susceptibility genes and a liability threshold, whereby a variety of combinations of predisposing variants may reach a specified level (e.g., 3 risk variants) to cause in combination the phenotype. In general, the importance of genetic modifiers and epigenetics is rapidly emerging, but little is known about these factors in the context of BAV/CAVD. Different BAV morphologies may reflect different combinations of shared genetic variants that carry different clinical risks, e.g. CAVD, thoracic aortic aneurysm and dissection, or associated CVM. It has been shown for example that RN BAV morphology is associated with a higher risk of developing valve disease and experiencing a cardiac event [5,22]. Together, patterns of predisposing genetic variants, which may be reflected in part by anatomical subtleties such as BAV morphology, may translate to variations in clinical disease states, suggesting major modifiers play a significant role in phenotype definition. Identifying these patterns may impact care, for example by facilitating the ability to consistently predict

opportunities for cell-based or molecular therapies for valve disease.

natural history [66,67].

ovascular diseases.

**CAVD is a latent phenotype, an injury or defect in valve maintenance.** Typically, aortic valve disease does not manifest until the fourth or fifth decade of life and often does not progress to require surgical intervention until a decade later. How can developmental defects be functional for so long, only to fail in adulthood? The prevailing view is that individuals with a genetic predisposition for CAVD require an additional "second" insult to trigger disease initiation and progression that otherwise would not have occurred. While many of the genes that have been implicated in CAVD effect valve development [61,62], they may have additional distinct roles in valve maintenance [63], that is, how the tissue responds over time to the hemodynamic demands of constant motion and changing physiology. Similarly, there are genes that do not have a role in valve development but may be necessary for valve homeostasis [35,63]. Indeed, CAVD has been labeled a "degenerative" condition for decades, and age-related "wear and tear" contributes to valve failure. For example, elastic fiber degradation occurs with advanced age and predisposes the individual to inflammation, which may contribute to CAVD acceler‐ ation in later life [64,65]. Equally important, however, are comorbid conditions such as CAD that may serve to be an injury, or second hit, in vulnerable aortic valve tissue. For example, in an individual with genetic variants predisposing specifically for CAVD, the presence of CAD

of cases of critical aortic stenosis in the newborn and is associated with hypoplastic left heart syndrome (HLHS), suggesting genetic ("severe" malformation) and environmental (flow perturbations) factors combine to result in disease manifestation [15,54,55]. Elucidation of the genetic basis of both BAV and CAVD will result in a reconciled classification system that integrates the molecular basis of cardiac development with the pathologic basis of disease in

**Genetic factors contributing to CAVD are numerous and relatively small.** Common complex traits are generally the result of numerous factors, each with a small additive effect and none necessary or sufficient to cause disease [56]. Coronary artery disease (CAD) and systemic hypertension (HTN) are well-described examples of this type of trait. While there is unequiv‐ ocal evidence that BAV with CAVD is a complex trait, it is not nearly as common as CAD or HTN, and is more strongly linked to developmental processes, therefore it is likely that BAV/ CAVD is an "intermediate" phenotype between the "rare single-gene" and "common com‐ plex" diseases. Importantly, this suggests that it is more likely to discover clinically useful patterns of variants associated with CAVD. Clinically, CAVD, CAD and HTN are considered discrete disease states, but there is a preponderance of epidemiologic and molecular evidence suggesting some pathogenesis is shared. Therefore, variants that have been identified studying individuals with CAD and HTN may inform risk assessment in patients with CAVD. Just as some clinical cardiovascular risk factors are common to all cardiovascular disease states, some genetic variants may pertain to predisposition of any cardiovascular disease depending on the aggregate risk (Figure 3). For example, the 10q24 locus has been identified in probands from BAV, CAD, HTN, thoracic aortic aneurysm (TAA) and intracranial aneurysm families [44, 57-60], suggesting the gene(s) in this region plays a role in each of these related cardiovascular phenotypes and therefore may be a general cardiovascular risk variant. It remains unclear whether a specified number of general cardiovascular risk variants are sufficient to cause any one disease, or more intuitively both *specific* and *general* disease variants are necessary.

**CAVD is a latent phenotype, an injury or defect in valve maintenance.** Typically, aortic valve disease does not manifest until the fourth or fifth decade of life and often does not progress to require surgical intervention until a decade later. How can developmental defects be functional for so long, only to fail in adulthood? The prevailing view is that individuals with a genetic predisposition for CAVD require an additional "second" insult to trigger disease initiation and progression that otherwise would not have occurred. While many of the genes that have been implicated in CAVD effect valve development [61,62], they may have additional distinct roles in valve maintenance [63], that is, how the tissue responds over time to the hemodynamic demands of constant motion and changing physiology. Similarly, there are genes that do not have a role in valve development but may be necessary for valve homeostasis [35,63]. Indeed, CAVD has been labeled a "degenerative" condition for decades, and age-related "wear and tear" contributes to valve failure. For example, elastic fiber degradation occurs with advanced age and predisposes the individual to inflammation, which may contribute to CAVD acceler‐ ation in later life [64,65]. Equally important, however, are comorbid conditions such as CAD that may serve to be an injury, or second hit, in vulnerable aortic valve tissue. For example, in an individual with genetic variants predisposing specifically for CAVD, the presence of CAD

a clinically meaningful manner.

178 Calcific Aortic Valve Disease

**Figure 3. Shared predisposing genetic risk variants in common cardiovascular diseases.** Cardiovascular diseases characterized by complex inheritance may have genetic variants specific to the clinical disease state, e.g., CAVD, CAD, HTN (yellow), as well as nonspecific genetic variants that may contribute to two (green) or three (blue) different cardi‐ ovascular diseases.

may initiate additional disease processes that incite CAVD (e.g. endothelial dysfunction). Taken together, a nonspecific cardiovascular insult in the context of a specific genetic predis‐ position for BAV may be necessary and sufficient for the manifestation of CAVD. As the genetic and developmental basis of valve malformation and disease is elucidated, opportunities for novel medical therapies will emerge and potentially preclude or delay the need for surgery. Defining regulation of valve tissue maintenance and homeostasis will provide exciting opportunities for cell-based or molecular therapies for valve disease.

**Complex inheritance is characterized by a liability threshold.** Polygenic conditions are characterized by a fixed number of susceptibility genes and a liability threshold, whereby a variety of combinations of predisposing variants may reach a specified level (e.g., 3 risk variants) to cause in combination the phenotype. In general, the importance of genetic modifiers and epigenetics is rapidly emerging, but little is known about these factors in the context of BAV/CAVD. Different BAV morphologies may reflect different combinations of shared genetic variants that carry different clinical risks, e.g. CAVD, thoracic aortic aneurysm and dissection, or associated CVM. It has been shown for example that RN BAV morphology is associated with a higher risk of developing valve disease and experiencing a cardiac event [5,22]. Together, patterns of predisposing genetic variants, which may be reflected in part by anatomical subtleties such as BAV morphology, may translate to variations in clinical disease states, suggesting major modifiers play a significant role in phenotype definition. Identifying these patterns may impact care, for example by facilitating the ability to consistently predict natural history [66,67].
