Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, Congenital Heart Disease

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1 Congenital Heart Disease Congenital Heart Disease and Other Heterotaxic Defects in a Large Cohort of Patients With Primary Ciliary Dyskinesia Marcus P. Kennedy, MD; Heymut Omran, MD; Margaret W. Leigh, MD; Sharon Dell, MD; Lucy Morgan, MD; Paul L. Molina, MD; Blair V. Robinson, MD; Susan L. Minnix, RN; Heike Olbrich, PhD; Thomas Severin, MD; Peter Ahrens, MD; Lars Lange, MD; Hilda N. Morillas, MD; Peadar G. Noone, MD; Maimoona A. Zariwala, PhD; Michael R. Knowles, MD Background Primary ciliary dyskinesia (PCD) is a recessive genetic disorder that is characterized by sinopulmonary disease and reflects abnormal ciliary structure and function. Situs inversus totalis occurs in 50% of PCD patients (Kartagener s syndrome in PCD), and there are a few reports of PCD with heterotaxy (situs ambiguus), such as cardiovascular anomalies. Advances in diagnosis of PCD, such as genetic testing, allow the systematic investigation of this association. Methods and Results The prevalence of heterotaxic defects was determined in 337 PCD patients by retrospective review of radiographic and ultrasound data. Situs solitus (normal situs) and situs inversus totalis were identified in 46.0% and 47.7% of patients, respectively, and 6.3% (21 patients) had heterotaxy. As compared with patients with situs solitus, those with situs abnormalities had more ciliary outer dynein arm defects, fewer inner dynein arm and central apparatus defects (P 0.001), and more mutations in ciliary outer dynein arm genes (DNAI1 and DNAH5; P 0.022). Seven of 12 patients with heterotaxy who were genotyped had mutations in DNAI1 or DNAH5. Twelve patients with heterotaxy had cardiac and/or vascular abnormalities, and most (8 of 12 patients) had complex congenital heart disease. Conclusions At least 6.3% of patients with PCD have heterotaxy, and most of those have cardiovascular abnormalities. The prevalence of congenital heart disease with heterotaxy is 200-fold higher in PCD than in the general population (1:50 versus 1:10 000); thus, patients with PCD should have cardiac evaluation. Conversely, mutations in genes that adversely affect both respiratory and embryological nodal cilia are a significant cause of heterotaxy and congenital heart disease, and screening for PCD is indicated in those patients. (Circulation. 2007;115: ) Key Words: defects heart defects, congenital lung pediatrics transposition of great vessels Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder, with a prevalence of 1 in ,2 Clinical disease reflects defective ciliary structure and function, and includes respiratory distress in term neonates, recurrent sinopulmonary infection, chronic otitis media, subfertility, and bronchiectasis. 2 Diagnosis is usually confirmed by studies of ciliary function and ultrastructure, but more recently diagnosis has been facilitated by immunohistochemistry of cilia and measurements of nasal nitric oxide. 2,3 Mutations that cause disease have been identified in 2 genes (DNAI1 and DNAH5) that code for ciliary outer dynein arm (ODA) proteins. 4,5 Mutations in these genes are found in 35% of all PCD patients, and in as many as 60% of PCD patients with defects in the ciliary ODA. 4,5 Editorial p 2793 Clinical Perspective p 2821 Abnormalities of thoraco-abdominal asymmetry occur in 50% of PCD patients. 1,2 The organs are usually a mirror image of normal, which is situs inversus totalis (SI; Kartagener s syndrome in PCD). SI occurs as a random phenomenon in PCD and reflects a loss of nodal ciliary function during embryogenesis. 6 9 The recognition of heterotaxy in several PCD patients provoked more careful consideration of situs abnormalities in PCD. There is no consensus on the definition and classification of heterotaxy. Some authors use situs ambiguus and heterotaxy interchangeably as any abnormality of thoraco-abdominal asymmetry other than SI, whereas other authors suggest that heterotaxy includes 2 groups, SI and situs ambiguus. 10,11 Received June 30, 2006; accepted February 22, From the University of North Carolina (M.P.K., M.W.L., P.L.M., B.V.R., S.L.M., H.N.M., P.G.N., M.A.Z., M.R.K.), Chapel Hill; University Hospital Freiburg (H. Omran, H. Olbrich, T.S.), Freiburg, Germany; The Toronto Hospital for Sick Children (S.D.), Toronto, Canada; Concord Hospital (L.M.), New South Wales, Australia; Darmstädter Kinderkliniken Prinzessin Margaret (P.A.), Darmstädt, Germany; and University Hospital Cologne (L.L.), Cologne, Germany. Correspondence to Dr Michael R. Knowles, Cystic Fibrosis/Pulmonary Research and Treatment Center, 7019 Thurston Bowles Bldg, CB7248, Chapel Hill, NC knowles@med.unc.edu 2007 American Heart Association, Inc. Circulation is available at DOI: /CIRCULATIONAHA

2 Kennedy et al PCD and Heterotaxy 2815 Figure 1. Situs anomalies in 337 patients with primary ciliary dyskinesia. There are a number of recognized subtypes of heterotaxy, which are determined on the basis of cardiac atrium anatomy; left (polysplenia syndrome) and right (asplenia syndrome) disorders of isomerism sequence. The anatomic abnormalities associated with left and right isomerism have been described, but an unusual combination of anatomic abnormalities can make it difficult to classify as left or right isomerism. 14 An important consequence associated with heterotaxy is complex cardiac defects. Heterotaxy, which includes L-transposition of the great arteries, is associated with at least 3% of congenital heart disease (CHD). 11 Earlier reports, which include autopsy series, suggested that the majority of patients with isomerism died in childhood because of CHD; however, interpretation of these reports alone leads to selection bias. 12,16 18 In contrast, patients with left and right isomerism have been identified with no functional cardiac defect and a normal life span Currently, 50% of patients with left isomerism with CHD survive to age ,23 Although a few case reports have noted that some PCD patients have heterotaxy, which includes CHD, 21,24 28 polysplenia, 21,29 31 and asplenia syndromes, 19,24 the prevalence of these anatomic variations in PCD is not known. We reviewed a large cohort of PCD patients to determine the prevalence of heterotaxy and CHD and to define the types of ciliary defects and genetic mutations in DNAI1 and DNAH5 among these patients. Methods Patients We performed a retrospective analysis of clinical and radiographic data of 337 PCD patients from the United States (n 147), Germany (n 128), Canada (n 36), and Australia (n 26) to identify patients with heterotaxy. Studies were performed under the auspices of respective Committees on the Protection of Rights to Human Figure 2. Defining situs anomalies in PCD with chest x-ray. Posterior-anterior chest radiographs of 47-year-old monozygotic twins with PCD demonstrate situs solitus (A) and situs inversus totalis (B). C, Posterior-anterior chest radiograph of a 30-year-old female with PCD demonstrates abdominal situs inversus. Genotyping identified 1 DNAH5 and no DNAI1 mutation. D, Posterior-anterior chest radiograph of a 4-year-old male with PCD demonstrates isolated dextrocardia. H indicates heart apex; L, liver; S, stomach. Reproduced from Noone et al 6 with permission from Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc. Copyright 1999.

3 2816 Circulation June 5, 2007 TABLE 1. Imaging Used to Classify Heterotaxy in 337 Patients With Primary Ciliary Dyskinesia Situs Solitus (n 155) Situs Inversus Totalis (n 161) Heterotaxy (n 21) Chest x-ray Echocardiogram Thoracic CT Abdominal imaging* Values expressed as %. CT indicates computed tomography. *CT thorax with upper abdominal images, CT abdomen, ultrasound abdomen, magnetic resonance imaging of abdomen, or surgical findings. Subjects. The diagnosis of PCD was confirmed by the presence of a compatible clinical phenotype and at least 1 confirmatory test, including (1) diagnostic abnormalities of ciliary structure by electron microscopy or mislocalization of axonemal dynein proteins with high-resolution immunofluorescence analysis (n 250), (2) abnormal ciliary beat pattern with high-speed video microscopy (n 56), and/or (3) nasal nitric oxide measurement (n 134). EM technique and interpretation, and high resolution immunofluorescence analysis of axonemal proteins, have been described. 2,3 Nasal production of nitric oxide was measured at 2 sites (USA and Canada), using either a Siever system (normal mean 1SD nl/min) or a NIOX system (normal 400 to 1000 ppb). 2 Genetic analyses of DNAI1 and DNAH5 had been previously performed in 161 subjects. 4,5 Methodology to Identify Heterotaxy Patients were ultimately subdivided into 3 distinct groups: situs solitus (SS; normal thoraco-abdominal asymmetry), SI (complete mirror image reversal of SS with no other defect) and heterotaxy (situs ambiguus [SA]; any thoracoabdominal asymmetry that differs from SS or SI). Heterotaxy was subdivided into 3 groups: left isomerism, right isomerism, and other (including isolated dextrocardia and abdominal SI). To define situs status, we used the chest x-ray for preliminary assessment: SS (left-sided cardiac apex and stomach bubble), SI (right-sided cardiac apex and stomach bubble), isolated dextrocardia (right-sided cardiac apex and left-sided stomach bubble), and abdominal SI (left-sided cardiac apex and right sided stomach bubble). To further determine classification of heterotaxy, we reviewed available computed tomography, magnetic resonance imaging, and abdominal sonographic and echocardiographic studies. If available studies did not allow classification, patients were contacted again to obtain relevant radiographic studies and surgical reports. Polysplenia was identified when the splenic mass was divided into fairly equal-sized masses that varied in number from 2 to 6 and ranged from 1 to 6 cm in diameter, which together approximated the mass of a normal spleen. 14,32 Statistical Analysis For the primary analysis, Fisher exact test was used to test for differences in the prevalence of 3 different types of ciliary defects and prevalence of mutations in DNAI1 and DNAH5 across groups of patients with SS, SI, and heterotaxy. Secondary analyses used Fisher exact test to compare pairs of situs groups (SS, SI, SA) and to compare patients with situs abnormalities to those with SS. A probability value of 0.05 was considered significant for all analyses; no adjustment was made for multiple comparisons. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written. Results Situs Status Of 337 PCD patients, SS was present in 46.0% and SI in 47.7%; in addition, 6.3% (21 patients) had heterotaxy (Figures 1 and 2). The imaging methodology used to classify these patients by situs group is summarized in Table 1. More than 65% and 76% of patients classified as SI or heterotaxy, respectively, had echocardiograms and abdominal imaging. Ciliary Defects There is a clear shift in the types of ciliary defects across the 3 groups classified by situs status (P 0.001) (Table 2). Specifically, there was a decreasing prevalence of inner dynein arm and central apparatus ciliary defects, as well as an increasing prevalence of ODA defects from SS to SI to heterotaxy. The higher prevalence of ODA defects in patients with situs abnormalities (SI plus heterotaxy) was strikingly different from SS (P 0.001). This indicates that ODA defects are more commonly involved (and inner dynein arm and/or central defects are less commonly involved) in PCD patients with abnormalities of organ development or location. TABLE 2. Types of Ciliary Defects and Genetic Mutations in 3 Groups of PCD Patients Who Had Characterization of Ciliary Defect and Genetic Analyses of 2 Cilia Genes (DNAI1 and DNAH5 ) Situs Solitus (n 122) Situs Inversus Totalis (n 112) Heterotaxy (n 16) No. % No. % No. % Ciliary defect (n 250 patients)* Outer dynein arm defect (n 165) Inner dynein arm defect, alone (n 69) Central apparatus defect (n 16) Genetic analyses of 2 cilia genes (n 161 patients) Mutations in DNAI1 or DNAH5 (n 60) 19 (70 tested for mutations) 27.1# 34 (79 tested for mutations) (12 tested for mutations) *Fisher exact test (P 0.001). Types of ciliary defects differed across the 3 situs groups. Of these 165 patients with outer dynein arm defects, 57 also had some inner dynein arm defect (n 24, 27, and 6 for situs solitus, situs inversus, and heterotaxy, respectively). Fisher exact test. Different from situs inversus (P 0.001); different from heterotaxy (P 0.008); different from situs inversus plus heterotaxy (P 0.001). Central pair microtubule defect or radial spoke defect. Fisher exact test (P 0.037). Prevalence of mutations differed across the 3 situs groups. At least 1 mutant allele (see text for details). #Fisher exact test (P 0.022). Different from situs inversus plus heterotaxy. 58.3

4 Kennedy et al PCD and Heterotaxy 2817 TABLE 3. Clinical Features of 21 Patients With PCD and Heterotaxy Patient Age Sex Heterotaxy NRD RP B RS OM Ciliary Defect Nasal NO (nl/min or ppb) Family History Genotype (4, 5) UNC F RI Y Y Y Y Y ODA IDA 20 nl/min No DNAI1 WT, DNAH5 EXC UNC M LI Y Y Y Y Y ODA 48 nl/min No DNAI1 WT G M LI Y Y Y Y N Dysmotile ND No ND G71 30 F LI Y Y Y Y Y ODA ND No DNAI1 2 MUT UNC F LI Y Y Y Y Y ODA 11 nl/min No DNAH5 1 MUT, DNAI1 WT UNC F LI Y Y Y Y Y ODA 46 nl/min No DNAH5 2 MUT CN2 15 F LI CHD N Y Y Y Y ODA 8 ppb Sister: PCD, SI ND CN3 14 M LI Y Y Y Y N ODA IDA 54 ppb No ND UNC F LI CHD Y Y Y Y Y ODA IDA 13 nl/min Aunt: TGA DNAI1 2 MUT G30 5 M LI CHD N Y N ND ND Immotile ND ND ND UNC340 5 F LI Y Y Y Y Y ODA IDA 12 nl/min No DNAH5 2 MUT UNC927 1 F LI N Y N Y Y Poor quality 14 nl/min Brother: LI CHD DNAI1 WT CN4 10 F Other: SS CHD Y Y Y Y Y ODA IDA 78 ppb No ND G19 4 M Other: ASI CHD Y Y N Y Y ODA ND No DNAH5 2 MUT G F Other: ASI Y Y Y Y Y ODA ND No DNAI1 1 MUT AU F Other: ASI N Y Y Y Y ODA ND Sister: PCD, SI DNAI1 WT G29 4 M Other: IDEX N Y N Y Y IDA ND No ND G103 1 M Other: IDEX Y N N Y Y Dysmotile ND No ND UNC919 9 M Other: SI CHD Y Y Y Y Y ODA ND Sister: PCD DNAI1 WT G246 4 M Other: SI CHD Y N N Y Y Dysmotile ND No ND UNC M Other: SI CHD Y Y N Y Y IDA 9 nl/min No ND ASI indicates abdominal situs inversus; B, bronchiectasis; EXC, excluded; F, female; IDA, inner dynein arm defect; IDEX, isolated dextrocardia; LI, left isomerism; M, male; MUT, mutation; Nasal NO, nasal nitric oxide (normal nl/min or 400 to 1000 ppb); ND, not performed; NRD, neonatal respiratory distress; OM, otitis media; poor quality, poor quality sample; RI, right isomerism; RP, recurrent pulmonary infection; RS, rhinosinusitis; TGA, transposition of great vessels; and WT, wild-type. Genetic Mutations Genetic testing of 161 PCD patients for mutations in 2 ciliary ODA genes (DNAI1 and DNAH5) paralleled the pattern of increasing prevalence of ciliary ODA defects across the 3 situs groups (P 0.037) (Table 2). Specifically, there was an increasing prevalence of mutations from SS to SI to heterotaxy. The higher prevalence of mutations in patients with situs abnormalities (SI plus heterotaxy) was different from SS (P 0.022). The distribution of DNAI1 versus DNAH5 mutations were similar across the 3 situs groups, as were the number of mutated alleles (patients with 2 mutations ranged from 71% to 84% in the 3 situs groups), and types of mutation (frameshift/stop alleles ranged from 83% to 92%). 4 5 Heterotaxy Patients There was an equal distribution of gender (11 females/10 males), and the mean age was 17 (range 1 to 54) years (Table 3). The clinical phenotype was consistent with PCD in all heterotaxy patients; 76% had respiratory distress as term neonates, and all adults (age 18 years) and 50% of pediatric patients (age 18 years) had bronchiectasis. Sixteen of the heterotaxy patients had characterization of the ciliary defect, and 14 patients had an ODA defect. Nasal nitric oxide was low in the 11 patients tested, consistent with PCD. Seven of 12 patients tested had at least 1 mutation in DNAI1 or DNAH5, and 5 patients had 2 mutations. 4 5 Three patients with heterotaxy had siblings with PCD (2 with Kartagener s syndrome) (Table 3). Another patient (UNC927) had a brother born preterm (32 weeks) who died at 2 days of life with left isomerism (polysplenia) and CHD. Other clinical features included pectus excavatum (2 of 21 patients) as per the Haller Index (Table 4). 33 Intestinal malrotation (left-sided appendectomy) was identified in only 1 patient (UNC875). Laterality Defect Subtypes The distribution of heterotaxic subtypes in 21 PCD patients is illustrated (Figure 1) and anatomic findings are summarized (Table 4). Eleven patients had left isomerism, which included 6 patients with polysplenia and cardiac and/or vascular anomalies (3 patients had CHD) (Figure 3, Table 4). One patient had right isomerism (Figure 4). 19 Nine patients had other heterotaxic anomalies (3 patients with SI plus CHD; 3 patients with abdominal SI; 2 patients with isolated dextrocardia; and 1 patient with SS with CHD) (Figure 2). Five patients had polysplenia, but without cardiovascular anomalies (2 patients with abdominal SI, 1 patient with SI, and 2 patients with SS). Cardiac and/or Vascular Malformations Twelve of the 21 PCD patients with heterotaxy had cardiac and/or vascular malformations. Four of these 12 patients had vascular anomalies alone (Tables 4 and 5), and 8 patients had complex cardiac anomalies that required surgery. In 3 patients with CHD and left isomerism, the cardiac defects included double outlet right ventricle and atrioventricular

5 2818 Circulation June 5, 2007 TABLE 4. Anatomy of 21 Patients With PCD and Heterotaxy Patient Heterotaxy Imaging Spleen Liver Heart Vascular Pulmonary* Other UNC373 RI CT, E, A ASP Middle Left IVC int, AZVC Normal Dextrogastria, Bil DU, HH UNC875 LI CT, E, A LPSP Right Left IVC int, HAZVC, Bil SVC Normal Intestinal malrotation G122 LI CT, A RPSP Left Left Normal Normal Dextrogastria G71 LI CT, A LPSP Right Left Normal Normal Normal UNC471 LI CT, E, A RPSP Left Left Normal Normal Dextrogastria UNC231 LI CT, E, A LPSP Right Left Normal Normal Pect Exc HI 3.0 CN2 LI CHD CT, E, A LPSP Right Left, LAI IVC int, AZVC Bil Hypart Dextrogastria CN3 LI CT, E, A RPSP Left Right Normal Reversed Dextrogastria UNC930 LI CHD CT, E, A RPSP Middle Left, LAI, AVSD, IVC int, AZVC, Bil SVC Bil Hypart Normal DORV, SPS, HB G30 LI CHD CT, E, A LPSP Right Right, LAI, CA ACOARC Bil Hypart Normal AVSD, DORV UNC340 LI CT, E, A RPSP Left Right IVC int, AZVC Reversed Dextrogastria UNC927 LI CT, E, A LPSP Right Left IVC int, AZVC Normal Pancreatic hypoplasia CN4 Other: SS CHD CT, E, A Left Right Tetralogy of Fallot Tetralogy of Fallot Normal Normal G19 Other: ASI CHD CT, E, A Right Left Left, ASD, VSD, LTGA Normal Bil Hypart Dextrogastria G725 Other: ASI CT, A Right Left Left Normal Normal Dextrogastria AU666 Other: ASI Und Left Left Normal Normal Dextrogastria G29 Other: IDEX E, A Left Right Right Normal Reversed Normal G103 Other: IDEX A Left Right Right Normal Reversed Normal UNC919 Other: SI CHD CT, E, A Right Left Right, VSD, LVOTob, Normal Reversed Dextrogastria SPS, LTGA G246 Other: SI CHD CT, E, A Right Left Right, LTGA Normal Reversed Dextrogastria UNC1005 Other: SI CHD CT, E, A Right Left ASD,VSD Normal Reversed Dextrogastria, Pect Exc HI 3.4 CT indicates computed tomography of chest; A, abdominal imaging (see text for details); ACOARC, aortic coarctation; ASD, atrial septal defect; ASI, abdominal situs inversus; ASP, asplenia; AVSD, atrioventricular septal defect; AZVC, azygos vein continuation; Bil, bilateral; CA, common atrium; DORV, double outlet right ventricle; DU, duplicate ureters; E, echocardiogram; HAZVC, hemiazygos vein continuation; HB, heart block; HH, hiatal hernia; Hypart, hyparterial bronchi, bilobed lungs; IDEX, isolated dextrocardia; IVC int, inferior vena cava interruption; LAI, left atrial isomerism; LI, left isomerism; LPSP, left-sided polysplenia; LTGA, L-transposition of great arteries; LVOTob, left ventricular outflow tract obstruction; Pect Exc, pectus excavatum; HI, Haller Index (normal 2.4) 33 ; RI, right isomerism; RPSP, right-sided polysplenia; SPS, subpulmonic stenosis; SVC, superior vena cava; Und, undefined; and VSD, ventricular septal defect. *Excludes pathology related to chronic airway disease. canal defects. In another 3 patients with CHD and SI, there were atrial and ventricular septal defects and L-transposition of the great arteries. Of the other 2 patients with CHD, one had abdominal situs inversus and atrial and ventricular septal defects, and 1 patient had SS and tetralogy of Fallot. There were 8 other patients with left isomerism who did not have complex cardiac malformations. Discussion The phenotype of PCD may be confused with other diseases, and diagnosis is often delayed. 34 The presence of SI often aids diagnosis because of its association with PCD (Kartagener s syndrome). However, even those patients with SI are frequently not diagnosed with PCD. Our retrospective study of laterality defects in a large PCD population indicates that heterotaxic anomalies are present in at least 6.3% of these patients. The prevalence (6.3%) is likely an underestimation, because echocardiogram and abdominal imaging are not routinely performed in PCD, and subtle anatomic abnormalities not recognized. The classification of heterotaxic syndromes remains controversial. 10,11,14 We attempted to characterize all heterotaxy (situs ambiguus) patients as right or left isomerism. However, 9 patients Figure 3. Left isomerism with polysplenia. Contrast-enhanced computed tomography scan of a 41-year-old male with PCD demonstrates features of left isomerism with polysplenia. A, Note bilateral superior vena cavas at the level of aortic arch (a). Right superior vena cava is enhanced after intravenous contrast material administration through a right antecubital vein. Left-sided superior vena cava indicated by white arrow. B, Upper abdomen image demonstrates left upper quadrant splenules (s).

6 Kennedy et al PCD and Heterotaxy 2819 Figure 4. Right isomerism with asplenia. Noncontrast-enhanced computed tomography scan of a 51-year-old female with PCD demonstrates features of right isomerism with asplenia. 19 A, Large azygous vein arch is noted, which compensates for the inferior vena cava interruption. B, Computed tomography scan through the upper abdomen demonstrates midline liver (L), dextrogastria (s), and absent spleen. Bilateral renal cysts (c) are also noted. Reproduced from Chmura et al 19 with permission from S. Karger AG, Basel. Copyright could not be characterized, despite abdominal and echocardiogram imaging in most of these patients. We classified 3 patients with SI and congenital heart disease as heterotaxy, because these patients did not have mirror image reversal of situs solitus. One patient classified as having right isomerism (asplenia, midline liver) had inferior vena cava interruption, although inferior vena cava interruption is unusual in right isomerism. 17,19,35 The respiratory phenotypes of the PCD patients with heterotaxy match those without heterotaxy. 2 Specifically, the majority of patients had respiratory distress at birth, typical sinopulmonary disease, and chronic otitis media (Table 3). Bronchiectasis was present in all adults and 50% of children. 2,36,37 Strikingly, there was a 200-fold higher prevalence of CHD related to heterotaxy in PCD (1 in 50 patients) versus the general population (CHD related to heterotaxy: 1 in patients). 38 PCD is not routinely cited as a cause of CHD. 39,40 We speculate that the diagnosis of PCD is not made in many PCD patients who also have heterotaxy and CHD. For instance, the diagnosis of PCD was not made in 1 patient (UNC930) until age 10 years and after cardiac surgery. The diagnosis of PCD must be considered in patients with CHD and heterotaxy, particularly in patients with recurrent respiratory symptoms. Conversely, patients diagnosed with PCD should have formal cardiac assessment, especially patients with heterotaxic anatomic defects such as polysplenia. More than half the PCD patients with heterotaxy had polysplenia (left isomerism; 11 of 21 patients), and 1 additional patient had asplenia. A spectrum of anatomic anomalies was seen in PCD patients with polysplenia or asplenia, which ranged from splenic anomaly alone to vascular anomalies to CHD and lung isomerism. The severity of polysplenia syndrome varied even within a single family, as 1 patient had vascular without cardiac anomalies, but a brother had complex CHD. The establishment of left-right axis in the embryo is complex. 11 Embryonic nodal dysfunction is 1 cause of randomization of left-right asymmetry and involves 2 distinct types of embryonic nodal cilia (motile and nonmotile sensory). 7,8,41 In PCD, it is hypothesized that SI reflects defective nodal ciliary motile function, 4,6,42 and our clinical data support that hypothesis; specifically, PCD patients can manifest heterotaxy, and siblings with PCD can have different situs anomalies. The spectrum of heterotaxic anomalies identified, such as CHD, is consistent with animal models of heterotaxy. 9,43 45 In fact, 40% of mice with mutations in an TABLE 5. Cardiovascular Abnormalities in 12 Patients With Primary Ciliary Dyskinesia and Heterotaxy Abnormality Left Isomerism (n 6) Right Isomerism (n 1) SS (n 1) SI (n 3) Abdominal SI (n 1) Systemic veins Bilateral superior vena cava Inferior vena cava drainage via azygous Inferior vena cava drainage via hemiazygous Great vessels Tetralogy of Fallot L-transposition of the great arteries Aortic coarctation Subpulmonic stenosis Intracardiac Left atrial isomerism Right atrial isomerism Atrial septal defect Common atrium Atrioventricular septal defect Ventricular septal defect Double outlet right ventricle Left ventricular outflow tract obstruction

7 2820 Circulation June 5, 2007 axonemal dynein heavy chain gene (lrd; iv/iv mice) show visceral, cardiac, and venous malformations of heterotaxy and small litter sizes because of intrauterine death. 9,45 The distribution of different types of ciliary defects and genetic mutations in PCD patients classified by situs status strongly support the concept that specialized embryonic nodal cilia play a key role in organ development and location. These nodal cilia are motile, even though they do not have the central apparatus (central pair microtubules or radial spokes); thus, the increasing prevalence of ODA defects (and decreasing prevalence of inner dynein arm and central apparatus defects) from SS to SI to heterotaxy is consistent with this concept. Likewise, the distribution of mutations in 2 genes (DNAI1 and DNAH5) that code for respiratory and nodal ciliary ODA proteins are consistent with this concept; ie, there is an increased prevalence of mutations from SS to SI to heterotaxy. Stated another way, genetic mutations that do not affect the outer ciliary microtubule doublets in the nodal cilia (such as central apparatus genes) are less likely to result in SI or heterotaxy. In support of this concept, mice deficient in Mdnah5 (murine homolog of DNAH5) also develop SI totalis and heterotaxy; these occur in association with ciliary immotility and recurrent respiratory infections. 46,47 Thus, PCD phenotypes with heterotaxy, such as polysplenia and CHD, are frequently associated with mutations in DNAI1 and DNAH5, and genetic testing is indicated in these patients. These observations broaden both the phenotypic spectrum of DNAI1 and DNAH5 mutations and our understanding of the genetic causes of heterotaxy, and have important clinical implications. It is unclear why the prevalence of heterotaxy in PCD is only one-tenth as common as SS or SI, and it does not seem likely that the prevalence of heterotaxy would change substantially, even if all PCD patients underwent full radiographic and ultrasound imaging. It is possible that humans (like mice) with heterotaxy and lifethreatening anomalies (such as CHD) may be dying in utero or in early life. 45 Mutations in other as yet unidentified ciliary genes likely cause PCD and may have consequences for embryonic node function and left-right asymmetry pathways at other steps. Maternal diabetes, paternal cocaine use, and retinoic acid deficiency have also been associated with heterotaxy; however, we have no data on these confounders in our PCD patients. 48 A previously unrecognized anomaly was the high prevalence of pectus excavatum ( 10%) in our PCD patients with heterotaxy compared with the general population (0.3%). 49 Although 1 PCD patient with pectus had previous cardiac surgery, the pectus excavatum was documented prior to surgery. Interestingly, pectus excavatum and CHD are described in other congenital disorders, such as Marfan syndrome and Noonan syndrome, and an association between pectus excavatum and SI has previously been identified. 50 At this point, it is uncertain if there is a genetic or pathophysiological link between pectus excavatum, heterotaxy, and PCD. In conclusion, we demonstrate that PCD is associated with a marked increase in the prevalence of heterotaxy with and without CHD. The association of ciliary motility defects and heterotaxy links ciliary dysfunction and CHD. Thus, cilia-related genes are excellent candidate genes for heterotaxy and CHD. Patients with SI or heterotaxic anomalies, particularly those with concomitant neonatal respiratory distress or chronic respiratory infections, are at risk to have an underlying defect in ciliary structure, function, and genetics, and should be evaluated for PCD and tested for genetic mutations in DNAI1 and DNAH5. Acknowledgments We are grateful to the patients and their families for their participation. We thank Mariana Schmajuk for help in data collection, Lisa Brown for editorial assistance, and Dr Fei Zou, PhD, for assistance with statistical analysis. We thank both the US Primary Ciliary Dyskinesia Foundation and Kartagener Syndrom und Primaere Ciliaere Dyskinesie e.v. in Germany. Sources of Funding This project was supported by grants from the National Institute of Health (NIH RR00046, RO1 HL071798, and 1U54 RR ) to Dr Knowles and HL04225 to Dr Noone. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. This project was also supported by grants from the Deutsche Forschungsgemeinschaft (SFB592, DFG Om 6/2, and DFG Om 6/4) to Dr Omran. Disclosures Dr Molina has received research support from the North Carolina Biotech Center. The other authors report no conflicts. References 1. Afzelius BA. A human syndrome caused by immotile cilia. Science. 1976;193: Noone PG, Leigh MW, Sannuti A, Minnix SL, Carson JL, Hazucha M, Zariwala MA, Knowles MR. Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir Crit Care Med. 2004;169: Fliegauf M, Olbrich H, Horvath J, Wildhaber JH, Zariwala MA, Kennedy M, Knowles MR, Omran H. Mislocalization of DNAH5 and DNAH9 in respiratory cells from patients with primary ciliary dyskinesia. Am J Respir Crit Care Med. 2005;171: Zariwala M, Leigh MW, Ceppa F, Kennedy MP, Noone PG, Carson LJ, Hazucha MJ, Lori A, Horvath J, Olbrich H, Loges NT, Bridoux AM, Pennaraun G, Duriez B, Escudier E, Mitchison HM, Chodhari EM, Chung MK, Morgan LM, De Iongh RU, Rutland J, Pradal U, Omran H, Amselem S, Knowles MR. Mutation analyses of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation. Am J Respir Crit Care Med. 2006;174: Hornef N, Olbrich H, Horvath J, Zariwala MA, Fliegauf M, Loges NT, Wildhaber J, Noone PG, Kennedy M, Antonarakis SE, Blouin JL, Bartoloni L, Nublein T, Ahrens P, Griese M, Kuhl H, Sudbrak R, Knowles MR, Reinhardt R, Omran H. DNAH5 mutations are a common cause of primary ciliary dyskinesia with outer dynein arm defects. Am J Respir Crit Care Med. 2006;174: Noone PG, Bali D, Carson JL, Sannuti A, Gipson CL, Ostrowski LE, Bromberg PA, Boucher RC, Knowles MR. Discordant organ laterality in monozygotic twins with primary ciliary dyskinesia. Am J Med Genet. 1999;82: Nonaka S, Shiratori H, Saijoh Y, Hamada H. Determination of left-right patterning of the mouse embryo by artificial nodal flow. Nature. 2002;418: Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, Kido M, Hirokawa N. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell. 1998;95: Supp DM, Witte DP, Potter SS, Brueckner M. Mutation of an axonemal dynein affects left-right asymmetry in inversus viscerum mice. Nature. 1997;389: Aylsworth AS. Clinical aspects of defects in the determination of laterality. Am J Med Genet. 2001;101: Zhu L, Belmont JW, Ware SM. Genetics of human heterotaxias. Eur J Hum Genet. 2006;14: Moller JH, Nakib A, Anderson RC, Edwards JE. Congenital cardiac disease associated with polysplenia: a developmental complex of bilateral leftsidedness. Circulation. 1967;36: Gilljam T, McCrindle BW, Smallhorn JF, Williams WG, Freedom RM. Outcomes of left atrial isomerism over a 28-year period at a single institution. J Am Coll Cardiol. 2000;36: Bartram U, Wirbelauer J, Speer CP. Heterotaxy syndrome: asplenia and polysplenia as indicators of visceral malposition and complex congenital heart disease. Biol Neonate. 2005;88: Ivemark BI. Implications of agenesis of the spleen on the pathogenesis of conotruncus anomalies in childhood: analysis of the heart malformations in the splenic

8 Kennedy et al PCD and Heterotaxy 2821 agenesis syndrome, with fourteen new cases. Acta Pediatr. 44(suppl 104):1 110, Peoples WM, Moller JH, Edwards JE. Polysplenia: a review of 146 cases. Pediatr Cardiol. 1983;4: Phoon CK, Neill CA. Asplenia syndrome-risk factors for early unfavorable outcome. Am J Cardiol. 1994;73: Winer-Muram HT, Tonkin IL. The spectrum of heterotaxic syndromes. Radiol Clin North Am. 1989;27: Chmura K, Chan ED, Noone PG, Zariwala M, Winn RA, Knowles MR, Iseman MD, Gardner EM. A middle-aged woman with recurrent respiratory infections. Respiration. 2005;72: Wolfe MW, Vacek JL, Kinard RE, Bailey CG. Prolonged and functional survival with the asplenia syndrome. Am J Med. 1986;81: Schidlow DV, Katz SM, Turtz MG, Donner RM, Capasso S. Polysplenia and kartagener syndromes in a sibship: association with abnormal respiratory cilia. J Pediatr. 1982;100: Gayer G, Apter S, Jonas T, Amitai M, Zissin R, Sella T, Weiss P, Hertz M. Polysplenia syndrome detected in adulthood: report of eight cases and review of the literature. Abdom Imaging. 1999;24: Lim JS, McCrindle BW, Smallhorn JF, Golding F, Caldarone CA, Taketazu M, Jaeggi ET. Clinical features, management, and outcome of children with fetal and postnatal diagnoses of isomerism syndromes. Circulation. 2005;112: Raman R, Al-Ali SY, Poole CA, Dawson BV, Carman JB, Calder L. Isomerism of the right atrial appendages: clinical, anatomical, and microscopic study of a long-surviving case with asplenia and ciliary abnormalities. Clin Anat. 2003;16: Engesaeth VG, Warner JO, Bush A. New associations of primary ciliary dyskinesia syndrome. Pediatr Pulmonol. 1993;16: Bitar FF, Shbaro R, Mroueh S, Yunis K, Obeid M. Dextrocardia and corrected transposition of the great arteries (I,D,D) in a case of Kartagener s syndrome: a unique association. Clin Cardiol. 1998;21: Tkebuchava T, Niederhauser U, Weder W, von Segesser LK, Bauersfeld U, Felix H, Lachat M, Turina MI. Kartagener s syndrome: clinical presentation and cardiosurgical aspects. Ann Thorac Surg. 1996;62: Sternick EB, Marcio Gerken L, Max R, Osvaldo Vrandecic M. Radiofrequency catheter ablation of an accessory pathway in a patient with Wolff- Parkinson-White and Kartagener s syndrome. Pacing Clin Electrophysiol. 2004; 27: Teichberg S, Markowitz J, Silverberg M, Aiges H, Schneider K, Kahn E, Daum F. Abnormal cilia in a child with the polysplenia syndrome and extrahepatic biliary atresia. J Pediatr. 1982;100: Gershoni-Baruch R, Gottfried E, Pery M, Sahin A, Etzioni A. Immotile cilia syndrome including polysplenia, situs inversus, and extrahepatic biliary atresia. Am J Med Genet. 1989;33: De Santis A, Morlupo M, Stati T, Lisi D, Pigna M, Antonelli M. Sonographic survey of the upper abdomen in 10 families of patients with immotile cilia syndrome. J Clin Ultrasound. 1997;25: Gayer G, Zissin R, Apter S, Atar E, Portnoy O, Itzchak Y. CT findings in congenital anomalies of the spleen. Br J Radiol. 2001;74: Daunt SW, Cohen JH, Miller SF. Age-related normal ranges for the Haller index in children. Pediatr Radiol. 2004;34: Coren ME, Meeks M, Morrison I, Buchdahl RM, Bush A. Primary ciliary dyskinesia: age at diagnosis and symptom history. Acta Paediatr. 2002;91: Ruscazio M, Van Praagh S, Marrass AR, Catani G, Iliceto S, Van Praagh R. Interrupted inferior vena cava in asplenia syndrome and a review of the hereditary patterns of visceral situs abnormalities. Am J Cardiol. 1998;81: Kennedy MP, Noone PG, Leigh MW, Zariwala MA, Minnix SL, Knowles MR, Molina PL. High-resolution CT of patients with primary ciliary dyskinesia. AJR Am J Roentgenol. 2007;188: Nadel HR, Stringer DA, Levison H, Turner JA, Sturgess JM. The immotile cilia syndrome: radiological manifestations. Radiology. 1985;154: Lin AE, Ticho BS, Houde K, Westgate MN, Holmes LB. Heterotaxy: associated conditions and hospital-based prevalence in newborns. Genet Med. 2000;2: Gelb BD. Genetic basis of congenital heart disease. Curr Opin Cardiol. 2004; 19: Pajkrt E, Weisz B, Firth HV, Chitty LS. Fetal cardiac anomalies and genetic syndromes. Prenat Diagn. 2004;24: McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell. 2003;114: McGrath J, Brueckner M. Cilia are at the heart of vertebrate left-right asymmetry. Curr Opin Genet Devel. 2003;13: Grinberg I, Millen KJ. The ZIC gene family in development and disease. Clin Genet. 2005;67: Otto EA, Schermer B, Obara T, O Toole JF, Hiller KS, Mueller AM, Ruf RG, Hoefele J, Beekmann F, Landau D, Foreman JW, Goodship JA, Strachan T, Kispert A, Wolf MT, Gagnadoux MF, Nivet H, Antignac C, Walz G, Drummond IA, Benzing T, Hildebrandt F. Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination. Nat Genet. 2003;34: Icardo JM, Sanchez de Vega MJ. Spectrum of heart malformations in mice with situs solitus, situs inversus, and associated visceral heterotaxy. Circulation. 1991; 84: Ibanez-Tallon I, Gorokhova S, Heintz N. Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum Mol Genet. 2002;11: Ibanez-Tallon I, Pagenstecher A, Fliegauf M, Olbrich H, Kispert A, Ketelsen UP, North A, Heintz N, Omran H. Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation. Hum Mol Genet. 2004;13: Kuehl KS, Loffredo C. Risk factors for heart disease associated with abnormal sidedness. Teratology. 2002;66: Mead J, Sly P, Le Souef P, Hibbert M, Phelan P. Rib cage mobility in pectus excavatum. Am Rev Respir Dis. 1985;132: Eroglu A, Kurkcuoglu IC, Karaoglanoglu N, Ikbal M. Pectus excavatum and situs inversus totalis: a new combination or a coincidence? Genet Couns. 2004;15: CLINICAL PERSPECTIVE Congenital heart disease (CHD) and abnormalities of organ anatomy and location (heterotaxy/situs ambiguus) are leading causes of morbidity in infants, and little is known about the underlying genetics of these disorders in humans. A few case reports have noted that some patients with primary ciliary dyskinesia (PCD), a genetic disorder of ciliary function, have heterotaxy with CHD and/or polysplenia, or asplenia syndromes. To better define the prevalence of these clinical disorders in PCD, we reviewed 337 well-characterized PCD patients from 4 specialized centers in the United States, Germany, Canada, and Australia. We determined that at least 6.3% (n 21) of these PCD patients had heterotaxy, which was largely associated with defects in the outer dynein arm of respiratory cilia and mutations in ciliary outer dynein arm genes (DNAI1 and DNAH5). Twelve of these patients with heterotaxy had cardiac and/or vascular abnormalities, and most (8 of 12 patients) had complex CHD that required surgery. The prevalence of CHD with heterotaxy was noted to be 200-fold higher in PCD than in the general population (1:50 versus 1:10 000), which indicates that patients with PCD should have formal cardiac evaluation. Conversely, genetic mutations that adversely affect respiratory and embryological nodal cilia are a significant cause of heterotaxy and CHD, and these patients should be evaluated for PCD.