Supplementary Information and Tables

1

Supplementary Information and Tables

Supplementary Information

RET Sequencing

RET exons and flanking regions in introns were sequenced. PCR primers (SupplementaryTable1) were selected based on one of two previous reports,20,21 or were designed manually and inspected using NetPrimer (Premier Biosoft International, Palo Alto, CA) or using Primer3 v.0.4.0.22 To facilitate cycle sequencing, M13 tags were added to the 5’ end of each primer. Each fragment was PCR-amplified (described below), cleaned-up using ExoSAP-IT (USB Corporation, Cleveland, OH), and sequenced in one direction using BigDye Terminator v.3.1 Cycle Sequencing chemistry kits (Applied Biosystems, Carlsbad, CA) on an ABI 3730 DNA Analyzer. Sequence chromatograms were analyzed using SeqScape v.2.1.1 or 2.5 (Applied Biosystems, Carlsbad, CA). Sequence variants were named and numbered by comparison to GenBank reference sequences NG_007489.1 and NM_020975.4 using standard nomenclature.23 Each rare sequence variant (MAF <0.05) and potentially pathogenic mutation was confirmed by sequencing in the opposite direction.

PCR Conditions

Each PCR reaction was carried out in a total volume of 25 µl, and consisted of an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 59, 61, 63, 67 or 68°C for 30 seconds (see also Supplementary Table 1), and elongation at 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. The exons 2-20 and 3' UTR PCR mixtures contained DNA Master HybProbe master mix (Roche, Indianapolis, IN), TaqStart antibody (Clontech Laboratories, Mountain View, CA), 2.5 mM MgCl2, and 10 µM of each primer. The exon 1 mixture was slightly different, in that it contained 3.5 mM MgCl2, 1M betaine and 2.5% formamide.

SNP Genotyping

Extracted DNA (≥30 ng) was whole-genome amplified in two separate rounds using a primer extension pre-amplification method. Both of the whole-genome amplifications for each individual were separately genotyped for each SNP. Genotyping was performed using KASPar chemistry, a competitive allele-specific PCR genotyping system. Quality control procedures involved the use of blank wells and repeat genotyping for approximately 4% of DNA samples.

Most SNPs were genotyped from whole-genome amplified DNA. SNP assays with high assay failure rates (>5%) were re-genotyped using non-amplified genomic DNA or directly sequenced.

All 42 SNPs genotyped were called successfully >95% of the time; the two RET exon 1 SNPs (rs10900296, rs10900297) sequenced in all cases and controls had 100% successful calls. There were no genotype discrepancies between samples from the two rounds of whole genome amplification. Only 1 of 4 620 genotypes was discordant upon repeat genotyping. There were three SNPs (rs1800858, rs1800861, rs2075912) sequenced in the RET gene (among all cases and 10 controls) that were also genotyped by KBiosciences (Herts, UK). Two cases had discordant genotypes for rs1800858 and one control had a discordant genotype for rs2075912.

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Supplementary Table 1. Primers for RET amplification

Exon / Forward Primer Sequence (5’>3’) / Reverse Primer Sequence (5’>3’) / Annealing Temp (°C) / Amplicon Size (bp)1 / Reference
1 / CTCCTCCCCAAGCCCCACC / CCCACGGCAAACAGAAAGG / 59 / 616 / This study
2 / AGCCTTATTCTCACCATCCC / ATAAGGGCGGCTTGAGGAAG / 63 / 406 / Chin et al 2008
3 / GGTTTACACCAGCCCTGGAG / TGTGTCAAGGGCTCGCAGAG / 61 / 489 / Chin et al 2008
4 / CTTCCCGAGGAAAGCGGCTG / CACGGACACTAAACCGACCG / 61 / 426 / Chin et al 2008
5 / CCTATGGGCTGTGTGGGACG / GAGCGAGCACCTCATTTCCT / 63 / 389 / F: This study;
R: Chin et al 2008
6 / CAGAGCAGCTTGGTGGTCA / AGTCTACTCTGTGCTGGTTGG / 63 / 382 / Chin et al 2008
7 / TCTACCCTCAGGCCATTACA / AACCATTTACTGCTGGGTCA / 59 / 516 / Ruiz-Ferrer et al 2006
8 / TGGTGCTGTTCCCTGTCC / CCACCGGTGCCATCGCCCCT / 67 / 295 / Ruiz-Ferrer et al 2006
9 / TAGAGGGGCAGGATCTGC / GCAACTCTGGCTGAAGTGC / 67 / 315 / Chin et al 2008
10 / AGAGAATGGTCAGTAGGGACACT / GGACCTCAGATGTGCTGTT / 61 / 564 / Chin et al 2008
11 / ATGAGGCAGAGCATACGCAG / TCCCTCCCTGGAAGGCAG / 61 / 433 / F: Chin et al 2008;
R: Ruiz-Ferrer et al 2006
12 / TTCTTCCTCCCCTGTCATCCT / TCTTCAGGGTCCCATGCTG / 61 / 340 / Ruiz-Ferrer et al 2006
13 / GCAGGCCTCTCTGTCTGAACTT / GGAGAACAGGGCTGTATGGA / 59 / 329 / Ruiz-Ferrer et al 2006
14 / AAGACCCAAGCTGCCTGAC / GCATGGTGGGCTAGAGTGTG / 67 / 424 / F: Ruiz-Ferrer et al 2006; R: This study
15 / CCCCCGGCCCAGGTCTCAC / GCTCCACTAATCTTCGGTATCTTT / 67 / 391 / Chin et al 2008
16 / CCTCCTTCCTAGAGAGTTAG / CCCCACTACATGTATAAGGG / 59 / 224 / Chin et al 2008
17 / GGCTCTGTGAGGGCCAGGT / GGGAGGGAATGCACACAGAT / 61 / 292 / F: Chin et al 2008;
R: Ruiz-Ferrer et al 2006
18 / GGCTGTCCTTCTGAGACC / ACTGCCCTGGGGTGAGGCT / 63 / 266 / Chin et al 2008
19 / TAGTTGTGGCACATGGCTTG / CAGAGCAGACTTTGGTTTTG / 59 / 346 / Chin et al 2008
20 / TGCACTTGAAGTTTTGGTTCTT / CTCAGAGCTCTTACCCGGTGT / 61 / 472 / Chin et al 2008
3’UTR-A / CCTAGCAGACATGCCACACC / CAATTCTGGGACTCCAACAGC / 68 / 690 / This study
3’UTR-B / CCGGGCCCACCACATCATC / AGCCCAAATTAGAGCCAGGTTACG / 61 / 868 / Chin et al 2008
3’UTR-C2 / GATGCGTGTGTAATAGAGCC / GAACATGATCTGGTTTAATGACC / 61 / 569 / This study

1Amplicon size includes an additional 33-bp due to the M13 tag added to each primer

2Approximately 90-bp of the 3’-most end of the 3’UTR was not captured by sequencing; there are no known polymorphisms in this region in dbSNP (build 130)

Supplementary Table 2. Candidate genes and SNPs

Gene
(location) / Gene name / Function / SNP1 / SNP location / Chromosomal location2
RET
(10q11.2) / ret proto-oncogene / Receptor tyrosine kinase that transduces signals for cell growth and development / rs10900296:GA / promoter / 43572507
rs10900297:CA / promoter / 43572511
rs1864410:CA / intron 1 / 43575625
rs2435357:CT / intron 1 / 43582056
rs1800858:GA (p.A45A) / exon 2 / 43595968
rs1800861:TG (p.L769L) / exon 13 / 43613843
rs2075912:CT / intron 19 / 43622217
ASCL1
(12q23.2) / achaete-scute complex homolog 1 (Drosophila) / Transcription factor involved in neuronal commitment and differentiation and in the generation of olfactory and autonomic neurons / rs9782:AG / 5’ UTR / 103351826
rs1391682:GA / 3’UTR / 103353525
rs2291854:CT / 3’UTR / 103353538
rs17450122:AG / 3’ near gene / 103354394
rs1874875:GC / 3’ near gene / 103354548
HOXB5
(17q21.3) / homeobox B5 / Transcription factor involved in lung and gut development / rs4793943:CG / 3’ near gene / 46666937
rs4793589:GC / 3’ near gene / 46667863
rs872760:TC / 3’UTR / 46668951
rs9299:AG / 3’UTR / 46669430
rs7406798:CT (p.Q175Q) / exon 1 / 46670520
rs1529334:TC / 5’ near gene / 46671792
L1CAM
(Xq28) / L1 cell adhesion molecule / Cell adhesion molecule involved in nervous system development, including neuronal migration and differentiation / rs4646266:CA / intron 19 / 153131957
rs5987173:GA (p.S769S) / exon 19 / 153132228
rs4646265:TC / intron 5 / 153137108
rs4646263:GA / intron 2 / 153140449
PHOX2B
(4p12) / paired-like homeobox 2b / Transcription factor involved in the development of several major noradrenergic neuron populations and the determination of neurotransmitter phenotype / rs11723860:GA / 3’UTR / 41746437
rs6826373:CT / 3’UTR / 41746460
rs2196822:AC / intron 2 / 41748511
rs6811325:CT / intron 1 / 41749668
rs4608840:CT / intron 1 / 41749938
PROK1
(1p21) / prokineticin 1 / Induces proliferation, migration, and fenestration in capillary endothelial cells derived from endocrine glands / rs12405277:AG / 5’ near gene / 110992681
rs1857512:GA / 5’ near gene / 110993349
rs4839391:GA / intron 1 / 110993990
rs884735:AT / intron 1 / 110994518
rs3795828:CT / intron 1 / 110996325
rs17628304:AC / intron 2 / 110996950
rs7534330:CT / intron 2 / 110997894
rs7513898:GA / intron 2 / 110998665
rs7514102:GA (p.V67I) / exon 3 / 110998854
rs1044837:CT / 3’UTR / 110999705
PROKR1
(2p13.1) / prokineticin receptor 1 / Receptor for prokineticin 1 / rs4854479:GC / 5’ near gene / 68871573
rs7570797:AG (p.S40G) / exon 2 / 68873071
rs12713655:AG / intron 2 / 68880286
rs4627609:TC / intron 2 / 68880436
rs6722313:TC (p.F314F) / exon 3 / 68882468
rs6731427:GA / 3’ near gene / 68884537
rs4854436:GA / 3’ near gene / 68884666

1Major allele is listed first

2Based on the human reference sequence: Genome Reference Consortium Human Build 37 (GRCh37.p5), February 2009

Abbreviation: UTR, untranslated region

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Supplementary Table 3. Minor allele frequencies of SNPs in candidate genes, amongHirschsprung’s disease cases with and without RET coding and splice-site variants.

SNP / RET coding and splice-site variants / P value1
Present
(N = 38) / Absent
(N = 263)
ASCL1
rs9782:AG / 0.276 / 0.335 / 0.31
rs1391682:GA / 0.026 / 0.029 / 0.91
rs2291854:CT / 0.250 / 0.192 / 0.24
rs17450122:AG / 0.079 / 0.061 / 0.54
rs1874875:GC / 0.197 / 0.224 / 0.60
HOXB5
rs4793943:CG / 0.118 / 0.141 / 0.60
rs4793589:GC / 0.118 / 0.141 / 0.60
rs872760:TC / 0.118 / 0.138 / 0.64
rs9299:AG / 0.431 / 0.488 / 0.36
rs7406798:C>T / 0.041 / 0.046 / 0.83
rs1529334:TC / 0.145 / 0.176 / 0.50
L1CAM (male)
rs4646266:CA / 0.043 / 0.047 / 0.93
rs5987173:GA / 0.000 / 0.016 / 0.54
rs4646265:TC / 0.304 / 0.238 / 0.49
rs4646263:GA / 0.435 / 0.363 / 0.50
L1CAM (female)
rs4646266:CA / 0.000 / 0.042 / -
rs5987173:GA / 0.000 / 0.007 / -
rs4646265:TC / 0.200 / 0.292 / 0.31
rs4646263:GA / 0.433 / 0.451 / 0.86
PHOX2B
rs11723860:GA / 0.194 / 0.224 / 0.58
rs6826373:CT / 0.351 / 0.388 / 0.54
rs2196822:AC / 0.270 / 0.354 / 0.16
rs6811325:CT / 0.276 / 0.356 / 0.13
rs4608840:CT / 0.276 / 0.356 / 0.17
PROK1
rs12405277:AG / 0.278 / 0.394 / 0.057
rs1857512:GA / 0.276 / 0.215 / 0.23
rs4839391:GA / 0.092 / 0.124 / 0.42
rs884735:AT / 0.184 / 0.146 / 0.39
rs3795828:CT / 0.184 / 0.251 / 0.20
rs17628304:AC / 0.289 / 0.372 / 0.16
rs7534330:CT / 0.026 / 0.013 / 0.38
rs7513898:GA / 0.408 / 0.481 / 0.23
rs7514102:GA / 0.303 / 0.406 / 0.083
rs1044837:CT / 0.171 / 0.114 / 0.15
PROKR1
rs4854479:GC / 0.184 / 0.208 / 0.63
rs7570797:AG / 0.224 / 0.105 / 0.0028
rs12713655:AG / 0.526 / 0.481 / 0.46
rs4627609:TC / 0.526 / 0.477 / 0.42
rs6731427:GA / 0.378 / 0.354 / 0.69
rs4854436:GA / 0.355 / 0.380 / 0.68
RET
rs10900296:GA / 0.355 / 0.492 / 0.025
rs10900297:CA / 0.342 / 0.279 / 0.26
rs1864410:CA / 0.329 / 0.458 / 0.035
rs2435357:CT / 0.351 / 0.471 / 0.052
rs1800858:GA / 0.342 / 0.473 / 0.032
rs1800861:TG / 0.368 / 0.346 / 0.70
rs2075912:CT / 0.289 / 0.306 / 0.77

1Chi-squared test for comparison of allele frequencies between Hirschsprung’s disease cases with and without RET coding and splice-site variants.

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Supplementary Table 4. RET SNP genotype and allele frequencies, by race/ethnicity1

SNP / All subjects / Non-Hispanic white / African-American / Hispanic / Asian
Case / Control / Case / Control / Case / Control / Case / Control / Case / Control
rs10900296:G>A
GG / 108 (35.9) / 721 (59.3) / 47 (28.3) / 381 (57.1) / 42 (68.9) / 184 (73.6) / 17 (34.0) / 117 (59.1) / 2 (9.1) / 35 (38.0)
GA / 100 (33.2) / 422 (34.7) / 56 (33.7) / 243 (36.4) / 17 (27.9) / 63 (25.2) / 20 (40.0) / 75 (37.9) / 6 (27.3) / 37 (40.2)
AA / 93 (30.9) / 72 (5.9) / 63 (38.0) / 43 (6.5) / 2 (3.3) / 3 (1.2) / 13 (26.0) / 6 (3.0) / 14 (63.6) / 20 (21.7)
MAF / 47.5 / 23.3 / 54.8 / 24.7 / 17.2 / 13.8 / 46.0 / 22.0 / 77.3 / 41.9
rs10900297:C>A
CC / 165 (54.8) / 428 (35.2) / 108 (65.1) / 260 (39.0) / 14 (23.0) / 66 (26.4) / 25 (50.0) / 60 (30.3) / 17 (77.3) / 42 (45.7)
CA / 99 (32.9) / 574 (47.2) / 45 (27.1) / 313 (46.9) / 31 (50.8) / 121 (48.4) / 17 (34.0) / 96 (48.5) / 5 (22.7) / 38 (41.3)
AA / 37 (12.3) / 213 (17.5) / 13 (7.8) / 94 (14.1) / 16 (26.2) / 63 (25.2) / 8 (16.0) / 42 (21.2) / 0 (0.0) / 12 (13.0)
MAF / 28.7 / 41.2 / 21.4 / 37.6 / 51.6 / 49.4 / 33.0 / 45.5 / 11.4 / 33.7
rs1864410:C>A
CC / 121 (40.2) / 768 (63.2) / 50 (30.1) / 385 (57.7) / 51 (83.6) / 218 (87.2) / 18 (36.0) / 126 (63.6) / 2 (9.1) / 35 (38.0)
CA / 90 (29.9) / 358 (29.5) / 54 (32.5) / 229 (34.3) / 9 (14.8) / 25 (10.0) / 20 (40.0) / 67 (33.8) / 6 (27.3) / 33 (35.9)
AA / 86 (28.6) / 64 (5.3) / 59 (35.3) / 38 (5.7) / 0 (0.0) / 2 (0.8) / 12 (24.0) / 4 (2.0) / 14 (63.6) / 20 (21.7)
MAF / 44.1 / 20.4 / 52.8 / 23.4 / 7.5 / 5.9 / 44.0 / 19.0 / 77.3 / 41.5
rs2435357:C>T
CC / 117 (38.9) / 767 (63.1) / 46 (27.7) / 380 (57.0) / 51 (83.6) / 222 (88.8) / 18 (36.0) / 126 (63.6) / 2 (9.1) / 35 (38.0)
CT / 91 (30.2) / 370 (30.5) / 55 (33.1) / 240 (36.0) / 8 (13.1) / 24 (9.6) / 21 (42.0) / 66 (33.3) / 6 (27.3) / 36 (39.1)
TT / 91 (30.2) / 71 (5.8) / 64 (38.6) / 45 (6.8) / 2 (3.3) / 2 (0.8) / 10 (20.0) / 4 (2.0) / 14 (63.6) / 20 (21.7)
MAF / 45.7 / 21.2 / 55.5 / 24.8 / 9.8 / 5.7 / 41.8 / 18.9 / 77.3 / 41.8
rs1800858:G>A
GG / 117 (38.9) / 758 (62.4) / 47 (28.3) / 377 (56.5) / 51 (83.6) / 219 (87.6) / 17 (34.0) / 122 (61.6) / 2 (9.1) / 36 (39.1)
GA / 93 (30.9) / 368 (30.3) / 56 (33.7) / 235 (35.2) / 8 (13.1) / 28 (11.2) / 22 (44.0) / 65 (32.8) / 6 (27.3) / 37 (40.2)
AA / 91 (30.2) / 71 (5.8) / 63 (38.0) / 45 (6.8) / 2 (3.3) / 2 (0.8) / 11 (22.0) / 6 (3.0) / 14 (63.6) / 18 (19.6)
MAF / 45.7 / 21.3 / 54.8 / 24.7 / 9.8 / 6.4 / 44.0 / 20.0 / 77.3 / 40.1
rs1800861:T>G
TT / 134 (44.5) / 716 (58.9) / 69 (41.6) / 376 (56.4) / 46 (75.4) / 192 (76.8) / 17 (34.0) / 116 (58.6) / 2 (9.1) / 27 (29.4)
TG / 124 (41.2) / 404 (33.3) / 75 (45.2) / 242 (36.3) / 14 (23.0) / 54 (21.6) / 27 (54.0) / 71 (35.9) / 7 (31.8) / 35 (38.0)
GG / 43 (14.3) / 89 (7.3) / 22 (13.3) / 48 (7.2) / 1 (1.6) / 3 (1.2) / 6 (12.0) / 11 (5.6) / 13 (59.1) / 27 (29.4)
MAF / 34.9 / 24.1 / 35.8 / 25.4 / 13.1 / 12.1 / 39.0 / 23.5 / 75.0 / 50.0
rs2075912:C>T
CC / 153 (50.8) / 800 (65.8) / 78 (47.0) / 435 (65.2) / 52 (85.3) / 201 (80.4) / 21 (42.0) / 126 (63.6) / 2 (9.1) / 32 (34.8)
TC / 113 (37.5) / 338 (27.8) / 71 (42.8) / 200 (30.0) / 8 (13.1) / 37 (14.8) / 25 (50.0) / 62 (31.3) / 8 (36.4) / 37 (40.2)
TT / 35 (11.6) / 48 (4.0) / 17 (10.2) / 19 (2.9) / 1 (1.6) / 2 (0.8) / 4 (8.0) / 7 (3.5) / 12 (54.6) / 20 (21.7)
MAF / 30.4 / 18.3 / 31.6 / 18.2 / 8.2 / 8.5 / 33.0 / 19.5 / 77.3 / 43.3

1Values for genotypes are N (%); values for minor allele frequency are %

Abbreviation: MAF, minor allele frequency
Supplementary Table 5. Genotype counts for SNPs in candidate genes for Hirschsprung’s disease1,2

SNP3 / All subjects / Non-Hispanic white / African-American / Hispanic / Asian
Case / Control / Case / Control / Case / Control / Case / Control / Case / Control
ASCL1
rs9782:A>G / 138/129/34 / 511/556/148 / 75/73/18 / 295/307/65 / 21/31/9 / 58/135/57 / 25/18/7 / 97/83/18 / 16/6/0 / 59/27/6
rs1391682:G>A / 284/17/0 / 1129/84/1 / 154/12/0 / 601/64/1 / 61/0/0 / 244/6/0 / 47/3/0 / 191/7/0 / 20/2/0 / 85/7/0
rs2291854:C>T / 192/98/11 / 758/398/59 / 113/49/4 / 414/227/26 / 31/25/5 / 132/95/23 / 29/19/2 / 139/51/8 / 17/5/0 / 69/21/2
rs17450122:A>G / 263/38/0 / 1046/161/8 / 141/25/0 / 548/114/5 / 56/5/0 / 244/6/0 / 45/5/0 / 169/28/1 / 19/3/0 / 78/12/2
rs1874875:G>C / 189/91/21 / 710/439/66 / 113/40/13 / 410/228/29 / 25/30/6 / 107/113/30 / 30/18/2 / 115/76/7 / 20/2/0 / 73/19/0
HOXB5
rs4793943:C>G / 226/67/8 / 904/282/26 / 114/48/4 / 454/189/22 / 56/5/0 / 212/37/0 / 36/10/4 / 151/44/3 / 19/3/0 / 81/10/1
rs4793589:G>C / 226/67/8 / 904/281/27 / 114/48/4 / 453/188/23 / 56/5/0 / 212/38/0 / 36/10/4 / 152/43/3 / 19/3/0 / 81/10/1
rs872760:T>C / 226/65/8 / 901/283/26 / 114/46/4 / 451/190/22 / 56/5/0 / 212/38/0 / 36/10/4 / 151/43/3 / 19/3/0 / 81/10/1
rs9299:A>G / 85/134/74 / 353/533/309 / 61/82/20 / 256/300/103 / 4/18/36 / 13/93/139 / 14/20/14 / 50/94/47 / 6/13/3 / 33/41/18
rs7406798:C>T / 272/23/2 / 1088/100/9 / 164/1/0 / 651/8/0 / 41/16/2 / 168/65/9 / 44/6/0 / 171/25/0 / 21/0/0 / 90/2/0
rs1529334:T>C / 204/79/11 / 823/346/37 / 105/50/7 / 420/213/28 / 48/11/0 / 176/69/3 / 31/14/4 / 142/52/4 / 19/3/0 / 81/9/1
L1CAM (male)
rs4646266:C>A / 203/10 / 603/20 / 113/1 / 330/3 / 41/4 / 137/8 / 32/5 / 89/7 / 16/0 / 42/2
rs5987173:G>A / 210/3 / 611/15 / 115/0 / 337/0 / 43/2 / 132/12 / 35/1 / 93/3 / 16/0 / 44/0
rs4646265:T>C / 160/52 / 469/155 / 110/4 / 322/13 / 13/31 / 39/107 / 20/17 / 61/33 / 16/0 / 42/2
rs4646263:G>A / 134/79 / 371/254 / 88/26 / 250/86 / 20/25 / 53/92 / 20/17 / 42/53 / 6/10 / 21/23
L1CAM (female)
rs4646266:C>A / 82/4/1 / 540/46/0 / 50/1/0 / 317/13/0 / 14/1/1 / 84/20/0 / 11/2/0 / 89/12/0 / 6/0/0 / 47/1/0
rs5987173:G>A / 85/1/0 / 567/15/1 / 50/0/0 / 330/0/0 / 15/1/0 / 86/13/1 / 13/0/0 / 100/2/0 / 6/0/0 / 48/0/0
rs4646265:T>C / 53/20/14 / 394/123/70 / 38/12/1 / 288/40/2 / 1/4/11 / 4/42/58 / 9/2/2 / 58/34/10 / 5/1/0 / 41/7/0
rs4646263:G>A / 29/37/20 / 229/242/116 / 23/22/5 / 184/123/23 / 1/8/7 / 19/38/47 / 3/5/5 / 21/57/24 / 2/1/3 / 4/22/22
PHOX2B
rs11723860:G>A / 178/98/15 / 665/416/72 / 114/43/4 / 399/212/24 / 24/28/7 / 93/112/31 / 26/19/4 / 118/59/11 / 13/7/0 / 49/31/6
rs6826373:C>T / 121/124/52 / 462/502/207 / 83/66/15 / 308/271/66 / 6/31/24 / 27/107/107 / 18/18/13 / 79/86/22 / 13/8/0 / 44/34/12
rs2196822:A>C / 133/124/40 / 548/502/162 / 90/61/12 / 347/266/52 / 8/33/19 / 53/117/79 / 20/21/9 / 97/82/19 / 14/8/0 / 46/34/12
rs6811325:C>T / 133/123/45 / 542/502/158 / 89/63/14 / 348/262/51 / 10/30/21 / 50/119/77 / 19/21/10 / 95/84/18 / 14/8/0 / 45/34/12
rs4608840:C>T / 136/122/43 / 558/505/152 / 91/61/14 / 350/267/50 / 10/31/20 / 59/118/73 / 20/21/9 / 98/83/17 / 14/8/0 / 46/34/12
PROK1
rs12405277:A>G / 116/34/45 / 455/545/198 / 54/81/28 / 219/328/106 / 39/18/1 / 148/88/12 / 19/18/13 / 60/78/59 / 4/16/2 / 26/46/20
rs1857512:G>A / 178/104/14 / 763/349/67 / 109/53/2 / 466/170/12 / 23/30/7 / 95/106/42 / 30/15/5 / 129/51/10 / 14/6/0 / 67/21/2
rs4839391:G>A / 232/64/4 / 953/229/28 / 134/31/0 / 563/96/5 / 53/8/0 / 205/40/4 / 31/16/3 / 119/61/17 / 13/8/1 / 60/30/2
rs884735:A>T / 213/80/5 / 861/271/46 / 108/52/4 / 433/184/34 / 48/12/0 / 212/25/2 / 39/10/1 / 144/42/4 / 16/6/0 / 67/18/5
rs3795828:C>T / 175/103/21 / 656/457/94 / 78/70/16 / 295/295/70 / 48/12/1 / 189/58/2 / 35/12/3 / 120/65/13 / 13/9/0 / 48/36/8
rs17628304:A>C / 121/140/38 / 452/554/198 / 57/82/26 / 223/330/110 / 39/21/1 / 141/91/14 / 20/21/8 / 61/81/54 / 5/15/2 / 25/47/19
rs7534330:C>T / 292/9/0 / 1156/46/5 / 165/1/0 / 663/3/0 / 54/7/0 / 204/34/5 / 49/1/0 / 189/9/0 / 22/0/0 / 92/0/0
rs7513898:GA / 84/149/67 / 311/572/330 / 56/78/32 / 215/337/114 / 10/33/18 / 33/100/117 / 14/21/14 / 38/85/74 / 4/16/2 / 24/45/23
rs7514102:G>A / 113/138/49 / 390/596/227 / 56/79/31 / 216/336/115 / 32/24/5 / 92/128/29 / 20/19/10 / 54/83/60 / 5/15/2 / 26/44/22
rs1044837:C>T / 231/67/3 / 902/275/33 / 131/35/0 / 537/119/8 / 41/18/2 / 138/93/18 / 40/9/1 / 149/43/5 / 17/5/0 / 71/19/2
PROKR1
rs4854479:G>C / 194/89/17 / 827/339/37 / 98/54/14 / 392/238/27 / 48/12/1 / 211/36/1 / 32/16/2 / 149/42/7 / 14/7/0 / 71/19/2
rs7570797:A>G / 237/56/8 / 947/212/49 / 147/19/0 / 601/59/4 / 30/25/6 / 89/115/42 / 38/10/2 / 161/34/3 / 20/2/0 / 89/3/0
rs12713655:A>G / 90/128/82 / 373/562/277 / 64/69/33 / 252/306/108 / 7/24/30 / 26/117/106 / 13/25/11 / 70/88/39 / 6/9/7 / 19/49/24
rs4627609:T>C / 91/129/81 / 375/567/273 / 64/70/32 / 253/310/104 / 7/24/30 / 27/116/107 / 14/25/11 / 70/90/38 / 6/9/7 / 19/49/24
rs6731427:G>A / 134/115/49 / 499/527/180 / 92/60/14 / 336/264/60 / 14/27/18 / 48/122/80 / 21/18/10 / 87/85/25 / 6/9/7 / 22/54/15
rs4854436:G>A / 126/122/52 / 458/514/240 / 50/75/41 / 155/339/170 / 45/14/2 / 190/53/7 / 18/23/8 / 64/82/52 / 12/9/1 / 48/37/7

1Genotype counts are numbers of subjects homozygous for major allele / heterozygous / homozygous for minor allele

2For SNPs in L1CAM, a gene on the X chromosome, values for males are numbers of subjects with each allele

3Major allele is listed first

Supplementary Table 6. Haplotype associations with Hirschsprung’s disease

Gene / SNPs in haplotype / Subject group / Haplotype / Control
frequency / Case frequency / Adjusted odds ratio
(95% confidence
interval)1 / P value
RET / rs10900296:G>A, rs10900297:C>A, rs1864410:C>A, rs2435357:C>T, rs1800858:G>A, rs1800861:T>G, rs2075912:C>T / All subjects / GACCGTC / 0.412 / 0.287 / Ref
ACCCGTC / 0.233 / 0.475 / 2.56 (2.06, 3.18) / 0.000
Non-Hispanic white / GCCCGTC / 0.378 / 0.238 / Ref
ACCCGTC / 0.247 / 0.548 / 3.04 (2.22, 4.18) / 6.4 x 10-12
African-American / GACCGTC / 0.494 / 0.516 / Ref
ACCCGTC / 0.138 / 0.156 / 1.00 (0.57, 1.78) / 0.990
Hispanic / GACCGTC / 0.455 / 0.330 / Ref
ACCCGTC / 0.139 / 0.020 / 0.24 (0.06, 0.98) / 0.047
ACCCGTC / 0.081 / 0.440 / 6.26 (3.28, 11.97) / 2.9 x 10-8
RET / rs10900296:G>A, rs10900297:C>A, rs1864410:C>A / Asian / ACA / 0.331 / 0.816 / Ref
GAC / 0.276 / 0.105 / 0.11 (0.03, 0.44) / 0.002
GCC / 0.196 / 0.079 / 0.08 (0.01, 0.48) / 0.006
ACC / 0.088 / 0.000 / 0.002 (0.001, 0.007) / 0.000
GAA / 0.061 / 0.000 / 0.03 (0.00, 0.69) / 0.028
HOXB5 / rs4793943:C>G, rs4793589:G>C, rs872760:T>C, rs9299:A>G, rs7406798:C>T, rs1529334:T>C / All subjects / CGTACT / 0.972 / 0.862 / Ref
GGTACT / 0.028 / 0.138 / 4.84 (3.31, 7.07) / 4.4 x 10-16
Hispanic / CGTACT / 0.874 / 0.820 / Ref
GCCACT / 0.076 / 0.180 / 2.36 (1.30, 4.26) / 0.005
ASCL1 / rs9782:A>G, rs1391682:G>A, rs2291854:C>T, rs17450122:A>G, rs1874875:G>C / African-American / GGCAC / 0.256 / 0.174 / Ref
AGCAG / 0.246 / 0.324 / 1.97 (1.05, 3.68) / 0.035
AGTAC / 0.045 / 0.100 / 3.44 (1.25, 9.46) / 0.017
AGTGG / 0.012 / 0.025 / 5.44 (1.68, 17.66) / 0.005
Hispanic / AGCAG / 0.547 / 0.517 / Ref
GGTAG / 0.032 / 0.077 / 3.00 (1.09, 8.28) / 0.034
AGTAC / 0.007 / 0.029 / 5.66 (1.02, 31.33) / 0.047

1Reference is most frequent haplotype in controls