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
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