Association of Functional (GA)n Microsatellite Polymorphism in the FLI1 Gene with Susceptibility to Human Systemic Sclerosis

Aya Kawasaki¹, Keita Yamashita ², Takashi Matsushita ³, Hiroshi Furukawa ⁴, Yuya Kondo ⁵, Naoko Okiyama ⁶, Shouhei Nagaoka ⁷, Kota Shimada ⁸, Shoji Sugii ⁹, Masao Katayama ¹⁰, Shunsei Hirohata ¹¹, Akira Okamoto ¹², Noriyuki Chiba ¹³, Eiichi Suematsu ¹⁴, Keigo Setoguchi ¹⁵, Kiyoshi Migita ¹⁶, Takayuki Sumida ⁵, Shigeto Tohma ¹⁷, Minoru Hasegawa ¹⁸, Yasuhito Hamaguchi ³, Shinichi Sato ¹⁹, Yasushi Kawaguchi ²⁰, Kazuhiko Takehara ³ and Naoyuki Tsuchiya ¹, ¹Molecular and Genetic Epidemiology Laboratory, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, ²Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, ³Department of Dermatology, Kanazawa University, Kanazawa, Japan, ⁴Molecular and Genetic Epidemiology Laboratory, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan, ⁵Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, ⁶Department of Dermatology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan, ⁷Yokohama Minami Kyousai Hospital, Yokohama, Kanagawa, Japan, ⁸Department of Rheumatic Diseases, Tokyo Metropoitan Tama Medica Center, Fuchu, ⁹Department of Rheumatic Diseases, Tokyo Metropoitan Tama Medica Center, Fuchu, Japan, ¹⁰National Hospital Organization Nagoya Medical Center, Nagoya, Japan, ¹¹Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine, Tatsuno, Japan, ¹²National Hospital Organization HImeji Medical Center, Himeji, Hyogo, Japan, ¹³National Hospital Organization Morioka Medical Center, Morioka, Iwate, Japan, ¹⁴National Hospital Organization Kyushu Medical Center, Fukuoka, Japan, ¹⁵Tokyo Metropolitan Cancer and Infectious Dease Center Komagome Hospital, Tokyo, Tokyo, Japan, ¹⁶Department of Rheumatology, Fukushima Medical University School of Medicine, Fukushima, Japan, ¹⁷National Hospital Organization Tokyo National Hospital, Kiyose, Japan, ¹⁸Department of Dermatology, Fukui University, Fukui, ¹⁹University of Tokyo Graduate School of Medicine, Department of Dermatology, Tokyo, Japan, ²⁰Department of Rheumatology, Tokyo Women’s Medical University, Tokyo, Japan

Meeting: 2019 ACR/ARP Annual Meeting

Keywords: genetics, polymorphism and transcription factor, Systemic sclerosis

Session Information

Date: Tuesday, November 12, 2019

Title: Genetics, Genomics & Proteomics Poster

Session Type: Poster Session (Tuesday)

Session Time: 9:00AM-11:00AM

Background/Purpose: Susceptibility genes which can account for the characteristic features of systemic sclerosis (SSc) such as fibrosis, vasculopathy and autoimmunity remain to be determined. A series of extensive studies by Asano and colleagues using genetically engineered mice convincingly demonstrated that deficiency of a transcription factor Fli1 can play a causal role in the development of SSc with all these characteristic features. Although previous genome-wide association studies (GWAS) have not reported association of single nucleotide polymorphisms (SNVs) in the FLI1 gene, FLI1 contains (GA)_n microsatellite in its regulatory region, which has been shown to be associated with expression level of FLI1. Because microsatellite polymorphisms are not usually captured by GWAS unless they are in tight linkage disequilibrium with SNVs, we thought it is possible that FLI1 microsatellite may partly account for the “missing heritability” of SSc. In this study, we directly genotyped FLI1 (GA)_n microsatellite and examined whether it is associated with susceptibility to SSc.

Methods: Genomic DNA from 639 Japanese SSc patients and 851 healthy controls (HCs) was genotyped for (GA)_n microsatellite using the fragment assay. In the discovery stage, the genotype distribution was compared between 479 patients and 573 HCs, and the cut-off repeat number for the susceptibility to SSc was determined by receiver operating characteristic (ROC) analysis to classify the alleles into L (long) and S (short) alleles. In the replication stage, 160 patients and 268 HCs were examined, and the data from both stages were combined using meta-analysis. Association with susceptibility and clinical characteristics was examined using logistic regression analysis with the adjustment for sex. FLI1 mRNA levels were determined using quantitative RT-PCR.

Results: In the discovery stage, genotype distribution of the (GA)_n microsatellite alleles in SSc patients showed a trend towards extension as compared with that in the HCs (Cochran-Armitage test for trend, P=0.045)(Figure 1). Based on the ROC analysis, (GA)n alleles with ≥22 repeats were collectively defined as L alleles, and alleles with ≤21 repeats as S alleles. The same trend was observed in the discovery and replication stages, and when combined, (GA)_n L alleles were significantly associated with susceptibility to SSc (P=5.0E-04, odds ratio[OR]=1.34, 95% confidence interval 1,14-1.58, additive model)(Table 1). Significant association was observed both in diffuse cutaneous and limited cutaneous SSc. Among the SSc, (GA)_n L alleles were significantly enriched in the patients with modified Rodnan total skin thickness score (mRSS)≥10 compared with those with mRSS< 10 (P=9.6E-03, OR=1.63). FLI1 mRNA level in the whole blood was significantly reduced in SSc patients when compared with HCs(Figure 2a). When the mRNA levels were compared between the carriers and non-carriers of (GA)_n L alleles among HCs, significant reduction was observed in individuals carrying (GA)_n L allele (Figure 2b).

Conclusion: FLI1 (GA)_n microsatellite alleles with extended repeat numbers were associated with lower FLI1 mRNA levels and susceptibility to human SSc.

Figure　1. Distribution of FLI1 -GA-n microsatellite alleles in the patients with systemic sclerosis and healthy controls in the discovery stage.
X and Y axes show the repeat number and allele frequency -%- of FLI1 -GA-n microsatellite, respectively. The distribution in SSc patients was significantly shifted to extended alleles when compared with healthy controls -Cochran-Armitage test for trend, P=0.045-. The dotted line shows the cutoff value for the susceptibility to SSc derived from the receiver operating characteristic -ROC- analysis

Figure 2. FLI1 mRNA expression in whole peripheral blood.
The band and quartiles in the boxplot diagrams indicate median -Q2- and interquartile range -IQR- of FLI1 mRNA expression levels. P values were calculated by Mann-Whitney U-test. -a- Patients with SSc and HCs -all genotypes combined-. HCs: n=19, Q2=1.07, IQR=0.23. SSc: n=8, Q2=0.76, IQR=0.24. -b- Healthy controls carrying -L alleles 1 or 2- and not carrying -L allele 0- -GA-n L alleles. -L allele 0: n=9, Q2=1.20, IQR=0.18, L allele 1or 2: n=10, Q2=0.97, IQR=0.14.-

Disclosure: A. Kawasaki, None; K. Yamashita, None; T. Matsushita, None; H. Furukawa, Ajinomoto Co., Inc., 8, Ayumi Pharmaceutical Corporation, 8, Bristol-Myers Squibb Co., 2, Daiichi Sankyo Co., Ltd., 8, Dainippon Sumitomo Pharma Co., Ltd., 8, Daiwa Securities Health Foundation, 2, Japan Research Foundation for Clinical Pharmacology, 2, Kato Memorial Trust for Nambyo Research, 2, Luminex Japan Corporation Ltd., 8, Mitsui Sumitomo Insurance Welfare Foundation, 2, Nakatomi Foundation, 2, Pfizer Japan Inc., 8, Takeda Pharmaceutical Company, 8, Takeda Science Foundation, 2, The Nakatomi Foundation, 2; Y. Kondo, None; N. Okiyama, None; S. Nagaoka, None; K. Shimada, None; S. Sugii, None; M. Katayama, None; S. Hirohata, None; A. Okamoto, None; N. Chiba, None; E. Suematsu, None; K. Setoguchi, None; K. Migita, None; T. Sumida, None; S. Tohma, Abbott Japan Co, Ltd., 2, Abbott Japan Co., Ltd., 2, AbbVie GK., 8, Asahi Kasei Pharma Corporation, 8, Astellas Pharma Inc, 2, Astellas Pharma Inc., 2, 8, Chugai Pharmaceutical Co, Ltd., 2, Chugai Pharmaceutical Co., Ltd., 2, 8, Eisai Co, Ltd., 2, Eisai Co., Ltd., 2, Merck Sharp and Dohme Inc., 2, Mitsubishi Tanabe Pharma Corporation, 2, 8, Ono Pharmaceutical Co., Ltd., 8, Pfizer Japan Inc, 2, Pfizer Japan Inc., 2, 8, Takeda Pharmaceutical Company Limited, 2, Teijin Pharma Limited, 2; M. Hasegawa, None; Y. Hamaguchi, None; S. Sato, None; Y. Kawaguchi, None; K. Takehara, None; N. Tsuchiya, Ayumi Pharmaceutical Corporation, 8, Bristol-Myers Squibb, 2, Bristol-Myers Squibb Co. Ltd, 2, Japan College of Rheumatology, 2, Japan Rheumatism Foundation, 2.

To cite this abstract in AMA style:

Kawasaki A, Yamashita K, Matsushita T, Furukawa H, Kondo Y, Okiyama N, Nagaoka S, Shimada K, Sugii S, Katayama M, Hirohata S, Okamoto A, Chiba N, Suematsu E, Setoguchi K, Migita K, Sumida T, Tohma S, Hasegawa M, Hamaguchi Y, Sato S, Kawaguchi Y, Takehara K, Tsuchiya N. Association of Functional (GA)n Microsatellite Polymorphism in the FLI1 Gene with Susceptibility to Human Systemic Sclerosis [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/association-of-functional-gan-microsatellite-polymorphism-in-the-fli1-gene-with-susceptibility-to-human-systemic-sclerosis/. Accessed .

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