- Email: [email protected]
Adrenergic receptor genotypes influence postoperative outcomes in infants in the Single-Ventricle Reconstruction Trial
Accepted Manuscript Adrenergic Receptor Genotypes Influence Post-operative Outcomes in Infants in the Single Ventricle Reconstruction Trial Ronand Ramroop, MD, George Manase, BSc, Danny Lu, MSc, Dorin Manase, BSc, Shan Chen, MSc, Richard Kim, MD, Teresa Lee, MD, William T. Mahle, MD, Kimberly McHugh, MD, Mike Mitchell, MD, Martin Tristani-Firouzi, MD, Stephanie B. Wechsler, MD, Nicole S. Wilder, MD, Victor Zak, PhD, Myriam Lafreniere-Roula, PhD, Jane W. Newburger, MD, J William Gaynor, MD, Mark W. Russell, MD, Seema Mital, MD PII:
S0022-5223(17)31360-0
DOI:
10.1016/j.jtcvs.2017.06.041
Reference:
YMTC 11717
To appear in:
The Journal of Thoracic and Cardiovascular Surgery
Received Date: 4 August 2016 Revised Date:
6 June 2017
Accepted Date: 20 June 2017
Please cite this article as: Ramroop R, Manase G, Lu D, Manase D, Chen S, Kim R, Lee T, Mahle WT, McHugh K, Mitchell M, Tristani-Firouzi M, Wechsler SB, Wilder NS, Zak V, Lafreniere-Roula M, Newburger JW, Gaynor JW, Russell MW, Mital S, Adrenergic Receptor Genotypes Influence Postoperative Outcomes in Infants in the Single Ventricle Reconstruction Trial, The Journal of Thoracic and Cardiovascular Surgery (2017), doi: 10.1016/j.jtcvs.2017.06.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Adrenergic Receptor Genotypes Influence Post-operative Outcomes in Infants in the Single
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Ventricle Reconstruction Trial
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Short title: Ramroop: Adrenergic genotypes and Norwood outcomes
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Ronand Ramroop1, MD, George Manase1, BSc, Danny Lu1, MSc, Dorin Manase1, BSc, Shan
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Chen2, MSc, Richard Kim3, MD, Teresa Lee4, MD, William T Mahle5, MD, Kimberly McHugh6,
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MD, Mike Mitchell7, MD, Martin Tristani-Firouzi8, MD, Stephanie B Wechsler9, MD, Nicole S
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Wilder10, MD, Victor Zak2, PhD, Myriam Lafreniere-Roula11, PhD, Jane W Newburger12, MD, J
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William Gaynor13, MD, Mark W Russell10, MD, Seema Mital1, MD
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Hospital for Sick Children, University of Toronto, Toronto, ON
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New England Research Institute, Watertown, MA
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Children’s Hospital of Los Angeles, Los Angeles, CA
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Columbia University Medical Center, New York, NY
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Emory University, Atlanta, GA
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Medical University of South Carolina, Charleston, SC
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Children’s Hospital of Wisconsin, Milwaukee, WI
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University of Utah School of Medicine, Salt Lake City, UT
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Duke University Medical Center, Durham, NC
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University of Michigan Health, Ann Arbor, MI
Cardiovascular Data Management Centre, Hospital for Sick Children, Toronto, ON Boston Children’s Hospital, Boston, MA Children’s Hospital of Philadelphia, Philadelphia, PA
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Conflict of Interest statement:
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The authors have no conflicts of interest.
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Sources of Funding:
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Supported by grants HL068269, HL068270, HL068279, HL068281, HL068285, HL068288,
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HL068290, HL068292, HL109778, and HL085057 from the National Heart, Lung, and Blood
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Institute.
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Corresponding author:
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Seema Mital, M.D
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Hospital for Sick Children
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555 University Avenue
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Toronto, Ontario M5G 1X8, Canada
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Phone: 416-813-7418
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Fax: 416-813-5857
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Email: [email protected]
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Clinical Trial Registry Number: NCT00115934
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Numbers and Dates of IRB Approvals*
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Hospital for Sick Children, 1000006285, 02/11/2005
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Children’s Hospital of Los Angeles, CCI-05-00038, 9/5/2008
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Columbia University Medical Center, AAAB0870, 1/12/2005
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Emory University, IRB00009510, 11/10/2005
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Medical University of South Carolina, HR18084, 5/6/2008
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Children’s Hospital of Wisconsin, CHW 05/29, 4/21/2005
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University of Utah School of Medicine, 13354, 2/5/2005
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Duke University Medical Center, Pro00004594, 4/5/2003
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University of Michigan Health, 41816, 9/17/2010
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Boston Children’s Hospital, 04-12-162, 12/13/2004
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Children’s Hospital of Philadelphia, IRB 05-004136, 1/5/2004
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SVR Biorepository at University of Michigan Health, HUM00031665, 9/3/2010
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*These are original approval dates of trial participating sites, and of the SVR biorepository
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located at University of Michigan. All IRB approvals are active.
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Glossary of Abbreviations
ADR- Adrenergic receptor
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CI- confidence interval
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ECMO- Extra-corporeal membrane oxygenation
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HLHS- Hypoplastic left heart syndrome
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HR- Hazard ratio
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LV- left ventricle
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MBTS- modified Blalock- Taussig shunt
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OR- Odds ratio
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PHN- Pediatric Heart Network
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RAAS- Renin- angiotensin- aldosterone system
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RV- Right ventricle
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RVEF- right ventricular ejection fraction
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RVPAS- Right ventricle to pulmonary artery shunt
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SVR trial- Single Ventricle Reconstruction Trial
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ABSTRACT
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Objectives: Adrenergic receptor (ADR) genotypes have been associated with adverse outcomes
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in heart failure. Our objective was to evaluate the association of ADR genotypes with post-
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Norwood outcomes in infants with hypoplastic left heart syndrome (HLHS).
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Methods: Infants with HLHS participating in the Pediatric Heart Network Single Ventricle
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Reconstruction Trial underwent genotyping for four single nucleotide polymorphisms in three
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ADR genes: ADRB1_231A/G, ADRB1_1165G/C, ADRB2_5318C/G and ADRA2A_2790C/T. The
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association of genotype with freedom from serious adverse events (SAE) (death, transplant, extra-
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corporeal membrane oxygenation, cardiopulmonary resuscitation, acute shunt failure, unplanned
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re-operations, or necrotizing enterocolitis) during 14 months follow-up was assessed using Cox
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regression and the association with post-Norwood complications was assessed using Poisson
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regression. Models were adjusted for clinical and surgical factors.
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Results: The study included 351 eligible patients (62% male; 83% White). The mean age at
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Norwood procedure was 5.6±3.6 days. 152 patients had SAEs during 14-month follow-up
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including 84 deaths and 10 transplants. ADRA2A_2790CC genotype had lower SAE-free survival
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compared to CT/TT genotypes during follow-up (Log rank test, p=0.02) and this association was
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independent of clinical and surgical risk factors (Adjusted Cox regression. HR 1.54 [1.04, 2.30]
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p=0.033). Post-Norwood complication rate did not differ by genotype.
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Conclusions: Infants with HLHS harboring ADR genotypes that are associated with higher
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catecholamine release or sensitivity had lower event-free survival after staged palliation. Excess
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catecholamine activation may adversely affect cardiovascular adaptation after the Norwood
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procedure. Future studies should explore if targeting adrenergic activation in those harboring risk
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genotypes can improve outcomes. (ClinicalTrials.gov number, NCT00115934)
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Central Message
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Variations in adrenergic receptor genes influence postoperative outcomes in infants with
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hypoplastic left heart syndrome undergoing staged palliation.
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Central Picture
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Kaplan Meier plot: Lower serious adverse event-free survival with ADRA2A_7790CC genotype
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(log rank test p-value=0.020). 7
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Perspective Statement
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Staged palliation for single ventricle lesions is associated with significant morbidity and mortality.
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Patients with adrenergic receptor genotypes associated with augmented catecholamine signaling
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had lower freedom from serious adverse events after Stage 1 palliation. Future studies should
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explore if targeting adrenergic activation in those harboring risk genotypes can improve outcomes.
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Single ventricle lesions comprise over 7% of all congenital heart defects with hypoplastic left
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heart syndrome (HLHS) being the most common lesion.1, 2 In HLHS, a single right ventricle (RV)
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acts as a systemic ventricle while the left ventricle (LV) is hypoplastic or rudimentary. Patients
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with HLHS require multiple palliative surgeries to separate the pulmonary and systemic
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circulations with the first staged palliation i.e. the Norwood procedure performed in the newborn
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period. Maintenance of systemic cardiac output after the Norwood procedure requires a balance
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between the systemic and pulmonary circulations and adequate RV function. Abnormal systemic
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and pulmonary vascular resistance and ventricular dysfunction are major contributors to
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maladaptation to a single ventricle physiology. In one analysis, up to 40% of patients with staged
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palliation were noted to have some degree of heart failure.3
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Single ventricle patients have fragile hemodynamics and are especially vulnerable after cardiac
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surgery. The post-operative physiological stress response involves a surge in endogenous
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catecholamine production which can have profound effects on cardiac function and vascular tone
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mediated by adrenergic receptors. Adrenergic receptors include presynaptic α2a and α2c receptors
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that inhibit norepinephrine release, cardiac β1 receptors that mediate chronotropic and inotropic
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response to norepinephrine, and β2 receptors that mediate vascular smooth muscle relaxation and
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response to β-blockers.4 Adrenergic upregulation is an important mechanism of cardiovascular
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adaptation during stress particularly in the infant where the circulation is highly catecholamine-
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dependent.5, 6 While acute upregulation is compensatory, chronic upregulation can have
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detrimental effects by increasing systemic and pulmonary vascular resistance, and increasing
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myocardial oxygen demands, resulting in adverse cardiac remodeling and ventricular
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dysfunction.4, 7-12 In children with dilated cardiomyopathy, α2 and β1 upregulation and β2
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downregulation genotypes were associated with worse ventricular function, heart failure
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progression and acute hemodynamic decompensation.13 The impact of adrenergic receptor
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genotypes on adaptation to hemodynamic and surgical stress in infants with HLHS undergoing
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staged palliation is not known. We hypothesized that genetic modulation of receptor activity
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would alter catecholamine response and thereby influence post-operative clinical course. The
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purpose of our study was to evaluate if genetic variants that upregulate adrenergic receptor
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activity adversely impact post-Norwood outcomes in infants with HLHS enrolled in the single
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ventricle reconstruction (SVR) trial.
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METHODS
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Study population
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This is an ancillary study that derives from the Pediatric Heart Network (PHN) SVR Trial, in
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which infants with HLHS (n=555) were enrolled from 15 North American centers between May
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2005 and July 2008. The details of the study design, inclusion criteria, study assessments as well
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as trial results have been previously published.14, 15 In brief, inclusion criteria for the SVR trial
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included a diagnosis of HLHS or a related single morphologic RV anomaly, planned Norwood
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procedure, and absence of a genetic or medical condition that would affect transplant-free
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survival. Infants were randomized to receive a modified Blalock-Taussig shunt (MBTS) versus a
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right ventricle – pulmonary artery shunt (RVPAS) as part of their Norwood procedure and were
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followed for 14 months with serial assessment. The study was approved by the local institutional
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review boards and informed consent for study participation was obtained from the parents or legal
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guardians. Data collection included patient demographics and baseline characteristics, operative
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variables, hospitalization course, clinical outcomes, and echocardiographic assessment of RV
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ejection fraction (RVEF). All echocardiograms were reviewed centrally by an independent
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observer at the echocardiography core laboratory.14, 15
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Genotyping
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Buccal swab epithelial cells were collected at enrollment or during Norwood hospitalization using
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CytoSoft cytology brushes (Medical Packaging Corporation, Camarillo, CA) after obtaining
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informed consent. Methods have been reported previously.16 Genomic DNA was extracted using a
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PureGene kit (Gentra Systems, Inc, Minneapolis, Minn) according to the manufacturer’s protocol.
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Samples were retrieved from the PHN Biorepository for genotyping. Participants in the SVR trial
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who did not consent to future testing of their samples were not included in the ancillary study. The
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ancillary study was approved by the PHN Ancillary Study Review Committee and by the
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University of Michigan Institutional Review Board.
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Adrenergic receptor single nucleotide polymorphisms (SNPs) were selected on the basis of
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previous association studies, functional effects, and population allele frequencies.13, 17,24
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Genotyping (Sequenom SNP) was performed for four SNPs in three adrenergic receptor (ADR)
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genes: a A/G missense variant at position 231 in ADRB1 resulting in a serine to glycine
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substitution (rs1801252), a C/G missense variant at position 1165 in ADRB1 resulting in a glycine
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to arginine substitution (rs1801253), a C/G missense variant at position 5318 in ADRB2 resulting
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in a glutamine to glutamic acid substitution (rs1042714) and a C/T 3’ untranslated region (UTR)
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variant at position 2790 in ADR2A (rs553668). Patients carrying a single copy of the variant
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allele or SNP (also known as minor allele) at the genetic locus were defined as having a
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heterozygous genotype, those with two copies of the variant allele as having a homozygous
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genotype, and those without a variant allele i.e. carrying only the normal allele (also known as
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major allele) were defined as having a wild-type genotype.
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Since variants in the renin-angiotensin-aldosterone signaling (RAAS) pathway genes have been
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previously associated with adverse ventricular remodeling in infants with single ventricle lesions,
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genotyping was also performed for four SNPs and one insertion-deletion variant in five RAAS
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genes. The following were considered RAAS-upregulation genotypes based on our previous
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work: AGT_CC, ACE_DD, AGTR1_CC, CYP11B2_CC, and CMA1_AA. The CYP11B2 and
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CMA1 variants were genotyped using Sequenom SNP genotyping and the AGTR1, AGT and the
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ACE insertion/deletion polymorphisms were genotyped using TaqMan primers and probes. For
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samples and SNPs which did not yield a valid result by Sequenom or TaqMan analysis, PCR
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amplification followed by Sanger sequencing was performed. The detailed genotyping methods,
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primers and probes for the Sequenom SNP analysis, the Taqman analysis and for the PCR
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amplification or Sanger sequencing are listed in Supplemental Methods and Supplemental
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Table 1.
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Statistical Analysis
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Allele and genotype frequencies were determined to calculate Hardy-Weinberg equilibrium. All
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demographic and clinical data were expressed as frequencies, mean with standard deviations, or
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medians with interquartile ranges (IQR). The primary outcome was a composite of any serious
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adverse event (SAE) during a 14-month follow-up after the Norwood procedure and included
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death, transplant, need for post-operative extra-corporeal membrane oxygenation (ECMO), post-
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operative cardiopulmonary resuscitation (CPR), acute shunt failure, unplanned re-operations or
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necrotizing enterocolitis.25, 26 Secondary outcomes included frequency of post-Norwood
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complications up to hospital discharge from stage 2 surgery, and RVEF during 14 months follow-
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up. Complications were defined as adverse events using the common terminology criteria for
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adverse events version 3.0 developed by the National Cancer Institute, NIH, but that did not meet
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criteria for SAEs.26 Since post-Norwood hospital and intensive care unit (ICU) length of stay were
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highly correlated with each other and with Norwood complication rate when analyzed using
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bootstrap Spearman rank correlation (data not shown), these outcomes were not analyzed
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separately.
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ADR genotype associations were analyzed by comparing outcomes in homozygous or
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heterozygous SNP carriers versus wild-types using a dominant model. Freedom from the primary
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outcome was described using the Kaplan-Meier method with stratification by genotype. The log-
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rank test was used to assess between-stratum differences. Cox proportional hazard models
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adjusted for covariates of interest were used to test the association of the genotypes with the
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primary outcome. The proportional hazards assumption was verified using cumulative Martingale
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process plots. Associations were quantified by hazard ratios (HRs) and reported along with their
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95% confidence intervals (CIs). P-values were calculated based on Wald’s statistics. Poisson
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regression adjusted for over-dispersion and for exposure time (number of days of hospitalization
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for Norwood procedure) was used to model the association of risk genotype with frequency of
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post-Norwood complications and results were presented as incidence rate ratios (IRR) with 95%
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confidence intervals. A mixed model was used to assess for association of genotype with RVEF
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during 14 months follow-up. All analyses were adjusted for gender, race, birth weight, gestational
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age, presence of aortic atresia or obstructed pulmonary venous return, presence of a genetic
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syndrome, age at Norwood procedure, total Norwood cardiopulmonary bypass time, deep
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hypothermic circulatory arrest time, type of shunt, and presence of a RAAS-upregulation
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genotype.27 The covariates selected were not found to exhibit significant collinearity. In
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regression models, when patients had missing data on the presence of identifiable syndromes, it
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was assumed that identifiable syndromes were not present. Observations with missing data in
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other variables were excluded from the models and this represented a very small percentage of the
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total cohort. All statistical analyses were performed using SAS v9.4.
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RESULTS
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Of 555 patients in the SVR trial, 436 provided DNA and 351 had complete genotype data (i.e.
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genotyping results for all 9 SNPs) and were included in the analysis (Figure 1). ADR genotype
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frequencies are shown in Table 1. All genotypes were in Hardy Weinberg equilibrium. Patient
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clinical characteristics and outcomes in the group with complete versus incomplete genotypes are
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shown in Table 2. The only significant difference was a higher incidence of death in the
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completely genotyped cohort compared to the incompletely genotyped cohort. Incomplete
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genotyping was the result of technical issues such as low quality or quantity of DNA. The results
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hereby focus on the 351 subjects with complete genotype data.
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We evaluated the association of ADR genotypes with the primary outcome of composite SAEs.
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Sixty-four percent patients had the ADRA2A_2790CC genotype (wild-type). Kaplan-Meier
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analysis showed that patients with the CC genotype had lower SAE-free survival during 14 month
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follow-up compared to CT/TT genotypes (log rank test: p=0.02) (Figure 2). This association was
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independent of RAAS genotype, clinical and surgical risk factors (Adjusted Cox regression: HR
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1.549 [1.04, 2.30] p=0.033). There was no synergistic adverse effect of multiple risk genotypes on
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outcomes.
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Tables 3 and 4 describe the incidence rate of Norwood complications by genotype groups.
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Overall, 79 percent patients had a post-Norwood complication with a median [95% CI] of 2 [1-4]
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complications per patient (Table 2). Complications by systems are shown in Table 5. The list of
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complications included for this trial have been previously published.26 Cardiac complications
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included arrhythmias, pericardial effusion, hypotension or hypertension, RV dysfunction, valvar
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insufficiency, and other. We evaluated the association of ADR genotypes with the rate of
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complications after the Norwood procedure using Poisson regression. All regression models were
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adjusted for demographic, clinical and surgical risk factors as well as for RAAS genotypes and the
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Poisson regression was adjusted for exposure time. There were no significant differences in
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incidence rates of Norwood complications by genotype (Figure 3). ADR genotypes were not
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associated with significant difference in RV function during 14 month follow-up (data not shown)
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as assessed by mixed effects models adjusted for clinical and surgical factors.
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DISCUSSION
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In an era of precision medicine, there is a growing recognition of the importance of identifying not
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just clinical predictors but also genetic predictors of outcomes that can be used to individualize
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care and improve the safety and efficacy of medical and surgical interventions based on the
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unique genome and phenome of a patient. Previous studies have identified genetic variants that
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increase susceptibility to neurodevelopmental outcomes and adverse ventricular remodeling in
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single ventricle patients undergoing staged palliation as well as in patients with other types of
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congenital heart disease undergoing surgical repair.16, 28, 29, 30, 31 In this study, we evaluated the
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association of genetic variants that increase ADR signaling with post-Norwood outcomes in
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infants with HLHS. Using genetic material collected during the trial, we demonstrated that the
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ADR genotype is an important predictor of outcomes of stage 1 palliation in infants with HLHS
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with lower serious adverse event-free survival in patients harboring ADR risk genotypes.
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Although further study is required, these findings may have implications for individualizing peri-
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operative management in this cohort based on genotype.
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We evaluated two SNPs in the ADRB1 gene and one in the ADRB2 gene. The ADRB1_389GG
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genotype has been previously associated with pediatric and adult heart failure and the
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ADRB1_231AA genotype is known to be associated with lower basal adenylate cyclase activity
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but increased sensitivity of the β1 receptor to norepinephrine with enhanced sympathetic blood
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pressure response.17, 18 Neither ADRB1 genotype nor the ADRB2 genotype were associated with
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post-operative complications or SAEs in our study.10
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The ADR2A receptor is important in vascular tone as well as metabolic responses like insulin
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release from pancreatic cells and adipocyte metabolism in humans. Genetic variations lead to
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alterations in G-protein coupling and in agonist-promoted receptor phosphorylation and
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desensitization thereby modulating response to catecholamines.12 The ADRA2A_2790CC
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genotype (wild-type) has been reported to cause reduced feedback inhibition of norepinephrine
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and failure to inhibit sympathetic tone. While we did not measure sympathetic tone, we found that
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patients with the CC genotype had lower freedom from SAEs during 14 month follow-up
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compared to CT/TT genotypes. One explanation for this finding is that a milieu of high circulating
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catecholamines in patients with the CC genotype may have resulted in increased vasomotor tone
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and an adverse effect on systemic output and organ perfusion, thereby increasing the risk of post-
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Norwood SAEs. The event-free survival curves diverged relatively sharply after the early post-
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operative period, at around 15 days (Figure 1). This suggests that while early events were more
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likely secondary to surgical complications which were similar in both genotype groups, later
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events were more likely influenced by genetic differences in adrenergic system-mediated
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adaptation to the Norwood circulation as well as recovery from complications. Overall, our
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findings suggest that genotypes associated with increased adrenergic neurohormonal activation
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and responsiveness may have a detrimental effect in the post-Norwood circulation.
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A limitation of our study is that only a small number of candidate SNPs were analyzed. The DNA
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in the trial was acquired via buccal swabs which required PCR amplification and did not yield
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sufficient quantity or quality of DNA for a more unsupervised approach using whole exome
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sequencing. Nonetheless, the SNPs were carefully selected based on prior associations with
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outcomes in other pediatric cardiac studies. Also, RV myocardial samples and detailed
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hemodynamic data were not collected during the Norwood procedure for the trial precluding
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assessment of tissue adrenergic receptor expression, and of potential influence of the genotypes on
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metabolic and hemodynamic function. Additionally, we were unable to evaluate the
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pharmacogenetic influences of the ADR genotypes with β-blocker response since less than 5%
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patients were receiving β-blocker therapy during follow-up. Also, the sample size of this study
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was reduced due to insufficient DNA or incomplete genotyping in the trial cohort. Nonetheless,
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the baseline characteristics of the genotyped and non-genotyped cohorts were similar excluding a
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survival bias in our study cohort.
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In summary, ADR genotypes that enhance catecholamine levels and/or sensitivity increase the
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risk of serious adverse events in patients who survive beyond the first two weeks after the
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Norwood procedure. Further studies are needed to delineate the biological and hemodynamic
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effects of these genotypes and to validate these findings in additional cohorts. Pharmacologic
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modification of adrenergic receptors has been shown to positively influence outcome in other
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heart failure cohorts. 21,22, 23 While there is no evidence to support empiric β-blocker use in HLHS
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patients, studies are needed to determine if pre-operative identification of these genetic subtypes
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can be used to target β-blockers and/or other forms of more aggressive systemic vasodilation post-
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Norwood to the subset with these risk genotypes.
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ACKNOWLEDGMENTS
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See Appendix (Supplementary Material) for a complete list of the Pediatric Heart Network
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Investigators.
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Hislop AA, Mak JCW, Kelly D, Reader JA, Barnes PJ, Haworth SG. Postnatal changes in
TE D
Whitsett JA, Noguchi A, Moore JJ. Developmental aspects of alpha- and beta-adrenergic
EP
Brouri F, Hanoun N, Mediani O, Saurini F, Hamon M, Vanhoutte PM et al.. Blockade of
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Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy and heart failure
Mann DL, Kent RL, Parsons B, Cooper G. Adrenergic effects on the biology of the adult
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Nikolaev VO, Moshkov A, Lyon AR, Miragoli M, Novak P et al.. Β2-adrenergic receptor
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redistribution in heart failure changes camp compartmentation. Science. 2010;327:1653-1657.
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induced myocardial necrosis: Morphology, quantification and regional distribution of acute
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contraction band lesions. Journal of Molecular and Cellular Cardiology. 1985;17:317-338.
364
12.
365
α2a- and β1-adrenergic receptors. Journal of Biological Chemistry. 2003;278:10770-10777.
366
13.
367
Adrenergic receptor genotype influences heart failure severity and beta-blocker response in
368
children with dilated cardiomyopathy. Pediatr Res. 2015;77:363-369.
369
14.
370
of shunt types in the norwood procedure for single-ventricle lesions. New England Journal of
371
Medicine. 2010;362:1980-1992.
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15.
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Design and rationale of a randomized trial comparing the Blalock–Taussig and right ventricle–
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pulmonary artery shunts in the Norwood procedure. The Journal of Thoracic and Cardiovascular
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Surgery. 2008;136:968-975.
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association of the apolipoprotein e ε2 allele with neurodevelopmental dysfunction after cardiac
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surgery in neonates and infants. The Journal of Thoracic and Cardiovascular Surgery.
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2014;148:2560-2568.
RI PT
Todd GL, Baroldi G, Pieper GM, Clayton FC, Eliot RS. Experimental catecholamine-
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Xu J, He J, Castleberry AM, Balasubramanian S, Lau AG, Hall RA. Heterodimerization of
M AN U
Reddy S, Fung A, Manlhiot C, Selamet Tierney ES, Chung WK, Blume E et al.
TE D
Ohye RG, Sleeper LA, Mahony L, Newburger JW, Pearson GD, Lu M et al. Comparison
EP
Ohye RG, Gaynor JW, Ghanayem NS, Goldberg CS, Laussen PC, Frommelt PC et al.
AC C
Gaynor JW, Kim DS, Arrington CB, Atz AM, Bellinger DC, Burt AA et al. . Validation of
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Small KM, Wagoner LE, Levin AM, Kardia SLR, Liggett SB. Synergistic polymorphisms
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of β1- and α2c-adrenergic receptors and the risk of congestive heart failure. New England Journal
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of Medicine. 2002;347:1135-1142.
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protein coupling domain of the human β1-adrenergic receptor. Journal of Biological Chemistry.
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1999;274:12670-12674.
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hypertrophy and fibrosis in mice at baseline but not after chronic pressure overload. Cardiovasc
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Res. 2010;86:432-444.
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fat cell function. J Lipid Res. 1993;34:1057-1091.
391
21.
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Adrenergic receptor genotype influences heart failure severity and β-blocker response in children
393
with dilated cardiomyopathy. Pediatric Research. 2015;77(2):363–369.
394
22.
395
adrenergic receptor genotype influences the effect of nonselective vs. selective beta-blockade on
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baroreflex function in chronic heart failure. International Journal of Cardiology.
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2011;153(2):230–232.
398
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399
impact of beta-adrenoreceptor gene polymorphisms on survival in patients with congestive heart
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failure. Eur J Heart Fail. 2005;7(6):966–973.
RI PT
Mason DA, Moore JD, Green SA, Liggett SB. A gain-of-function polymorphism in a g-
M AN U
SC
Gilsbach R, Schneider J, Lother A, et al. Sympathetic α2-adrenoceptors prevent cardiac
Lafontan M, Berlan M. Fat cell adrenergic receptors and the control of white and brown
TE D
Reddy S, Fung A, Manlhiot C, Tierney ES, Chung WK, Blume E et al. (2015).
AC C
EP
Truijen J, de Peuter OR, Kim Y, Bogaard B, Wouter EK, Kamphuisen PW et al. Beta2-
de Groote P, Lamblin N, Helbecque N, Mouquet F, Mc Fadden E, Hermant, X et al. The
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Sawczuk M, Maciehewska-Karlowska A, Cieszczyk P. A single nucleotide polymorphism
402
rs553668 in the adra2a gene and the status of polish elite endurance athletes. Trends in Sport
403
Sciences. 2013;1:30-35.
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of death in the Single Ventricle Reconstruction trial. Journal of thoracic and Cardiovascular
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Surgery. 2012:144(4):907-914
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surgical trial for complex congenital heart disease: The Pediatric Heart Network experience.
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Journal of Thoracic and Cardiovascular Surgery. 2011;142:531-7.
410
27.
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hospital morbidity and mortality after the Norwood procedure: A report from the Pediatric Heart
412
Network Single Ventricle Reconstruction trial. Journal of Thoracic and Cardiovascular Surgery.
413
2012;144:882-995
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28.
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angiotensin-aldosterone genotype influences ventricular remodeling in infants with single
416
ventricle. Circulation. 2011;123:2353-2362.
417
29.
418
polymorphisms influence progression of pediatric hypertrophic cardiomyopathy. Hum Genet.
419
2007;122:515-523.
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30.
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Patient genotypes impact survival after surgery for isolated congenital heart disease. The Annals
422
of Thoracic Surgery. 2014;98:104-111.
RI PT
Ohye R, Schonbeck J, Eghtesady P, Laussen P, Pizarro C et al. Cause, timing, and location
M AN U
SC
Virzi L, Pemberton V, Ohye R, Tabbutt S, Lu M et al. Reporting adverse events in a
TE D
Tabbutt S, Ghanayem N, Ravishankar C, Sleeper L, Cooper D et al. Risk factors for
EP
Mital S, Chung WK, Colan SD, Sleeper LA, Manlhiot C, Arrington CB et al. Renin-
AC C
Kaufman B, Auerbach S, Reddy S, Manlhiot C, Deng L, Prakash A et al. Raas gene
Kim DS, Kim JH, Burt AA, Crosslin DR, Burnham N, McDonald-McGinn DM et al.
23
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31.
Jeewa A, Manickaraj AK, Mertens L, Manlhiot C, Kinnear C, Mondal T et al. Genetic
424
determinants of right-ventricular remodeling after Tetralogy of Fallot repair. Pediatr Res.
425
2012;72:407-413.
AC C
EP
TE D
M AN U
SC
RI PT
423
24
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426
FIGURE LEGENDS
427
Figure 1: A consort flow diagram showing the outcomes in the complete and incompletely
429
genotyped groups from patients randomized in the single ventricle reconstruction (SVR) trial.
RI PT
428
430
Figure 2: Kaplan Meier survival curve showing survival free from composite primary outcome
432
stratified by ADRA2A_2790 genotype. The shaded bands represent 95% confidence intervals.
433
Subjects with ADRA2A_2790CC genotype had lower survival free from serious adverse events
434
(SAE) compared to subjects with CT/TT genotypes (p=0.02).
M AN U
SC
431
435
Figure 3: Forest plot of incidence rate ratios (IRRs) of Norwood complications by genotype. The
437
IRR is the ratio of the expected number of complications per patient per day of Norwood
438
hospitalization in each genotype group. There were no significant differences in the IRRs of
439
Norwood complications by genotype.
AC C
EP
TE D
436
25
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Table 1. Adrenergic receptor variant frequencies Gene (SNP
Nucleotide
Amino acid
Function by
SNP
HWE
ID)
substitution
substitution
genotype
carrier
p
frequency
value
32%
0.717
ADRB1
231 A/G
Ser49Gly
(rs1801252)
(missense)
GG: Higher basal adenylate cyclase
SC
activity and lower
RI PT
440
isoproterenol
1165 C/G
Gly389Arg
(rs1801253)
(missense)
ADRB2
5318 C/G
(rs1042714)
(missense)
Gln27Glu
EP AC C
CC: Increases β1
44%
0.818
62%
0.972
36%
0.918
receptor sensitivity
TE D
ADRB1
M AN U
sensitivity
GluGlu: Enhances response to β adrenergic receptor antagonist
ADRA2A
2790 C/T (3'
CC: Increased
(rs553668)
UTR)
norepinephrine release
441
ADR, adrenergic receptor; HWE, Hardy Weinberg equilibrium; SNP, single nucleotide
442
polymorphism
26
ACCEPTED MANUSCRIPT
Table 2. Patient characteristics and outcomes Characteristics
N
Complete
Incomplete p†
N
genotyping
Gender, Male (%)
351
220 (62.7%)
85
Race, White (%)
351
288 (82.1%)
85
Birth weight (kg)
351
3.13 ± 0.53
85
Gestational age (weeks)
344
38 ± 1
83
RI PT
genotyping
Identifiable syndrome (%)
150
22 (14.7%)
19
3 (15.8%) 1.00
Aortic atresia (%)
351
215 (61.3%)
85
50 (58.8%) 0.71
Obstructed PVR (%)
351
10 (2.8%)
85
1 (1.2%) 0.70
Age at Norwood (days)
351
6± 4
85
6 ± 4 0.12
Age at stage II palliation (days)
276
162 ± 53
75
164 ± 77 0.78
Total CPB time (mins)
351
139 ± 51
85
140 ± 43 0.86
324
33 ± 21
76
31 ± 18 0.27
351
170 (48.4%)
85
44 (51.8%) 0.63
351
183 (52.1%)
85
43 (50.6%) 0.81
Median hospital LOS (days)
351
24 (15-42)
85
22 (15-32) 0.35
Median ICU LOS (days)
255
15 (9-33)
52
13 (9-24) 0.65
Patients with complications (%)
351
278 (79.2%)
85
63 (74.1%) 0.31
Number of complications per patient
351
3±3
85
2 ± 3 0.25
Pre-stage II RVEF (%)
195
44 ± 9
56
43 ± 7 0.47
MBTS (%)
EP
RVPAS (%) Norwood course
56 (65.9%) 0.62 68 (80.0%) 0.44
3.13 ± 0.53 0.96 38 ± 2 0.38
SC
M AN U
TE D
DHCA time (mins)
AC C
443
14 months follow-up
27
ACCEPTED MANUSCRIPT
351
84 (23.9%)
85
Norwood hospitalization (%)
84
36 (42.9%)
11
4 (36.3%)
After Norwood discharge (%)
84
48 (57.1%)
11
7 (63.6%)
351
10 (2.8%)
85
Norwood hospitalization (%)
10
6 (60%)
2
After Norwood discharge (%)
10
4 (40%)
2
Serious adverse events (%)
351
152 (43.3%)
85
14 months RVEF (%)
168
43 ± 8
48
2 (2.4%) 1.00
RI PT
Transplants (%)
11 (12.9%) 0.028
1 (50%) 1 (50%)
29 (34.1%) 0.14
SC
Deaths (%)
42 ± 7 0.52
444
†
445
PVR, pulmonary venous return; CPB, cardiopulmonary bypass; DHCA, deep hypothermic
446
circulatory arrest; MBTS, modified Blalock-Taussig shunt; RVPAS, Right ventricle to pulmonary
447
artery shunt; LOS, Length of stay; ICU, intensive care unit; IQR, interquartile range; RVEF, Right
448
ventricular ejection fraction.
AC C
EP
TE D
M AN U
p values were calculated using either Fisher exact test or Student t test.
28
ACCEPTED MANUSCRIPT
Table 3. Incidence rate of Norwood complications by ADR genotype. Incidence rate of Norwood complications (Number/subject/day of hospitalization) [95% CI] AA GA/GG 0.09 [0.08, 0.10] 0.08 [0.07, 0.09]
ADRB1 (rs1801253)
CC 0.07 [0.06, 0.09]
CG/GG 0.09 [0.08, 0.1]
ADRB2 (rs1042714)
CC 0.09 [0.08, 0.09]
CG/GG 0.08 [0.07, 0.10]
ADRA2A (rs553668)
CC 0.08 [0.07, 0.10]
CT/TT 0.08 [0.07, 0.10]
0.38
0.07
0.70
0.93
M AN U
450
P-value
RI PT
Gene (SNP ID) ADRB1 (rs1801252)
SC
449
451
1
452
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP
453
ADR, adrenergic receptor; SAE, serious adverse event; CI, confidence interval
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
AC C
EP
TE D
454
29
ACCEPTED MANUSCRIPT
Table 4. Incidence rate ratio of Norwood complications by ADR genotype P-value†
AA1 vs. GA/GG
Incidence rate ratio [ 95% CI] of Norwood complications 1.004 [0.774, 1.302]
ADRB1 (rs1801253)
CC1 vs. CG/GG
0.969 [0.779, 1.206]
0.78
ADRB2 (rs1042714)
CC1 vs. CG/GG
0.814 [0.638, 1.037]
0.10
ADRA2A (rs553668)
CC1 vs. CT/TT
1.077 [0.875, 1.367]
0.55
Gene (SNP ID) ADRB1 (rs1801252)
Genotype
0.98
RI PT
455
SC
456 †
Poisson regression adjusted for covariates of interest and exposure time.
458
1
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
459
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP
460
SAE, serious adverse event; CI, confidence interval
AC C
EP
TE D
M AN U
457
30
ACCEPTED MANUSCRIPT
461
Table 5. Frequency of post-Norwood complications by systems
Post-Norwood complications by system
239 (23.3%)
Gastro-intestinal
100 (9.8%)
Hematologic
84 (8.2%)
Infectious disease
233 (22.7%)
SC
RI PT
Cardiac
Neurological
41 (4.0%)
35 (3.4%)
M AN U
Renal Respiratory
249 (24.3%)
Vascular
5 (0.5%) 39 (3.8%)
AC C
EP
TE D
Other
462
N=1025
31
ACCEPTED MANUSCRIPT
463
Figure 1. Consort diagram
Not analyzed (Incomplete genotype data) (n=85)
TE D
Analyzed (Complete genotype data) (n=351)
464
Serious adverse events (n=28; 33%)
AC C
EP
Serious adverse events (n=149; 42%)
DNA unavailable for genetic study (n=119)
M AN U
DNA available for genetic study (n=436)
SC
RI PT
Randomized to SVR trial (n=555)
32
ACCEPTED MANUSCRIPT
Figure 2. Freedom from serious adverse events post Norwood by ADRA2A genotype
466
AC C
EP
TE D
M AN U
SC
RI PT
465
33
ACCEPTED MANUSCRIPT
467
Figure 3. Incidence rate ratios of post-Norwood complications by ADR genotypes
M AN U
SC
RI PT
468
469
AC C
EP
TE D
470
34
ACCEPTED MANUSCRIPT
Table 1. Adrenergic receptor variant frequencies Nucleotide
Amino acid
Function by
SNP
HWE
ID)
substitution
substitution
genotype
carrier
p
frequency
value
32%
0.717
ADRB1
231 A/G
Ser49Gly
(rs1801252)
(missense)
GG: Higher basal adenylate cyclase
SC
activity and lower
RI PT
Gene (SNP
isoproterenol
1165 C/G
Gly389Arg
(rs1801253)
(missense)
ADRB2
5318 C/G
(rs1042714)
(missense)
Gln27Glu
EP AC C
CC: Increases β1
44%
0.818
62%
0.972
36%
0.918
receptor sensitivity
TE D
ADRB1
M AN U
sensitivity
GluGlu: Enhances response to β adrenergic receptor antagonist
ADRA2A
2790 C/T (3'
CC: Increased
(rs553668)
UTR)
norepinephrine release
ADR, adrenergic receptor; HWE, Hardy Weinberg equilibrium; SNP, single nucleotide polymorphism
ACCEPTED MANUSCRIPT
Table 2. Patient characteristics and outcomes Characteristics
Complete
N
Incomplete p†
N
Gender, Male (%)
351
220 (62.7%)
85
Race, White (%)
351
288 (82.1%)
85
Birth weight (kg)
351
3.13 ± 0.53
85
Gestational age (weeks)
344
38 ± 1
83
RI PT
genotyping
Identifiable syndrome (%)
150
22 (14.7%)
19
3 (15.8%) 1.00
Aortic atresia (%)
351
215 (61.3%)
85
50 (58.8%) 0.71
Obstructed PVR (%)
351
10 (2.8%)
85
1 (1.2%) 0.70
Age at Norwood (days)
351
6± 4
85
6 ± 4 0.12
Age at stage II palliation (days)
276
162 ± 53
75
164 ± 77 0.78
Total CPB time (mins)
351
139 ± 51
85
140 ± 43 0.86
324
33 ± 21
76
31 ± 18 0.27
351
170 (48.4%)
85
44 (51.8%) 0.63
351
183 (52.1%)
85
43 (50.6%) 0.81
AC C
genotyping
Median hospital LOS (days)
351
24 (15-42)
85
22 (15-32) 0.35
Median ICU LOS (days)
255
15 (9-33)
52
13 (9-24) 0.65
Patients with complications (%)
351
278 (79.2%)
85
63 (74.1%) 0.31
Number of complications per patient
351
3±3
85
2 ± 3 0.25
Pre-stage II RVEF (%)
195
44 ± 9
56
43 ± 7 0.47
MBTS (%)
EP
RVPAS (%) Norwood course
14 months follow-up
68 (80.0%) 0.44
3.13 ± 0.53 0.96 38 ± 2 0.38
SC
M AN U
TE D
DHCA time (mins)
56 (65.9%) 0.62
ACCEPTED MANUSCRIPT
351
84 (23.9%)
85
Norwood hospitalization (%)
84
36 (42.9%)
11
4 (36.3%)
After Norwood discharge (%)
84
48 (57.1%)
11
7 (63.6%)
351
10 (2.8%)
85
Norwood hospitalization (%)
10
6 (60%)
2
After Norwood discharge (%)
10
4 (40%)
2
Serious adverse events (%)
351
152 (43.3%)
85
14 months RVEF (%)
168
43 ± 8
48
1 (50%) 1 (50%)
29 (34.1%) 0.14 42 ± 7 0.52
p values were calculated using either Fisher exact test or Student t test.
M AN U
†
2 (2.4%) 1.00
RI PT
Transplants (%)
11 (12.9%) 0.028
SC
Deaths (%)
PVR, pulmonary venous return; CPB, cardiopulmonary bypass; DHCA, deep hypothermic circulatory arrest; MBTS, modified Blalock-Taussig shunt; RVPAS, Right ventricle to pulmonary artery shunt; LOS, Length of stay; ICU, intensive care unit; IQR, interquartile range;
AC C
EP
TE D
RVEF, Right ventricular ejection fraction.
ACCEPTED MANUSCRIPT
Table 3. Frequency of SAEs during the first 14 months of follow-up and incidence rate of Norwood complications by ADR genotype. Proportion of subjects
P-value
Incidence rate of Norwood
(SNP ID)
with at least one SAE in
complications
the first 14 months
(Number/subject/day of
P-value
RI PT
Gene
hospitalization) [95% CI] GA/GG
ADRB1
CC1
CG/GG
(rs1801253) 85/195 (44%)
ADRB2
CC1
67/156 (43%)
CG/GG
(rs1042714) 58/133 (43%)
ADRA2A
CC1
(rs553668)
107/226 (47%)
0.36
0.91
94/218 (43%)
1.00
EP
CT/TT
45/125 (36.0%)
0.04
GA/GG
0.09 [0.08, 0.10]
0.08 [0.07, 0.09]
CC
CG/GG
0.07 [0.06, 0.09]
0.09 [0.08, 0.1]
CC
CG/GG
0.09 [0.08, 0.09]
0.08 [0.07, 0.10]
CC
CT/TT
0.08 [0.07, 0.10]
0.08 [0.07, 0.10]
AC C
1
44/111 (40%)
M AN U
(rs1801252) 108/240 (45%)
AA
SC
AA1
TE D
ADRB1
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP ADR, adrenergic receptor; SAE, serious adverse event; CI, confidence interval
0.38
0.07
0.70
0.93
ACCEPTED MANUSCRIPT
Table 4. Comparison of occurrence of at least one SAE and incidence rate of Norwood complications during 14-month follow-up by ADR genotype Genotype
(SNP ID)
Incidence rate ratio
Odds ratio [95%
P-
CI] of SAE in first
value† [ 95% CI] of
Pvalue††
RI PT
Gene
Norwood
14 months
complications
GA/GG
ADRB1
CC1 vs.
(rs1801253)
CG/GG
ADRB2
CC1 vs.
(rs1042714)
CG/GG
ADRA2A
CC1 vs.
(rs553668)
CT/TT
†
0.62
1.004 [0.774, 1.302]
0.98
0.564 [0.205, 1.555]
0.27
0.969 [0.779, 1.206]
0.78
1.036 [0.346, 3.105]
0.95
0.814 [0.638, 1.037]
0.10
1.785 [1.040, 3.063]
0.036
1.077 [0.875, 1.367]
0.55
SC
(rs1801252)
0.732 [0.215, 2.498]
M AN U
AA1 vs.
TE D
ADRB1
Logistic regression adjusted for covariates of interest. All subjects who did not experience an
††
Poisson regression adjusted for covariates of interest and exposure time.
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
AC C
1
EP
SAE were followed for at least 14 months.
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP SAE, serious adverse event; CI, confidence interval
ACCEPTED MANUSCRIPT
Table 5. Frequency of post-Norwood complications by systems
Post-Norwood complications by system
N=1025 239 (23.3%)
Gastro-intestinal
100 (9.8%)
Hematologic
84 (8.2%)
Infectious disease
233 (22.7%)
SC
RI PT
Cardiac
Neurological
41 (4.0%)
35 (3.4%)
M AN U
Renal Respiratory
249 (24.3%)
Vascular
5 (0.5%) 39 (3.8%)
AC C
EP
TE D
Other
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0022-5223(17)31360-0
DOI:
10.1016/j.jtcvs.2017.06.041
Reference:
YMTC 11717
To appear in:
The Journal of Thoracic and Cardiovascular Surgery
Received Date: 4 August 2016 Revised Date:
6 June 2017
Accepted Date: 20 June 2017
Please cite this article as: Ramroop R, Manase G, Lu D, Manase D, Chen S, Kim R, Lee T, Mahle WT, McHugh K, Mitchell M, Tristani-Firouzi M, Wechsler SB, Wilder NS, Zak V, Lafreniere-Roula M, Newburger JW, Gaynor JW, Russell MW, Mital S, Adrenergic Receptor Genotypes Influence Postoperative Outcomes in Infants in the Single Ventricle Reconstruction Trial, The Journal of Thoracic and Cardiovascular Surgery (2017), doi: 10.1016/j.jtcvs.2017.06.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Adrenergic Receptor Genotypes Influence Post-operative Outcomes in Infants in the Single
2
Ventricle Reconstruction Trial
3
Short title: Ramroop: Adrenergic genotypes and Norwood outcomes
RI PT
4
Ronand Ramroop1, MD, George Manase1, BSc, Danny Lu1, MSc, Dorin Manase1, BSc, Shan
6
Chen2, MSc, Richard Kim3, MD, Teresa Lee4, MD, William T Mahle5, MD, Kimberly McHugh6,
7
MD, Mike Mitchell7, MD, Martin Tristani-Firouzi8, MD, Stephanie B Wechsler9, MD, Nicole S
8
Wilder10, MD, Victor Zak2, PhD, Myriam Lafreniere-Roula11, PhD, Jane W Newburger12, MD, J
9
William Gaynor13, MD, Mark W Russell10, MD, Seema Mital1, MD
M AN U
SC
5
10 1
Hospital for Sick Children, University of Toronto, Toronto, ON
12
2
New England Research Institute, Watertown, MA
13
3
Children’s Hospital of Los Angeles, Los Angeles, CA
14
4
Columbia University Medical Center, New York, NY
15
5
Emory University, Atlanta, GA
16
6
Medical University of South Carolina, Charleston, SC
17
7
Children’s Hospital of Wisconsin, Milwaukee, WI
18
8
University of Utah School of Medicine, Salt Lake City, UT
19
9
Duke University Medical Center, Durham, NC
20
10
21
11
22
12
23
13
AC C
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University of Michigan Health, Ann Arbor, MI
Cardiovascular Data Management Centre, Hospital for Sick Children, Toronto, ON Boston Children’s Hospital, Boston, MA Children’s Hospital of Philadelphia, Philadelphia, PA
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Conflict of Interest statement:
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The authors have no conflicts of interest.
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Sources of Funding:
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Supported by grants HL068269, HL068270, HL068279, HL068281, HL068285, HL068288,
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HL068290, HL068292, HL109778, and HL085057 from the National Heart, Lung, and Blood
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Institute.
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Corresponding author:
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Seema Mital, M.D
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Hospital for Sick Children
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555 University Avenue
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Toronto, Ontario M5G 1X8, Canada
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Phone: 416-813-7418
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Fax: 416-813-5857
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Email: [email protected]
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Clinical Trial Registry Number: NCT00115934
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Numbers and Dates of IRB Approvals*
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Hospital for Sick Children, 1000006285, 02/11/2005
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Children’s Hospital of Los Angeles, CCI-05-00038, 9/5/2008
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Columbia University Medical Center, AAAB0870, 1/12/2005
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Emory University, IRB00009510, 11/10/2005
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Medical University of South Carolina, HR18084, 5/6/2008
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Children’s Hospital of Wisconsin, CHW 05/29, 4/21/2005
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University of Utah School of Medicine, 13354, 2/5/2005
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Duke University Medical Center, Pro00004594, 4/5/2003
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University of Michigan Health, 41816, 9/17/2010
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Boston Children’s Hospital, 04-12-162, 12/13/2004
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Children’s Hospital of Philadelphia, IRB 05-004136, 1/5/2004
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SVR Biorepository at University of Michigan Health, HUM00031665, 9/3/2010
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*These are original approval dates of trial participating sites, and of the SVR biorepository
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located at University of Michigan. All IRB approvals are active.
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Glossary of Abbreviations
ADR- Adrenergic receptor
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CI- confidence interval
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ECMO- Extra-corporeal membrane oxygenation
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HLHS- Hypoplastic left heart syndrome
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HR- Hazard ratio
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LV- left ventricle
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MBTS- modified Blalock- Taussig shunt
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OR- Odds ratio
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PHN- Pediatric Heart Network
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RAAS- Renin- angiotensin- aldosterone system
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RV- Right ventricle
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RVEF- right ventricular ejection fraction
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RVPAS- Right ventricle to pulmonary artery shunt
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SVR trial- Single Ventricle Reconstruction Trial
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ABSTRACT
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Objectives: Adrenergic receptor (ADR) genotypes have been associated with adverse outcomes
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in heart failure. Our objective was to evaluate the association of ADR genotypes with post-
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Norwood outcomes in infants with hypoplastic left heart syndrome (HLHS).
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Methods: Infants with HLHS participating in the Pediatric Heart Network Single Ventricle
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Reconstruction Trial underwent genotyping for four single nucleotide polymorphisms in three
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ADR genes: ADRB1_231A/G, ADRB1_1165G/C, ADRB2_5318C/G and ADRA2A_2790C/T. The
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association of genotype with freedom from serious adverse events (SAE) (death, transplant, extra-
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corporeal membrane oxygenation, cardiopulmonary resuscitation, acute shunt failure, unplanned
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re-operations, or necrotizing enterocolitis) during 14 months follow-up was assessed using Cox
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regression and the association with post-Norwood complications was assessed using Poisson
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regression. Models were adjusted for clinical and surgical factors.
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Results: The study included 351 eligible patients (62% male; 83% White). The mean age at
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Norwood procedure was 5.6±3.6 days. 152 patients had SAEs during 14-month follow-up
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including 84 deaths and 10 transplants. ADRA2A_2790CC genotype had lower SAE-free survival
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compared to CT/TT genotypes during follow-up (Log rank test, p=0.02) and this association was
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independent of clinical and surgical risk factors (Adjusted Cox regression. HR 1.54 [1.04, 2.30]
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p=0.033). Post-Norwood complication rate did not differ by genotype.
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Conclusions: Infants with HLHS harboring ADR genotypes that are associated with higher
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catecholamine release or sensitivity had lower event-free survival after staged palliation. Excess
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catecholamine activation may adversely affect cardiovascular adaptation after the Norwood
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procedure. Future studies should explore if targeting adrenergic activation in those harboring risk
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genotypes can improve outcomes. (ClinicalTrials.gov number, NCT00115934)
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Central Message
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Variations in adrenergic receptor genes influence postoperative outcomes in infants with
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hypoplastic left heart syndrome undergoing staged palliation.
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Central Picture
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Kaplan Meier plot: Lower serious adverse event-free survival with ADRA2A_7790CC genotype
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Perspective Statement
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Staged palliation for single ventricle lesions is associated with significant morbidity and mortality.
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Patients with adrenergic receptor genotypes associated with augmented catecholamine signaling
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had lower freedom from serious adverse events after Stage 1 palliation. Future studies should
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explore if targeting adrenergic activation in those harboring risk genotypes can improve outcomes.
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Single ventricle lesions comprise over 7% of all congenital heart defects with hypoplastic left
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heart syndrome (HLHS) being the most common lesion.1, 2 In HLHS, a single right ventricle (RV)
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acts as a systemic ventricle while the left ventricle (LV) is hypoplastic or rudimentary. Patients
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with HLHS require multiple palliative surgeries to separate the pulmonary and systemic
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circulations with the first staged palliation i.e. the Norwood procedure performed in the newborn
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period. Maintenance of systemic cardiac output after the Norwood procedure requires a balance
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between the systemic and pulmonary circulations and adequate RV function. Abnormal systemic
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and pulmonary vascular resistance and ventricular dysfunction are major contributors to
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maladaptation to a single ventricle physiology. In one analysis, up to 40% of patients with staged
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palliation were noted to have some degree of heart failure.3
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Single ventricle patients have fragile hemodynamics and are especially vulnerable after cardiac
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surgery. The post-operative physiological stress response involves a surge in endogenous
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catecholamine production which can have profound effects on cardiac function and vascular tone
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mediated by adrenergic receptors. Adrenergic receptors include presynaptic α2a and α2c receptors
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that inhibit norepinephrine release, cardiac β1 receptors that mediate chronotropic and inotropic
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response to norepinephrine, and β2 receptors that mediate vascular smooth muscle relaxation and
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response to β-blockers.4 Adrenergic upregulation is an important mechanism of cardiovascular
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adaptation during stress particularly in the infant where the circulation is highly catecholamine-
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dependent.5, 6 While acute upregulation is compensatory, chronic upregulation can have
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detrimental effects by increasing systemic and pulmonary vascular resistance, and increasing
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myocardial oxygen demands, resulting in adverse cardiac remodeling and ventricular
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dysfunction.4, 7-12 In children with dilated cardiomyopathy, α2 and β1 upregulation and β2
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downregulation genotypes were associated with worse ventricular function, heart failure
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progression and acute hemodynamic decompensation.13 The impact of adrenergic receptor
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genotypes on adaptation to hemodynamic and surgical stress in infants with HLHS undergoing
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staged palliation is not known. We hypothesized that genetic modulation of receptor activity
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would alter catecholamine response and thereby influence post-operative clinical course. The
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purpose of our study was to evaluate if genetic variants that upregulate adrenergic receptor
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activity adversely impact post-Norwood outcomes in infants with HLHS enrolled in the single
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ventricle reconstruction (SVR) trial.
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METHODS
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Study population
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This is an ancillary study that derives from the Pediatric Heart Network (PHN) SVR Trial, in
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which infants with HLHS (n=555) were enrolled from 15 North American centers between May
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2005 and July 2008. The details of the study design, inclusion criteria, study assessments as well
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as trial results have been previously published.14, 15 In brief, inclusion criteria for the SVR trial
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included a diagnosis of HLHS or a related single morphologic RV anomaly, planned Norwood
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procedure, and absence of a genetic or medical condition that would affect transplant-free
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survival. Infants were randomized to receive a modified Blalock-Taussig shunt (MBTS) versus a
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right ventricle – pulmonary artery shunt (RVPAS) as part of their Norwood procedure and were
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followed for 14 months with serial assessment. The study was approved by the local institutional
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review boards and informed consent for study participation was obtained from the parents or legal
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guardians. Data collection included patient demographics and baseline characteristics, operative
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variables, hospitalization course, clinical outcomes, and echocardiographic assessment of RV
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ejection fraction (RVEF). All echocardiograms were reviewed centrally by an independent
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observer at the echocardiography core laboratory.14, 15
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Genotyping
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Buccal swab epithelial cells were collected at enrollment or during Norwood hospitalization using
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CytoSoft cytology brushes (Medical Packaging Corporation, Camarillo, CA) after obtaining
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informed consent. Methods have been reported previously.16 Genomic DNA was extracted using a
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PureGene kit (Gentra Systems, Inc, Minneapolis, Minn) according to the manufacturer’s protocol.
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Samples were retrieved from the PHN Biorepository for genotyping. Participants in the SVR trial
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who did not consent to future testing of their samples were not included in the ancillary study. The
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ancillary study was approved by the PHN Ancillary Study Review Committee and by the
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University of Michigan Institutional Review Board.
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Adrenergic receptor single nucleotide polymorphisms (SNPs) were selected on the basis of
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previous association studies, functional effects, and population allele frequencies.13, 17,24
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Genotyping (Sequenom SNP) was performed for four SNPs in three adrenergic receptor (ADR)
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genes: a A/G missense variant at position 231 in ADRB1 resulting in a serine to glycine
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substitution (rs1801252), a C/G missense variant at position 1165 in ADRB1 resulting in a glycine
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to arginine substitution (rs1801253), a C/G missense variant at position 5318 in ADRB2 resulting
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in a glutamine to glutamic acid substitution (rs1042714) and a C/T 3’ untranslated region (UTR)
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variant at position 2790 in ADR2A (rs553668). Patients carrying a single copy of the variant
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allele or SNP (also known as minor allele) at the genetic locus were defined as having a
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heterozygous genotype, those with two copies of the variant allele as having a homozygous
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genotype, and those without a variant allele i.e. carrying only the normal allele (also known as
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major allele) were defined as having a wild-type genotype.
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Since variants in the renin-angiotensin-aldosterone signaling (RAAS) pathway genes have been
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previously associated with adverse ventricular remodeling in infants with single ventricle lesions,
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genotyping was also performed for four SNPs and one insertion-deletion variant in five RAAS
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genes. The following were considered RAAS-upregulation genotypes based on our previous
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work: AGT_CC, ACE_DD, AGTR1_CC, CYP11B2_CC, and CMA1_AA. The CYP11B2 and
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CMA1 variants were genotyped using Sequenom SNP genotyping and the AGTR1, AGT and the
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ACE insertion/deletion polymorphisms were genotyped using TaqMan primers and probes. For
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samples and SNPs which did not yield a valid result by Sequenom or TaqMan analysis, PCR
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amplification followed by Sanger sequencing was performed. The detailed genotyping methods,
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primers and probes for the Sequenom SNP analysis, the Taqman analysis and for the PCR
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amplification or Sanger sequencing are listed in Supplemental Methods and Supplemental
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Table 1.
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Statistical Analysis
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Allele and genotype frequencies were determined to calculate Hardy-Weinberg equilibrium. All
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demographic and clinical data were expressed as frequencies, mean with standard deviations, or
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medians with interquartile ranges (IQR). The primary outcome was a composite of any serious
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adverse event (SAE) during a 14-month follow-up after the Norwood procedure and included
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death, transplant, need for post-operative extra-corporeal membrane oxygenation (ECMO), post-
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operative cardiopulmonary resuscitation (CPR), acute shunt failure, unplanned re-operations or
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necrotizing enterocolitis.25, 26 Secondary outcomes included frequency of post-Norwood
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complications up to hospital discharge from stage 2 surgery, and RVEF during 14 months follow-
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up. Complications were defined as adverse events using the common terminology criteria for
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adverse events version 3.0 developed by the National Cancer Institute, NIH, but that did not meet
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criteria for SAEs.26 Since post-Norwood hospital and intensive care unit (ICU) length of stay were
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highly correlated with each other and with Norwood complication rate when analyzed using
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bootstrap Spearman rank correlation (data not shown), these outcomes were not analyzed
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separately.
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ADR genotype associations were analyzed by comparing outcomes in homozygous or
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heterozygous SNP carriers versus wild-types using a dominant model. Freedom from the primary
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outcome was described using the Kaplan-Meier method with stratification by genotype. The log-
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rank test was used to assess between-stratum differences. Cox proportional hazard models
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adjusted for covariates of interest were used to test the association of the genotypes with the
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primary outcome. The proportional hazards assumption was verified using cumulative Martingale
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process plots. Associations were quantified by hazard ratios (HRs) and reported along with their
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95% confidence intervals (CIs). P-values were calculated based on Wald’s statistics. Poisson
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regression adjusted for over-dispersion and for exposure time (number of days of hospitalization
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for Norwood procedure) was used to model the association of risk genotype with frequency of
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post-Norwood complications and results were presented as incidence rate ratios (IRR) with 95%
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confidence intervals. A mixed model was used to assess for association of genotype with RVEF
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during 14 months follow-up. All analyses were adjusted for gender, race, birth weight, gestational
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age, presence of aortic atresia or obstructed pulmonary venous return, presence of a genetic
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syndrome, age at Norwood procedure, total Norwood cardiopulmonary bypass time, deep
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hypothermic circulatory arrest time, type of shunt, and presence of a RAAS-upregulation
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genotype.27 The covariates selected were not found to exhibit significant collinearity. In
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regression models, when patients had missing data on the presence of identifiable syndromes, it
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was assumed that identifiable syndromes were not present. Observations with missing data in
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other variables were excluded from the models and this represented a very small percentage of the
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total cohort. All statistical analyses were performed using SAS v9.4.
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RESULTS
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Of 555 patients in the SVR trial, 436 provided DNA and 351 had complete genotype data (i.e.
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genotyping results for all 9 SNPs) and were included in the analysis (Figure 1). ADR genotype
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frequencies are shown in Table 1. All genotypes were in Hardy Weinberg equilibrium. Patient
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clinical characteristics and outcomes in the group with complete versus incomplete genotypes are
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shown in Table 2. The only significant difference was a higher incidence of death in the
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completely genotyped cohort compared to the incompletely genotyped cohort. Incomplete
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genotyping was the result of technical issues such as low quality or quantity of DNA. The results
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hereby focus on the 351 subjects with complete genotype data.
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We evaluated the association of ADR genotypes with the primary outcome of composite SAEs.
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Sixty-four percent patients had the ADRA2A_2790CC genotype (wild-type). Kaplan-Meier
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analysis showed that patients with the CC genotype had lower SAE-free survival during 14 month
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follow-up compared to CT/TT genotypes (log rank test: p=0.02) (Figure 2). This association was
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independent of RAAS genotype, clinical and surgical risk factors (Adjusted Cox regression: HR
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1.549 [1.04, 2.30] p=0.033). There was no synergistic adverse effect of multiple risk genotypes on
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outcomes.
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Tables 3 and 4 describe the incidence rate of Norwood complications by genotype groups.
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Overall, 79 percent patients had a post-Norwood complication with a median [95% CI] of 2 [1-4]
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complications per patient (Table 2). Complications by systems are shown in Table 5. The list of
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complications included for this trial have been previously published.26 Cardiac complications
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included arrhythmias, pericardial effusion, hypotension or hypertension, RV dysfunction, valvar
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insufficiency, and other. We evaluated the association of ADR genotypes with the rate of
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complications after the Norwood procedure using Poisson regression. All regression models were
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adjusted for demographic, clinical and surgical risk factors as well as for RAAS genotypes and the
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Poisson regression was adjusted for exposure time. There were no significant differences in
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incidence rates of Norwood complications by genotype (Figure 3). ADR genotypes were not
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associated with significant difference in RV function during 14 month follow-up (data not shown)
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as assessed by mixed effects models adjusted for clinical and surgical factors.
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DISCUSSION
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In an era of precision medicine, there is a growing recognition of the importance of identifying not
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just clinical predictors but also genetic predictors of outcomes that can be used to individualize
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care and improve the safety and efficacy of medical and surgical interventions based on the
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unique genome and phenome of a patient. Previous studies have identified genetic variants that
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increase susceptibility to neurodevelopmental outcomes and adverse ventricular remodeling in
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single ventricle patients undergoing staged palliation as well as in patients with other types of
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congenital heart disease undergoing surgical repair.16, 28, 29, 30, 31 In this study, we evaluated the
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association of genetic variants that increase ADR signaling with post-Norwood outcomes in
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infants with HLHS. Using genetic material collected during the trial, we demonstrated that the
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ADR genotype is an important predictor of outcomes of stage 1 palliation in infants with HLHS
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with lower serious adverse event-free survival in patients harboring ADR risk genotypes.
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Although further study is required, these findings may have implications for individualizing peri-
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operative management in this cohort based on genotype.
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We evaluated two SNPs in the ADRB1 gene and one in the ADRB2 gene. The ADRB1_389GG
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genotype has been previously associated with pediatric and adult heart failure and the
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ADRB1_231AA genotype is known to be associated with lower basal adenylate cyclase activity
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but increased sensitivity of the β1 receptor to norepinephrine with enhanced sympathetic blood
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pressure response.17, 18 Neither ADRB1 genotype nor the ADRB2 genotype were associated with
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post-operative complications or SAEs in our study.10
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The ADR2A receptor is important in vascular tone as well as metabolic responses like insulin
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release from pancreatic cells and adipocyte metabolism in humans. Genetic variations lead to
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alterations in G-protein coupling and in agonist-promoted receptor phosphorylation and
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desensitization thereby modulating response to catecholamines.12 The ADRA2A_2790CC
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genotype (wild-type) has been reported to cause reduced feedback inhibition of norepinephrine
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and failure to inhibit sympathetic tone. While we did not measure sympathetic tone, we found that
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patients with the CC genotype had lower freedom from SAEs during 14 month follow-up
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compared to CT/TT genotypes. One explanation for this finding is that a milieu of high circulating
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catecholamines in patients with the CC genotype may have resulted in increased vasomotor tone
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and an adverse effect on systemic output and organ perfusion, thereby increasing the risk of post-
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Norwood SAEs. The event-free survival curves diverged relatively sharply after the early post-
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operative period, at around 15 days (Figure 1). This suggests that while early events were more
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likely secondary to surgical complications which were similar in both genotype groups, later
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events were more likely influenced by genetic differences in adrenergic system-mediated
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adaptation to the Norwood circulation as well as recovery from complications. Overall, our
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findings suggest that genotypes associated with increased adrenergic neurohormonal activation
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and responsiveness may have a detrimental effect in the post-Norwood circulation.
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A limitation of our study is that only a small number of candidate SNPs were analyzed. The DNA
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in the trial was acquired via buccal swabs which required PCR amplification and did not yield
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sufficient quantity or quality of DNA for a more unsupervised approach using whole exome
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sequencing. Nonetheless, the SNPs were carefully selected based on prior associations with
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outcomes in other pediatric cardiac studies. Also, RV myocardial samples and detailed
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hemodynamic data were not collected during the Norwood procedure for the trial precluding
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assessment of tissue adrenergic receptor expression, and of potential influence of the genotypes on
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metabolic and hemodynamic function. Additionally, we were unable to evaluate the
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pharmacogenetic influences of the ADR genotypes with β-blocker response since less than 5%
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patients were receiving β-blocker therapy during follow-up. Also, the sample size of this study
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was reduced due to insufficient DNA or incomplete genotyping in the trial cohort. Nonetheless,
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the baseline characteristics of the genotyped and non-genotyped cohorts were similar excluding a
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survival bias in our study cohort.
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In summary, ADR genotypes that enhance catecholamine levels and/or sensitivity increase the
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risk of serious adverse events in patients who survive beyond the first two weeks after the
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Norwood procedure. Further studies are needed to delineate the biological and hemodynamic
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effects of these genotypes and to validate these findings in additional cohorts. Pharmacologic
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modification of adrenergic receptors has been shown to positively influence outcome in other
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heart failure cohorts. 21,22, 23 While there is no evidence to support empiric β-blocker use in HLHS
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patients, studies are needed to determine if pre-operative identification of these genetic subtypes
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can be used to target β-blockers and/or other forms of more aggressive systemic vasodilation post-
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Norwood to the subset with these risk genotypes.
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ACKNOWLEDGMENTS
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See Appendix (Supplementary Material) for a complete list of the Pediatric Heart Network
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Investigators.
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fat cell function. J Lipid Res. 1993;34:1057-1091.
391
21.
392
Adrenergic receptor genotype influences heart failure severity and β-blocker response in children
393
with dilated cardiomyopathy. Pediatric Research. 2015;77(2):363–369.
394
22.
395
adrenergic receptor genotype influences the effect of nonselective vs. selective beta-blockade on
396
baroreflex function in chronic heart failure. International Journal of Cardiology.
397
2011;153(2):230–232.
398
23.
399
impact of beta-adrenoreceptor gene polymorphisms on survival in patients with congestive heart
400
failure. Eur J Heart Fail. 2005;7(6):966–973.
RI PT
Mason DA, Moore JD, Green SA, Liggett SB. A gain-of-function polymorphism in a g-
M AN U
SC
Gilsbach R, Schneider J, Lother A, et al. Sympathetic α2-adrenoceptors prevent cardiac
Lafontan M, Berlan M. Fat cell adrenergic receptors and the control of white and brown
TE D
Reddy S, Fung A, Manlhiot C, Tierney ES, Chung WK, Blume E et al. (2015).
AC C
EP
Truijen J, de Peuter OR, Kim Y, Bogaard B, Wouter EK, Kamphuisen PW et al. Beta2-
de Groote P, Lamblin N, Helbecque N, Mouquet F, Mc Fadden E, Hermant, X et al. The
22
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401
24.
Sawczuk M, Maciehewska-Karlowska A, Cieszczyk P. A single nucleotide polymorphism
402
rs553668 in the adra2a gene and the status of polish elite endurance athletes. Trends in Sport
403
Sciences. 2013;1:30-35.
404
25.
405
of death in the Single Ventricle Reconstruction trial. Journal of thoracic and Cardiovascular
406
Surgery. 2012:144(4):907-914
407
26.
408
surgical trial for complex congenital heart disease: The Pediatric Heart Network experience.
409
Journal of Thoracic and Cardiovascular Surgery. 2011;142:531-7.
410
27.
411
hospital morbidity and mortality after the Norwood procedure: A report from the Pediatric Heart
412
Network Single Ventricle Reconstruction trial. Journal of Thoracic and Cardiovascular Surgery.
413
2012;144:882-995
414
28.
415
angiotensin-aldosterone genotype influences ventricular remodeling in infants with single
416
ventricle. Circulation. 2011;123:2353-2362.
417
29.
418
polymorphisms influence progression of pediatric hypertrophic cardiomyopathy. Hum Genet.
419
2007;122:515-523.
420
30.
421
Patient genotypes impact survival after surgery for isolated congenital heart disease. The Annals
422
of Thoracic Surgery. 2014;98:104-111.
RI PT
Ohye R, Schonbeck J, Eghtesady P, Laussen P, Pizarro C et al. Cause, timing, and location
M AN U
SC
Virzi L, Pemberton V, Ohye R, Tabbutt S, Lu M et al. Reporting adverse events in a
TE D
Tabbutt S, Ghanayem N, Ravishankar C, Sleeper L, Cooper D et al. Risk factors for
EP
Mital S, Chung WK, Colan SD, Sleeper LA, Manlhiot C, Arrington CB et al. Renin-
AC C
Kaufman B, Auerbach S, Reddy S, Manlhiot C, Deng L, Prakash A et al. Raas gene
Kim DS, Kim JH, Burt AA, Crosslin DR, Burnham N, McDonald-McGinn DM et al.
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Jeewa A, Manickaraj AK, Mertens L, Manlhiot C, Kinnear C, Mondal T et al. Genetic
424
determinants of right-ventricular remodeling after Tetralogy of Fallot repair. Pediatr Res.
425
2012;72:407-413.
AC C
EP
TE D
M AN U
SC
RI PT
423
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426
FIGURE LEGENDS
427
Figure 1: A consort flow diagram showing the outcomes in the complete and incompletely
429
genotyped groups from patients randomized in the single ventricle reconstruction (SVR) trial.
RI PT
428
430
Figure 2: Kaplan Meier survival curve showing survival free from composite primary outcome
432
stratified by ADRA2A_2790 genotype. The shaded bands represent 95% confidence intervals.
433
Subjects with ADRA2A_2790CC genotype had lower survival free from serious adverse events
434
(SAE) compared to subjects with CT/TT genotypes (p=0.02).
M AN U
SC
431
435
Figure 3: Forest plot of incidence rate ratios (IRRs) of Norwood complications by genotype. The
437
IRR is the ratio of the expected number of complications per patient per day of Norwood
438
hospitalization in each genotype group. There were no significant differences in the IRRs of
439
Norwood complications by genotype.
AC C
EP
TE D
436
25
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Table 1. Adrenergic receptor variant frequencies Gene (SNP
Nucleotide
Amino acid
Function by
SNP
HWE
ID)
substitution
substitution
genotype
carrier
p
frequency
value
32%
0.717
ADRB1
231 A/G
Ser49Gly
(rs1801252)
(missense)
GG: Higher basal adenylate cyclase
SC
activity and lower
RI PT
440
isoproterenol
1165 C/G
Gly389Arg
(rs1801253)
(missense)
ADRB2
5318 C/G
(rs1042714)
(missense)
Gln27Glu
EP AC C
CC: Increases β1
44%
0.818
62%
0.972
36%
0.918
receptor sensitivity
TE D
ADRB1
M AN U
sensitivity
GluGlu: Enhances response to β adrenergic receptor antagonist
ADRA2A
2790 C/T (3'
CC: Increased
(rs553668)
UTR)
norepinephrine release
441
ADR, adrenergic receptor; HWE, Hardy Weinberg equilibrium; SNP, single nucleotide
442
polymorphism
26
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Table 2. Patient characteristics and outcomes Characteristics
N
Complete
Incomplete p†
N
genotyping
Gender, Male (%)
351
220 (62.7%)
85
Race, White (%)
351
288 (82.1%)
85
Birth weight (kg)
351
3.13 ± 0.53
85
Gestational age (weeks)
344
38 ± 1
83
RI PT
genotyping
Identifiable syndrome (%)
150
22 (14.7%)
19
3 (15.8%) 1.00
Aortic atresia (%)
351
215 (61.3%)
85
50 (58.8%) 0.71
Obstructed PVR (%)
351
10 (2.8%)
85
1 (1.2%) 0.70
Age at Norwood (days)
351
6± 4
85
6 ± 4 0.12
Age at stage II palliation (days)
276
162 ± 53
75
164 ± 77 0.78
Total CPB time (mins)
351
139 ± 51
85
140 ± 43 0.86
324
33 ± 21
76
31 ± 18 0.27
351
170 (48.4%)
85
44 (51.8%) 0.63
351
183 (52.1%)
85
43 (50.6%) 0.81
Median hospital LOS (days)
351
24 (15-42)
85
22 (15-32) 0.35
Median ICU LOS (days)
255
15 (9-33)
52
13 (9-24) 0.65
Patients with complications (%)
351
278 (79.2%)
85
63 (74.1%) 0.31
Number of complications per patient
351
3±3
85
2 ± 3 0.25
Pre-stage II RVEF (%)
195
44 ± 9
56
43 ± 7 0.47
MBTS (%)
EP
RVPAS (%) Norwood course
56 (65.9%) 0.62 68 (80.0%) 0.44
3.13 ± 0.53 0.96 38 ± 2 0.38
SC
M AN U
TE D
DHCA time (mins)
AC C
443
14 months follow-up
27
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351
84 (23.9%)
85
Norwood hospitalization (%)
84
36 (42.9%)
11
4 (36.3%)
After Norwood discharge (%)
84
48 (57.1%)
11
7 (63.6%)
351
10 (2.8%)
85
Norwood hospitalization (%)
10
6 (60%)
2
After Norwood discharge (%)
10
4 (40%)
2
Serious adverse events (%)
351
152 (43.3%)
85
14 months RVEF (%)
168
43 ± 8
48
2 (2.4%) 1.00
RI PT
Transplants (%)
11 (12.9%) 0.028
1 (50%) 1 (50%)
29 (34.1%) 0.14
SC
Deaths (%)
42 ± 7 0.52
444
†
445
PVR, pulmonary venous return; CPB, cardiopulmonary bypass; DHCA, deep hypothermic
446
circulatory arrest; MBTS, modified Blalock-Taussig shunt; RVPAS, Right ventricle to pulmonary
447
artery shunt; LOS, Length of stay; ICU, intensive care unit; IQR, interquartile range; RVEF, Right
448
ventricular ejection fraction.
AC C
EP
TE D
M AN U
p values were calculated using either Fisher exact test or Student t test.
28
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Table 3. Incidence rate of Norwood complications by ADR genotype. Incidence rate of Norwood complications (Number/subject/day of hospitalization) [95% CI] AA GA/GG 0.09 [0.08, 0.10] 0.08 [0.07, 0.09]
ADRB1 (rs1801253)
CC 0.07 [0.06, 0.09]
CG/GG 0.09 [0.08, 0.1]
ADRB2 (rs1042714)
CC 0.09 [0.08, 0.09]
CG/GG 0.08 [0.07, 0.10]
ADRA2A (rs553668)
CC 0.08 [0.07, 0.10]
CT/TT 0.08 [0.07, 0.10]
0.38
0.07
0.70
0.93
M AN U
450
P-value
RI PT
Gene (SNP ID) ADRB1 (rs1801252)
SC
449
451
1
452
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP
453
ADR, adrenergic receptor; SAE, serious adverse event; CI, confidence interval
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
AC C
EP
TE D
454
29
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Table 4. Incidence rate ratio of Norwood complications by ADR genotype P-value†
AA1 vs. GA/GG
Incidence rate ratio [ 95% CI] of Norwood complications 1.004 [0.774, 1.302]
ADRB1 (rs1801253)
CC1 vs. CG/GG
0.969 [0.779, 1.206]
0.78
ADRB2 (rs1042714)
CC1 vs. CG/GG
0.814 [0.638, 1.037]
0.10
ADRA2A (rs553668)
CC1 vs. CT/TT
1.077 [0.875, 1.367]
0.55
Gene (SNP ID) ADRB1 (rs1801252)
Genotype
0.98
RI PT
455
SC
456 †
Poisson regression adjusted for covariates of interest and exposure time.
458
1
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
459
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP
460
SAE, serious adverse event; CI, confidence interval
AC C
EP
TE D
M AN U
457
30
ACCEPTED MANUSCRIPT
461
Table 5. Frequency of post-Norwood complications by systems
Post-Norwood complications by system
239 (23.3%)
Gastro-intestinal
100 (9.8%)
Hematologic
84 (8.2%)
Infectious disease
233 (22.7%)
SC
RI PT
Cardiac
Neurological
41 (4.0%)
35 (3.4%)
M AN U
Renal Respiratory
249 (24.3%)
Vascular
5 (0.5%) 39 (3.8%)
AC C
EP
TE D
Other
462
N=1025
31
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463
Figure 1. Consort diagram
Not analyzed (Incomplete genotype data) (n=85)
TE D
Analyzed (Complete genotype data) (n=351)
464
Serious adverse events (n=28; 33%)
AC C
EP
Serious adverse events (n=149; 42%)
DNA unavailable for genetic study (n=119)
M AN U
DNA available for genetic study (n=436)
SC
RI PT
Randomized to SVR trial (n=555)
32
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Figure 2. Freedom from serious adverse events post Norwood by ADRA2A genotype
466
AC C
EP
TE D
M AN U
SC
RI PT
465
33
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467
Figure 3. Incidence rate ratios of post-Norwood complications by ADR genotypes
M AN U
SC
RI PT
468
469
AC C
EP
TE D
470
34
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Table 1. Adrenergic receptor variant frequencies Nucleotide
Amino acid
Function by
SNP
HWE
ID)
substitution
substitution
genotype
carrier
p
frequency
value
32%
0.717
ADRB1
231 A/G
Ser49Gly
(rs1801252)
(missense)
GG: Higher basal adenylate cyclase
SC
activity and lower
RI PT
Gene (SNP
isoproterenol
1165 C/G
Gly389Arg
(rs1801253)
(missense)
ADRB2
5318 C/G
(rs1042714)
(missense)
Gln27Glu
EP AC C
CC: Increases β1
44%
0.818
62%
0.972
36%
0.918
receptor sensitivity
TE D
ADRB1
M AN U
sensitivity
GluGlu: Enhances response to β adrenergic receptor antagonist
ADRA2A
2790 C/T (3'
CC: Increased
(rs553668)
UTR)
norepinephrine release
ADR, adrenergic receptor; HWE, Hardy Weinberg equilibrium; SNP, single nucleotide polymorphism
ACCEPTED MANUSCRIPT
Table 2. Patient characteristics and outcomes Characteristics
Complete
N
Incomplete p†
N
Gender, Male (%)
351
220 (62.7%)
85
Race, White (%)
351
288 (82.1%)
85
Birth weight (kg)
351
3.13 ± 0.53
85
Gestational age (weeks)
344
38 ± 1
83
RI PT
genotyping
Identifiable syndrome (%)
150
22 (14.7%)
19
3 (15.8%) 1.00
Aortic atresia (%)
351
215 (61.3%)
85
50 (58.8%) 0.71
Obstructed PVR (%)
351
10 (2.8%)
85
1 (1.2%) 0.70
Age at Norwood (days)
351
6± 4
85
6 ± 4 0.12
Age at stage II palliation (days)
276
162 ± 53
75
164 ± 77 0.78
Total CPB time (mins)
351
139 ± 51
85
140 ± 43 0.86
324
33 ± 21
76
31 ± 18 0.27
351
170 (48.4%)
85
44 (51.8%) 0.63
351
183 (52.1%)
85
43 (50.6%) 0.81
AC C
genotyping
Median hospital LOS (days)
351
24 (15-42)
85
22 (15-32) 0.35
Median ICU LOS (days)
255
15 (9-33)
52
13 (9-24) 0.65
Patients with complications (%)
351
278 (79.2%)
85
63 (74.1%) 0.31
Number of complications per patient
351
3±3
85
2 ± 3 0.25
Pre-stage II RVEF (%)
195
44 ± 9
56
43 ± 7 0.47
MBTS (%)
EP
RVPAS (%) Norwood course
14 months follow-up
68 (80.0%) 0.44
3.13 ± 0.53 0.96 38 ± 2 0.38
SC
M AN U
TE D
DHCA time (mins)
56 (65.9%) 0.62
ACCEPTED MANUSCRIPT
351
84 (23.9%)
85
Norwood hospitalization (%)
84
36 (42.9%)
11
4 (36.3%)
After Norwood discharge (%)
84
48 (57.1%)
11
7 (63.6%)
351
10 (2.8%)
85
Norwood hospitalization (%)
10
6 (60%)
2
After Norwood discharge (%)
10
4 (40%)
2
Serious adverse events (%)
351
152 (43.3%)
85
14 months RVEF (%)
168
43 ± 8
48
1 (50%) 1 (50%)
29 (34.1%) 0.14 42 ± 7 0.52
p values were calculated using either Fisher exact test or Student t test.
M AN U
†
2 (2.4%) 1.00
RI PT
Transplants (%)
11 (12.9%) 0.028
SC
Deaths (%)
PVR, pulmonary venous return; CPB, cardiopulmonary bypass; DHCA, deep hypothermic circulatory arrest; MBTS, modified Blalock-Taussig shunt; RVPAS, Right ventricle to pulmonary artery shunt; LOS, Length of stay; ICU, intensive care unit; IQR, interquartile range;
AC C
EP
TE D
RVEF, Right ventricular ejection fraction.
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Table 3. Frequency of SAEs during the first 14 months of follow-up and incidence rate of Norwood complications by ADR genotype. Proportion of subjects
P-value
Incidence rate of Norwood
(SNP ID)
with at least one SAE in
complications
the first 14 months
(Number/subject/day of
P-value
RI PT
Gene
hospitalization) [95% CI] GA/GG
ADRB1
CC1
CG/GG
(rs1801253) 85/195 (44%)
ADRB2
CC1
67/156 (43%)
CG/GG
(rs1042714) 58/133 (43%)
ADRA2A
CC1
(rs553668)
107/226 (47%)
0.36
0.91
94/218 (43%)
1.00
EP
CT/TT
45/125 (36.0%)
0.04
GA/GG
0.09 [0.08, 0.10]
0.08 [0.07, 0.09]
CC
CG/GG
0.07 [0.06, 0.09]
0.09 [0.08, 0.1]
CC
CG/GG
0.09 [0.08, 0.09]
0.08 [0.07, 0.10]
CC
CT/TT
0.08 [0.07, 0.10]
0.08 [0.07, 0.10]
AC C
1
44/111 (40%)
M AN U
(rs1801252) 108/240 (45%)
AA
SC
AA1
TE D
ADRB1
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP ADR, adrenergic receptor; SAE, serious adverse event; CI, confidence interval
0.38
0.07
0.70
0.93
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Table 4. Comparison of occurrence of at least one SAE and incidence rate of Norwood complications during 14-month follow-up by ADR genotype Genotype
(SNP ID)
Incidence rate ratio
Odds ratio [95%
P-
CI] of SAE in first
value† [ 95% CI] of
Pvalue††
RI PT
Gene
Norwood
14 months
complications
GA/GG
ADRB1
CC1 vs.
(rs1801253)
CG/GG
ADRB2
CC1 vs.
(rs1042714)
CG/GG
ADRA2A
CC1 vs.
(rs553668)
CT/TT
†
0.62
1.004 [0.774, 1.302]
0.98
0.564 [0.205, 1.555]
0.27
0.969 [0.779, 1.206]
0.78
1.036 [0.346, 3.105]
0.95
0.814 [0.638, 1.037]
0.10
1.785 [1.040, 3.063]
0.036
1.077 [0.875, 1.367]
0.55
SC
(rs1801252)
0.732 [0.215, 2.498]
M AN U
AA1 vs.
TE D
ADRB1
Logistic regression adjusted for covariates of interest. All subjects who did not experience an
††
Poisson regression adjusted for covariates of interest and exposure time.
Major allele: AA, CC, CC, CC genotypes at the four loci represent wild-type genotypes;
AC C
1
EP
SAE were followed for at least 14 months.
remainder are polymorphic genotypes i.e. heterozygotes or homozygotes for the SNP SAE, serious adverse event; CI, confidence interval
ACCEPTED MANUSCRIPT
Table 5. Frequency of post-Norwood complications by systems
Post-Norwood complications by system
N=1025 239 (23.3%)
Gastro-intestinal
100 (9.8%)
Hematologic
84 (8.2%)
Infectious disease
233 (22.7%)
SC
RI PT
Cardiac
Neurological
41 (4.0%)
35 (3.4%)
M AN U
Renal Respiratory
249 (24.3%)
Vascular
5 (0.5%) 39 (3.8%)
AC C
EP
TE D
Other
AC C
EP
TE D
M AN U
SC
RI PT
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