Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke
Authors: Hiwasa Takaki , Wang Hao , Goto Ken-ichiro , Mine Seiichiro , Machida Toshio , Kobayashi Eiichi , Yoshida Yoichi , Adachi Akihiko , Matsutani Tomoo , Sata Mizuki , Yamagishi Kazumasa , Iso Hiroyasu , Sawada Norie , Tsugane Shoichiro , Kunimatsu Mitoshi , Kamitsukasa Ikuo , Mori Masahiro , Sugimoto Kazuo , Uzawa Akiyuki , Muto Mayumi , Kuwabara Satoshi , Kobayashi Yoshio , Ohno Mikiko , Nishi Eiichiro , Hattori Akiko , Yamamoto Masashi , Maezawa Yoshiro , Kobayashi Kazuki , Ishibashi Ryoichi , Takemoto Minoru , Yokote Koutaro , Takizawa Hirotaka , Kishimoto Takashi , Matsushita Kazuyuki , Kobayashi Sohei , Nomura Fumio , Arasawa Takahiro , Kagaya Akiko , Maruyama Tetsuro , Matsubara Hisahiro , Tomiita Minako , Hamanaka Shinsaku , Imai Yushi , Nakagawa Tomoo , Kato Naoya , Terada Jiro , Matsumura Takuma , Katsumata Yusuke , Naito Akira , Tanabe Nobuhiro , Sakao Seiichiro , Tatsumi Koichiro , Ito Masaaki , Shiratori Fumiaki , Sumazaki Makoto , Yajima Satoshi , Shimada Hideaki , Shirouzu Mikako , Yokoyama Shigeyuki , Kudo Takashi , Doi Hirofumi , Iwase Katsuro , Ashino Hiromi , Li Shu-Yang , Kubota Masaaki , Tomiyoshi Go , Shinmen Natsuko , Nakamura Rika , Kuroda Hideyuki , Iwadate Yasuo 
Published: June 2021

Abstract
Background
Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS.

Methods
Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay.

Results
The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297–311 of DIDO1, 426–440 of FOXJ2, and 607–621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case–control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS.

Conclusions
Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.