IKZF1 rs4132601 and rs11978267 gene polymorphisms and paediatric systemic lupus erythematosus; relation to lupus nephritis

The demographic factors, the socioeconomic status and the ethnicity of populations are important players that determine the incidence, the prevalence and the spectrum of systemic lupus erythematosus (SLE) clinical presentations in different populations. Therefore, the purpose of the present research was to investigate the possible association between the Ikaros family zinc finger 1 gene (IKZF1) rs4132601 and rs11978267 single nucleotide polymorphisms (SNPs) and SLE susceptibility and clinical presentations including lupus nephritis (LN) among Egyptian paediatric patients. After DNA extraction from Ethylenediaminetetraacetic acid (EDTA) blood samples for 104 paediatric SLE (pSLE) patients and 286 healthy controls, the investigated SNPs (IKZF1 rs4132601 and rs11978267) were genotyped using TaqMan‐Real‐time Polymerase chain reaction (PCR). The G allele, GG and GT genotypes of IKZF1 rs4132601 were associated with pSLE (pc<.001, OR 2.97, 3.2 and 2.25, respectively). The GG and GA haplotype were more frequent in pSLE patients than other haplotypes (pc<.001, OR 3.47 and pc = .004, OR = 2.8, respectively). The studied SNPs have no impact on the distinctive features of pSLE. The rs4132601 TG genotype was significantly associated with proliferative LN (pc = .03) The IKZF1 rs4132601 can be considered a risk factor for SLE in the cohort of Egyptian children. The TG genotype of the IKZF1 rs4132601 may predispose to proliferative LN.

with proliferative LN (p c = .03)The IKZF1 rs4132601 can be considered a risk factor for SLE in the cohort of Egyptian children.The TG genotype of the IKZF1 rs4132601 may predispose to proliferative LN.

K E Y W O R D S
Egyptian, Ikaros family zinc finger 1 gene rs4132601 and rs11978267, paediatric SLE, single nucleotide polymorphisms

BACKGROUND
Systemic lupus erythematosus (SLE) is a severe and chronic inflammatory autoimmune disease.It is characterized by breakdown of self-tolerance, dysregulated immune response and multiple organ affection.SLE clinical presentation is complex and heterogeneous with different and highly varied laboratory findings.In contrast to adult SLE, juvenile or paediatric SLE (pSLE) has sudden onset, aggressive course, higher rates of organ affection with more tissue damage (Ghodke-Puranik & Niewold, 2015;Hammad et al., 2017;Iwamoto & Niewold, 2017;Levy & Kamphuis, 2012;Mosaad et al., 2018).Lupus nephritis (LN) is a dangerous complication in SLE and in most cases, LN progresses to end stage renal disease.Moreover, the differences in race or ethnicity affect the prevalence of LN in different areas of the world (Almaani et al., 2017;Yu et al., 2017).
The exact immunopathogenesis of SLE is still unclarified but it is known to be multifactorial.It is usually attributed to the interplay between genetic and environmental factors.It is documented that patients with SLE from different ethnicities exhibit differences in incidence, prevalence, severity, clinical presentation, genetic and immunologic features (Iwamoto & Niewold, 2017;Ko et al., 2012;Sanchez et al., 2011;Sharma et al., 2015).
Ikaros family zinc finger 1 gene (IKZF1) gene encodes for Ikaros family zinc finger 1 protein.It is a transcription factor which plays a significant role in the regulation of lymphocyte proliferation and differentiation (Georgopoulos et al., 1994) and self-tolerance through its regulation of signalling of B-cell receptor (Wojcik et al., 2007).Hu et al. (2013) genome-wide association study (GWAS) on Han population reported an association between IKZF1 gene polymorphism and SLE.Also, Hu et al. (2011) demonstrated low expression of IKZF1 mRNA in patients with SLE, which may be correlated with SLE pathogenesis.
An association between IKZF1 gene variants was reported with SLE clinical feature (malar rash), laboratory findings (haematological such as RBCs, haemoglobin level and platelets) and the expression and serum level of cytokines (such as CD40 ligand and granzyme B).These findings provide evidence for the role which may be played by IKZF1 single nucleotide polymorphisms (SNPs) in pathophysiology, development and progression of SLE (Chen et al., 2020).Therefore, and to the best of our knowledge, this may be the first time in Egyptian children to investigate the possible association between the IKZF1 rs4132601 and rs11978267 SNPs and SLE susceptibility and clinical presentations including LN.

Participants
Two The samples were collected from patients in the period from 2008 to 2017.SLE diagnosis was done following the criteria of American College of Rheumatology 1997 (Hochberg, 1997) and the Systemic Lupus International Collaborating Clinics 2012 (Petri et al., 2012).

Patients and controls information
pSLE patients with any evidence of LN such as the presence of persistent proteinuria >0.5 g/day or the presence of cellular casts were undergoing percutaneous ultrasound guided needle biopsy before starting treatment.Pathological evaluation and classification were done by the same expert pathologist according to the International Society of Nephrology/Renal Pathology Society classification criteria (Weening et al., 2004)

Real-time PCR typing of IKZF1 rs4132601 and rs11978267
The GeneJET DNA extraction kits (Thermo Scientific, lot 00138029) were used for the extraction of DNA from EDTA blood samples.
The NanoDrop 2000 (Thermo Scientific) was used to determine the concentration and purity of the extracted DNA in each sample.The chromosomal location is Ch 7p12.2 and site of the studied SNPs was 50,402,906 for the rs4132601 and was 50,398,606 for the rs11978267.The TaqMan SNP genotyping assays (Catalog number: 435,1379 and435,1379, Applied Biosystem, Foster)  was used for typing of genotypes and alleles of the SNPs.

Statistical analysis
Revision All tests were 2-sided and a p-value < .05 was considered statistically significant.

Demographic data
The current study was conducted on 104 pSLE.Their mean age was 11.29 years.They were 99 males (95.2%) and 5 females (4.8%) and 282 healthy control subjects of matched age and gender (Table 1).

Association of IKZF1 polymorphisms with pSLE susceptibility, biopsy findings and survival
The G allele, GG and GT genotypes of IKZF1 rs4132601 were present in significant higher risk frequency in patients with pSLE (p c < .001).
The odd ratios for the G allele and G-containing genotypes genotype (GG and GT) were 2.97, 3.2 and 2.25, respectively.This means that any individual carrying such an allele or genotype will be two to three times more susceptible to developing SLE than individuals without such variants.The dominant TG + GG (p c < .001and OR = 2.446), the recessive GG (p c < .001and OR = 2.1) and the over dominant TG (p c < .001and OR = 1.66) genetic models are confirming the results.As regards IKZF1 rs11978267, non-significant risk associations were found (Table 3).
We analysed the frequency of IKZF1 haplotypes in pSLE patients and healthy controls and data is presented in   4).Patients with the GG genotype of rs4132601 were more liable to have higher grade I (p = .027),patients with TT genotype were more liable to have higher grade II (p = .045),whereas patients with heterozygous TG genotype were more liable to have higher grade IV of LN (p = .029).Unfortunately, after Bonferroni correction, the observed significance was omitted (p c > .05)(Table 5).
The IKZF1 rs4132601 and rs11978267 were not considered predictors of clinical data in all studied cases.The prediction of OS in pSLE patients was done using the Cox regression test, and the covariates used were patient's age, sex and genotypes.None of the covariates can be used for the prognosis of OS (data not shown).Non-significant association was found with the OS between rs4132601 and rs11978267 genotypes (data not shown).

Comparison of genetic variability in studied SNPs in Egyptian healthy controls and published data in TopMed and gnomAD projects concerning main ethnic groups
The frequency of SNPs alleles in the studied Egyptian cohort is comparable to the other reported data in the TopMed (<0.001 and 0.003, respectively), genomAD (<0.001 and 0.002, respectively), European (0.002 and <0.001, respectively), African (0.004 and 0.013, respectively), East Asian (0.024 and 0.043, respectively) and American (<0.001 and 0.001, respectively) (Table 6).

DISCUSSION
A combination of factors is needed for SLE to develop in an individual.A triad of factors is usually needed, suitable genetic background, dysregulated immune system and exposure to certain environmental factors.
The demographic factors, the socioeconomic status and the ethnicity of populations are important players that determine the incidence, the prevalence and the spectrum of clinical manifestations of SLE in different populations (Danchenko et al., 2006).To reveal the genetic susceptibility for SLE, many GWAS and case-control genetic studies have been performed across the world (Deng & Tsao, 2014;Ghodke-Puranik & Niewold, 2015, 2013).Recent reported data have linked more than 60 genetic loci to be associated with the risk to develop SLE and to affect clinical presentation and outcome, Huamn Leukocyte antigen (HLA) showed the strongest association.Other susceptibility loci included many non-HLA genes.However, some of these genes have been located within or near to the genes of the immune system either innate or adaptive.However, other genes have no relation to immune system function or pathways and even have not been linked to the SLE pathogenesis (Ghodke-Puranik & Niewold, 2015).
In the present work, we investigated the possible association between IKZF1 rs4132601 and rs11978267 The prevalence of LN varies across the world.It was reported to be 29% in white population, 69% in black, 61% in Hispanic and 40%-82% in Asian (Bastian et al., 2002;Jakes et al., 2012).Moreover, the early developments of LN, aggressive course and worse outcome were observed in black and Hispanic SLE patients (Almaani et al., 2017;Burgos et al., 2011).
The haplotype-based association methods are a more powerful genetic tool when evaluating the susceptibility risk than the allelebased methods.This is more applicable for disease susceptibility when the genetic variant is originating and predisposing to the disease independently of each other (Morris & Kaplan, 2002).Patients carrying the haplotypes GG (p c < .001,OR 3.47 and 95% CI 1.78-6.76)and GA (p c = .004,OR 2.8 and 95% CI 1.552-5.124)are more prone to develop the disease than patients who carrying other haplotypes (Table 3).
Although the studied SNPs have no impact on the different clinical, haematologic or immunologic features of SLE, the proliferative LNs are more liable by about twofolds to develop in children with the TG genotype of rs4132601 (p c = .03)(Table 4).balance toward Th1, thereby increasing the production of IFN-gamma (Hu et al., 2013, Thomas et al., 2010).Peripheral blood mononuclear cells from patients with SLE showed a significantly reduced expression of messenger RNA of IKZF1 (p < .001)(Lee et al., 2014).Therefore, the reduced expression of IKZF1 may play a role in SLE immunopathogenesis.It may modulate signal pathways involved in SLE, such as STAT4 (signal transducers and activators of transcription 4) and IFN.IKZF1 was found to bind to the STAT4 promoter and to be involved in the regulation of STAT4 in human T cells (Yap et al., 2005).

CONCLUSION
The IKZF1 rs4132601 can be considered a risk factor for SLE in the cohort of Egyptian children.The TG genotype of the IKZF1 rs4132601 may predispose to proliferative LN.
groups of Egyptian individuals were included in the present work: pSLE and healthy control individuals.The pSLE consisted of 104 patients; their mean (± SD) age was 11.3(± 3.5) years and ranged from 4 to 17 years.They were 99 females (95.2%) and 5 males (4.8%).The median age of onset was 6.0 years and the median disease duration was 4.5 years.The pSLE patients and their samples were collected from different clinics (Paediatric Nephrology, Rheumatology and Rehabilitation and Dermatology) in Mansoura University Hospitals, Egypt.
were used to genotype the SNPs.The IKZF1 rs4132601 polymorphism is G/T, transversion substitution and the sequence is as follows: TGC AAT CAC AGA GAA AGA TGC GCC T [G/T] ATC CAA GTT AAT ATC TCT AAG GTG A (VIC/FAM).The IKZF1 rs11978267 polymorphism is A/G, transition substitution and the sequence is as follows: GGG AAG GAA TTA TCC ATG CAA TCA C [A/G] CAT AAA CTT CTA CCT ACC CTC CCC T (VIC/FAM).Twenty microliter of PCR reaction mix for qPCR analysis was prepared as follow: 10.0 µL TaqMan universal master mix II with UNG, 2× (Applied Biosystem, lot 4440042), 1.0 µL TaqMan assay 20× and 5.0 µL DNA template +4 µL RNase-free water.For each sample, 20 µL of PCR reaction mix was transferred to the 48-well reaction plate.The plate was sealed using appropriate cover and was briefly centrifuged and loaded into the real-time PCR (Step one, Applied Biosystem).Reaction conditions were carried out with the following cycling stages: stage I for UNG incubation at 60 • C for 30 s; stage II for polymerase activa-tion at 95 • C for 10 min; stage III for PCR and includes 40 cycles of denaturation at 95 • C for 15 s and annealing/extension at 60 • C for 90 s and finally stage IV 60 • C for 30 s. Allelic discrimination was carried out by measuring fluorescence intensity at the end point.The Safety Data Sheet (SDS) software version 1.7 (Applied Biosystems, Foster) , coding and tabulation of data were done by Statistical package for Social Science (IBM Corp. Released 2017.IBM SPSS Statistics for Windows, Version 25.0.: IBM Corp.).Non-numerical data were presented as frequency and percentage.The relationship between two qualitative variables was assessed by Chi-square test and by Fisher's exact test when the relationship between the variables is less than 5 in more than 20% of cells.The Kruskal-Wallis test was used for comparison of non-parametric data in more than two groups.Bonferroni correction was used to account for multiple comparisons to reduce the likelihood of Type I errors.Goodness of fit test was used to evaluate for Hardy-Weinberg equilibrium expectations.Regression analysis,the odds ratio and 95% confidence interval were calculated.Overall survival (OS) was estimated using the Kaplan-Meier method.Differences between survival curves were assessed for significance using the log-rank test.The HaploView program (version 4.2) was applied to estimate the haplotypes(Barrett et al., 2005), which uses the expectation maximization algorithm.Comparison of the result of healthy controls in the present study with the data in TopMed and gnomAD project database was done by Pairwise FST statistics using the GenALEx 6.502.
different haplotypes: TA, TG, GA and GG.In healthy controls, the TA haplotype showed the highest frequency and the GG haplotype showed the lowest frequency.We observed that the frequencies of GA and GG haplotypes were statistically higher in children's patients with SLE (p c = .004and p c < .001,respectively).The GG and GA haplotypes increased the susceptibility risk by about three folds in pSLE patients (OR = 2.81 and OR = 3.47, respectively).The frequency of the alleles and genotypes of the IKZF1 rs4132601 and rs11978267 were not associated with any clinical, haematologic or immunologic features in pSLE patients (data not shown).Children with the TG genotype of rs4132601 were more liable to have proliferative LN (p c = .03and OR = 2.53).Over-dominant genetic model confirmed the same results (p c = .022and OR = 2.2) (Table Chen et al. (2020) reported a significantly higher level of CD40L in SLE patients with the GG genotype of the IKZF1 rs4132601.In SLE patients, an aberrant expression of CD40L on B cells and monocytes and an overexpression on T lymphocytes subsets were reported.It is known that the CD40-CD40L interaction is one of the mechanisms of central tolerance induction and, thereby, the development of autoimmunity.Therefore, IKZF1 may be involved in the development of SLE by maintaining serum elevated levels of CD40L in patients with the GG genotype.On the other hand, individuals carrying the GG genotype of IKZF1 rs4917014 had lower serum level of granzyme B. The granzyme B is important for the cleavage of autoantigen and, hence, the synthesis of pathogenic autoantibodies due to the appearance of new immunogenic epitopes.It can be assumed that prevention of granzyme B overexpression by the GG genotype may affect the development of SLE(Kok et al., 2017).At the same time, immune mechanism involved in the development of SLE includes complement down regulation and type one IFN upregulation.Westra et al. (2013) reported that IKZF1 gene variants influence the regulation of both immune mediators.IKZF1 blocks the production of T-bet and directs the Th1/Th2 Demographic data of controls and features of paediatric SLE (pSLE) patients.
). IKZF1 rs4132601 is composed of T and G alleles and the T allele is the wild one.IKZF1 rs11978267 is composed of A and G alleles and the A allele is the wild one.Both SNPs are located within IKAROS family zinc finger 1 (IKZF1) gene on chromosome 7p12.2andare about 4 kb apart.TA B L E 1

Table 3 .
There are four TA B L E 3 Comparison of Ikaros family zinc finger 1 gene (IKZF1) rs4132601 and rs11978267 genotypes, alleles and haplotypes between paediatric SLE (pSLE) patients and controls.
Comparison of Ikaros family zinc finger 1 gene (IKZF1) genotypes and alleles between proliferative and non-proliferative lupus nephritis (LN) cases.Comparison of biopsy results according to rs4132601 and rs11978267 genotypes in studied lupus nephritis (LN) cases.Comparison of genetic variability in studied single nucleotide polymorphisms (SNPs) in Egyptian healthy controls and published data in TopMed and gnomAD projects concerning main ethnic groups.
TA B L E 4 c , p value corrected by Bonferroni correction.P value is significant if less than .05*P value is significant if less than .05TA B L E 6 SNPs and the risk to develop SLE and clinical presentation especially nephritis in a cohort of Egyptian children from East Delta, Egypt (Table 1).Similar findings were reported by Abdel-Nabi and Abdel-Noor (2018).They inves-tigated the prevalence, pattern of clinical manifestations and organ damage in adult and juvenile Egyptian SLE patients from Middle Delta, Lee et al. (2014)11;Han et al., 2009;Wang et al., 2013) in Chinese Han population.They reported a significant risk association between the rs4917014 of IKZF1 and renal nephritis (p = .02andOR=1.13).Graham et al., 2011;Han et al., 2009;Wang et al., 2013).To reveal the effect of ethnicity on the SLE risk loci,Lee et al. (2014)conducted a GWAS on Korean females with SLE.They classified the genes associated with SLE into three groups; genes associated with SLE in both populations (Caucasians and Asian specific genes); genes associated with SLE in Caucasians only and not replicated in Asian or Koreans (Caucasians specific genes); and genes associated with SLE in Asian only (Asian specific).Although they confirmed the association between IKZF1 and SLE in Koreans; however, they concluded that the IKZF1 locus is more likely to be Asian-specific locus.
(Taylor et al., 2008)s(SLEGEN) et al., 2008).SLE is known to be a heterogeneous disease with different genotype-phenotype patterns (such as the association between STAT4 and LN)(Taylor et al., 2008).To explore the relation between SLE sub-phenotypes and the five new discovered loci (TNIP1, SLC15A4, ETS1, RasGRP3 and IKZF1), He et al.(