Psychiatry Research 219 (2014) 690–692

Contents lists available at ScienceDirect

Psychiatry Research journal homepage: www.elsevier.com/locate/psychres

Brief report

Association between VNTR polymorphism in promoter region of prodynorphin (PDYN) gene and heroin dependence Khyber Saify a, Iraj Saadat a,b, Mostafa Saadat a,b,n a b

Department of Biology, College of Sciences, Shiraz University, Shiraz 71454, Iran Institute of Biotechnology, Shiraz University, Shiraz, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 1 January 2014 Received in revised form 17 June 2014 Accepted 26 June 2014 Available online 5 July 2014

Within the core promoter region of prodynorphin (PDYN), a 68-bp sequence was found to occur as a polymorphism element, either singular or as tandemly repeated two, three or four times. We report the sequence of a novel allele (5-repeats). Our study revealed the existence of an ancestral nucleotide (A) at 29th position of the VNTR in human. In total, 442 heroin addicts and 799 controls were included in this study. The present findings revealed a male-limited association between VNTR polymorphism and heroin dependence risk. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Heroin dependence PDYN VNTR

1. Introduction Studies suggest that genetic factors have a strong impact on substance-dependence traits. Understanding of the molecular mechanisms of the etiology of heroin dependence is crucial for improving the prevention and treatment programs. Genes that could be risk factors for heroin dependence have not been consistently identified. Prodynorphin (PDYN; OMIM: 131340) is the precursor of the dynorphin related peptides. The PDYN plays an important role in several complex traits such as drug abuse (Schwarzer, 2009). Within the core promoter region of PDYN, a 68-bp sequence was found to occur as a polymorphism element, either singular or as tandemly repeated two, three or four times (Zimprich et al., 2000). This VNTR has an important role in regulation of PDYN expression. Each repeat contains a putative activator protein transcription factor binding site. Alleles with three or four repeats, have an approximately 50% greater level of transcriptional activity compared to alleles with 1–2 repeats (Zimprich et al., 2000; Rouault et al., 2011). There is one study reported that the 3 and 4 repeats of VNTR increase heroin dependence risk (Wei et al., 2011). However another study did not found any significant association (Zimprich et al., 2000). The aims of this study are to introduce sequence of a novel allele for the VNTR polymorphism and study the

n Corresponding author at: Department of Biology, College of Sciences, and Institute of Biotechnology, Shiraz University, Shiraz 71454, Iran. Fax: þ98 711 2280926. E-mail addresses: [email protected], [email protected] (M. Saadat).

http://dx.doi.org/10.1016/j.psychres.2014.06.048 0165-1781/& 2014 Elsevier Ireland Ltd. All rights reserved.

association between this polymorphism and heroin dependence susceptibility.

2. Materials and methods This study was performed in Shiraz (southern Iran) on 442 heroin addicts (42 females and 400 males) and 799 controls (137 females and 662 males). The patients were in methadone maintenance and all of them reported heroin as their primary drug of choice. Control individuals were blood donors, who declared that they did not suffer substance abuse. The mean 7S.D. of age of the patients and controls were 38.3 7 10.0 and 38.5 7 12.7 years, respectively (t ¼0.3, d.f. ¼1239, and P¼ 0.718). Considering the high heterogeneity of the Iranian population (Rafiee et al., 2010), the participants were selected from Persian Muslims (Caucasians) living in Shiraz (southern Iran). Informed consent was obtained from each subject before the study, which was approved by the institutional review board of our university. Genomic DNA was isolated from EDTA treated blood samples. Conventional PCR amplification was performed using following primers: forward primer 50 -AGCAATCAGAGGTTGAAGTTGGCAGC-30 and reverse primer 50 -GCACCAGGCGGTTAGGTAGAGTTGTC-30 . The PCR conditions consisted of an initial denaturation step of 94 1C for 5 min, followed by 30 cycles of 94 1C for 30 s, 62 1C for 45 s and 72 1C for 45 s, a final extension of 72 1C for 5 min. The PCR products were subjected to 2.5% agarose gel electrophoresis. Alleles containing 1, 2, 3, or 4 repeats, produced PCR amplicons of 379, 447, 515, and 583 bp, respectively. For quality control, 15% of randomly selected samples were repeated to verify genotyping results and 100% concordance was found. The alleles of the VNTR polymorphism were divided into two groups, L and H reflecting the relatively low expressed (1–2 repeats) and high expressed (3 and more repeats) alleles, respectively. PCR amplification for selected samples was performed and the 651 bp bands were purified by DNA gel extraction kit (geneJET Gel Eextraction kit) from Thermo Scientific, (K0691) and sequencing by BIONEER (Korea), the sequence results were compared to each other and to the genomic database, using the BLAST tool on the NCBI website.

K. Saify et al. / Psychiatry Research 219 (2014) 690–692

3. Results A new band (651 bp) above the band of the 4R allele was detected in two persons. According to the sequencing results, the newly identified sequences were the same and contained a new fragment similar to previously identified cis regulatory elements in the promoter region of the PDYN (Fig. 1). This allele was named 5R allele. This allele has very low frequency in our population (2 alleles from 2396 alleles). Table 1 shows the genotype distribution of the VNTR polymorphism between cases and controls. Table 2 shows the distribution of the genotypes according the L and H allele groups. The genotypic frequencies of the polymorphism in controls (χ2 ¼ 3.21, d.f.¼1, and P¼ 0.073) and patients (χ2 ¼0.26, d.f.¼1, and P¼ 0.607) were consistent with the Hardy–Weinberg equilibrium distribution. In overall, the HL genotypes increased the risk of heroin dependence, in comparison with the HH genotypes (OR ¼1.45, 95%CI: 1.13–1.87, and P¼ 0.003). However, there is no significant association between the LL genotypes and the risk of heroin dependence (OR ¼1.42, 95%CI: 0.97–2.09, and P ¼0.071). Considering that men are more vulnerable to addiction when compared to women, the analyses were performed on each gender group separately. Among male subjects, the HL (OR ¼1.44, 95%CI: 1.10– 1.88, and P¼ 0.007) and LL (OR ¼1.54, 95%CI: 1.02–2.31, and P ¼0.037) genotypes increased the risk of heroin dependence, in comparison with the HH genotypes. The risk of heroin dependence increased as a function of number of the L allele (χ2 ¼7.87, and P ¼0.005). Among females, the HL and LL genotypes had no significant effect on heroin addiction risk, in comparison with the HH genotypes (Table 2).

4. Discussion According to sequencing results, the newly identified sequences were the same and contained a new fragment similar to previously identified cis regulatory elements in the promoter region of the PDYN. Therefore, we identified five kinds of alleles, 1–5 copies of the 68-bp tandem repeat, of the polymorphism in the promoter region of the PDYN. It should be noted that previously, Rouault et al. (2011), reported that they found only one 5R allele among their subjects,

691

but they did not report the sequence of the 5R allele. In Fig. 1, the gray nucleotides shows single nucleotide polymorphism on repeats of the study VNTR that reported previously (Rockman et al., 2005; Rouault et al., 2011). However, the selected nucleotide by red color (29th nucleotide) indicates a new polymorphism in the VNTR. To the best of our knowledge, this polymorphism was not reported previously. It should be noted that during the course of evolution, at this position, “A” is highly conserved among Chimpanzee, Bonobo, Gorilla, Orangutan, Baboon, Pig-tailed macaque and Rhesus monkey (Rockman et al., 2005). Our present study revealed the existence of this ancestral nucleotide (“A” at 29th position) in human. At present time, the biological significance of this polymorphism is not clear. The L alleles have a lower promoter activity of the PDYN compared with the H alleles (Zimprich et al., 2000; Rouault et al., 2011). The present study shows that the L allele is a genetic risk factor for vulnerability to heroin dependence. It should be noted that the male-limited associations between genetic polymorphisms and other traits were reported. For example, the DRD2 A1 allele associated with alcohol dependence only in male subjects (Limosin et al., 2002). Few studies investigated the association between heroin dependence and PDYN VNTR polymorphism with inconsistent results (Zimprich et al., 2000; Wei et al., 2011).

Table 1 Genotypic distribution of PDYN VNTR polymorphism in control and heroin dependence cases stratified by gender. Genotypes/Alleles

1/1 1/2 1/3 2/2 2/3 2/4 2/5 3/3 3/4 3/5 4/4

Males

Females

Controls

Cases

Controls

Cases

1 6 6 62 252 8 0 302 23 1 1

0 3 2 49 175 10 1 152 8 0 0

0 1 2 14 42 6 0 68 4 0 0

0 0 0 2 19 1 0 18 2 0 0

Fig. 1. Sequence of 68-bp VNTR in promoter region of the human PDYN. The putative recognition sites for the AP-1 transcription factor were shown (underline) within the 68 bp repeat element. The selected nucleotides by yellow color are previously identified polymorphisms. The selected red color nucleotide showed the newly identified polymorphism. These sequences also show the 5 copies of cis regulatory elements in the promoter region of PDYN (4 previously identified and 1 newly identified elements). The primers used for amplifications were shown (underline).

692

K. Saify et al. / Psychiatry Research 219 (2014) 690–692

Cases N (%)

OR

95% CI

P-value

399 (49.9) 316 (39.6) 84 (10.5)

180 (40.7) 208 (47.1) 54 (12.2)

1.0 1.45 1.42

– 1.13–1.87 0.97–2.09

– 0.003 0.071

1114 (69.7) 484 (30.3)

568 (64.3) 316 (35.7)

1.0 1.28

– 1.07–1.52

– 0.005

(α ¼0.05), d.f. ¼ 2, Lambda ¼9.64, and an effect size of 0.2; a minimum sample of 241 females would be necessary. Considering that in the present study 179 females had participated; when we stratified our participants by gender, the present analyses on females may have been statistically underpowered; 4) The present findings may not be generalizable, because our participants belonged to the same population. Considering the fact that ethnicity may influence the observed associations in multifactorial diseases (Saadat and Ansari-Lari, 2009), replication of this study in other countries is recommended.

327 (49.4) 266 (40.2) 69 (10.4)

160 (40.0) 188 (47.0) 52 (13.0)

1.0 1.44 1.54

– 1.10–1.88 1.02–2.31

– 0.007 0.037

Acknowledgments

920 (69.5) 404 (30.5)

508 (63.5) 292 (36.5)

1.0 1.30

– 1.08–1.57

– 0.004

72 (52.6) 50 (36.5) 15 (10.9)

20 (47.6) 20 (47.6) 2 (4.8)

1.0 1.44 0.48

– 0.70–2.95 0.10–2.27

– 0.319 0.355

194 (70.8) 80 (29.2)

60 (71.4) 24 (28.6)

1.0 0.97

– 0.56–1.66

– 0.912

Table 2 Association between PDYN VNTR polymorphism and risk of heroin dependence. Genotypes/ Alleles Both genders HH HL LL Alleles H L Males HH HL LL Alleles H L Females HH HL LL Alleles H L

Controls N (%)

Considering the fact that male-limited association between this polymorphism and risk of heroin dependence, the discrepancy between studies, at least in part might be interpreted by the sex proportion of the participants (cases and controls). Finally, the present study has the following limitations: 1) The associations found could be related more to the progression and severity of heroin dependence than vulnerability to this disorder, because patients had a long history of heroin use; 2) The malelimited association must be considered as a preliminary finding, because more males than females participated in the present study; 3) Using the GPOWER software (version 3.1.3), to detect a real difference in genotypic frequency with a power of 0.80

The authors are indebted to the participants for their close cooperation. This study was supported by Shiraz University. References Limosin, F, Gorwood, P, Loze, JY, et al., 2002. Male limited association of the dopamine receptor D2 gene TaqI A polymorphism and alcohol dependence. American Journal of Medical Genetics 112, 343–346. Rafiee, L, Saadat, I, Saadat, M, 2010. Glutathione S-transferase genetic polymorphisms (GSTM1, GSTT1 and GSTO2) in three Iranian populations. Molecular Biology Reports 37, 155–158. Rockman, MV, Hahn, MW, Soranzo, N, et al., 2005. Ancient and recent positive selection transformed opioid cis-regulation in humans. PLoS Biology 3, e387. Rouault, M, Nielsen, DA, Ho, A, et al., 2011. Cell-specific effects of variants of the 68-base pair tandem repeat on prodynorphin gene promoter activity. Addiction Biology 16, 334–346. Saadat, M, Ansari-Lari, M, 2009. Polymorphism of XRCC1 (at codon 399) and susceptibility to breast cancer, a meta-analysis of the literatures. Breast Cancer Research Treatment 115, 137–144. Schwarzer, C, 2009. 30 years of dynorphins - new insights on their functions in neuropsychiatric diseases. Pharmacology & Therapeutics 123, 353–370. Wei, SG, Zhu, YS, Lai, JH, et al., 2011. Association between heroin dependence and prodynorphin gene polymorphisms. Brain Research Bulletin 85, 238–242. Zimprich, A, Kraus, J, Woltje, M, et al., 2000. Allelic variation in the human prodynorphin gene promoter alters stimulus-induced expression. Journal of Neurochemistry 74, 472–477.

Association between VNTR polymorphism in promoter region of prodynorphin (PDYN) gene and heroin dependence.

Within the core promoter region of prodynorphin (PDYN), a 68-bp sequence was found to occur as a polymorphism element, either singular or as tandemly ...
1MB Sizes 0 Downloads 5 Views