Endothelin‐1 rs9296344 associates with the susceptibility of childhood primary nephrotic syndrome

Abstract Background Recently, the rs5370 single nucleotide polymorphisms (SNPs) of Endothelin‐1 (EDN1) showed association with the susceptibility of childhood primary nephrotic syndrome (CPNS). This study aims to investigate potential relationships between other EDN1 SNPs and CPNS. Methods Seven SNPs (rs5370, rs10478723, rs1476046, rs1800541, rs2070698, rs2071942, and rs9296344) of the EDN1 gene were genotyped in 579 CPNS patients and 586 age‐matched healthy children. Then, we analyzed potential associations of the six SNPs with susceptibility of CPNS by using rs5370 as a conditional variant in a logistic regression model. SNP‐SNP interaction analysis was performed to investigate the joint effects of the seven SNPs in the pathogenesis of CPNS. Results Independent with rs5370, only rs9296344 significantly associated (T vs C, odds ratio [OR] = 0.71, 95% confidence interval [CI] = 0.57‐0.88, P = .001) with the susceptibility of CPNS. Meanwhile, no joint effect among the analyzed seven SNPs was discovered in this study. Conclusions This study discovered that C allele of rs9296344 on EDN1 is a novel independent risk factor for CPNS.


| INTRODUC TI ON
Childhood nephrotic syndrome is a group of symptoms that indicate kidney damage, particularly damage to the glomeruli, the tiny units within the kidney where blood is filtered, and results in the release of too much protein from the body into the urine. 1,2 Childhood primary nephrotic syndrome (CPNS) is the most common type of childhood nephrotic syndrome. 1,3 There are around 16 ~ 18 CPNS patients in every 100 000 children. 4 The cause of CPNS is not known in most cases. However, recent studies suggested that genetic factors, such as single nucleotide polymorphisms (SNPs), might contribute to the susceptibility of CPNS. [5][6][7][8] Endothelin-1 (EDN1) encodes a preproprotein that is proteolytically processed to generate a secreted peptide that belongs to the endothelin/sarafotoxin family. This peptide is a potent vasoconstrictor, and its cognate receptors are therapeutic targets in the treatment of pulmonary arterial hypertension. The overexpression of EDN1 associated with pathological kidney phenotypes, such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis, and leads to a progressive decrease in glomerular filtration rate. 9  To study the potential rs5370-independent relationships between other SNPs of EDN1 and susceptibility of CPNS, we genotyped seven SNPs (rs5370, rs10478723, rs1476046, rs1800541, rs2070698, rs2071942, and rs9296344) of EDN1 and used rs5370 as conditional variant in the regression model to analyze the potential association independent of rs5370.

| Participants
Childhood primary nephrotic syndrome patients and age-matched healthy child participants were enrolled in between July 1, 2014, and October 31, 2018, in Xuzhou Children's Hospital. All participants are in Chinese Han population. A full rationalization of the procedure was conducted to legal guardians of all participants to obtain written consent. The performance of this study was approved by the ethics committee of Xuzhou Children's Hospital following the principle of the Helsinki Declaration.

| Polymerase chain reaction (PCR) and DNA sequencing
Blood samples were drawn from CPNS patients and age-matched healthy child participants to extract DNA with the QIAamp DNA Blood Mini Kit (Qiagen). PCR was used to capture selected SNPs from DNA. PCR system for 50 μL was as follows: The reaction mixture contained 2 μL DNA, 2 μL primers, 4 μL dNTP, 5 μL PCR buffer, and 0.25 μL Taq enzyme (Takara), and then, the water was added to make up a volume of 50 μL. The reaction conditions were set as follows: 94°C for 5 minutes; 39 amplification cycles, 30 seconds at 94°C, 45 seconds at 53.7°C, and 50 seconds at 72°C; and ultimate extension lasted for 5 minutes at 72°C. All the six SNPs (rs10478723, rs1476046, rs1800541, rs2070698, rs2071942, and rs9296344) in EDN1 were selected with minor allele frequencies (MAF) >0.1 in Chinese population according to SNP data in dbSNP database (www. ncbi.nlm.nih.gov/SNP). Among the six SNPs, rs9296344 locates on the 3'UTR region, while the others locate on intron region. The sequences of PCR products were obtained by using an ABI PRISM 3730 genetic analyzer (Applied Biosystems).

| Statistical analysis
The PLINK software was used to determine the association of single SNP loci in genotype and allele frequencies with CPNS, as well as the P-value of the Hardy-Weinberg equilibrium (HWE). The rs5370 was used as conditional variant in the regression model to analyze the potential associations independent of rs5370. The risk of alleles was estimated by the odds ratio (OR) with 95% confidence intervals (CIs). Linkage disequilibrium (LD) between SNPs was measured by D' and R 2 using Haploview software. P-values are two-tailed with the threshold of .05 for statistical significance.
All statistical analyses were conducted by using R (version 3.5.2).
The SNP-SNP interaction was performed by using GMDR software, 11 which is the software for detecting gene-gene and geneenvironment interactions underlying complex traits. P-value <.05 was selected as significant results.

| C allele of rs9296344 associates with the susceptibility of CPNS
There were 579 CPNS patients (272 boys and 307 girls with a mean age ± SD of 8.56 ± 4.01 years) and 586 age-matched healthy children (277 boys and 309 girls with a mean age ± SD of 8.80 ± 4.10 years) involved in this study ( Table 1). The SNP rs5370 and other six SNPs

| Linkage disequilibrium (LD)
Linkage disequilibrium analysis of the seven SNPs in CPNS patients and age-matched healthy children revealed that only weak (D' <0. 4) or median LD (0.4 < D' <0.6) interactions existed in these analyzed SNPs (Table 3, Figure 1). No strong LD (D' >0.6) interactions were discovered among these SNPs, which means that these SNPs act independently in both CPNS patients and age-matched healthy child population. Therefore, we did not perform further haplotype analysis in this study.

TA B L E 3
The linkage disequilibrium analysis of analyzed SNP pairs in studied populations F I G U R E 1 Linkage disequilibrium analysis of analyzed SNPs in patients and healthy controls from 1 SNP locus to 7 SNP loci were investigated in this study.
Models with best testing balanced accuracy in every loci size all had rs9296344. But no significant joint effects were discovered in this study (Table 4). This revealed that rs9296344 may play its role in CPNS independently. Endothelin-1 encodes a preproprotein, which associated with pathological kidney phenotypes, such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis, and leads to a progressive decrease in glomerular filtration rate. 9 The association of EDN1 rs5370 G > T gene polymorphism revealed the potential relationship between EDN1 gene polymorphism and the pathogenesis of CPNS. In this study, we discover a novel susceptibility SNP rs9296344 in 579 CPNS patients and 586

| D ISCUSS I ON
healthy controls of Chinese Han population, which is a larger population than previous studies. 5 19,20 The association of rs9296344 C allele implied that it may affect the expression of EDN1 gene, since overexpression of EDN1 is associated with pathological kidney phenotypes in CPNS. 9 A potential mechanism is that the rs9296344 C allele may increase the susceptibility of CPNS by increasing the expression of

EDN1.
In general, this study reported that rs9296344 C allele on EDN1 associates with the susceptibility of CPNS.