Cystatin C gene polymorphism is associated with the incidence of PHN in herpes zoster patients Zili Gong1, Chunmei Luo2,Li Wang1 ,Wenjie Zi1 # 1:Department of Neurology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400 037, China. 2:Department of Orthopedics, Xinqiao Hospital, Third Military Medical University, 183 Xinqiao S treet, Shapingba District,Chongqing 400037, China Address for correspondence and reprints: Dr. Wenjie Zi: Mailing address: Department of Neurology, Xinqiao Hospital, Third Military Medic al University, Chongqing, 400037, China; E-mail address: [email protected] . Suggested reviewer: 1. Cui W, Department of Anesthesiology, The First Affiliated Hospital, China Medical University, 110001 NO,155 of Nanjingbei Street, Heping District, Shenyang, Liaoning Province, People's Republic of China. [email protected] 2. Dr. Frank Booz. Department of Anesthesiology, Tulane University school of medicine, USA, [email protected] 1 ABSTRACT Background and aim: Cystatin C level in the cerebrospainal fluid has been reported to be associated with several pain conditions, but little is know about the gene polymorphism of Cystatin C in this field. This study is to investigate if the gene polymorphism of Cystatin C is related to the postherpetic neuralgia (PHN) in patients with herpes zoster. Methods: A total of 776 patients were enrolled into this study, of which 76 developed the PHN and 709 did not. Two gene polymorphism of Cystatin C, namely, the substitution of A for G at +148 (+148G/A ) and the substitution of G for C at -82 (–82G/C ) of the Cystatin C gene were determined. Results: We found that the +148G/A polymorphism of Cystatin C gene significantly affect the incidence of PHN in patients with herpes zoster. The +148AA genotype is associated with higher risk for developing PHN 6 month after the disappearance of skin lesion. Additionally, this genotype is related to the pain severity in these patients developing PHN. In contrast, the polymorphism at –82G/C was associated with the PHN incidence and the pain severity in these patients. Conclusion: The close association between the +148G/A gene polymorphism of Cystatin C gene and PHN susceptibility suggests that this genetic variant can be used as a molecular marker to predict PHN in patients with herpes zoster. Keywords: Cystatin C, polymorphism, postherpetic neuralgia, herpes zoster 2 Introduction Herpes zoster is a common disease characterized by belts of vesicular eruptions on the body, particularly on the face, chest and abdomen. Generally, the skin lesions heal within 4 to 6 weeks. However, in some patients (about 5–10%) develop postherpetic neuralgia (PHN) which means persistent pain over 4 weeks or more after healing of vesicular eruptions [1] [2]. PHN causes physical disability, emotional distress and interference with daily activities and sleep. Once established, postherpetic neuralgia is particularly difficult to treat and is often resistant to conventional analgesics [3]. The neural mechanism of PHN is still unclear. Some factors, including age, vesicular eruption severity and host immunological status are considered as risk factors for incidence of PHN [4] [5]. Only a few recent studies revealed that the host genetic background is also associated with PHN development [6] [7]. But the candidate gene associated with the PHN remains to be determined. Cystatin C is a 13-kDa protein that consists of 120 amino acids encoded by a 7.3-kb gene located on chromosome 20 [8] [9]. Cystatin C is reported to be closely associated with the incidence of death and cardiovascular events, kidney failure and neurodegeneration. In addition, the association between Cystatin C with pain condition was revealed. A study in rats demonstrated an increase in expression Cystatin C in the spinal cord during acute peripheral inflammation, suggesting this protein may be involved in the pathogenes is of persistent pain [10]. A subsequent study in women suggested that prolonged labor pain resulted in increased cystatin C concentrations in cerebrospinal fluid, and that this could be used as a biomarker for pain [10]. Also, cystatin C in cerebrospinal fluid is up-regulated in elderly patients with chronic osteoarthritis pain [11]. So far, more than 100 single-nucleotide polymorphisms (SNPs) have been identified in cystatin C gene. Among these SNPs, two gene polymorphism, namely, the substitution of A for G at +148 (+148G/A, rs1064039) and the substitution of G for C at -82 (–82G/C, rs5030707) have been reported to have functional significance. The +148G/A is located in the 5′-untranslated region of the gene and it can affect the promoter activity of cystatin C gene [12]. The +148G/A is located in the coding region that regulates the cystatin secretion [13]. 3 A previous study showed that recombinant cystatin C treatment inhibits herpes simplex virus (HSV) replication [14]. In this study, we found that the –82G/C polymorphism is closely related to the incidence of PHN in patents with herpes zoster. Compared to the measurement of cystatin protein level in cerebrospainal fluid (CSF), the SNP determination is simple, faster, more reliable and almost without any danger. Our study suggests that the cystatin C gene polymorphism at +148G/A may be used as a marker for the PHN development in patients with herpes zoster. 4 Methods Patient enrollment This study included consecutive patients diagnosed with herpes zoster and treated at our department from Jan 2006 to Jan 20012. All the patients were treated with anti-virus and if necessary, analegics. All patients were followed up for more than 6 months to determine whether they developed PHN or not. The diagnosis of PHN is defined as a constant pain that persists for at least more than 3 months after the disappearance of vesicular eruptions [15]. We excluded patients who did not respond to the standard anti-virus agent therapy (including Acyclovir, Valacyclovir or Famciclovir ) and failed to heal within 8 weeks, patients with skin bacterial infection and patients received immunosuppressive agents. Also patients with psychological conditions (such as depression and anxiety) and diabetic mellitus were strictly excluded from this study. So, a total of 776 patients were enrolled into this study, of which 76 developed the PHN and 709 did not. The degree of pain was checked using Barrow Neurological Institute scores (BNI score) [16]. DNA extraction and cystatin genotyping Genomic DNA was isolated using a QIAamp DNA Blood Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer's protocol. The exon 1 of cystatin C gene, including exon-intron boundaries, was amplified using the following primer pair: forward: 5'-GCGGGTCCTCTCTATCTAGC-3' and reverse: 5'-GCCGGGGCTTCGGACCTGCG-3'. The + 148 G/A polymorphism of cystatin C were determined using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) with SstII. In unclear samples, direct sequencing of exon 1 was (5'-ATCTAGCTCCAGCCTCTCG-3') and performed using both internal forward reverse (5'-TGCTGGCTTTGTTGTACTCG -3') primers. Statistical analysis The Fisher's exact or Chi-square test was used to compare the frequency distribution of age, gender, smoking status between patients with or without PHN, if appropriate. A chi square test was performed to assess Hardy–Weinberg equilibrium between two groups based on allelic and genotypic frequencies. We performed multivariate logistic regression analys is to estimate the 5 effect of cystatin C polymorphisms on development of PHN in the presence of other known prognostic factors, including age, sex, smoking status, skin lesion Duration and skin lesion location. The odds ratios (OR) and 95% confidence intervals (CIs) were calculated. Analyses were performed using the software SPSS 16.0 (SPSS Inc., Chicago, IL, USA). All P values were two-sided, and a P value < 0.05 was considered significant. 6 Results The characteristics of patients with and without PHN are listed in Table 1. The patients with PHN had a higher percentage of current smokers (P < 0.001). Besides, the patients with PHN were older than those without (P=0.016), while the sex distribution, skin lesion duration and location, pain severity were similar (both P > 0.05). Table 1 The characteristics of patients and controls Characteristics Mean age [17] Patients with PHN Patients without PHN n=67 n=709 58.3 (39.7-78.8) 55.4(37.2-79.1) P value 0.016 Sex Male 33 49.25% 351 49.51% Female 34 50.75% 358 50.49% Never smo ker 10 14.93% 231 32.58% Ever smoker 25 37.31% 311 43.86% Current s moker 32 47.76% 167 23.55% 0.934 Smoking status Skin lesion Durati on 13.4 (9-15) 12.4 (8-16) <0.001 0.064 (days) Skin lesion location Chest 29 43.28% 387 44.13% Abdominal 38 56.72% 490 55.87% The genotype distribution of cystatin C between patients with PHN and those without are presented in Table 2. The cystatin C genotype frequencies in the two groups were in Hardy–Weinberg equilibrium (both P > 0.05). Patients with PHN had a higher prevalence of +148AA genotype than those without (52.24% vs. 25.95%, P < 0.001). For allele analyses, Patients with PHN had higher +148A short allele frequency than those without PHN (69.40% vs. 51.76%, P = 0.036). Multivariate regression analyses showed that the +148AA genotype carriers had a significantly higher risk for PHN development after adjustments with age, sex, smoke and lesion location (adjusted OR = 3.361, P < 0.001). In contrast, the polymorphism at –82G/C variants were not significant different between patients with PHN and those without (both P > 0.05, Table 2). 7 0.078 Table 2 The genotype distribution of cystatin C gene polymorphism between Patients with PHN and those without Genotype –82G/C Patients Patients wi th PHN wi thout PHN n=67 n=709 Adjusted OR 95%CI Adjusted P GG 19 28.36% 206 29.06% 1.000 0.556 GC 31 46.27% 333 46.97% 1.009 0.546 1.833 0.916 CC 17 25.37% 170 23.98% 1.084 2.151 0.823 G 69 51.49% 745 52.54% 1.000 C 65 48.51% 673 47.46% 1.043 +148G/A GG 9 13.43% 159 22.43% 1.000 GA 23 34.33% 366 51.62% 1.110 0.502 2.453 0.776 AA 35 52.24% 184 25.95% 3.361 1.567 7.205 <0.001 G 41 30.60% 684 48.24% 1.000 A 93 69.40% 734 51.76% 2.114 0.732 1.486 0.881 1.443 3.097 0.036 OR, odds ratios ; 95% CI, 95 % confidence intervals Next we studied the association between the genotype distribution of cystatin C and the pain severity of PHN patients. According to their BNI scale, patients with PHN were divided into two groups: PHN with BNI grade (IV-V) and PHN with BNI grade scores (I-III). As shown in Table 3, the +148AA carriers had a significantly higher percentage of having high BNI scale than AG and AA carriers. Multivariate regression analyses revealed that the +148AA genotype is associated with pain severity after adjustments with age, sex, smoke and lesion location in patents with PHN (adjusted OR = 6.875, P = 0.012). 8 Table 3. The genotype distri bution of cystatin C gene pol ymorphism between patients having PHN stratified by the pain severity PHN with Genotype –82G/C +148G/A PHN with BNI BNI grade grade (IV-V ) scores(I-III) Adjusted OR 95%CI Adjusted P GG 8 26.67% 9 24.32% 1.000 GC 16 53.33% 19 51.35% 0.947 0.297 3.027 0.927 CC 6 20.00% 9 24.32% 0.750 0.184 3.057 0.688 G 32 53.33% 37 50.00% 1.000 C 28 46.67% 37 50.00% 1.000 0.442 1.730 0.701 GG 3 10.00% 12 32.43% 1.000 GA 15 50.00% 18 48.65% 3.333 0.791 14.052 0.091 AA 12 40.00% 7 18.92% 6.857 1.424 33.009 0.012 G 21 35.00% 42 56.76% 1.000 A 39 65.00% 32 43.24% 2.438 1.208 4.919 0.012 OR, odds ratios; 95% CI, 95 % confidence intervals 9 Discussion In the present study, we reported the +148G/A polymorphism of cystatin C gene significantly affects the incidence of PHN in patients with herpes zoster. The +148AA genotype is associated with higher risk for developing PHN 6 month after the disappearance of skin lesion. In addition, this genotype is related to the pain severity in these patients developing PHN. The close association between the +148G/A gene polymorphism of Cystatin C gene and PHN susceptibility suggests that this genetic variant can be used as a molecular marker to predict PHN in patients with herpes zoster. As a major complication of herpes zoster in human, PHN occurs particularly in elderly patients, which causes fatigue, sleep disturbance, anorexia, depression and impaired activities in daily living. Older age, vesicular eruption severity and acute herpes zoster severity are considered as risk factors for prolonged PHN. Previous studies reported the association of HLA-A*3303-B*4403-DRB1*1302 haplotype with PHN in Japanese population. The frequency of HLA-A*3303-B*4403-DRB1*1302 haplotype was significantly higher in the PHN patients than in the healthy controls (P = 0.0039) [18][19][20]. In Caucasian population, the apolipoprotein E (APOE) genotype is associated with the susceptibility to HPN [21]. The authors reported that the APOE- 4 4 frequency is significantly higher for shingles sufferers with PHN disorder [21]. Another study suggests that human cytochrome P4502D6 (CYP2D6) gene is a genetic predictor for PHN [6]. Our study is the first to report the positive association between the polymorphism of Cystatin C gene and development of PHN in Chinese patients. Changes of cystatin C expression level in animal models of neurodegeneration and human central neurological disorders have been reported [10] [22]. . The role of cystatin C gene expression in pain was reported in the lumbar spinal cord of rats following induction of acute hindlimb inflammation, where message RNA for cystatin C was significantly increased [22]. In a clinical study, Mannes et al. collected CSF from pregnant women and found that the cystatin C protein expression was significantly higher in the labor pain group than in the control group without labor pain [10]. Cystatin C prevents degeneration of rat nigral dopaminergic neurons [23]. However, 10 Eisenach et al. examined CSF samples from patients with labor pain, cesarean section, and chronic neuropathic pain, and observed no significant differences in cystatin C expression among these patient [24]. Thus, the role of cystatin C in pain is still to be determined. To date, the role of cystatin C gene polymorphism is mostly studied in central neurological conditions. Cystatin C gene polymorphisms are significantly associated with the likelihood of deep white matter hyperintensity detected by T2-weighted MR imaging. Substitution of A for G at +148 of the Cystatin C gene decreased the extracellular availability of cystatin C in vitro, which results in the activation of protease activity [25] [26]. our study is the first to report the role of cystatin C in peripheral neuropathy PHN. Our data shows that the +148G/A is the one associated with the incidence of PHN in patients with herpes zoster. Compared to the measurement of cystatin C protein level in CSF, the gene polymorphism determination is simple, faster, more reliable and almost without any danger. Our study suggests that the cystatin C gene polymorphism at +148G/A may be used as a marker for the PHN development. Several limitation of our study was the relatively small sample size, especially the patients with PHN. Secondly, this study enrolled only Chinese patients. Thus a duplicate study in a different ethical population is needed to validate our finding. 11 References [1] Watson PN. Postherpetic neuralgia. Clin Ev id (Online). 2010; 2010. 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