The Impact of CNVs on Outcomes for Infants with Single ...circgenetics.ahajournals.org/content/circcvg/early/2013/09/09/CIRC... · The Impact of CNVs on Outcomes for Infants with

DOI: 10.1161/CIRCGENETICS.113.000189

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The Impact of CNVs on Outcomes for Infants with Single Ventricle Heart Defects

Running title: Carey et al.; CNVs and Single Ventricle Defect Outcomes

Abigail S. Carey, MD1*; Li Liang, PhD2*; Jonathan Edwards, BS1; Tracy Brandt, PhD2;

Hui Mei, PhD2; Andrew J. Sharp, PhD2; Daphne T. Hsu, MD5; Jane W. Newburger MD, MPH6;

Richard G. Ohye, MD7; Wendy K. Chung, MD, PhD8; Mark W. Russell, MD9;

Jill A. Rosenfeld, MS, CGC10; Lisa G. Shaffer, PhD11; Michael K. Parides, PhD4;

Lisa Edelmann, PhD2**; Bruce D. Gelb, MD1,2,3**

1Mindich Child Health & Development Institute, Depts of 2Genetics and Genomic Sciences, 3Pediatrics,4Health Evidence & Policy, Icahn School of Medicine at Mount Sinai, New York; 5Pediatric Cardiology,

The Children’s Hospital at Montefiore, Bronx, NY; 6Dept of Cardiology, Children’s Hospital Boston, Boston, MA; 7Dept of Cardiac Surgery, Section of Pediatric Cardiovascular Surgery, University of

Michigan Medical School, Ann Arbor, MI; 8Dept of Pediatrics, Columbia University Medical Center,New York, NY; 9Division of Pediatric Cardiology, C.S. Mott Children’s Hospital, University of

Michigan, Ann Arbor, MI; 10Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WA; 11Paw Print Genetics, Genetic Veterinary Sciences, Spokane, WA

*contributed equally as first author / **senior author

Correspondence:

Bruce D. Gelb, M.D.

Icahn School of Medicine at Mount Sinai

One Gustave Levy Place, Box 1040

New York, NY 10029

Tel.: (212)824-8938

Fax: (212)241-3310

E-mail: [email protected]

Journal Subject Codes: [41] Pediatric and congenital heart disease, including cardiovascular surgery, [89] Genetics of cardiovascular disease, [109] Clinical genetics, [146] Genomics

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Abstract

Background - Human genomes harbor copy number variants (CNVs), regions of DNA gains or

losses. While pathogenic CNVs are associated with congenital heart disease (CHD), their impact

on clinical outcomes is unknown. This study sought to determine whether pathogenic CNVs

among infants with single ventricle (SV) physiology were associated with inferior

neurocognitive and somatic growth outcomes.

Methods and Results - Genomic DNAs from 223 subjects of two National Heart, Lung, and

Blood Institute-sponsored randomized clinical trials with infants with SV CHD and 270 controls

from The Cancer Genome Atlas project were analyzed for rare CNVs >300 kb using array

comparative genomic hybridization. Neurocognitive and growth outcomes at 14 months from the

CHD trials were compared among subjects with and without pathogenic CNVs. Putatively

pathogenic CNVs, comprising 25 duplications and 6 deletions, had a prevalence of 13.9%,

significantly greater than the 4.4% rate of such CNVs among controls. CNVs associated with

genomic disorders were found in 13 cases but no control. Several CNVs likely to be causative of

SV CHD were observed, including aberrations altering the dosage of GATA4, MYH11, and

GJA5. Subjects with pathogenic CNVs had worse linear growth, and those with CNVs associated

with known genomic disorders had the poorest neurocognitive and growth outcomes. A minority

of children with pathogenic CNVs were noted to be dysmorphic on clinical genetics

examination.

Conclusions - Pathogenic CNVs appear to contribute to the etiology of SV forms of CHD in at

least 10% of cases, are clinically subtle but adversely affect outcomes in children harboring

them.

Key words: copy number variant, congenital cardiac defect, outcome, hypoplastic left heart syndrome

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Congenital heart disease (CHD) care has advanced remarkably over the past 40 years.1 This has

shifted focus to decreasing morbidity and improving neurologic and developmental outcomes,

which are affected by genetic factors. For example, aneuploidies and genomic lesions such as

22q11.2 deletions, are associated with poorer neurocognitive outcomes. For most cases of CHD,

however, the underlying genetic basis remains unknown. Identification of causative genetic

factors could help to explain a larger percent of the variance in CHD outcomes, and might be

useful for patient management as well as clinical trial design.

Copy number variants (CNVs), DNA gains or losses greater than 1000 base pairs,2 are

detectable due to recent advances in molecular cytogenetics, particularly in microarray-based

methods. Using high-resolution, whole-genome scanning methods, it has become evident that a

significant proportion of the normal healthy human genome harbors benign CNVs.3, 4 A smaller

subset of CNVs, more often large and de novo, are considered pathogenic and are increasingly

associated with disease such as schizophrenia and intellectual and developmental disabilities

(IDD).5-8

Pathogenic CNV prevalence in CHD has been estimated at 5-15%.9-17 Two small-to-

modest-sized studies of patients with hypoplastic left heart syndrome (HLHS), a single ventricle

(SV) form of CHD, have suggested that CNV frequency is not increased for that heart lesion.12, 17

CNVs appear to be present at higher rates among patients with CHD plus extracardiac or

developmental abnormalities,9-12 although roles for de novo CNVs causing isolated CHD have

also been observed.13, 14 No prior study of CNVs has included careful follow-up of outcomes,

particularly growth and neurocognitive development, in children with CHD.

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Methods

Study Cohort

Study participants from the Pediatric Heart Network’s Infants with Single Ventricle (ISV) and

Single Ventricle Reconstruction (SVR) trials were combined into a single cohort based on the

similarity of their demographics, assessment tools and CHD lesions as previously described.18, 19

Additional subject demographics are provided in the Supplement. Genomic DNAs (gDNAs) and

outcome data were provided anonymously through the New England Research Institute. The

protocol was deemed exempt by the Mount Sinai Institutional Review Board.

Genomic DNA Samples

The quality and quantity of gDNA for each sample was examined via NanoDropTM (Thermo

Fisher Scientific, Waltham, MA, USA). For partially degraded or insufficient (<500 ng) samples

as determined by agarose gel electrophoresis, whole genome amplification (WGA) was

performed using the GenomePlex Complete WGA Kit (Sigma-Aldrich, St. Louis, MO, USA) as

described by the manufacturer.

Array Comparative Genomic Hybridization (aCGH)

aCGH was performed on microarrays according to the manufacturer's instructions (Agilent

Human CGH 1 × 244A; Agilent Technologies, Santa Clara, CA, USA). The data were analyzed

with DNA Analytics 5.0.14 software (Agilent Technologies) via the Aberration Detection

Method-1 algorithm with a sensitivity threshold of 6.0 and a data filter rejecting aberrations with

less than five probes with a log2 ratio ± 0.25.

CNV Characterization

CNVs were considered pathogenic if they were >300 kb in size, contained genes20 and were

either novel or well-established as abnormal21, 22 (Figure 1). Novelty was determined by

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ned bybybby agagagagaarossee gegegelll llelelece troppophohohohoresiiiisss, whwhwhwh lolollee gegenooomemmeme aaampmppliililifififificacatititition ((((WGWGWGWGA)A)A) wasas

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comparing CNVs to the Database of Genomic Variants (DGV)

(http://projects.tcag.ca/variation/). CNVs were deemed polymorphisms if there was >50%

overlap with CNVs already catalogued in the DGV, unless they were well established in the

literature as associated with a genomic disorder. Putatively pathogenic CNVs were also checked

against CNV data from 2500 controls released by the Eichler group23 and against a set of

pathogenic CNVs identified from a proprietary database of >40,000 individuals, most of whom

had IDD, at Signature Genomic Laboratories (SGL). CNVs that were observed infrequently or

deemed pathogenic in the SGL system24 were designated as pathogenic for our study.

CNV Confirmation

Putatively pathogenic CNVs were confirmed via quantitative qPCR using the Universal Probe

Library (UPL; Roche, Indianapolis, IN) system (details in the Supplement).

Pathogenic CNV Inheritance

For 12 SVRII subjects harboring pathogenic CNVs, one or both parental gDNAs were available

(n=19) and analyzed for those CNVs with qPCR.

Normal Controls

Two-hundred-seventy controls were obtained from The Cancer Genome Atlas Project (TCGA).

We used aCGH data from peripheral blood gDNAs from subjects with solid tumors, either

glioblastoma multiforme (GBM) or ovarian cancer (OV). Of note, an aged-matched control

group would have been inferior as some neonates are destined to have IDD, often related to

pathogenic CNVs. Adults enrolling in cancer clinical trials provide a better “healthy”

comparison for the outcomes of interest. We eliminated the possibility of missing CNVs

associated with early lethality by interrogating DGV and the SGL database.

The TCGA blood gDNAs were analyzed at the Dana Farber Cancer Institute using a

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RII subjects harboring pathogenic CNVs, one or both parental gDNAs were avai

anal ed for those CNVs ith qPCR

pathohohohoggenenenicicicc CCCNVNVNVVs were confirmed via quauauantn itative qPCR uusisisising the Universal Pro

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custom 415K array (Agilent Technologies), which included 230,000 of the 244,000 probes used

for the CHD cases. Reference DNA was from Promega, as for the CHD cases. The raw aCGH

data were re-analyzed identically to the cases. All pathogenic CNVs (>300 kb) identified in CHD

cases and TCGA controls would have been called using the other array.

Neurocognitive and Somatic Growth Outcomes

Subjects were dichotomized into subgroups of those with pathogenic CNVs (referred to as

genotype+) and those without pathogenic CNVs (referred to as genotype-), and compared

according to entry characteristics (sex, race/ethnicity, birth weight, and cardiac diagnoses).

Subgroups were also assessed with respect to neurocognitive function using the Mental

Developmental Index (MDI) and Psychomotor Developmental Index (PDI) measured with the

Bayley Scales of Infant Development II at 14 months of age. Growth outcomes at 14 months of

age were compared (weight-, height-, and head circumference-for-age Z-scores and weight-for-

height Z-scores). For SVRII subjects, clinical or research genetic evaluations at 14 months of

age were used for subgroup analysis.25

Statistical Analysis

Comparisons of demographic characteristics between the subgroup of 223 subjects included in

this study and the larger cohort from which they were derived, and of those with and without

CNV were based on Fisher’s exact tests for categorical measures and t-tests for continuous

measures. Normality was tested using the Shapiro-Wilk test and confirmed by the observed

distribution. Secondary analyses with other subgroups were not adjusted for the multiplicity of

comparisons. Z scores for height, weight and head circumference for age were calculated from

the measured values using the National Health and Nutrition Examination Survey

(http://www.cdc.gov/nccdphp/dnpa/growthcharts/resources/sas.htm). T-tests were used to

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ental Index (MDI) and Psychomotor Developmental Index (PDI) measured with

ales of Infant Development II at 14 months of age. Growth outcomes at 14 month

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compare neurocognitive and somatic growth measurements among those with CNV gains and

losses and those without CNVs. All tests were performed at the 0.05 level.

Results

We studied 82 ISV-only subjects, 113 SVRII-only subjects, and 28 subjects enrolled in both

trials. Among these 223 subjects, 147 were male (66%). Racial composition was 85% Caucasian,

11% African-American, 4% other or unknown. All enrolled subjects had SV forms of CHD, 76%

being HLHS. No statistically significant difference in demographic characteristics was noted

between our subjects derived from the ISV and SVRII cohorts and the larger cohorts from which

they were obtained.

Pathogenic CNVs were detected in 31 subjects (13.9%, Table 1). The median sizes of the

twenty-five duplications and six deletions were 674 kb and 1.5 Mb, respectively. Twenty-nine

CNVs were successfully confirmed by qPCR. Two other CNVs, for which there was insufficient

DNA, were confirmed after whole-genome amplification using aCGH with a 105K Agilent

array. The demographic characteristics, including birth weight, gestational age, sex, race, and

ethnicity, were not significantly different among these 31 subjects with pathogenic CNVs

compared to the 192 subject without such CNVs (Supplementary Table 1). Cardiac anatomy also

did not differ with genotype (HLHS: genotype+ 84% vs. genotype- 75%, not significant).

Among the TCGA controls, 164 had glioblastoma multiforme and 106 had ovarian

cancer, resulting in a female predominance (64.5%). The racial composition was 87% Caucasian,

6.6% African-American, 6.4% other or unknown. Pathogenic CNVs were observed in 12

individuals (4.4%, Supplementary Table 2). The median sizes of the nine duplications and three

deletions were 675 kb and 326.6 kb, respectively.

A statistically significant increase in pathogenic CNVs was observed in the CHD

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population relative to the TCGA cohort (13.9% vs. 4.4%, p=0.0003 from the Fisher’s exact test).

The ratios of pathogenic gain and loss CNVs were similar between the CHD and TCGA cohorts

(CHD, 25:6; TCGA 9:3, p = 0.69).

Analysis of parental gDNAs of 12 SVRII subjects harboring pathogenic CNVs revealed

inherited lesions in 3/7 for whom both parents were assessed and 3/5 for whom one parent was

analyzed (Table 1).

To examine possible effects of pathogenic CNVs on health outcomes, neurocognitive and

somatic growth measurements were compared between the genotype+ and genotype– cohorts

(Table 2; details by subject with pathogenic CNV, Supplementary Table 3). Genotype+ subjects

were significantly shorter by an average of 0.65 z-score (p=0.031). No other significant

difference was observed. For the genotype+ subgroups with gain or loss CNVs (Table 2), the

PDI scores were significantly lower among children who harbored loss CNVs than those in the

genotype- cohort (p=0.032). The MDI scores also trended lower among those with deletions but

did not achieve statistical significance in this small cohort (p = 0.29).

Among the 31 pathogenic CNVs found among the CHD subjects, 13 (42%) have

previously been associated with genomic disorders (Table 1). The demographics of this group

did not differ from the genotype- cohort (Supplementary Table 1) and 77% had HLHS. As

compared to the genotype- cohort, the 11 children with known CNVs for whom 14-month

outcomes were available had the worst outcomes with globally reduced neurocognitive

development (MDI and PDI) as well as the slowest growth.

To determine the sensitivity of clinical examination in detecting children with CHD who

harbored pathogenic CNVs, we reviewed the data from 116 subjects from the SVR study

genotyped in our study who had been evaluated by a clinical geneticist. Four children (3.4%)

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were significantly lower among children who harbored loss CNVs than those in

cohort (p=0.032). The MDI scores also trended lower among those with deletion

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were ee sisisisigngngng iiificcanantlttltly llolowew r ammamamong chhchchilililildrdrdrd enen whohohoh hhhharararboboboreeedddd lllolossss CCCCNVNVNVNVsss ttthanan ttthohohh sese iiin

cohort ((p=pp 0.032)2)2)2). ThThThhe MDMDMDI II scores alslll o trenddded d lower among gg hhthose with deletion

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were diagnosed with known genetic syndromes and an additional 29 (25%) were observed to

have one or more dysmorphic features and/or extra-cardiac malformations. Of interest, none of

the 14 subjects with a putatively pathogenic CNV who had a clinical genetic evaluation was

diagnosed with a syndrome and only three (21%) had dysmorphic features or extra-cardiac

malformations. Seven of those 14 subjects harbored CNVs previously associated with genomic

disorders; two of those had dysmorphic features or extra-cardiac malformations.

The outcomes for the 18 subjects with a genetic syndrome or a putatively pathogenic

CNV were worse than those for the 69 children without genetic abnormalities, dysmorphic

features, or extra-cardiac malformations with lower PDI scores, weights and lengths (Table 3).

The outcomes for the 29 individuals with dysmorphic features and/or extra-cardiac

malformations but without a genetic diagnosis or pathogenic CNV did not differ from the 69

subjects without genetic abnormalities, dysmorphic features or extra-cardiac malformations.

Inclusion of the subjects with only dysmorphic features or extra-cardiac malformations with

those without genetic abnormality provide the same conclusions about the inferior outcomes for

the children with a genetic syndrome or pathogenic CNV.

Discussion

Here, we provide a case-control study of the role of pathogenic CNVs in the etiology of SV heart

defects, particularly HLHS, and the first study to relate pathogenic CNVs to formal outcome

assessments for children with CHD but without a known syndrome. Based on our results, SV

forms of CHD are associated with a 10% excess of pathogenic CNVs, which may underestimate

their importance. For the ISV and SVR trials, gDNAs were procured after the Stage II

operations. Since the 12-month mortality in the SVR trial was nearly 30%, we were unable to

assess CNV frequency in most subjects who died during these trials. A birth cohort study is

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onnnnssss but withouuttt a gegeene ititiicc c diagaagnooossis oroor patthhogegenininicc cc CNCNCNVV didd d nooot difffffererere frrrommm ththththee 6

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needed to determine pathogenic CNV prevalence among those not surviving. We also suspect

that some CNVs <300 kb, ignored in the present study, may be pathogenic. They were not

included because public databases are far less populated for smaller CNVs, making separation of

benign polymorphic CNVs from pathogenic ones less accurate.

Although we were only able to analyze a limited number of parents, it appears that

roughly 50% of the pathogenic CNVs are inherited. Three of the six inherited pathogenic CNVs

altered a region on chromosome 16p13.1, an established genomic disorder with variable

expressivity and incomplete penetrance.26 Since parents were not examined, we cannot exclude

that those harboring these pathogenic CNVs had subtle phenotypes.

The 4.4% rate of pathogenic CNVs among the controls has plausible explanations. As

noted above, CNVs associated with genomic disorders are incompletely penetrant. An excess of

second-site CNVs has been found among individuals with CNVs associated with variable

phenotypes.27 While no large second-site CNV was observed in our study, other mutations were

not excluded. Lastly, CNVs labeled as pathogenic could also be false positives.

With our secondary analyses, we examined the outcomes among several subgroups such

as subjects with pathogenic gain or loss CNVs, known genomic lesions, etc. A limitation of this

study is that these subgroups were small, underpowering those analyses. Specifically, secondary

comparisons were not adjusted for multiple hypothesis testing.

The most commonly identified pathogenic CNVs in our CHD cohort were three

overlapping duplications and a deletion at chromosome 16p13.1 (Figures 2 and 3), CNVs

previously associated with IDD, neuropsychiatric disorders, aortic dissection and other forms of

CHD.28-31 Compared to 8329 controls from a recent study,5 16p13.1 duplications were

significantly enriched in our CHD cohort (p=0.004). Among the eight genes in the region,

mined, we cannot eeeexcxxx

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MYH11 and ABCC6 are of interest because MYH11 mutations are associated with aortic

aneurysm and bicuspid aortic valve and ABCC6 mutations cause pseudoxanthoma elasticum.29,

32, 33 In addition to possible etiologic relevance for CHD, parents carrying loss CNVs may be at

risk for aortic aneurysm.

We identified gain and loss CNVs altering GATA4 dosage. GATA4 point mutations cause

CHD,34 and deletions also altering neighboring genes underlie CHD with IDD.35, 36 Our subject

with the GATA4 deletion had a PDI score of 53, >1 standard deviation below the mean for our

CHD cohort. Gain CNVs altering GATA4 gains have also been associated with CHD, including

HLHS, and may be associated with IDD37, 38

We observed a subtelomeric loss CNV at 12p13.33 (Figure 2), which has been associated

with IDD with or without CHD.39 The subject harboring this CNV had a PDI score of 50.

We identified three nearly identical duplications at 16p11.2, which have been implicated

in IDD and neuropsychiatric disease.40-42 While the PDI score in one subject harboring this CNV

was 50, outcomes were not inferior in the other two. Interestingly, deletions at 16p11.2 have also

been associated with aortic valvular defects.43

We detected two distal 1q21.1 duplication CNVs, which have previously been associated

with CHD, particularly non-syndromic tetralogy of Fallot,13, 44 and poor neurocognitive and

growth outcomes.45 For the one child with this CNV with outcome results, there was marked

global neurocognitive delay (MDI and PDI scores of 77 and 54, respectively) and poor growth

(weight- and height-for-age Z scores of -3.1 and -7.4, respectively).

Lastly, one subject with double-inlet left ventricle harbored a de novo duplication altering

22q11.2, which has been linked to cardiac and extra-cardiac abnormalities46, 47 and partially

overlapped with the proximal region of the 22q11 distal deletion syndrome.48, 49 This individual

ated with CHD, incncncnclulll

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identified three nearly identical duplications at 16p11.2, which have been impli

s

tcomes ere not inferior in the other t o Interestingl deletions at 16p11 2 ha

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had a PDI score of 56.

The most interesting novel CNV was the 18p11.31-p11.2 duplication, which overlapped

with a duplication in a patient with HLHS from the SGL database. Among the 40,000 individuals

in that database, only 53 are known to have HLHS. PTPRM, the sole gene residing within the

overlapping region for these two CNVs, appears to be relevant for cardiac development based on

its expression pattern. At mouse embryonic day (E) 9.5, Ptprm is expressed in the endocardium,

dorsal aortic and branchial arch endothelia as well as portions of the brain;50 at E14.5, it is

expressed most highly in the ascending aorta and at lower levels in the developing brain and

lung.51 Thus, PTPRM gains appear to be a new genomic lesion underlying HLHS.

With regard to the types of pathogenic CNVs detected, disproportionately more gains

were observed than losses. In the general population, deletion CNVs exceed duplications by a

2:1 ratio.2 Among individuals with neurocognitive disorders, de novo losses outnumber

duplications by 3:1.52 Similarly, children with CHD plus extra-cardiac abnormalities have an

excess of deletions over duplications. Of interest, an excess of gain CNVs over losses was

observed in children with isolated tetralogy of Fallot.13 In our study, the most striking adverse

effects on neurocognitive development and growth were observed among the children with

CNVs previously associated with genomic disorders, which included gains and losses with a

predominance of the former.

The subgroup of genotyped SVR subjects phenotyped by clinical geneticists provided

interesting insights. Children harboring putatively pathogenic CNVs were not clinically obvious

as none was diagnosed with a syndrome and dysmorphic features or extra-cardiac malformations

were not enriched. Most strikingly, more than 70% of the children with CNVs previously

associated with genomic disorders had no dysmorphic feature or extra-cardiac anomaly. Of note,

e developing brainnnn aaaan

ying HLHLHLHLHSHSHSHS.

h regard to the types of pathogenic CNVs detected, disproportionately more gain

ved than losses. In the general population, deletion CNVs exceed duplications

A

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13

these CNVs would have been declared as abnormalities if clinical testing had been performed.

The findings from this study support the routine use of CNV testing in newborns with SV

forms of CHD to enable better prognostication and early intervention. Similarly, the poorer

linear growth associated with all pathogenic CNVs, the worse neurocognitive outcomes with

deletions, and particularly the globally poor outcomes with CNVs associated with known

genomic disorders could impact clinical trial outcomes depending on the designated endpoints.

Thus, future CHD clinical trials might benefit from an incorporation of CNV status when

determining entry criteria or randomization strategies.

Acknowledgments: We thank the ISV and SVRII study families for their participation and the National Heart, Lung, and Blood Institute (NHLBI)-Pediatric Heart Network for sponsorship of the ISV, SVR and SVRII studies and enabling this ancillary study.

Funding Sources: This project was funded by R21 HL104243 from NIH to L.E. and B.D.G. A.S. was funded by the Doris Duke Clinical Research Fellowship at the Icahn School of Medicine at Mount Sinai. J.E. was funded by a fellowship from the Sarnoff Cardiovascular Research Foundation. The project was supported in part by the NIH-funded Pediatric Heart Network.

Conflict of Interest Disclosure: JAR is an employee of Signature Genomic Laboratories, a subsidiary of PerkinElmer, Inc.

References

1. Pierpont ME, Basson CT, Benson DW, Jr., Gelb BD, Giglia TM, Goldmuntz E, et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation. 2007;115:3015-3038.

2. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global variation in copy number in the human genome. Nature. 2006;444:444-454.

3. Conrad DF, Pinto D, Redon R, Feuk L, Gokcumen O, Zhang Y, et al. Origins and functional impact of copy number variation in the human genome. Nature. 2010;464:704-712.

4. Park H, Kim JI, Ju YS, Gokcumen O, Mills RE, Kim S, et al. Discovery of common Asian

dgments: We thank the ISV and SVRII study families for their participation ande h

V

o Gut Mount Sinai. J.E. was funded by a fellowship from the Sarnoff Cardiovasculao t

dgmentntsss: WeWW ththhank the ISV and SVRII studu y families for ththheir participation andeaaaartrtrtr ,,, , LLungg,,,, aanddd d Blood Institute (NHLBI)-P-P-Pediatric Heaeae rt NNetwork for sponsorsh

VRRRR and SVRIIII I I ssstudududieieieess ananand d d ennnababablilll nng tttthiihh s aanccillllarary y ststudududdyy.

ourcececessss:: ThThThThiss pprorojejeje tctct was fffununununded ddd bybyby RRRR2121211 HHHL1L1L1L 044040424224243333 frfrfrfrommomom NNNIHIHIHIH totooo LLL EEE.E. anandddd B.BBB DDD.Gunded by the DDoooro isiss DDDDukukukkeee ClClCllininininiciciccalalalal RRRReseseseseaeaarcrcrch h h FeFeFeF llowowowowshshshshipipipi aaaattt t thththheee IcIcIcIcahahahahnn n School of t Mount Sinai. JJJ.E.EE was fffunded d ddd bbybyb a fffelllllllowshihhh p pp frfff om thehhh SSSarnoffffffff Cardiovasculaoooundation.n. TTTThehehe pppprororoojejjejectctctt wwwwasassas ssssupupuppopopoportrttrtedeeded iiiin nn n papaapartrrr bbby y yy thtththeee NNNNIHHHIH----fufufufundndndndededded PPPededdediaiaiai trt ic Hearrt



ownloaded from



14

copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing. Nat Genet. 2010;42:400-405.

5. Cooper GM, Coe BP, Girirajan S, Rosenfeld JA, Vu TH, Baker C, et al. A copy number variation morbidity map of developmental delay. Nat Genet. 2011;43:838-846.

6. Rosenfeld JA, Ballif BC, Torchia BS, Sahoo T, Ravnan JB, Schultz R, et al. Copy number variations associated with autism spectrum disorders contribute to a spectrum ofneurodevelopmental disorders. Genet Med. 2010;12:694-702.

7. Walsh T, McClellan JM, McCarthy SE, Addington AM, Pierce SB, Cooper GM, et al. Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science. 2008;320:539-543.

8. Weiss LA, Shen Y, Korn JM, Arking DE, Miller DT, Fossdal R, et al. Association between microdeletion and microduplication at 16p11.2 and autism. N Engl J Med. 2008;358:667-675.

9. Thienpont B, Mertens L, de Ravel T, Eyskens B, Boshoff D, Maas N, et al. Submicroscopic chromosomal imbalances detected by array-CGH are a frequent cause of congenital heart defects in selected patients. Eur Heart J. 2007;28:2778-2784.

10. Breckpot J, Thienpont B, Peeters H, de Ravel T, Singer A, Rayyan M, et al. Array comparative genomic hybridization as a diagnostic tool for syndromic heart defects. J Pediatr.2010;156:810-817, 817 e811-817 e814.

11. Richards AA, Santos LJ, Nichols HA, Crider BP, Elder FF, Hauser NS, et al. Cryptic chromosomal abnormalities identified in children with congenital heart disease. Pediatr Res.2008

12. Payne AR, Chang SW, Koenig SN, Zinn AR, Garg V. Submicroscopic chromosomal copy number variations identified in children with hypoplastic left heart syndrome. Pediatr Cardiol.2012;33:757-763.

13. Greenway SC, Pereira AC, Lin JC, DePalma SR, Israel SJ, Mesquita SM, et al. De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet. 2009;41:931-935.

14. Erdogan F, Larsen LA, Zhang L, Tumer Z, Tommerup N, Chen W, et al. High frequency of submicroscopic genomic aberrations detected by tiling path array comparative genome hybridisation in patients with isolated congenital heart disease. J Med Genet. 2008;45:704-709.

15. Hitz MP, Lemieux-Perreault LP, Marshall C, Feroz-Zada Y, Davies R, Yang SW, et al. Rare copy number variants contribute to congenital left-sided heart disease. PLoS Genet.2012;8:e1002903.

16. Soemedi R, Wilson IJ, Bentham J, Darlay R, Topf A, Zelenika D, et al. Contribution of

al. Association beeeetwtwtwtweMMMMedededed. 2020202008080808;3;3;3;358585858:6:6:6:667676767-6-6-66777

N et aaaallll SuSuSuSubmbmbmbmicicccrorororoscscscom d

p

oi

dmal abnormalities identified in children ith congenital heart disease P di t R

mal imimimimbabababalalalal ncncncess ddddetected by array-CGH ararareee a frequent causee ooof congenital heart dpaaaatiiiients. EuEuEur HeHeHeHearrrrtttt JJJJ. 20222 0707707;2;2;2;28::8::2727272 78888-2-22784.4. JJJJJ

ottt JJJ, Thienpnpnponnt B, Peeeteeersrs H,,, ded RRavvveell T, SSingngn eer AA, RaRRayyyyyyyann MM,, etee all. ArAArA raaay e genononoomimimim c hyybrbrb idiididizizi tatatioii n asasass a diagngngnosososostititicc ttoto lolol ffforroror synynyndrrrommomomicici hhhheeartrtrtrt dddefefeffecctstst . J JJ PePPP didd10-817, 817 e8888111111-8-8-8-8171717 eeee8181814.4.4.4

dds AA, Sanantotototos ss LJLJLJLJ,, NiNiNNichhhhololololsss HAHAHAHA, CrCCrCrididdidereerer BBBBP,P,PP, EEEEldddererrer FFFF,F,F, HHHauauauausesseser r r r NSNSNNS, , , etetet aaaallll. . . CrCC yptic ll bab laliitiie idid itififi ded ii hchililddr iithh iit lal hh rt ddiis PP didi t RR by guest on M


Dow

nloaded from



15

global rare copy-number variants to the risk of sporadic congenital heart disease. Am J Hum Genet. 2012;91:489-501.

17. Tomita-Mitchell A, Mahnke DK, Struble CA, Tuffnell ME, Stamm KD, Hidestrand M, et al.Human gene copy number spectra analysis in congenital heart malformations. Physiol Genomics.2012;44:518-541.

18. Hsu DT, Zak V, Mahony L, Sleeper LA, Atz AM, Levine JC, et al. Enalapril in infants with single ventricle: results of a multicenter randomized trial. Circulation. 2010;122:333-340.

19. Ohye RG, Sleeper LA, Mahony L, Newburger JW, Pearson GD, Lu M, et al. Comparison of shunt types in the Norwood procedure for single-ventricle lesions. N Engl J Med.2010;362:1980-1992.

20. Cruz GBGoUS. Human (Homo sapiens) Genome Browser Gateway http://genome.ucsc.edu/cgi-bin/hgGateway?hgsid=331446057&clade=mammal&org=Human&db=hg18

21. Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet. 2010;86:749-764.

22. Kaminsky EB, Kaul V, Paschall J, Church DM, Bunke B, Kunig D, et al. An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities. Genet Med. 2011;13:777-784.

23. Itsara A, Cooper GM, Baker C, Girirajan S, Li J, Absher D, et al. Population analysis of large copy number variants and hotspots of human genetic disease. Am J Hum Genet. 2009;84:148-161.

24. Shaffer LG, Dabell MP, Rosenfeld JA, Neill NJ, Ballif BC, Coppinger J, et al. Referral patterns for microarray testing in prenatal diagnosis. Prenat Diagn. 2012;32:611.

25. Newburger JW, Sleeper LA, Bellinger DC, Goldberg CS, Tabbutt S, Lu M, et al. Early developmental outcome in children with hypoplastic left heart syndrome and related anomalies: the single ventricle reconstruction trial. Circulation. 2012;125:2081-2091.

26. Grayton HM, Fernandes C, Rujescu D, Collier DA. Copy number variations in neurodevelopmental disorders. Prog Neurobiol. 2012;99:81-91.

27. Girirajan S, Rosenfeld JA, Coe BP, Parikh S, Friedman N, Goldstein A, et al. Phenotypic heterogeneity of genomic disorders and rare copy-number variants. N Engl J Med.2012;367:1321-1331.

28. Ingason A, Rujescu D, Cichon S, Sigurdsson E, Sigmundsson T, Pietilainen OP, et al. Copy number variations of chromosome 16p13.1 region associated with schizophrenia. MolPsychiatry. 2011;16:17-25.

ayy mamamal&l&l&orororg=g=g=HuHuHumamaman&n&n&dbdbdb===

Carterrrr NNNNPPPP etetetet aaaallll CoCoCoConsnnc tn

so

Aer variants and hotspots of human genetic disease Am J Hum Genet 2009;84:14

chromomomososososomommomal mmmmicroarray is a first-tier clilililininical diagnostic tttesesesst for individuals witntatatal ll l disabililililitiessss or cocococongngnggennitittitalalalal anononomamamalililiesee . AmAmAm JJ HHHHumumuum GGGGenenenete .. 20222 10000;8;8;8;86:666 749-99 7676764.444

skykyky EEEEB, Kauuul V,V,, Paaschalalall ll J,J CChuuhurcch DMDDM, BBunknknkee BB, Kunuunig DD, etet aaal. AnAnAn evivividencnnce-o estatattablblbllisisisish hh the ee fufufuncncnctititiionononalll aaandndndnd clinininin caacacallll sisisigngngngnifififficicici ananancccce oooof fff cococopypypypy nnnumumumumbebebeberrr r vvvvariananantsttsts iiiinnn

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AAA, Cooper GGGGM,M,MM, BBBBakakakkereee CCCC, ,, GiGiiGiririririrararajaaaan n n n S,S,SS, LLLiiii J,JJ,J, AAAAbsbsbsb heheheher r r D,D,D, eteee aaaallll... PoPoPoPopupupupulalalalatititionononon aaanalysis ooer va iriantts a dnd hh totsp tots fof hhuman gen tetiic ddiisease AAm JJ HHum GGen tet 20200909 8;844:1414 by guest on M


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16

29. Kuang SQ, Guo DC, Prakash SK, McDonald ML, Johnson RJ, Wang M, et al. Recurrent chromosome 16p13.1 duplications are a risk factor for aortic dissections. PLoS Genet.2011;7:e1002118.

30. Nagamani SC, Erez A, Bader P, Lalani SR, Scott DA, Scaglia F, et al. Phenotypic manifestations of copy number variation in chromosome 16p13.11. Eur J Hum Genet.2011;19:280-286.

31. Ullmann R, Turner G, Kirchhoff M, Chen W, Tonge B, Rosenberg C, et al. Array CGH identifies reciprocal 16p13.1 duplications and deletions that predispose to autism and/or mental retardation. Hum Mutat. 2007;28:674-682.

32. Varadi A, Szabo Z, Pomozi V, de Boussac H, Fulop K, Aranyi T. ABCC6 as a target in pseudoxanthoma elasticum. Curr Drug Targets. 2011;12:671-682.

33. Zhu L, Vranckx R, Khau Van Kien P, Lalande A, Boisset N, Mathieu F, et al. Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus. Nat Genet. 2006;38:343-349.

34. Garg V, Kathiriya IS, Barnes R, Schluterman MK, King IN, Butler CA, et al. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature.2003;424:443-447.

35. Devriendt K, Matthijs G, Van Dael R, Gewillig M, Eyskens B, Hjalgrim H, et al. Delineation of the critical deletion region for congenital heart defects, on chromosome 8p23.1. Am J Hum Genet. 1999;64:1119-1126.

36. Wat MJ, Shchelochkov OA, Holder AM, Breman AM, Dagli A, Bacino C, et al.Chromosome 8p23.1 deletions as a cause of complex congenital heart defects and diaphragmatic hernia. Am J Med Genet A. 2009;149A:1661-1677.

37. Yu S, Zhou XG, Fiedler SD, Brawner SJ, Joyce JM, Liu HY. Cardiac defects are infrequent findings in individuals with 8p23.1 genomic duplications containing GATA4. Circ Cardiovasc Genet. 2011;4:620-625.

38. Barber JC, Bunyan D, Curtis M, Robinson D, Morlot S, Dermitzel A, et al. 8p23.1 duplication syndrome differentiated from copy number variation of the defensin cluster at prenatal diagnosis in four new families. Mol Cytogenet. 2010;3:3.

39. Macdonald AH, Rodriguez L, Acena I, Martinez-Fernandez ML, Sanchez-Izquierdo D, Zuazo E, et al. Subtelomeric deletion of 12p: Description of a third case and review. Am J Med Genet A. 2010;152A:1561-1566.

40. Degenhardt F, Priebe L, Herms S, Mattheisen M, Muhleisen TW, Meier S, et al. Association between copy number variants in 16p11.2 and major depressive disorder in a German case-control sample. Am J Med Genet B Neuropsychiatr Genet. 2012;159B:263-273.

hihihiieueueueu FFFF, etetetet aaaallll. MuMuMuMuttatatatitititionnnnneurysyy //m//aortttticiii ddddisisisisssssec

,c u4

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, KaKaKaathiriya ISISISI ,,, BaBBB rnrnnrnesesees RRRR, ScSccSchlhlhlh ututututererermamamaman nn MKK, Kiiingngngng IIIN,N,N,N, BBButuu lelelerr rr CAAAA,,,, etetetet aaaal. GAGAGAGATATATAA4 caaaausssse human cconnngenenniiiti alal hhhheartrtrt dddeeefeectsss aand rreveaeal ananaa innnteeracttiononon wwwith TBTBTBTBX5X5X55. NaNaNatu443333-444447.

ndt K, Matthijs s s s G,G,G,G VVVVananan DDDDaeaeaeel l l l R,R,RR, GGGGewewewwililili lilig gg M,M,M,, EEEEysysyskekekek nsnsnsns BBB, HjHjHjH alalala grgrgrimimimm H, et al. Delincal deletion regggion fffof r congggeniiti allll hhheart ddddefffects,,, on chrhhh omosome 8p88p8 23.1. Am J Hu999;64:11199-1-11-11212126.6.66.



ownloaded from



17

41. Horev G, Ellegood J, Lerch JP, Son YE, Muthuswamy L, Vogel H, et al. Dosage-dependent phenotypes in models of 16p11.2 lesions found in autism. Proc Natl Acad Sci U S A.2011;108:17076-17081.

42. Rosenfeld JA, Coppinger J, Bejjani BA, Girirajan S, Eichler EE, Shaffer LG, et al. Speech delays and behavioral problems are the predominant features in individuals with developmental delays and 16p11.2 microdeletions and microduplications. J Neurodev Disord. 2010;2:26-38.

43. Ghebranious N, Giampietro PF, Wesbrook FP, Rezkalla SH. A novel microdeletion at 16p11.2 harbors candidate genes for aortic valve development, seizure disorder, and mild mental retardation. Am J Med Genet A. 2007;143A:1462-1471.

44. Soemedi R, Topf A, Wilson IJ, Darlay R, Rahman T, Glen E, et al. Phenotype-specific effect of chromosome 1q21.1 rearrangements and GJA5 duplications in 2436 congenital heart disease patients and 6760 controls. Hum Mol Genet. 2012;21:1513-1520.

45. Rosenfeld JA, Traylor RN, Schaefer GB, McPherson EW, Ballif BC, Klopocki E, et al.Proximal microdeletions and microduplications of 1q21.1 contribute to variable abnormal phenotypes. Eur J Hum Genet. 2012;20:754-761.

46. Brunet A, Gabau E, Perich RM, Valdesoiro L, Brun C, Caballin MR, et al. Microdeletion and microduplication 22q11.2 screening in 295 patients with clinical features of DiGeorge/Velocardiofacial syndrome. Am J Med Genet A. 2006;140:2426-2432.

47. Yang YH, Hu YL, Zhu XY, Mo XM, Wang DJ, Yao JC, et al. Diagnosis of 22q11 deletion and duplication in congenital heart disease by multiplex ligation dependent probe amplification. Zhongguo Dang Dai Er Ke Za Zhi. 2009;11:892-896.

48. Ben-Shachar S, Ou Z, Shaw CA, Belmont JW, Patel MS, Hummel M, et al. 22q11.2 distal deletion: a recurrent genomic disorder distinct from DiGeorge syndrome and velocardiofacial syndrome. Am J Hum Genet. 2008;82:214-221.

49. Coppinger J, McDonald-McGinn D, Zackai E, Shane K, Atkin JF, Asamoah A, et al.Identification of familial and de novo microduplications of 22q11.21-q11.23 distal to the 22q11.21 microdeletion syndrome region. Hum Mol Genet. 2009;18:1377-1383.

50. Koop EA, Lopes SM, Feiken E, Bluyssen HA, van der Valk M, Voest EE, et al. Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice. Int J Dev Biol. 2003;47:345-354.

51. Mouse atlas project www.emouseatlas.org

52. Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, et al. Strong association of de novo copy number mutations with autism. Science. 2007;316:445-449.

BC, KlKlKlKlopockikikiki EEEE, etetetet aaaalll.o variiiiabababablelelele aaaabnbnbnbnorororormamamam ll

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Table 1: Pathogenic CNVs detected among 223 subjects with isolated single ventricle heart defects

Subject Location Start Stop Size (kb) CN Status Inheritance92022989 1p31.1 76420271 76809986 390 Loss Novel UnknownGT04009571 1q21.1 145009267 146318068 1309 Gain Known Unknown92027535/GT04007822 1q21.1 145009267 147203477 2194 Gain Known De novo

92026280/GT04009083 2q13 111108466 112819206 1711 Gain Novel Unknown

GT04013352 2q14.1 114390707 114975263 585 Gain Novel Not maternal

GT04008671 3p14.1 65445598 66643726 1198 Gain Novel Unknown92026850/GT04007792

4q35.1-q35.2 186295054 188154168 1859 Loss Novel Maternal

92022561 5q12.3 6435972 65164354 328 Gain Novel Unknown 92124531 8p23.1 8131939 11898119 3766 Loss Known Unknown 92022496 8p23.1 10791639 11898119 1106 Gain Known Unknown 92120189 9q21.31 81376647 83149610 1773 Gain Novel Unknown GT04006712 9q21.32 83460453 83972537 512 Loss Novel Unknown 92024262 10q25.3 116304612 116870123 566 Gain Novel Unknown 92021139 12p13.33 551096 2543903 1993 Loss Known Unknown 92024760/GT04010341

16p13.12-p13.11 14669540 16190029 1520 Gain Known Maternal

GT04011611 16p13.11 14817506 16515223 1698 Gain Known PaternalGT04012411 16p13.11 14955977 16199882 1244 Loss Known PaternalGT04011421 16p13.11 15398828 16166985 768 Gain Known De novo92020315 16p12.3 16701973 17654093 952 Gain Novel Unknown 92124101 16p11.2 29500084 30107008 607 Gain Known Unknown 92020074 16p11.2 29500084 30107008 607 Gain Known Unknown GT04011942 16p11.2 29560300 30265521 705 Gain Known De novo92023830 16q23.1 74558771 75090033 531 Gain Novel Unknown GT04010673 17p13.1 9922263 10359076 437 Gain Novel Unknown 92021026 17p12 13709239 14218834 510 Gain Novel Unknown 92121321/GT04008312

18p11.31-p11.23 6837549 8088466 1251 Gain Novel Not paternal

92029147 20q11.21 29297070 29971177 674 Gain Novel Unknown

GT04010851 22q11.21-q11.22 20294861 20753261 458 Gain Known De novo

GT04009061 Xp22.2 10709385 11255827 546 Gain Novel MaternalGT04013021 Xq24 117055728 117415164 359 Gain Novel Paternal92021913 Xq28 148573118 149245260 672 Gain Novel Unknown

Human Genome Build 36.1 (hg18) used for genomic coordinates. CN, copy number. Novel, aberration has not been previously reported in CHD patients. Known, aberration reported in genomic disorders with CHD.

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q9q9q9q9q2122121 33.3222 83460453 83972537 512 Losssss Novel Unkn1101 q25.3 11111636366304040446111222 11111686868700001212121 3 56566566666 GaGaGaininnin NoNoNoNovevvv l UnUUU kn12p13.33 55100009969 252254399903 19199993333 Losss Knowowowo n UnUnUnkn1611 p13.1222-p1p1p1p13.3.3.3.1111111 1414446695555404040 1666619191 00000002999 151515202020 GaGGG iin Knowowown MaMM te

16p13.11 141414818175757506060 16161651515252522323 1616169888 GaGaGaininin KnKKK own Pate16p13.11 1414141495959559595959777777 1616161619191998989898828282 11124444 LoLoLoLossssss KKnKK own Pate161616p1p1p1333.111111 151515393939888888282828 161616161616696969858585 767676888 GaGaGaininin KnKnKnowowownnn DeDeDe nnn16p12.33 16161670707 191919733 17171765654040409393 959595222 GaGaGainininn NoNoNovevevelll Unknn



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Table 2: Fourteen-month outcomes for subgroups by genotype

N‡ MDI PDI Weight Z Length Z HC ZGenotype- 192 91.4 (17.0) 77.6 (19.1) -0.61

(1.15)-1.00(1.37)

-0.18(1.32)

Genotype+ 31 90.6 (17.8) 71.4 (20.4) -0.61(1.37)

-1.65*(1.82)

-0.16(1.66)

Gain CNV 25 92.5 (16.3) 74.7 (20.9) -0.80(1.24)

-1.81*(1.87)

-0.30(1.66)

Loss CNV 6 83.2 (23.1) 59.1* (13.5) 0.16(1.77)

-0.94(1.55)

0.46(1.70)

Known CNV 11 79.8* (18.3) 56.8† (7.7) -0.87(1.57)

-1.89*(2.16)

-0.43(1.69)

*, p < 0.05 compared to Genotype-; †, p < 0.005 compared to Genotype-; Abbreviations: MDI, Mental Developmental Index; PDI, Psychomotor Developmental Index; Weight Z, Weight-for-Age Z Score at 14 months; Length Z, Length-for-Age Z score at 14 months; HC Z, Head Circumference Z Score at 14 months. All data shown as mean (standard deviation). ‡Size of each cohort. Incomplete data for outcomes resulted in lower N’s.

Table 3: Fourteen-month outcomes for subgroups based on genetic examination

N‡ MDI PDI Weight Z Length Z HC ZCNV- Syndrome-Dysmorphic- Extracardiac-

69 89.1(18.0)

77.5(20.2)

-0.71(1.07)

-1.13(1.32)

-0.34(1.24)

CNV+ 14 85.4(20.1)

65.1*(17.6)

-0.94(0.88)

-1.61(1.08)

-0.04(1.38)

CNV+ or Syndrome 18 83.2(18.7)

67.9*(19.4)

-1.19*(1.11)

-1.99*(1.73)

-0.16(1.27)

Dysmorphic/Extracardiac 29 89.3(18.1)

78.1(20.0)

-0.63(1.25)

-1.18(1.30)

-0.10(1.28)

CNV-Syndrome-

98 89.4(17.5)

77.8(19.8)

-0.73(1.10)

-1.18(1.43)

-0.25(1.32)

*, p < 0.05 compared to CNV-/Syndrome-/Dysmorphic-/Extracardiac-;Abbreviations: MDI, Mental Developmental Index; PDI, Psychomotor Developmental Index; Weight Z, Weight-for-Age Z Score at 14 months; Length Z, Length-for-Age Z score at 14 months; HC Z, Head Circumference Z Score at 14 months. All data shown as mean (standard deviation). ‡Size of each cohort. Incomplete data for outcomes resulted in lower N’s.

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Figure Legends:

Figure 1: Workflow for aCGH studies. Starting with the available cohorts from the Infants with

Single Ventricle and the Single Ventricle Reconstruction-Extension trials of the Pediatric Heart

Network, the flow of statistical and genetic analyses is outlined. All pathogenic CNVs identified

were successfully confirmed via secondary methods.

Figure 2: Pathogenic deletion and duplication CNVs likely causative for CHD defects. (A) 1.5-

Mb deletion CNV at 12p13.33 affecting 223 consecutive probes. (B) 1.6-Mb duplication CNV at

16p13.12-p13.11 affecting 124 consecutive probes and altering MYH11. For each 244K array

clone, the Cy3/Cy5 signal intensity ratios are plotted. The green and red dots correspond to log2

-0.25 25 (gain), respectively. All chromosomes are depicted according

to hg18.

Figure 3: Schematic illustration of the 16p13.1 region. The smallest region of overlap (SRO)

between the three duplications (shown in blue) and the deletion (shown in red) at 16p13.1 is 758

kb. This region includes eight genes, including MYH11 and ABCC6. MYH11 encodes the smooth

muscle myosin, heavy chain.

( )

.6-MbMbMbMb ddddupupupuplilililicacacacatitititionononon CCCCN

p13.11 affecting 124 consecutive probes and altering MYH11 For each 244K arr

Cy3/Cy5 signal intensity ratios are plotted. The green and red dots correspond to

- r

p13.111 afafaffef ctcc inng g g g 124 consecutive probes ananand altering MYH1H1111. For each 244K arr

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Lisa G. Shaffer, Michael K. Parides, Lisa Edelmann and Bruce D. GelbHsu, Jane W. Newburger, Richard G. Ohye, Wendy K. Chung, Mark W. Russell, Jill A. Rosenfeld, Abigail S. Carey, Li Liang, Jonathan Edwards, Tracy Brandt, Hui Mei, Andrew J. Sharp, Daphne T.

The Impact of CNVs on Outcomes for Infants with Single Ventricle Heart Defects

Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2013 American Heart Association, Inc. All rights reserved.

TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

published online September 10, 2013;Circ Cardiovasc Genet.

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SUPPLEMENTAL MATERIAL

Supplemental Methods

ISV Study Participants

Infants were included in the ISV study if they were less than 45 days of age, had SV physiology,

and had stable systemic and pulmonary blood flow. Infants were excluded if they were

determined to be: small for gestational age, premature, had a chromosomal abnormality or

recognizable syndrome, pulmonary atresia with an intact ventricular septum, or severe illness

requiring medical intervention (e.g. mechanical ventilatory support, inotropic support, renal

failure, etc.)

We obtained genomic DNAs (gDNAs) that had been banked as part of the ISV protocol.

These gDNAs were extracted from peripheral leukocytes and procured late in the course of the

trial. Therefore, subjects who did not complete the ISV protocol, often due to death, did not have

banked gDNA for our analysis.

SVR Study Participants

Infants were enrolled in the SVR study if they were diagnosed with SV CHD and had a planned

Norwood procedure, generally performed in the early neonatal period. SVR exclusion criteria

included patients with anatomy identified peri-operatively that rendered one or the other shunt

technically impossible or those with any major congenital anomaly that could adversely affect

survival (e.g. the development of end-organ damage.)

Of note, the SVR protocol included procurement of buccal swabs from participants prior to

surgery for genetic analysis. Because those samples resulted in low-quality gDNA that could not

be used for our study, we obtained banked gDNA samples from SVR participants who enrolled

in the subsequent SVR extension study (SVRII). Thus, our study did not have gDNAs for any

subject (29% overall) who died during SVR.

Array Comparative Genomic Hybridization

Array CGH was performed on microarrays according to the manufacturer's instructions (Agilent

Human CGH 1 × 244A; Agilent Technologies, Santa Clara, CA, USA). Five-hundred nanograms

of experimental and gender-matched reference DNAs (Promega, Madison, WI, USA) were

digested with AluI and RsaI restriction endonucleases (Promega) and fluorescently labeled with

cyanine 5-dCTP (Cy-5; experimental) and cyanine 3-dCTP (Cy-3; reference). Labeled

experimental and reference DNAs were purified, combined, denatured, pre-annealed and

hybridized to the microarrays in a rotating oven (20 rpm) at 65 °C for 40 h. All quality control

metrics passed, or the results were repeated. The data were analyzed with DNA Analytics 5.0.14

software (Agilent Technologies) via the Aberration Detection Method-1 algorithm with a

sensitivity threshold of 6.0 and a data filter rejecting aberrations with less than five probes with a

log2 ratio ± 0.25.

CNV Confirmation

Putatively pathgenic CNVs were confirmed via quantitative PCR (qPCR) by using Universal

Probe Library (UPL; Roche, Indianapolis, IN) system. UPL probes and primers were selected

using the ProbeFinder v2.45 software (Roche, Indianapolis, IN). qPCR was performed on an

ABI Prism™ 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Each

sample was analyzed in triplet in a 10 ml reaction mixture (100 nM probe, 200 nM each primer,

1x Platinum Quantitative PCR SuperMix-Uracil-DNA-Glycosylase (UDG) with ROX from

Invitrogen, and 4 ng genomic DNA). The values were evaluated using Sequence Detection

Software v2.2 (Applied Biosystems, CA). The relative copy number was calculated as a ratio of

the gDNA level for a tested gene to the gDNA level for a reference gene in the same sample.

With respect to internal control genes, two genes were used (CFGR3, DENND1B, and/or GLRX2

for CNVs residing on autosomes; KAL1 and ARHGAP6 for CNVs residing on chromosome

X).The relative copy number of a given gene in tested samples was compared with that in the

normal control.

Supplemental Table 1. Demographics of CHD subcohorts

Mean Birth Weight (g)

Mean Gestational

Age (months) Male % White % Hispanic % CNV- 3195 38.5 64.1 88.0 17.4 CNV+ 3136 38.4 77.4 87.1 16.1 Genomic Disorder CNV 3085 38.2 69.2 84.6 15.4

Supplemental Table 2: Pathogenic CNVs detected among 270 TCGA controls Proband Location Start Stop Size (kb) CN Status 1970 1p31.1 71940551 72256560 316 Loss Novel 1396 2p25.3 803179 1836309 1033 Loss Novel 1669 4q35.1 184797544 185322222 525 Gain Novel 2526 5q23.2 125109297 125910677 801 Gain Novel 1582 7p22.2 2146793 2451255 304 Gain Novel 1084 9q33.1 118962396 121086819 2124 Gain Novel 1809 10q24.2 99885068 100211673 327 Loss Novel 1599 10q25.1 107737574 109762101 2025 Gain Novel 1601 12q24.32 126798090 127473136 675 Gain Novel 1800 16p12.1-p11.2 27177103 27654583 477 Gain Novel 2632 17q12 28983763 29962842 979 Gain Novel 1982 19q13.41 58726417 59037213 311 Gain Novel

Supplemental Table 3: Neurocognitive and growth outcomes for subjects with pathogenic CNVs Proband Location CN MDISCORE PDISCORE ZWEI ZLEN ZWFL ZHC

92022989 1p31.1 Loss 116 82 0.44 -0.87 1.07 1.74 GT04009571 1q21.1 Gain 77 54 -3.1 -7.44 2.4 -1.46 92027535/ GT04007822 1q21.1 Gain N/A N/A N/A N/A N/A N/A

92026280/ GT04009083 2q13 Gain 105 110 1.15 -0.13 1.54 0.88

GT04013352 2q14.1 Gain 80 53 -2.15 -3.63 -0.37 -1.21 GT04008671 3p14.1 Gain 86 52 -0.59 -1.95 0.41 1.43 92026850/ GT04007792 4q35.1-q35.2 Loss 90 60 -0.57 -2.64 0.93 1.38

92022561 5q12.3 Gain N/A N/A N/A N/A N/A N/A 92124531 8p23.1 Loss 86 53 2.64 0.72 2.94 0.05 92022496 8p23.1 Gain N/A N/A -0.34 -1.79 0.64 -3.78 92120189 9q21.31 Gain N/A N/A 1.2 -2.04 2.72 -0.38 GT04006712 9q21.32 Loss N/A N/A N/A N/A N/A N/A 92024262 10q25.3 Gain 80 100 0.95 1.72 0.34 -1.64 92021139 12p13.33 Loss 69 50 -2.22 -2.36 -1.54 -2.35 92124101 16p11.2 Gain N/A N/A -0.28 -1.3 0.4 1.44 92020074 16p11.2 Gain 101 64 -1.16 -2.45 0.03 -0.07 GT04011942 16p11.2 Gain 50 50 -1.94 -2.85 -0.66 0.46 92020315 16p12.3 Gain 106 96 0.34 0.63 0.12 1.05 GT04011611 16p13.11 Gain N/A N/A N/A N/A N/A N/A GT04012411 16p13.11 Loss 55 50 0.51 0.46 0.36 1.47 GT04011421 16p13.11 Gain 90 62 -0.41 -0.83 -0.07 -1.17 92024760/ GT04010341 16p13.12-p13.11 Gain 103 100 -2.33 -2.95 -1.23 -0.97

92023830 16q23.1 Gain 112 106 -1.68 -2.34 -0.74 -0.44 92021026 17p12 Gain 97 68 0.47 -0.99 1.19 1.55 GT04010673 17p13.1 Gain 65 50 -0.96 -2.4 0.26 -2.59 92121321/ GT04008312 18p11.31-p11.23 Gain 95 72 -1.33 -1.11 -1.13 -0.82

92029147 20q11.21 Gain 97 74 -2.57 -4.32 -0.33 -2.55 GT04010851 22q11.21-q11.22 Gain 95 56 -1.99 -1.83 -1.56 1.47 GT04009061 Xp22.2 Gain 99 94 -0.73 -0.96 -0.39 -0.86 GT04013021 Xq24 Gain 112 70 -0.04 -0.61 0.29 -0.19 92021913 Xq28 Gain 108 88 0.16 -0.18 0.31 3.35

CN, copy number. ZWEI, z–score for weight-for-age at 14 months. ZLEN, z-score for length-for-age at 14 months. ZWFL, z-score weight for length at 14 months. ZHC, z-score for head circumference-for-age at 14 months. N/A indicates not available.

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