Single-Nucleotide Nr3C1 Gene Polymorphisms Affect Glucocorticosteroid Treatment Ecacy In Patients With Pemphigus Vulgaris

We


Abstract
Background in recent years, the effectiveness of glucocorticosteroid (GC) treatment has become a central issue when managing patients with pemphigus vulgaris (PV). Polymorphisms in the gene encoding the nuclear receptor subfamily 3, group C, member 1 (NR3C1) protein (the GC receptor) may explain the observed variations in treatment e cacy. We aim to evaluate the effects of 10 SNPs (rs 17209237, rs11745958, rs7701443, rs33388, rs41423247, rs6189, rs6190, rs6195, rs6196, and rs6198) in Vietnamese PV patients.
Methods we studied 10 sites in the NR3C1 gene (selected using published data) and sought correlations between single nucleotide polymorphisms (SNPs) in these regions and the clinical responses to GCs in 15 PV patients. Whole blood samples from all patients were collected in tubes containing ethylenediamine tetraacetic acid (EDTA) and were genotyped using TaqMan SNP Genotyping assay.

Results
Of the 10 sites in the NR3C1 gene, SNPs were detected in 6 (rs17209237, rs11745958, rs7701443, rs41423247, rs33388, and rs6196); the genotypes rs17209237 AA, rs11745958 CC, and rs6196 AG may be associated with a need for a lower total GC dose; rs17209237 AA and rs6196 AG with shorter times to commencement of tapering; and rs17209237 AA and rs11745958 CC with shorter times to attainment of 50 and 25% Pemphigus Disease Activity Index scores. Conclusions NR3C1 gene variations may predict GC e cacy in PV patients. However, larger, randomized controlled trials are required.

Background
Pemphigus vulgaris (PV) is a blistering autoimmune disease typically affecting the skin and mucosa. The incidence of PV varies ethnically, ranging from 0.76 to 16.1 new cases per million subjects per year [1], [2].
In the pre-corticosteroid era, the mortality rate was 70% [3]. In the time since such drugs were approved in the 1950s, corticosteroids have been the backbone of PV treatment and have reduced mortality to about 30% [4]. Although many other immunosuppressive agents are available, glucocorticosteroids (GCs) remain the rst-line treatment according to most PV management guidelines [5], [6], [7]. Although GC sideeffects are of concern, GC e cacy has become increasingly recognized. In patients with diseases Page 3/13 responding to GCs, some always exhibit "GC resistance"; they require higher drug doses for longer times, or other immunosuppressive agents.

Subjects
We enrolled 15 patients treated between December 2018 and June 2019 as inpatients of the Department of Dermatology and Venereology of Ho Chi Minh City Hospital. All subjects were diagnosed with PV based on clinical and histological ndings or direct immuno uorescence. All exhibited active disease (new skin or mucosal blisters or erosions) and required a return to the initial dose of oral GCs (usually about 1 mg/kg). All subjects gave written informed consent; this study was approved by the ethics committee of the University of Medicine and Pharmacy of Ho Chi Minh City. Patients were monitored to the time of the rst tapering dose. The PDAI was used to assess disease severity in week 1 and every 1-2 weeks thereafter until the PDAI scores attained 50 and 25%. We recorded the time from the rst GC dose to the rst tapering dose and the total amount of GCs prescribed. Patients with systemic diseases, who could not receive optimal-dose GC (such as patients with liver or renal failure), those who received immunosuppressive agents other than GCs within the prior 6 months, and patients who did not adhere to treatment, were excluded.
Statistical analysis EPI DATA ver. 3.1 and STATA ver. 14.0 software were used to manage and analyze all data. Possible associations between NR3C1 polymorphisms and GC e cacy were explored using the Kruskal-Wallis and Wilcoxon rank-sum tests. The signi cance level was set to p < 0.05.

In silico analysis
An in silico tool was used to search for functional SNPs in linkage disequilibrium (LD) with other SNPs of the HapMap JPT and CHB populations [19]. ESE nder (http://rulai.cshl.edu/cgibin/tools/ESE3/ese nder.cgi?process=home) was used to evaluate whether SNPs lay in exonic splicing enhancers (ESEs). ESEs bind Ser/Arg-rich proteins (SR proteins) that play multiple roles prior to mRNA splicing.

Characteristics of the subjects
We enrolled 15 PV patients, 2 (13.33%) males and 13 (86.67%) females, of mean age 49.33 ± 16.10 years. The mean PDAI score on the rst hospital day was 44.2 (± 15.45). Five patients (33.3%) had moderate disease (PDAI score 15-45) and 10 (66.66%) severe disease (PDAI score > 45) [20]. The median GC commencement dose was 1.00 (0.98-1.02) mg/kg/day, the minimum 0.89 mg/kg/day, and the maximum 1.16 mg/kg/day. Ten subjects remained on their starting doses to the time of start of tapering but the remaining ve (all with severe disease) required higher doses. GC e cacy Table 1 lists the clinical responses to GCs in terms of PDAI score changes and GC doses. We recorded the times required to attain 50 and 25% of the initial PDAI scores; the median values were 23 and 36 days, respectively. One patient required only 7 days to attain the 25% PDAI score but another 91 days. The median time to tapering was 1 month, but one patient required more than 3 months.

Associations between NR3C1 SNPs and GC e cacy
We found signi cant positive correlations between SNPs rs17209237, rs11745958, and rs6196 ( Table 3).
The correlations between the total amounts of GC required and the genotypes of the six SNPs are shown in Fig. 1. For rs17209237, the total amount of GCs (mg), the time to the tapering dose (days), and the time to the PDAI 50% score of the genotype AA group were lower than those of the genotype non-AA group (p < 0.05), re ecting better GC e cacy in the AA group. For rs11745958, the total amount of GCs (mg), the time to the tapering dose (days), and the time to the PDAI 50% score of the genotype CC group were lower than those of the genotype non-CC group (p < 0.05), re ecting better GC e cacy in the CC group. For rs6196, the total amount of GCs (mg) and the time to the tapering dose (days) of the genotype AG group were lower than those of the genotype AA group (p < 0.05), re ecting better GC e cacy in the AG group.     SNPs were in LD (r 2 > 0.8) in the CHB and JPT populations. Of these, rs6194 was predicted to be functional by the SNPinfo Web Server. Although rs6196 was predicted to exhibit no function, rs6194 affected splicing; this may be of functional signi cance.

Discussion
We found correlations between three SNPs (rs17209237, rs117458958, and rs6196) and the clinical response to GCs. rs17209237 and rs117458958, located in introns, were rst shown to correlate with GC e cacy in PV patients by Fang et al. [17]. However, of the 17 SNPs for each position in LD (r 2 > 0.8) in the CHB and JPT populations, none was predicted to be functional; further research is needed.
We are the rst to report an association between rs6196 and GC e cacy in PV patients. Rs6196 AA patients scored higher on all four indices of GC e cacy. The rs6196 genotype signi cantly affected the time to tapering (days) and total amount of GCs (mg) (p = 0.0429 and 0.0208, respectively). Rs6196 AA patients with idiopathic nephrotic syndrome were at increased risk of steroid resistance [16]. Rs6196 lies in exon 9α of the transcriptionally active form of GCs [21]. Rs6196 SNP is a synonymous codon substitution. In silico, rs6196 was predicted to have no function. However, this SNP and rs6194 are located in ligand-binding domains important in terms of protein-protein interactions [22]. Niu et al. [23] investigated the functional impacts of common NR3C1 SNPs (including rs6196 and rs6194) in COS-1 cells; the protein expression levels were higher than that of the wild type.
None of the 15 PV patients exhibited the rs6189, rs6190, rs6195, or rs6198 SNPs associated with GC e cacy in patients with in ammatory bowel disease and rheumatoid arthritis [10], [11], [12], [15]; our sample size may have been too small. A Chinese study found signi cant relationships between rs17209237, rs11745958, rs33388, and rs7701443 and GC e cacy in PV patients [17]. We also identi ed these SNPs; the genotype and allele frequencies were similar to those in Chinese patients. We found that rs17209237 (AA) and rs11745958 (CC) were "protective" in terms of times to the 50 and 25% PDAI scores (days), GC dose required (mg/kg), time to tapering, and average daily GC dose (mg/kg/day). Fang et al. [17] found correlations between rs33388 and rs7701443 and GC e cacy. Although we also identi ed these polymorphisms, we failed to detect any correlation with GC e cacy.
Rs41423247 featured the genotypes CC and GC (53 and 47%). Rs41423247 (Bcl1) correlated with GC e cacy in patients with in ammatory bowel disease and rheumatoid arthritis [10], [11], [12], [15]. In Chinese PV patients, Fang et al. [17] failed to nd an SNP at this site; rs41423247 in PV patients is rst reported in the present study. However, we found no signi cant association between this SNP and any measure of GC e cacy.

Conclusion
SNPs in NR3C1 may affect GC e cacy in PV patients. We found signi cant correlations between rs17209237, rs11745958, and rs6196 and several aspects of the clinical response to GCs. Further study is required. Figure 1 Correlations between SNP genotypes and total amounts of GCs required (mg). A: A marginal correlation between rs17209237 genotypes and total GC amounts (p=0.0666). B, C: Signi cant associations between