Supplementary data

Polymorphism / Primer sequences / Primer annealing / FRET probe sequences
rs13412102 / CAGCTCCTTTCATTCCCAC
CTTCCCTCTCTCCTTCACC / 60 °C / LC640-ACCCTGCCCTAGAACGTAC
GACACTTCCTTCAATAAATCTCTCATGTCT-FL
rs12471449 / GCTGGGTTGGAATCTGC
TAGCACTTATCACCTCTGATATCTTAG / 60 °C / CATACCTATAAAGCACTTACTGTG-FL
LC610-GCAAGACACCGTTGTCAGTATTAATTC
rs6431660 / GTATTTTGGTTAATTTTAGGTGTAG
GCTGCATATTCCAAGCAC / 57 °C / CAGATAGAAATGTTTTCAGTTTTC-FL
LC670-GGATGAATTTAGACGAAAATGGTGAAAC
rs1441090 / GAGGGAAGAAGGAACCATAGAT
CTTGGGCATTCACACAGGT / 60 °C / LC640-GAGACCCCCAACCCTTCA
GGAGGCAGCTCTTGCTCATTAAAGCTGGT-FL
rs2289472 / GAATATGTTTCCCACTTAGTTCC
ATTGTGAGGTTAGTTGTCTAATGC / 57 °C / LC610-GTTGGGTTTCTCTTGGCT
CCCACTGTTTTCTGGCAGTTTGGTACTG-FL
rs2241880 / ATTTGTCTTTATGTTATTTCTTAGGAGACG
GTAACAAATTTTGTCCTCTGAAACTA / 60 °C / LC610-CCAGGATGAGCATCCACATTGT GTAGCTGGTACCCTCACTTCTTTACCAGA-FL
rs2241879 / TTTGCCCCATCCCTCAT
GCCTTGTGTGTCTTCGTAAGTAT / 60 °C / CCAGGCTCTGTCACCATATCA -FL
LC670-GCGTGGTAGGGTTCGGGGCT
rs3792106 / GTTCATCTTCAATGTGTCGAGC
CTTTTACTAAGCTGAGAGCTGACTAC / 60 °C / TGGCTTTACAAAAGTCTAAACC-FL
LC640-CAATACAACATACCCTCATTTTCCAACC
rs4663396 / AACAGAGGAAGTAATGTTAAGATGA
GCAGATCCCTTGAGGTCA / 60 °C / LC670-GATCACACTACTATACCCCACAC
GAAGGCGGAGGTTGCAGTGAGCTG-FL

Supplementary data, Table S1. Primer sequences, FRET probe sequences, and primer annealing temperatures used for genotyping ATG16L1 variants. Note: FL: Fluorescein, LC610: LightCycler-Red 610; LC640: LightCycler-Red 640; LC670: LightCycler-Red 670. The polymorphic position within the sensor probe is underlined. A phosphate is linked to the 3'-end of the acceptor probe to prevent elongation by the DNA polymerase in the PCR.

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Polymorphism / Primer sequences
rs13412102 / CAGCTCCTTTCATTCCCAC AGGCTGAGAGTCTTCCTACC
rs12471449 / CAGCAGGAGATCTGAGAGATACA TAGCACTTATCACCTCTGATATCTTAG
rs6431660 / CCATCCTTGCTTTGTAACAGT AGAACGGCTGCATATTCC
rs1441090 / GAGGGAAGAAGGAACCATAGAT CTTGGGCATTCACACAGGT
rs2289472 / GCTTACTAAAGAGGAGGGGAG GTCAGAAATTGTGAAGTTAGTTAGC
rs2241880 / ATTTGTCTTTATGTTATTTCTTAGGAGACG GTAACAAATTTTGTCCTCTGAAACTA
rs2241879 / TTTGCCCCATCCCTCAT
TGCCTTGTGTGTCTTCGT
rs3792106 / GTTCATCTTCAATGTGTCGAGC CTTTTACTAAGCTGAGAGCTGACTAC
rs4663396 / AACAGAGGAAGTAATGTTAAGATGA
GCCTGTAATCCCAGCAC

Supplementary data, Table S2. Primer sequences used for the sequence analysis of ATG16L1 variants.

IL23R variant / ATG16L1 variant / P value
rs1004819 / rs6431660 / 0.01
rs1004819 / rs3792106 / 0.02
rs2201841 / rs12471449 / 0.02
rs2201841 / rs6431660 / 0.02
rs2201841 / rs2241879 / 0.02
rs2201841 / rs3792106 / 0.02
rs1343151 / rs12471449 / 0.01
rs10889677 / rs12471449 / 0.03
rs10889677 / rs6431660 / 0.01
rs10889677 / rs2241880 / 0.02
rs10889677 / rs2241879 / 0.01
10889677 / rs3792106 / 0.02
rs10889677 / rs4663396 / 0.02
rs1495965 / rs13412102 / 0.03

Supplementary data, Table S3. Several ATG16L1 variants demonstrated weak epistasis to certain IL23R variants. However, none of the calculated P-values remained significant after correction for multiple testing.

Supplementary data: Methods

Genotyping of the ATG16L1 variants

NineATG16L1 SNPs (rs13412102, rs12471449, rs6431660, rs1441090, rs2289472, rs2241880 (=T300A), rs2241879, rs3792106, rs4663396) were genotyped by PCR and melting curve analysis using a pair of fluorescence resonance energy transfer (FRET) probes in a LightCycler® 480 Instrument (Roche Diagnostics, Mannheim, Germany). The donor fluorescent molecule (fluorescein) at 3'-end of the sensor probe (or the anchor probe in the case of rs13412102,rs1441090, rs2289472, rs2241880 and rs4663396) is excited at its specific fluorescence excitation wavelength (533 nm) and the energy is transferred to the acceptor fluorescent molecule at the 5'-end (LightCycler Red 610, 640 or 670) of the anchor probe (or the sensor probe in the case of rs13412102,rs1441090, rs2289472, rs2241880 and rs4663396). The specific fluorescence signal emitted by the acceptor molecule is detected by the optical unit of the LightCycler 480 Instrument. The sensor probe is exactly matching to one allele of each SNP, preferentially to the rarer allele, whereas in the case of the other allele there is a mismatch resulting in a lower melting temperature. The total volume of the PCR was 5 µl containing 25 ng of genomic DNA, 1 x Light Cycler 480 Genotyping Master (Roche Diagnostics), 2.5 pmol of each primer and 0.75 pmol of each FRET probe (TIB MOLBIOL, Berlin, Germany). In the case of rs1441090, the concentration of the forward primer was reduced to 0.5 pmol. Partly, two SNPs were analyzed in a multiplex reaction, the combinations were: rs13412102 + rs12471449, rs6431660 + rs2289472, rs2241880 + rs4663396 and rs2241879 + rs3792106. The PCR comprised an initial denaturation step (95°C for 10 min) and 45 cycles (95°C for 10 sec, primer annealing temperature as given in the Supplementary data (Table S1) for 10 sec, 72°C for 15 sec). The melting curve analysis comprised an initial denaturation step (95°C for 1 min), a step rapidly lowering the temperature to 40 °C and holding for 2 min, and a heating step slowly (1 acquisition/°C) increasing the temperature up to 80 °C and continuously measuring the fluorescence intensity. The results of melting curve analysis have been confirmed by analyzing two patient samples for each possible genotype using sequence analysis. For sequencing the total volume of the PCR was 100 µl containing 250 ng of genomic DNA, 1 x PCR-buffer (Qiagen, Hilden, Germany), a final MgCl2 concentration of 2 mM, 0.5 mM of a dNTP-Mix (Sigma, Steinheim, Germany), 2.5 units of HotStar Plus Taq™ DNA polymerase (Qiagen) and 10 pmol of each primer (TIB MOLBIOL). The PCR comprised an initial denaturation step (95°C for 5 min), 35 cycles (denaturation at 94 °C for 30 sec, primer annealing at 55 °C for 30 sec, extension at 72 °C for 30 sec) and a final extension step (72 °C for 10 min). The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and sequenced by a commercial sequencing company (Sequiserve, Vaterstetten, Germany). All sequences of primers and FRET probes and primer annealing temperatures used for genotyping and for sequence analysis are given in Tables S1 and S2.

Supplementary data:Figure Legends

Supplementary Figure S1. Regulation of ATG16L1 mRNA expression by TLR ligands and proinflammatory cytokines in intestinal epithelial cells in vitro. (A) After stimulation of HT-29 cells with ligands to TLR2 (Heat-killed Lysteria monocytogenes, HKLM), TLR4 (lipopolysaccharide, LPS), TLR5 (flagellin, flag), TLR6 (diacetylated synthetic lipoprotein that represents the N-terminal part of the 44-kDa lipoprotein LP44 from Mycoplasma salivarium, FSL-1) or TLR9 (CpG), ATG16L1 mRNA expression is variably upregulated with the TLR9 and TLR2 ligands demonstrating the strongest effect as determined by quantitative PCR analysis. (B) Stimulation with TNF- (50 ng/mL), IL-1 (10 ng/mL) or IFN- (1000 U/mL) for the indicated time intervals results in slight upregulation of ATG16L1 mRNA expression in HT-29 cells which was highest following IL-1 treatment. (C) In contrast, both TNF- (50 ng/mL) and LPS (1 µg/mL) significantly up-regulated IL-8 mRNA expression in HT-29 cells confirming the overall responsiveness of this cell line to TLR ligands and proinflammatory cytokines.

Supplementary Figure S2. Intestinal ATG16L1 mRNA expression in a murine model of ileitis and Crohn’s disease. (A) No significant differences in ATG16L1 mRNA expression were observed in colonic and ileal epithelial cells of 18 week old TNFARE heterozygous mice (n=9) compared to wildtype mice (n=9) as determined by quantitative PCR. In contrast TNF- mRNA expression is significantly up-regulated in TNFARE heterozygous mice. mRNA expression levels were normalized to expression in ileal epithelial cells from wildtype mice which was arbitrarily set as 1.0. (B)Relative ATG16L1 and IL-8 mRNA expression in inflamed colonic lesions compared to uninflamed tissue of patients with CD (n=12) representing a total of 48 biopsies (n=24 from inflamed intestinal tissue, n=24 from uninflamed tissue).

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