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

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 JAG1 mutation in the proband. Subjects were considered for inclusion in the present analysis if they had been tested for JAG1 mutations as previously described.^3,8 They were included if either a JAG1 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 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×velocity²), 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 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.

JAG1 Mutation Analysis

Analysis of JAG1 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 al¹ 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 al¹ 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 JAG1 mutation and, within the cohort of subjects with branch PA anomalies, between different phenotypic variables using nonparametric analysis (χ² or Fisher’s exact test). Genotype-phenotype analysis was then performed within the cohort of subjects with a JAG1 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.

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 JAG1 mutations. Adequate cardiac data were obtained for 200 of these individuals (90%), including 154 (77%) with a JAG1 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 JAG1 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 JAG1 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). 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 JAG1 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 JAG1 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 JAG1 mutation. None of the other anatomic variables differed according to the presence or absence of a JAG1 mutation.

Among the 154 subjects with a JAG1 mutation, there was no correlation between the type or location of the JAG1 mutation and the frequency or type of cardiovascular malformation. In fact, there was considerable variability in cardiovascular phenotype among subjects with JAG1 mutations of all types and locations and even among probands and affected parents and siblings with identical mutations.

There were 12 subjects with a JAG1 mutation who did not meet our criteria for AGS. All except 1 of these subjects were evaluated for a JAG1 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 8.3±9.2 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 JAG1 mutation and 22% (n=10) of those without. During follow-up, 14 (7%) subjects (12 with and 2 without a JAG1 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.

Cardiovascular Phenotype

This study analyzes the cardiovascular phenotype in 200 subjects with a JAG1 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 (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 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 JAG1 mutation was identified in 154 (77%), which is similar to the 60% to 75% frequency reported in other studies of JAG1 mutations in individuals with AGS.⁵ We have hypothesized that the identification of JAG1 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 JAG1 mutation.⁵ Of note, however, is that there were small but significant differences in the PA phenotype between subjects with and without a JAG1 mutation, raising the possibility that AGS is attributable to mutations in JAG1 functional domains outside of the open-reading frame or to different genetic mechanisms in at least some individuals without an identified JAG1 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 JAG1 mutation, there was no correlation between the type or location of the JAG1 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 JAG1 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 JAG1 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.

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 JAG1 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 JAG1 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 JAG1 mutation.