FOXP3 variants are independently associated with transforming growth factor B1 plasma levels in female patients with inflammatory bowel disease

Inoue, Cláudia Junko; Flauzino, Tamires; Goncalves, Beatriz Piantoni; Paula, Jaqueline Costa Castardo de; Galvão, Talita Cristina; Miyazaki, Paula Kikuchi; Alcantara, Camila Cataldi de; Westmore, Lucilene Rosa e Silva; Lozovoy, Marcell Alysson Batisti; Reiche, Edna Maria Vissoci; Simão, Andréa Name Colado

doi:10.1016/j.clinsp.2022.100084

Cláudia Junko Inoue

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

Outpatient Clinic of Gastroenterology, Hospital Universitário, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0003-2873-9918

Tamires Flauzino

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0001-6658-8810

Beatriz Piantoni Goncalves

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

Outpatient Clinic of Gastroenterology, Hospital Universitário, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-9559-1442

Jaqueline Costa Castardo de Paula

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0003-2726-7242

Talita Cristina Galvão

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-2937-2356

Paula Kikuchi Miyazaki

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-8783-3237

Camila Cataldi de Alcantara

Laboratory of Research in Applied Immunology, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-9181-3925

Lucilene Rosa e Silva Westmore

Outpatient Clinic of Gastroenterology, Hospital Universitário, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-9868-2059

Marcell Alysson Batisti Lozovoy

Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-4023-9548

Edna Maria Vissoci Reiche

Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0001-6507-2839

Andréa Name Colado Simão ^**Corresponding author. E-mail address:deiname@uel.br (A.N.C. Simão).

Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, Universidade Estadual de Londrina, Londrina, PR, Brazil

http://orcid.org/0000-0002-2073-6782

*Corresponding author. E-mail address:deiname@uel.br (A.N.C. Simão).

Authors’ contributions

Study Design: Andréa Simão, Edna Reiche, Marcell Lozovoy, Lucilene Westmore.

Data Collection and Experiments: Claudia Inoue, Tamires Flauzino, Beatriz Piantoni, Jaqueline Castardo, Talita Galvão, Paula Kikuchi, Camila Cataldi.

Statistical Analysis: Tamires Flauzino, Andréa Simão, Edna Reiche.

Data Interpretation: Claudia Inoue, Tamires Flauzino Marcell Lozovoy, Andréa Simão.

Manuscript Preparation: Claudia Inoue, Tamires Faluzino, Andréa Simão.

Literature Search: Claudia Inoue, Marcell Lozovoy, Edna Reiche, Andréa Simão, Lucilene Westmore.

Conflicts of interest

The authors declare no conflicts of interest.

Figures (3)
Tables (3)

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Fig. 1
Cytokine plasma levels in patients with Inflammatory Bowel Disease (IBD), Ulcerative Colitis (UC), Crohn’s Disease (CD) and controls. A) Transforming Gowth Factor beta 1 (TGF-β1) plasma levels in IBD, UC and CD patients compared to controls; B) Interleukin 10 (IL-10) plasma levels in IBD, UC and CD patients compared to controls; results were expressed as median and confidence interval 95%; p-value adjusted by age, ethnicity, body mass index, smoking, and treatment.

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Fig. 2
Cytokine plasma levels according to the haplotypes of FOXP3-924 A>G (rs2232365) and FOXP3-3279 C>A (rs3761548) variants in Inflammatory Bowel Disease (IBD) patients. A) Transforming Growth Factor beta 1 (TGF-β1) plasma levels in G/A recessive haplotype in IBD, Ulcerative Colitis (UC), and Crohn’s Disease (CD) female patients; B) TGF-β1 plasma levels in G/C dominant haplotype in IBD, UC and CD female patients; C) Interleukin 10 (IL-10) plasma levels in G/A recessive haplo-type in IBD, UC, and CD female patients; D) IL-10 plasma levels in G/C dominant haplotype model in IBD, UC, and CD female patients; results expressed as median and 95% confidence interval; p-value adjusted by age, ethnicity, body mass index, smoking, and treatment. Haplotype models: G/A recessive (GAGA vs. A/C, A/A, and G/C carriers) and G/C dominant (G/C carriers vs. A/C, A/A, and G/A carriers). Yes, presence of haplotype; No, other haplotypes

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Fig. 3
Endoscopic and clinical activity according to the haplotypes of FOXP3-924 A>G (rs2232365) and FOXP3-3279 C>A (rs3761548) variants in Inflammatory Bowel Disease (IBD) patients. A) Endoscopic activity in Ulcerative Colitis (UC) patients was evaluated by Mayo endoscopic score in G/A recessive haplotype model; B) Clinical activity in UC patients was evaluated by Partial Mayo Index Score G/A recessive haplotype model; C) Endoscopic activity in Crohn’s Disease (CD) patients was evaluated by Crohn’s disease endoscopic index of severity (CDEIS) in G/C dominant haplotype model; D) Clinical activity in CD patients was evaluated by Crohn's Disease Activity Index (CDAI) in G/C dominant haplotype model. Haplotype models: G/A recessive (GAGA vs. A/C, A/A, and G/C carriers) and G/C dominant (G/C carriers vs. A/C, A/A, and G/A carriers). Yes, Presence of haplotype; No, Other haplotypes.

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Table 1
Endoscopic and clinical parameters of female patients with Inflammatory Bowel Disease (IBD).

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Table 2
Distribution of FOXP3-924 G>A (rs2232365) and FOXP3-3279 C>A (rs3761548) genotypes and allelic frequencies among Brazilian patients with Inflammatory Bowel Disease (IBD) and controls.

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Table 3
Distribution of rs2232365 -924 (G>A) and rs3761548 -3279 (C>A) FOXP3 haplotypes among Brazilian patients with Inflammatory Bowel Disease (IBD) and controls.

Characteristics	Ulcerative Colitis (n = 60)	Crohn Disease (n = 50)
Endoscopic Activity^a a Endoscopic activity in UC patients was evaluated by Mayo endoscopic score as remission (0), mild activity (1), moderate (2), and severe (3). In CD patients by Crohn’s Disease Endoscopic Index of Severity (CDEIS) and classified in remission (< 3), mild (3‒8), moderate (9‒12), and severe (> 12).
Remission	26 (43.3)	31 (62.0)
Mild	10 (16.7)	8 (16.0)
Moderate	17 (28.3)	5 (10.0)
Severe	7 (11.7)	6 (12.0)
Clinical Activity^b b Clinical activity in UC patients was evaluated by Partial Mayo Index Score: remission (< 2), mild activity (2‒4), moderate (5‒7), and severe (> 7). In CD patients, clinical activity was assessed by Crohn's Disease Activity Index (CDAI) and the results were classified according to the activity status: remission (≤ 150), mild (151‒219), moderate (220‒450), and severe or very severe activity (> 450).
Remission	34 (56.7)	30 (60.0)
Mild	22 (36.7)	19 (38.0)
Moderate	3 (5.0)	1 (2.0)
Severe	1 (1.7)	0 (0.0)
Treatment
Sulfasalazine or 5-aminisalicylic acid (Yes/No)	55 (91.7) / 5 (8.3)	9 (18.0) / 41 (82.0)
Corticosteroids (Yes/No)	8 (13.3) / 52 (86.7)	5 (10.0) / 45 (90.0)
TNF-α inhibitors (Yes/No)	11 (18.3) / 49 (81.7)	30 (60.0) / 20 (40.0)
Azathioprine or methotrexate (Yes/No)	17 (28.3) /43 (71.7)	34 (68.0) / 16 (32.0)

	Controls (n = 154)	IBD (n = 110)	UC (n = 60)	CD (n = 50)	Controls × IBD (p-value)^* * p-value adjusted by age, ethnicity, body mass index, and smoking.	Controls × UC (p-value)^* * p-value adjusted by age, ethnicity, body mass index, and smoking.	Controls × CD (p-value)^* * p-value adjusted by age, ethnicity, body mass index, and smoking.
rs2232365 -924 (G>A)
Allele model
G	170 (55.2)	120 (54.5)	70 (58.3)	50 (50.0)	Reference	Reference	Reference
A	138 (44.8)	100 (45.5)	50 (41.7)	50 (50.0)	0.214	0.892	0.056
Co-dominant model
GG	44 (28.9)	29 (26.4)	18 (30.0)	11 (22.0)	Reference	Reference	Reference
GA	82 (53.9)	62 (56.4)	34 (56.7)	28 (56.0)	0.302	0.505	0.202
AA	26 (17.1)	19 (17.2)	8 (13.3)	11 (22.0)	0.193	0.914	0.047^a a OR = 3.147, 95% CI 1.015-9.758, p = 0.047.
Dominant model
GG	44 (28.9)	29 (26.4)	18 (30.0)	11 (22.0)	Reference	Reference	Reference
GA+AA	108 (71.1)	81 (73.6)	42 (70.0)	39 (78.0)	0.219	0.610	0.105
Recessive model
GG+GA	126 (82.9)	91 (82.7)	52 (86.7)	39 (78.0)	Reference	Reference	Reference
AA	26 (17.1)	19 (17.2)	8 (13.3)	11 (22.0)	0.368	0.627	0.097
rs3761548 -3279 (C>A)
C	215 (69.8)	140 (63.6)	73 (60.8)	67 (67.0)	Reference	Reference	Reference
A	93 (30.2)	80 (36.4)	47 (39.2)	33 (33.0)	0.478	0.287	0.896
Co-dominant
CC	69 (44.8)	45 (40.9)	24 (40.0)	21 (42.0)	Reference	Reference	Reference
CA	77 (50.0)	50 (45.5)	25 (41.7)	25 (50.0)	0.794	0.800	0.962
AA	8 (5.2)	15 (13.6)	11 (18.3)	4 (8.0)	0.141	0.053	0.510
Dominant model
CC	69 (44.8)	45 (40.9)	24 (40.0)	21 (42.0)	Reference	Reference	Reference
CA+AA	85 (55.2)	65 (59.1)	36 (60.0)	29 (58.0)	0.891	0.779	0.934
Recessive model
CC+CA	146 (94.8)	95 (86.4)	49 (81.7)	46 (92.0)	Reference	Reference	Reference
AA	8 (5.2)	15 (13.6)	11 (18.3)	4 (8.0)	0.104	0.041^b b OR = 3.221, 95% CI 1.050-9.876, p = 0.041.	0.482

Haplotypes (rs2232365/ rs3761548)	Controls, n (%)	IBD, n (%)	OR (95% CI)	p^a a p-value adjusted by age, ethnicity, body mass index, and smoking. Haplotype models: A/C dominant (A/C carriers vs. A/A, G/C, and G/A carriers), A/C recessive (ACAC vs. A/A, G/C, and G/A carriers), G/A dominant (G/A carriers vs. A/C, A/A, and G/C carriers), G/A recessive (GAGA vs. A/C, A/A, and G/C carriers), and G/C dominant (G/C carriers vs. A/C, A/A, and G/A carriers).	UC, n (%)	OR (95% CI)	p^a a p-value adjusted by age, ethnicity, body mass index, and smoking. Haplotype models: A/C dominant (A/C carriers vs. A/A, G/C, and G/A carriers), A/C recessive (ACAC vs. A/A, G/C, and G/A carriers), G/A dominant (G/A carriers vs. A/C, A/A, and G/C carriers), G/A recessive (GAGA vs. A/C, A/A, and G/C carriers), and G/C dominant (G/C carriers vs. A/C, A/A, and G/A carriers).	CD, n (%)	OR (95% CI)	p^a a p-value adjusted by age, ethnicity, body mass index, and smoking. Haplotype models: A/C dominant (A/C carriers vs. A/A, G/C, and G/A carriers), A/C recessive (ACAC vs. A/A, G/C, and G/A carriers), G/A dominant (G/A carriers vs. A/C, A/A, and G/C carriers), G/A recessive (GAGA vs. A/C, A/A, and G/C carriers), and G/C dominant (G/C carriers vs. A/C, A/A, and G/A carriers).
A/C dominant	110 (72.8)	80 (72.7)	1.270 (0.679 - 2.377)	0.454	42 (70.)	1.038 (0.499 - 2.160)	0.920	38 (76.0)	1.628 (0.692 - 3.825)	0.264
A/C recessive	28 (18.5)	19 (17.3)	1.275 (0.622 - 2.615)	0.507	8 (13.3)	0.764 (0.304 - 1.921)	0.764	11 (22.0)	1.934 (0.780 - 4.792)	0.154
G/A dominant	82 (54.3)	65 (59.1)	1.077 (0.614 - 1.890)	0.796	36 (60.0)	1.163 (0.598 - 2.262)	0.656	29 (58.0)	1.065 (0.505 - 2.247)	0.869
G/A recessive	5 (3.3)	14 (12.7)	4.003 (1.100 - 14.56)	0.035	11 (18.3)	6.107 (1.609 - 23.18)	0.008	3 (6.0)	2.215 (0.303 - 16.20)	0.433
G/C dominant	67 (44.4)	39 (35.5)	0.606 (0.341 - 1.077)	0.088	22 (36.7)	0.789 (0.400 - 1.555)	0.494	17 (34.0)	0.432 (0.196 - 0.951)	0.037

[1] *Corresponding author. E-mail address:deiname@uel.br (A.N.C. Simão).

Brasil

Brasil

FOXP3 variants are independently associated with transforming growth factor B1 plasma levels in female patients with inflammatory bowel disease

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