Association of microRNA 17 host gene variant (rs4284505) with susceptibility and severity of systemic lupus erythematosus

Abstract Objective MicroRNAs are large family clusters of small noncoding RNAs that implicated in genetic and epigenetic regulation of several immunological processes and pathways. As an epigenetic modifier, the microRNA 17‐92 cluster host gene (MIR17HG) has been shown to regulate the expression of genes involved in systemic lupus erythematosus (SLE) pathway. This study aimed to explore the association of MIR17HG (rs4284505; A>G) variant with SLE development and phenotype in a sample of the Eastern Mediterranean population. Methods A total of 326 participants (163 patients with SLE and 163 healthy controls) were enrolled in this study. The different genotypes of the MIR17HG (rs4284505) variant were characterized using the TaqMan real‐time polymerase chain reaction technique. Association with the available clinical and laboratory data, including the systemic lupus erythematosus disease activity index (SLEDAI), was also executed. Results The MIR17HG (rs4284505) variant showed a protective effect against developing SLE under heterozygote (A/G vs A/A; odds ratio [OR] = 0.10, 95% confidence interval [CI] = 0.05‐0.20, P < 0.001) and dominant (A/G+G/G vs A/A; OR = 0.39, 95% CI = 0.25‐0.61, P < .001) models. This association was consistent even after SLE stratified by lupus nephritis. In contrast, rs4284505 (G/G) genotype conferred increased susceptibility to SLE (G/G vs A/A+A/G; OR = 2.15, 95% CI = 1.31‐3.53, P = .002). Moreover, the rs4284505 variant showed a statistically significant association with mucocutaneous lesions and SLEDAI scores (all P < .05). Conclusion This study is the first one to explore that the MIR17HG rs4284505 is associated with SLE risk; (A/G) genotype conferred a protective effect, while the (G/G) genotype showed increased susceptibility to SLE and association with the disease severity in the study population.


| INTRODUCTION
The systematic lupus erythematosus (SLE) is a multisystem autoimmune disease with diverse clinical presentations. 1 The pathogenesis of SLE is complex and undefined, with evidence of the interaction of several genetic and environmental causes. 2 MicroRNAs (miRNAs) are short, evolutionary conserved, single-stranded noncoding RNAs with approximately 22 nucleotides in length derived from large primary transcript encoded by their host genes. 3 They have several genetic and epigenetic regulatory functions, modulating diverse cellular processes, and molecular pathways at the posttranscriptional level. 4 The implication of miRNAs in immune response was evident in several studies, [5][6][7][8] and their deregulation was associated with multiple autoimmunity-induced pathological entities, including SLE. 3,9,10 Some "miRNAs-containing" primary transcripts can yield a cluster of several and different miRNAs, rather than single miRNA, usually working in concert. 3 One of the best-studied miRNA clusters is the "miR-17-92 family," which encoded by miR-17 host gene (MIR17HG) and including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1. 11 This gene cluster is located on the plus strand of chromosome 13 (13q13. 3) and spanned about 49 411 bases. 12 Although this cluster was reported to be involved in cellular proliferation, angiogenesis, and survival, 4,13 other essential roles in multisystem processes, including the normal development, and the innate and acquired immune response among others, have been emerged. 11,14 Furthermore, the upregulation of this cluster in splenic T cells has been found to induce abnormal B cell activation and inflammatory T cell production by targeting the Bim gene in murine models of lupus. 15 Our in silico analysis also revealed MIR17HG could target several immune-related genes involved in SLE development and progression ( Figure 1). [16][17][18] As most genetic variants (ie, single-nucleotide polymorphisms [SNPs]) related to SLE risk were identified to be located in noncoding regions of the genome, 19 the study of MIR17HG cluster variants could enhance our understanding of its role in SLE pathogenesis. The most common SNP of this gene cluster is rs4284505 (G>A), which identified within the first intron of the MIR17HG. This variant has been associated with several diseases, 4,12,13,20,21 but its impact on SLE development and severity has not been assessed. In this sense, the authors were inspired to investigate the association of MIR17HG (rs4284505) variant with the development of SLE and with the available clinical and laboratory data in a sample of the Middle Eastern population.  22 At the same time, the diagnosis of lupus nephritis was made as previously described. 23 This study was conducted following the Helsinki Declaration guidelines, and the research ethics committee of the Faculty of Medicine, Suez Canal University, approved it. The controls were collected from the healthy blood donors attending the university hospital blood banks. They were matched in age and sex with patients with SLE. Individuals having a history of autoimmune illnesses, endocrine diseases, and any other chronic diseases were excluded. All enrolled subjects were asked to sign written consent before taking part. The basic characteristics and clinical features of patients with SLE were extracted from the medical health records. The assessment of disease activity was completed according to the systemic lupus erythematosus disease activity index (SLEDAI). 24

| Blood sampling and laboratory measurements
Upon establishing this study, 5 mm of peripheral blood were collected and allocated into two parts; the first part was gathered into a vacutainer tube containing ethylenediamine tetraacetic acid anticoagulant for purposes of genetic and hematological analysis, while the other part was centrifuged to isolate the serum for immunological and biochemical measurements. The estimation of complete blood count including hemoglobin, white blood cells (WBCs) count, neutrophil %, lymphocyte %, red blood cells count, hematocrit, mean cell volume (MCV), and platelet count was performed utilizing an automated cell counter (CELL-DYN 1700; Abbott Diagnostics). The assessment of biochemical measurements involving complement 3 (C3), C4, C-reactive protein, alanine transaminase, aspartate transaminase, creatinine, urea, and albumin was measured using automated biochemical analyzer Cobas c501 (Roche Diagnostics, Manheim, Germany). The indirect immunofluorescence technique was applied for the specification of antinuclear antibodies (ANA) and anti-double-stranded DNA antibodies (Anti-dsDNA) (Bio-Rad Laboratories, CA). F I G U R E 1 MIR17HG targets in systemic lupus erythematosus (SLE). SLE is an autoimmune disease featured by increased production of autoantibodies that are specific for self-antigens, resulting in a wide range of clinical presentations. A, The miR-17-92 host gene family enclose a cluster of six microRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1). Functional enrichment pathway analysis using Diana lab tools (http://diana.imis.athena-innovation.gr/DianaTools/index.php) revealed that these miRNAs could target at least 18 genes in the SLE pathway (KEGG ID: hsa05322, P = 1.179199e−43). B, The miR-17-92 clustertargeted genes (red-colored boxes) code for the different histone family members (ie, H2A, H2B, H3, and H4), which are implicated in autoantigen clearance and tolerance mechanism and the major histocompatibility complex class II (MHCII), which is implicated in antigen presentation mediated by MHCII. All the previous stages of antigen processing and autoantibodies generation lead to tissue injury and end-organ damage, 16 (http://www.kegg.jp/kegg/kegg1.html). C3, complement 3; dsDNA, double-stranded DNA; IFN-γ, interferon-γ; IgG, immunoglobulin G; IL-10, interleukin 10; JAK-STAT, Janus kinase-signal transducer and activator of transcription; KEGG, Kyoto Encyclopedia of Genes and Genomes; MIR17HG, microRNA 17-92 cluster host gene; NMDAR, N-methyl-D-aspartate receptor; snRNP, small nuclear ribonucleoproteins; TCR, T cell receptor; TNF-α, tumor necrosis factor-α 2.3 | DNA extraction and allelic discrimination of MIR17HG rs4284505 variant Genomic DNA from nucleated WBCs of whole blood was extracted utilizing the Mini Kit of QIAamp DNA extraction designated for blood samples (Catalog #51104; Qiagen, Hilden, Germany). The NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc, Wilmington, DE) was utilized for estimation of the quantity and purity of genomic DNA. The intronic variant (rs4284505; A>G) of MIR17HG was characterized and genotyped by the TaqMan allelic discrimination polymerase chain reaction (PCR) using StepOne Real-Time (Applied Biosystems). The protocol used was managed blindly with a final volume of 20 μL containing 20 ng of DNA template, 1 μL of TaqMan SNP Genotyping Assay Mix (assays ID: C__26557482_10; Applied Biosystems, Waltham), and 10 μL of TaqMan Universal PCR Master Mix. Proper negative controls were applied in each run. 25 About 10% of the samples were randomly selected for assay replication, and the results yield 100% concordance.

| Statistical analysis
The processing of statistical analysis of the MIR17HG rs4284505 variant was performed using the SPSS program version 26.0 as well as R Language programming, version 4.0.0 and RStudio, version 1.3.959. The calculations of allelic and genotypic frequencies were executed using the Fischer exact test with χ 2 , as previously described. 26 The estimation of the Hardy-Weinberg equation and the analysis of the MIR17HG rs4284505 variant among patients with SLE and healthy controls was computed utilizing SNPstats (www.SNPstats.org). Logistic regression analysis was assessed to determine the adjusted odds ratio (OR) with a 95% confidence interval (CI) of multiple genetic association models. 27 The evaluation of principal component analysis (PCA) was conducted utilizing the "factoextra" and "FactoMineR" packages. P < .05 was adapted to be statistically significant.

| Basic characteristics of the study population
The main characteristics of the SLE patients with and without lupus nephritis are summarized in Table 1. A total of 93 (57.1%) SLE patients with lupus nephritis (85 females and 8 males) and 70 (42.9%) SLE patients without lupus nephritis (62 females and 8 males) were included in this study. Their mean age (SD) at diagnosis was 36.4 (9.7) and 34.5 (9.4), respectively. Of the 163 patients with SLE, 58 (36.5%) had a positive family history of SLE. As depicted in Table 1, there was no significant difference regarding age at diagnosis (P = .21), sex (P = .60), positive

| Genotypic and allelic frequencies of MIR17HG (rs4284505) variant
The Hardy-Weinberg equilibrium among healthy controls was in concurrence with observed equilibrium (P = .402) ( Table 2). The minor allele frequency (MAF; G allele) of the MIR17HG variant was 0.40 among patients with SLE, while the MAF (G allele) among healthy controls was 0.44. Moreover, the common allele (A) frequency of the whole study population was 58%, while the MAF (G) was 42% (Figure 2A). The most predominant genotype among patients with SLE was A/A genotype, accounting for 56% of patients, while the most frequent genotype among healthy controls was G/A genotype, accounting for 46% of controls ( Figure 2B). The MIR17HG allele frequencies in the present study were comparable with those related to the African and East Asian populations ( Figure 2C). Upon construction, the PCA among patients with SLE, all variables showed no clear distinct demarcation of SLE patients with different genotypes ( Figure 2D).

| DISCUSSION
miRNAs are large family clusters of tiny noncoding RNAs that are implicated in genomic and epigenomic regulation. 20 The primary function of miRNAs is to target the designated mRNAs causing their instability and/or degradation, mediating their inhibitory effects. 28 Accumulating evidence identified various genetic variants within the MIR17HG gene, including (rs4284505; g.91349218A>G), (rs17735387; g.91353800G>T), (rs1428; g.91354516C>A), (rs7336610; g.91352883C>T), and (rs72640333; g.91352673T>A) that are destined to be correlated with different disorders involving multiple myeloma, 13 breast cancer, 4 multiple sclerosis, 21 and periapical lesions. 20 Until now, to the best of our knowledge, this is the first study investigating the association of MIR17HG (rs4284505; A>G) variant with the development of SLE among a sample of Middle Eastern population. Our findings indicated that the A/G genotype revealed a protective effect against the development of SLE, and this protective effect remains significant after stratifying the patients according to the presence of lupus nephritis. In contrast, the G/G genotype conferred an increased susceptibility of SLE development. Interestingly, the substitution of A to G has been found to disrupt a regulatory region upstream to the MIR17HG cluster, affecting multiple transcription factors (TF)-binding elements (Table S1) with subsequent change in the type of activated genes. In this sense, having activated TF for genes with A as well as genes with G allele simultaneously might synergistically promote the expression of protective genes, and A/G genotype carriers become less susceptible to the disease. In contrast, having two copies of the G allele might promote the expression of genes that increase susceptibility to the disease and increase the severity. Moreover, due to the functional relationship of the intronic miRNA (eg, MIR17HG) with its host gene, this miRNA family may have an individual negative regulatory feedback by directly targeting and regulating the host transcripts, mediating some roles in disease phenotype at the transcriptomic level. 29 Further functional studies will be required to confirm these findings.
Accumulating evidence has uncovered the potential role of the MIR17HG cluster with the macrophage colonystimulating factor in promoting CD34 + hematopoietic progenitor cell differentiation into monocytes in human cord blood. 30 In addition, the upregulation of this cluster in the DN1 stage has been associated with increased proliferation and survival of CD4 + T cells, which mediates the autoimmunity. 14 By targeting BIM and PTEN genes, which play a critical role in immune tolerance mechanism, in B/T lymphocyte precursors and memory CD8 + T cell, respectively, miR-17-92 family was associated with B and T cell development and autoimmunity. 31,32 MIR17HG transcriptomic signature also was reported to be involved in the pro-B cell transition to pre-B regulating B cells generated in the bone marrow during the early stages of development. 3 By analogy to other types of autoimmune disease, earlier studies also support the key role MIR17HG cluster play in controlling the inflammatory process and disease activity through regulation of the "Apoptosis Signal-Regulating Kinase 1 (ASK1) signalosome," a central actor in the inflammatory pathways activated during rheumatoid arthritis. 33,34 Collectively, all the above evidence confirm the role which MIR17HG play in autoimmunity and tissue damage (Figure 1), supporting the potential association of the specified genetic variant with SLE development and severity, as reflected in association of the risky genotype (GG) with a higher frequency of mucocutaneous manifestations and a higher SLEDAI score than the other genotypes in the study population. The current work also observed that the MAF of rs4284505*G accounted for 42% of the study population. Comparing with the 1000 genome project indicated that our MAF has resembled with East Asian population (46%), but entirely disagreed with African (30%), American (61%), European (59%), and South Asian (62%).
Similarly, a recent report studied the association of MIR17HG (rs4284505) variant with the susceptibility for multiple myeloma, revealed that the G/G genotype observed increased risk with the developing of multiple myeloma and the A/G genotype revealed a poor prognosis with the outcome of the disease. 13 On the contrary, another study investigated the association of MIR17HG (rs4284505) variant with the risk of persistent apical periodontitis and observed no significant association of Note: Data are presented as frequency (percentage) or mean ± SD. χ 2 and one-way ANOVA tests were used. Bold values indicate that P < .05 and considered as statistically significant. Abbreviations: ANOVA, analysis of variance; ALT, alanine transaminase; AST, aspartate transaminase; C3, complement 3; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HCT, hematocrit; MCV, mean cell volume; RBC, red blood cell; SLEDAI, systemic lupus erythematosus disease activity index; WBC, white blood cell.
this variant under recessive and dominant models with the progression of the disease. 20 Also, we encountered a single study that revealed a protective effect of the MIR17HG rs4284505*A allele with the development of breast carcinoma among the Australian population. 4 These conflicting findings confirm the previous notion that the genetic variants often exert their effect in "a celltype-specific and context-dependent manner." 19,35,36 Additional contributing factors could be due to the ethnicity, environmental, and geographical factors, as well as the variability in the sample size and methodology of the different studies.

| CONCLUSION
In conclusion, the MIR17HG (rs4284505) variant was associated with SLE development among Egyptian subjects. The MIR17HG A/G genotype conferred a protective effect with the progression of SLE, while the MIR17HG G/G genotype associated with increased susceptibility to SLE development and severity.