Validation of association of the apolipoprotein E ε2 allele with neurodevelopmental dysfunction after cardiac surgery in neonates and infants

Abstract

Objective

Apolipoprotein E (APOE) genotype is a determinant of neurologic recovery after brain ischemia and traumatic brain injury. The APOE ε2 allele has been associated with worse neurodevelopmental (ND) outcome after repair of congenital heart defects (CHD) in infancy. Replication of this finding in an independent cohort is essential to validate the observed genotype-phenotype association.

Methods

The association of APOE genotype with ND outcomes was assessed in a combined cohort of patients with single-ventricle CHD enrolled in the Single Ventricle Reconstruction and Infant Single Ventricle trials. ND outcome was assessed at 14 months using the Psychomotor Development Index (PDI) and Mental Development Index (MDI) of the Bayley Scales of Infant Development-II. Stepwise multivariable regression was performed to develop predictive models for PDI and MDI scores.

Results

Complete data were available for 298 of 435 patients. After adjustment for preoperative and postoperative covariates, the APOE ε2 allele was associated with a lower PDI score (P = .038). Patients with the ε2 allele had a PDI score approximately 6 points lower than those without the risk allele, explaining 1.04% of overall PDI variance, because the ε2 allele was present in only 11% of the patients. There was a marginal effect of the ε2 allele on MDI scores (P = .058).

Conclusions

These data validate the association of the APOE ε2 allele with adverse early ND outcomes after cardiac surgery in infants, independent of patient and operative factors. Genetic variants that decrease neuroresilience and impair neuronal repair after brain injury are important risk factors for ND dysfunction after surgery for CHD. (J Thorac Cardiovasc Surg 2014;148:2560-8)

A dramatic reduction in mortality after surgical correction of congenital heart defects (CHD) in recent years has been accompanied by increasing recognition of adverse neurodevelopmental (ND) outcomes in some survivors.¹ Evaluation of children after neonatal repair of CHD demonstrates a pattern of ND sequelae characterized by cognitive impairment, speech and language abnormalities, impaired visual-spatial and visual-motor skills, attention deficit hyperactivity disorder, motor delays, and learning disabilities.^2-4

Cerebral ischemia in the perioperative period has been proposed as a primary mechanism of central nervous system (CNS) injury. Management strategies during the operation, including type of support (deep hypothermic circulatory arrest [DHCA] or continuous cardiopulmonary bypass [CPB]), hemodilution, degree of cooling, and blood gas management, have been implicated as factors in postoperative ND dysfunction. These potential risk factors do not fully explain the frequency or pattern of ND dysfunction. ^5-8 There is significant interindividual variation in developmental outcome, even among children with the same cardiac defect, suggesting that other patient-specific factors may be important determinants of CNS injury.⁶

Genetic polymorphisms that increase susceptibility to neurologic injury are potentially important modifiers of ND outcome in children with CHD. Apolipoprotein E (APOE) genotype has been shown to have an important role as a determinant of neurologic recovery after CNS ischemia, intracerebral hemorrhage, and traumatic brain injury.⁹ A previous study demonstrated an association between the APOE ε2 allele and postoperative ND disabilities in neonates and infants undergoing cardiac surgery with CPB.¹⁰ In that study, early ND outcomes were assessed with the Bayley Scales of Infant Development-II (BSID-II).

Most initial reports of genotype-phenotype associations are not subsequently validated. Ideally, validation should be performed in a similar population with similar environmental exposures. The phenotype assessed in the validation study should be the same as the initial report and similar measures used. In the current study, a combined cohort of patients from the Infant Single Ventricle (ISV) and Single Ventricle Reconstruction (SVR) trials was used to validate the association of the APOE ε2 allele with ND outcomes after surgery for CHD in infancy. For both the ISV and SVR trials, the study population consisted of infants with single-ventricle CHD who underwent surgery in the first year of life and in whom an ND evaluation was performed at 14 months of age using the BSID-II.^7,8,11-13

METHODS

Study Population

The study population includes patients from both the ISV and SVR trials performed by the Pediatric Heart Network (PHN). The inclusion and exclusion criteria, study designs, and the participating centers for both studies have been described previously.^12,13 The study populations contain many similarities that justify their grouping for the current study. In brief, inclusion criteria for the ISV study included (1) single-ventricle–type CHD (except pulmonary atresia with intact ventricular septum), (2) planned superior vena caval to pulmonary artery connection, and (3) absence of a genetic or medical condition that would affect growth. Inclusion criteria for the SVR trial included (1) a diagnosis of hypoplastic left heart syndrome (HLHS) or a related single, morphologic, right ventricular anomaly, (2) planned Norwood procedure, and (3) absence of a genetic or medical condition that would affect transplant-free survival. The primary outcomes and ND outcomes for both studies have been published previously. ^7,8,13 For the ISV trial, DNA was extracted from peripheral blood samples and testing of renin-angiotensin-aldosterone systempathway variants was performed.¹⁴ DNAsamples were stored for future testing in the PHN biorepository in accordance with the informed consent process. Samples were sent to the University of Michigan for APOE genotyping. Participants in the ISV study who did not consent to future testing of their samples were not included in the current study. For the SVR trial, the participants gave consent for APOE genotyping. Sampleswere identified by the PHN Data Coordinating Center to verify that participants who were enrolled in both studies were only represented once in the current study.

Genotyping

Two methods were used to establish APOE genotype. The ε2 allele consists of a cysteine at positions 112 and 158; ε3 consists of a cysteine at position 112 and an arginine at position 158; ε4 consists of an arginine at both positions. These alleles result in 6 possible genotypes: 3 homozygous genotypes (ε2/ε2, ε3/ε3, and ε4/ε4) and 3 heterozygous genotypes (ε2/ε3, ε2/ε4, and ε3/ε4).

For the SVR participants, buccal swab epithelial cells were collected using CytoSoft cytology brushes (Medical Packaging Corporation, Camarillo, Calif) after obtaining informed consent. Genomic DNA was extracted using a PureGene kit (Gentra Systems, Inc, Minneapolis, Minn) according to the manufacturer’s protocol. Genotype analysis for the presence of the single nucleotide polymorphisms that specify a cysteine and/or an arginine residue at codons 112 and 158 of the APOE gene, which define the ε2, ε3, and ε4 alleles, was performed using a TaqMan assay and an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif) as described by Koch and colleagues.¹⁵ Reactions were carried out in 96-well microtiter plates in the ABI Prism 7000 Sequence Detection System. The assay volume of 22 μL consisted of 11 μL of 23 TaqMan Universal PCR Master Mix (Applied Biosystems), 75 nmol/L each forward and reverse primers, 25 nmol/L each allele specific probe, and 2 μL of DNA (rv30-100 ng). Amplification involved 40 cycles of denaturation at 95°C for 15 seconds and primer annealing and extension at 60° C for 1 minute. Patient samples were run along with controls representing all 3 possible genotypes (ie, Cys/Cys, Cys/Arg, and Arg/Arg) as well as a no-template control. Postamplification analysis of the genotypes at each codon was performed using the allelic discrimination analysis module of the ABI 7000 Sequence Detection System. The genotype of the patient was deduced from the combined results at amino acid positions 112 and 158. DNA samples were available for 398 SVR participants and genotyping was successful for 397. However, for 90 samples, there was no consent to use the DNA for secondary studies.

The ISV samples were amplified by polymerase chain reaction (PCR) and the amplicons were subjected to Sanger sequencing. PCR primers were designed to produce an amplicon containing the sequence differences that are associated with the APOE ε2, ε3, and ε4 variants. PCR primers were selected using the LaserGene Primer Select Program (DNASTAR Inc, Madison, Wis). Forward (5⁰-CCG CCC CAT CCC AGC CCT TCT CC) and reverse (5⁰-TCC GGC TGC CCA TCT CCT CCA TCC) primers were selected. A 12-μL PCR reaction was set up for each patient and control DNA sample. Each 12-μL PCR reaction contained 1 μL of 10-40 ng genomic patient or control DNA, 1 μL of 1.5 pmol forward primer, 1 μL of 1.5 pmol reverse primer, 3 μL of DNase/RNase free water, and 6 μL of HotStarTaq polymerase mixture (Qiagen, Inc, Valencia, Calif). PCR DNA amplification was performed on a thermal cycler (Bio-Rad MyCycler, Bio-Rad Life Sciences, Hercules, Calif) using 96-well plates. A touchdown PCR protocol was used as follows: initial denaturation at 94° C for 15 minutes, followed by 24 cycles with an annealing temperature decreasing 0.7° C per cycle, starting at 72° C for 30 seconds; denaturation at 94° C for 30 seconds, and extension at 72° C for 1 minute. An additional 32 cycles were added: 94° C for 30 seconds, 55° C for 30 seconds, 72° C for 1 minute, with a final extension of 72°C for 10 minutes. EachPCR product was diluted with 40 μL of distilled water and submitted for sequencing. Sanger sequencing was performed by the University of Michigan DNA Sequencing Core facility using an Applied Biosystems DNA Sequencer (Model 3730 XL). Each chromatogram was visually inspected using Applied Biosystems Sequence Scanner (v1.0) software to determine genotypes. Twenty-eight samples were genotyped using both methods with 100% concordance of genotypes with all genotype combinations represented. DNAwas available for 160 patients and adequate genotypes were obtained in 150. Genotyping was unsuccessful in 10 patients because of low sample quantity or quality.

Neurodevelopmental Evaluation

In both trials, ND testing with the BSID-II was performed at 14 months by a designated study-site psychologist certified by the PHN’s neuropsychological testing consultant (DCB). The BSID-II was administered in English or Spanish depending on the dominant language spoken at home. The BSID-II offers a standardized assessment of cognitive and motor development for children aged 1 to 42 months.¹⁶ It yields 2 scores: the Psychomotor Development Index (PDI) and the Mental Development Index (MDI). The PDI assesses control of gross muscle function, including crawling and walking, as well as fine muscle skills necessary for prehension, use of writing instruments, and imitation of hand movements. The MDI assesses memory, problem solving, early number concepts, generalization, vocalizations, and language and social skills. The mean ± standard deviation is 100 ± 15 in the normative population for both scores. Patients who were too impaired to complete neurodevelopmental testing were assigned a score of 50. The PDI score is usually more severely affected in infant survivors of cardiac surgery than the MDI score.^5,7

Statistical Analyses

All analyses were performed in R (http://www.r-project.org/). APOE genotypes were classified into 3 groups as follows: ε2 (ε2ε2 or ε2ε3), ε3 (ε3ε3), or ε4 (ε4ε3 or ε4ε4). Participants with ε2ε4 genotype (n = 9) were excluded from the analyses because the alleles have opposing effects in adults and cannot be placed in either the ε2 or ε4 groups. Stepwise linear regression analysis was used to select from the numerous demographic, clinical, and genetic variables entering the model those that have the greatest contribution to the model as measured by Akaike’s information criterion (AIC). The variables included in the stepwise regression model were surgical center, gender, a dummy variable for race with the white subgroup (the majority racial group) as the reference group, HLHS, gestational age, birth weight, presence of a genetic anomaly, post-Norwood length of stay (LOS) or LOS after neonatal palliative surgery, bypass time during palliative surgery, aortic crossclamp time, DHCA time, weight at stage 2 surgery, height at stage 2 surgery, head circumference at stage 2 surgery, post-stage 2 surgery LOS, weight/height/head circumference at 14 months, maternal education, and number of serious adverse events until 14 months of age. Model comparison was performed using AIC, beginning with a base model that included only surgical site and presence of genetic syndrome. Only specific demographic, clinical, and genetic variables that improved model prediction of the outcome of either MDI or PDI at 14 months were retained in each respective final regression model. A subgroup analysis was performed for nonsyndromic participants (n = 232) to ensure that APOE ε2 genotype effects were consistent when excluding participants with chromosomal or other genetic anomalies.

RESULTS

The ISV trial randomized 230 patients. The SVR trial randomized 549 patients. There were 66 patients enrolled in both studies resulting in 713 unique participants for the combined cohort. There was no overlap with the original discovery cohort. In the ISV trial, 92% of survivors returned and had valid BSID-II scores.⁸ In the SVR trial, the follow-up rate for the BSID-II examination among transplant-free survivors was 86%.⁷ Overall, the ND evaluation was completed in 435 patients. Complete data, including APOE genotype, were available for 298 patients, who form the study cohort (Figure 1). The most common reason for missing data was lack of APOE genotype. The only significant differences in patient and management variables between patients with complete versus incomplete data were a greater proportion of Hispanic patients and a greater frequency of serious events in the group with complete data (Table 1). Overall, the cohort with complete data was predominately male (65%) and white (65%), very similar to the composition of the initial discovery cohort.^5,17 The predominant diagnosis was HLHS (77%). The ε2 allele was present in 11% of the cohort. APOE genotype was in Hardy-Weinberg equilibrium, with a rejection cutoff of P < 10⁻⁴. For the entire cohort, the median PDI was 76.9 (range, 50-120) and the median MDI was 90.6 (range, 50-132).

FIGURE 1.

The study cohort. ISV, Infant Single Ventricle trial; SVR, Single Ventricle Reconstruction trial; APOE, apolipoprotein E.

TABLE 1.

Comparison between patients with complete and incomplete data

	Complete data (n = 298)	Incomplete data (n = 137)	P value
Patient variables
Gender, n (%)
Male	193 (64.8)	85 (62.0)	.59
Female	105 (35.2)	52 (38.0)
Race/ethnicity, n (%)
White	196 (65.8)	104 (75.9)	.035
Black	25 (8.4)	24 (17.5)	.084
Hispanic	64 (21.5)	9 (6.6)	5.32 × 10−5
Other	13 (4.3)	0	.012
Hypoplastic left heart syndrome, n (%)	230 (77.2)	112 (81.8)	.32
Gestational age, wk ± SD	38.3 ± 1.49	38.4 ± 1.38	.52
Birth weight, kg ± SD	3.198 ± 0.516	3.162 ± 0.527	.51
Genetic or chromosomal syndrome, n (%)	47 (15.8)	31 (22.6)	.11
Maternal education, n (%)*
Elementary school	8 (2.7)	1 (0.9)	.14
Junior high	4 (1.3)	1 (0.9)
Some high school	23 (7.7)	8 (6.7)
High school or GED equivalent	46 (15.4)	33 (27.7)
Some college or 2-y vocational school	95 (31.9)	35 (29.4)
4-y college	84 (28.2)	30 (25.2)
Graduate degree	38 (12.8)	11 (9.2)
APOE genotype
ε2 (ε2/ε3 or ε2/ε2)	33 (11.1)
ε3 (ε3/ε3)	178 (59.7)
ε4 (ε3/ε4 or ε4/ε4)	87 (29.2)
ε2/ε4	0	9 (6.6)
Stage 1
Cardiopulmonary bypass time, min ± SD	130.4 ± 53.4	139.1 ± 51.4	.11
Crossclamp time, min ± SD	49.85 ± 25.1	53.6 ± 24.5	.15
DHCA use, n (%)	255 (85.6)	121 (88.3)	.45
Post-Norwood procedure LOS, d ± SD	30.8 ± 30.4	31.9 ± 29.3	.73
Stage 2
Pre-stage 2 weight, kg ± SD	5.89 ± 1.07	5.92 ± 1.04	.79
Pre-stage 2 height, cm ± SD	61.6 ± 4.30	62.03 ± 4.24	.34
Pre-stage 2 head circumference, cm ± SD	39.9 ± 2.34	40.3 ± 2.20	.10
Post-stage 2 LOS, d ± SD	13.4 ± 23.0	12.4 ± 11.9	.55
Neurodevelopmental evaluation
Weight at 14 mo, kg ± SD	9.22 ± 1.31	9.38 ± 1.28	.27
Height at 14 mo, cm ± SD	74.7 ± 3.76	75.2 ± 4.13	.20
Head circumference at 14 mo, cm ± SD	45.9 ± 1.98	45.7 ± 2.09	.46
Serious adverse events until 14 mo, n ± SD	0.71 ± 1.38	0.47 ± 0.89	.028
MDl score at 14 mo ± SD	90.6 ± 16.2	90.6 ± 17.3	.98
PDI score at 14 mo ± SD	76.9 ± 19.2	73.8 ± 18.7	.12

SD, Standard deviation; GED, General Educational Development; DHCA, deep hypothermic circulatory arrest; LOS, length of stay; MDI, Mental Development Index; PDI, Psychomotor Development Index; APOE, apolipoprotein E.

^*Information on maternal education collected on 119 patients in the incomplete data subset.

In the overall cohort, after adjustment for preoperative and postoperative covariates, the APOE ε2 allele was associated with a lower PDI score (P = .038). Other significant predictors of the PDI score were study center, height at the time of ND evaluation, postoperative LOS during the Norwood hospitalization or neonatal palliation, presence of a genetic syndrome, head circumference at the time of stage 2 surgery, and the number of serious adverse events up to the neurodevelopmental evaluation (Table 2). The overall model explained 24.9% of the variability in the PDI scores. After removing the syndromic patients (n = 66), the association of the APOE ε2 allele with the PDI score became less significant (P = .06). After covariate adjustment, patients with the APOE ε2 allele would be predicted to have a PDI score that was approximately 6 points lower than those without the risk allele, explaining 1.04% of overall PDI variance.

TABLE 2.

Stepwise regression results for Bayley-II PDI score at 14 months of age (n = 298)

Variable	Beta coefficient ± SE	% PDI explained	P value
Site	—	10.87	.00037
Height at 14 mo (cm)	1.27 ± 0.303	8.46	3.28 × 10−5
Post-Norwood LOS (d)	−0.137 ± 0.0394	5.34	.00062
Presence of genetic syndrome	−7.09 ± 2.54	1.93	.0057
Pre-stage 2 surgery head circumference (cm)	1.068 ± 0.458	1.18	.021
APOE e2 genotype	−6.50 ± 3.11	1.04	.038
Number of serious adverse events	−1.70 ± 0.972	0.89	.081
Gender (female)	2.98 ± 2.14	0.49	.16

SE, Standard error; PDI, Psychomotor Development Index; LOS, length of stay; APOE, apolipoprotein E.

For the overall cohort, there was a marginal effect of APOE ε2 allele on MDI scores (P = .058). Significant predictors of the MDI score were study center, birth weight, postoperative LOS during the stage 2 hospitalization, presence of a genetic syndrome, race other than white, female gender, and head circumference at the time of ND evaluation (Table 3). The overall model explained 31.4% of the variability in the MDI scores. After removing the syndromic patients (n = 66), the association of the APOE ε2 allele with the MDI score became more significant (P = .02). The presence of the APOE ε2 allele accounted for 0.84% of overall MDI variation. After covariate adjustment, nonsyndromic patients with the APOE ε2 allele would be predicted to have a MDI score that was approximately 7 points lower than those without the risk allele.

TABLE 3.

Stepwise regression results for Bayley-II MDI score at 14 months of age (n = 298)

Variable	Beta coefficient ± SE	% MDI explained	P value
Site	—	15.46	1.18 × 10−7
Post-Norwood LOS (d)	−0.0955 ± 0.0391	7.83	.015
Birth weight (g)	0.00623 ± 0.00172	4.19	.00035
Presence of genetic syndrome	−6.35 ± 2.20	2.58	.0043
Post-stage 2 LOS (d)	−0.215 ± 0.0815	1.79	.0909
Race other	−16.41 ± 5.46	1.70	.0029
Gender (female)	5.15 ± 1.89	1.09	.0068
Head circumference at 14 mo (cm)	0.966 ± 0.485	0.83	.048
APOE ε2 genotype	−5.15 ± 2.70	0.84	.058
Maternal education	0.965 ± 0.686	0.46	.14

SE, Standard error; MDI, Mental Development Index; LOS, length of stay; APOE, apolipoprotein E.

DISCUSSION

These findings validate the previously reported association of the APOE ε2 allele with early ND disability after cardiac surgery in neonates and infants.^5,10 There are multiple reports of genotype-phenotype associations in many populations that have failed replication in indepenent studies.^17-19 To our knowledge, this represents the first time that a genotype-phenotype association modifying postoperative outcomes after surgery for CHD has been replicated in an independent cohort. Replication of the initial finding is necessary to establish the validity of a genotype-phenotype association.^17-19 The impact of APOE genotype on an individual patient’s ND outcomes is significant. After adjustment for covariates, patients with the å2 allele are predicted to have PDI and MDI 6 to 7 points lower than those without the risk allele, similar to the magnitude of the effect in the discovery cohort.⁵ This is a significant decline and represents more than a 1/3 standard deviation decrease in the scores. However, APOE genotype explained only approximately 1% of the variation in MDI and PDI scores in the overall cohort, which is less than the variation explained by other factors such as site, postoperative LOS, growth, and presence of genetic syndromes. The small overall impact likely relates to the relatively low frequency of APOE ε2, which was present in only 11% of the cohort.

ApoE-containing lipoproteins are the primary lipid transport vehicles in the CNS and have an important role in mobilization and redistribution of cholesterol and phospholipids during remodeling of neuronal membranes.⁹ The gene for the human APOE is located on chromosome 19 and codes for a 299 amino acid protein. There is increasing evidence that APOE protein is important for neuronal repair. There are 3 common isoforms of APOE protein (E2, E3, and E4), which are encoded by 3 alleles (ε2, ε3, and ε4, respectively) and vary by single amino acid substitutions at 2 sites. The normal function of APOE within neurons is believed to be maintenance of microtubular integrity and stabilization of the neuronal cytoskeleton.^9,20 Mechanisms by which APOE may modify CNS injury include protection against oxidative stress, modulation of the glial response to inflammation, and a direct neurotropic effect on injured neurons. APOE synthesis is upregulated by astrocytes and oligodendrocytes after CNS injury or cerebral ischemia. Studies in APOE knockout mice have shown that APOE deficiency worsens neuronal injury after cerebral ischemia and compromises the blood-brain barrier after CNS injury.^9,20,21 A recent study using brain biopsies from patients with epilepsy found that neurons from patients with the APOE ε4 allele are less resilient to the chronic excitation of epilepsy.²² Neurons from patients with APOE ε3ε3 demonstrated more beneficial responses to hyperexcitability, neuroinflammation, and neuronal DNA damage. APOE genotype may also modulate white matter development. Westlye and colleagues²³ evaluated white matter microstructure in healthy adults. Carriers of either the ε2 or ε4 allele demonstrated regional decreases in fractional anisotropy. This finding, and the known role of APOE in cholesterol transport, support a functional role for APOE in myelin-related processes in the brain. Trachtenberg and colleagues²⁴ assessed functional connectivity in cognitively normal adults using functional magnetic resonance imaging and found different patterns of connectivity based on APOE genotype. They speculate that APOE genotype has an intrinsic effect on the development and differentiation of functional networks in the brain.

In adults, strong associations have been validated between the APOE genotype and Alzheimer disease, as well as recovery after traumatic brain injury (TBI).^20,25,26 In adults, the APOE ε4 allele is associated with an increased risk of Alzheimer disease and worse recovery after TBI. However, multiple studies have demonstrated that the effects of APOE genotype on recovery after brain injury are not the same in the immature developing brain as in the aging brain. In contrast to adults, the ε2 allele is associated with worse outcomes and the ε4 allele is protective after a variety of types of brain injury in infants and children. APOE genotype has been shown to modify the adverse effects of lead exposure in children. In a study of lead exposure by Wright and colleagues,²⁷ the APOE ε4 allele was associated with a 4.4 point higher score on the MDI. The negative effects of lead exposure on the MDI were 4-fold greater for ε2 and ε3 carriers compared with ε4 carriers. Chronic diarrhea with malnutrition is associated with deficits in cognition and executive function.^28-30 In children who have multiple diarrheal episodes early in life, the ε4 allele is associated with better visual working memory and semantic fluency. However, Gelfand and colleagues³¹ found that the ε4 allele is associated with an increased risk of perinatal arterial ischemic stroke. In addition, the data on the impact of APOE genotype on the risk of cerebral palsy (CP) is mixed. Braga and colleagues³² reported a higher prevalence of APOE ε2 carriers in patients with CP and, in a study by Kuroda and colleagues,³³ APOE ε2 carriers had a 12-fold increased risk of CP and ε4 carriers a 5-fold increase. Wu and colleagues³⁴ also described an increased prevalence of the APOE ε4 allele in patients with CP; however, the association was not significant after correction for multiple comparisons. A recent study from Australia which evaluated 35 candidate genes and the risk of CP found no association between APOE genotype and CP.³⁵

There are limitations to this study. Because this is a secondary analysis, data including APOE genotype, are incomplete for some patients. Nonetheless, our study has important strengths, which are necessary for validation of a genotype-phenotype association. Our validation cohort population was similar to the discovery cohort, had similar environmental exposures, and the same phenotype was assessed using the same instrument. Specifically, both the discovery and validation cohorts consist of neonates and infants with CHD undergoing surgery with CPB early in life. The ethnic makeup of the 2 cohorts is similar, reducing the risk of population stratification altering the results. Both the discovery and validation studies assessed the phenotype of early ND outcomes using the BSID-II, and the magnitude of the effect of the APOE ε2 allele on PDI and MDI scores was similar in both cohorts.

In summary, this study provides evidence confirming the previous report that the APOE ε2 allele is associated with worse early ND outcomes after cardiac surgery in neonates and infants. This APOE genotype–environment interaction demonstrates that genetic variants that impair neuroresilience and CNS recovery may explain some of the interindividual variation in developmental outcome after surgery for CHD. However, it would be premature to begin routine clinical testing of APOE genotype, as the impact is small and the knowledge would not alter clinical management. These findings support the need for studies to delineate the mechanisms by which APOE genotype modulates brain recovery and investigate potential therapies. Future studies of neurologic outcome and clinical trials of new neuroprotective strategies should include risk stratification for APOE genotype. In addition, further studies are needed to identify other genetic variants that modify the risk of brain injury and ND disability.

Abbreviations and Acronyms

AIC

Akaike’s information criterion

ApoE

apolipoprotein E

BSID-II

Bayley Scales of Infant Development-II

CHD

congenital heart defects

CNS

central nervous system

CP

cerebral palsy

CPB

cardiopulmonary bypass

DHCA

deep hypothermic circulatory arrest

HLHS

hypoplastic left heart syndrome

ISV

Infant Single Ventricle trial

LOS

length of stay

MDI

Mental Development Index

ND

neurodevelopmental

PCR

polymerase chain reaction

PDI

Psychomotor Development Index

PHN

Pediatric Heart Network

SVR

Single Ventricle Reconstruction trial

TBI

traumatic brain injury