Kerkel et al., Supplementary Tables and Figures Table S1. Summary of 16 SNP tagged loci from the combined 50K and 250K MSNP screens with independently validated ASM. PBL, total peripheral blood leukocytes; CD34+, hematopoietic stem cells; NBM, normal bone marrow. US, upstream; DS, downstream. ASM is graded qualitatively as strong (++) or weak to moderate (+) based on the pre digestion/pcr assays. SNP array Affymetrix probeset SNP ID 50K XbaI SNP_A 1675862 rs1042073 3 Chr. Tissue(s) with validated ASM PBL, placenta, kidney, brain 1 Gene region ASM Genotypedependent LTF, exon 13 ++ (frequent) yes 250K SNP_A 1796232 rs3844442 19 PBL, liver CYP2A7, 27 kb US of exon 1 ++ (frequent) yes 250K SNP_A 2085305 rs12205095 6 PBL, CD34+ VNN1, 2kb US of exon 1 ++ (frequent) yes 50K XbaI SNP_A 1721658 rs9258170 6 PBL, placenta, kidney, brain LA F, 2kb US of exon 1 ++ (frequent) yes 250K SNP_A 2225326 rs11161318 15 PBL MAGEL2, 0.5kb DS ++ (frequent) no 250K SNP_A 4294342 rs1438846 2 PBL, CD34+ EN1 MARCO, intergenic + (rare) no 250K SNP_A 1862698 rs9951893 18 PBL, NBM PLPP BCL2, intergenic + (frequent) yes 250K SNP_A 1895330 rs4925109 17 PBL RAI1, intron 2 + to ++ (frequent) 250K SNP_A 2243309 rs443731 X placenta EFNB1, 11kb DS + (frequent) yes 250K SNP_A 2052954 rs6969642 7 PBL DNAJB6, intron 1 + to ++ (frequent) 250K SNP_A 4287008 rs12873012 13 PBL s.493062 /// POU4F1 ++ (frequent) yes 250K SNP_A 4250374 rs9366927 6 PBL FGD2 PIM1, intergenic ++ (frequent) yes 250K SNP_A 1859378 rs6494120 15 PBL, CD34+ GCNT3, 9kb US of exon 1 ++ (frequent) yes 250K SNP_A 4286211 rs17162992 7 placenta TCRB V5 1 region, 2kb DS ++ (rare) no 250K SNP_A 2065646 rs943049 13 PBL, NBM PARP4 ATP12A, intergenic ++ (frequent) yes 250K SNP_A 4303161 rs206337 13 placenta CG018, intron 1 ++ (frequent) no yes yes
Table S2. Genotype dependence of allele specific mrna expression (ASE) of the CYP2A7 gene. Genotypes are shown for 8 SNPs distributed from exon 2a to exon 6 of the CYP2A7 gene in 17 livers heterozygous for the rs3815710 SNP, which was used for determining ASE (see Fig. 2 of the main text). For the most informative SNP, rs3815706 (in bold), the association of the T/T genotype with unbiased allelic expression (C=A) or slightly more expression of the A allele (C<A) was significant at p=.01 by Fisher s exact test. The ASE score, assigned as C=A or C<A, score = 1; C>>A, score = 2, was used for classifying the cases into 2 groups for this statistical test. Liver ID ASE rs3815710 Exon 2b ASE score rs10425176 a Exon 2a A Ile/T Phe aa 61 rs10425169 Exon 2a G Arg/A Cys aa 64 rs3815711 Exon 2b A Val/G Ala aa 117 rs3815710 Exon 2b C Arg/A Leu aa 128 rs3815709 Exon 2b rs3815708 Exon 2b A Ser/C Ala aa 131 rs3815706 Intron 3 rs2261144 Exon 6 G Thr/A Met aa 368 16 C<A 1 A/T A/G A/G A/C A/G A/C T/T A/G 17 C=A 1 A/T A/G A/G A/C A/G A/C T/T A/G 1 C=A 1 A/T A/G A/G A/C A/G A/C T/T A/G 13 C=A 1 A/T A/G A/G A/C A/G A/C T/T A/G 12 C=A 1 A/T A/G A/G A/C A/G A/C T/T A/G 14 C=A 1 A/T A/G A/G A/C A/G A/C T/T A/G 11 C=A 1 A/T A/G A/G A/C A/G A/C T/T A/G 18 C>>A 2 A/T A/G A/G A/C A/G A/C G/T A/A 19 C>>A 2 A/T A/G A/G A/C A/G A/C G/T A/A 4 C>>A 2 A/T A/G A/G A/C A/G A/C T/T A/A 10 C>>A 2 A/T A/G A/G A/C A/G A/C G/T A/A 7 C>>A 2 A/T A/G A/G A/C A/G A/C G/T A/G 20 C>>A 2 A/T A/G A/G A/C A/G A/C T/T A/G 3 C>>A 2 A/T A/G A/G A/C A/G A/C G/T A/G 2 C>>A 2 A/T A/G A/G A/C A/G A/C G/T A/G 8 C>A 2 A/T A/G A/G A/C A/G A/C G/T A/A 9 C>A 2 A/T A/G A/G A/C A/G A/C T/T A/G 2
Table S3. PCR primers and conditions used in this study. Primers for validating ASM by paii pre digestion/pcr. Gene/Region Index SNP RFLP or indel SNP Forward Primer Reverse Primer Anneal. Temp ( o C) LTF rs1042073 TCTCTGCACCCTTCACACTG ACTCCCCGTGTTCCTTCTCT 63.4 54.4 788 BsrDI LAF a rs3998799 CGTGAACCCGGGACGCAGA GACCCCAACCTCCGCGTCT 67.8 58.8 994 XbaI PLPP_BCL2 rs9951893 CAAAAGGCCCCACTTCCTAT CCTTTCCTCTGCCAACTCAG 61 52 554 SspI EN1_MARCOS rs1438846 TGCTCAGCTTTGCCAGTAGA ATGAAGTTTGCCCAGGTGAC 65 56 369 infi GCNT3 rs6494120 AGGAAAGATGGGGGTTGGTTAG AGCCTGGTAGAAGCAAGATGGAG 63 58 528 BstNI POU4F1 rs12873012 CTGCTAATTGAACCCAGACA TGATGGGTGTGTATAAATCCA 55 50 1307 BsrBI MAGEL2 rs11161318 CAGTTCATTCCCTTGTCCCTTTC ATAACCAGGTGGTCCCGCTTC 63 58 409 BtsI DNAJB6 rs6969642 CACTTTGAAGCCCACATTCTCG TCCCATCCAAGCACTAACCAGG 62 57 649 RsaI VNN1 rs12205095 rs6569826 GTTGTGGTTGGCTGGTTCTCC GGTTGGCATTTTTCCTTCCCC 63 58 615 TaqI PARP4 ATP12A rs943049 ACATGAACCTGTGGGCTACC TGCACGGTCTTTATTTGTGG 56 51 391 BfaI EFNB1 rs443731 CATGAGGCTTCTAGGGCACAAC ACTGGGGAGTCAGAGACTGTTC 65 60 705 BsmI RAI1 rs4925109 ACCTGGGCACTCAGCAAAC AAGCCAGATGCGACAGGGATTG 64 55 418 indiii TCRB rs17162992 GCAGACCTAACGGAATCTGC AGCCCCCATTCTTTCACAC 58 53 427 BfaI CYP2 cluster index region rs3844442 rs34724660 GCTGTGCCGTCATCTCCTTTTTAG TACTCTCAACAATCCCCCAACCCG 65 56 511 sequence CYP2A7 exon 2b b n.a. rs34039986 TTTCCTTCTCCTGCCCCCGCACTC TCTCTTTCTCTCCCCACTTCCTAC 64 58 546 sizing gel FGD2 PIM1 rs9366927 GCATGTCCCTGGATGAC GTGGAGGCTTTTTCCAG 54 51 334 sequence CG018 rs206337 GGACCACTTCCTCTACTGAACAG GCAGCCCGCTTTGAAAATCTG 65 61 593 sequence a Restriction analysis of PCR products showed 1 polymorphic paii site between these LA F primers; however ASM affects multiple CpGs in this region as indicated by bisulfite sequencing (Supplementary Fig. S2). b There is 1 polymorphic paii site between these PCR primers; however ASM affected at least 1 of the 2 non polymorphic paii sites that are also between the primers and multiple additional CpGs, as indicated by bisulfite sequencing (Fig. 2 in main text). Primers for making the LTF exon 13 Southern blotting probe. size (bp) RFLP Gene/Region Index SNP Forward Primer Reverse Primer Anneal. Temp ( o C) size (bp) LTF exon 13 rs1042073 TGTGACTGAGCTCCATCAGGA CCTTCACACTGGTAAATAGTGC 62 57 606 3
Primers for bisulfite conversion/pcr. The corresponding unconverted sequences are shown to allow reference to the genomic sequence. Gene/Region SNP Forward Primer Reverse Primer Anneal. Temp( o C) LAF converted rs3998799 GGGGTTAAAGTAATAGTTTTTATATATAAG TAATAAAATCTTAATAACCCCTAAATTATC 48 39 384 LAF unconverted rs3998799 GGCCAAAGTAACAGTTTTTACACAT AGAATCTTGGTAACCCCTGAATTA n.a. n.a. VNN1 converted rs12195735 GGGAAAGGGAAAATTTTAGTTTTA ATCAACAATTTAAAACCAACCTA 57 48 284 VNN1 unconverted rs12195735 GGGAAAGGGAAAATCCCAGCTCTA GTCAGCAATTTGAGACCAGCCTG n.a. n.a. CYP2A7 Ex2b converted a rs3815710 TTAGGAGGTAGGGTTTTGTTGAGTT; AGGGTTTTGTTGAGTTAAATTTTT TTAAAAAACCCCTTAAAACTAATCC 60 51 387 Product (bp) CYP2A7 Ex2b unconverted rs3815710 CCAGGAGGCAGGGCCTTGTTGAGCC; AGGGCCTTGTTGAGCCAAATTCCC TTGGGGAGCCCCTTGGAACTGGTCC n.a. n.a. CYP2A7 index SNP region rs34724660 AAGGGAGTATTAGTTGGTGGTTAA CTCCCTCCCAAAACATAAAAATATT 57 48 303 CYP2A7 index SNP region unconverted rs34724660 AAGGGAGTATCAGCTGGTGGCTCAA CTCCCTCCCAGGGCATAGAAGTGTT a Semi nested bisulfite PCR: outer forward primer; inner forward primer. PCR products were successfully obtained using both nested and non nested PCR strategies. Primers for analyzing allele specific mrna expression (ASE). Initial sequencing of PCR products generated using outer primers confirmed that all primer binding sites were free of SNPs. Gene SNP FOR Primer REV Primer Anneal. Temp( o C) VNN1 cdna rs2294757 CCAGCTTACGTGGCAATTTT GCCACCAGTTTTCCTTGAGA 62 57 506 VNN1 genomic rs2294757 TAATGCCAAAGCCTCCTCAC GCAGCCCTAGCGAACTAATG 60 55 372 VNN2 cdna rs1883613 CCTCAGGTGAACTGGATTCCG GGTTTGATGGTGGTGGCGTAG 63 54 680 VNN2 genomic rs1883613 GATACCGTGTGGGAAGCATT GGTAACGTGCCACGAGTTTT 58 53 373 VNN3 cdna rs2294759 TACCCTGCAGCTGTTGACTG CGTTTCCAGGTCAGTGGTTT 58 53 303 VNN3 genomic rs2294759 ATGTTGCTTGTAGGGAGTGGAATC GCTTTTGAAACCTGAACCTGGC 64 55 641 CYP2A7 genomic rs3815710 TATTGGCGCCTGCGGGTGTGGA GGAGCCCCTTGGAACTGGTC 64 58 469 CYP2A7 cdna rs3815710 GAACACAGAGCACATATGT TCCTGAGGGTTGGAGAAGAAGC 61 52 1083 n.a. n.a. Product (bp) **All primer binding sites were checked for the presence of SNPs. 4
Figure S1. Analysis of trios showing that ASM in LTF intron 13 is not due to parental imprinting. Trios of parental blood DNA and placentas (fetal surface) were analyzed by the genomic pre-digestion/pcr/rflp assay (arrowheads, PCR primers). The parental DNAs were amplified by PCR without pre-digestion. The placental DNAs were amplified either without predigestion or after pre-digestion with paii, as indicated, and the PCR products were then digested with BsrDI to score the RFLP corresponding to SNP rs1042073. The parental origin of the methylated allele is opposite in the 2 trios, excluding parental imprinting. Figure S2. ASM upstream of the LA-F gene detected by 50K MSNP and validated by predigestion RFLP and bisulfite sequencing. A, hybridization data from 50K XbaI MSNP indicating ASM within the XbaI fragment containing SNP rs9258170, 3kb upstream of LA-F. The signal for the B-allele drops out in the +paii genomic representation. B, The upper panel shows predigestion/pcr/rflp validating ASM near SNP rs9258170. Genomic DNA was digested with paii or not digested, followed by PCR with the indicated primers (see map in part C). The PCR products were digested with XbaI to score the RFLP corresponding to SNP rs3998799. Lanes (a) and (b) are from homozygotes. In both of the heterozygous samples and in additional individuals (Table 1) the C-allele selectively drops out with paii pre-digestion, indicating relative hypomethylation., paii. The lower panel shows ASM in this region validated by analysis of the rs9278271 indel polymorphism. DNA samples were analyzed by paii pre-digestion/pcr followed by electrophoresis of the PCR products on 2.5% Metaphor agarose gels. Pre-digestion of the genomic DNA with paii (+) leads to a reduced or absent lower band indicating relative hypermethylation of the upper allele. This size polymorphism in the PCR product is dominated by rs9278271 but the product also includes smaller indels (rs9256969 and rs9278269), accounting for the complex size variations among individuals. See Table 1 of main text for complete data. C, 5
Validation of ASM and assessment of additional CpG dinucleotides by bisulfite conversion/pcr/sequencing. The DNA sample was from a placenta (fetal surface). Each row represents 1 sequenced clone; with open circles being unmethylated CpG s and filled circles methylated CpG s. The 2 alleles (above and below the line) were distinguished by 2 SNPs (dashes), which eliminate 2 CpG s but do not affect the paii sites. The allele above the line is relatively hypermethylated, with the greatest differential methylation seen in the upstream half of the sequence. Figure S3. ASM downstream of the imprinted MAGEL2 gene and in the PLPP-BCL2 intergenic region detected by 250K MSNP and validated by pre-digestion/pcr and RFLP/sequencing analysis. A, hybridization data from 250K MSNP showing ASM within the fragment containing SNP rs11161318 located 0.5 kb downstream of MAGEL2. In this heterozygous blood DNA sample the signal for the A-allele drops out in the +paii genomic representation and the allele call converts from AB to BB. B, Validation of ASM at paii sites near SNP rs11161318 by genomic pre-digestion/pcr/rflp and direct sequencing. PBL 1 and 2 are from homozygotes; PBL 3, 4 and 5 are from heterozygotes. In one heterozygote the A-allele drops out with paii pre-digestion, indicating relative hypomethylation of this allele, while in the other 2 heterozygotes the C-allele is relatively hypomethylated (see complete data in Table 1 of main text). This result is consistent with the known imprinting of MAGEL2, in which parent-of-origin, not DNA sequence, dictates the epigenetic asymmetry., paii. C, hybridization data from 250K MSNP indicating ASM within the fragment containing SNP rs9951893, located approximately in the center of the 250 kb PLPP-BCL2 intergenic region., paii. D, Validation of ASM at paii sites near SNP rs9951893 by genomic pre-digestion/pcr/rflp (SspI) and direct sequencing. PBL 1 and 2 are from homozygotes. In both of the heterozygotes, PBL 3 and 4, the band 6
corresponding to the T-allele is reduced with paii pre-digestion of the genomic DNA, indicating relative hypomethylation of this allele. Figure S4. ASM in the POU4F1 downstream region and GCNT3 upstream region detected by 250K MSNP and validated by pre-digestion/pcr/rflp and sequencing. A, hybridization data from 250K MSNP indicating ASM within the fragment containing SNP rs9318544, in a region of conserved sequence motifs within an intron of the long RNA transcript Unigene s.493062, overlapping the POU4F1 gene., paii; B, BsrBI. B, Validation of ASM at paii sites near the index SNP rs9318544 in PBL DNA from 2 heterozygotes by genomic pre-digestion/pcr/rflp (BsrBI) and direct sequencing. The sequence is shown for one of the 2 cases, through a second SNP in the same fragment (map in panel A). C, hybridization data from 250K MSNP indicating ASM within the fragment containing SNP rs6494120, located 9 kb upstream of GCNT3. In this PBL DNA the signal for the B-allele drops out in the +paii genomic representation and the allele call converts from AB to AA. D, Validation of ASM at paii sites near SNP rs6494120 by genomic pre-digestion/pcr/rflp (BstNI) and direct sequencing. PBL samples 2 and 4 are from homozygotes. In both of the heterozygotes (PBL 1 and 3) the C-allele drops out with paii predigestion of the genomic DNA, indicating relative hypomethylation of this allele., paii. Figure S5. ASM in the PARP4- ATP12A intergenic region and CG018 intron 1 detected by 250K MSNP and validated by pre-digestion/pcr/rflp and sequencing. A, hybridization data from 250K MSNP indicating ASM within the fragment containing SNP rs943049., paii. B, Validation of ASM at paii sites near SNP rs943049 by genomic pre-digestion/pcr/rflp (BfaI) and direct sequencing. PBL1 and PBL2 are from homozygotes; PBL3 is from a heterozygote and shows strong ASM. C, hybridization data from 250K MSNP indicating ASM within the fragment containing SNP rs206337 in CG018 intron 1., paii. D, Validation of ASM at paii 7
sites near SNP rs206337 by genomic pre-digestion/pcr/sequencing. Placentas 1 and 2 are from homozygotes; Placentas 3 5 are from heterozygotes. 8
Figure S1 C allele T allele Paternal Trio A: Paternal allele methylated Maternal Trio B: Maternal allele methylated rs1042073: BsrDI RFLP X B* B X R LTF ex13 R 200 bp
Figure S2 A. 50K XbaI array: LAF upstream region, rs9258170 C. ASM PCR (Panel B) kidney digest: XbaI alone SNP call: AB X Index SNP rs9258170 rs9278271, rs3998799 X* X XbaI +paii AA.5 kb bisulfite PCR (23 CpG) LA F ex1 ex2 B. paii paii PBL 1 PBL 2 a b + + paii pre digest C allele G allele rs3998799: XbaI RFLP Ki 1 Ki 2 Ki 3 Plac 1 Plac 2 + + + + + paii predigest rs9278271 : indel
Figure S3 A. B. 250K array: PBL digest: alone SNP call: AB PBL 1 2 3 4 5 + + + + + paii pre digest C allele A allele l +paii BB rs11161318 BtsI* MAGEL2, rs11161318 MAGEL2 0.5 kb DS of poly A site 100 bp C. D. 250K array: PBL digest: SNP call: PBL alone AB 1 2 3 + + + 4 + paii pre digest C allele T allele +paii AA rs9951893 SspI* PLPP BCL2 intergenic 150 bp
Figure S4 A. B. 250K array: PBL digest: SNP call: alone AB PBL1 PBL2 paii predigest: + + C allele +paii AA T allele l rs12873012 B* rs12873012 POU4F1 US region 200 bp C. D. 250K array: PBL Digest: SNP call: PBL alone AB paii pre digest: 1 2 3 4 + + + + T allele +paii AA C allele rs6494120 BstNI* GCNT3 US region 100 bp
Figure S5 A. B. 250K array: PBL digest: SNP call: PBL1 PBL2 PBL3 paii alone AB + + + pre digest G allele A allele +paii BB rs943049 BfaI* PARP4 ATP12A 100 bp C. D. 250K array: Placenta digest: SNP call: Placenta alone AB 1 2 3 4 5 +paii BB rs206337 paii pre digest CG018 intron 1 150 bp