alexa Identification of Cerebral Infarction-Specific Antibody Markers from Autoantibodies Detected in Patients with Systemic Lupus Erythematosus | OMICS International
ISSN-2155-9929
Journal of Molecular Biomarkers & Diagnosis

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Identification of Cerebral Infarction-Specific Antibody Markers from Autoantibodies Detected in Patients with Systemic Lupus Erythematosus

Ken-ichiro Goto1,2, Takao Sugiyama3, Ryutaro Matsumura4, Xiao-Meng Zhang2, Risa Kimura2, Akiko Taira2, Emiko Arita2, Katsuro Iwase2, Eiichi Kobayashi5, Yasuo Iwadate5, Naokatsu Saeki5, Masahiro Mori6, Akiyuki Uzawa6, Mayumi Muto6, Satoshi Kuwabara6, Minoru Takemoto7, Kazuki Kobayashi7, Harukiyo Kawamura7, Ryoichi Ishibashi7, Ken-ichi Sakurai7, Masaki Fujimoto7, Koutaro Yokote7, Takashi Nakayama8, Jun-ya Harada8, Yoshio Kobayashi8, Mikiko Ohno9, Hirotoshi Chin9, Eiichiro Nishi9, Toshio Machida10, Yo Iwata11, Seiichiro Mine12, Ikuo Kamitsukasa13, Takeshi Wada14, Akiyo Aotsuka14, Kaoru Katayama15, Yuriko Kikkawa15, Kenro sunami16, Hirotaka Takizawa17, Rika Nakamura2,18, Go Tomiyoshi2,18, Natsuko Shinmen2,18, Hideyuki Kuroda18 and Takaki Hiwasa2*

1Department of Orthopedics, National Hospital Organization, Chiba-East-Hospital, Chiba, Japan

2Department of Biochemistry, Chiba University, Graduate School of Medicine, Chiba, Japan

3Department of Rheumatology, Shimoshizu National Hospital, Chiba, Japan

4Department of Rheumatology, National Hospital Organization, Chiba-East-Hospital, Chiba, Japan

5Department of Neurological Surgery, Chiba University, Graduate School of Medicine, Chiba, Japan

6Department of Neurology, Chiba University, Graduate School of Medicine, Chiba, Japan

7Department of Clinical Cell Biology and Medicine, Chiba University, Graduate School of Medicine, Chiba, Japan

8Department of Cardiovascular Medicine, Chiba University, Graduate School of Medicine, Chiba, Japan

9Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan

10Department of Neurosurgery, Chiba Cerebral and Cardiovascular Center, Chiba, Japan

11Department of Cardiovascular Medicine, Chiba Cerebral and Cardiovascular Center, Chiba, Japan

12Department of Neurological Surgery, Chiba Prefectural Sawara Hospital, Chiba, Japan

13Department of Neurology, Chiba Rosai Hospital, Chiba, Japan

14Department of Internal Medicine, Chiba Aoba Municipal Hospital, Chiba, Japan

15Department of Neurosurgery, Narita Red Cross Hospital, Chiba, Japan

16Chiba Medical Center, Department of Neurosurgery, Chiba, Japan

17Port Square Kashiwado Clinic, Kashiwado Memorial Foundation, Chiba, Japan

18Medical Project Division, Research Development Center, Fujikura Kasei Co, Saitama, Japan

*Corresponding Author:
Takaki Hiwasa
Department of Biochemistry and Genetics Chiba University
Graduate School of Medicine Inohana 1-8-1
Chuo-ku, Chiba 260-8670, Japan
Tel: +81-432262541
E-mail:
[email protected]

Received Date: January 05, 2015; Accepted Date: January 28, 2015; Published Date: February 02, 2015

Citation: Goto K, Sugiyama T, Matsumura R, Zhang XM, Kimura R, et al. (2015) Identification of Cerebral Infarction-Specific Antibody Markers from Autoantibodies Detected in Patients with Systemic Lupus Erythematosus. J Mol Biomark Diagn 6:219. doi: 10.4172/2155-9929.1000219

Copyright: © 2015 Goto K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease which may be caused by development of the autoantibodies. On the other hand, SLE is a high-risk group of atherosclerosis, so it is possible that some of autoantibodies in SLE are the result of atherosclerosis-related diseases such as cerebral infarction (CI), cardiovascular disease (CVD) and diabetes mellitus (DM).

Methods: The initial screening of autoantibodies was performed using the protein array method. AlphaLISA was used to analyze the serum antibody levels using synthetic polypeptides as antigens.

Results: After the initial screening using protein array, we identified 67 antigens that were recognized by IgG antibodies in sera of patients with SLE. In the second screening, 170 peptides derived from amino acid sequences of 67 antigens were synthesized and used as antigens for analysis of serum antibody levels by AlphaLISA. The antibody levels for ten peptides were significantly higher in the sera of patients with SLE than in those of healthy donors. Further AlphaLISA analysis of sera of patients with CI, CVD or DM revealed that the serum antibody levels for four peptides derived from SOSTDC1, CTNND1, CLDND1 and CCNG2 were elevated in patients as compared to those of healthy donors.

Conclusions: Serum antibody levels against peptide antigens of SOSTDC1, CTNND1, CLDND1 and CCNG2 are useful markers for diagnosis of the progression of CI, CVD and/or DM.

Keywords

Systemic lupus erythematosus; Cerebral infarction; Cardiovascular disease; Diabetes mellitus; Antibody biomarker

Introduction

Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder characterized by damage to multiple organ systems caused by the production of many autoantibodies, generation of immune complexes, and activation of the complement system [1-3]. Dysfunction of T cells and accelerated activation of B cells in SLE patients [4] enables the development of various autoantigens such as the anti-nuclear antibody [5]. SLE specific autoantibodies thus far reported were the anti-Sm antibody [6], anti-double-stranded DNA antibody [7], anti-U1RNP antibody [8], anti-SSA/Ro antibody [9,10] and the anti-P ribosomal protein antibody [11], yet the pathogenic role of these antibodies remains to be proven.

Accelerated atherosclerotic diseases have been recognized as major causes of mortality in SLE. In the study of large case series of patients with SLE, 6-20% and 4-15% of deaths were due to cardiovascular disease (CVD) and cerebrovascular disease, respectively [12-14]. To estimate the onset risk of accelerated atherosclerosis in SLE patients, several markers have been introduced including C-reactive protein [15], lipoprotein (a) [16], homocysteine [17], inflammatory cytokines [18,19], yet the satisfactory results have not been obtained.

On the other hand, recent studies have revealed that specific autoantibodies exist in the sera of patients with atherosclerosis, such as autoantibodies for phospholipid (Antiphospholipid syndrome) [20,21], apolipoprotein A-1 [22] and oxidized low-density lipoprotein [23]. We have also reported that the antibody levels against RPA2 were associated with the onset of ischemic stroke [24]. These antibody markers might be useful for evaluation of the onset of lethal atherosclerotic disease in patients with SLE.

In the present study, we have comprehensively screened autoantigens which were recognized by IgG antibodies in the sera of patients with SLE by the protein array method. We then selected and identified autoantigens specific for cerebral infarction (CI), CVD and/or diabetes mellitus (DM).

Materials and Methods

Patients and healthy donor sera

This study was approved by the Local Ethical Review Board of the Chiba University, Graduate School of Medicine as well as that of the National Hospital Organization, Shimoshizu Hospital and Chiba-East hospitals. Sera were collected from patients after they had given written informed consent. Each serum sample was centrifuged at 3,000 x g for 10 min, and then the supernatant was stored at -80°C until use.

The serum samples of SLE were obtained from Shimoshizu Hospital, and those of CI and transient ischemic attack (TIA) were obtained from Sawara Hospital, Rosai Hospital, Aoba Hospital and Chiba Medical Center. Samples of CVD and DM were obtained from Chiba University Hospital, and those of healthy donors were from Chiba University, Kashiwado Clinic and Fujikura Kasei Co.

Protein array screening

Initial screening was performed using ProtoArrays® Human Protein Microarrays v4.0 (Thermo Fisher Scientific, Waltham, MA), which were loaded with 9,480 species of proteins. A total of 11 sera, 6 from patients and 5 from healthy donors, were used to detect antigens recognized specifically by IgG antibodies in the sera of patients.

Peptide synthesis

Three epitope sites in the candidate antigen proteins were predicted using the program ProPred (http://www.imtech.res.in/raghava/propred/). N-terminal biotinylated 15mer peptides without purification were synthesized and used in the second screening. For the third screening, synthetic peptides were purified by HPLC. The purity of each peptide was determined to be higher than 90%.

AlphaLISA (Amplified Luminescence Proximity Homogeneous Assay)

To evaluate the serum antibody levels, AlphaLISA was used. AlphaLISA was performed in 384-well microtiter plates (white opaque OptiPlate™ from Perkin Elmer) containing 2.5 μL of 1/100-diluted serum and 2.5 μL of biotinylated synthetic peptides (400 ng/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500, and 0.05% Proclin-300). The reaction mixture was incubated at room temperature for 6-8 h, then anti-human IgG-conjugated acceptor beads (2.5 μL at 40 μg/mL) and streptavidin-conjugated donor beads (2.5 μL at 40 μg/mL) were added and incubated at room temperature in the dark for another 1 - 14 days. The plate was read on an EnSpire Alpha microplate reader (PerkinElmer).

Statistical analyses

Fisher's exact (two-sided) probability test and the Mann-Whitney U test were used to determine the significance of the differences between the two groups. All statistical analyses were carried out using the GraphPad Prism 5 (GraphPad Software, La Jolla, CA). P values lower than 0.05 were considered statistically significant.

Results

Initial screening of SLE-specific antigens by protein array

By using protein microarrays loading with 9,480 proteins, we examined 6 sera from SLE patients and 5 sera from healthy controls to identify SLE-associated antigens. Sixty-seven proteins such as SOSTDC1, CTNND1 were selected as antigens by reacting with more than 5 sera from SLE patients and not with any of the sera from healthy donors (Table 1). These proteins may include not only antigens specific for SLE but also those specific for the complication such as CI, CVD and DM.

Name
ZIC4 C9orf32
SDHB DKFZp762
MGC17553 SOSTDC1
RARS2 RNPC3
IPO11 CDC45L
SLC25A24 ZNF649
OTX1 ABAT
MKRN2 CLIC5
KCNS3 H2AFY
ATP6V0A1 TOP3B
SIAH1 KIAA0391
TAS2R13 SND1
FGF23 PCDHA7
TFAM ZNF449
CLDND1 PPAN
MAFG AGPAT6
CCNG2 CTRB1
ACTL6B MAPK13
APEX1 ORC3L
PARP15 MAP4K4
CLK1 RAPGEF4
KIF12 SIRT1
CTNND1 VEGFD
TCF7L2 ZNF187
MYBBP1A TUFM
ANK1 MIER3
C15orf15 ERp27
PRKCH C3orf37
ENG HAPLN1
NOLA1 RNF32
RPS15A GLCE
C15orf15 UXS1
RBMS3 ETV3
CSNK1A1  

Table 1: List of Protein array-selected antigens recognized by serum antibodies of SLE patients.

Second screening using crude peptides

The amino acid sequences of the 170 peptides shown in Table 2 were predicted as epitope sites of 67 candidate antigen proteins selected in the first screening. In the second screening, these 170 peptides were synthesized and used as antigens for analysis of the serum antibody levels by AlphaLISA. The serum levels of eight peptides (No. 55, 57, 63, 79, 87, 88, 113 and 128) were significantly higher in SLE, CI and/or CVD patients than in healthy controls, and these peptides were selected as useful markers.

No. Name Sequence No. Name Sequence No. Name Sequence No. Name Sequence
1 ZIC4-3 YKTSLVMRKRLRLYR 44 SIAH1-267 FAENGNLGINVTISM 87 MYBBP1A-1134 LYWQAMKTLGVQRPK 130 KIAA0391-180 KYLYLCVFHMQTSEV
2 ZIC4-185 FKAKYKLVNHIRVHT 45 TAS2R13-30 INCIDWVSKRELSSV 88 MYBBP1A-1306 IRSPSLLQSGAKKKA 131 KIAA0391-478 DDPFLLYATLHSGNH
3 ZIC4-269 RGCDKCYTHPSSLRK 46 TAS2R13-110 KIASFSSPAFLYLKW 89 ANK1-34 CFVLKHIHQELDKEL 132 SND1-423 INIAEALVSKGLATV
4 SDHB-238 FSLYRCHTIMNCTRT 47 TAS2R13-179 VKFTMTMFSLTPFTV 90 ANK1-99 TKKIIRKVVRQIDLS 133 PCDHA7-172 LSPNEYFFLDVPTSN
5 MGC17553-17 PSKENWFRQLRSQAV 48 TAS2R13-284 GNAKLRQAFLLVAAK 91 C15orf15-22 VRNDCKVFRFCKSKC 134 PCDHA7-789 PNPDWRYSASLRAGM
6 MGC23985-18 LTCYADDKPDKPDDK 49 FGF23-6 LRLWVCALCSVCSMS 92 PRKCH-130 YDHFVANCTLQFQEL 135 ZNF449-69 ILELLVLEQFLTILP
7 RARS2-2 ACGFRRAIACQLSRV 50 FGF23-40 IHLYTATARNSYHLQ 93 PRKCH-171 TLTGSFTEATLQRDR 136 PPAN-69 RMTLQLIKVQEGVGE
8 RARS2-179 GLLGTGFQLFGYEEK 51 FGF23-85 ITGVMSRRYLCMDFR 94 PRKCH-360 SRSTLRRQGKESSKE 137 AGPAT6-45 LYMKSLLKIFAWATL
9 RARS2-359 QMLKIMGYDWAERCQ 52 FGF23-131 QYHFLVSLGRAKRAF 95 ENG-98 VLSVNSSVFLHLQAL 138 AGPAT6-171 RYCFLLPLRIALAFT
10 RARS2-402 LRMLQNMASIKTTKE 53 TFAM-5 RSMWGVLSALGRSGA 96 ENG-473 SFVQVRVSPSVSEFL 139 AGPAT6-368 MVTYLLRMMTSWAIV
11 RARS2-500 QHLLRFDEVLYKSSQ 54 TFAM-38 LPRWFSSVLASCPKK 97 ENG-583 TSKGLVLPAVLGITF 140 CTRB1-229 GIVSWGSDTCSTSSP
12 IPO11-52 HTLDINVRWLAVLYF 55 TFAM-231 LRRTIKKQRKYGAEE 98 NOLA1-123 FYFSVKLSENMKASS 141 MAPK13-189 RAPEVILSWMHYNQT
13 IPO11-143 RQHRALLTFYHVTKT 56 CLDND1-12 ACVLSLISTIYMAAS 99 RPS15A-2 VRMNVLADALKSINN 142 ORC3L-212 SPPVVVILKDMESFA
14 IPO11-215 LKVLRKLTVNGFVEP 57 CLDND1-69 FRYNGTVGLWRRCIT 100 RPS15A-31 SKVIVRFLTVMMKHG 143 ORC3L-297 QFPFKINEKVLQVLT
15 IPO11-320 CMNLIKMIVKNYAYK 58 CLDND1-177 HLLAGLCTLGSVSCY 101 RPS15A-99 FGFIVLTTSAGIMDH 144 ORC3L-410 MNYFLVLRCLHKFTS
16 IPO11-526 DQDLVVRIETATTLK 59 MAFG-34 VRELNQHLRGLSKEE 102 C15orf15-23 RNDCKVFRFCKSKCH 145 MAP4K4-40 VEVVGNGTYGQVYKG
17 IPO11-579 HVLHVLSCVIERVNM 60 CCNG2-84 LDRFLALMKVKPKHL 103 RBMS3-130 PTNLYISNLPISMDE 146 MAP4K4-152 GLAHLHIHHVIHRDI
18 IPO11-708 KIINGYIFLSSTEFL 61 CCNG2-130 QCKCTASDIKRMEKI 104 CSNK1A1-13 VGGKYKLVRKIGSGS 147 RAPGEF4-44 PLRPANTITKVPSEK
19 SLC25A24-113 QSLQTLGLTISEQQA 62 CCNG2-181 SLDKLEAQLKACNCR 105 CSNK1A1-109 TMKTVLMLADQMISR 148 RAPGEF4-391 MMHCVFMPNTQLCPA
20 SLC25A24-248 RSLWRGNGTNVIKIA 63 CCNG2-231 KKHSKINDTEFFYWR 106 CSNK1A1-277 LRQLFRILFRTLNHQ 149 RAPGEF4-583 DVSVFTTLTINGRLF
21 SLC25A24-389 LGCGALSSTCGQLAS 64 CCNG2-270 WIVSRRTAQNLHNSY 107 C9orf32-156 SLRPNGIIVIKDNMA 150 RAPGEF4-680 QFWVVTEICLCSQLS
22 SLC25A24-430 LFRRIISKEGIPGLY 65 ACTL6B-146 FFLCKTAVLTAFANG 108 C9orf32-184 CRDLDVVRRIICSAG 151 RAPGEF4-841 SYVRQLNVIDNQRTL
23 SLC25A24-444 YRGITPNFMKVLPAV 66 ACTL6B-376 KLIASNSTMERKFSP 109 DKFZp762-2 LHSMSRLLSTKPSSI 152 SIRT1-26 MTLWQIVINILSEPP
24 OTX1-68 REEVALKINLPESRV 67 APEX1-168 VTAYVPNAGRGLVRL 110 DKFZp762-142 KWLISPVKIVSRPTI 153 SIRT1-246 VIGSSLKVRPVALIP
25 MKRN2-109 LRDRNLSGMAERKTQ 68 APEX1-189 DEAFRKFLKGLASRK 111 DKFZp762-365 PHFQGFQKLPSSPLG 154 VEGFD-155 NTSTSYISKQLFEIS
26 MKRN2-300 PSVYWVEDQNKKNEL 69 APEX1-250 ADSFRHLYPNTPYAY 112 SOSTDC1-9 YLLPLACILMKSCLA 155 VEGFD-173 TSVPELVPVKVANHT
27 MKRN2-367 QGTVRFFNSVRLWDF 70 APEX1-271 MNARSKNVGWRLDYF 113 SOSTDC1-156 KITVVTACKCKRYTR 156 ZNF187-274 CQKAFRLNSHLAQHV
28 MKRN2-402 GDLFMHLSGVESSEP 71 PARP15-9 FLHNIVVVSNCFYFQ 114 RNPC3-234 VLFGKPMVVQFARSA 157 TUFM-25 FLLQGLLRLLKAPAL
29 KCNS3-76 FRYVLNFYYTGKLHV 72 PARP15-382 YVVRVLTGVFTKGRA 115 CDC45L-15 QSQRVLLFVASDVDA 158 TUFM-408 KFNLILRQPMILEKG
30 KCNS3-171 IWIRMENPAYCLSAK 73 CLK1-75 EYRNDYTQGCEPGHR 116 CDC45L-196 TSSAMVMFELAWMLS 159 MIER3-359 NILNSFTASDLTALT
31 KCNS3-193 SVVLASIVAMCVHSM 74 CLK1-115 SKHRIHHSTSHRRSH 117 CDC45L-459 LFSRPASLSLLSKHL 160 MIER3-507 FISAHALHQHAALHS
32 KCNS3-240 RLAAAPCQKKFWKNP 75 KIF12-102 LYISRQTAQQMPSVD 118 CDC45L-488 LLPLVMAAPLSMEHG 161 ERp27-74 ILHSMVQKFPGVSFG
33 ATP6V0A1-128 LKFILRKTQQFFDEM 76 KIF12-203 CVSPSAQCLPETLST 119 ZNF649-480 QGKSPVNMVTVAMVA 162 ERp27-159 VIQIHLLLIMNKASP
34 ATP6V0A1-214 YVHKSVFIIFFQGDQ 77 KIF12-218 LRYASRAQRVTTRPQ 120 ABAT-370 FRPNAPYRIFNTWLG 163 C3orf37-169 DNWRLLTMAGIFDCW
35 ATP6V0A1-269 QMVLNQTEDHRQRVL 78 CTNND1-134 VRLLRKARDMDLTEV 121 ABAT-387 SKNLLLAEVINIIKR 164 HAPLN1-261 FYYLIHPTKLTYDVA
36 ATP6V0A1-297 VRKMKAIYHTLNLCN 79 CTNND1-211 LRNVSSERSEARRKL 122 ABAT-446 DDSIRNKLILIARNK 165 RNF32-29 LQLRNLSVADHSKTQ
37 ATP6V0A1-426 RESRILSQKNENEMF 80 CTNND1-480 NKSGNRSEKEVRAAA 123 CLIC5-41 ILWLKGVVFNVTTVD 166 RNF32-187 IKCVTRIQAYWRGCV
38 ATP6V0A1-494 LRGNPVLQLNPALPG 81 CTNND1-525 NNASRSQSSHSYDDS 124 CLIC5-212 TGLWRYLKNAYARDE 167 GLCE-17 CALFTLVTVLLWNKC
39 ATP6V0A1-526 TNKLTFLNSFKMKMS 82 TCF7L2-162 SNKVPVVQHPHHVHP 125 H2AFY-42 LWLKGVVFNVTTVDL 168 GLCE-504 PSSFVLNGFMYSLIG
40 ATP6V0A1-774 LFFFFTAFATLTVAI 83 TCF7L2-334 KEMRAKVVAECTLKE 126 H2AFY-214 LWRYLKNAYARDEFT 169 UXS1-134 VSDLVNGLVALMNSN
41 SIAH1-43 VCFDYVLPPILQCQS 84 MYBBP1A-251 LKMAASSVKKDRKLP 127 TOP3B-301 LNTVEMLRVASSSLG 170 ETV3-124 NYPFINIRSSGKIQT
42 SIAH1-182 QSCFGFHFMLVLEKQ 85 MYBBP1A-395 VRFLSPPALQGYVAW 128 TOP3B-628 HRFMKYIQAKPSRLH  
43 SIAH1-211 LIGTRKQAENFAYRL 86 MYBBP1A-2036 KTLSMREVRSCFEDP 129 KIAA0391-4 YLFGIRSFPKLWKSP  

Table 2: List of amino acid sequences of synthetic peptides used for the second screening. A total of 170 peptides were predicted as epitopes derived from 67 antigen proteins. The selected useful antigen peptides are shown in bold. Numbers of peptide names represent the first amino acid number of the original proteins.

Third screening using purified peptides

We then obtained highly purified biotinylated polypeptides, SOSTDC1-156, CTNND1-211, CLDND1-69, CCNG2-231, TFAM-231, TOP3B-628, MYBBP1A-1134 and MYBBP1A-1306. The sera of HD and SLE patients used for AlphaLISA were obtained from Shimoshizu Hospital. Serum antibodies against SOSTDC1-156, CTNND1-211, TOP3B-628 and MYBBP1A-1306 showed significantly high levels in patients with SLE as compared to those in HD (Table 3). Other peptides showed no apparent difference, probably because the peptides were selected based on the difference between CI and HD in the second screening.

    SOSTDC1-156 CTNND1-211 CLDND1-69 CCNG2-231 TFAM-231 TOP3B-628 MYBBP1A-1134 MYBBP1A-1306
HD Average 1730 2302 4518 2053 3374 1799 1739 2505
SD 442 884 2,329 492 2,882 367 433 676
Cut-off value 2,613 4,071 9,176 3,036 9,139 2,532 2,606 3,858
Total No. 111 111 111 111 111 111 111 111
Positive No. 5 6 5 5 1 5 6 3
Positive (%) 4.50% 5.40% 4.50% 4.50% 0.90% 4.50% 5.40% 2.70%
SLE Average 2,211 3,049 3,780 2,994 4,283 2,319 2,149 3,215
SD 968 2,992 2,606 5,308 4,169 2,321 1,974 3,101
Total No. 84 84 84 84 84 84 84 84
Positive No. 13 8 4 10 1 11 10 11
Positive (%) 15.50% 9.50% 4.80% 11.90% 1.20% 13.10% 11.90% 13.10%
P (vs HD) 0.000048 0.029 - 0.109 0.089 0.045 0.065 0.042

Table 3: Comparison of serum antibody levels between HD and SLE patients examined by AlphaLISA. Shown are average, SD, cut-off values (average + 2SD), total sample numbers, the number of positive sera of which the antibody levels were higher than the cut-off value, and the positive rate (%) of HD; average, SD, total sample number, number of positive sera of which the antibody levels were higher than the cut-off value, and the positive rate (%) of SLE patients; and P value of comparison between HD and SLE patients. P values lower than 0.05 and positive rates higher than 10% were marked in bold.

The antibody levels of most peptides were higher in patients with CI (Rosai Hospital and Aoba Hospital) or CVD (Chiba University Hospital and Kyoto University Hospital) than in HD (Table 4). In particular, the levels against SOSTDC1-156 and CLDND1-69 showed obvious differences between CI and HD. On the other hand, CTNND1-211 and CLDND1-69 showed large differences between CVD and HD. When the cut-off value was determined as the average + 2SD of healthy donors, the positivity of CTNND1-211 in CVD was 12.5%. We then examined another set of patients with CI (Narita Red Cross Hospital, Chiba Medical Center and Chiba University Hospital) together with DM (Chiba University Hospital). The differences between CI and HD were reproduced for SOSTDC1-156 and CLDND1-69, and CCNG2-231, and TOP3B-628 also showed clear differences (Table 5). By comparing between HD and patients with DM, CCNG2-231 showed the most obvious difference, although most of other peptides showed similar differences.

    SOSTDC1-156 CTNND1-211 CLDND1-69 CCNG2-231 TFAM-231 TOP3B-628 MYBBP1A-1134 MYBBP1A-1306
HD Average 2970 2233 2948 1804 4694 2386 2074 3808
  SD 1,187 739 1,691 442 1,392 757 703 1,060
  Cut-off value 5,344 3,711 6,331 2,688 7,479 3,900 3,479 5,928
  Total No. 128 128 127 128 125 128 127 128
  Positive No. 6 6 3 7 5 6 6 7
  Positive (%) 4.70% 4.70% 2.40% 5.50% 4.00% 4.70% 4.70% 5.50%
CI Average 3,549 2,529 3,587 1,936 5,133 2,619 2,292 4,672
  SD 1,241 1,227 1,941 413 1,508 941 902 5,267
  Total No. 128 128 128 128 125 127 128 128
  Positive  No. 13 6 10 9 11 9 8 12
  Positive (%) 10.20% 4.70% 7.80% 7.00% 8.80% 7.10% 6.30% 9.40%
  P (vs. HD) 0.00017 0.02 0.0055 0.015 0.018 0.03 0.033 0.071
CVD Average 3,260 2,815 3,624 1,948 5,084 2,358 2,278 4,136
  SD 1,076 876 1,568 454 1,514 901 740 1,114
  Total No. 128 128 128 128 124 128 128 128
  Positive No. 7 16 9 7 4 6 8 7
  Positive (%) 5.50% 12.50% 7.00% 5.50% 3.20% 4.70% 6.30% 5.50%
  P (vs. HD) 0.042 2.7E-08 0.0011 0.011 0.036 - 0.025 0.016

Table 4: Comparison of serum antibody levels among HD, CI patients and CVD patients examined by AlphaLISA.

    SOSTDC1-156 CTNND1-211 CLDND1-69 CCNG2-231 TFAM-231 TOP3B-628 MYBBP1A-1134 MYBBP1A-1306
HD Average 4823 1833 7307 3989 15203 7546 3680 5356
  SD 1,793 179 3,501 1,226 5,761 1,690 2,469 1,039
  Cut-off value 8,409 2,190 14,308 6,442 26,725 10,926 8,618 7,435
  Total No. 137 137 137 137 136 136 136 136
  Positive No. 7 4 5 7 2 6 6 7
  Positive (%) 5.10% 2.90% 3.60% 5.10% 1.50% 4.40% 4.40% 5.10%
CI Average 5,686 1,870 8,917 4,638 16,009 8,753 3,555 5,876
  SD 1,949 229 4,494 1,421 4,614 5,002 1,374 1,616
  Total No. 139 139 139 139 139 139 139 139
  Positive  No. 11 8 14 12 1 12 3 16
  Positive (%) 7.90% 5.80% 10.10% 8.60% 0.70% 8.60% 2.20% 11.50%
  P (vs HD) 0.00016 0.137 0.001 0.000063 0.202 0.0078 - 0.0017
CVD Average 5,565 1,938 9,274 4,580 17,035 9,013 4,117 5,845
  SD 2,033 397 5,780 1,418 3,718 5,739 2,160 1,451
  Total No. 108 108 108 108 108 108 108 108
  Positive No. 9 9 13 8 4 14 5 12
  Positive (%) 8.30% 8.30% 12.00% 7.40% 3.70% 13.00% 4.60% 11.10%
  P (vs HD) 0.0032 0.012 0.0022 0.00071 0.003 0.011 0.143 0.0036

Table 5: Comparison of serum antibody levels among HD, CI patients and DM patients examined by AlphaLISA.

Validation test for acute CI and TIA

We further examined the serum antibody levels using another set of sera from patients with aCI (Sawara Hospital) as well as those who has experienced TIA. The serum antibody levels to SOSTDC1-156 were higher in TIA and aCI patients as compared with those of HD (Figure 1). The levels to CTNND1-211, CLDND1-69 and CCNG2-231 were elevated at the aCI stage but not at the TIA stage.

molecular-biomarkers-diagnosis-Serum-antibody

Figure 1: Serum antibody levels examined by AlphaLISA. The levels in the sera of 228 CI patients, 44 TIA patients and 137 HD were measured by AlphaLISA using synthetic antigen peptides, SOSTDC1-156, CTNND1-211, CLDND1-69 and CCNG2-231. The box-whisker plots display the 25th, 50th and 75th percentiles. The upper and lower cross bars of the box represent 90th and 10th percentiles, respectively. Values higher than 90th percentile or lower than 10th percentile are marked by dots. P values between HD and TIA or CI patients were calculated using the Mann- Whitney U test.

Correlation analysis

We then performed Spearman correlation analysis between the antibody levels and the information of the subject persons including gender, age, height, weight, BMI, maximum intima-media thickness (IMT), blood test data and lifestyle such as smoking, alcohol intake, and work and exercise habits. Data from more than 400 patients were analyzed. The levels of SOSTDC1-156 showed a positive correlation with age, max IMT, complication of hypertension and smoking habit but reverse correlation with working and Chinese tea drinking habits (Table 6). Correlation with IMT represents that this marker reflects atherosclerosis. The levels of CTNND1-211 showed reverse correlation with weight and BMI.

  SOSTDC1-156 CTNND1-211 bCLDND1-69 bCCNG2-231
  r value P value r value P value r value P value r value P value
Gender -0.079 0.0408 0.019 0.6341 -0.019 0.6226 0.057 0.1448
Age 0.182 <0.0001 0.157 <0.0001 0.102 0.0089 0.057 0.142
Height -0.062 0.1131 -0.054 0.1639 -0.009 0.8115 -0.028 0.477
Weight -0.008 0.8351 -0.125 0.0013 -0.065 0.0932 -0.044 0.2595
Body mass index 0.039 0.3227 -0.111 0.0043 -0.074 0.0586 -0.027 0.4849
Intima media thickness (IMT) 0.218 <0.0001 0.117 0.0127 0.04 0.392 0.019 0.6819
Diabetes 0.11 0.0045 0.013 0.7397 -0.036 0.3614 0.017 0.6708
Hypertension 0.16 <0.0001 0.066 0.0919 0.038 0.3346 0.035 0.3678
Albumin/globulin ratio 0.011 0.7883 -0.005 0.9026 -0.001 0.9827 0.066 0.0962
Aspartate transaminase 0.004 0.9241 0.009 0.8197 0.016 0.6736 -0.011 0.7763
Alanine transaminase -0.013 0.7353 0.015 0.7042 -0.051 0.1903 -0.006 0.8714
Alkaline phosphatase 0.046 0.2624 0.007 0.8733 -0.031 0.4473 -0.042 0.2991
Lactate dehydrogenase -0.016 0.6972 0.061 0.1269 -0.015 0.7089 0.025 0.5356
Total billirubin 0.046 0.2502 -0.017 0.6647 0.026 0.5049 0.015 0.7068
Choline esterase -0.039 0.3834 0.009 0.8342 0.018 0.6893 -0.001 0.9749
gamma-GTP 0.027 0.4988 -0.004 0.9311 -0.019 0.6432 0.003 0.94
Total protein -0.044 0.2729 -0.073 0.0656 -0.011 0.7861 0.002 0.9596
Albumin -0.024 0.5439 -0.065 0.0994 -0.013 0.7397 0.054 0.1743
Blood urea nitrogen -0.019 0.6331 -0.038 0.3306 0 0.9916 -0.04 0.3009
Creatinin 0.01 0.7904 -0.021 0.5848 0.023 0.5547 -0.007 0.8603
Estimated glomerular filtration rate -0.004 0.9326 0.023 0.5866 -0.01 0.806 -0.004 0.9214
Uric acid -0.019 0.669 0.03 0.5104 0.011 0.7992 0.025 0.5729
Amylase -0.084 0.0875 -0.015 0.754 0.017 0.7362 -0.074 0.1322
Total cholesterol -0.067 0.1131 0.033 0.4346 -0.054 0.203 -0.022 0.5983
HDL cholesterol -0.002 0.9599 0 0.9931 0.038 0.434 0.087 0.0694
Triglyceride -0.031 0.5086 0.013 0.7773 -0.028 0.5419 -0.035 0.4594
Na -0.001 0.9811 0.002 0.95 0.003 0.937 0.077 0.0507
K -0.025 0.5245 0.043 0.2779 0.031 0.4397 0.058 0.1393
Cl 0.005 0.8985 0.061 0.1236 0.007 0.868 0.036 0.3583
C-reactive protein 0.047 0.3018 -0.046 0.3182 0.056 0.2241 -0.05 0.2732
LDL cholesterol -0.119 0.0275 0.043 0.4254 -0.07 0.194 -0.091 0.0913
White blood cell 0.015 0.7028 -0.041 0.2992 0.03 0.4382 -0.036 0.3629
Red blood cell -0.005 0.9062 -0.049 0.211 0.03 0.4471 -0.009 0.8185
Hemoblobin 0.013 0.7468 -0.059 0.136 0.034 0.3896 0.007 0.8621
Hematocrit 0.017 0.6567 -0.047 0.2325 0.039 0.3262 0.031 0.4225
Mean cell volume 0.072 0.0647 0.026 0.5057 -0.005 0.896 0.049 0.2145
Mean corpuscular hemoglobin 0.05 0.1988 -0.018 0.6498 0.009 0.8229 -0.002 0.9513
Mean corpuscular hemoglobin concentration -0.021 0.6015 -0.07 0.0737 0.019 0.6314 -0.067 0.0891
Red cell dstribution width 0.021 0.5894 -0.011 0.7837 -0.002 0.9551 -0.03 0.4462
Platelet -0.031 0.4254 -0.027 0.4944 0.027 0.4896 0.01 0.802
Mean platelet volume 0.005 0.8969 0.025 0.5186 -0.019 0.6356 0.025 0.5217
Procalcitonin -0.02 0.6023 -0.016 0.6912 0.035 0.3677 0.031 0.4303
Platelet distribution width -0.002 0.9667 -0.002 0.9601 -0.037 0.3433 0.006 0.8785
Blood sugar 0.047 0.2467 0.011 0.7832 -0.069 0.0909 -0.063 0.1215
HbA1c 0.016 0.7264 0.015 0.7405 -0.067 0.131 -0.042 0.3409
Smoking habit 0.152 <0.0001 -0.058 0.1368 -0.01 0.8047 -0.036 0.3532
Alcohol drinking habit 0.058 0.1386 -0.053 0.1762 0.029 0.4552 -0.033 0.3934
Green tea drinking habit -0.017 0.6664 0.018 0.6377 -0.014 0.7178 0.054 0.169
Coffee drinking habit -0.064 0.1022 -0.005 0.8913 -0.008 0.8346 0.021 0.5904
Chinese tea drinking habit -0.083 0.0323 -0.025 0.5245 -0.023 0.5616 0.003 0.944
Working habit -0.137 0.0005 -0.073 0.0659 -0.024 0.5457 -0.057 0.1472
Exercise habit -0.029 0.484 -0.011 0.7888 -0.024 0.5572 -0.049 0.2309

Table 6: Correlation analysis between antibody marker levels and the subject’s information. Shown are correlation coefficients (r) and P values calculated by Spearman's analysis. Significant correlations are marked in bold.

Discussion

There are various types of autoantibodies in the sera of SLE patients due to the dysfunction of T cells and the accelerated activation of B cells. Available data suggest that young women with SLE are at a substantially increased risk of AMI, congestive heart failure, and cerebrovascular accidents [12-14]. If autoantibodies develop during the progress of CI and CVD, they can be amplified in patients with SLE due to their dysregulated immune systems. Thus, we performed the first screening using SLE sera and then the second and third screenings using CI and CVD samples. Through the first screening by protein array method followed by second screening using crude peptide antigens and validation tests using three sets of control HD and patients' sera, we identified SOSTDC1, CTNND1, CLDND1 and CCNG2 as novel useful markers for the diagnosis of atherosclerosis-related diseases such as CI, CVD and DM.

The following information is known for these selected markers: SOSTDC1/sclerostin domain containing 1 (Accession No.: NM_015464) is a member of bone morphogenetic protein (BMP) of TGF-β superfamily [25,26]. It works as a BMP antagonist and suppresses cell proliferation, differentiation or cell death induced by BMP. BMPs also play important parts in the development of atherosclerosis [27]. CTNND1/catenin (cadherin-associated protein), delta 1 (Accession No.: NM_001085458) is a member of the Armadillo protein family and mediates the signaling from the cell-adhesion molecule cadherin onto cells [28]. CLDND1/claudin domain containing 1 (Accession No.: NM_001040181) contains the domain of claudin which is involved in tight junction, but its function is not known [29]. CCNG2/cyclin G2 (Accession No.: NM_004354): It is a member of the cyclin family and induced by DNA damaging agents [30].

The positivity was approximately 10% and 13% at most. Multiple factors can affect the progress of CI, CVD and DM. Spearman correlation analysis between the antibody levels and the information of the patients revealed that the levels of SOSTDC1-156 but not of CTNND1-211, CLDND1-69 or CCNG2-231 are correlated with IMT, hypertension and smoking (Table 6). Thus, the SOSTDC1-156 marker can predict atherosclerotic CI caused by hypertension and/or smoking habit. There are many causes that affect the progress of CI, and each antibody marker may be associated with a respective cause of CI. Thus, the positivity of each maker cannot be expected to particularly high. The development of an increasing number of such antibody markers may make the prediction of the onset of CI at a strong possibility.

We used the sera of patients with CI within two weeks of onset. Various antigens appear immediately after the onset of CI whereas the antibodies are not produced until two weeks later. Thus, the antibodies specifically detected in sera immediately after the onset are known to have been present prior to the onset. By measuring the levels of these antibodies, it is possible to predict the onset, i.e., serum antibody markers can be prediction markers for the onset of CI.

In most cases, CI is not induce suddenly but mediated frequently by health issues such as TIA and asymptomatic CI. When small infarctions occur, it is possible for antigens to leak out from infarction lesions. Repeated exposure to such antigens may raise the antibodies to detectable levels. In fact, the antibody levels against SOSTDC1-156 were found to be higher in TIA patients than that of those in HD (Figure 1). The antibody levels of CCNG2-231 were highly associated with DM (Table 5), and therefore, it may be useful for the early diagnosis of DM. If the levels of both SOSTDC1-156 and CCNG2-231 were high, the patient might suffer from CI caused by DM. CTNND1-211 and CLDND1-69 may contribute to diagnose CVD. Application of these biomarkers for the clinical use is very important and the early development of the diagnosis kit is expected.

Acknowledgment

The authors thank Prof. Masaki Takiguchi (Chiba University, Graduate School of Medicine) for valuable discussion. This work was partly supported by Grants-in-Aid of Japan Science and Technology Agency (Exploratory Research No. 14657335) and Ministry of Health, Labour and Welfare, and a grant from SEISHIN Medical Research Foundation.

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