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(A) Sample Contribution to the PLS Model SUPPLEMENTARY DATA Supplementary Figure 1. Correlation of levels of auto-antibodies with disease duration. (A) Sample contribution to the PLS model. The bars represent the distances of individual samples to the PLS model, which represent the contribution of individual samples to the separation between T1DM and T2DM/NGT captured by the PLS model. A small distance indicates high contribution of the corresponding sample to the separation captured by the PLS model. The numbers in parenthesis in the x- axis labels of T1DM samples represent the disease durations in months. (B) Scatterplot of disease duration and sample contribution to the separation between T1DM and T2DM/NGT captured by the PLS model (i.e. distances of samples to the PLS model). The distances showed no significant correlation (r = –0.465 and P = 0.070) with disease durations of the individual samples. ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1566/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 2. Expression of EEF1A1 and UBE2L3 in human tissues. Immunohistochemistry of tissue array showed that (A) EEF1A1 and (B) UBE2L3 were expressed in skin, liver, kidney, stomach, intestine, lung, adrenal gland, and thyroid gland as well as pancreas. Original magnification 400. 헑 ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1566/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 3. Detection of EEF1A1 and UBE2L3 auto-antibodies by immunoblotting. Three pairs of T1DM patient samples were selected from the second cohort based on their ELISA absorbance values categorized in three groups: high: >1.3, medium: <1.3 and >0.5, and low: <0.5 absorbance at 450 nm (A450nm). Immunoblots of EEF1A1 and UBE2L3 auto-antibodies of T1DM and NGT samples immunoprecipitated with EEF1A1-flag or UBE2L3-flag antigen are shown. (A) Heavy chain (HC) and light chain (LC) of EEF1A1-AAb from the T1DM sera with EEF1A1-AAb ELISA A450nm values of 2.4 and 1.8 (lane 1 and 2), 1.0 and 0.9 (lane 3 and 4), and 0.2 and 0.3 (lane 5 and 6). (B) HC and LC of UBE2L3-AAb from T1DM sera with UBE2L3-AAb ELISA A450nm values of 1.3 and 1.4 (lane 1 and 2), 0.6 and 0.5 (lane 3 and 4), and 0.3 and 0.2 (lane 5 and 6). Immunoblots of NGT with auto-antibody ELISA A450nm value of 0.2 in both EEF1A1 and UBE2L3 are shown in lane 8 and the control immunoprecipitates of human IgG are shown in lane 7 (A and B). Supplementary Figure 4. Correlation of auto-antibody concentration estimated between ELISA and immunoprecipitation methods for EEF1A1-AAb and UBE2L3-AAb.Scatterplot depicting relative fold changes in A450nm of auto-antibody levels measured by ELISA (y-axis) compared to relative fold changes in density of auto-antibody levels measured by the immunoprecipitation method (x-axis). There are a significant correlation between them in both EEF1A1-AAb (r = 0.779, P = 0.039; A) and UBE2L3-AAb (r = 0.940, P = 0.002; B). Relative fold changes of auto-antibody levels were normalized to the levels of NGT. ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1566/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 5. Positive correlation between the concentrations of EEF1A1-AAb and UBE2L3-AAb in the second cohort. The level of the auto-antibodies showed a significant correlation between EEF1A1-AAb and UBE2L3-AAb (r = 0.65, P<0.001). Supplementary Figure 6. Auto-antibodies involved in T1DM-related pathophysiology in the pancreatic β-cells: insulin secretion, calcium ion homeostasis, cytokines, and glucose metabolism. The columns and rows in the heat map represent samples and auto-antibodies, respectively. Yellow and blue represent the increase and decrease in abundances, respectively. Color bar represents the gradient of the auto- scaled log2-intensities.See the legend of Fig. 1B for the descriptions of the heat map. ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1566/-/DC1 SUPPLEMENTARY DATA Supplementary Table 1. Previously reported auto-antigen in T1DM and their availability in ProtoArray. Number of Signal with T1DM vs. T1DM Gene ProtoArray † T1DM Protein Header Reference Array ID Z>1.64 (NGT+T2DM) vs. Symbol (Y/N)* vs. NGT‡ NGT T1DM T2DM ‡ T2DM‡ glutamate decarboxylase 2 Baekkeskov et B11R21C09 0 7 0 <0.001 <0.001 0.001 GAD2 (pancreatic islets and brain, Y al.,1990 (1) 65kDa) B11R21C10 0 7 0 <0.001 <0.001 0.001 Palmer et al., B19R03C13 0 0 0 1 1 1 INS insulin Y 1983 (2) B19R03C14 0 0 0 1 1 1 Taniguchi et B19R14C17 0 0 0 1 1 1 CA2 carbonic anhydrase II (CA2) Y al., 2003 (3) B19R14C18 0 0 0 1 1 1 dopa decarboxylase (aromatic Rorsman et al., B14R21C03 0 0 0 1 1 1 DDC Y L-amino acid decarboxylase) 1995 (4) B14R21C04 0 0 0 1 1 1 heat shock 60kDa protein 1 Jones et al., B25R03C21 1 0 0 0.380 0.237 1 HSPD1 Y (chaperonin) 1990 (5) B25R03C22 2 0 0 0.380 0.237 1 regenerating islet-derived 3 Gurr et al., B18R05C03 0 0 0 0.528 0.628 0.500 REG3A Y alpha 2002 (6) B18R05C04 0 0 0 0.528 0.628 0.500 SLC30A8 solute carrier family 30 (zinc Wenzlau et al., N - (ZnT8A) transporter), member 8 2007 (7) Bottazo et al., ICA1 islet cell autoantigen 1 N - 1974 (8) PTPRN protein tyrosine phosphatase, Rabin et al., N - (IA-2A) receptor type, N 1994 (9) Castano et al., CPH carboxypeptidase H N - 1991 (10) PTPRN2 protein tyrosine phosphatase, Kawasaki et N - (IA-2beta) receptor type, N polypeptide 2 al., 1996 (11) solute carrier family 2 Inman et al., SLC2A2 (facilitated glucose N - 1993 (12) transporter), member 2 topoisomerase (DNA) II alpha Chang et al., TOP2A N - 170kDa 1996 (13) For the known auto-antibodies, the numbers of samples with Z >1.64 and P-values from M-tests were shown. *Whether auto-antigen is included in ProtoArray 5.0 platform. Y, included in ProtoArray; N, not included in ProtoArray †Z-score defined by (spot intensity - the averaged intensity of all probes) / the standard deviation of all probes ‡P-values from M-test T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; NGT, subjects with normal glucose tolerance ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1566/-/DC1 SUPPLEMENTARY DATA Supplementary Table 2. Sixty nine auto-antibody candidates. T1DM vs. (NGT+T2DM) T1DM vs. NGT T1DM vs. T2DM NGT+ Gene Symbol Protein Header Database ID Array ID T1DM T1DM NGT T1DM T2DM T2DM P-value† P-value† P-value† Count* Count* Count* Count* Count* Count* GAD2 Glutamate decarboxylase 2 NM_000818.1 B11R21C09 7 0 3.35E-05 7 0 3.55E-04 8 0 0.001 Coiled-coil domain-containing c7orf53 NM_182597.1 B31R08C07 5 0 0.001 5 0 0.005 5 0 0.022 transmembrane protein C7orf53 PRKCA protein kinase C, alpha (PRKCA) NM_002737.1 B21R02C15 5 0 0.001 5 0 0.005 5 0 0.022 cytoplasmic polyadenylation element CPEB1 BC035348.1 B30R20C01 4 0 0.017 6 0 0.007 2 0 0.242 binding protein 1 (CPEB1) ATP-binding cassette, sub-family F (GCN20), member 2 (ABCF2), nuclear ABCF2 NM_007189.1 B02R15C05 3 0 0.017 3 0 0.045 3 0 0.113 gene encoding mitochondrial protein, transcript variant 1 ATG16L1 Autophagy-related protein 16-1 NM_017974.2 B32R21C17 3 0 0.017 3 0 0.045 3 0 0.113 chromosome 2 open reading frame 47 C2orf47 BC017959.1 B41R05C05 3 0 0.017 3 0 0.045 3 0 0.113 (C2orf47) development and differentiation DDEFL1 BC060786.1 B07R02C21 3 0 0.017 3 0 0.045 3 0 0.113 enhancing factor-like 1 (DDEFL1) EEF1A1 Elongation factor 1-alpha 1 BC094687.1 B20R19C01 3 0 0.017 5 0 0.005 3 0 0.113 EXOSC5 exosome component 5 (EXOSC5) BC007742.1 B45R14C21 3 0 0.017 3 0 0.045 3 0 0.113 HES6 Transcription cofactor HES-6 NM_018645.3 B39R21C09 3 0 0.017 3 0 0.045 3 0 0.113 HIG1 domain family, member 2A HIGD2A NM_138820.1 B27R15C09 3 0 0.017 3 0 0.045 3 0 0.113 (HIGD2A) IGKC immunoglobulin kappa constant (IGKC) BC070334.1 B28R06C05 3 0 0.017 5 0 0.005 3 0 0.113 potassium inwardly-rectifying channel, KCNJ1 subfamily J, member 1 (KCNJ1), NM_153764.1 B16R08C11 3 0 0.017 3 0 0.045 3 0 0.113 transcript variant rom-k2 Smith-Magenis syndrome chromosome SMCR7 NM_139162.2 B25R03C15 3 0 0.017 3 0 0.045 3 0 0.113 region candidate gene 7 protein ubiquitin-conjugating enzyme E2L 3 UBE2L3 NM_198157.1 B46R08C03 3 0 0.017 3 0 0.045 3 0 0.113 (UBE2L3), transcript variant 2 ZNF434 zinc finger protein 434 (ZNF434) BC002859.1 B26R09C05 3 0 0.017 3 0 0.045 3 0 0.113 hypothetical protein MGC31957 MGC31957 BC005043.1 B04R08C11 6 1 0.001 6 0 0.001 6 1 0.041 (MGC31957) acidic (leucine-rich) nuclear ANP32B phosphoprotein 32 family, member B NM_006401.1 B18R03C13 6 1 0.003 6 0 0.001 6 0 0.110 (ANP32B) erythrocyte membrane protein band 4.9 EPB49 BC006318.1 B16R02C11 4 1 0.017 3 0 0.045 3 0 0.113 (dematin) (EPB49) FLJ21908 RNA polymerase II-associated protein 3 BC056415.1 B35R19C11 4 1 0.017 3 0 0.045 4 0 0.051 FLT3LG fms-related tyrosine kinase 3 ligand PHC1705 B40R16C01 4 1 0.017 4 0 0.015 3 0 0.113 ©2014 American Diabetes Association.
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