Contribution of LOC105371267 and MRPS30-DT Genetic Polymorphisms in IgA Nephropathy Among Chinese Han Population

Background: IgA nephropathy (IgAN) is the common primary glomerulonephritis worldwide. Genetic factors have been reported to take essential part in IgAN progression. This study was designed to investigate the association between LOC105371267and MRPS30-DT with IgAN risk among Chinese. Methods: 6 SNPs were genotyped. A logistic recession model was used to calculate the effects of candidate SNPs on IgAN. The SNP-SNP interaction was analyzed by MDR. Results: We observed only LOC105371267 had relationships with IgAN. The results indicated an association between the genotype “CC” and the decreased IgAN risk (OR = 0.44, p = 0.014). The stratication analysis at age ≥ 35 showed that rs3931698 contributed to the IgAN susceptibility in “GT” (OR = 1.78, p = 0.038), while rs8044565 signicantly showed a decreasing-risk effect with IgAN (“T”, OR = 0.59, p = 0.006; “CC”, OR = 0.15, p = 0.015; “CC-CT”, OR = 0.59, p = 0.023; Log-additive, OR = 0.56, p = 0.005). Rs8044565 was correlated with the decreased susceptibility of IgAN in male (“CC”, OR = 0.27, p = 0.006) and in Lee’s grade ≥ III (“CC”, OR = 0.46, p = 0.046). We found rs8044565 was related to systolic blood pressure and urinary casts, and rs3852740 had a relationship with Serum C3 and hemoglobin (p < 0.05). Conclusion: The present study rst demonstrated that the SNPs in lncRNAs might be related to IgAN.


Introduction
With the development of modern society, especially for the change of dietary habits, chronic kidney disease with increased morbidity and mortality has attracted much attention nowadays. Immunoglobulin A nephropathy (IgAN) is a kind of autoimmune disease, accounting for 45.3% -54.3% of primary glomerulonephritis and remains a leading cause of endstage renal disease (ESRD) in China [1,2]. IgAN is characterized by a single histopathological criterion of pre-dominant IgA deposits on kidney biopsy, however, renal biosy is invasive with limitations in assessing disease activity only at the time of biopsy, which could lead to inconclusive ndings and decisions [3,4]. To date, it is gradually recognized that genetic factors play a crucial role in the development of IgAN and it may serve as potential diagnostic indicators [5][6][7].
As researched revealed that greater than 70% of genome is transcribed and that vast majority of transcribed DNA encodes long non-coding RNAs (lncRNAs) [8,9]. LncRNAs are important class of noncoding RNA that are characterized by their length longer than 200nt. Accumulating evidence have suggested that lncRNAs take essential part in diverse pathological settings, including cancer, cardiovascular disease, and pathogenesis of kidney disease [9,10]. Recent studies have also reported the relationship between lncRNAs and various kidney disease [11][12][13], but few about IgAN [14,15]. Guo et al. used high-throughput RNA sequencing and qRT-PCR to test the exosomes isolated from plasma of IgAN patients and their healthy rst-degree relative. The results revealed that exsomal lncRNA-G21551 was down-regulated in IgAN patients, indicating its potential to serve as a non-invasive biomarker for IgAN [14]. In the study of Zuo et al, peripheral blood mononuclear cells were collected from both IgAN patients and healthy controls to identify differentially expressed lncRNAs and mRNAs by microarray analysis and quantitative polymerase chain reaction. Their results demonstrated that differentially expressed lncRNAs and mRNAs may have a role in the development of IgAN [15]. However, there are not any genetic polymorphism research about IgAN. Thus, we designed the current study to investigate association between single nucleotide polymorphisms (SNPs) and lncRNAs.
Loc105371267, located on chromosome 16, is a lncRNA involved in the p53 network which is hardly researched. It was reported that p53 upregulation in renal resident cells may be linked to the pathogenesis of progressive IgAN [16], but the role of Loc105371267 to IgAN susceptibility remains unclear. Additionally, MRPS30-DT on chromosome 5 is broadly expressed in breast, kidney and other tissues [17]. Until now, no data have been found on the relationship between MRP30-DT and IgAN.
Therefore, in the current study, we will conduct a case-control study to identify the association between IgAN susceptibility and six SNPs in the Loc105371267and MRP30-DT in the Chinese Han population. The study aims to identify the potential role of these SNPs in IgAN.

Study participants
The current study was included 836 unrelated subjects including 413 IgAN patients and 423 geographically ethnicitymatched healthy subjects who were collected from Xi'an Hospital of Traditional Chinese Medicine. All patients must meet the diagnostic criteria which tested by renal biopsy and the patients with other autoimmune diseases or secondary IgAN were excluded. [18]. The healthy subjects were collected from the physical examination center at the same period. The clinical information of participants were collected, including age, gender, serum albumin (ALB) level, creatinine (CREA) level, Urine red blood cell (URBC) count, hemoglobin (HB), serum uric acid (UA), brinogen (FIB), and pathological grade (Lee's classi cation).
We designed this protocol in compliance with the the Ethics Committee of the Xi'an Hospital of Traditional Chinese Medicine and the guidelines of the Declaration of Helsinki. All participants were provided and signed up the written informed consent.

Selection and genotyping of SNPs
We identi ed six SNPs in LOC105371267 and MRP30-DT with a minor allele frequency (MAF) > 0.05 in the 1000 Genomes Projects (http://www.internationalgenome.org/). Fasting peripheral blood of all participants were collected in anticoagulant tubes and stored at -80 ℃. We extracted DNA by using the whole blood genomic DNA extraction kit (GoldMag, China) in accordance with manufacture's protocol provided, and the DNA content was measured by spectrometry (NanoDrop 2000 spectrophotometer, Thermo Scienti c, USA). Multiplexed SNP MassEXTEND assay was designed by Agena MassARRAY Assay Design Software (version 3.0, Agena Bioscience, USA). Moreover, Agena MassARRAY RS100 was used to detect SNP genotyping. Data were analyzed using Agena Typer Software (version 4.0, Agena Bioscience, USA).

Statistical analysis
SPSS software (version 20.0) was used for data analysis. The independent sample T-test or χ2 test was used to examine the differences of basic parameters between the cases and controls. Hardy-Weinberg equilibrium (HWE) was tested by χ2 test for each SNP selected in the current study. The IgAN risk associated with genotyping was estimated by odds ratios (ORs) with 95% con dence intervals (CIs) for ve different genetic models. The difference in clinical characteristics among different genotypes was analyzed using the ANOVA test. The SNP-SNP interactions in the risk of IgAN were analyzed by multifactor dimensionality reduction (MDR) (version 3.0.2). For all test, a two-tailed p-value < 0.05 was considered statistically signi cant.

Results
Basic characteristic of the participants The current study was included 413 IgAN patients (267 males and 146 females) and 423 healthy controls (275 males and 148 females). The mean age of cases and controls were 33.21 ± 12.07 and 33.34 ± 10.11 years, and there were no signi cant differences in age and gender between cases and controls group (p = 0.861, p = 0.942, respectively). Demographic and clinical characteristics were listed in Table 1, including age, gender, pathological grade, urine red blood cell (RBC), urine casts, serum albumin (ALB), creatinine (CREA), serum uric acid (UA), hemoglobin (HB) and brinogen (FIB). Signi cant differences were observed in urine RBC, urine casts, ALB, CREA, UA, HB and FIB between two groups (all p < 0.001).

SNP-SNP interactions
We used MDR analysis to assess the effect of SNP-SNP interaction among 4 selected SNPs in LOC105371267 (Table 8). In total, we found a three-locus mode including rs8044565, rs3852740, rs111577197 were the best model (cross-validation consistency = 9/10, testing balanced accuracy = 0.464, p = 0.006). Obviously, there were interactions between locus and locus presented in a dendrogram and the Fruchterman-Reingold in Figure 1 (A and B, respectively).

Discussion
IgAN is a complex autoimmune disease with pathogenesis needed to be clari ed. Accumulating evidence indicated that genetic and environmental factors take essential part in the development of IgAN. Previous studies revealed that some genetic variations, such as FCRL3, DRB1 and DEFA [19][20][21], were signi cantly associated with the risk of IgAN, but are few reported in long non-coding RNA (lncRNA) which have attracted much attention for their functions in gene regulation nowadays [22]. More importantly, it was reported that lncRNA was associated with IgAN, however, there has been found no data to demonstrate the genetics polymorphisms about IgAN.
We designed this case-control study to detect the association between genetic polymorphisms in two lncRNAs and the susceptibility to IgAN. The results revealed that only Loc105371267 had an association with IgAN and rs8044565 variants in Loc105371267 might serve as a potential protective factor to IgAN in the overall. Interestingly, our further strati ed analysis showed that LOC105371267 rs3931698 variants was associated with the susceptibility to IgAN in the subgroup ≤ 35 years, while LOC105371267 rs8044565 variants reduced the risk of IgAN. We also found that rs8044565 was decreased the risk of IgAN in females as well as it was signi cantly associated with Lee's grade. In view of the complicated pathogenic factors of IgAN, SNP-SNP interaction studies may help discover the risk factors for IgAN [23]. Accordingly, we analyzed the potential SNP-SNP interactions in LOC105371267 by MDR. The analysis indicated a strong interaction between the rs8044565, rs3852740 and rs111577197 regarding association with IgAN. To the best of our knowledge, it is the rst time to demonstrate the effects of the relationships between these SNPs in lncRNA and the IgAN risk.
Loc105371267, located on chromosome 16, is a p53-regulated lncRNA which remains unclear in IgAN risk. In recent years, evidence has emerged that dysfunction of the p53 network is associated with the development of autoimmune disease [24][25][26]. Thus, it is signi cance to detect the association between LOC105371267 and IgAN. In current study, we rstly investigated the association between 4 polymorphisms in the LOC105371267 with IgAN risk among Chinese Han population. The results provided an evidence that rs8044565 in LOC105371267 was signi cantly associated with reduced IgAN risk in different genetic models. Therefore, it may serve as an important protective role for IgAN which needed to be further veri ed.
Given the current aging society in China, age in IgAN patients is an important factor to consider. Previous cohort study in 2019 indicated that the mean age at diagnosis of IgAN was 32.9 years [3]. Thus, to detect the genetics effect of age in IgAN, we did an analyzation strati ed by age at 35 years, it showed that genetype "GG" in LOC105371267 rs3931698 was contributed to the IgAN susceptibility (OR = 1.78, 95% CI = 1.03-3.07), p = 0.038) in the group of age ≥ 35 years, while LOC105371267 rs8044565 signi cantly association with the reduced IgAN risk at the same subgroup. At the same time, the genotype "CC" in rs8044565 showed the decreasing risk-effect with IgAN (OR = 0.27, 95% CI = 0.11-0.69, p = 0.006). It probably revealed that rs8044565 in LOC105371267 might perform a protective effect in the group of male who were older than 35 years.
Besides, several studies have showed that SNPs have strong susceptibility to IgAN in Lee's grade [27][28][29]. In the current study, we observed that the genotype "CC" (OR = 0.46, 95% CI = 0.21-0.98, p = 0.046) in LOC105371267 rs8044565 was signi cantly related with the reduced IgAN risk under the strati cation of Lee's grade > III. Clinical characteristics can be regarded as indicators for IgAN [30]. Thus, we determined the correlation between SNPs and clinical characteristics in IgAN. We observed that LOC105371267 rs8044565 was related to diastolic blood pressure, urinary casts and LOC105371267 rs3852740 was related to serum C3 and hemoglobin. We speculate that SNPs in LOC105371267 may correlate with clinical indicators, which needed to verify by investigating the more indicators in the further study.
Several intrinsic limitations to our study should be considered. First, the selection bias in this case-control study was its hospital-based design, and it may not be representative of the general population. Second, environmental exposure was not available, which limited us to further analyze the potential interaction of gene-environment on IgAN risk. Importantly, further functional assay of our present study provided scienti c evidence about LOC105371267 with IgAN in the future study.

Conclusion
To summarize, the current study investigated the SNPs in lncRNA LOC105371267 and MRPS30-DT with the risk of IgAN. The results revealed that only LOC105371267 variants signi cantly associate with the IgAN risk and rs8044565 may be a protective factor which need to be further veri ed in the larger samples study. Notably, our results was rstly detected the relationship between lncRNA and IgAN risk. Besides, a new insight for the molecular mechanism in the development of IgAN was provided.

Declarations
Ethics approval and consent to participate This study strictly obeyed the World Medical Association Declaration of Helsinki, which was also approved by the Ethical Committee of the Xi'an Hospital Of Traditional Chinese Medicine. Written informed consent was obtained from each study participant.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.         p values were calculated using χ 2 tests. p < 0.05 indicates statistical signi cance. Figure 1 There were interactions between locus and locus presented in a dendrogram and the Fruchterman-Reingold in Figure 1 (A and B, respectively).