Analysis of Cardiovascular Phenotype and Genotype-Phenotype Correlation in Individuals With a JAG1 Mutation and/or Alagille Syndrome

Transcription

1 Analysis of Cardiovascular Phenotype and Genotype-Phenotype Correlation in Individuals With a Mutation and/or Alagille Syndrome Doff B. McElhinney, MD; Ian D. Krantz, MD; Lynn Bason, MS; David A. Piccoli, MD; Karan M. Emerick, MD; Nancy B. Spinner, PhD; Elizabeth Goldmuntz, MD Background Cardiovascular anomalies are among the most common features of Alagille syndrome (AGS). Mutations of are found in most individuals with AGS. This study was undertaken to determine the spectrum of cardiovascular phenotypes associated with a mutation and/or AGS, investigate potential genotype-phenotype correlations, and begin to correlate clinical outcome with genetic pathogenesis. Methods and Results We reviewed the records of 200 individuals with a mutation or AGS. A total of 187 (94%) subjects had evidence of cardiovascular involvement. Cardiovascular anomalies were identified by imaging in 150 subjects (75%), and 37 (19%) had a peripheral pulmonary stenosis murmur with either a normal echocardiogram or no imaging study. Of the 150 subjects with anomalies confirmed by imaging, right-sided anomalies were present in 123 and left-sided anomalies in 22, with both in 12. Seventeen subjects had other anomalies. The most common abnormality was stenosis/hypoplasia of the branch pulmonary arteries (PAs), which was documented by imaging (n 111) or inferred from a peripheral pulmonary stenosis murmur (n 41) in 76% of subjects. Tetralogy of Fallot was present in 23 subjects and was accompanied by pulmonary atresia in 8. Branch PA phenotype differed between individuals with and without a mutation. Among subjects with a mutation, there was no correlation between the type or location of mutation and the frequency or type of cardiovascular anomaly. Conclusions More than 90% of individuals with a mutation or AGS have cardiovascular anomalies, with branch PA stenosis the most common abnormality. Cardiovascular phenotype does not correlate with the type or location of mutation. (Circulation. 2002;106: ) Key Words: cardiovascular disease genetics tetralogy of Fallot Alagille syndrome (AGS) is characterized by a constellation of phenotypic features that includes a paucity of interlobular bile ducts, cholestasis, cardiovascular anomalies, vertebral anomalies (typically butterfly vertebrae), ocular anomalies (predominantly anterior chamber defects and retinal pigmentary abnormalities), and a characteristic facies (consisting of a triangular face and chin, with a prominent forehead, deep-set eyes, hypertelorism, flat midface, and straight long nose). 1,2 Mutations or deletions of the gene, which encodes a ligand in the Notch signaling pathway, have been identified in 60% to 75% of individuals with AGS. 3 5 mutations have also been discovered in individuals with only one or two features of AGS and in relatives of individuals with AGS who themselves have few or no overt phenotypic manifestations of AGS. 6 8 There does not seem to be any correlation between the type or location of abnormality and phenotypic penetrance or severity. 5 Congenital heart disease is one of the diagnostic criteria for AGS. 1 In previously published series, documented cardiovascular anomalies or a murmur consistent with stenosis/hypoplasia of the branch pulmonary arteries (PAs) have been identified in 85% to 97% of individuals with AGS. 1,2,9 Nonetheless, the spectrum of cardiovascular phenotypes associated with AGS is not well characterized, particularly given that a low percentage of previous study subjects had undergone imaging studies. 10 More than 200 individuals have been screened for mutations in the gene as part of an ongoing study at The Children s Hospital of Philadelphia to characterize the molecular basis of AGS. To define precisely the spectrum of cardiovascular phenotypes in individuals with a mutation or AGS, to ascertain whether a genotype-phenotype correlation exists with regard to cardiovascular anatomy, and to begin to correlate clinical outcome with genetic pathogen- Received May 30, 2002; revision received August 23, 2002; accepted August 24, From the Divisions of Cardiology (D.B.E., E.G.), Human Genetics (I.D.K., L.B., N.B.S.), and Gastroenterology and Nutrition (D.A.P.), The Children s Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pa, and Division of Gastroenterology (K.M.E.), Children s Memorial Hospital, Chicago, Ill. Dr McElhinney is now at the Department of Cardiology, Children s Hospital, Boston, Mass. Correspondence to Elizabeth Goldmuntz, MD, Division of Cardiology, The Children s Hospital of Philadelphia, Abramson Research Center 702A, 3516 Civic Center Blvd, Philadelphia, PA goldmuntz@ .chop.edu 2002 American Heart Association, Inc. Circulation is available at DOI: /01.CIR

2 2568 Circulation November 12, 2002 TABLE 1. Primary Cardiovascular Anomalies Among 200 Subjects With a Mutation and/or AGS (%) Primary Cardiovascular Anomaly (n 200) Cardiovascular anomalies as defined by imaging modalities 150 (75) 119 (77) 31 (67) Right-sided anomalies 110 (55) 93 (60) 17 (37) Tetralogy of Fallot 23 (12) 20 (13) 3 (7) Valvar pulmonary stenosis 15 (8) 11 (7) 4 (9) Branch PA stenosis 70 (35) 60 (39) 10 (22) Pulmonary atresia, intact ventricular septum 1 (1) 1 (1) 0 (0) Truncus arteriosus 1 (1) 1 (1) 0 (0) Left-sided anomalies 13 (7) 9 (6) 4 (9) Valvar AS 4 (2) 4 (3) 0 (0) Bileaflet aortic valve without stenosis 2 (1) 1 (1) 1 (2) Supravalvar AS 2 (1) 1 (1) 1 (2) Coarctation of the aorta 4 (2) 2 (1) 2 (4) Sinus of Valsalva aneurysm 1 (1) 1 (1) 0 (0) Other anomalies 27 (14) 17 (10) 10 (22) Ventricular septal defect 10 (5) 6 (4) 4 (9) Atrial septal defect 10 (5) 7 (5) 3 (7) Unbalanced atrioventricular septal defect 1 (1) 0 (0) 1 (2) Patent ductus arteriosus 2 (1) 1 (1) 1 (2) Left SVC, absent right SVC 1 (1) 1 (1) 0 (0) Right aortic arch 1 (1) 1 (1) 0 (0) Anomalous left coronary artery from the PA 1 (1) 1 (1) 0 (0) Pulmonary vein stenosis 1 (1) 0 (0) 1 (2) esis, we analyzed the cardiovascular features in the segment of this cohort who had a defined mutation in the gene or had no identifiable mutation but met the clinical criteria, as specified below, for the diagnosis of AGS. Methods Subjects Subjects were drawn from a database of individuals enrolled in an ongoing program at The Children s Hospital of Philadelphia on the genetic pathogenesis of AGS; some have been included in previous publications. 2,3,5,6,8,11 The program s database includes individuals with suspected AGS and relatives of probands who were enrolled after the detection of a mutation in the proband. Subjects were considered for inclusion in the present analysis if they had been tested for mutations as previously described. 3,8 They were included if either a mutation was identified or if they met the clinical criteria for AGS (defined below). Written informed consent was obtained for all subjects according to a protocol approved by the Institutional Review Board for the Protection of Human Subjects at The Children s Hospital of Philadelphia. Cardiovascular Phenotype and Definitions Cardiovascular phenotype was ascertained by authors of this study (D.B.M. and E.G.) on review of the following records, when (n 154) AGS Without a (n 46) Normal or no cardiovascular imaging 50 (25) 35 (23) 15 (33) PPS murmur without documented anomalies 37 (19) 27 (18) 10 (22) Normal echocardiogram 26 (13) 19 (12) 7 (15) No cardiovascular imaging 11 (6) 8 (5) 3 (7) No PPS murmur with normal or no imaging 13 (7) 8 (5) 5 (11) applicable: notes and letters from the consulting cardiologist, ECG reports, echocardiogram reports, cardiac catheterization reports, operative notes, and autopsy summaries. Cardiovascular anomalies were ascertained either by physical examination reported by a cardiologist or by additional imaging studies. Cardiovascular phenotype was stratified according to primary and secondary anomalies. In subjects with multiple anomalies, the primary anomaly was considered to be that for which intervention was performed or was most likely to be performed. In subjects with a cardiovascular complex, such as tetralogy of Fallot (TOF), the typical components of the complex were not listed separately as primary and secondary anomalies. Cardiovascular anomalies were also categorized as right-sided, left-sided, or neither right- nor left-sided ( other ), as summarized in Table 1. Branch PA stenosis/hypoplasia (the terms stenosis and hypoplasia are used without specific differentiation) was defined as one or more of the following: a documented pressure gradient 10 mm Hg by cardiac catheterization, a gradient estimated at 15 mm Hg by Doppler echocardiography using the simplified Bernoulli equation (Pressure 4 velocity 2 ), obvious stenosis/hypoplasia of one or both branch PAs visualized by cross-sectional echocardiography or angiocardiography, or surgical or transcatheter intervention on the branch PAs. Branch PA anomalies were characterized according to extent (ie, discrete, diffuse, or discontinuous), severity (ie, mild or moderate to severe), and sidedness (ie, unilateral or bilateral) of the stenosis. Discrete PA stenosis was defined as no more than 2

3 McElhinney et al Mutation and Cardiovascular Phenotype 2569 documented stenoses in the branch PA supplying either lung, whereas diffuse PA stenosis/hypoplasia was defined as extensive hypoplasia of the PA tree or bilateral multilevel stenosis/hypoplasia observed on angiography. Branch PA stenosis was classified as moderate to severe (single category) if the pressure gradient into at least one branch PA was 30 mm Hg, a qualitative interpretation by the cardiologist performing the diagnostic procedure was recorded as moderate or severe stenosis/hypoplasia and a documented pressure gradient was not available, or intervention was performed on one or both branch PAs. Otherwise, the stenosis was considered mild. To be considered bilateral PA stenosis, both branch PAs had to meet the criteria for stenosis, defined above. For the purposes of this study, the term peripheral pulmonary stenosis (PPS) referred to the presence of a PPS murmur without documented branch PA stenosis by imaging. If a typical PPS murmur (systolic ejection murmur audible over the precordium with radiation into the axillae or back) was noted on examination, the phenotype was defined according to echocardiographic or angiographic findings, as summarized above. If a PPS murmur was noted by a cardiologist, but no abnormalities were seen on imaging or no imaging studies were performed, the phenotype was categorized as PPS murmur, normal imaging or PPS murmur, no imaging, respectively. The gradations of severity for other obstructive anomalies (eg, valvar pulmonary or aortic stenosis) are defined in the appropriate table or section of the Results. Mutation Analysis Analysis of genotype was performed in all subjects, as described previously. 3,8 Subjects were initially screened with fluorescence in situ hybridization (YACs 940d11 and 881h20 used as probes) to identify whole-gene deletions. If fluorescence in situ hybridization demonstrated the normal complement of 2 alleles, single-strand conformation polymorphism electrophoresis was performed to detect intragenic mutations or deletions. In subjects with band shifts identified on electrophoresis, the mutation was characterized by direct sequencing of the corresponding coding region and exon-intron boundaries. Definition of Alagille Syndrome The criteria specified by Alagille et al 1 for the diagnosis of AGS require biopsy-proven paucity of interlobular bile ducts, along with 3 of the following 5 features: chronic cholestasis, cardiovascular anomalies (including a PPS murmur detected by a cardiologist), vertebral anomalies, ocular anomalies, and characteristic facies (see the introduction). For this study, we adopted a modified definition of AGS, such that 3 of the features specified by Alagille et al 1 were necessary to meet the criteria for AGS, with biopsy-proven paucity of interlobular bile ducts a nonessential feature. The diagnosis of AGS was assigned after review of records by an attending geneticist (I.D.K.). Data Analysis Data are presented as the number of subjects with a particular anatomic feature or diagnosis. The frequencies of diagnoses and anatomic variables were compared between subjects with and without a mutation and, within the cohort of subjects with branch PA anomalies, between different phenotypic variables using nonparametric analysis ( 2 or Fisher s exact test). Genotype-phenotype analysis was then performed within the cohort of subjects with a mutation, with genotypic variables including the type (whole gene deletion, protein-truncating mutation, missense mutation, splice-site mutation) and location of mutation. Because of the large number of subjects for whom the parent-of-origin of the mutation was undetermined (see below), genotype-phenotype analysis with respect to this variable was not performed. Because of the small number of subjects without AGS according to our definition, comparison between subjects with and without AGS was not performed. Results are presented as ORs with 95% CIs. Results Subjects Documentation of cardiovascular phenotype was requested from the referring physician or family of 222 individuals in the aforementioned database, all of whom were tested for mutations. Adequate cardiac data were obtained for 200 of these individuals (90%), including 154 (77%) with a mutation and 188 (94%) who met our criteria for AGS. The study cohort consisted of these 200 subjects, whereas those with inadequate cardiovascular data were omitted from the analysis. Among the 154 subjects with a mutation, 142 (92%) met our criteria for AGS, 127 were probands enrolled in the study with suspected AGS, and 27 were relatives without previously suspected AGS (in some cases, there were multiple probands in a single kindred). Cardiovascular Phenotype Of the 200 subjects in the study cohort, 196 (98%) were evaluated by a cardiologist, whereas the other 4 were reported to have no murmur on physical examination by at least one physician. Echocardiography was performed in 180 (90%) subjects, and 59 (30%) underwent both echocardiography and cardiac catheterization, including 33% of subjects (n 51) with a mutation and 17% of subjects without (n 8). Of the 200 subjects with adequate cardiac data, 187 (94%) had some form of cardiovascular involvement. A wide variety of cardiovascular anomalies were diagnosed by imaging in 150 (75%) subjects, and distal branch PA anomalies were suspected in 37 (19%) subjects who had a PPS murmur with either a normal echocardiogram or no imaging studies. Of the 150 subjects with anomalies documented by imaging, 105 had a single anomaly and 45 had multiple anomalies. Tables 1 and 2 list the primary and secondary cardiovascular diagnoses, respectively. Right-sided anomalies were documented in 123 subjects (62% of 200) and left-sided anomalies were documented in 22 (11% of 200), 12 of whom had both right- and left-sided anomalies. Among the 150 subjects with documented cardiovascular abnormalities, right ventricular hypertrophy was diagnosed by electrocardiography or cardiac imaging in 69 (35%). No primary arrhythmias were noted on review of electrocardiograms. Among the 50 subjects with either normal or no imaging studies, 37 (19% of 200) had a PPS murmur and either a normal echocardiogram (n 26) or no cardiac imaging (n 11). Of the 26 subjects with a PPS murmur but a normal echocardiogram, 2 had electrocardiographic features of right ventricular hypertrophy. The remaining 13 (7% of 200) subjects did not have a murmur and had either a normal echocardiogram (n 4) or no imaging study (n 9). Abnormalities of the branch PAs were identified by imaging in 111 subjects (56%). Murmurs suggesting branch PA anomalies without documented stenosis/hypoplasia on imaging studies were heard by a cardiologist in 41 additional subjects (20%), including 4 with and 37 without other cardiovascular anomalies (Table 3). Of the 111 subjects with branch PA anomalies detected by imaging, 55 (50%) had isolated anomalies of the branch PAs and 56 (50%) had associated cardiovascular malformations (including TOF).

4 2570 Circulation November 12, 2002 TABLE 2. Secondary Diagnoses Among 45 Subjects With Multiple Cardiovascular Anomalies Mutation (n 32) AGS Without a Mutation (n 13) Secondary Cardiovascular Anomaly (n 45) Right-sided anomalies Valvar pulmonary stenosis* Branch PA stenosis* Double-chambered right ventricle Left-sided anomalies Valvar AS Bileaflet aortic valve without stenosis Supravalvar AS Other anomalies Ventricular septal defect* Atrial septal defect Patent ductus arteriosus* Left SVC, absent right SVC Left SVC, normal right SVC Biventricular fibrosis/calcification Because some subjects had multiple secondary anomalies within a category (ie, right-sided, left-sided, other), the sums of subcategories (eg, other anomalies ) do not necessarily equal the total number of subjects listed in the category. Moreover, because some subjects had primary and secondary diagnoses in the same category (eg, 2 left-sided anomalies), the sum of subjects listed under a category in Tables 1 and 2 may not equal the total number of subjects with anomalies in that category. *Values do not include subjects with TOF or truncus arteriosus, in whom these anomalies are either a component of the primary lesion or frequently present. Diagnosed at autopsy. Subjects with associated cardiovascular malformations were significantly more likely to have severe PA stenosis (OR 3.1 [95% CI, 1.4 to 7.0], P 0.006) and bilateral PA stenosis (OR 1.2 [95% CI, 1.0 to 1.4], P 0.05) than those without. Both of these differences were heavily influenced by the number of subjects with TOF and were no longer significant when subjects with TOF were excluded from the analysis. Twenty-three (12%) of our subjects had TOF, phenotypic details of which are described in Table 4. The pulmonary valve was stenotic in 14 subjects, atretic in 8, and absent in 1. The aortic arch was left-sided in those for whom aortic arch anatomy was specified, of which 2 had an aberrant right subclavian artery. An additional 20 (10%) individuals (without TOF) had abnormalities of the pulmonary valve, as detailed in Table 5. In this subset, the pulmonary valve was stenotic in 19 subjects and atretic with an intact ventricular septum in 1. Left-sided cardiovascular anomalies were present in 22 subjects and were associated with additional cardiac defects in 13, as summarized in Table 6. Valvar and supravalvar aortic anomalies as well as coarctation of the aorta were identified. Details of subjects with other intracardiac anomalies, including atrial and ventricular septal detects, are summarized in Table 7. Genotype-Phenotype Analysis Mutations or deletions of were present in 154 (77%) subjects, including 12 with a deletion of the entire gene, 104 with an intragenic frameshift mutation, 24 with an intragenic missense mutation, and 14 with a splice-site consensus sequence alteration. Mutations were de novo in 46 subjects, maternally inherited in 21, paternally inherited in 19, and undetermined in 64. Individuals with a mutation had a significantly higher frequency of branch PA anomalies (OR 2.1 [95% CI, 1.1 to 4.0], P 0.03), bilateral branch PA anomalies (OR 2.2 [95% CI, 1.1 to 4.5], P 0.02), and diffuse stenosis/hypoplasia of the PAs (OR 12.5 [95% CI, 1.7 to 94], P 0.001) than individuals with AGS but no mutation. None of the other anatomic variables differed according to the presence or absence of a mutation. Among the 154 subjects with a mutation, there was no correlation between the type or location of the mutation and the frequency or type of cardiovascular malformation. In fact, there was considerable variability in cardiovascular phenotype among subjects with mutations of all types and locations and even among probands and affected parents and siblings with identical mutations. There were 12 subjects with a mutation who did not meet our criteria for AGS. All except 1 of these subjects were evaluated for a mutation after identification of a mutation in a child or sibling with AGS. Cardiovascular anomalies were documented in 3 of these subjects (bilateral branch PA stenosis in 2 and a sinus of Valsalva aneurysm in 1) and suspected on the basis of a PPS murmur in 3, 2 of whom had a normal echocardiogram. The remaining 6 subjects without AGS had a normal cardiac physical examination, of which 2 had a normal echocardiogram. Clinical Outcome Cross-sectional cardiovascular follow-up data were available for 148 subjects (74%) at an average of years after the earliest documentation of cardiovascular evaluation that we were able to obtain. At least 1 cardiovascular intervention was performed in 23% (n 46) of subjects, including 23% (n 36) of those with a mutation and 22% (n 10) of those without. During follow-up, 14 (7%) subjects (12 with and 2 without a mutation) were reported to have died from cardiovascular causes, including 10 with TOF (43% of subjects with TOF), 2 with isolated severe branch PA stenosis, 1 with truncus arteriosus, and 1 with a sinus of Valsalva aneurysm. Of the 10 subjects with TOF who died of cardiovascular causes, 6 had TOF and pulmonary atresia (75% of 8), 3 had TOF and pulmonary stenosis (21% of 14), and 1 had TOF and absent pulmonary valve. Among subjects with branch PA stenosis (not including those with TOF), serial echocardiographic data were available in 55. The follow-up echocardiograms did not demonstrate significant progression of the branch PA stenosis in any subject; 5 subjects (4 with severe stenosis initially and 1 with mild stenosis) had small decreases (10 to 15 mm Hg) in the severity of branch PA obstruction, and 1 with mild stenosis initially had a small increase in the severity of obstruction.

5 McElhinney et al Mutation and Cardiovascular Phenotype 2571 TABLE 3. Branch PA Anatomy in Study Cohort (n 154) AGS Without a (n 46) (%) (n 200) Branch PA anomalies identified by imaging 111 (56) 91 (59) 20 (43) Isolated PA anomalies 55 (50) 47 (52) 8 (40) Extent of PA stenosis/hypoplasia Discrete 46 (84) 38 (81) 8 (100) Diffuse 9 (16) 9 (19) 0 (0) Discontinuous branch PAs 0 (0) 0 (0) 0 (0) Severity of PA stenosis/hypoplasia Mild 27 (49) 24 (51) 3 (37) Moderate to severe 28 (51) 23 (49) 5 (63) Sidedness of PA stenosis/hypoplasia Bilateral 44 (80) 38 (81) 6 (75) Unilateral 11 (20) 9 (19) 2 (25) Left PA stenosis only 10 (91) 8 (89) 2 (100) Right PA stenosis only 1 (9) 1 (11) 0 (0) Associated cardiovascular anomalies* 56 (50) 44 (48) 12 (60) Extent of PA stenosis/hypoplasia Discrete 28 (50) 17 (39) 11 (92) Diffuse 24 (43) 23 (52) 1 (8) Discontinuous branch PAs 4 (7) 4 (9) 0 (0) Severity of PA stenosis/hypoplasia Mild 13 (23) 11 (25) 2 (17) Moderate to severe 43 (77) 33 (75) 10 (83) Sidedness of PA stenosis/hypoplasia Bilateral 51 (91) 42 (95) 9 (75) Unilateral 5 (9) 2 (5) 3 (25) Left PA stenosis only 5 (100) 2 (100) 3 (100) Right PA stenosis only 0 (0) 0 (0) 0 (0) Branch PA anomalies suspected from PPS murmur without 41 (21) 30 (19) 11 (24) documentation by imaging Abnormal intracardiac anatomy PPS murmur with normal PAs on echocardiogram Normal intracardiac anatomy PPS murmur with normal PAs on echocardiogram PPS murmur with no cardiac imaging No PPS murmur or documentation of branch PA anomalies 48 (24) 33 (21) 15 (31) Abnormal intracardiac anatomy Normal or no imaging Percentages are based on the immediately preceding stratum. *Includes subjects with TOF. In all subjects with discontinuous branch PAs, the distal PA tree was diffusely hypoplastic, but these individuals are not included in the diffuse category. Discussion Cardiovascular Phenotype This study analyzes the cardiovascular phenotype in 200 subjects with a mutation or AGS. Cardiovascular anomalies were defined by imaging in 75% of subjects. An additional 19% of subjects had a PPS murmur on examination by a cardiologist but either had a normal echocardiogram or did not undergo cardiovascular imaging. If these individuals are considered by examination to have some degree of stenosis/hypoplasia of the PA tree that might not be detected by routine echocardiography, then 94% of individuals studied have evidence of cardiovascular involvement. The most frequently affected segment of the cardiovascular system was the branch PA tree, with anomalies of the branch PAs documented by imaging

6 2572 Circulation November 12, 2002 TABLE 4. s of Subjects With TOF (%) (n 23) (n 20) AGS Without a (n 3) Pulmonary valve Stenosis 14 (61) 11 (55) 3 (100) Atresia 8 (35) 8 (40) 0 (0) Absent 1 (4) 1 (5) 0 (0) Pulmonary blood supply* Diffuse PA hypoplasia 15 (65) 14 (70) 1 (33) Discontinuous branch PAs 4 (17) 4 (25) 0 (0) MAPCAs 8 (35) 8 (40) 0 (0) Aortic arch sidedness Left 16 (100) 14 (100) 2 (100) Right 0 (0) 0 (0) 0 (0) Aortic arch branching pattern Normal 14 (88) 12 (86) 2 (100) Aberrant right subclavian artery 2 (12) 2 (14) 0 (0) Abnormal coronary artery pattern 2 (9) 2 (10) 0 (0) Valvar AS 3 (13) 3 (15) 0 (0) MAPCAs indicates major aortopulmonary collateral arteries. *These categories are not mutually exclusive: all individuals with discontinuous branch PAs had MAPCAs and diffuse PA hypoplasia. Data not available for all subjects. (n 111) or inferred from a PPS murmur (n 41) in 76% of subjects. There are several particularly interesting findings of this study. First, although AGS has been considered a disease of TABLE 5. s of Subjects With Anomalies of the Pulmonary Valve* Mutation (n 12) AGS Without a Mutation (n 8) (n 20) Valvar pulmonary atresia with intact ventricular septum Valvar pulmonary stenosis Isolated valvar pulmonary stenosis Associated with other anomalies Branch PA stenosis Ventricular septal defect Atrial septal defect Patent ductus arteriosus Coarctation of the aorta Bileaflet aortic valve Pulmonary vein stenosis Severity of stenosis Moderate/severe ( 40mm Hg) Mild ( 40 mm Hg) *Does not include subjects with TOF. Multiple associated anomalies were present in some subjects, so the number of individual associated lesions do not sum to the number of subjects with associated anomalies. the right side of the heart, left-sided cardiovascular anomalies were noted in 11% of our study population, including 12 (6%) subjects with both left- and right-sided defects, an extremely uncommon combination. Second, the frequency of severe forms of TOF, particularly TOF with pulmonary atresia and major aortopulmonary collateral arteries, was substantially higher than in the general population of individuals with TOF, where only 20% have pulmonary atresia. 12 Third, all of the individuals in this study with TOF for whom data were available regarding the sidedness and branching pattern of the aortic arch had a left-sided (ie, normal) aortic arch and only 2 had an abnormal aortic arch branching pattern. In contrast, abnormal sidedness or branching of the aortic arch is common among individuals with TOF and a chromosome 22q11 deletion. 13 Finally, 4 of the 200 subjects (2%) were found to have an absent right superior vena cava, which is otherwise extremely rare. 14,15 Genotype-Phenotype Considerations Of the 200 subjects in this study, a mutation was identified in 154 (77%), which is similar to the 60% to 75% frequency reported in other studies of mutations in individuals with AGS. 5 We have hypothesized that the identification of mutations in only 60% to 75% of individuals with AGS is attributable to technical limitations of the testing methods presently used rather than a separate pathogenesis for AGS in individuals without an identified mutation. 5 Of note, however, is that there were small but significant differences in the PA phenotype between subjects with and without a mutation, raising the possibility that AGS is attributable to mutations in functional domains outside of the open-reading frame or to different genetic mechanisms in at least some individuals without an identified mutation. Correlation of these cardiac findings with other features of AGS, as well as additional molecular analysis, will be required to clarify this issue. Within the cohort of subjects with a mutation, there was no correlation between the type or location of the mutation and the presence or type of cardiovascular anomaly. This finding is not surprising, given that cardiovascular status can vary markedly between family members sharing the same mutation. 5 Moreover, most mutations are predicted to result in loss of protein function. 5,16 The variable phenotypic expression of a mutation in the cardiovascular system suggests that additional epigenetic factors or genetic background influence the final cardiac phenotype. Clinical Implications Although details of cardiovascular clinical outcome were only available for 74% of the study cohort, our analysis reveals several important findings. Among 23 subjects with TOF, 10 (43%) were known to have died from cardiovascular causes, including 6 of 8 with TOF and pulmonary atresia. The apparently poor outcome among individuals with a mutation and TOF with pulmonary atresia is striking and warrants additional investigation, because it may have implications for clinical decision making in this subset of individuals.

7 McElhinney et al Mutation and Cardiovascular Phenotype 2573 TABLE 6. s of Subjects With Left-Sided Cardiovascular Anomalies (n 22) Mutation (n 17) AGS Without a Mutation (n 5) Isolated left-sided anomalies Left-sided anomalies with associated anomalies Associated left-sided anomalies* Associated right-sided anomalies* Associated septal anomalies only Specific anomalies Valvar AS Mild stenosis Moderate-severe stenosis Isolated valvar AS Valvar AS associated with other anomalies Supravalvar AS Isolated supravalvar AS Supravalvar AS associated with other anomalies Coarctation of the aorta Isolated coarctation of the aorta Coarctation of the aorta associated with other anomalies Bileaflet aortic valve without stenosis Isolated bileaflet aortic valve Bileaflet aortic valve associated with other anomalies *Two subjects had both left- and right-sided associated anomalies. Associated right-sided anomalies included TOF (n 3), branch PA stenosis (n 8), and valvar pulmonary stenosis (n 2, both with branch PA stenosis as well). All subjects with mild valvar AS had pressure gradients 25 mm Hg, and all subjects with moderate-severe valvar AS had pressure gradients 50 mm Hg. All subjects with supravalvar AS had pressure gradients 35 mm Hg. Equally important, among 55 subjects with branch PA stenosis (not including those with TOF) and serial imaging studies, there were no cases in which the severity of the branch PA stenosis increased substantially over time. There were small changes in 10% of these subjects, which consisted of a decrease in the stenotic gradient in all but 1 case. These findings may be helpful in counseling and decision-making for individuals with AGS or a mutation and branch PA stenosis. Potential Biases There does not seem to be any systematic bias regarding the composition of our study cohort. However, it is possible that individuals with a mutation who have no or few phenotypic features and have no relatives with overt AGS may be underrepresented in our study group. A survival bias is also possible, because individuals with severe forms of cardiovascular disease may be underrepresented due to neonatal or early infant mortality. One of the most significant biases affecting this study is the limited number of subjects in whom the distal PA tree was imaged. To definitively characterize the PA anatomy in subjects with AGS, evaluation of the distal PA tree by angiography or MRI would be necessary, because echocardiography only provides images of the proximal branch PAs. Two of the subjects in our series with a PPS murmur but no branch PA stenosis/hypoplasia by echocardiography had electrocardiographic evidence of right ventricular hypertrophy, which suggests significant obstruction in the distal PA tree. Only 30% of subjects in this series underwent imaging of the distal pulmonary vascular tree, many of whom had TOF. Thus, our characterization of PA anatomy and ascertainment of PA anomalies, especially diffuse involvement, is incomplete, and our determination of discrete versus diffuse PA involvement is most likely biased. From a practical point of view, this is inevitable, because there is no clinical indication for invasive imaging studies in most individuals with AGS or a mutation. Conclusions These findings should assist the cardiologist, hepatologist, and pediatrician in counseling families and providing appropriate evaluation and follow-up for patients with AGS or a mutation. The phenotypic spectrum should remind the cardiologist to take additional family history and be observant for signs of AGS, especially in individuals with distal branch PA disease. Finally, detailed examination of the cardiovascular phenotype in this cohort will allow for future studies correlating clinical outcome with genetic pathogenesis. Acknowledgments This work was supported by National Institutes of Health grants P50 HL62177 (to Drs Spinner and Goldmuntz), RO1 DK53104 (to Dr

8 2574 Circulation November 12, 2002 TABLE 7. s of Subjects With Other Intracardiac Anomalies* Mutation AGS Without a Mutation Atrial septal defect (ASD) Isolated ASD ASD associated with other anomalies Branch PA stenosis Valvar pulmonary stenosis Valvar AS Ventricular septal defect Ventricular septal defect (VSD) Isolated VSD VSD associated with other anomalies Branch PA stenosis Valvar pulmonary stenosis Atrial septal defect Patent ductus arteriosus Double-chambered right ventricle Pulmonary vein stenosis Type of VSD Perimembranous Muscular Subarterial (conoseptal hypoplasia) Data not available Atrioventricular septal defect (unbalanced) *Does not include subjects with TOF, pulmonary atresia with intact ventricular septum, or truncus arteriosus. Multiple associated anomalies were present in some subjects, so the numbers of individual associated lesions do not sum to the numbers of subjects with associated anomalies. Spinner), and KO8 DK02541 (to Dr Krantz) and a grant from The Fred and Suzanne Biesecker Foundation and Pediatric Liver Center (to Dr Piccoli). We would like to thank the subjects and families that participated in this study, the referring physicians that provided information, and Raymond Colliton for his work on genotyping. References 1. Alagille D, Estrada A, Hadchouel M, et al. Syndromic paucity of interlobular bile ducts (Alagille syndrome or arteriohepatic dysplasia): review of 80 cases. J Pediatr. 1987;110: Emerick KM, Rand EB, Goldmuntz E, et al. Features of Alagille syndrome in 92 patients: frequency and relation to prognosis. Hepatology. 1999;29: Li L, Krantz ID, Deng Y, et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet. 1997;16: Oda T, Elkahloun AG, Pike BL, et al. Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat Genet. 1997;16: Spinner NB, Colliton RP, Crosnier C, et al. Jagged1 mutations in Alagille syndrome. Hum Mutat. 2001;17: Krantz ID, Smith R, Colliton RP, et al. Jagged1 mutations in patients ascertained with isolated congenital heart defects. Am J Med Genet. 1999;84: Eldadah ZA, Hamosh A, Biery NJ, et al. Familial tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet. 2001;10: Colliton RP, Bason L, Lu FM, et al. Mutation analysis of Jagged1 () in Alagille syndrome patients. Hum Mutat. 2001;17: Deprettere A, Portmann B, Mowat AP. Syndromic paucity of the intrahepatic bile ducts: diagnostic difficulty; severe morbidity throughout early childhood. J Pediatr Gastroenterol Nutr. 1987;6: Silberbach M, Lashley D, Reller MD, et al. Arteriohepatic dysplasia and cardiovascular malformations. Am Heart J. 1994;127: Krantz ID, Colliton RP, Genin A, et al. Spectrum and frequency of Jagged1 () mutations in Alagille syndrome patients and their families. Am J Hum Genet. 1998;62: Ferencz C, Loffredo CA, Correa-Villasenor A, et al. Perspectives in Pediatric Cardiology Volume 5: Genetic and Environmental Risk Factors of Major Cardiovascular Malformations: The Baltimore-Washington Infant Study Armonk, NY: Futura; Goldmuntz E, Clark BJ, Mitchell LE, et al. Frequency of 22q11 deletions in patients with conotruncal defects. J Am Coll Cardiol. 1998;32: Lucas RV, Krabill KA. Abnormal systemic venous connections. In: Emmanouilides GC, Riemenschneider TA, Allen HD, et al, eds. Moss and Adams Heart Disease in Infants, Children, and Adolescents. 5th ed. Baltimore: Williams & Wilkins; 1995: Bartram U, Van Praagh S, Levine JC, et al. Absent right superior vena cava in visceroatrial situs solitus. Am J Cardiol. 1997;80: Morrissette JD, Colliton RP, Spinner NB. Defective intracellular transport and processing of missense mutations in Alagille syndrome. Hum Mol Genet. 2001;10: