Learning to Crawl: Determining the Role of Genetic Abnormalities on Postoperative Outcomes in Congenital Heart Disease

Background Our cardiac center established a systematic approach for inpatient cardiovascular genetics evaluations of infants with congenital heart disease, including routine chromosomal microarray (CMA) testing. This provides a new opportunity to investigate correlation between genetic abnormalities and postoperative course. Methods and Results Infants who underwent congenital heart disease surgery as neonates (aged ≤28 days) from 2015 to 2020 were identified. Cases with trisomy 21 or 18 were excluded. Diagnostic genetic results or CMA with variant of uncertain significance were considered abnormal. We compared postoperative outcomes following initial congenital heart disease surgery in patients found to have genetic abnormality to those who had negative CMA. Among 355 eligible patients, genetics consultations or CMA were completed in 88%. A genetic abnormality was identified in 73 patients (21%), whereas 221 had negative CMA results. Genetic abnormality was associated with prematurity, extracardiac anomaly, and lower weight at surgery. Operative mortality rate was 9.6% in patients with a genetic abnormality versus 4.1% in patients without an identified genetic abnormality (P=0.080). Mortality was similar when genetic evaluations were diagnostic (9.3%) or identified a variant of uncertain significance on CMA (10.0%). Among 14 patients with 22q11.2 deletion, the 2 mortality cases had additional CMA findings. In patients without extracardiac anomaly, genetic abnormality was independently associated with increased mortality (P=0.019). CMA abnormality was not associated with postoperative length of hospitalization, extracorporeal membrane oxygenation, or >7 days to initial extubation. Conclusions Routine genetic evaluations and CMA may help to stratify mortality risk in severe congenital heart disease with syndromic or nonsyndromic presentations.

C ongenital heart disease (CHD) causes significant infant morbidity and mortality. 1 Chromosomal microarray (CMA) is a genome-wide test that identifies copy-number variants (CNVs) including deletions or duplications or regions of homozygosity. CMA can identify genetic syndromes that have high penetrance of CHD, such as 22q11.2 deletion and 7q11.23 deletion (Williams syndrome). CMA can also identify more rare genomic disorders with CHD association such as 8p23.1 duplication, 16p11.2 deletion, or 15q11.2 (BP1-BP2) deletion. [2][3][4] Pathogenic CNVs have been reported in patients with extracardiac anomalies (ECAs) as well as in those with isolated CHD. 5 As we have recently described in detail, in 2014 the cardiovascular genetics (CVG) program at Riley Hospital for Children at Indiana University Health implemented a clinical algorithm for the genetic evaluation of infants hospitalized with CHD, which included routine CMA testing of patients with any class of severe CHD. 6 Prior literature has suggested, to varying degrees, that genetic diagnoses are associated with worse postoperative outcomes in infants. 7,8 The understanding of CNVs that cause CHD has advanced rapidly, and the routine clinical application of CMA has increased identification of CNVs in this population. However, the use of CMA is still variable between different pediatric cardiac centers. Therefore, current understanding of the impact of CNVs on surgical outcomes in the era of clinical CMA testing is incomplete and unable to be ascertained from current multi-institutional surgical outcomes databases. Our standardized and routine performance of CVG evaluations at our center provides a new opportunity to understand these risks. Therefore, the objective of this study was to determine the impact of genetic abnormalities including CNVs on early postoperative outcomes following neonatal CHD surgery.

Transparency and Openness
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Study Population and Data Collection
Infants undergoing cardiac surgery for CHD from January 1, 2015 to March 1, 2020 at age ≤28 days were identified through the Society of Thoracic Surgeons (STS) Congenital Heart Surgery Database at Indiana University Health. Cardiac transplants and patent ductus arteriosus ligations were excluded. Each patient's first cardiac surgery entered into the database was selected for analysis. Demographic and clinical data were primarily collected from STS data. Data entered into the noncardiac congenital anatomic abnormalities field of the STS database were included as ECAs. Intubation prior to CHD surgery was defined as intubation that occurred >24 hours before surgery. The STS-European Association for Cardio-Thoracic Surgery (STAT) mortality risk scores (range from 1 to 5) were designated per STS protocol. 9 The STS defined operative mortality as death that occurred during the same hospitalization or within 30 days of the operation. The number of days of intubation was calculated from the time of operation to initial extubation. Patients who died before postoperative day 8 were excluded from extubation analysis. Patients who died before hospital discharge were not included in the analysis of length of postoperative hospitalization.

Genetics Evaluations
The records of genetic testing completion, results, and interpretation were collected from the electronic medical record along with information from the genetics consultation, if performed. Clinical genetic testing was performed in clinical laboratories using standard methods or in-house as previously described. 6 CMA was performed routinely per the clinical algorithm. The indications for additional molecular gene testing were determined by the consulting clinical geneticist. Geneticists and genetic counselors reviewed all testing reports, including clinical interpretation of CNVs, as part of standard inpatient consultation practices. CMA and molecular genetic testing results were classified as (1) normal, (2) variants of uncertain significance (VUSs), or (3) diagnostic (ie, pathogenic or likely pathogenic) according to established guidelines. 10,11 For this study, the definition of genetic abnormality was (1) CMA abnormality, including CNVs classified as diagnostic or VUS or (2) genetic syndrome identified by a geneticist clinically or with additional genetic testing at the time

CLINICAL PERSPECTIVE
What Is New?
• The routine application of formal inpatient cardiovascular genetics evaluations for neonates undergoing congenital heart disease surgery facilitated analysis of early postoperative outcomes in a genetically well-defined clinical population. • Genetic abnormalities that likely would not have been otherwise detected were associated with mortality in patients with syndromic or nonsyndromic presentations.
What Are the Clinical Implications?
• More widespread adoption of routine genetic evaluation practices in neonates with severe congenital heart disease will be critical to define the clinical impact of genetic abnormalities and foster tailored care that improves outcomes.

Human Subjects
The study was approved by the institutional review board at Indiana University (Protocol ID: 1408953015) and used a waiver of informed consent. Study procedures followed were in accordance with institutional guidelines.

Rates of Genetic Evaluations and Yields
Neonatal CHD surgery (aged ≤28 days) was performed in 355 patients. Clinical genetics consultation or CMA testing was performed in 311 patients (88%). Forty-nine patients (14%) had molecular testing sent during the hospitalization in which the neonatal CHD surgery was performed (Table S1). A genetic abnormality was identified in 73 patients (21% of the cohort), and 221 without an identified genetic abnormality had a CMA confirming absence of CNV (62%) (Figure 1). Sixtyone patients did not have a genetic abnormality or CMA sent (17%). These included 17 patients who had genetics consultation without CMA (5%) and 44 patients who did not have either consultation or CMA (12%). Taken together, implementation of the clinical algorithm was largely successful over the course of 5 years.

Genetic Findings and Baseline Characteristics
An abnormal CNV was identified in 68 patients, including 38 with diagnostic CNV and 30 with a VUS (Table 1). Of these, the genetic diagnosis was first identified prenatally in 3 patients (22q11.2 deletion, Turner syndrome, and Recombinant 8 syndrome). Four additional patients were diagnosed with single-gene disorders based on molecular testing sent in addition to CMA, and 1 patient was given a clinical diagnosis of a genetic syndrome ( Table 2). The most frequent genetic diagnoses were 22q11.2 deletion syndrome (N=14), 15q11.2 (BP1-BP2) deletion (N=4), Turner syndrome  (N=3), 16p11.2 deletion (N=3), and CHARGE syndrome (N=3). We compared the baseline characteristics between 73 total patients with genetic abnormality and the 221 who had a negative CMA. Genetic abnormality was associated with increased frequency of prematurity and presence of ECA and lower weight at CHD surgery (Table 3). CHD surgery with CPB was less frequent in patients with genetic abnormality. Meanwhile, the STAT mortality risk categories were similar between the 2 groups; overall >70% of the operations were STAT risk 4 or 5. Taken together, comorbidities were increased in patients with genetic abnormalities, whereas the cardiac operations had similar classification of mortality risk between groups.

Postoperative Outcomes
Genetic abnormality was not associated with longer duration of postoperative hospitalization, requirement for intubation for >7 days postoperatively, or need for extracorporeal membrane oxygenation (ECMO) postoperatively ( . The operative mortality rate in patients with genetic abnormality was approximately 2 times higher than for those without genetic abnormality (9.6% versus 4.1%), although this difference was not statistically significant (P=0.080 using the Fisher exact test). The genetic abnormalities that were identified in mortality cases are shown in Table 5. These included 4 patients diagnosed with well-characterized genetic syndromes and 3 with CNVs that were classified as VUS. The mortality rate of patients with diagnostic genetic evaluation (4/43=9.3%) was similar to patients with VUS on CMA (3/30=10.0%), although the surgeries in the VUS group were less commonly on CPB and had fewer STAT risk 4 or 5 operations (

Analysis of Patients Without Extracardiac Anomalies
The presence of ECAs has been previously associated with poorer surgical outcomes. 7 In the present study, patients with genetic abnormality were more likely to have an ECA (Table 3). To determine the impact of genetic abnormality independent of ECA, the cohort was subclassified according to the presence or absence of ECAs. Genetic abnormality was identified in 20.1% of the 174 patients without ECAs (Table 7). In patients without ECAs, a genetic abnormality was associated with prematurity and lower weight at CHD surgery, whereas CPB and STAT categories did not significantly differ (Table S2). Genetic abnormality was not associated with postoperative length of hospitalization, need for postoperative ECMO, or intubation for >7 days postoperatively (  (Figure 2). In contrast to these findings, among the 120 patients who had an ECA, the operative mortality rate was similar between those with genetic abnormality (3/38 patients, 7.9%) and without genetic abnormality (6/82 patients, 7.3%). This result suggests that in patients with ECA, the interrelationship between ECA and genetic abnormality likely complicates the ability to discriminate their relative effects on mortality. Meanwhile, the findings provide evidence that genetic abnormality significantly impacts outcome in the absence of ECAs. This strengthens the rationale for performing genetics evaluation in all patients with severe CHD regardless of ECA status.

DISCUSSION
This study was performed in a pediatric cardiac center where CVG evaluations were performed systematically over a 5-year period. Genetic abnormalities were identified frequently in neonates undergoing CHD surgery. Patients with diagnostic genetic findings or VUS on CMA had similar increases in the rates of operative mortality. CMA identified genetic abnormalities that likely would not have been otherwise detected and were associated with mortality. These included CNVs in patients with apparently isolated CHD and additional CMA findings in both patients with 22q11.2 deletion who did not survive. We did not identify significant associations between genetic abnormality and the secondary postoperative outcomes that were measured, including length of stay, need for ECMO, or prolonged intubation.

With Increased Identification of Genetic Abnormalities in CHD, There Is A Need to Understand Their Clinical Impact in the Context of Systematic Evaluation
The current study demonstrates that clinical CVG algorithms have high yields. The observed rate of genetic abnormality in the present study is similar to results from 2 different centers where similar algorithms were successfully implemented by CVG providers. 12,13 Prior studies have investigated the association between genetic abnormalities and postoperative survival during earlier eras when CMA testing was infrequently performed. 7,14-20 The overall conclusion from these studies was that, with some exceptions, genetic diagnoses generally increased the risk for poorer outcomes including mortality and other secondary outcomes. However, the interpretation of this literature was complicated by inconsistency in genetics evaluations within study cohorts; variable definitions of what constitutes a genetic abnormality (sometimes including ECA without a specific genetic diagnosis); accuracy of genetic data collection, which can be fraught because of the inherent complexity of genetic testing and interpretation; and lack of documentation of what testing or evaluations were performed, including whether normal cases had negative testing. Since then, there has emerged more advanced genetic testing, increased understanding of genetic causes of CHD, and clinical initiatives to implement more comprehensive genetics evaluations. 21 Several recent studies have used broad genetic testing technologies to investigate the clinical impact of genetic abnormalities in CHD and compared survival among patients who underwent the same type of genetic testing. These studies are summarized along with their main findings in Table 8. [22][23][24] For the present study, our center's concerted and sustained clinical initiative to perform CVG evaluation and genetic testing of critically ill infants with CHD has helped to minimize some of the challenges faced by these prior studies. First, our CVG service was well integrated within the intensive care unit, and clinical geneticist consultations and testing were performed soon after birth. This decreased the likelihood of missed genetic diagnoses in operative mortality cases, minimizing survival bias. Only 1 mortality case did not receive CVG consultation or testing. The testing performed in this study was clinical and a routine practice, and therefore did not face obstacles inherent to research enrollment and testing, which likely increased the ascertainment of eligible patients. Importantly, we can report not only the number of patients who were excluded for not having CMA, which was relatively infrequent, but also describe their characteristics. Our systematic clinical approach to CVG evaluations and testing likely decreased selection bias, while also presenting a real-world application of genetic screening and risk assessment in the clinical setting.

Cardiac Considerations for Study Inclusion and Early Versus Late Survival Outcomes
CHD is morphologically and physiologically heterogeneous, and the timing, frequency, and complexity of surgical interventions variable. The studies included in Table 8 selected the types of CHDs to varying degrees.
The sole cardiac criterion for eligibility in the present study was requirement of neonatal CHD surgery. We analyzed outcomes that occurred during the hospitalization when the initial CHD surgery took place. Because the focus was on early postoperative survival, the impact of the type of operation on outcome was controlled using the STAT category in multivariable analysis and by performing subanalysis restricted to STAT category 4 or 5 operations. The statistical rationale for using cohorts with heterogeneous types of CHD is that adequate sample sizes are needed to analyze the impact of rare genetic abnormalities. Also,  the known genetic causes of CHD have variable expressivity and present with a range of different types of CHD. Imposing stringent criteria for types of CHD in the study of outcomes may decrease the ability to identify the risks that are associated with rare genetic diagnoses that have variable CHD presentations. There are conceptual differences for analyzing the impact of genetic abnormalities on early postoperative survival, such as in the present study, versus their impact on longitudinal survival in other studies. In the early postoperative period, factors that decrease cardiorespiratory reserve or alter fluid balance regulation via involvement of renal or lymphatic systems are critically important. Patients are closely monitored for infection, neurological changes such as seizures, and arrhythmia. In the ambulatory setting, development of  these complications may be more difficult to identify and treat promptly. Congenital aerodigestive anomalies that are successfully repaired or palliated to achieve hospital discharge may have risk for later complications and chronic comorbidity. Increased pulmonary artery pressure secondary to chronic respiratory insufficiency or vascular anomalies (congenital or maladaptive) can negatively impact longitudinal outcomes, particularly in palliated single-ventricle physiologies that depend on passive pulmonary blood flow. Thus, genetic abnormalities may differentially impact acute or longitudinal outcomes, depending on the associated phenotypes and systems involved.

Genetic Considerations for Study Inclusion
The current study includes all patients with an identified genetic diagnosis except for trisomy 21 or 18. Syndromic conditions, such as 22q11.2 deletion and CHARGE syndrome, were included to understand the overall impact of genetic abnormalities that were identified via our center's inpatient CVG algorithm. We excluded trisomy 21 because, unlike other genetic syndromes with strong CHD association, trisomy 21 is routinely diagnosed during standard prenatal and postnatal care, large studies have demonstrated favorable outcomes, and trisomy 21 is more common than other rare genomic disorders. [25][26][27] Trisomy 18 was excluded because of the known high lethality that is largely independent of CHD.

CNVs Classified as VUSs May Impact Clinical Outcomes in CHD
The clinical testing and interpretations in our study were performed by CVG experts, and abnormal CMA results included diagnostic variants and VUSs. The similar mortality rates between patients with diagnostic results and VUSs suggest that some VUSs may impact early postoperative survival. From their specified criteria, it is likely that several CNVs included as abnormal in the studies of Kim et al and Boskovski et al (Table 8) would be classified as VUSs clinically. These results together raise an interesting possibility that genetic variants may not need to be independently or definitively causative of CHD to be clinically impactful. Previous studies in 22q11.2 deletion syndrome have predominantly determined that overall postoperative mortality is not greatly increased. 17,18,[28][29][30][31] In the current study, we found that among 14 cases with 22q11.2 deletion, the only 2 operative mortalities occurred in cases who also had additional CMA findings. These data, albeit small in numbers, lead to a hypothesis that additional genetic abnormalities significantly impact survival in 22q11.2 deletion. Genome-wide testing with CMA may not only help to identify pathogenic CNVs at greater resolution than traditional fluorescence in situ hybridization (FISH), 5 but also may identify such additional variants that are clinically important postoperatively. Larger data sets to test this hypothesis may become available as more clinical centers adopt routine CMA testing.
Genetic Abnormalities Are Common in Apparently Isolated CHD Cases and Are Clinically Significant As expected, genetic abnormalities were more frequent in patients with ECAs. However, 20% of patients without an ECA were found to have a genetic abnormality, which is comparable to prior observations in a similarly evaluated cohort. 12 In the current study, a genetic abnormality was associated with increased mortality in patients without ECA. Kim et al excluded patients with multiple ECAs, and found that CNVs were associated with decreased survival. Similarly, the genetic abnormalities defined in Boskovski et al were associated with decreased survival in patients who did not have ECAs. These data, taken together, including the current results acquired from a strictly clinical setting, indicate that routinely testing patients with critical CHD clinically for CNVs can identify increased risk for poor outcomes, including when a genetic diagnosis may not be otherwise suspected.

Most Patients With Pathogenic CNVs Survived Neonatal CHD Surgery
Although association with increased mortality was observed, the majority of patients with genetic abnormality, including pathogenic CNVs, survived neonatal CHD surgery. Survivors included all 3 patients with Turner syndrome, all 3 with 16p11.2 deletion, and both with 8p23.1 duplication. Boskovski et al identified 15q11.2 deletion as having increased mortality risk. 24 In the present study, all 4 neonates with 15q11.2 deletion survived, including 2 with STAT risk category 5 procedures (both Norwood operations) and 1 with STAT risk category 4 (total anomalous pulmonary venous return repair). A 15q11.2 deletion may be an example of a condition that can be successfully managed postoperatively but may predispose to complications that decrease longer-term survival. Another pertinent finding in this study was that the duration of hospitalization, need for ECMO, and prolonged postoperative intubation were not increased in patients with genetic abnormality. It is possible that the specific type of CHD or operation (eg, Norwood procedure) has a stronger effect on these outcomes than on mortality. Analysis of these outcomes may also be confounded by mortalities that occur shortly after an operation.
The limitations of this study include that it was retrospective and single center. Overall, the mortality rate was low, which limited statistical power. Requiring CNVs to be ruled out with CMA, the early postnatal timing of evaluations, and the standardized approach to evaluation increase specificity and clinical validity. A minority of patients were excluded from the primary analyses because CMA was not completed. Approximately 50% of these excluded patients had surgery in 2015, which was early in the implementation of the CVG clinical algorithm. By the years 2018 to 2020, only 5 of the 150 (3.3%) eligible cases did not have CMA. Thus, the integration of routine CVG evaluation into pediatric cardiac critical care settings took a short period of time and was successful. This study was clinical and did not include research-based genetic testing. Parental testing for CNVs that were classified as VUSs was pursued only when thought to be highly informative. The ability to complete parental testing for VUSs during the patient's initial hospitalization was limited by practical factors such as parental sample availability, as well as uninsured financial costs. The indications and feasibility of parental testing or additional genetic testing are often reconsidered in outpatient CVG follow-up. This report ascertained all neonates who underwent surgery and does not include cases where comfort care was chosen or were unable to be stabilized preoperatively, which were rare. The sample size of our study was relatively small, which may lead to relatively wide confidence intervals for a few covariates (eg, STAT). We further estimated the variance inflation factors for all covariates in the multiple logistic regression model. The values ranged between 1.03 and 1.45 and did not suggest collinearity among covariates.
In conclusion, this study provides a novel perspective of the impact of genetic abnormalities on early postoperative outcomes when CVG consultations and genetic testing are systematically performed. The first clinical implication of this study is that CVG evaluations of neonates with CHD have demonstrable yield and identify genetic abnormalities that would not otherwise be suspected and should be routine. Second, genetic abnormalities in patients without ECA or additional genetic findings in patients with 22q11.2 deletion may have increased risk for postoperative mortality, and adjusting clinical management accordingly may improve outcomes. The consensus statement recommending CMA as a first-tier test in patients who have congenital anomalies was first published in 2010. 32 It has required time and resources to incorporate these guidelines faithfully into the clinical care of infants with critical CHD. Given recent evidence supporting the use of whole genome sequencing in critically ill neonates, 33 we anticipate that new guidelines may be on the near horizon that would apply to infants with critical CHD. In this article, we have discussed the challenges associated with the study of genetic abnormalities and outcomes in CHD, which apply to research and clinical cohorts. Understanding the nature and depth of clinical genetic evaluation and testing that was completed for all included patients is of paramount importance, if not a prerequisite, for studies that seek to determine the clinical impact of genetic abnormalities on outcomes in CHD. Concerted clinical efforts and collaboration to adopt genomic testing practices quickly in a standardized manner between centers may be the most efficient path toward understanding the clinical impact of genetic abnormalities in CHD. By learning to crawl in this manner we will develop the fundamental basis to walk and run. Table S1. Types and frequencies of molecular genetic testing sent during the hospitalization in which the neonatal CHD surgery was performed (N=355).  The column headed NA were patients without genetic diagnosis or CMA testing and were not included in the statistical analysis.