Table S1. Complete Classic Heterotaxy Case Inclusions/Exclusions

Case Definition: Major congenital heart disease (CHD) + another anomaly of situs
Examples of Major/Qualifying CHD:
Atrioventicular Canal Defect
Truncus Arteriosus
Tetralogy of Fallot
Tricuspid/Mitral Atresia
Double Outlet Right Ventricle
Hypoplastic Left Heart Syndrome
Pulmonic Stenosis/Atresia
Aortic stenosis/coarctation
Interrupted Aortic Arch
L or D Transposition of the Great Arteries
Total Anomalous Pulmonary Vascular Return
Dextrocardia
Major VSD
Thoracic Venous Anomalyb / Examples of situs anomalies:
Situs inversus (unspecified)
Situs inversus totalis
Situs inversus abdominis
Situs inversus thoracis
Situs Ambiguus
Heterotaxy
Asplenia
Polysplenia
Examples of isolated Minor CHD not meeting inclusion:
Aortic Insufficiency
Tricuspid/Mitral Insufficiency
Atrial Septal Defect
Patent Foramen Ovale
Patent Ductus Arteriosus
Peripheral Pulmonic Stenosis / Absolute exclusions:
Isolated situs inversus totalisa
Isolated asplenia
Isolated polysplenia
Isolated situs inversus thoracis or abdominis
Kartagener Syndrome
Primary Ciliary Dyskinesia
Isolated Dextrocardia
Isolated Transposition of the Great Arteries / Other Exclusions:
Trisomy 13, 18, 21
22q11 deletion syndrome
Turner Syndrome
Teratogenic Syndrome
Arthrogryposis
Microcephaly
Schizencephaly
Various facial anomalies
Major limb anomalies

aDextrocardia + situs inversus abdominis assumed to represent complete situs inversus

bThoracic Venous Anomaly –one case with persistent left superior vena cava and interrupted inferior vena cava

Table S2. Sequencing primers and amplification and cycling conditions.

Gene / Amplicon / F Primer sequence (5'>3')a / R Primer sequence (5'>3')a / Amplicon Size (bp) / Annealing Temp (°C) / PCR Reagents or Cycling Condition Differences
CFC1 / Exon 1 / TGTTTTACATCAGGGATTGGTC / GTGGCTCATGCACTACGATTTA / 461 / 55 / N/A
CFC1 / Exons 2&3 / CCTACAAGTTACAAAATGATTGCCTTA / GCTCTCCTTGGATTTTATCCTG / 586 / 55 / N/A
CFC1 / Exon 4 / CCCTAACAAGCAGGGTTTACCT / GCCGCATTCACTGAGAAGAC / 537 / 53 / 5% DMSO
CFC1 / Exon 5 / CTACTGCGAGCATGACCAGAG / CCAATGAATGCGACTTTCTTC / 564 / 53 / 5% DMSO + 0.8% Taq antibody; Touchdown PCR
CFC1 / Exon 6 / CCAGGGACAGAGCCTAGTGAG / TGTGAAAGGAAGGTGCTAACTG / 589 / 55 / N/A
ACVR2B / Exon 2 / AATTAAGAGGTGTGGGACGGATGG / TCCTCAGGGCTAAGGTAGGACAAA / 567 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 3 / TTCCTTGGCTTTGGGTCTCCTGTA / ACAGAGCCCTGTGTCAAACAGTC / 428 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exons 4&5 / TTCTTTGGCTTGGAGCGCTTGG / ACTAGGCCCAGTAAGGCTCACTTT / 636 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 6 / TACAAATGCTGAAGCTGGGAGGCT / AATGTCCACACCTATGGCAGGAGT / 462 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 7 / TCCTGGCCTCATCCATCTTCCATA / GAGTCAGCACTTCATGTGGCCATT / 531 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 8 / TTCCATCTGAAGTGCACTGAGGGA / TACACTGCCTTCCTGGTCTTGGTT / 501 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 9 / GATGCTGTGACCTCTTGTAACTGT / TCCTCAAAGGGCAGCATGTACTCA / 447 / 59 / 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 10 / GCCTTCCTGCGCATTGACATGTAT / GCCCAAACCATACAAGTGCCCAAA / 530 / 55 / 1.5 mM MgCl2 + 0.8% Taq antibody; Touchdown PCR
ACVR2B / Exon 11 / GCAGGCATGCTGATTCCTGAAGTT / GTGATACAGTAGAGCAATATGCTGGT / 597 / 55 / 0.8% Taq antibody; Touchdown PCR
NODAL / Exon 1 / AACTAGGTCCTCCGCCAGCTTT / GGAGACACCCGCAGAGCTA / 570 / 55 / 0.8% Taq antibody; Touchdown PCR
NODAL / Exon 2A / TCCAGCAAGAGCTATGGTGGTT / CATAAGGAGCACATTGGTGGCA / 482 / 55 / N/A
NODAL / Exon 2B / TTCACTGTCACTTTGTCCCAGGTC / TGGAGGTGCTTGAGTAACTGTG / 599 / 55 / N/A
NODAL / Exon 3 / ACAAGCACAGAGCTAAAGGTAGT / AGTAAAGAGAACATCCAGCCAGCC / 607 / 55 / 0.8% Taq antibody
FOXH1 / Exon 1 / TCCAGATTAACCAAAACCTGCT / AGAAGGGTCGTGCCTTGAG / 550 / 61 / 0.8% Taq antibody; Touchdown PCR
FOXH1 / Exon 2 / TATGCCAGTGGGACCAGTCCTGA / TCACGTCGACCGCCCAGAAGTT / 484 / 55 / 0.8% Taq antibody; Touchdown PCR
FOXH1 / Exon 3A / TACGAGGGCTGGAAAGACTCCATT / AGGCCTCTCAGAAGAGGGAAGG / 526 / 55 / 0.8% Taq antibody; Touchdown PCR
FOXH1 / Exon 3B / AACTTCTGGGCGGTCGACGTGAG / TGGCTGACCCAAACGTCGTAGAT / 725 / 55 / 0.8% Taq antibody; Touchdown PCR
FOXH1 / Exon 3C / TCCGTCAACCAGCCCTGCCTACT / AAACAGGCATGACAGACCAGGGTGA / 399 / 59 / 0.8% Taq antibody; Touchdown PCR
ZIC3 / Exon 1A / AGGAGACTCTTGCAGTGACGGA / CAGAAACTCGCGCGTTGAGTTGAA / 529 / 55 / N/A
ZIC3 / Exon 1B / GGCGCACGATCTATCTTCAGGC / TGACATGTGTCACCAGCTCATGC / 662 / 57 / N/A
ZIC3 / Exon 1C / TGAACATGGGAGTGAACGTGGC / ATGGTCACTGACAGCGCTTCTT / 538 / 57 / 0.8% Taq antibody
ZIC3 / Exon 2 / TACGCTCTTCATTTCTCTCCAGGG / AATGGGTTCAAGGTGTCACTGCTG / 468 / 61 / 0.8% Taq antibody
ZIC3 / Exon 3 / CCGTAACACCAGTCTTAGGAGCAA / TGCAATTAACAAGATGTAGCCCT / 523 / 57 / N/A

aForward and reverse primers included 5' M13 tags to facilitate cycle sequencing (not shown and not included in size of amplicons).

N/A=Not applicable; bp=base pairs.

Table S3. TaqMan copy number assay details.

CNV Locusa / Assay ID / Gene / Target Coordinates / # Cases Tested
(N=74) / # Controls Tested
(N=138)
1q23.1 / Hs00817935_cn / OR6K3 / Chr.1:158,687,085 / 74 / 12
Hs06569276_cn / - / Chr.1:158,717,880 / 74 / 12
Hs06603435_cn / - / Chr.1:158,727,717 / 74 / 12
Hs00533922_cn / MNDA / Chr.1:158,819,007 / 74 / 138
1q42.3 / Hs06575556_cn / - / Chr.1:235,177,040 / 74 / 138
Hs05768103_cn / - / Chr.1:235,189,486 / 74 / 12
Hs06584666_cn / - / Chr.1:235,247,139 / 74 / 12
2p24.1 / Hs02105776_cn / GDF7 / Chr.2:20,867,383 / 74 / 12
Hs05825259_cn / C2orf43 / Chr.2:20,996,093 / 74 / 4
Hs05850146_cn / - / Chr.2:21,588,783 / 74 / 138
2p13.2 / Hs04649181_cn / - / Chr.2:72,255,683 / 74 / 138
Hs04615341_cn / - / Chr.2:72,270,068 / 74 / 12
3p26.1 / Hs05893284_cn / SUMF1 / Chr.3:4,435,474 / 74 / 12
Hs04788790_cn / ITPR1 / Chr.3:4,646,166 / 74 / 138
Hs06619372_cn / EGOT, ITPR1 / Chr.3:4,793,217 / 74 / 12
3q28 / Hs06670818_cn / FGF12 / Chr.3:192,026,981 / 74 / 20
Hs04763916_cn / FGF12 / Chr.3:192,228,841 / 74 / 138
4q25 / Hs04836354_cn / RPL34 / Chr.4:109,550,756 / 74 / 12
Hs04846665_cn / - / Chr.4:109,559,234 / 74 / 12
Hs05929890_cn / - / Chr.4:109,563,467 / 74 / 12
Hs05951969_cn / OSTC / Chr.4:109,572,416 / 74 / 138
4q26 / Hs04844278_cn / - / Chr.4:117,138,372 / 74 / 12
Hs07542683_cn / - / Chr.4:117,367,829 / 74 / 12
Hs03087805_cn / - / Chr.4:117,544,887 / 74 / 20
Hs03244541_cn / - / Chr.4:117,612,844 / 74 / 138
5q21.2 / Hs06076524_cn / - / Chr.5:103,328,839 / 74 / 138
Hs05972411_cn / - / Chr.5:103,420,676 / 74 / 138
5q34 / Hs06110497_cn / - / Chr.5:162,651,398 / 74 / 138
Hs05996442_cn / - / Chr.5:162,670,912 / 74 / 12
7q11.21-7q11.22 / Hs03627857_cn / STAG3L4 / Chr.7:66,783,803 / 74 / 12
Hs03648563_cn / - / Chr.7:66,804,316 / 74 / 12
7q11.2 / Hs05012331_cn / - / Chr.7:67,109,352b / 74 / 12
Hs04952721_cn / - / Chr.7:67,474,902 / 74 / 138
9p23 / Hs06863077_cn / - / Chr.9:13,665,202 / 74 / 12
Hs05081944_cn / - / Chr.9:13,848,182 / 74 / 138
10q26.13-telomere / Hs03760991_cn / DHX32 / Chr.10:127,545,013 / 74 / 12
Hs02592062_cn / FOXI2 / Chr.10:129,536,041 / 74 / 138
Hs01199874_cn / DPYSL4 / Chr.10:134,004,291 / 74 / 12
Hs02224597_cn / NKX6-2 / Chr.10:134,598,475 / Probe Excluded
11q22.3 / Hs06320095_cn / - / Chr.11:109,670,132 / 74 / 138
Hs06314459_cn / - / Chr.11:109,801,196 / 74 / 12
13q21.31 / Hs05303389_cn / - / Chr.13:62,874,380 / 74 / 12
Hs06368525_cn / - / Chr.13:62,989,711 / 74 / 138
19q13.42 / Hs07122892_cn / ZNF331 / Chr.19:54,064,656 / 74 / 12
Hs00752003_cn / DPRX / Chr.19:54,140,214 / 74 / 138
20p12.3-20p12.2 / Hs07173529_cn / CRLS1 / Chr.20:6,020,153 / 74 / 12
Hs01969121_cn / BMP2 / Chr.20:6,749,120 / 74 / 12
Hs03058564_cn / PAK7 / Chr.20:9,518,068 / 74 / 138
21q22.3 / Hs01792272_cn / TSPEAR / Chr.21:45,919,505 / 74 / 12
Hs01861771_cn / UBE2G2 / Chr.21:46,189,714 / 74 / 138
22q13.2 / Hs04503915_cn / SHISA8 / Chr.22:42,310,474 / 74 / 138
Hs02770843_cn / WBP2NL / Chr.22:42,423,163 / 74 / 12

aAt least one assay per CNV locus was run in all 138 control subjects.

bIt was initially suspected that 6 smaller CNVs called by pennCNV in case 2 (2 smaller CNVs were called by cnvPartition) at this locus actually represented one large CNV that had been artificially split into smaller CNVs by the calling algorithms. However, this assay demonstrated the presence of 2 alleles at this locus, indicating the presence of at least two separate duplications.

Table S4. Rare variantsaidentified in CFC1, ACVR2B, NODAL, FOXH1, and ZIC3among 19heterotaxy cases with CNVs of interest.

Gene / DNA changec / Protein changec / dbSNP rs IDb / dbSNP MAFb / Case ID (s)
CFC1 / c.61A>C
c.75C>T
c.140G>A
c.433G>A
c.588C>A / p.N21H
p.S25S
p.R47Q
p.A145T
p.P196P / rs199607550
N/A
rs201431919
rs199715380
N/A / 0.0444
N/A
0.046
N.D.
N/A / 19
1
19
2, 10, 18
19
ACVR2B / c.666+5G>A
c.993C>T
c.1075-5C>T / N/A
p.S331S
N/A / rs187763364
rs2228012
rs115155428 / 0.0037
0.0037
0.0055 / 9
9
2
NODAL / None
FOXH1 / c.-88_c.-76del13
c.-54C>T
c.47C>T / N/A
N/A
p.S16L / N/A
rs187197849
rs180724802 / N/A
0.0087
0.0005 / 10
11
13
ZIC3 / None

aNovel or rare (defined as global dbSNP MAF ≤ 0.05) variants, in the coding region, promoter region or within 10 nucleotides of exons, and not detected in control subjects screened in this study are included in the table. All variants were heterozygous and none are known to be pathogenic.

bProvided when available.

cVariants described by comparison to reference sequences NG_008148.1 and NP_115934.1 (CFC1), NG_011791.1 and NP_001097.2 (ACVR2B), NG_030003.1 and NP_003914.1 (FOXH1).

MAF = Global minor allele frequency, N/A = Not applicable, N.D. = Not determined