Sequencing analysis of the human glucocorticoid receptor (NR3C1) gene in multiple sclerosis patients
Introduction
Accumulating evidence suggests that patients with multiple sclerosis (MS) display increased hypothalamic-pituitary-adrenal (HPA) axis activity, while the lack of their response to the combined Dex-CRH suppression test [5], [25] implicates an impaired negative feedback and a loss of sensitivity to glucocorticoids in the central nervous system. In addition, patients with MS may also demonstrate peripheral glucocorticoid (GC) resistance, as determined by decreased ex vivo dexamethasone inhibition of lipopolysaccharide (LPS)-induced interleukin (IL)-6 or tumor necrosis factor (TNF)-α production in peripheral blood lymphocytes compared with healthy individuals [4]. Notably, central and peripheral GC sensitivity have been associated with clinical MS disease characteristics and clinical improvement [4], [24], [25]. The human glucocorticoid receptor (hGR, hGRα and hGRβ isoforms) is a key regulator of the negative feedback mechanism and of the peripheral GC sensitivity, therefore a defect in the number and/or function of hGR could be a cause for the impaired HPA function and GC resistance in peripheral blood lymphocytes in patients with MS. Various mutations or polymorphism(s) of the hGR gene are reported to alter hGR activity, resulting in enhanced or diminished GC sensitivity [7]. However, no mutation data of the entire hGR (NR3C1) gene in patients with MS have been, so far, reported. Existing studies have investigated various hGR gene polymorphisms in MS patients and correlated these polymorphisms, with disease progression, susceptibility and aggressiveness. van Winsen et al. [23] have demonstrated that among the hGR gene polymorphisms N363S (rs6195), ER22/23EK (rs6189) and the BclI (rs41423247), the ER22/23EK polymorphism is associated with a more aggressive MS phenotype. In a subsequent study, none of the haplotypes of hGR gene (TthIIIΙ (rs10052957), ER22/23EK, BclI, N363S and GR-9β (rs6198) were associated with disease susceptibility, age at onset or onset type [24]; however, MS patients carrying the haplotype 6 (TthIIIΙ, ER2223EK, and GR-9β) have a more rapid disease progression, possibly due to the presence of ER22/23EK and not of GR-9β G and TthIIIΙ polymorphisms [24]. Finally, van Winsen et al. [22] showed that the variants N363S, BclI or ER22/23EK were not associated with GC sensitivity in patients with MS.
To further elucidate the role of hGR gene in MS, we investigated the presence of gene alterations in the entire coding region of hGR in MS patients of variable clinical status, such as clinical isolated syndrome (CIS), relapsing/remitting multiple sclerosis (RRMS) and secondary progressive multiple sclerosis (SPMS). We also investigated the association(s) of alterations in the hGR gene with severity of disease, response to glucocorticoid (GC) treatment and clinical improvement.
Section snippets
Patients
Sixty Caucasian Greek patients fulfilling the revised McDonald criteria for MS or possible MS [13] were included in the study.
Twenty patients (8 males, 12 females) had CIS suggestive of MS, while 22 (4 males, 18 females) had RRMS and 18 patients (9 males, 9 females) had SPMS disability that was assessed through the Expanded Disability Severity Scale (EDSS).
Thirty six out of sixty patients (14/20 CIS, 10/22 RRMS, 12/18 SPMS) were in relapse. These patients received corticosteroids intravenously
Results
Sequencing the coding region of the hGR gene did not reveal the presence of mutation(s) in any of the sixty patients with MS examined. However, three previously described polymorphisms were detected: i) the p.N363S (rs6195), ii) the p.N766N (rs6196) and iii) an intronic variant 16 bp upstream of the exon 5 acceptor splice site, c.1469-16G > T (rs6188).
Testing for heterozygote deficit by application of a score (U) test showed that MS population was in Hardy-Weinberg Equilibrium (p = 0.304).
Discussion
The current study was designed firstly to provide evidence for hGR gene alterations in patients with MS of different disease subtypes, such as CIS, RRMS and SPMS, and secondly to possibly correlate these hGR gene alterations with the severity of disease, response to GC therapy and subsequent clinical improvement. To the best of our knowledge, this is the first time that the entire hGR coding sequence was screened for the presence of mutations and polymorphisms in patients with MS. Utilizing
Declaration of interest
There is no conflict of interest.
Funding
We acknowledge the financial support from the National Kapodistrian University of Athens (Grant number 70/3/9601).
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