FaceBase Kick-Off MeetingNov 15-16, 2009

Bethesda

Oral Clefts: Moving from Genome Wide Studies Toward Functional Genomics

TH Beaty forAlan F Scott, Ingo Ruczinski, Hong Kai Ji, Kung Yee Liang

Johns Hopkins University

Starting point: Genome wide association study (GWAS) of oral clefts• Cleft consortium supported by U01-DE-004425

(2007-2009)– Part of GENEVA collaboration

• Murray (UIowa), Marazita (UPittsburgh), Munger (USU), Wilcox/Lie (NIEHS/UBergen)

• Case-parent trio design to test for – Gene effects– Gene-environment interaction– Parent of origin effects

Isolated, non-syndromic cleft cases & their parents from 13 populations

Table 1: Case-parent trios from 13 recruitment sites in the International Cleft Consortium Recruitment Site CL Trios

Complete(Incomplete)

CLP Trios Complete (Incomplete)

CP Trios Complete (Incomplete)

Total Trios Complete (Incomplete)

Utah 68(16) 96(20) 52(12) 216 (48)Norway 106(4) 174(8) 107(3) 387 (15)Korea 19(0) 40(2) 5(0) 64 (2)Maryland 19(12) 71(42) 25(18) 115 (72)Pittsburgh 26(2) 70(28) 11(4) 107 (34)Singapore 15(1) 45(7) 53(4) 113 (12)Taiwan 42(4) 176(11) 74(5) 292 (20)Iowa 16(9) 29(11) 24(17) 69 (37)Denmark 6(15) 15(12) 5(8) 26 (35)Philippines 0(0) 94(4) 0(0) 94 (4)WuHan 39(3) 136(9) 42(3) 219 (15)*Shandong Prov. 54(21) 129(70) 30(8) 215 (101)*Western China 43(3) 63(3) 38(2) 144 (8)Total 453(90) 1138(227) 466(84) 2060 (403)*

*total includes probands of indeterminate cleft type: 2 in WuHan; 3 in Shandong Prov.

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

2

4

6

8

10

12

14

16

-log1

0(O

bser

ved

p)

1908 CL/P Trios

Genome wide

significant

Genome wide search turns up several

“interesting” genes/regions

– but strength of evidence

varies between

Europeans & Asians

CHR 8q24: Estimated genotypic relative risk (GRR) & 95%CI under an additive model obtained from a conditional logistic regression model for 78 SNPs in chr. 8q24 showing genome wide significance. The SNP showing the strongest signal was rs987525 (denoted by the star) is identical to that reported by 2 previous studies.

Asian European

Statistical signal from 78 SNPs in 8q24 and pairwise LD for A) CL/P case-parent trios of European ancestry; B) CL/P case-parent trios of Asian ancestry.

A B

Recognized Candidate Genes: IRF6Estimated OR(case) for 7 SNPs in IRF6 fit under an additive model plus minor allele and its overall frequency and frequency among parents of European and Asian CL/P cases

SNP Loc OR(case) 95%CI p-value MA* Overall MAF

Euro Freq.

Asian Freq.

rs1044516 208026237 0.755 (0.677,0.842) 4.29E-07 A 0.349 0.175 0.514rs2073485 208029417 0.689 (0.615,0.771) 7.86E-11 A 0.299 0.174 0.423rs2013162 208035307 0.705 (0.636,0.782) 2.82E-11 A 0.427 0.353 0.514rs2236906 208038108 1.098 (0.987,1.223) 0.086 A 0.329 0.407 0.256rs861020 208043734 1.432 (1.274,1.609) 1.62E-09 G 0.241 0.239 0.230

rs17389541 208053795 0.899 (0.775,1.043) 0.161 G 0.138 0.201 0.087rs10863790 208054670 0.580 (0.504,0.667) 1.91E-14 C 0.192 0.019 0.363*the target allele was defined as the minor allele (MA) among parents of European ancestry

Genes identified from GWAS

1. Statistical evidence requires follow-up2. Direct investigation of genes & regions

showing through sequencing3. Next generation sequencing techniques

are evolving rapidly

Evaluating target capture for Next Gen Sequencing

1. Long range PCR techniques– We sequenced 40kb of IRF6 in 4 individuals and

compared results to SNP genotypes2. Multiplex PCR (RainDance Technology)

– 4000 exons for CEPH samples3. Agilent Sure Select (RNA baits 55K per

assay for 2Mb of sequence)4. DNA hybridization chip (Febit, Germany

500kb-1Mb)

RainDance Technology Chr. 1 SNPsPosition Gene Reference Multiplex FREQ Coverage Variant 1 Variant 2 1000 GEN SEQ FOR dbSNP

hg18 Filtered counts counts GENOTYPE NA128783,318,218 PRDM16 T C 99.3 144 143 - C rs870124 (missense)3,318,519 PRDM16 C C/T 60.9/39.1 46 28 18 C/T C/T rs2493292 (missense)3,319,244 PRDM16 C C/T 50.0/50.0 12 6 6 - C/T rs24932913,331,969 PRDM16 G G/T 52.3/47.7 44 23 21 - G/T rs413038613,332,664 PRDM16 G G/T 65.8/31.6 38 25 12 G/T G/T rs8701716,106,679 CHD5 A G 96.7 91 88 A/G G rs27943586,106,779 CHD5 A G/A 65.6/34.4 32 21 11 A/G G rs22730416,108,119 CHD5 G A/G 50.0/46.7 30 15 14 A/G A rs22730376,111,133 CHD5 G A 84.6 26 22 - A rs22730346,111,366 CHD5 G C 81.1 37 30 G/C C rs22730336,118,080 CHD5 G A 91.3 23 21 A/G A rs22505046,126,853 CHD5 A G 100 15 15 A/G G rs94351026,133,770 CHD5 G A 89.3 28 25 A/G A rs94347116,138,323 CHD5 C C/G 52.8/47.2 161 85 76 C/G C rs120743696,180,443 RPL22 G G/A 63.6/36.4 11 7 4 A/G A/G rs2294713

11,127,645 FRAP1 C T/C 63.6/36.4 22 14 8 C/T T rs105707917,243,739 SDHB T C 100 11 11 C/T C rs1207302823,081,512 EPHB2 G G/A 59.7/40.3 134 80 54 A/G A/G rs3606987927,558,650 MAP3K6 G C 100 21 21 G/C C rs752155243,584,662 MPL G A/G 53.3/46.7 8 7 15 A/G A/G rs176067043,587,451 MPL T T/C 63.0/37.0 27 17 10 - T rs83999543,808,059 PTPRF G A 76 25 19 A/G A rs94351343,843,278 PTPRF A G 82.9 35 29 A/G G rs1737190343,847,814 PTPRF T C 95.2 21 20 C/T C rs51762243,851,998 PTPRF T C 92.6 81 75 C/T C rs60354243,856,094 PTPRF T C 96.2 105 101 C/T C rs64136543,856,106 PTPRF G A 97.5 81 79 G/A A rs64135143,858,667 PTPRF T C 100 20 20 C/T C rs56863943,860,083 PTPRF C T 98 100 98 C/T T rs67348545,570,092 MUTYH C C/G 56.4/43.6 39 22 17 C/G C/G rs3219489 (GLN-HIS)45,571,142 MUTYH T C 100 22 22 C/T C rs321948747,462,429 TAL1 C C/G 60.0/40.0 10 6 4 C/G C/G rs3408721065,083,802 JAK1 G G/C 50.9/48.1 108 55 52 C/G C/G rs373713985,514,509 BCL10 G C 96.2 26 25 - C rs273559485,514,600 BCL10 G C 95.9 121 116 C/G C rs11576939

110,683,967 RBM15 A G 94.7 94 89 - G rs814771143,641,953 PDE4DIP C A/C 52.9/47.1 17 9 8 - A/C rs2762867143,642,297 PDE4DIP T T/C 54.2/45.8 24 13 11 - C/T rs2147326 (LYS-GLU)143,706,015 PDE4DIP C C/A 54.1/44.3 368 199 163 - C/A rs1664022143,706,218 PDE4DIP C T 78.4 102 80 - C/T rs2442640143,787,211 PDE4DIP G C/G 61.4/38.6 83 51 32 - C/G rs1663853153,428,691 MUC1 C C/T 51.4/48.6 105 54 51 C/T C/T rs4072037155,018,672 PRCC C G 88.6 35 31 C/G G rs2274503155,112,398 NTRK1 C C/T 66.1/33.9 56 37 19 C/T C/T rs72698669155,112,857 NTRK1 G G/A 59.0/41.0 78 46 32 G/A G/A rs6334155,115,418 NTRK1 C C/T 67.9/32.1 28 19 9 C/T C/T rs2768756155,115,432 NTRK1 T C 86.7 15 13 - C rs2768757155,115,619 NTRK1 C C/T 64.0/36.0 125 80 45 - C/T rs6337155,816,690 IRTA1 T C 83.9 31 26 C/T C rs10489673155,825,746 IRTA1 C T 86 43 37 - T rs11582663 (ARG-GLN)155,834,375 IRTA1 A G 100 14 14 - G rs1361889163,035,812 PBX1 A A/C 62.1/31.8 66 44 21 - A NEW173,565,924 TNR T T/C 54.1/45.4 196 106 89 C/T C/T rs2027867173,590,069 TNR A A/G 58.8/41.2 34 20 14 A/G A/G rs2301432173,621,751 TNR C G/C 58.8/41.2 17 10 7 C/G G rs859436173,622,014 TNR A G 92.3 13 12 A/G G rs859437200,543,117 LGR6 C T 90.7 75 68 C/T T rs896551200,553,829 LGR6 T C 71.9 32 23 C/T C rs788795 (VAL-ALA)200,554,160 LGR6 G A 81 58 47 - A rs788794200,554,436 LGR6 T C/T 60/40 20 12 8 C/T C rs788793212,624,851 PTPN14 A G/A 60/40 10 6 4 - A rs3013446212,704,795 PTPN14 C T 100 10 10 C/T T rs10864100225,248,546 CDC42BPA C C/T 57.6/42.4 33 19 14 C/T C/T rs3795451225,248,656 CDC42BPA G A 99 104 103 G/A A rs2802269 (ALA-VAL)225,249,198 CDC42BPA A G 100 34 34 A/G G rs1045247226,466,389 OBSCN T T/C 56.7/43.3 97 55 42 - C/T rs55971985226,468,670 OBSCN A G/A 51.3/48.7 78 40 38 - G/A rs1620111226,468,744 OBSCN A G/A 56/44 50 28 22 G/A G/A rs1771487 (GLN-ARG)226,469,131 OBSCN C T/C 56/44 50 28 22 T/C C/T rs2776853226,471,291 OBSCN T T/C 53.2/46.8 124 66 58 C/T C/T rs3795777226,478,729 OBSCN T C/T 68.2/31.8 22 15 7 C/T C/T rs1771482226,478,931 OBSCN G G/A 62.5/37.5 56 35 21 G/A G/A rs1757152226,510,941 OBSCN T G/T 61.1/38.9 18 11 7 G/T G/T rs7532144226,511,188 OBSCN T A 97.4 38 37 T/A A rs7532342 (VAL-ASP)226,531,465 OBSCN C C/G 58.6/41.4 145 85 60 C/G C/G rs936885226,537,669 OBSCN C C/T 53.3/46.7 15 8 7 C/T C/T rs41303079226,546,905 OBSCN A G/A 56/44 50 28 22 A/G A/G rs1150908226,561,413 OBSCN G A/G 51.9/48.1 81 42 39 G/A G/A rs435776 (GLY-ARG)226,563,676 OBSCN A G/A 65/35 20 13 7 G/A G/A rs379620226,570,300 OBSCN A A/G 51.7/48.3 29 15 14 G/A G/A rs1150912 (HIS-ARG)226,571,095 OBSCN T T/C 69.7/30.3 66 46 20 - C rs1188732 (CYS-ARG)226,571,293 OBSCN C C/T 52.9/47.1 34 18 16 - T rs11810627 (ARG-TRP)226,572,291 OBSCN C G/C 51.4/48.6 105 54 51 G/C C rs1188729 (SER-CYS)226,572,322 OBSCN T C/T 56.7/43.3 104 59 45 C/T C/T rs1188728226,576,050 OBSCN A G/A 51.9/46.6 131 68 61 G/A G/A rs373610 (ASP-GLY)226,588,670 OBSCN A T/A 63.6/36.4 11 7 4 - A/T rs520552226,590,241 OBSCN G G/A 55/45 20 11 9 G/A G/A rs1757160226,591,542 OBSCN G G/A 55.7/44.3 70 39 31 G/A G/A rs3795807226,591,584 OBSCN C C/A 54.9/44.6 175 96 78 C/A C/A rs3795808226,593,109 OBSCN G G/A 63.6/36.4 33 21 12 G/A G/A rs58480216226,593,288 OBSCN T T/C 63.8/36.2 196 125 71 - C/T rs505629

Comparing sequence to

1000 genomes genotypes &

sequence

Position Gene Reference

Multiplex FREQ Coverage

Variant 1

Variant 2

1000 GEN

SEQ FOR dbSNP

hg18 Filtered counts counts GENO-TYPE

NA12878

3,318,218 PRDM16 T C 99.3 144 143 - C rs870124 (missense)

3,318,519 PRDM16 C C/T 60.9/39.1 46 28 18 C/T C/T rs2493292 (missense)

3,319,244 PRDM16 C C/T 50.0/50.0 12 6 6 - C/T rs2493291

3,331,969 PRDM16 G G/T 52.3/47.7 44 23 21 - G/T rs41303861

3,332,664 PRDM16 G G/T 65.8/31.6 38 25 12 G/T G/T rs870171

6,106,679 CHD5 A G 96.7 91 88 A/G G rs2794358

6,106,779 CHD5 A G/A 65.6/34.4 32 21 11 A/G G rs2273041

6,108,119 CHD5 G A/G 50.0/46.7 30 15 14 A/G A rs2273037

6,111,133 CHD5 G A 84.6 26 22 - A rs2273034

6,111,366 CHD5 G C 81.1 37 30 G/C C rs2273033

6,118,080 CHD5 G A 91.3 23 21 A/G A rs2250504

6,126,853 CHD5 A G 100 15 15 A/G G rs9435102

6,133,770 CHD5 G A 89.3 28 25 A/G A rs9434711

6,138,323 CHD5 C C/G 52.8/47.2 161 85 76 C/G C rs12074369

Magnified view of RDT results

MAFB on chr 20q11: A total 18 SNPs in the 3’UTR 20kb from MAFB showed genome wide significance (left). Estimated genotypic relative risk (GRR) & 95%CI under an additive model are shown over the region of signal.

MAFB

ABCA4 on chr 1q22 spans 150kb & showed multiple SNPs attaining genome wide significance ( left). Estimated GRR from a conditional logistic regression model & 95%CI for each SNP in this region.

AB

CA

4

Hopkins FaceBase Project

• Follow genes of interest by Next Gen Sequencing– This will include

• Genes controlling risk alone• Genes involved in GxE interaction with common maternal

exposures• Genes exhibiting parent-of-origin effects that may represent

genomic imprinting

• Analyze copy number variants using monomorphic markers in regions of CNV available from this marker panel