1. FaceBase Kick-Off Meeting Nov 15-16, 2009 Bethesda Oral Clefts: Moving from Genome Wide Studies Toward Functional Genomics TH Beaty for Alan F Scott, Ingo Ruczinski, Hong Kai Ji, Kung Yee Liang Johns Hopkins University

2. 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

3. 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 SiteCL Trios Complete (Incomplete) CLP Trios Complete (Incomplete) CP Trios Complete (Incomplete) Total Trios Complete (Incomplete) Utah68(16)96(20)52(12)216 (48) Norway106(4)174(8)107(3)387 (15) Korea19(0)40(2)5(0)64 (2) Maryland19(12)71(42)25(18)115 (72) Pittsburgh26(2)70(28)11(4)107 (34) Singapore15(1)45(7)53(4)113 (12) Taiwan42(4)176(11)74(5)292 (20) Iowa16(9)29(11)24(17)69 (37) Denmark6(15)15(12)5(8)26 (35) Philippines0(0)94(4)0(0)94 (4) WuHan39(3)136(9)42(3)219 (15)* Shandong Prov.54(21)129(70)30(8)215 (101)* Western China43(3)63(3)38(2)144 (8) Total453(90)1138(227)466(84)2060 (403)* *total includes probands of indeterminate cleft type: 2 in WuHan; 3 in Shandong Prov.

4. Genome wide significant Genome wide search turns up several “interesting” genes/regions

5. – but strength of evidence varies between Europeans & Asians

6. 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

7. 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

8. Recognized Candidate Genes: IRF6 Estimated 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 SNPLocOR(case)95%CIp-valueMA*Overall MAF Euro Freq. Asian Freq. rs10445162080262370.755(0.677,0.842)4.29E-07A 0.3490.1750.514 rs20734852080294170.689(0.615,0.771)7.86E-11A 0.2990.1740.423 rs20131622080353070.705(0.636,0.782)2.82E-11A 0.4270.3530.514 rs22369062080381081.098(0.987,1.223)0.086A 0.3290.4070.256 rs8610202080437341.432(1.274,1.609)1.62E-09G 0.2410.2390.230 rs173895412080537950.899(0.775,1.043)0.161G 0.1380.2010.087 rs108637902080546700.580(0.504,0.667)1.91E-14C 0.1920.0190.363 *the target allele was defined as the minor allele (MA) among parents of European ancestry

10. Genes identified from GWAS 1.Statistical evidence requires follow-up 2.Direct investigation of genes & regions showing through sequencing 3.Next generation sequencing techniques are evolving rapidly

11. 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 genotypes 2.Multiplex PCR (RainDance Technology) – 4000 exons for CEPH samples 3.Agilent Sure Select (RNA baits  55K per assay for 2Mb of sequence) 4.DNA hybridization chip (Febit, Germany  500kb-1Mb)

13. RainDance Technology Chr. 1 SNPs Comparing sequence to 1000 genomes genotypes & sequence

14. PositionGeneRefer ence MultiplexFREQCove rage Varian t 1 Varian t 2 1000 GEN SEQ FORdbSNP hg18Filteredcounts GENO- TYPE NA12878 3,318,218PRDM16TC99.3144143-Crs870124 (missense) 3,318,519PRDM16CC/T60.9/39.1462818C/T rs2493292 (missense) 3,319,244PRDM16CC/T50.0/50.01266-C/Trs2493291 3,331,969PRDM16GG/T52.3/47.7442321-G/Trs41303861 3,332,664PRDM16GG/T65.8/31.6382512G/T rs870171 6,106,679CHD5AG96.79188A/GGrs2794358 6,106,779CHD5AG/A65.6/34.4322111A/GGrs2273041 6,108,119CHD5GA/G50.0/46.7301514A/GArs2273037 6,111,133CHD5GA84.62622-Ars2273034 6,111,366CHD5GC81.13730G/CCrs2273033 6,118,080CHD5GA91.32321A/GArs2250504 6,126,853CHD5AG10015 A/GGrs9435102 6,133,770CHD5GA89.32825A/GArs9434711 6,138,323CHD5CC/G52.8/47.21618576C/GCrs12074369 Magnified view of RDT results

15. 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

16. 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. ABCA4

17. 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