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1 Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References
2 1. Supplementary Table legends Suppl. Table 1.) Primers for IL1, IL1RA and IL1RB sequencing. The numbers after the gene identification refer to the exon. Exons exceeding a length > 7 bp were subdivided into several PCR products. PCR conditions for exon amplification are specified in reference 1. *Primers were used for long-range PCR amplification of exon 4 and 5 of IL1 gene. Suppl. Table 2.) Bioinformatic analysis on identified IL1 and IL1R mutations. The impact of IL1 and IL1R mutations with amino acid substitutions on protein structure and function was assessed using SIFT 2 and PolyPhen 3. Bioinformatic analysis with respect to the 3'UTR mutation (c.*c52t) using ESEfinder 4 indicates that the mutation targets a putative SR-protein binding site.
3 2. Supplementary Figure legends Suppl. Fig. 1.) Molecular details of - and R-deficient patients. A.) Chromatograms of Sanger DNA sequencing show mutations detected in individual patients compared with the sequence in healthy donor controls. The mutated codon is underlined. B.) Long-range PCR results revealing a genomic deletion with an approximate size of 1 kb in the IL1RB gene of patient 16. C.) Chromatograms demonstrating the precise location of the genomic deletion (spanning from intron 3 to exon 5) identified in patient 16 compared to the sequence in a healthy donor. BP, breakpoint. Suppl. Fig. 2.) Defective R-mediated signal transduction in R-deficient patients. A.) B.) Western blot analysis revealing defective STAT3 signal transduction (pstat3, Tyr75) in identified R-deficient patients upon stimulation with. GAPDH was used as loading control. Western blot results of patient 3 before HSCT have been previously shown in reference 1. ELISA results showing abrogated inhibition of TNF-! secretion in PBMCs isolated from individual R-deficient patients upon costimulation with LPS and (Healthy donors: black bars, ient: grey bars). ELISA data on patient 1 and patient 3 before HSCT have been previously shown in reference 1. Suppl. Fig. 3.) Determination of the hematopoietic chimerism in R-deficient patients after HSCT. Hematopoietic chimerism in transplanted patients was quantified by Real-time PCR analysis, allowing for detection of differences between recipient and donor at a single nucleotide polymorphism. Data shown represent the percentage of donor cells in PBMCs.
4 Suppl. Fig. 4.) Reconstitution of -mediated signal transduction in transplanted R-deficient patients. A.) Western blot analysis reveals induction of STAT3 phosphorylation at the Tyr75 residue in PBMCs from transplanted R-deficient patients. GAPDH served as a loading control. B) ELISA results showing reconstitution of -mediated inhibition of TNF-! secretion in LPS-/-costimulated patient s PBMCs after HSCT (Healthy donors: black bars, ient: grey bars).
5 3. Supplementary Tables
6 primer identification primer sequence (5' - 3') IL1 (NM_572) IL1 1F IL1 1R IL1 2F IL1 2R IL1 3F IL1 3R IL1 4F* IL1 4R IL1 5_1F IL1 5_1R IL1 5_2F IL1 5_2R* GGG AAA CCT TGA TTG TGG C CGC AGG AGG AGG GTT CTT ATA G AAA TCT CTA AAT GAA AGG GCA TC GAA ACA AAC CCA ATT CCC TG GGA TCG CTA GAA CCA AGC TG GAG TGT CCC TGC TGG TCT G ACC AGC TTG TCC CCT AAG TG GAA TGG GGC CTA TTG AGT CC CAT GAG CAT GAG GGA GGG TCC GAG ACA CTG GAA GGT G ATC TTG TCT CTG GGC TAG GG TGC AGA ATT CAT TCA CCC AC IL1RA (NM_1558) IL1RA 1F IL1RA 1R IL1RA 2F IL1RA 2R IL1RA 3F IL1RA 3R IL1RA 4F IL1RA 4R IL1RA 5F IL1RA 5R IL1RA 6F IL1RA 6R IL1RA 7_1F IL1RA 7_1R IL1RA 7_2F IL1RA 7_2R IL1RA 7_3F IL1RA 7_3R IL1RA 7_4F IL1RA 7_4R IL1RA 7_5F IL1RA 7_5R IL1RA 7_6F IL1RA 7_6R GAC AGT GGT TCC CCG TCC CAC TGG ATG GAG AAC TTT AAT GG GAA CCT CCC TTT CTT CTT TGG AGG CAG GTA TCT TCC CAT GC GGC CTC TTG CGT CTC CC GCA GAC ATG GTG AGC TAT GG ATT CTG GAG GCA AAG TCT CG AGT TCC CAA TGG CAC ACA AG CTA AAG GCC CAC CAG CTC TC ACG CGT TTT GGA TTG CAC AAT GGA TTT CAT GGG ACC AG ACT GGC TGG GAG GAA AAG AG CGA GCT CTC CTC CTG GG CCT CAG GTA ACC CTG GAA TG TGA CAG TGG CAT TGA CTT AGT TC GTC CAG GCA GAG GAG CAG CCT GGG CAG CTT TAA CTC AG AGG TTC CCC ATG TGA CCA TC GCT GAA GTC AGC TCA GAC CC CAG TGC CCA GTG GCT TAT C TCA TAA CTC AGC CCT TTG GG AAG AGT ACT TTG GCA GAG GGG AAG GCA GTT CAG TCC ACA GG TCC CAC ACA TTA TTA CCC TCC IL1RB (NM_628) IL1RB 1F IL1RB 1R IL1RB 2F IL1RB 2R IL1RB 3F IL1RB 3R IL1RB 4F IL1RB 4R IL1RB 5F IL1RB 5R IL1RB 6F IL1RB 6R IL1RB 7_1F IL1RB 7_1R IL1RB 7_2F IL1RB 7_2R IL1RB 7_3F IL1RB 7_3R AGG GTA AAG AAG ACC CTC AAA CCT AGT TGC GTC TCA GCA G GGA GAA CCA AGT GCT GGA TG CAG ACT CCC TTC CTC CTG TG TTA ACA CAG TTT CCA CTC CCG AAG GCC ATC CAT TTG TGG TCC GTG GAC TAA TTG TTC TGC AGT CCA TAA GGT GCT GCC AC AAG TCT AAA ACG GCT ATT ATC ACT G AGC TGG AAT TTG AGT TGG ATG GGC TCT GTT TTC AGG GAT TG CAT GTT GTC TGG AAT TGG GC TCC AGC CAG GAG TTC TGT G TCC AGC CAG GAG TTC TGT G CTC CCA GAC CCT GGA CTT AG TCA CTT TGT CAC CCA GGC GAT GGC GCA TGC CTA TAA TC TGG ACA TCA AGA TGG CAA AC Supplementary Table 1
7 ient Mutation [Gene; Genotype] PolyPhen Score Prediction of function effect of human nssnps SIFT Score Prediction of function effect of human nssnps 3 IL1RA, Ex 4: c.g421a, p.gly141arg 1 probably damaging.2 affects protein function 4 IL1RA, Ex 3: c.c251t, p.thr84ile 1 probably damaging.1 affects protein function 5 IL1RA, Ex 3: c.c31t, p.arg11trp 1 probably damaging affects protein function 7 IL1RA, Ex 2: c.a17a/g, p.tyr57tyr/cys 1 probably damaging affects protein function IL1RA, Ex 3: c.c349c/t,p.arg117arg/cys 1 probably damaging.6 tolerated 8 IL1RB, Ex 3: c.g197a, p.cys66tyr 1 probably damaging.1 affects protein function 12 IL1RA, Ex 4: c.t56c, p.ile169thr.725 possibly damaging.1 tolerated 13, 14, 15 IL1, Ex 5: c.g458a; p.gly153asp 1 probably damaging affects protein function 9 9 IL1RB, 3'UTR: c.*c52t: mutation targets a putative high-scoring SR-protein binding site (ESEfinder 4 ) Supplementary Table 2
8 4. Supplementary Figures
9 A 1,2,6 C A A T A C T G A A A A A A C C A A T A C T G G A A A A A C IL1RB, Exon 4 c.g477a, p.trp159x 3 IL1RA, Exon 4 c.g421a, p.gly141arg A T C C T C A G G A A G A T T A T C C T C G G G A A G A T T 4 IL1RA, Exon 3 c.c251t, p.thr84ile G A C C T T A T C G C A G T G G A C C T T A C C G C A G T G 5 IL1RA, Exon 3 c.c31t, p.arg11trp A G A G T G T G G G C T G T G A G A G T G C G G G C T G T G 7 IL1RA, Exon 2 c.a17a/g, p.tyr57tyr/cys A C C T G C T R T G A A G T G A C C T G C T A T G A A G T G IL1RA, Exon 3 c.c349c/t, p.arg117arg/cys A A C A C C Y G C T T C T C T A A C A C C C G C T T C T C T 8 IL1RB, Exon 3 c.g197a, p.cys66tyr G A T A A A T A C A T G A A T G A T A A A T G C A T G A A T 9 IL1RB, 3'UTR c.*c52t C A T C T C A T A T C T G C C C A T C T C A C A T C T G C C 1 IL1RB, Exon 5 c.g611g/a, p.trp24trp/x G G G G A A T R G A G T G A G G G G G A A T G G A G T G A G A T G G C C T M G G T C T T C A T G G C C T C G G T C T T C IL1RB, Exon 6 c.c689c/a, p.ser23ser/x 11 IL1RB, Exon 5 c.g611a; p.trp24x G G G G A A T A G A G T G A G G G G G A A T G G A G T G A G 12 IL1RA, Exon 4 c.t56c; p.ile169thr T A T G A G A C T G C C A T T T A T G A G A T T G C C A T T 13,14,15 IL1, Exon 5 c.g458a; p.gly153asp G A G A A A G A C A T C T A C G A G A A A G G C A T C T A C Supplementary Figure 1A
10 B C bp Mot 16 BP T T C T T T T T T T C T T T C T T T C T T T C T T T T IL1RB Intron 3 IL1RB Exon 5 BP A T T G T G T T C A A G T T C G A G G G T T T C T T C 16 IL1RB c _574del Intron 3 BP Exon 5 T T C T T T T T T T C T T T C G A G G G T T T C T T C Supplementary Figure 1B/C
11 p-stat3 (Tyr75) p-stat3 (Ser727) STAT3 GAPDH p-stat3 (Tyr75) p-stat3 (Ser727) STAT3 GAPDH Supplementary Figure 2A
12 Healthy Donor ient TNF-! [pg/ml] TNF-! [pg/ml] Supplementary Figure 2B
13 Chimerism [%] time [days] time [days] time [days] time [days] time [days] Supplementary Figure 3
14 p-stat3 (Tyr75) p-stat3 (Ser727) STAT3 GAPDH Supplementary Figure 4A
15 Healthy Donor ient TNF-! [pg/ml] Supplementary Figure 4B
16 5. Supplementary References 1. Glocker EO, Kotlarz D, Boztug K, et al. Inflammatory bowel disease and mutations affecting the interleukin-1 receptor. N Engl J Med 29;361: Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res 22;12: Ramensky V, Bork P, Sunyaev S. Human non-synonymous SNPs: server and survey. Nucleic Acids Res 22;3: Cartegni L, Wang J, Zhu Z, et al. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 23;31:
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CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'
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