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TP53 and p21 (CDKN1A) polymorphisms and the risk of systemic lupus erythematosus

Abstract

Background

The p53 and p21 proteins are important regulators of cell cycle and apoptosis and may contribute to autoimmune diseases, such as systemic lupus erythematosus (SLE). As genetic polymorphisms may cause changes in protein levels and functions, we investigated associations of TP53 and p21 (CDKN1A) polymorphisms (p53 72 G > C—rs1042522; p53 PIN3—rs17878362; p21 31 C > A—rs1801270; p21 70 C > T—rs1059234) with the development of systemic lupus erythematosus (SLE) in a Southeastern Brazilian population.

Methods

Genotyping of 353 female volunteers (cases, n = 145; controls, n = 208) was performed by polymerase chain reaction, restriction fragment length polymorphism and/or DNA sequencing. Associations between TP53 and p21 polymorphisms and SLE susceptibility and clinical manifestations of SLE patients were assessed by logistic regression analysis.

Results

Protective effect was observed for the genotype combinations p53 PIN3 A1/A1 -p21 31 C/A, in the total study population (OR 0.45), and p53 PIN3 A1/A2-p21 31 C/C, in non-white women (OR 0.28). In Whites, p53 72 C-containing (OR 3.06) and p53 PIN3 A2-containing (OR 6.93) genotypes were associated with SLE risk, and higher OR value was observed for the combined genotype p53 72 G/C-p53 PIN3 A1/A2 (OR 9.00). Further, p53 PIN3 A1/A2 genotype was associated with serositis (OR 2.82), while p53 PIN3 A2/A2 and p53 72 C/C genotypes were associated with neurological disorders (OR 4.69 and OR 3.34, respectively).

Conclusions

Our findings showed that the TP53 and p21 polymorphisms included in this study may have potential to emerge as SLE susceptibility markers for specific groups of patients. Significant interactions of the TP53 polymorphisms with serositis and neurological disorders were also observed in SLE patients.

Highlights

  • The polymorphisms TP53 rs1042522 (G > C) and TP53 rs17878362 (16 bp Del/Ins) were associated with SLE risk in whites.

  • In whites, the combined genotype TP53 rs1042522 GC- TP53 rs17878362 A1A2 and the haplotype TP53 rs1042522 C-rs17878362 A2 represented higher SLE risk.

  • Combination of TP53 rs17878362 (16 bp Del/Ins) and p21 rs1801270 (C > A) protected against SLE in non-white women.

  • TP53 and p21 (CDKN1A) polymorphisms may be SLE susceptibility markers for specific groups.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown in self-tolerance, dysregulation of lymphocytes number and subsets in association with an increased autoantibody production [11 Balomenos D. Cell cycle regulation and systemic lupus erythematosus. In: Lahita RG, editor. Systemic lupus erythematosus. 5th ed. London: Academic Press; 2011. p. 191-8. https://doi.org/10.1016/B978-0-12-374994-9.10011-7.
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, 22 Sharabi A, Tsokos GC. T cell metabolism: new insights in systemic lupus erythematosus pathogenesis and therapy. Nat Rev Rheumatol. 2020;16:100-12. https://doi.org/10.1038/s41584-019-0356-x.
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]. Although the interaction of genetic, immunological and environmental factors is widely accepted, this complex pathophysiological process is not fully understood [33 Pan L, Lu MP, Wang JH, Xu M, Yang SR. Immunological pathogenesis and treatment of systemic lupus erythematosus. World J Pediatr. 2020;16:19-https://doi.org/10.1007/s12519-019-00229-3.
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]. There is a central role for an excess of autoantigens derived mainly from altered programmed cell-death mechanisms like NETosis, pyroptosis and apoptosis [44 Gupta S, Kaplan MJ. The role of neutrophils and NETosis in autoimmune and renal diseases. Nat Rev Nephrol. 2016;12:402-13. https://doi.org/10.1038/nrneph.2016.71.
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] in association with a deficient clearance of extracellular vesicles associated autoantigens [55 Gaipl US, Munoz LE, Grossmayer G, Lauber K, Franz S, Sarter K, et al. Clearance deficiency and systemic lupus erythematosus (SLE). J Autoimmun. 2007;28:114-21. https://doi.org/10.1016/jjaut.2007.02.005.
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, 66 Zhao Y, Wei W, Liu ML. Extracellular vesicles and lupus nephritis—new insights into pathophysiology and clinical implications. J Autoimmun. 2020;115:102540. https://doi.org/10.1016/j.jaut.2020.102540.
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].

It has already been suggested that apoptosis plays an important role in the elimination of autoreactive lymphocytes in SLE patients. Cell cycle control, in its turn, is responsible for maintaining lymphocyte homeostasis as it participates in lymphocyte differentiation, effector function, memory development, tolerance induction, and apoptosis [55 Gaipl US, Munoz LE, Grossmayer G, Lauber K, Franz S, Sarter K, et al. Clearance deficiency and systemic lupus erythematosus (SLE). J Autoimmun. 2007;28:114-21. https://doi.org/10.1016/jjaut.2007.02.005.
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]. Therefore, defects in the regulation of apoptotic process and cell cycle may cause loss of tolerance, generating autoimmune responses [11 Balomenos D. Cell cycle regulation and systemic lupus erythematosus. In: Lahita RG, editor. Systemic lupus erythematosus. 5th ed. London: Academic Press; 2011. p. 191-8. https://doi.org/10.1016/B978-0-12-374994-9.10011-7.
https://doi.org/10.1016/B978-0-12-374994...
].

One of the major regulators of cell cycle and apopto- sis is the p53 tumor suppressor protein. The protein p53 acts as a transcription factor and regulates the expression of genes involved in several cellular processes, including cell cycle arrest, DNA repair and activation of apoptosis [77 Takatori H, Kawashima H, Suzuki K, Nakajima H. Role of p53 in systemic autoimmune diseases. Crit Rev Immunol. 2014;34:509-16. https://doi.org/10.1615/CritRevImmunol.2014012193.
https://doi.org/10.1615/CritRevImmunol.2...
]. In SLE patients, it has been observed high levels of p53 expression and anti-p53 antibodies, with a significant correlation between p53 levels and SLE activity index [88 Kovacs B, Patel A, Hershey JN, Dennis GJ, Kirschfink M, Tsokos GC. Antibodies against p53 in sera from patients with systemic lupus erythematosus and other rheumatic diseases. Arthritis Rheum. 1997;40:980-2. https://doi.org/10.1002/art.1780400531.
https://doi.org/10.1002/art.1780400531...
].

The TP53 gene, which encodes the p53 protein, is located on chromosome 17 (17p13.1), contains 11 exons, and its expression is controlled by two promoter regions giving rise to 12 different p53 protein isoforms [99 Joruiz SM, Bourdon JC. p53 isoforms: key regulators of the cell fate decision. Cold Spring Harb Perspect Med. 2016;6:a026039. https://doi.org/10.1101/cshperspect.a026039.
https://doi.org/10.1101/cshperspect.a026...
]. This gene has numerous polymorphisms, and some of them are known to alter protein function [1010 Bouaoun L, Sonkin D, Ardin M, Hollstein M, Byrnes G, Zavadil J, et al. TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data. Hum Mutat. 2016;37:865-76. https://doi.org/10.1002/humu.23035.
https://doi.org/10.1002/humu.23035...
]. Two TP53 polymorphisms have been more widely studied. The single nucleotide polymorphism (SNP) rs1042522 is characterized by substitution of guanine to cytosine at the second position of the codon 72, which is located in exon 4 of the TP53 gene [NM_000546.5(TP53):c.215C > G (p.Pro72Arg)]. Experimental evidence suggests that the p53Arg (CGC codon) variant is more effective in activating the apoptosis pathway in altered cells than the p53Pro (CCC codon) variant, which, in turn, showed to be more efficient in the activation of cell cycle arrest [1111 Thomas M, Kalita A, Labrecque S, Pim D, Banks L, Matlashewski G. Two polymorphic variants of wild-type p53 differ biochemically and biologically. Mol Cell Biol. 1999;19:1092-100. https://doi.org/10.1128/mcb.19.2.1092.
https://doi.org/10.1128/mcb19.2.1092...
]. The second polymorphism, rs17878362, is a 16 base pairs duplication in intron 3 of the TP53 gene (NM_000546.5(TP5 3):c.96 + 25_96 + 40ACCTGGAGGGCTGGGG(1_2)- intron variant) [1212 Senturk E, Manfredi JJ. p53 and cell cycle effects after DNA damage. Methods Mol Biol. 2013;962:49-61. https://doi.org/10.1007/978-1-62703-236-0_4.
https://doi.org/10.1007/978-1-62703-236-...
]. The 16 bp duplication allele is associated with lower levels of p53 transcript, which might be due to modification of mRNA processing [1313 Gemignani F, Moreno V, Landi S, Moullan N, Chabrier A, Gutierrez- Enriquez S, et al. A TP53 polymorphism is associated with increased risk of colorectal cancer and with reduced levels of TP53 mRNA. Oncogene. 2004;23:1954-6. https://doi.org/10.1038/sj.onc.1207305.
https://doi.org/10.1038/sj.onc.1207305...
].

One of the mechanisms by which p53 acts on cell cycle arrest is through the transcriptional activation of the gene encoding the p21 protein, a kinase dependent cyclin inhibitor (CDKI). In DNA damaged cells, the p21 protein acts on different cyclin/CDK complexes, promoting cell cycle arrest at the G1/S and G2/M checkpoints [1212 Senturk E, Manfredi JJ. p53 and cell cycle effects after DNA damage. Methods Mol Biol. 2013;962:49-61. https://doi.org/10.1007/978-1-62703-236-0_4.
https://doi.org/10.1007/978-1-62703-236-...
]. p21 inhibits DNA synthesis by binding to proliferating cell nuclear antigen, and also affects key molecules of the apoptotic process, such as p53, and contributes to cell senescence [1414 Lawson BR, Baccala R, Song J, Croft M, Kono DH, Theofilopoulos AN. Deficiency of the cyclin kinase inhibitor p21(WAF- 1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity. J Exp Med. 2004;199:547-57. https://doi.org/10.1084/jem.20031685.
https://doi.org/10.1084/jem.20031685...
]. In addition, the p21 protein seems to be associated with susceptibility to autoimmune diseases, especially to SLE [1515 Kong EK, Chong WP, Wong WH, Lau CS, Chan TM, Ng PK, et al. p21 gene polymorphisms in systemic lupus erythematosus. Rheumatology (Oxford). 2007;46:220-6. https://doi.org/10.1093/rheumatology/kel210.
https://doi.org/10.1093/rheumatology/kel...
].

The p21 protein is encoded by the p21 (WAF1, CIP, CDKN1A) gene, which is located in the chromosomal region 6p21.2 [1616 El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppresion. Cell. 1993;75:817-https://doi.org/10.1016/0092-8674(93)90500-P.
https://doi.org/10.1016/0092-8674(93)905...
]. Changes in the p21 (CDKN1A) gene sequence may result in p21 inactivation, which in turn, may lead to an increased activation and proliferation of self-reactive T cells, and apoptosis [1717 Santiago-Raber ML, Lawson BR, Dummer W, Barnhouse M, Koundouris S, Wilson CB, et al. Role of cyclin kinase inhibitor p21 in systemic autoimmunity. J Immunol. 2001;67:4067-74. https://doi.org/10.4049/jimmunol.167.7.4067.
https://doi.org/10.4049/jimmunol.167.7.4...
]. More than 40 polymorphisms have already been identified in the p21 gene. One of the most studied polymorphism is rs1801270, characterized by a C to A substitution at the third base of codon 31 of the p21 (CDKN1A) gene (NM_000389.5(CDKN1A):c.93C > A (p.Ser31Arg)). This SNP results in the substitution of serine (AGC codon) by arginine (AGA codon) in a conserved region of the p21 protein [1818 Li G, Liu Z, Sturgis EM, Shi Q, Chamberlain RM, Spitz MR, Wei Q. Genetic polymorphisms of p21 are associated with risk of squamous cell carcinoma of the head and neck. Carcinogenesis. 2005;26:1596-602. https://doi.org/10.1093/carcin/bgi105.
https://doi.org/10.1093/carcin/bgi105...
]. The second most studied polymorphism, rs1059234, is a C to T substitution at 20 nucleotides after the stop codon at exon 3 in the 3’ untranslated region of the gene (NM_000389.5(CDKN1A):c.*20C > T). By altering mRNA stability and inducing its faster degradation, this SNP leads to a change in the p21 protein level [1818 Li G, Liu Z, Sturgis EM, Shi Q, Chamberlain RM, Spitz MR, Wei Q. Genetic polymorphisms of p21 are associated with risk of squamous cell carcinoma of the head and neck. Carcinogenesis. 2005;26:1596-602. https://doi.org/10.1093/carcin/bgi105.
https://doi.org/10.1093/carcin/bgi105...
].

In recent years, associations of multiple genes and susceptibility to SLE have been analyzed, and among these candidate genes are p21 (CDKN1A) and TP53 [1515 Kong EK, Chong WP, Wong WH, Lau CS, Chan TM, Ng PK, et al. p21 gene polymorphisms in systemic lupus erythematosus. Rheumatology (Oxford). 2007;46:220-6. https://doi.org/10.1093/rheumatology/kel210.
https://doi.org/10.1093/rheumatology/kel...
, 1919 Yang J, Zhu JM, Wu S, Li J, Wang MR, Wang TT, Lu YW. Association study between the TP53 rs1042522G/C polymorphism and susceptibility to systemic lupus erythematosus in a Chinese Han population. Rheumatol Int. 2017;37:523-9. https://doi.org/10.1007/s00296-017-3662-0.
https://doi.org/10.1007/s00296-017-3662-...
]. The number of studies published so far is still small, and there are no reports of studies evaluating TP53 and p21 (CDKN1A) polymorphisms in Brazilian SLE patients. Taking all this into consideration, the objective of this work was to evaluate possible associations of TP53 rs1042522 (G > C), TP53 rs17878362 (16 bp Del/Ins), p21 rs1801270 (C > A) and p21 rs1059234 (C > T) polymorphisms with risk of systemic lupus erythematosus, some SLE clinical manifestations and/or age at onset of symptoms.

Material and methods

Study population

The population included in the group of cases was composed of women with SLE (n = 145) according to the American College of Rheumatology (ACR) criteria [2020 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40:1725. https://doi.org/10.1002/art.1780400928.
https://doi.org/10.1002/art.1780400928...
] who were selected sequentially during routine outpatient consultations at the Rheumatology Department of the State University of Rio de Janeiro. The control group included healthy women without any complain (n = 208) who attended for a routine outpatient clinic at the same institution. Sociodemographic data and clinical manifestations were obtained with a standardized questionnaire and medical records review. The presence of any autoimmune disease was excluded after anamnesis and physical examination performed by an experienced rheumatologist. Skin color/ethnicity was established by self-classification as phenotypic proxy for ancestry previously proved to have a high concordance in Southeastern Brazil [2121 Pena SD, Di Pietro G, Fuchshuber-Moraes M, Genro JP, Hutz MH, Kehdy FS, et al. The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected. PLoS ONE. 2011;6:e17063. https://doi.org/10.1371/journal.pone.0017063.
https://doi.org/10.1371/journal.pone.001...
]. All participants signed an informed consent previously approved by the University Hospital Ethics Committee (#321 and #909).

DNA extraction and genotyping of polymorphisms in the TP53 and p21 (CDKN1A) genes

A total of 5 mL of peripheral blood from all participants was collected in Vacutainer tubes containing ethylenedi- amine tetraacetic acid (EDTA) and transported on ice to the Molecular Biology Laboratory, Department of Bio- chemistry/IBRAG/UERJ, where molecular analyses were performed.

DNA was extracted from mononuclear cells following a salting out method described by Vargas-Torres et al. [2222 Vargas-Torres SL, Portari EA, Klumb EM, Guillobel HCR, Camargo MJ, Russomano FB, et al. Association of CDKN2A polymorphisms with the severity of cervical neoplasia in a Brazilian population. Biomarkers. 2014;19:121-7. https://doi.org/10.3109/1354750X.2014.881419.
https://doi.org/10.3109/1354750X.2014.88...
].

The polymorphisms rs1042522 and rs17878362 in the TP53 gene were analyzed by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. A primer pair (Forward: 5’GAGACCTGTGGGGAAGCGAA-3’ and Reverse: 5’GGAAGCCAGCCCCTCAG-3’) was specifically designed for amplification of the genomic region containing both TP53 gene polymorphisms and generated a 476 or 492 bp fragment depending, respectively, on the absence or presence of 16 bp duplication in the intron 3 region.

The PCR reaction mixture was composed of 1x PCR Buffer (BIOTOOLS), 2 mM MgCl2 (BIOTOOLS), 15 pmol of each oligonucleotide (P53324R and P5334F), 65 μΜ dNTPs (dATP, dCTP, dGTP, dTTP-PHARMA- CIA) 0.15 U Taq DNA polymerase (BIOTOOLS) and about 200 ηg genomic DNA in a final volume of 30 μL adjusted with sterile deionized water.

The amplification reaction was performed in a thermocycler (model MJ96 + Biocycler) using the following program: an initial denaturation step at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 45 s, primer pairing at 61 °C for 45 s and extension at 72 °C for 45 s; and a final step extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis on 6% polyacrylamide gels followed by ethidium bromide staining, which allowed the identification of the three TP53 rs17878362 (16 bp Del/Ins) genotypes composed of alleles without (A1) and with (A2) 16 bp duplication.

The TP53 rs1042522 (G > C) polymorphism was analyzed by digestion of PCR products (7 μL) with 2 U of the BstUI enzyme (BioLabs) in a final volume of 12 μL adjusted with sterile deionized water. Samples were incubated at 60 °C for approximately 16 h and then elec- trophoresed on 2% agarose gels stained with ethidium bromide.

The two p21 (CDKN1A) gene polymorphisms were analyzed by PCR-RFLP techniques using the endonucleases Alw26I (Thermo Scientific), for rs1801270 (C > A), and PstI (INVITROGEN), for rs1059234 (C > T), as previously described by Vargas-Torres et al. (2016) [2323 Vargas-Torres SL, Portari EA, Silva AL, Klumb EM, Guillobel HCR, Camargo MJ, et al. Roles of CDKN1A gene polymorphisms (rs1801270 and rs1059234) in the development of cervical neoplasia. Tumour Biol. 2016;37:10469-78. https://doi.org/10.1007/s13277-016-4850-3.
https://doi.org/10.1007/s13277-016-4850-...
].

Genotyping identification was performed by two independent researchers. In order to validate the genotyping results, 10% of the total samples were reanalyzed using PCR or PCR-RFLP techniques. In addition, representative samples of each genotype were confirmed by direct sequencing of PCR products using an ABI 3130 sequencing platform (Applied Biosystems), following the manufacturer's instructions.

Statistical analysis

Deviation from the Hardy-Weinberg equilibrium was estimated within the study groups (cases and controls) by using the chi-square (χ2) test.

Associations between TP53 and p21 (CDKN1A) polymorphisms and SLE susceptibility in the whole population and in skin color/ethnicity-stratified subgroups were assessed by logistic regression analysis using four genetic models, codominant, dominant, recessive, and overdominant. Combinatorial interactions of the four studied polymorphisms, linkage disequilibrium, haplo-type frequency estimation (Expectation maximization algorithm), and haplotype association analyses were also performed. The magnitude of association between TP53 and p21 (CDKN1A) polymorphisms and systemic lupus erythematosus was estimated by calculating odds ratio (OR) and 95% confidence interval (CI). Interaction analysis between the TP53 and p21 polymorphisms and some clinical manifestations of SLE and age at onset of symptoms were performed. Reference categories were the most frequent allele for each polymorphism, the homozygous genotypes for these alleles [2424 Iniesta R, Guino E, Moreno V. Analisis estadistico de polimorfismos gene- ticos en estudios epidemiologicos. Gac Sanit. 2005;19:333-41. https://doi.org/10.1157/13078029.
https://doi.org/10.1157/13078029...
], the combination of these homozygous genotypes, and haplotypes formed by the most frequent variants. Adjusted OR was calculated controlling for age and/or skin color/ethnicity.

Statistical analyses were performed using the software GraphPad Prism version 6.05 (GraphPad Software, Inc., San Diego, CA) and the SNPStats program (Institut Català d’Oncologia, Barcelona, Spain) [2525 Sole X, Guino E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006;22:1928-9. https://doi.org/10.1093/bioinformatics/btl26.
https://doi.org/10.1093/bioinformatics/b...
], a webtool for genetic association analysis. A p value below 0.05 was considered statistically significant.

Results

Characteristics of study population

The mean ± standard deviation and median age of patients (n = 145) and controls (n = 208) at the time they were included in the study were 39.7 ± 11.2 [Median (2575%): 39.0 (31.0-46.5)] and 37.6 ± 10.5 years [Median (25-75%): 40.0 (29.0-45.5)], respectively. The study population was composed of 76% Afro descendant (cases: n = 95; controls: n = 171) and 34% Whites (cases: n = 50; controls: n = 35), mostly of European ancestry. The most frequent clinical manifestations in patients with SLE were polyarthritis (87.4%), malar rash (75.5%), photosensitivity (73.9%), hematological abnormalities (76.1%) and glomerulonephritis (73.1%). The mean ± standard deviation age at the onset of symptoms was 27.1 ± 10.6 years.

Association of TP53 and p21 (CDKN1A) polymorphisms with SLE risk

All samples were genotyped with respect to at least one of the four polymorphisms, TP53 rs1042522 (G > C), TP53 rs17878362 (16 bp Del/Ins), p21 rs1801270 (c > a), and p21 rs1059234 (C > T). PCR or PCR-RFLP patterns of genotypes corresponding to the four analyzed polymorphisms and their confirmation by DNA sequencing can be seen in Fig. 1. Genotype distribution of the four polymorphisms was in Hardy-Weinberg equilibrium in both study groups, cases and controls (p > 0.05) (Table 1).


Fig. 1 Molecular analysis of polymorphisms in the TP53 and p21 (CDKN1A) genes. Left: A TP53 rs1042522 (G > C). Photography of an ethidium bromide-stained agarose gel (2%) showing PCR-fistUI RFLP patterns. Lane 1: 50 bp ladder (INVITROGEN); lanes 2, 3 and 4: genotypes rs1042522 CC (one fragment of 492 or 476 bp), rs1042522 GC (three fragments of 492 or 476, 305 or 284 and 187 bp) and rs1042522 GG (two fragments of 305 or 284 bp and 187 bp), respectively. B TP53 rs17878362 (16 bp Del/Ins). Photography of an ethidium bromide-stained polyacrylamide gel (8%) showing PCR patterns. Lane 1: 50 bp ladder (INVITROGEN); lanes 2, 3 and 4: genotypes rs17878362 A1A1 (one fragment of 492 bp), rs17878362 A1A2 (two fragments of 492 and 476 bp) and rs17878362 A2A2 (one fragment of 476 bp), respectively. C p21 rs1801270 (C > A). Photography of an ethidium bromide-stained polyacrylamide gel (8%) showing PCR-A/w26I RFLP patterns. Lane 1: 50 bp ladder (INVITROGEN); lanes 2, 3 and 4: genotypes rs1801270 CC (two fragments of 105 and 74 bp), rs1801270 CA (three fragments of 179, 105 and 74 bp) and rs1801270 AA (one fragment of 179 bp), respectively. D p21 rs1059234 (C >T) polymorphism. Photography of an ethidium bromide-stained polyacrylamide gel (8%) showing PCR-PstI RFLP patterns. Lane 1: 50 bp ladder (INVITROGEN); lanes 2, 3 and 4: genotypes rs1059234 CC (two fragments of 115 and 68 bp), rs1059234 CT (three fragments of 183, 115 and 68 bp) and rs1059234 TT (one fragment of 183 bp). Right: A-D DNA sequencing—electropherograms corresponding to different genotypes. The polymorphic sites are indicated

No significant results were observed in comparative analyzes of genotype and allele distributions in the groups of cases and controls regardless the genetic model used (Table 1).

Table 1
Comparative analysis of genotype and allele distributions of the four polymorphisms, TP53 rs1042522 (G > C), TP53 rs17878362 (16 bp Del/Ins), p21 rs1801270 (C > A) and p21 rs1059234 (C > T), between the groups of cases and controls using different genetic models

Concerning skin color/ethnicity-classification there was no significant difference for the p21 polymorphisms (data not shown). However, both polymorphisms in the TP53 gene showed significant results in the subgroup of white women (Table 2). Statistically relevant differences were observed in the distribution of TP53 rs1042522 (G > C) polymorphism between cases and controls, in both codominant (GC vs. GG: adjusted OR 2.73; 95% CI 1.02-7.34; p = 0.041) and dominant (GC + CC vs. GG: adjusted OR 3.06; 95% CI 1.17-8.04; p = 0.021) models. Regarding the TP53 rs17878362 (16 bp Del/Ins), the A1A2 genotype (codominant model: adjusted OR 5.22; 95% CI 1.33-20.47; p = 0.003) and A2-containing genotypes (dominant model: adjusted OR 6.93; 95% CI 1.81-26.51; p = 0.001 and overdominant: adjusted OR 4.47; 95% CI 1.15-17.33; p = 0.018) were more prevalent among cases in comparison with controls. Still among whites, the alleles rs1042522 C and rs17878362 A2 were more frequent in the case group than in controls (OR 2.23; 95% CI 1.10-4.54; p = 0.037 and OR 7.00; 95% CI 1.99-24.66; p < 0.001, respectively).

Table 2
Analysis of interaction between the TP53 rs1042522 (G > C) and TP53 rs17878362 (16 bp Del/Ins) polymorphisms, skin color/ ethnicity and development of SLE

Association of combined genotypes and haplotypes regarding the TP53 and p21 (CDKN1A) polymorphisms with SLE risk

Results of genotype combination analysis are shown in Table 3. Considering the entire study population, the TP53 rs17878362 A1A1-p21 rs1801270 CA combined genotype was less frequent in the case group than in controls (OR 0.45; 95% CI 0.23-0.87; p = 0.018). In non-white individuals, the frequency of the genotype combination rs17878362 A1A2-rs1801270 CC was lower among SLE patients (OR 0.28; 95% CI 0.10-0.80; p = 0.015).

Table 3
Genotype interactions of the TP53 rs 1042522 (G>C), TP53 rs 17878362 (16 bp Del/Ins), p21 rs1801270 (C>A) and p21 rs 1059234 (C>T) polymorphisms between the group and subgroups of cases and controls stratified by skin color/ethnicity

In contrast, among Whites the prevalence of the TP53 rs1042522 GC- TP53 rs17878362 A1A2 combined genotype was significantly higher in SLE patients as compared with controls (OR 9.00; 95% CI 1.70-47.62; p = 0.008).

Linkage disequilibrium was observed between the two TP53 (rs1042522 and rs17878362) polymorphisms (D = 0.1011, D’ = 0.9246, r = 0.5215, p < 0.001), and the two SNPs (rs1801270 and rs1059234) in the p21 gene (D = 0.1403, D’ = 0.8518, r = 0.825, p < 0.001). No statistically significant difference was observed between hap- lotype frequencies of cases and controls in the entire study population. However, haplotype frequency estimation considering skin color/ethnicity-stratified subgroups revealed that in Whites the haplotype rs17878362 A2-rs1042522 C was more frequent among SLE patients in comparison with controls (OR 9.67, 95% CI 2.0246.28; p = 0.006) (Table 4).

Table 4
Haplotype frequency estimation and haplotype association analysis, with respect to the TP53 polymorphisms, rs1042522 (G > C) and rs17878362 (16 bp Del/Ins), and the SNPs in the p21 (CDKN1A) gene, rs1801270 (C > A) and rs1059234 (C > T), in the entire study sample and in the groups stratified according skin color/ethnicity

Association of genotypes of TP53 and p21 (CDKN1A) polymorphisms with clinical manifestations of SLE and age at onset of symptoms

Interaction analysis between the TP53 and p21 (CDKN1A) polymorphisms and some clinical manifestations of SLE patients was also investigated. Statistically significant results were not found with respect to cutaneous-articular manifestations, hematological and immunological disorders and nephritis (data not shown). Notably, serosistis was more prevalent among SLE patients carrying the TP53 rs17878362 A1A2 genotype (overdominant model: OR 2.82; 95% CI 1.18-6.74; p = 0.021) (Table 5). Neuropsychiatric disorders (seizures and psychosis) were more frequent in SLE patients carrying the TP53 rs17878362 A2A2 genotype (codominant model: OR 4.69; 95% CI 1.04-21.24; p = 0.054) and in the TP53 rs1042522 C/C genotype carriers (codominant model: OR 5.82; 95% CI 1.46-23.17; p = 0.015 and recessive model: OR 3.34; 95% CI 1.17-9.55; p = 0.031) (Table 5). The TP53 alleles rs17878362A2 and p53 rs1042522 C were more frequent in SLE patients with neuropsychiatric manifestations (OR 2.15; 95% CI 1.064.35; p = 0.048 and OR 2.23; 95% CI 1.18-4.29; p = 0.015, respectively). No association of TP53 and p21 polymorphisms with age at onset of symptoms was observed (data not shown).

Table 5
Interaction analysis between the polymorphisms TP53 rs17878362 (16 bp Del/Ins) and TP53 rs1042522 (G > C) and clinical manifestations (serositis and neuropsychiatric disorders) of SLE patients

Discussion

In the present work, the TP53 rs1042522 (G > C) and TP53 rs17878362 (16 bp Del/Ins) polymorphisms were associated with risk of SLE in the subgroup of white women (Table 2). The rs1042522 C allele carriers and rs17878362 A2 allele carriers presented two-, and seven-fold higher risk of developing SLE (p = 0.037 and p < 0.001, respectively).

To date, we could not find any association studies regarding the TP53 rs17878362 (16 bp Del/Ins) polymorphism and SLE or the TP53 rs1042522 (G > C), p21 rs1801270 (C > A) and p21 rs1059234 (C > T) polymorphisms and the development of SLE in Brazilian populations. Moreover, the few published studies related to the importance of TP53 and p21 polymorphisms for the development of SLE present discordant results [1515 Kong EK, Chong WP, Wong WH, Lau CS, Chan TM, Ng PK, et al. p21 gene polymorphisms in systemic lupus erythematosus. Rheumatology (Oxford). 2007;46:220-6. https://doi.org/10.1093/rheumatology/kel210.
https://doi.org/10.1093/rheumatology/kel...
, 1919 Yang J, Zhu JM, Wu S, Li J, Wang MR, Wang TT, Lu YW. Association study between the TP53 rs1042522G/C polymorphism and susceptibility to systemic lupus erythematosus in a Chinese Han population. Rheumatol Int. 2017;37:523-9. https://doi.org/10.1007/s00296-017-3662-0.
https://doi.org/10.1007/s00296-017-3662-...
, 2626 Onel KB, Huo D, Hastings D, Fryer-Biggs J, Crow MK, Onel K. Lack of Association of the TP53 Arg72Pro SNP and the MDM2 SNP309 with systemic lupus erythematosus in Caucasian, African American, and Asian children and adults. Lupus. 2009;18:61-6. https://doi.org/10.1177/0961203308094558.
https://doi.org/10.1177/0961203308094558...
2929 Piotrowski P, Lianeri M, Mostowska M, Wudarski M, Chwalinska-Sadowska H, Jagodzinski PP Contribution of polymorphism in codon 72 of p53 gene to systemic lupus erythematosus in Poland. Lupus. 2008;17:148-51. https://doi.org/10.1177/0961203307084722.
https://doi.org/10.1177/0961203307084722...
]. A meta-analysis on association between TP53 polymorphisms and SLE showed significant results only after stratification by ethnicity, confirming the rs1042522 C allele as risk factor for SLE development in Asians (Koreans) [3030 Lee Y, Bae SC, Choi SJ, Ji JD, Song GG. Associations between the p53 codon 72 polymorphisms and susceptibility to systemic lupus erythematosus and rheumatoid arthritis: a meta-analysis. Lupus. 2012;21:430-7. https://doi.org/10.1177/0961203311434941.
https://doi.org/10.1177/0961203311434941...
]. We are aware that some studies describing Brazilian genetic structure have reported divergences between self-identified skin color and genetic ancestry [3131 Barbosa FB, Cagnin NF, Simioni M, Farias AA, Torres FR, Molck MC, et al. Ancestry informative marker panel to estimate population stratification using genome-wide human array. Ann Hum Genet. 2017;81:225-33. https://doi.org/10.1111/ahg.12208.
https://doi.org/10.1111/ahg.12208...
, 3232 Pereira FDSCF, Guimaraes RM, Lucidi AR, Brum DG, Paiva CLA, Alvarenga RMP A systematic literature review on the European, African and Amerindian genetic ancestry components on Brazilian health outcomes. Sci Rep. 2019;9:8874. https://doi.org/10.1038/s41598-019-45081-7.
https://doi.org/10.1038/s41598-019-45081...
]. However, most clinical and genetic studies all around the world employ self-declaration of skin color [3333 Braun-Prado K, Petzl-Erler ML. Programmed cell death 1 gene (PDCD1) polymorphism and pemphigus foliaceus (fogo selvagem) disease susceptibility. Genet Mol Biol. 2007;30:314-21. https://doi.org/10.1590/S1415-47572007000300003.
https://doi.org/10.1590/S1415-4757200700...
], and this criterion previously proved to have a high concordance in the Southeastern region of Brazil [2121 Pena SD, Di Pietro G, Fuchshuber-Moraes M, Genro JP, Hutz MH, Kehdy FS, et al. The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected. PLoS ONE. 2011;6:e17063. https://doi.org/10.1371/journal.pone.0017063.
https://doi.org/10.1371/journal.pone.001...
].

The divergence of susceptibility genes found among various ethnicities may be related to the influence of local environmental factors, which may interfere with epigenetic modifications [33 Pan L, Lu MP, Wang JH, Xu M, Yang SR. Immunological pathogenesis and treatment of systemic lupus erythematosus. World J Pediatr. 2020;16:19-https://doi.org/10.1007/s12519-019-00229-3.
https://doi.org/10.1007/s12519-019-00229...
,2828 Sanchez E, Sabio JM, Callejas JL, Ramon E, Haro M, Jimenez-Alonso J, et al. Study of a functional polymorphism in the p53 gene in systemic lupus erythematosus: lack of replication in a Spanish population. Lupus. 2006;15:658-61. https://doi.org/10.1177/0961203306070986.
https://doi.org/10.1177/0961203306070986...
,3434 Mazzone R, Zwergel C, Artico M, Taurone S, Ralli M, Greco A, Mai A. The emerging role of epigenetics in human autoimmune disorders. Clin Epigenet. 2019;11:34. https://doi.org/10.1186/s13148-019-0632-2.
https://doi.org/10.1186/s13148-019-0632-...
, 3535 Surace AEA, Hedrich CM. The role of epigenetics in autoimmune/inflammatory disease. Front Immunol. 2019;10:1525. https://doi.org/10.3389/fimmu.2019.01525.
https://doi.org/10.3389/fimmu.2019.01525...
]. Besides, allele distributions of the above four polymorphisms vary widely among different populations and may have different impact on susceptibility for SLE [3636 Hu Z, Li X, Qu X, He Y, Ring BZ, Song E, Su L. Intron 3 16 bp duplication polymorphism of TP53 contributes to cancer susceptibility: a metaanalysis. Carcinogenesis. 2010;31:643-7. https://doi.org/10.1093/carcin/bgq018.
https://doi.org/10.1093/carcin/bgq018...
, 3737 Sucheston L, Witonsky DB, Hastings D, Yildiz O, Clark VJ, Rienzo A, et al. Natural selection and functional genetic variation in the p53 pathway. Hum Mol Genet. 2011;20:1502-8. https://doi.org/10.1093/hmg/ddr028.
https://doi.org/10.1093/hmg/ddr028...
]. Particularly, Brazilian population presents a mixture of genetic ancestry, mostly European and African, which may partially explain the present results [3232 Pereira FDSCF, Guimaraes RM, Lucidi AR, Brum DG, Paiva CLA, Alvarenga RMP A systematic literature review on the European, African and Amerindian genetic ancestry components on Brazilian health outcomes. Sci Rep. 2019;9:8874. https://doi.org/10.1038/s41598-019-45081-7.
https://doi.org/10.1038/s41598-019-45081...
].

Even though a single polymorphism may represent a valuable biomarker of a complex disease, such as SLE, it is believed that multiple small-effect genetic variants influence disease susceptibility [3838 Liu N, Zhang K, Zhao H. Haplotype-association analysis. Adv Genet. 2008;60:335-405. https://doi.org/10.1016/S0065-2660(07)00414-2.
https://doi.org/10.1016/S0065-2660(07)00...
]. Therefore, genotype combination and haplotype association analyses were also performed to assess the combined effect of the four polymorphisms in the TP53 and p21 (CDKN1A) genes on the risk of SLE.

Some genotype combinations were absent in some groups or subgroups stratified according to skin color/ ethnicity (Table 3). These findings are not unexpected, since some individual genotypes were found at a very low frequency in our study sample (Tables 1 and 22 Sharabi A, Tsokos GC. T cell metabolism: new insights in systemic lupus erythematosus pathogenesis and therapy. Nat Rev Rheumatol. 2020;16:100-12. https://doi.org/10.1038/s41584-019-0356-x.
https://doi.org/10.1038/s41584-019-0356-...
). Noteworthy, a combined effect of both TP53 polymorphisms seems to exist among Whites, since the rs1042522 GC- rs17878362 A1A2 combined genotype were ninefold more frequent in cases than in controls (p = 0.008) (Table 3). Although a significant difference does exist, due to the limited sample size when the total study population was stratified according to skin color/ethnicity, the statistical power is not strong enough to establish the true association with SLE development.

It has been previously shown that the p53 Arg (rs1042522 G allele) variant has a greater ability to activate apoptosis pathways, while the p53Pro (rs1042522 C allele) variant demonstrates greater efficacy in promoting cell cycle arrest [1111 Thomas M, Kalita A, Labrecque S, Pim D, Banks L, Matlashewski G. Two polymorphic variants of wild-type p53 differ biochemically and biologically. Mol Cell Biol. 1999;19:1092-100. https://doi.org/10.1128/mcb.19.2.1092.
https://doi.org/10.1128/mcb19.2.1092...
], a mechanism that may be associated with SLE development [11 Balomenos D. Cell cycle regulation and systemic lupus erythematosus. In: Lahita RG, editor. Systemic lupus erythematosus. 5th ed. London: Academic Press; 2011. p. 191-8. https://doi.org/10.1016/B978-0-12-374994-9.10011-7.
https://doi.org/10.1016/B978-0-12-374994...
, 22 Sharabi A, Tsokos GC. T cell metabolism: new insights in systemic lupus erythematosus pathogenesis and therapy. Nat Rev Rheumatol. 2020;16:100-12. https://doi.org/10.1038/s41584-019-0356-x.
https://doi.org/10.1038/s41584-019-0356-...
]. Besides, removal of self-reactive B and T cells in SLE patients and mice with lupus-like syndrome could be impaired by defective apoptotic processes [3939 Chae BS, Shin TY. Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune response in pristane-induced lupus mice. Arch Pharm Res. 2007;30:191-8. https://doi.org/10.1007/BF02977694.
https://doi.org/10.1007/BF02977694...
, 4040 Tiller T, Tsuiji M, Yurasov S, Velinzon K, Nussenzweig MC, Wardemann H. Autoreactivity in human IgG memory B cells. Immunity. 2007;26:205-13. https://doi.org/10.1016/j.immuni.2007.01.009.
https://doi.org/10.1016/j.immuni.2007.01...
]. This might support the hypothesis that the p53 Pro variant may reduce the clearance of auto-reactive lymphocytes in patients with SLE by reducing the efficiency of apoptosis [4141 Sakamuro D, Sabbatini P, White E, Prendergast GC. The polyproline region of p53 is required to activate apoptosis but not growth arrest. Oncogene. 1997;15:887-98. https://doi.org/10.1038/sj.onc.1201263.
https://doi.org/10.1038/sj.onc.1201263...
]. On the other hand, the rs17878362 A2 allele is associated with lower levels of p53 transcription [4242 Park JS, Lim MA, Cho ML, Ryu JG, Moon YM, Jhun JY, et al. p53 controls autoimmune arthritis via STAT-mediated regulation of the Th17 cell/Treg cell balance in mice. Arthritis Rheum. 2013;65:949-59. https://doi.org/10.1002/art.37841.
https://doi.org/10.1002/art.37841...
]. Thus, the suggested combined effect of TP53 polymorphisms could be explained by a lower expression of the p53 protein and the presence of the p53 Pro variant, which is less effective in apoptosis process.

On the other hand, TP53 rs17878362 A1A1-p21 rs1801270 CA (OR 0.45) and TP53 rs17878362 A1A2- p21 rs1801270 CC (OR 0.28) combined genotypes showed protective effects on SLE development in the entire study sample and in the subgroup of non-white women, respectively. Of note, the allele TP53 rs17878362 A2 has been associated with lower constitutive levels of p53 mRNA in lymphoblast cell lines, suggesting that this polymorphism may interfere in mRNA splicing, thus influencing transcript stability [1313 Gemignani F, Moreno V, Landi S, Moullan N, Chabrier A, Gutierrez- Enriquez S, et al. A TP53 polymorphism is associated with increased risk of colorectal cancer and with reduced levels of TP53 mRNA. Oncogene. 2004;23:1954-6. https://doi.org/10.1038/sj.onc.1207305.
https://doi.org/10.1038/sj.onc.1207305...
]. A consequence of that includes changes in the p53 protein activity, which may negatively alter its response to apoptosis activation, cell cycle arrest, and DNA repair, all recognized as important mechanisms for the development of SLE. Regarding the p21 rs1801270 (C > A) polymorphism, previous studies reported that both p21 variants exhibit similar activity in relation to CDK inhibition and tumor suppressor activity, although A allele (Arg) seems to be associated with a decrease in mRNA expression [4343 Su L, Sai Y, Fan R, Thurston SW, Miller DP, Zhou W, et al. p53 (codon 72) and p21 (codon 31) polymorphisms alter in vivo mRNA expression of p21. Lung Cancer. 2003;40:259-66. https://doi.org/10.1016/S0169-5002(03)00081-3.
https://doi.org/10.1016/S0169-5002(03)00...
]. In addition, p21 deficiency proved to be associated with loss of immune tolerance in mice [4444 Arias CF, Ballesteros-Tato A, Garcia MI, Martin-Caballero J, Flores JM, Martinez-A C, Balomenos D. p21 CIP1/WAF1 controls proliferation of activated/memory T cells and affects homeostasis and memory T cell responses. J Immunol. 2007;178:2296-306. https://doi.org/10.4049/jimmunol.178.4.2296.
https://doi.org/10.4049/jimmunol.178.4.2...
]. Taken together, the protection against SLE attributed to these genotype combinations cannot be explained by the combined influence of the individual genotypes on transcript levels, and/or levels and functions of proteins. However, it is important to emphasize that both p53 and p21 proteins may play different roles, with p21 not always acting as a downstream signaling component of p53 [4545 Kim J, Bae S, An S, Park JK, Kim EM, Hwang SG, Kim WJ, Um HD. Cooperative actions of p21WAF1 and p53 induce slug protein degradation and suppress cell invasion. EMBO Rep. 2014;15:1062-8. https://doi.org/10.15252/embr.201438587.
https://doi.org/10.15252/embr.201438587...
] and are involved in several pathways [4646 Engeland K. Cell cycle regulation: p53-p21 -RB signaling. Cell Death Differ. 2022;29:946-60. https://doi.org/10.1038/s41418-022-00988-z.
https://doi.org/10.1038/s41418-022-00988...
] that could be responsible for the observed effect.

In this study, a statistically significant difference was also observed for distribution of the TP53 haplotypes between white SLE patients and controls, with the haplo- type rs17878362 A2-rs1042522 C being associated with risk of SLE development (OR 9.67) (Table 4). This estimated risk effect is higher than the effects observed for the individual alleles rs1042522 C (OR 2.23) and rs17878362 (OR 7.00) and corroborate the risk of SLE associated with the rs1042522 GC/rs17878362 A1A2 combined genotype. To determine if the polymorphism are in cis or in trans other methods, such as NGS (Next Generation Sequencing) or genotyping of mother-father-child trios followed by robust bioinformatics tools have to be used [4747 Dilernia D, Amin P, Flores J, Stecenko A, Sorscher E. Mutation profiling of the c.1521_1523delCTT (p.Phe508del, F508del) cystic fibrosis transmembrane conductance regulator allele using haplotype-resolved long-read next generation sequencing. Hum Mutat. 2022;43:595-603. https://doi.org/10.1002/humu.24352.
https://doi.org/10.1002/humu.24352...
, 4848 Wakita S, Hara M, Kitabatake Y, Kwatani K, Kurahashi, Hashizume R. Experimental method for haplotype phasing across the entire length of chromosome 21 in trisomy 21 cells using a chromosome elimination technique. J Hum Genet. 2022;67:565-72. https://doi.org/10.1038/s10038-022-01049-6.
https://doi.org/10.1038/s10038-022-01049...
]. We believe that our findings may pave the way for further research in this area.

In this work, the relationship between the studied polymorphisms and clinical characteristics of SLE patients was also observed. The SNP TP53 rs1042522 (G > C) was associated with risk for both serositis and neuropsychiatric manifestations, while the TP53 rs17878362 (16 bp Del/Ins) polymorphism was associated only with neuropsychiatric disorders. Serositis is a common manifestation related to lupus, and its pathogenesis is still not well described. It has been proposed that the appearance of serositis may be associated with an inflammatory response resulting from an abnormal influx of Ca2+ [4949 Andrews BS, Arora NS, Shadforth MF, Goldberg SK, Davis JS 4th. The role of immune complexes in the pathogenesis of pleural effusions. Am Rev Respir Dis. 1981;124:115-20. https://doi.org/10.1164/arrd.1981.124.2.115.
https://doi.org/10.1164/arrd.1981.124.2....
]. Although increasing number of genetic associations with pathways involved in innate and adaptive immunity systems has been observed [5050 Bentham J, Morris DL, Graham DSC, Pinder CL, Tombleson P, Behrens TW. Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus. Nat Genet. 2015;47:1457-64. https://doi.org/10.1038/ng.3434.
https://doi.org/10.1038/ng.3434...
], more genetic studies concerning neuropsychiatric disorders are needed.

In conclusion, we found associations of the TP53 (rs1042522 and rs17878362) and p21 (rs1801270) polymorphisms, individually or in combination, with SLE development, particularly in skin color/ethnicity- stratified subgroups. In addition, interaction analysis revealed that TP53 polymorphisms might be associated with serositis and neuropsychiatric disorders in SLE patients. Our results suggest that ethnic variations of these polymorphisms should be considered in association studies for complex diseases, such as SLE, as widely shown by different authors [3030 Lee Y, Bae SC, Choi SJ, Ji JD, Song GG. Associations between the p53 codon 72 polymorphisms and susceptibility to systemic lupus erythematosus and rheumatoid arthritis: a meta-analysis. Lupus. 2012;21:430-7. https://doi.org/10.1177/0961203311434941.
https://doi.org/10.1177/0961203311434941...
, 3131 Barbosa FB, Cagnin NF, Simioni M, Farias AA, Torres FR, Molck MC, et al. Ancestry informative marker panel to estimate population stratification using genome-wide human array. Ann Hum Genet. 2017;81:225-33. https://doi.org/10.1111/ahg.12208.
https://doi.org/10.1111/ahg.12208...
, 3535 Surace AEA, Hedrich CM. The role of epigenetics in autoimmune/inflammatory disease. Front Immunol. 2019;10:1525. https://doi.org/10.3389/fimmu.2019.01525.
https://doi.org/10.3389/fimmu.2019.01525...
, 5151 Sebastiani GD, Galeazzi M. Immunogenetic studies on systemic lupus erythematosus. Lupus. 2009;18:878-83. https://doi.org/10.1177/0961203309106918.
https://doi.org/10.1177/0961203309106918...
]. To our knowledge, this is the first study to investigate associations of TP53 and p21 gene polymorphisms with the development of SLE in a Brazilian population. Our findings may provide the basis for further studies on association of TP53 and p21 polymorphisms with the development of SLE in different and larger populations, since these polymorphisms may have potential to emerge as SLE susceptibility markers for specific groups of patients.

  • Funding
    CNPq—National Council for Scientific and Technological Development (# 476116/2009-0). FAPERJ—Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (# E-26/170.922/2004 and # E-26/111.334/2011).
  • Declarations
    Ethics approval and consent to participate
    The Research Ethics Committee of the Pedro Ernesto University Hospital approved the projects that involved the selection of members of both groups (#321 and #909). All participants signed an informed consent form agreeing to participate in the study.
  • Consent for publication
    All participants signed an informed consent form, and they were told that the results would be used for education and publishing purposes, but their names would not be revealed.
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    Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Availability of data and materials

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Acknowledgements

Not applicable.

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Publication Dates

  • Publication in this collection
    23 Oct 2023
  • Date of issue
    2023

History

  • Received
    08 Mar 2023
  • Accepted
    03 Aug 2023
  • Published
    21 Aug 2023
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