American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 151C:307 – 317 (2009) A R T I C L E Disorders of Left–Right Asymmetry: Heterotaxy and Situs Inversus MARDI J. SUTHERLAND AND STEPHANIE M. WARE* Cilia function is critical to the development of proper organ laterality. Primary ciliary dyskinesia (PCD) causes randomization of situs. Heterotaxy, or situs ambiguus, is an abnormal arrangement of the thoracic and abdominal organs that results in congenital anomalies. Animal models and developmental biological approaches have defined pathways required during embryogenesis for proper left–right pattern formation. New candidates for genetic causes of human laterality disorders have emerged from recent studies on the assembly, transport, and signaling functions of cilia at the node as well as identification of cilia within the developing heart. There is evidence that deleterious genetic variants within one or more developmental pathways may disrupt signaling in a synergistic or combinatorial fashion to cause congenital anomalies. The molecular pathways underlying PCD and heterotaxy are being discovered at a rapid pace, and there is increasing recognition of the overlap between these two categories of laterality disorders and their relationship to isolated cardiovascular malformations. This review focuses on the clinical manifestations, molecular mechanisms, and human genetics of these disorders of laterality. ß 2009 Wiley-Liss, Inc. KEY WORDS: cilia; heterotaxy; left–right asymmetry; situs inversus; primary ciliary dyskinesia How to cite this article: Sutherland MJ, Ware SM. 2009. Disorders of left–right asymmetry: Heterotaxy and situs inversus. Am J Med Genet Part C Semin Med Genet 151C:307–317. INTRODUCTION Heterotaxy and situs inversus are disorders of laterality in which the internal organs do not have their typical pattern of asymmetry. In situs inversus, the internal organ arrangement is mirror image of normal anatomy, termed situs solitus. Baillie first described situs inversus in 1793. One hundred forty years later, Kartagener , a pulmonoloStephanie M. Ware, M.D., Ph.D. is an Assistant Professor in the Divisions of Molecular Cardiovascular Biology, Human Genetics, and Cardiology at Cincinnati Children’s Hospital Medical Center. Her laboratory investigates the genetic basis of cardiac disease in children. She is board certified in Pediatrics and Clinical Genetics. Mardi J. Sutherland is a graduate student in the Molecular and Developmental Biology graduate program at Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine. *Correspondence to: Stephanie M. Ware, M.D., Ph.D., Cincinnati Children’s Hospital Medical Center, 240 Albert Sabin Way, MLC 7020, Cincinnati, OH 45229. E-mail: email@example.com DOI 10.1002/ajmg.c.30228 Published online 27 October 2009 in Wiley InterScience (www.interscience.wiley.com) ß 2009 Wiley-Liss, Inc. gist, reported four patients with the triad of sinusitis, bronchiectasis, and situs inversus. In the 1970s, the relationship between situs inversus and ciliary abnormalities was established when Afzelius reported cilia immotility in infertile males, half of whom had Kartagener’s triad [Afzelius, 1976]. His In the 1970s, the relationship between situs inversus and ciliary abnormalities was established when Afzelius reported cilia immotility in infertile males, half of whom had Kartagener’s triad. His speculation that cilia played a role in determining laterality would require several more decades to prove. speculation that cilia played a role in determining laterality would require several more decades to prove. Primary ciliary dyskinesia (PCD), previously known as immotile cilia syndrome, is one of the most widely recognized ciliopathies. Kartagener syndrome (OMIM #244400), in which situs is randomized, is a subset of PCD and accounts for approximately 20% of these cases. PCD has an estimated prevalence of 1:15,000. Heterotaxy, or situs ambiguus, is an abnormal arrangement of the thoracic and/or abdominal viscera. It is characterized by multiple congenital malformations, with the major morbidity and mortality resulting from complex cardiovascular malformations. A variety of descriptive (e.g., asplenia or polysplenia syndrome, left or right isomerism) or eponymous (e.g., Ivemark syndrome) designations arose in an attempt to precisely classify constellations of clinical and phenotypic features of patients with laterality disorders. In reality, both clinical observation and molecular investigation indicate that exceptions are the rule, and the 308 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) designations encompass an overlapping spectrum. It is therefore more precise and clinically useful to use the term heterotaxy or situs ambiguus followed by the exact anatomic description. The International Nomenclature Committee for Pediatric and Congenital Heart Disease recently published cardiac evaluation and nomenclature recommendations for heterotaxy, and details of the cardiac and extra-cardiac anomalies have been reviewed in detail previously [Zhu et al., 2006; Jacobs et al., 2007; Ware and Belmont, 2008]. Heterotaxy accounts for approximately 3% of all congenital heart defects. ‘‘Classic’’ heterotaxy, in which typical cardiac defects are seen in conjunction with visceral situs anomalies, has an The International Nomenclature Committee for Pediatric and Congenital Heart Disease recently published cardiac evaluation and nomenclature recommendations for heterotaxy. estimated prevalence of 1:10,000 [Lin et al., 2000]. Because of the wide phenotypic spectrum identified in laterality disorders, it is likely that this underestimates the true prevalence. It is now clear, for example, that gene mutations that cause classic forms of heterotaxy can also result in isolated cardiovascular malformations without evidence of visceral anomalies [Megarbane et al., 2000; Goldmuntz et al., 2002; Ware et al., 2004]. Similarly, patients with PCD can have cardiovascular malformations with or without dextrocardia. A recent study by Kennedy et al.  demonstrates that 6.3% of patients with PCD have heterotaxy. These data illustrate the spectrum of disease caused by abnormal left– right development during embryogenesis. DIAGNOSIS AND CLINICAL MANAGEMENT Patients with PCD frequently have neonatal respiratory distress, but the diagnosis of a ciliopathy may not be made until months or years later. Classically, patients with PCD have significant problems with mucociliary clearance, leading to sinusitis, persistent otitis media, or bronchiectasis (Table I). Situs inversus or heterotaxy may occur. ARTICLE Infertility is reported in greater than 50% of PCD males due to decreased or absent sperm motility. Cystic fibrosis shares many overlapping features and needs to be ruled out. The standard for diagnosis is transmission electron microscopy for analysis of ciliary ultrastructure. Common findings include abnormalities of the outer or inner dynein arms (most frequent), radial spokes, central microtubules, or absence of cilia. Although abnormal electron microscopy confirms the diagnosis of PCD, normal electron microscopy does not rule out the diagnosis. Additional methods are being developed to better analyze cilia structure and function, including analysis of ciliary motility on high-speed videomicroscopy, analysis of mucociliary clearance, demonstration of decreased nasal nitric oxide levels, or immunohistochemical analysis of the outer dynein arms [Zariwala et al., 2007]. Strict clinical diagnostic criteria have not yet been developed for PCD [Leigh et al., 2009]. As more data regarding the overlap of clinical features between heterotaxy and PCD emerge, diagnostic recommendations can be formalized (Table I). Heterotaxy is most frequently diagnosed in the newborn period due TABLE I. Clinical Features and Diagnosis of PCD and Heterotaxy PCD Clinical features Variable age of presentation Neonatal respiratory distress Situs inversus or heterotaxy Chronic sinusits, otitis media, or bronchiectasis Persistent cough, nasal congestion, or respiratory infection Male infertility/impaired sperm motility Diagnostic evaluation Pedigree Ultrastructural analysis (TEM) of cilia Consider chromosomes and chromosome microarray analysis Gene mutation analysis (DNAH5, DNAI1) Imaging studies as necessary TEM, transmission electron microscopy. Heterotaxy Presentation in infancy in classic form Congenital heart disease, dextrocardia, or mesocardia Malrotation, microgastria, abdominal situs anomalies Biliary atresia Vertebral or rib anomalies Asplenia or polysplenia Pedigree Imaging studies to define anatomy (echocardiogram/cardiac MRI; head ultrasound or brain MRI; upper gastrointestinal series; abdominal ultrasound; chest X-ray and spine films; consideration of hepatobiliary scintigraphy) Chromosomes and chromosome microarray analysis Consider gene mutation analysis (ZIC3) ARTICLE to cyanotic congenital heart disease. These patients require an extensive diagnostic evaluation and multiple imaging studies in order to define their anatomy (Table I). While it may not be practical to perform all of the diagnostic evaluations in a critically ill infant, the spectrum of potential anomalies needs to be recognized, particularly in the case of sudden decompensation. All patients should have chromosomes and chromosome microarray analysis due to associations with chromosome abnormalities, as described in more detail below. Strong consideration should be given to ZIC3 mutation testing particularly in males. The prevalence of PCD in patients with heterotaxy remains to be determined. Nevertheless, consideration should be given to evaluation for PCD since respiratory and pulmonary management could be optimized in an attempt to improve the higher than expected surgical morbidity and mortality in this patient group. The development of improved methods for PCD diagnosis, as described above, is important to better define the patient subsets and direct management and therapy. AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) (LPM), (5) signaling from the LPM to organ primordia. Heterotaxy may result from defects in any of these five steps. Isolated dextrocardia or congenital heart defects related to abnormal laterality can also result from abnormalities at various stages of left–right patterning. In contrast, situs inversus caused by PCD usually results from defects in signaling at the node involving cilia structure and function. Signaling Upstream of the Node The early stage of left–right patterning occurs before cilia are established at the node, a transient embryonic organizer. 309 This involves processes including planar cell polarity in which asymmetric gradients are established within a cell, across a sheet of cells and ultimately throughout the embryo [Aw and Levin, 2009]. The signaling pathways and molecular mechanisms underlying these events during left–right patterning are not yet well defined, and these early steps of asymmetry may vary by model organism. There is some evidence that asymmetry is first established as intracellular gradients [Palmer, 2004; Vandenberg, 2009]. No pathogenic mutations have been described at this early stage of axis formation in humans with laterality defects. ETIOLOGY AND MOLECULAR MECHANISMS The left–right axis is established early in development subsequent to the development of anterior–posterior and dorsal–ventral axes. A basic understanding of the embryonic tissues and molecular events necessary for left–right axis formation is beneficial for initiating a diagnostic work-up, understanding the genetic basis of disease, and assessing recurrence risk. Recent work with model organisms has offered a detailed view of the steps taken to establish asymmetry in the vertebrate. Figure 1 illustrates five steps for left–right patterning: (1) signaling upstream of the node, (2) signaling at the node involving cilia structure and function, (3) leftward flow breaking bilateral symmetry at the node, (4) transfer of asymmetric gene expression from the node to the lateral plate mesoderm Figure 1. Overview of left–right asymmetry in the mouse embryo. Before the establishment of the node or cilia, there is evidence for early intracellular asymmetry involving mechanisms such as planar cell polarity (PCP). PCP also plays a later role in the proper orientation of cilia at the node. In the second stage, the node and functioning motile and sensory cilia form. Although the genetic control of node morphogenesis is largely unknown, Notch signaling is necessary for both proper node structure as well as later asymmetric nodal expression. Foxj1 is a master regulator of ciliogenesis, and intraflagellar transport is required for cilia assembly. Centrally located monocilia are motile and peripheral monocilia lack left–right dynein and are immotile. In stage 3, the movement of cilia at the node creates a leftward flow of morphogens. FGF signaling triggers the release of Shh and retinoic acid. An increase in cytoplasmic calcium occurs. Nodal expression is initially bilateral, but becomes asymmetrically expressed on the left, providing molecular evidence of left–right asymmetry. In stage 4, Nodal signal is propagated from the node to the LPM where asymmetric gene expression is established. The Nodal signal transduction pathway occurs when the TGF-b ligand Nodal is cleaved to its active form. It requires EGF-CFC co-receptors to bind to Type I/II activin receptors. Lefty proteins interfere with binding of the mature ligand. After binding, phosphorylated Smad complexes translocate to the nucleus where they bind DNA with co-factors such as FoxHI. Through this pathway, Nodal can activate Lefty1, Lefty2, Pitx2 and its own expression in the LPM. In stage 5, asymmetric signaling is propagated from the LPM to organ primordia in order for proper morphogenesis to occur. IFT, intraflagellar transport; LPM, lateral plate mesoderm; PCP, planar cell polarity. 310 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) Signaling at the Node Involving Cilia Structure and Function Although the existence of an organizing left-right coordinator has been documented in all vertebrate species studied to date, its structure varies. In mouse, this structure is known as the ventral node; in chick, Henson’s node; in zebrafish, kupffer’s vesicle (KV); in Xenopus, the gastrocoel roof plate (GRP); and in rabbit, the posterior notochordal plate (PNC) [Essner et al., 2002; Okada et al., 2005; Lee and Anderson, 2008]. Although the existence of an organizing left-right coordinator has been documented in all vertebrate species studied to date, its structure varies. In mouse, this structure is known as the ventral node; in chick, Henson’s node; in zebrafish, kupffer’s vesicle (KV); in Xenopus, the gastrocoel roof plate (GRP); and in rabbit, the posterior notochordal plate (PNC). Additionally, the coordinating structure has recently been examined in pig. The organizer is required for proper axis formation prior to organogenesis. In mouse, the node is present between 7.5 and 8.5 days post coitum, which is equivalent to Carnegie stages 7–10 in humans. The node structure in pig and chick begins with bilateral symmetry, but later in development exhibits leftward displacement and asymmetric gene expression. These structural and molecular changes result from cell migration to the left of the node [Gros et al., 2009]. In mouse, rabbit, and zebrafish, the organizing structures contain monociliated cells with motile cilia which generate a ‘‘nodal flow.’’ Experimental evidence in mouse and zebrafish has linked the Notch pathway with both node formation and downstream activation of asymmetric Nodal gene expression at the node [Krebs et al., 2003; Przemeck et al., 2003; Takeuchi et al., 2007]. Leftward fluid flow at the node results from the clockwise rotational motion of the cilia creating a unidirectional flow. In many vertebrate species examined, including rabbit, fish, and mouse, cilia are detected as being tilted toward the posterior axis [Nonaka et al., 2002; Okada et al., 2005]. This posterior tilt is predicted as a necessary component of the leftward unidirectional flow at the node, which is essential for proper left– right asymmetry [Nonaka et al., 1998; Okada et al., 1999]. There are two populations of cilia located at the mouse node [McGrath et al., 2003]. These consist of the There are two populations of cilia located at the mouse node. These consist of the central motile cilia and the peripheral sensory cilia. central motile cilia and the peripheral sensory cilia. Typical motile cilia contain nine microtubule doublets surrounding two central microtubules and are referred to as 9 þ 2 motile cilia. Motile cilia of the node lack the two central microtubules and are referred to as 9 þ 0 motile cilia. Sensory cilia (9 þ 0) also lack the central microtubules but, in addition, lack dynein arms and are immotile. Proteins necessary for ciliary movement are specific to motile cilia, and include dynein proteins which make up the inner and outer dynein arms along the microtubules [Ibanez-Tallon et al., 2003; McGrath et al., 2003; Inglis et al., 2006; Satir and Christensen, 2007]. Motile cilia of the node also contain the left–right dynein protein (Dnahc11 gene, homologous to human DNAH11), which is required for their functional motility [Supp et al., 1997, 1999; Okada et al., 1999; McGrath et al., 2003]. ARTICLE Motile cilia of the node also contain the left–right dynein protein (Dnahc11 gene, homologous to human DNAH11), which is required for their functional motility. Recent data have provided insight into the genetic program required for motile ciliogenesis. Foxj1, a forkhead transcription factor, is expressed in tissues with motile cilia. Foxj1 is required for induction of gene expression necessary for motile ciliogenesis [Stubbs et al., 2008; Yu et al., 2008] in multiple species including zebrafish, mice, and Xenopus [Chen et al., 1998; Brody et al., 2000; Zhang et al., 2004; Stubbs et al., 2008; Yu et al., 2008]. Thus, Foxj1 is acting as an upstream regulator of cilia formation and function at the organizer [Whitsett and Tichelaar, 1999; Zhang et al., 2004; Stubbs et al., 2008]. Intraflagellar transport (IFT) is important for cilia formation, structural function and motility, and signaling function. It is the mechanism responsible for delivering proteins necessary for ciliary formation and function from the cytoplasm of the cell to the cilia. This process involves anterograde transport, toward the tip of the ciliary structure, Intraflagellar transport (IFT) is important for cilia formation, structural function and motility, and signaling function. It is the mechanism responsible for delivering proteins necessary for ciliary formation and function from the cytoplasm of the cell to the cilia. ARTICLE performed by plus-end directed motor protein complexes involving kinesins, and retrograde transport, directed toward the base of the cilium, carried out by minus-end directed motor protein complexes involving dyneins [Rosenbaum and Witman, 2002]. Studies of loss of function in mouse models demonstrate the necessity of these proteins for left–right patterning [Supp et al., 1997; Nonaka et al., 1998; Marszalek et al., 1999; Takeda et al., 1999; Rana et al., 2004]. Recent IFT studies in mouse have also uncovered the critical role for cilia in signal transduction in a number of developmental pathways including Sonic hedgehog and Wnt pathways [Huangfu et al., 2003; Huangfu and Anderson, 2005, 2006; Liu et al., 2005; Davis et al., 2006; Singla and Reiter, 2006; Corbit et al., 2008; Jopling and Izpisua Belmonte, 2009]. Studies in zebrafish and Xenopus have also linked FGF signaling with the intraflagellar pathway and thus, ciliogenesis [Hong and Dawid, 2009; Neugebauer et al., 2009]. New intracellular roles for molecular motor proteins apart from their role in ciliogenesis are also being examined in left–right asymmetry [Armakolas and Klar, 2007; Levin and Palmer, 2007]. Leftward Flow Breaks Bilateral Symmetry at the Node In mouse, Nodal, a ligand of the TGF-b signaling pathway, is first expressed symmetrically in crown cells surrounding the node, followed by asymmetrical leftsided expression. The development of asymmetric gene expression at the node is the first molecular evidence of asymmetry and is critical for subsequent left-sided gene expression in the LPM. Two theories exist involving the mechanism by which information is transferred by the leftward flow of cilia. The first involves secretion of morphogenetic proteins at the node into the extracellular fluid. FGF signaling triggers the release of particles carrying morphogens Sonic hedgehog and retinoic acid, which are forced to the left of the node inducing a cascade of gene AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) expression [Okada et al., 2005; Hirokawa et al., 2006]. There is also evidence that the leftward flow of extracellular fluid causes bending of the peripheral immotile cilia surrounding the node [McGrath et al., 2003]. An asymmetric calcium flux is subsequently induced and may trigger left-sided signaling. Both of these pathways seem likely to play a role, but the precise mechanism by which asymmetric Nodal expression is induced at the node is not known. Transfer of Asymmetric Gene Expression From the Node to the LPM The role of the Nodal signal transduction pathway in vertebrate left–right asymmetry is a common pathway in all vertebrates studied thus far and has been extensively described [Palmer, 2004; Raya and Belmonte, 2004; Levin, 2005; Levin and Palmer, 2007]. The majority of known human heterotaxy mutations have been discovered in this pathway using a candidate gene approach. However, mutations within this pathway cause heterotaxy in a minority of cases, indicating that novel genetic etiologies remain to be determined (Table II). After left-sided expression of Nodal is established at the mouse node, the signal is propagated to the left LPM where Nodal, in the presence of an EGF-CFC co-factor, signals through Type I and Type II receptors. Nodal can activate two antagonists, Lefty-1, expressed in the midline, and Lefty-2, expressed in the LPM (homologous to human LEFTYA and LEFTYB, respectively). The activation of these Nodal antagonists limits Nodal expression and inhibits the transfer of left-sided gene expression across the midline of the embryo. The role of the midline in the establishment of left–right asymmetry was recently reviewed [Lee and Anderson, 2008]. Pitx2, a homeobox transcription factor, is also activated by Nodal and is asymmetrically expressed in the LPM as well as various organs. This molecular asymmetry is conserved across vertebrates. 311 Signaling From the LPM to Organ Primordial The asymmetric expression of Pitx2 is initiated by Nodal signaling but persists after the disappearance of these signals in the LPM [Shiratori et al., 2001]. Pitx2 is involved in organogenesis in the heart, gut, and lung and demonstrates asymmetric expression in developing organ primordia. Recent work examining the looping of the linear gut tube [Davis et al., 2008; Kurpios et al., 2008], found that Pitx2 and Isl2 act upstream of asymmetric cellular morphology and regulate expression of adhesion molecules. Information about cardiac specific targets during looping morphogenesis is currently lacking and needs further investigation. Interestingly, recent work has identified cilia within the developing heart [Van der Heiden et al., 2006; Slough et al., 2008] and suggested a potential role in cardiac development distinct from their role in left–right asymmetry. In addition, Interestingly, recent work has identified cilia within the developing heart and suggested a potential role in cardiac development distinct from their role in left–right asymmetry. there is evidence that cilia are acting as sensors for endothelial shear stress produced by the flow of fluid through vessels [Iomini et al., 2004]. Preliminary work suggests involvement of cilia in nitric oxide production and endothelial cell dysfunction [Van der Heiden et al., 2008; AbouAlaiwi et al., 2009]. Further investigation into these additional roles for cilia in the cardiovascular system may lead to a better understanding of the pathogenesis of heterotaxy and PCD. New Approaches and Advantages Using Model Organisms Recently, new approaches in the study of left–right asymmetry using model 312 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE TABLE II. Gene Mutations Identified in Human Laterality Disorders Gene Heterotaxy NODAL ZIC3 CFC1 Mutation prevalence Refs. 5–10% 1% in sporadic; >75% in familial X-linked 6–21% Mohapatra et al. , Roessler et al.  Ware et al.  Bamford et al. , Roessler et al. , Selamet Tierney et al.  Robinson et al.  Roessler et al.  Peeters et al. [2003, 2006] Kosaki et al. [1999a] Karkera et al. , Roessler et al.  Kosaki et al. [1999b] Watanabe et al.  CRELD1 FOXH1 SESN1 LEFTY A GDF1 ACVR2B NKX2.5 PCD DNAH5 DNAI1 <1% Unknown <1% 1.6% 2.1% 2.4% Unknown DNAI2 DNAH11 TXNDC3 RPGR KTU Heterotaxy þ PCD DNAI1 or DNAH5 1.9% Unknown 2.1% Unknown 1.8% organisms have emerged. Given that the core components of the Nodal signaling pathway are conserved across species, it is useful to exploit the advantages that each model organism provides to study the establishment of left–right asymmetry. For example, a recent zebrafish mutagenesis study [Zhang et al., 2009] using forward genetic screening followed by massively parallel sequencing identified a gene, Megf8, involved in the propagation of the Nodal signal from the node to the LPM. This is a relatively fast and cost efficient method to identify new pathways and genes implicated in left–right development. Additionally, Xenopus offers the advantage of combinatorial knockdown of genes using morpholinos as well as right- and left-side specific knockdown of genes. These processes can be observed and directly manipulated with dorsal explants in which unidirectional flow can be established. The advantages of this system have recently been reviewed [Levin and Palmer, 2007; Blum et al., 2009]. While differences in left–right patterning 15–28% 2–10% 58% Failly et al. , Hornef et al. , Olbrich et al.  Failly et al. , Zariwala et al. , Zariwala et al.  Guichard et al. , Pennarun et al.  Loges et al.  Bartoloni et al. , Schwabe et al.  Duriez et al.  Moore et al.  Omran et al.  Kennedy et al.  exist between model organisms, there are also core conserved mechanisms involved in the establishment of asymmetry, and exploiting the strengths of a variety of model organisms will undoubtedly uncover new pathways as well as new roles for previously discovered genes. GENETICS OF PCD AND HETEROTAXY In the majority of cases, situs inversus associated with PCD is an autosomal recessive condition. The differential would include other ciliopathies in which situs inversus can be found, including Bardet–Biedl syndrome. Heterotaxy most often occurs as a sporadic condition. Complex inheritance or autosomal dominant inheritance with reduced penetrance and variable expressivity are presumed genetic mechanisms. Familial clustering of heterotaxy has been documented, with pedigrees suggestive of autosomal recessive and autosomal dominant inheritance [Vitale et al., 2001; Belmont et al., 2004; Zhu et al., 2006; Wessels et al., 2008]. X-linked inheritance is well documented in heterotaxy and is caused by mutations in ZIC3. A large number of genetic syndromes and chromosome abnormalities have been associated with heterotaxy, including aneuploidies, complex chromosomal rearrangements, and microdeletions [Bisgrove et al., 2003; Ware and Belmont, 2008; Song et al., 2009]. For example, patients with trisomy 13 or trisomy 18 have been reported. A number of submicroscopic chromosomal deletions, including 22q11.2 (DiGeorge/VCFS syndrome), have been identified in patients with heterotaxy. Bisgrove et al.  review unbalanced translocations, terminal deletions, inversions, and more complex chromosomal rearrangements associated with heterotaxy. Analysis of genes located within submicroscopic chromosomal imbalances in patients with abnormal left–right asymmetry should be a useful tool in uncovering novel genes and pathways in laterality defects. ARTICLE X-Linked Inheritance An X-linked form of PCD has been described in association with retinitis pigmentosa, resulting from mutations in RPGR (OMIM #300455). The visual abnormalities are associated with hearing loss, sinusitis, and chronic recurrent respiratory tract infections, and abnormalities of the ciliary axoneme have been identified by electron microscopy [Zito et al., 2003; Moore et al., 2006]. The Xlinked form of heterotaxy, HTX-1 (OMIM #306955) is caused by mutations in a zinc finger transcription factor, ZIC3 [Ferrero et al., 1997; Gebbia et al., 1997]. Approximately 1% of sporadic heterotaxy cases (male and female) are caused by mutations in ZIC3, and affected females have been described with point mutations or with chromosomal translocations [Megarbane et al., 2000; Ware et al., 2004; Fritz et al., 2005; Tzschach et al., 2006; Chhin et al., 2007]. In X-linked pedigrees, ZIC3 mutations are commonly identified (Table II). Clinical testing is available. The mutations cause loss of function and, in some cases, result in abnormal subcellular localization and trafficking [Bedard et al., 2007]. Loss of function of ZIC3 has been correlated with a range of phenotypes ranging from classic heterotaxy to isolated cardiovascular malformations typical of abnormal heart looping, such as double outlet right ventricle, L-transposition of the great arteries, and ventricular inversion. There is evidence in model organisms that ZIC3 acts upstream of Nodal signaling at the node, but its precise developmental function is not yet known [Purandare et al., 2002; Ware et al., 2006]. Autosomal Dominant Inheritance Heterotaxy patients without mutations in ZIC3 have been screened for mutations in genes involved in the conserved Nodal signal transduction pathway. Given the importance of the Nodal pathway in establishing left–right asymmetry, it is not surprising that mutations in components of this pathway, including NODAL itself, have been identified AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) in human heterotaxy (Table II) [Kosaki et al., 1999a,b; Bamford et al., 2000; Goldmuntz et al., 2002; Selamet Tierney et al., 2007; Roessler et al., 2008; Mohapatra et al., 2009]. Mutations have been identified in genes encoding the ligand (NODAL), ligand co-receptor (CFC-1), receptor (ACVRIIB), transcriptional co-activator (FOXH1), and midline inhibitor (LEFTYA) within the Nodal signal transduction pathway (Table II). Recently, NODAL variants were investigated in 269 patients with heterotaxy or isolated cardiovascular mutations thought to be associated with irregular looping of the heart. Missense mutations were observed in 14 of the unrelated patients [Mohapatra et al., 2009]. Functional analysis of the mutant proteins showed a decrease in Nodal signaling. Similarly, functional analysis of mutations in Cryptic, a gene encoding an epidermal growth factor family protein, CFC1, which functions as a NODAL co-receptor, revealed abnormal cellular localization of the mutant protein [Bamford et al., 2000]. In addition, mutations in CFC1 have been identified in patients with transposition of the great arteries but without extra-cardiac anomalies [Goldmuntz et al., 2002]. ACVRIIB mutations were identified in 3 of 126 patients with left–right anomalies and mutations in LEFTYA were discovered in 2 patients [Kosaki et al., 1999a,b]. In the latter case, each patient inherited the mutant allele from an unaffected carrier parent, indicating incomplete penetrance. Candidate gene analysis has also focused on GDF1, a gene expressed in the node encoding a TGF-b ligand. In 375 patients, 8 potential disease-causing mutations were discovered [Karkera et al., 2007]. With over 100 candidate genes identified through mouse models of left–right patterning defects, it is likely that significant genetic heterogeneity will be found in human heterotaxy. To date, the majority of genes identified have either not yet been tested in larger heterotaxy populations (e.g., NKX2.5, CRELD1), or have been found to have mutations at low frequency [Watanabe 313 et al., 2002; Robinson et al., 2003]. For example, positional cloning of a region involved in a reciprocal translocation in one patient identified SESN1, a gene involved in left–right patterning. Functional analysis of this protein revealed that it plays a role in activating Nodal signaling. However, the prevalence of disease causing mutations in this gene is less than 1% based on an initial screen of a heterotaxy cohort [Peeters et al., 2003, 2006]. Efforts are ongoing to identify novel genes that cause or contribute to the heterotaxy phenotype. Mapping studies in two different families with autosomal dominant inheritance with reduced penetrance [Vitale et al., 2001; Wessels et al., 2008] have identified a locus on chromosome 6p encompassing approximately 12 Mb and a small number of strong candidate genes. Interestingly, two of the candidates are dynein heavy chain genes, while another is a kinesin gene. Autosomal Recessive Inheritance PCD disorders are characterized by abnormalities of ciliary structure and function and are most commonly autosomal recessive. Mutations in DNAH5 are the most commonly identified cause In addition, it has recently been shown that patients with PCD have a higher risk for congenital heart disease, similar to patients with heterotaxy, or situs ambiguus. of PCD [Olbrich et al., 2002], with mutations in DNAI1 [Pennarun et al., 1999] and DNAH11 [Pan et al., 1998; Bartoloni et al., 2002] also contributing. All are outer dynein arm components which are essential for ciliary function at the node. Mutations in these genes result in randomized situs, leading to situs inversus in approximately 50% of 314 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) patients [Noone et al., 2004]. In addition, it has recently been shown that patients with PCD have a higher risk for congenital heart disease, similar to patients with heterotaxy, or situs ambiguus [Kennedy et al., 2007]. Complex Inheritance Approximately 10% of patients with heterotaxy have a family history of a close relative with a congenital heart defect. To date, the majority of mutations identified have been shown to have reduced penetrance and variable expressivity, indicating multifactorial causation. X-linked heterotaxy, caused by mutations in ZIC3, is the best understood genetic cause of heterotaxy. Interestingly, mutations in this gene can cause classic heterotaxy or isolated congenital heart defects. In one family, three of nine female carriers exhibited situs inversus [Gebbia et al., 1997; Ware et al., 2004]. In vitro analyses of these ZIC3 mutations indicate similar levels of functional abnormalities, suggesting that the range of phenotypes results from genetic modifiers, environmental modifiers, epigenetic effects, or developmental stochasticity. There are reports to support each of these mechanisms. For example, in a small number of cases, mutations in two or more genes within the Nodal signal transduction pathway, or in NODAL and ZIC3 in a female patient, have been identified. In addition, a common NODAL polymorphism that reduces gene activity has been identified. These findings support the hypothesis that common or rare deleterious genetic variants within developmental pathways may disrupt signaling in a synergistic or combinatorial fashion to cause congenital anomalies [Gebbia et al., 1997; Roessler et al., 2008]. Environmental modifiers such as maternal diabetes, maternal cocaine use, and monozygotic twinning have all been associated with heterotaxy spectrum defects [Kuehl and Loffredo, 2002]. Stochastic levels of Nodal expression have been reported in inbred mouse lines deficient for Gdf3, a TGF-b ligand that acts in a Nodal-related pathway [Chen et al., 2006]. Taken together, these mechanisms likely account for the significant degree of the variability within the heterotaxy/congenital heart disease/PCD population. CONCLUSIONS AND FUTURE DIRECTIONS The molecular players involved in PCD and heterotaxy are being discovered at a rapid pace and there is increasing recognition of the overlap between these two categories of laterality disorders. Over 100 genes have been identified as important in left–right asymmetry in animal models. Combined with recent The molecular players involved in PCD and heterotaxy are being discovered at a rapid pace and there is increasing recognition of the overlap between these two categories of laterality disorders. Over 100 genes have been identified as important in left–right asymmetry in animal models. studies on the ciliome, the number of candidates for laterality disorders is quite large [Inglis et al., 2006]. This heterogeneity, combined with a prediction of reduced penetrance and variable expressivity, makes clinical molecular diagnostics challenging. Further clinical insight into the relationship between situs inversus, situs ambiguus, and isolated cardiovascular malformations within the heterotaxy spectrum will be important for clinical management and diagnosis. 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