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IL17A and IL17RA gene polymorphisms in Fanconi anemia

Abstract

Fanconi anemia is a rare autosomal recessive disease. In this disease, cytokine pathways can induce the bone marrow failure that is observed in individuals with Fanconi anemia. Interleukin IL-17 exhibits a protective effect in organisms because it induces neutrophil recruitment and shows a pathological role in several models of autoimmune diseases, periodontal disease, cancer, allograft rejection, and graft versus host disease. Polymorphisms in the IL17A and IL17RA genes were evaluated from DNA in saliva, comparing individuals with or without Fanconi anemia, using models of genotypic transmission (additive, dominant, and recessive). Polymorphisms in the IL17A and IL17RA genes (rs2241044 [C allele], rs879577 [C allele], rs9606615 [T allele], and rs2241043 [C allele]) were risk factors for developing Fanconi anemia. We also performed an analysis of gene markers with clinical variables in the Fanconi group. Polymorphisms in the IL17A gene (rs3819025 [A allele] and rs2275913 [G allele], respectively) were associated with an age of less than 20 years (p = 0.026; RP 0.65) and the female sex (p = 0.043; RP 0.88). The IL17RA gene was also associated with age and the presence of leukoplakia (a potentially malignant oral disorder). An age of less than 20 years was associated with rs917864 (T allele; p = 0.036; RP 0.67). The presence of leukoplakia was associated with rs17606615 (T allele; p = 0.042; RP 0.47). To our knowledge, this is the first study that associates IL17A and IL17RA gene polymorphisms with Fanconi anemia and examines rs2241044 polymorphisms in scientific literature thus far.

Fanconi Anemia; Interleukin-17; Receptors, Interleukin-17

Introduction

Fanconi anemia (FA) is a rare autosomal recessive disease that is usually characterized by progressive bone marrow failure, congenital abnormalities, and a striking predisposition to the development of hematological malignancies and solid tumors.11. Alter BP, Giri N, Savage SA, Rosenberg PS. Cancer in the National Cancer Institute inherited bone marrow failure syndrome cohort after fifteen years of follow-up. Haematologica. 2018 Jan;103(1):30-9. https://doi.org/10.3324/haematol.2017.178111
https://doi.org/10.3324/haematol.2017.17...
Androgens can be used to treat the bone marrow failure, but hematopoietic stem cell transplantation (HSCT) is the only curative treatment for the hematological complications related to this disease.22. Bonfim C, Ribeiro L, Nichele S, Bitencourt M, Loth G, Koliski A, et al. Long-term survival, organ function, and malignancy after hematopoietic stem cell transplantation for Fanconi anemia. Biol Blood Marrow Transplant. 2016 Jul;22(7):1257-63. https://doi.org/10.1016/j.bbmt.2016.03.007
https://doi.org/10.1016/j.bbmt.2016.03.0...
Although results after HSCT have improved dramatically over the past decade, HSCT is associated with an increased risk of developing solid tumors in patients with FA.11. Alter BP, Giri N, Savage SA, Rosenberg PS. Cancer in the National Cancer Institute inherited bone marrow failure syndrome cohort after fifteen years of follow-up. Haematologica. 2018 Jan;103(1):30-9. https://doi.org/10.3324/haematol.2017.178111
https://doi.org/10.3324/haematol.2017.17...
,33. Latour RP, Porcher R, Dalle JH, Aljurf M, Korthof ET, Svahn J, et al. Allogeneic hematopoietic stem cell transplantation in Fanconi anemia: the European Group for Blood and Marrow Transplantation experience. Blood. 2013 Dec;122(26):4279-86. https://doi.org/10.1182/blood-2013-01-479733
https://doi.org/10.1182/blood-2013-01-47...
The risks of developing head and neck cancer, for example, are estimated to be 500–700 times likelier in patients with FA undergoing HSCT versus patients in the general population.22. Bonfim C, Ribeiro L, Nichele S, Bitencourt M, Loth G, Koliski A, et al. Long-term survival, organ function, and malignancy after hematopoietic stem cell transplantation for Fanconi anemia. Biol Blood Marrow Transplant. 2016 Jul;22(7):1257-63. https://doi.org/10.1016/j.bbmt.2016.03.007
https://doi.org/10.1016/j.bbmt.2016.03.0...
,44. Velleuer E, Dietrich R. Fanconi anemia: young patients at high risk for squamous cell carcinoma. Mol Cell Pediatr. 2014 Dec;1(1):9. https://doi.org/10.1186/s40348-014-0009-8
https://doi.org/10.1186/s40348-014-0009-...
Furthermore, oral squamous cell carcinoma (OSCC) is the most common type of cancer that develops in FA patients.55. Kutler DI, Auerbach AD, Satagopan J, Giampietro PF, Batish SD, Huvos AG, et al. High incidence of head and neck squamous cell carcinoma in patients with Fanconi anemia. Arch Otolaryngol Head Neck Surg. 2003 Jan;129(1):106-12. https://doi.org/10.1001/archotol.129.1.106
https://doi.org/10.1001/archotol.129.1.1...
The mechanisms underlying the development of cancer in this population have not been fully investigated nor reported in the literature.66. Furquim CP, Pivovar A, Amenábar JM, Bonfim C, Torres-Pereira CC. Oral cancer in Fanconi anemia: review of 121 cases. Crit Rev Oncol Hematol. 2018 May;125(125):35-40. https://doi.org/10.1016/j.critrevonc.2018.02.013
https://doi.org/10.1016/j.critrevonc.201...

Neutrophil recruitment is one of the main functions associated with interleukin IL-17. To induce neutrophil recruitment, innate cells producing IL-17 are strategically positioned to quickly recruit neutrophils and thus detect lesions in and infections of the mucosa77. Abusleme L, Moutsopoulos NM. IL-17: overview and role in oral immunity and microbiome. Oral Dis. 2017;23(7):854-65. https://doi.org/10.1111/odi.12598
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,88. Iwakura Y, Nakae S, Saijo S, Ishigame H. The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunol Rev. 2008 Dec;226(1):57-79. https://doi.org/10.1111/j.1600-065X.2008.00699.x
https://doi.org/10.1111/j.1600-065X.2008...
. This initial production of IL-17 is necessary for neutrophil recruitment and resistance to infections. Innate IL-17-producing cells not only interact with pathogens during infection, but are also critical in physiological conditions that contain microbial flora and maintain mucosal homeostasis.77. Abusleme L, Moutsopoulos NM. IL-17: overview and role in oral immunity and microbiome. Oral Dis. 2017;23(7):854-65. https://doi.org/10.1111/odi.12598
https://doi.org/10.1111/odi.12598...
,88. Iwakura Y, Nakae S, Saijo S, Ishigame H. The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunol Rev. 2008 Dec;226(1):57-79. https://doi.org/10.1111/j.1600-065X.2008.00699.x
https://doi.org/10.1111/j.1600-065X.2008...

In the tumor microenvironment, the inflammatory response involves the recruitment and activation of neutrophils to sites through the release of chemokines and cytokines, including that of IL-17.99. Silva RN, Dallarmi LB, Araujo AK, Alencar RC, Mendonça EF, Silva TA, et al. Immunohistochemical analysis of neutrophils, interleukin-17, matrix metalloproteinase-9, and neoformed vessels in oral squamous cell carcinoma. J Oral Pathol Med. 2018/07/18. 2018;47(9):856-63. https://doi.org/10.1111/jop.12762
https://doi.org/10.1111/jop.12762...
Silva et al.99. Silva RN, Dallarmi LB, Araujo AK, Alencar RC, Mendonça EF, Silva TA, et al. Immunohistochemical analysis of neutrophils, interleukin-17, matrix metalloproteinase-9, and neoformed vessels in oral squamous cell carcinoma. J Oral Pathol Med. 2018/07/18. 2018;47(9):856-63. https://doi.org/10.1111/jop.12762
https://doi.org/10.1111/jop.12762...
observed increases in gene and protein expression of IL-17 in patients with oral cancer, and this may be because IL-17 is involved in the initial activation of the immune system and improvement of inflammation induced by cancer. A previous study reported that the increased release of IL-17 by immunocytes that infiltrate tumors can promote the progression of the neoplastic processes observed in OSCC.1010. Garley M, Jablonska E, Grabowska SZ, Piotrowski L. IL-17 family cytokines in neutrophils of patients with oral epithelial squamous cell carcinoma [Internet]. Neoplasma. 2009;56(2):96-100. https://doi.org/10.4149/neo_2009_02_96
https://doi.org/10.4149/neo_2009_02_96...

From what is known, no previous studies associating gene polymorphisms of IL17A and IL17RA in a large sample of patients with FA exist. The authors hypothesize that single nucleotide polymorphisms (SNPs) in the IL17A and IL17RA genes are associated with the development of Fanconi anemia.

Methodology

Sample and data collection

The study was approved by the Human Research Ethics Committee of the Complexo Hospital de Clínicas da Universidade Federal do Paraná (Nº 2469.076/2011-04). Subjects were divided into two groups: the case group was composed of 98 individuals with FA who participated in the IV Brazilian FA Meeting (Curitiba, Brazil). The control group was composed of 30 healthy individuals from the southern region of Brazil who visited the Dental Clinic of the Pontifícia Universidade Católica do Paraná (PUCPR) for routine consultation. Subjects completed personal, medical, and dental history questionnaires, and (within a protocol approved by an Institutional Review Board) signed a consent form after being advised of the nature of the study (Human Research Ethics Committee of PUCPR; Nº 25141813.4.0000.0020).

The inclusion criteria for the case group included individuals who were previously diagnosed with FA in their referral services (assessed by salivary samples). No exclusion criteria were listed for this group. Inclusion criteria for the control group included individuals being over 18 years of age. The exclusion criteria included individuals with any systemic or dental comorbidity (such as periodontitis or odontogenic infections), or having some form of kinship with individuals who have or have had FA.

All participants, or guardians, signed an informed consent form, and answered a questionnaire regarding sociodemographic information and medical history. In the case group, clinical variables (such as HSCT, graft versus host disease [GVHD], and leukoplakia statuses) were also collected.

For each subject, unstimulated saliva was collected in a sterile Falcon tube for 5 minutes, and 500 μL aliquots were frozen at -80º C and stored until analysis.

DNA extraction

DNA was collected from salivary samples obtained from patients. The obtained buccal epithelial cells were subjected to centrifugation, at 2600 g for 10 minutes, to be sedimented. This process resulted in a supernatant (which was discarded) and a cell pellet, where the pellet was resuspended in a 1300 μL extraction buffer (10 mM Tris-HCl [pH 7.8], 5 mM EDTA, 0.5% SDS). Ten microliters of proteinase K (20 mg/mL) were added to the solution, remaining overnight at 65º C. DNA purification was performed by adding 10 M ammonium acetate, precipitated with isopropanol, and resuspended with 50 μL of 10 mM Tris (pH 7.6) and 1 mM EDTA.1111. Aidar M, Line SR. A simple and cost-effective protocol for DNA isolation from buccal epithelial cells. Braz Dent J. 2007;18(2):148-52. https://doi.org/10.1590/S0103-64402007000200012
https://doi.org/10.1590/S0103-6440200700...
The obtained DNA was stored at -20º C.

Genotyping

The tag SNP markers of the IL17A and IL17RA genes were selected according to functional relevance criteria reported in qualified articles. In this search, aspects such as the quality and impact of the journals were considered. Grouping articles, in which a similarity in the specific pathology addressed in the article in question was observed, was also performed. After this initial selection, we tried to confirm the relevance of these tag SNPs in the SNPinfo1212. National Institute of Environmental Health Sciences. SNPinfo Web Server. Year [cited 2021 Nov 2]. Available from: https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html
https://snpinfo.niehs.nih.gov/snpinfo/sn...
NIH web page. Two tag SNPs (rs3819025 and rs2275913) for the IL17A gene and six tag SNPs (rs2241044, rs9606615, rs2241049, rs917864, rs879577, and rs2241043) for the IL17RA gene were selected. The polymorphisms were genotyped by the technique of real-time PCR (Applied Biosystems 7500 Real-Time PCR System–Waltham, Massachusetts).

Statistical analysis

Nominal variables were expressed as number and frequency, and were analyzed by Pearson’s chi-square or Fisher’s exact tests. Continuous variables were expressed as mean ± standard deviation, and were analyzed by Mann-Whitney U tests. Values of p < 0.05 were considered significant. Bonferroni correction was done for multiple comparison testing, and p-values < 0.002 were considered significant for genotype associations. For the multivariate analysis, a binary logistic regression model was used, and only variables with a p-value of < 0.20 were included at the beginning of the composition of the first model; thereafter, variables with a p-value > 0.05 were excluded for the composition of the final model. Haploview 4.2 (MIT—Harvard broad institute) software was used to estimate the Hardy–Weinberg equilibrium, estipulate the associated or reference allele for each tag SNP. SPSS (IBM - Armonk, New York) version 20.0 was used for all statistical analyses.

Results

Clinical and sociodemographic variables

Both the case and control groups were composed mainly of females. The mean ages of individuals in the control and case groups were 43.8 ± 13.9 and 20.2 ± 7.8, respectively. Other clinical characteristics of individuals in the case group are shown in Table 1.

Table 1
Sociodemographic and clinical characteristics of the study population.

Genetic analysis

Polymorphisms for models of genotypic transmission (additive, dominant, and recessive) were evaluated for the control and case groups. Furthermore, the allele frequencies in the control group were found to be in Hardy-Weinberg equilibrium.

Polymorphisms in the IL17A (rs3819025) and IL17RA (rs2241044, rs9606615, rs2241049, rs917864, rs879577, and rs2241043) genes showed a statistically significant difference when analyzed between the control and case groups for additive, dominant, and recessive models (Tables 2 and 3). After Bonferroni correction was performed, only five SNPs in the IL17RA gene (rs9606615, rs2241049, rs917864, rs879577, and rs2241043) maintained significance (p < 0.002).

Table 2
Genotypic analysis in the additive model of the tag SNPs in the IL17 and IL17RA genes.
Table 3
Genotypic analysis for the dominant and recessive models of the IL17A and IL17RA genes.

The adjusted final model for logistic regression, which compared individuals with or without FA, showed that FA was associated with four polymorphisms (rs2241044 [p = 0.011], rs9606615 [p < 0.001], rs879577 [p = 0.001], rs2241043 [p < 0.001]).

We also performed a nested analysis of gene markers with clinical variables in the case group. Polymorphisms in the IL17A gene showed an association of FA regarding an age of less than 20 years, the rs3819025 (A allele) SNP (p = 0.026; RP 0.65), and the female sex (rs2275913 [G allele; p = 0.043; RP 0.88]). The IL17RA gene was also associated with age and the presence of leukoplakia. An age of less than 20 years was associated with the rs917864 (T allele) SNP (p = 0.036; RP 0.67). The presence of leukoplakia was associated with the rs17606615 (T allele) SNP (p = 0.042; RP 0.47). Other variables (such as HSCT, GVHD, and subject relation) were not associated with gene markers of IL17A and IL17RA.

Discussion

The aim of this study was to investigate whether polymorphisms in the IL-17 (IL17A and IL17RA) genes, where IL-17 protein expression is linked to important physiological functions, could be associated with FA. In our results, we found that some polymorphisms were risk factors for the development of FA.

The loss of function of the FA/BRCA pathway in FA affects DNA repair mechanisms. Abnormally high levels of several proinflammatory cytokines, such as tumor necrosis factor alpha and interferon gamma, can contribute to disease progression and are associated with increased apoptosis.1313. Zhang QS, Deater M, Phan N, Marcogliese A, Major A, Guinan EC, et al. Combination therapy with atorvastatin and celecoxib delays tumor formation in a Fanconi anemia mouse model. Pediatr Blood Cancer. 2019 Jan;66(1):e27460. https://doi.org/10.1002/pbc.27460
https://doi.org/10.1002/pbc.27460...
The effect on cytokine pathways can favor the bone marrow failure that is observed in FA patients. Our results showed that IL17A and IL17RA gene polymorphisms were associated with FA, in a large sample of FA patients.

The relationship between IL-17 and immune-mediated diseases is already well-reported in the literature.1414. Medrano LM, García-Magariños M, Dema B, Espino L, Maluenda C, Polanco I, et al. Th17-related genes and celiac disease susceptibility. PLoS One. 2012;7(2):e31244. https://doi.org/10.1371/journal.pone.0031244
https://doi.org/10.1371/journal.pone.003...

15. Park JS, Park BL, Kim MO, Heo JS, Jung JS, Bae DJ, et al. Association of single nucleotide polymorphisms on Interleukin 17 receptor A (IL17RA) gene with aspirin hypersensitivity in asthmatics [Internet]. Hum Immunol. 2013 May;74(5):598-606. https://doi.org/10.1016/j.humimm.2012.11.002
https://doi.org/10.1016/j.humimm.2012.11...

16. Lew BL, Cho HR, Haw S, Kim HJ, Chung JH, Sim WY. Association between IL17A/IL17RA gene polymorphisms and susceptibility to alopecia areata in the Korean population. Ann Dermatol. 2012 Feb;24(1):61-5. https://doi.org/10.5021/ad.2012.24.1.61
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17. McGovern DP, Rotter JI, Mei L, Haritunians T, Landers C, Derkowski C, et al. Genetic epistasis of IL23/IL17 pathway genes in Crohn’s disease. Inflamm Bowel Dis. 2009 Jun;15(6):883-9. https://doi.org/10.1002/ibd.20855
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18. Kunisato T, Watanabe M, Inoue N, Okada A, Nanba T, Kobayashi W, et al. Polymorphisms in Th17-related genes and the pathogenesis of autoimmune thyroid disease. Autoimmunity. 2018 Nov;51(7):360-9. https://doi.org/10.1080/08916934.2018.1534963
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19. Wu TW, Chou CL, Chen YC, Juang YL, Wang LY. Associations of common genetic variants on IL-17 genes and carotid intima-media thickness. J Atheroscler Thromb. 2018 Nov;25(11):1156-67. https://doi.org/10.5551/jat.44453
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20. Catanoso MG, Boiardi L, Macchioni P, Garagnani P, Sazzini M, De Fanti S, et al. IL-23A, IL-23R, IL-17A and IL-17R polymorphisms in different psoriatic arthritis clinical manifestations in the northern Italian population. Rheumatol Int. 2013 May;33(5):1165-76. https://doi.org/10.1007/s00296-012-2501-6
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-2121. Batalla A, Coto E, González-Lara L, González-Fernández D, Gómez J, Aranguren TF, et al. Association between single nucleotide polymorphisms IL17RA rs4819554 and IL17E rs79877597 and Psoriasis in a Spanish cohort. J Dermatol Sci. 2015 Nov;80(2):111-5. https://doi.org/10.1016/j.jdermsci.2015.06.011
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Associations with other diseases (such as periodontal disease, cancer, allograft rejection, and GVHD) have also been studied.1010. Garley M, Jablonska E, Grabowska SZ, Piotrowski L. IL-17 family cytokines in neutrophils of patients with oral epithelial squamous cell carcinoma [Internet]. Neoplasma. 2009;56(2):96-100. https://doi.org/10.4149/neo_2009_02_96
https://doi.org/10.4149/neo_2009_02_96...
,2222. Espinoza JL, Takami A, Onizuka M, Kawase T, Sao H, Akiyama H, et al. A single nucleotide polymorphism of IL-17 gene in the recipient is associated with acute GVHD after HLA-matched unrelated BMT. Bone Marrow Transplant. 2011 Nov;46(11):1455-63. https://doi.org/10.1038/bmt.2010.325
https://doi.org/10.1038/bmt.2010.325...

23. Espinoza JL, Takami A, Nakata K, Onizuka M, Kawase T, Akiyama H, et al. A genetic variant in the IL-17 promoter is functionally associated with acute graft-versus-host disease after unrelated bone marrow transplantation. PLoS One. 2011;6(10):e26229. https://doi.org/10.1371/journal.pone.0026229
https://doi.org/10.1371/journal.pone.002...

24. Karimi MH, Salek S, Yaghobi R, Ramzi M, Geramizadeh B, Hejr S. Association of IL-17 gene polymorphisms and serum level with graft versus host disease after allogeneic hematopoietic stem cell transplantation. Cytokine. 2014 Sep;69(1):120-4. https://doi.org/10.1016/j.cyto.2014.05.011
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25. Lee YC, Chung JH, Kim SK, Rhee SY, Chon S, Oh SJ, et al. Association between interleukin 17/interleukin 17 receptor gene polymorphisms and papillary thyroid cancer in Korean population. Cytokine. 2015 Feb;71(2):283-8. https://doi.org/10.1016/j.cyto.2014.11.011
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26. Avadhani AV, Parachuru VP, Milne T, Seymour GJ, Rich AM. Multiple cells express interleukin 17 in oral squamous cell carcinoma. J Oral Pathol Med. 2017 Jan;46(1):39-45. https://doi.org/10.1111/jop.12465
https://doi.org/10.1111/jop.12465...

27. Gorczynski RM. IL-17 Signaling in the tumor microenvironment. Adv Exp Med Biol. 2020;1240:47-58. https://doi.org/10.1007/978-3-030-38315-2_4
https://doi.org/10.1007/978-3-030-38315-...
-2828. Li C, Zhao Y, Zhang W, Zhang W. Increased prevalence of TH17 cells in the peripheral blood of patients with head and neck squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112(1):81-9. https://doi.org/10.1016/j.tripleo.2010.11.032
https://doi.org/10.1016/j.tripleo.2010.1...
IL-17 is present in the tumor bed of most solid tumors and in hematopoietic neoplasms, which is why these studies highlight the IL-17/IL-17R axis as being a potentially new immunotherapeutic target.2727. Gorczynski RM. IL-17 Signaling in the tumor microenvironment. Adv Exp Med Biol. 2020;1240:47-58. https://doi.org/10.1007/978-3-030-38315-2_4
https://doi.org/10.1007/978-3-030-38315-...

The study by Silva et al.99. Silva RN, Dallarmi LB, Araujo AK, Alencar RC, Mendonça EF, Silva TA, et al. Immunohistochemical analysis of neutrophils, interleukin-17, matrix metalloproteinase-9, and neoformed vessels in oral squamous cell carcinoma. J Oral Pathol Med. 2018/07/18. 2018;47(9):856-63. https://doi.org/10.1111/jop.12762
https://doi.org/10.1111/jop.12762...
demonstrated that IL-17 immunolocation in OSCC samples was positive in the cytoplasm of neoplastic cells, where some samples showed high IL-17 counts in comparison to healthy individuals. Similar to the IL-17 expression observed in the tumor stroma, an extensive percentage of IL-17 immunostaining was observed in the cytoplasm of OSCC neoplastic cells. The results by Silva et al.99. Silva RN, Dallarmi LB, Araujo AK, Alencar RC, Mendonça EF, Silva TA, et al. Immunohistochemical analysis of neutrophils, interleukin-17, matrix metalloproteinase-9, and neoformed vessels in oral squamous cell carcinoma. J Oral Pathol Med. 2018/07/18. 2018;47(9):856-63. https://doi.org/10.1111/jop.12762
https://doi.org/10.1111/jop.12762...
still showed an expression of IL-17, and IL-17 mRNA expression was significantly higher in patients with OSCC versus healthy controls. Other authors2626. Avadhani AV, Parachuru VP, Milne T, Seymour GJ, Rich AM. Multiple cells express interleukin 17 in oral squamous cell carcinoma. J Oral Pathol Med. 2017 Jan;46(1):39-45. https://doi.org/10.1111/jop.12465
https://doi.org/10.1111/jop.12465...
have also demonstrated that the spatial distribution of IL-17+ cells suggests specific interactions between various types of cells that express IL-17 and other cells in the tumor microenvironment, implying that IL-17+ cells likely play a role in oral carcinogenesis. In other studies,1010. Garley M, Jablonska E, Grabowska SZ, Piotrowski L. IL-17 family cytokines in neutrophils of patients with oral epithelial squamous cell carcinoma [Internet]. Neoplasma. 2009;56(2):96-100. https://doi.org/10.4149/neo_2009_02_96
https://doi.org/10.4149/neo_2009_02_96...
,2828. Li C, Zhao Y, Zhang W, Zhang W. Increased prevalence of TH17 cells in the peripheral blood of patients with head and neck squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112(1):81-9. https://doi.org/10.1016/j.tripleo.2010.11.032
https://doi.org/10.1016/j.tripleo.2010.1...
some researcher demonstrated that IL-17 levels were higher in the blood serum of patients with OSCC versus healthy controls, where this difference was associated with advanced stages of tumor invasion.2828. Li C, Zhao Y, Zhang W, Zhang W. Increased prevalence of TH17 cells in the peripheral blood of patients with head and neck squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112(1):81-9. https://doi.org/10.1016/j.tripleo.2010.11.032
https://doi.org/10.1016/j.tripleo.2010.1...

OSCC is the most common tumor in patients with FA. Failure in DNA repair mechanisms is the most likely reason for the development of cancer in these patients, where the risk of OSCC development increases after HSCT.11. Alter BP, Giri N, Savage SA, Rosenberg PS. Cancer in the National Cancer Institute inherited bone marrow failure syndrome cohort after fifteen years of follow-up. Haematologica. 2018 Jan;103(1):30-9. https://doi.org/10.3324/haematol.2017.178111
https://doi.org/10.3324/haematol.2017.17...
,2929. Niraj J, Färkkilä A, D’Andrea AD. The Fanconi anemia pathway in cancer. Annu Rev Cancer Biol. 2019 Mar;3(1):457-78. https://doi.org/10.1146/annurev-cancerbio-030617-050422
https://doi.org/10.1146/annurev-cancerbi...
Our results demonstrate that some polymorphisms in the IL17A and IL17RA genes have alleles that are risk factors for the development of FA. In nested analyses, we also found that leukoplakia was associated with FA (rs9606615 [T allele]). Leukoplakia is a potentially malignant oral disorder. Leukoplakia was observed in 12% of patients with FA in a cohort of 138 patients, with a higher prevalence at a mean age of 16 years.3030. Cavalcanti LG, Lyko KF, Araújo RLF, Amenábar JM, Physician CB, Torres-Pereira CC. Oral leukoplakia in patients with Fanconi anaemia without hematopoietic stem cell transplantation. Pediatr Blood Cancer. 2015;62(6):1024-6. https://doi.org/10.1002/pbc.25417
https://doi.org/10.1002/pbc.25417...
Future longitudinal studies should evaluate the results of these polymorphisms and their associations with important clinical characteristics (bone marrow failure, HSCT, clinical presentation of potentially malignant lesions [such as leukoplakia], and OSCC). Such studies will help contribute to the understanding of aspects involved in the pathophysiology of FA – particularly, the process of carcinogenesis and the tropism of oral carcinogenesis.

In the nested analysis, we also observed an association between the rs2275913 (G allele) and rs917864 (T allele) SNPs with an age of less than 20 years (this association was also observed in the control group; Table 3). Female sex was also associated with the rs2275913 (G allele) SNP (where this association was not observed in the control group). In their review of 121 cases of OSCC in FA, Furquim et al.66. Furquim CP, Pivovar A, Amenábar JM, Bonfim C, Torres-Pereira CC. Oral cancer in Fanconi anemia: review of 121 cases. Crit Rev Oncol Hematol. 2018 May;125(125):35-40. https://doi.org/10.1016/j.critrevonc.2018.02.013
https://doi.org/10.1016/j.critrevonc.201...
observed a prevalence higher than 75% in patients with OSCC who were younger than 25 years of age, but these results were not statistically different to the prevalence of OSCC and sex. Conversely, female patients tended to develop more second primary tumors than did male patients.

After Bonferroni correction, only five SNPs (rs9606615, rs2241049, rs917864, rs879577, and rs2241043) in the IL17RA gene maintained significance levels, and the adjusted final model for logistic regression showed association to four polymorphisms (rs9606615 [p < 0.001], rs879577 [p = 0.001], rs2241043 [p < 0.001], and rs2241044 [p = 0.011]).

The 1000Genome database was used to search the minimum allele frequency (MAF) for significant tag SNPs. The C allele in the rs9606615 SNP was 34.5% lower than our results (57%) for the same SNP. For the rs9606615 SNP, the T allele (89.5%) displayed a higher frequency. Other studies found that this polymorphism was significantly associated with asthma,1515. Park JS, Park BL, Kim MO, Heo JS, Jung JS, Bae DJ, et al. Association of single nucleotide polymorphisms on Interleukin 17 receptor A (IL17RA) gene with aspirin hypersensitivity in asthmatics [Internet]. Hum Immunol. 2013 May;74(5):598-606. https://doi.org/10.1016/j.humimm.2012.11.002
https://doi.org/10.1016/j.humimm.2012.11...
psoriatic arthritis,2020. Catanoso MG, Boiardi L, Macchioni P, Garagnani P, Sazzini M, De Fanti S, et al. IL-23A, IL-23R, IL-17A and IL-17R polymorphisms in different psoriatic arthritis clinical manifestations in the northern Italian population. Rheumatol Int. 2013 May;33(5):1165-76. https://doi.org/10.1007/s00296-012-2501-6
https://doi.org/10.1007/s00296-012-2501-...
autoimmune thyroid disease,1818. Kunisato T, Watanabe M, Inoue N, Okada A, Nanba T, Kobayashi W, et al. Polymorphisms in Th17-related genes and the pathogenesis of autoimmune thyroid disease. Autoimmunity. 2018 Nov;51(7):360-9. https://doi.org/10.1080/08916934.2018.1534963
https://doi.org/10.1080/08916934.2018.15...
and carotid intima-media thickness.1919. Wu TW, Chou CL, Chen YC, Juang YL, Wang LY. Associations of common genetic variants on IL-17 genes and carotid intima-media thickness. J Atheroscler Thromb. 2018 Nov;25(11):1156-67. https://doi.org/10.5551/jat.44453
https://doi.org/10.5551/jat.44453...

For the rs879577 SNP, we found a higher frequency of the C allele (67.6%) in the FA group. In the 1000Gnenome database, this missense variant, presenting 27.1% for T allele MAF. The same gene polymorphism was found in other studies assessing alopecia,1616. Lew BL, Cho HR, Haw S, Kim HJ, Chung JH, Sim WY. Association between IL17A/IL17RA gene polymorphisms and susceptibility to alopecia areata in the Korean population. Ann Dermatol. 2012 Feb;24(1):61-5. https://doi.org/10.5021/ad.2012.24.1.61
https://doi.org/10.5021/ad.2012.24.1.61...
psoriasis,2121. Batalla A, Coto E, González-Lara L, González-Fernández D, Gómez J, Aranguren TF, et al. Association between single nucleotide polymorphisms IL17RA rs4819554 and IL17E rs79877597 and Psoriasis in a Spanish cohort. J Dermatol Sci. 2015 Nov;80(2):111-5. https://doi.org/10.1016/j.jdermsci.2015.06.011
https://doi.org/10.1016/j.jdermsci.2015....
Crohn’s disease,1717. McGovern DP, Rotter JI, Mei L, Haritunians T, Landers C, Derkowski C, et al. Genetic epistasis of IL23/IL17 pathway genes in Crohn’s disease. Inflamm Bowel Dis. 2009 Jun;15(6):883-9. https://doi.org/10.1002/ibd.20855
https://doi.org/10.1002/ibd.20855...
and papillary thyroid câncer.2525. Lee YC, Chung JH, Kim SK, Rhee SY, Chon S, Oh SJ, et al. Association between interleukin 17/interleukin 17 receptor gene polymorphisms and papillary thyroid cancer in Korean population. Cytokine. 2015 Feb;71(2):283-8. https://doi.org/10.1016/j.cyto.2014.11.011
https://doi.org/10.1016/j.cyto.2014.11.0...

The rs2241043 SNP showed a higher frequency in the C allele (85.3%) in the FA group, but in the 1000Genome database, the MAFs for the C and T alleles were 53.7% and 46.3%, respectively. In other studies that focused on psoriatic arthritis2020. Catanoso MG, Boiardi L, Macchioni P, Garagnani P, Sazzini M, De Fanti S, et al. IL-23A, IL-23R, IL-17A and IL-17R polymorphisms in different psoriatic arthritis clinical manifestations in the northern Italian population. Rheumatol Int. 2013 May;33(5):1165-76. https://doi.org/10.1007/s00296-012-2501-6
https://doi.org/10.1007/s00296-012-2501-...
and carotid intima-media thickness,1919. Wu TW, Chou CL, Chen YC, Juang YL, Wang LY. Associations of common genetic variants on IL-17 genes and carotid intima-media thickness. J Atheroscler Thromb. 2018 Nov;25(11):1156-67. https://doi.org/10.5551/jat.44453
https://doi.org/10.5551/jat.44453...
significant results for this polymorphism were not found. However, Park et al.1515. Park JS, Park BL, Kim MO, Heo JS, Jung JS, Bae DJ, et al. Association of single nucleotide polymorphisms on Interleukin 17 receptor A (IL17RA) gene with aspirin hypersensitivity in asthmatics [Internet]. Hum Immunol. 2013 May;74(5):598-606. https://doi.org/10.1016/j.humimm.2012.11.002
https://doi.org/10.1016/j.humimm.2012.11...
demonstrated that rs2241043 gene polymorphisms decrease the risk of aspirin-exacerbated respiratory diseases.

The MAF for the C allele was 42.3% in the rs2241044 SNP. This marker, together with the prior SNP, appeared in multivariate analyses. For the rs2241044 SNP, we found a higher frequency (79.1%) of the C allele in individuals with FA. Medrano et al.1414. Medrano LM, García-Magariños M, Dema B, Espino L, Maluenda C, Polanco I, et al. Th17-related genes and celiac disease susceptibility. PLoS One. 2012;7(2):e31244. https://doi.org/10.1371/journal.pone.0031244
https://doi.org/10.1371/journal.pone.003...
included the rs2241044 SNP in their study on celiac disease, although this SNP was not analyzed in their sample. Thus, no other study analyzing this gene polymorphism has been conducted.

In their study on IL17A gene polymorphisms in GVHD, Karimi et al.2424. Karimi MH, Salek S, Yaghobi R, Ramzi M, Geramizadeh B, Hejr S. Association of IL-17 gene polymorphisms and serum level with graft versus host disease after allogeneic hematopoietic stem cell transplantation. Cytokine. 2014 Sep;69(1):120-4. https://doi.org/10.1016/j.cyto.2014.05.011
https://doi.org/10.1016/j.cyto.2014.05.0...
found a higher frequency of the GG genotype (rs2275913) in patients who developed GVHD after unrelated HSCT versus patients who did not present with GVHD. In addition, after multivariate analysis, the researchers found a higher frequency in men with GVHD when compared to men without GVHD. AA genotype (rs2275913) results were associated with a higher chance for the development of severe versus mild grades of GVHD. We did not find rs2275913 as being a risk factor for FA. In the increased frequency of the G allele (rs3819025), after adjustment by multivariate analysis, they found a higher frequency of GVHD in mild degrees. Thus, they suggest IL17A as a marker for predicting GVHD after unrelated allografts. In our results, patients with FA presented with a higher frequency of the rs3819025 (G allele) SNP when compared with control patients. In addition, 82.3% of individuals with FA underwent HSCT and 41.1% manifested GVHD (Table 1), but in the nested analysis, we did not observe an association of FA with HSCT or GVHD.

In two studies, Espinoza et al.2222. Espinoza JL, Takami A, Onizuka M, Kawase T, Sao H, Akiyama H, et al. A single nucleotide polymorphism of IL-17 gene in the recipient is associated with acute GVHD after HLA-matched unrelated BMT. Bone Marrow Transplant. 2011 Nov;46(11):1455-63. https://doi.org/10.1038/bmt.2010.325
https://doi.org/10.1038/bmt.2010.325...
,2323. Espinoza JL, Takami A, Nakata K, Onizuka M, Kawase T, Akiyama H, et al. A genetic variant in the IL-17 promoter is functionally associated with acute graft-versus-host disease after unrelated bone marrow transplantation. PLoS One. 2011;6(10):e26229. https://doi.org/10.1371/journal.pone.0026229
https://doi.org/10.1371/journal.pone.002...
found a higher frequency of the AA genotype (rs2275913) associated with severe grades of GVHD. Our results did not demonstrate rs2275913 as a risk factor for the development of FA. In another association, the researchers claim that the donor’s IL17A genotype does not influence the results of HSCT. Though individuals in our sample underwent HSCT (82.3%) and manifested GVHD (41.1%), a bias in comparison with these studies exists, since they do not report whether the underlying disease (with indication for HSCT) was a consequence of FA. In the nested analyses, we also did not observe an association with donor relation, HSCT, or GVHD.

Our results present new observations regarding FA that have never been previously described. We found that IL17A and IL17RA gene polymorphisms contribute significantly as risk factors for the development FA. We also demonstrated an association of FA with a potentially malignant oral disorder, leukoplakia, bringing a new perspective to studies that search for answers to the understanding of OSCC carcinogenesis in FA patients. Clearly, these results should be confirmed with more extensive studies and associated with clinical variables (such as the phenotypic characteristics of individuals, specificities of treatment, variables related to HSCT [mainly GVHD], and potentially malignant oral disorder).

Conclusion

To conclude, IL17A and IL17RA gene polymorphisms, including the SNPs rs2241044 (C allele), rs879577 (C allele), rs9606615 (T allele), and rs2241043 (C allele), were associated with the development of FA. When comparing individuals with or without FA, an association between age (rs3819025 [A allele]; rs917864 [T allele]), sex (rs2275913 [G allele]) and leukoplakia (rs17606615 [T allele]) were also associated with the development of FA, as observed via nested analyses.

Acknowledgments

We appreciate Coordenação de Aperfeiçoamento de Pessoal de Nível Superior of Brazil’s government for encouraging scholarships to the Post Graduate Program in Dentistry of UFPR.

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Publication Dates

  • Publication in this collection
    13 Feb 2023
  • Date of issue
    2023

History

  • Received
    2 Dec 2021
  • Accepted
    18 Aug 2022
  • Reviewed
    30 Aug 2022
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