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ANKK1 gene polymorphisms in forensic autopsies of methamphetamine intoxication fatalities
Legal Medicine 33 (2018) 6–9
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Short Communication
DRD2/ANKK1 gene polymorphisms in forensic autopsies of methamphetamine intoxication fatalities
T
⁎
Aya Matsusuea, , Takaki Ishikawab, Tomoya Ikedab, Naoto Tanib, Hisatomi Arimac, Brian Watersa, Kenji Haraa, Masayuki Kashiwagia, Mio Takayamaa, Natsuki Ikematsua, Shin-ichi Kuboa a
Department of Forensic Medicine, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, Osaka 545-8585, Japan c Department of Preventive Medicine and Public Health, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fatal methamphetamine intoxication DRD2/ANKK1 Taq1A –141C Ins/Del
Dopamine D2 receptor/ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1) gene polymorphisms have been associated with responses to psychotropic drugs and addiction. We analyzed two DRD2/ANKK1 polymorphisms, Taq1A and –141C Ins/Del, in 37 fatal methamphetamine (MA) intoxication cases and 235 control cases in which MA and psychotropic drugs were not detected. The association among polymorphism, cause of death, and cerebrospinal fluid (CSF) dopamine concentration was evaluated. The Taq1A polymorphism distribution in the fatal MA intoxication cases differed from in the controls (P = 0.030) with a significantly high A1/A1 + A1/A2 genotype frequency. No significant associations were observed between –141C Ins/Del polymorphisms and MA intoxication cases or between DRD2/ANKK1 polymorphisms and CSF dopamine concentrations. Our findings suggest that the DRD2/ANKK1 Taq1A polymorphism is associated with susceptibility to fatal MA intoxication.
1. Introduction Forensic autopsy often identifies methamphetamine (MA) as the cause of fatal intoxication. MA is a central nervous system stimulant, and its dependence is a serious public health issue worldwide. Individual variability in response to drugs has also been widely reported [1]. MA blood concentrations vary widely in cases of fatal MA intoxication; however, these levels can be lower than the lethal concentration and still be fatal. Therefore, the effects of a MA depend on the genetic differences of the individuals. The dopamine D2 receptor (DRD2), a key receptor that mediates dopamine-associated brain functions, is targeted by all antipsychotic drugs [2]. Many polymorphisms of the DRD2 gene have been identified [3,4]. Taq1A and –141C Ins/Del are located in the DRD2/ANKK1 gene cluster [5] and the 5′-flanking region of the DRD2 gene [6], respectively. The effects of these polymorphisms on response to antipsychotic drugs have been previously investigated [7,8], and significant associations between the Taq1A polymorphism and substance dependence have been reported [9–12]. In this study, we analyzed two DRD2/ANKK1 gene polymorphisms, Taq1A and –141C Ins/Del, in autopsies of fatal MA intoxication cases
⁎
and control cases where MA and psychotropic drugs were not detected. We also examined the influence of DRD2/ANKK1 gene polymorphisms on fatal MA intoxication and dopamine concentrations in cerebrospinal fluid (CSF). 2. Materials and methods 2.1. Autopsy cases The cases were divided into the following two groups, as shown in Table 1: fatal MA intoxication and controls in which neither MA nor psychotropic drugs were involved. In addition to MA, psychotropic, antihistamine, and/or antitussive drugs were also involved in fatal MA intoxication cases. MA intoxication was defined as death caused by MA without pathological evidence of any other cause. 2.2. Analytical procedures The drugs were analyzed by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry. Blood samples were collected from the right cardiac chambers, and serum was
Corresponding author. E-mail address: [email protected] (A. Matsusue).
https://doi.org/10.1016/j.legalmed.2018.04.005 Received 14 November 2017; Received in revised form 20 March 2018; Accepted 20 April 2018 Available online 22 April 2018 1344-6223/ © 2018 Elsevier B.V. All rights reserved.
Legal Medicine 33 (2018) 6–9
A. Matsusue et al.
Table 1 Case profiles. Case
Number of cases (male/female)
Age (median)
Postmortem interval (day) (median; h)
MA concentration in RHB (µmol/ 100 mL) (median)
AMP concentration in RHB (µmol/ 100 mL) (median)
Fatal methamphetamine intoxication
37 (29/8) 235 (145/90)
24–62 (43) 0–94 (57)
< 0.5–8 (34) < 0.5–14 (28)
0.044–8.00 (0.64)
0–1.20 (0.10)
−
−
Control
Control: Cases where MA and psychotropic drugs were not detected. Abbreviations: MA, methamphetamine; AMP, amphetamine; RHB, right heart blood.
MA intoxication cases or control cases. The genotype frequencies of Taq1A and -141C Ins/Del polymorphisms were compared between fatal MA intoxication and control cases using genotype, dominant, recessive, and allele models. The frequencies of Taq1A polymorphisms in fatal MA intoxication cases differed from those in the control cases. The frequency of the A1/A1 + A1/A2 and A2/A2 genotypes were higher and lower, respectively, in fatal MA intoxication cases than in control cases (p = 0.030). The genotype and allele frequencies of the –141C Ins/Del polymorphisms did not differ significantly between fatal MA intoxication and control cases. The effects of the DRD2/ANKK1 genotype on CSF dopamine concentration are shown in Table 3. The median CSF dopamine concentrations were 33.0 ng/mL in the fatal MA intoxication cases and 17.1 ng/mL in the control cases. As the minor allele frequency was low, the associations between CSF dopamine concentrations and the A1/ A2 + A1/A1 versus the A2/A2 genotypes of Taq1A, and Del/Ins + Del/ Del versus Ins/Ins genotypes of –141C Ins/Del polymorphism were examined. No significant associations between CSF dopamine concentration and Taq1A or –141C Ins/Del genotype were found in any of the autopsy case groups.
separated immediately by centrifugation. CSF (approximately 2–10 mL) was collected using aseptic syringe from the great cistern in the base of the brain at the time of opening of the skull. CSF was subsequently stored at −20 °C until analysis. Dopamine concentrations were measured as previously described [13] from 24 MA intoxication cases and 73 controls within 4 days of death. The deproteinizing solution was prepared by diluting 60% perchloric acid solution to 6% with purified water. This deproteinized solution (0.3 mL) was mixed well with 0.6 mL of the CSF, to which 120 µL of 1.5 M sodium acetate was added, followed by centrifugation and separation of the supernatant, resulting in the sample for further analysis. Next, 500 µL of the deproteinized sample was injected for high performance liquid chromatography and dopamine was measured using the CA test “TOSOH” (Tosoh, Tokyo, Japan) following the manufacturer’s instructions. We did not include hospital death patients who underwent resuscitation. 2.3. Genotyping Peripheral blood was obtained at autopsy, and genomic DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The DRD2/ANKK1 Taq1A polymorphisms were assessed using a Custom TaqMan® SNP Genotyping Assay (Applied Biosystems, Foster City, CA). Polymerase chain reaction (PCR) was performed using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, USA) in 25-μL reaction aliquots containing 11.25 μL of genomic DNA (1–20 ng), 12.5 μL of 2 × TaqMan® genotyping master mix, and 1.25 μL of 40 × TaqMan® SNP genotyping assay mix. The reactions were performed with one cycle at 95 °C for 10 min, 40 cycles at 92 °C for 15 s and 58 °C for 90 s. Allelic discrimination was carried out using CFX Manager software (Bio-Rad, Hercules, USA). The DRD2/ANKK1 –141C Ins/Del polymorphisms were determined as described by Grzywacz et al. [14]. Genotypes of several samples were confirmed by direct sequencing.
4. Discussion This case-control study investigated the association between DRD2/ ANKK1 polymorphisms and fatal MA intoxication. There were significant differences in Taq1A polymorphism genotype frequency between the fatal MA intoxication cases and control cases. In the dominant model, the A1/A1 + A1/A2 genotype was more likely to be found in fatal MA intoxication cases than the A2/A2 genotype (OR = 2.47; 95%CI = 1.12–5.46). The Taq1A polymorphism is located 10 kb downstream of the DRD2 gene and results in a Glu-to-Lys (Glu713Lys) substitution within ANKK11 [5]. The A1 allele of the Taq1A polymorphism is associated with reduced brain DRD2 density [15]. The Taq1A polymorphism was not found within the DRD2 coding region; however, it is likely located in a regulatory region of the DRD2 gene, or linked to the DRD2 functional polymorphism or regulatory region. The Taq1A polymorphism has been reported as associated with decreased aggregation of the ANKK1 protein [16], and it may influence ANKK1 activity and subsequently affect DRD2 expression or the DRD2 signal transduction pathway. The association between Taq1A polymorphism and neuropsychiatric disorders and addiction has been well studied. Meta-analyses found that the Taq1A A1 allele was associated with risk of attention deficit hyperactivity disorder [17] and the Taq1A polymorphism significantly decreased the risk of schizophrenia susceptibility in Asian populations [18]. The Taq1A A1 allele has also been reported to increase the risk of addictive behaviors including alcohol [10,12], cocaine [11], opioid [9], and cannabinoid [19] dependence. The Taq1A A1 carriers (A1/ A1 + A1/A2) were found to be more frequent in a group of MA abusers than in healthy controls [20]; however, other studies did not find an association between Taq1A polymorphism and MA dependence [21,22] or MA abuse [23,24]. Ujike et al. reported that the A1/A1 genotype was
2.4. Statistical analysis Hardy–Weinberg equilibrium analysis was performed using the SNPAlyze software Ver. 8.0.2 (Dynacom, Chiba, Japan), and differences in genotype and allele distribution were assessed using Fisher's exact test. The CSF dopamine concentrations and DRD2/ANKK1 genotypes were compared via nonparametric Wilcoxon rank-sum tests using SAS statistical software (SAS Enterprise Guide, Cary, NC, USA). Differences were considered significant at p-values < 0.05. 3. Results Thirty-seven fatal MA intoxication and 235 control cases were genotyped for both DRD2/ANKK1 polymorphisms. Table 2 shows the results of the allele and genotype frequency analyses for Taq1A and –141C Ins/Del polymorphisms, and the associations of Taq1A and –141C Ins/Del genotypes with fatal MA intoxication. There were no significant deviations from the Hardy–Weinberg equilibrium in fatal 7
Legal Medicine 33 (2018) 6–9
A. Matsusue et al.
Table 2 Frequencies of Taq1A and –141C Ins/Del genotypes in fatal methamphetamine intoxication and control cases, and their association with fatal methamphetamine intoxication. C: cases where methamphetamine and psychotropic drugs were not detected, MA: fatal methamphetamine intoxication cases. SNP
C
Taq1A
Genotype A1/A1 A1/A2 A2/A2 Dominant A2/A2 A1/A1 + A1/A2 Recessive A2A2 + A1/A2 A1/A1 Allele A1 A2
–141C Ins/Del
Genotype Ins/Ins Ins/Del Del/Del Dominant Ins/Ins Del/Del + Ins/Del Recessive Ins/Ins + Ins/Del Del/Del Allele Ins Del
MA
Taq1A A2/A2 A1/A2 + A1/A1
(%)
n
(%)
30 101 104
(12.77) (42.98) (44.26)
5 23 9
(13.51) (62.16) (24.32)
0.050
104 131
(44.26) (55.74)
9 28
(24.32) (75.68)
− 0.030
1.00 (reference) 2.47 (1.12–5.46)
205 30
(87.23) (12.77)
32 5
(86.49) (13.51)
− 0.798
1.00 (reference) 1.07 (0.39–2.95)
161 309
(34.26) (65.74)
33 41
(44.59) (55.41)
− 0.091
1.00 (reference) 0.65 (0.39–1.06)
158 65 12
(67.23) (27.66) (5.11)
26 10 1
(70.27) (27.03) (2.70)
1.000
158 77
(67.23) (32.77)
26 11
(70.27) (29.73)
− 0.851
1.00 (reference) 0.87 (0.41–1.85)
223 12
(94.89) (5.11)
36 1
(97.30) (2.70)
− 1.000
1.00 (reference) 0.52 (0.07–4.09)
381 89
(81.06) (18.94)
62 12
(83.78) (16.22)
− 0.633
1.00 (reference) 0.83 (0.43–1.60)
of relapse of substance abuse such as of MA [29]. Although the effects of the Taq1A polymorphism on fatal MA intoxication are not clear, the Taq1A polymorphism likely affects dopamine signaling and MA dependence. The frequencies of –141C Ins/Del polymorphisms in fatal MA intoxication cases did not differ significantly. The –141C Del-positive genotypes, Del/Del and Del/Ins, were risk factors for the rapid-onset of methamphetamine psychosis within 3 years after beginning abuse [22]. However, the –141C Ins/Del polymorphism was not associated with fatal MA intoxication in this study. Thus, the –141C Ins/Del polymorphism is likely not related to death by MA intoxication. The CSF dopamine concentration was not associated with Taq1A or –141C Ins/Del polymorphisms in any of the study cases. Postmortem serum, pericardial fluid, CSF, and urine dopamine concentrations have previously been demonstrated to be higher in cases of fatal intoxication, such as by MA and psychotropic drugs, than in cases with other causes of death [13,30]. However, there was no significant association between Taq1A or –141C Ins/Del polymorphisms and CSF dopamine concentrations in MA intoxication cases. These results suggest that the Taq1A and –141C Ins/Del polymorphisms did not affect CSF dopamine concentrations in our fatal MA intoxication cases. This study was limited by its small sample size. Further studies are required to evaluate the effects of the DRD2/ANKK1 genotype on drug intoxication with a larger sample of fatal MA intoxication cases.
–141C Ins/Del Ins/Ins Del/Ins + Del/Del
MA
n
Median (range)
P-value
n
Median (range)
P-value
31
18.8 (8.07–152) 15.8 (0.005–265)
0.262
8
25.1 (5.22–63.2) 36.0 (1.72–198)
0.878
18.5 (1.10–265) 12.3 (0.005–76.5)
0.217
33.0 (1.72–198) 28.6 (3.72–127)
0.943
42
44 29
16
19 5
Odds ratio (95% CI)
n
Table 3 Dopamine concentrations (ng/ml) in autopsy specimens of cerebrospinal fluid from fatal methamphetamine intoxication and control cases. C: cases where methamphetamine and psychotropic drugs were not detected, MA: fatal methamphetamine intoxication cases. C
P-value
a negative risk factor for prolonged MA psychosis after therapy and complication of spontaneous relapse after remission [22]. The A1 allele of Taq1A decreases the DRD2 density, and individuals with a lower DRD2 density have a lower risk for prolonged MA psychosis and spontaneous relapse [22]. Striatal DRD2 binding potential was lower among carriers of the A1 allele [25]. The Taq1A A1 allele was associated with fatal MA intoxication in this study, but it is unclear how reduced DRD2 density affects fatal MA intoxication. The primary mechanism of action of MAs is on the dopamine system in the brain, resulting in high abuse potential and neurotoxic effects [26]. MA, alcohol, and cocaine abusers have significantly lower than normal DRD2 availability, and the consequent effect on neuronal function in the orbitofrontal cortex has been associated with loss of control and compulsive drug intake in addicted subjects [27]. The A1 Taq1A allele has been described as a reward gene [28], and Blum K et al. found that low DRD2 density and polymorphism were associated with an increased risk
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Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Short Communication
DRD2/ANKK1 gene polymorphisms in forensic autopsies of methamphetamine intoxication fatalities
T
⁎
Aya Matsusuea, , Takaki Ishikawab, Tomoya Ikedab, Naoto Tanib, Hisatomi Arimac, Brian Watersa, Kenji Haraa, Masayuki Kashiwagia, Mio Takayamaa, Natsuki Ikematsua, Shin-ichi Kuboa a
Department of Forensic Medicine, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, Osaka 545-8585, Japan c Department of Preventive Medicine and Public Health, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fatal methamphetamine intoxication DRD2/ANKK1 Taq1A –141C Ins/Del
Dopamine D2 receptor/ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1) gene polymorphisms have been associated with responses to psychotropic drugs and addiction. We analyzed two DRD2/ANKK1 polymorphisms, Taq1A and –141C Ins/Del, in 37 fatal methamphetamine (MA) intoxication cases and 235 control cases in which MA and psychotropic drugs were not detected. The association among polymorphism, cause of death, and cerebrospinal fluid (CSF) dopamine concentration was evaluated. The Taq1A polymorphism distribution in the fatal MA intoxication cases differed from in the controls (P = 0.030) with a significantly high A1/A1 + A1/A2 genotype frequency. No significant associations were observed between –141C Ins/Del polymorphisms and MA intoxication cases or between DRD2/ANKK1 polymorphisms and CSF dopamine concentrations. Our findings suggest that the DRD2/ANKK1 Taq1A polymorphism is associated with susceptibility to fatal MA intoxication.
1. Introduction Forensic autopsy often identifies methamphetamine (MA) as the cause of fatal intoxication. MA is a central nervous system stimulant, and its dependence is a serious public health issue worldwide. Individual variability in response to drugs has also been widely reported [1]. MA blood concentrations vary widely in cases of fatal MA intoxication; however, these levels can be lower than the lethal concentration and still be fatal. Therefore, the effects of a MA depend on the genetic differences of the individuals. The dopamine D2 receptor (DRD2), a key receptor that mediates dopamine-associated brain functions, is targeted by all antipsychotic drugs [2]. Many polymorphisms of the DRD2 gene have been identified [3,4]. Taq1A and –141C Ins/Del are located in the DRD2/ANKK1 gene cluster [5] and the 5′-flanking region of the DRD2 gene [6], respectively. The effects of these polymorphisms on response to antipsychotic drugs have been previously investigated [7,8], and significant associations between the Taq1A polymorphism and substance dependence have been reported [9–12]. In this study, we analyzed two DRD2/ANKK1 gene polymorphisms, Taq1A and –141C Ins/Del, in autopsies of fatal MA intoxication cases
⁎
and control cases where MA and psychotropic drugs were not detected. We also examined the influence of DRD2/ANKK1 gene polymorphisms on fatal MA intoxication and dopamine concentrations in cerebrospinal fluid (CSF). 2. Materials and methods 2.1. Autopsy cases The cases were divided into the following two groups, as shown in Table 1: fatal MA intoxication and controls in which neither MA nor psychotropic drugs were involved. In addition to MA, psychotropic, antihistamine, and/or antitussive drugs were also involved in fatal MA intoxication cases. MA intoxication was defined as death caused by MA without pathological evidence of any other cause. 2.2. Analytical procedures The drugs were analyzed by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry. Blood samples were collected from the right cardiac chambers, and serum was
Corresponding author. E-mail address: [email protected] (A. Matsusue).
https://doi.org/10.1016/j.legalmed.2018.04.005 Received 14 November 2017; Received in revised form 20 March 2018; Accepted 20 April 2018 Available online 22 April 2018 1344-6223/ © 2018 Elsevier B.V. All rights reserved.
Legal Medicine 33 (2018) 6–9
A. Matsusue et al.
Table 1 Case profiles. Case
Number of cases (male/female)
Age (median)
Postmortem interval (day) (median; h)
MA concentration in RHB (µmol/ 100 mL) (median)
AMP concentration in RHB (µmol/ 100 mL) (median)
Fatal methamphetamine intoxication
37 (29/8) 235 (145/90)
24–62 (43) 0–94 (57)
< 0.5–8 (34) < 0.5–14 (28)
0.044–8.00 (0.64)
0–1.20 (0.10)
−
−
Control
Control: Cases where MA and psychotropic drugs were not detected. Abbreviations: MA, methamphetamine; AMP, amphetamine; RHB, right heart blood.
MA intoxication cases or control cases. The genotype frequencies of Taq1A and -141C Ins/Del polymorphisms were compared between fatal MA intoxication and control cases using genotype, dominant, recessive, and allele models. The frequencies of Taq1A polymorphisms in fatal MA intoxication cases differed from those in the control cases. The frequency of the A1/A1 + A1/A2 and A2/A2 genotypes were higher and lower, respectively, in fatal MA intoxication cases than in control cases (p = 0.030). The genotype and allele frequencies of the –141C Ins/Del polymorphisms did not differ significantly between fatal MA intoxication and control cases. The effects of the DRD2/ANKK1 genotype on CSF dopamine concentration are shown in Table 3. The median CSF dopamine concentrations were 33.0 ng/mL in the fatal MA intoxication cases and 17.1 ng/mL in the control cases. As the minor allele frequency was low, the associations between CSF dopamine concentrations and the A1/ A2 + A1/A1 versus the A2/A2 genotypes of Taq1A, and Del/Ins + Del/ Del versus Ins/Ins genotypes of –141C Ins/Del polymorphism were examined. No significant associations between CSF dopamine concentration and Taq1A or –141C Ins/Del genotype were found in any of the autopsy case groups.
separated immediately by centrifugation. CSF (approximately 2–10 mL) was collected using aseptic syringe from the great cistern in the base of the brain at the time of opening of the skull. CSF was subsequently stored at −20 °C until analysis. Dopamine concentrations were measured as previously described [13] from 24 MA intoxication cases and 73 controls within 4 days of death. The deproteinizing solution was prepared by diluting 60% perchloric acid solution to 6% with purified water. This deproteinized solution (0.3 mL) was mixed well with 0.6 mL of the CSF, to which 120 µL of 1.5 M sodium acetate was added, followed by centrifugation and separation of the supernatant, resulting in the sample for further analysis. Next, 500 µL of the deproteinized sample was injected for high performance liquid chromatography and dopamine was measured using the CA test “TOSOH” (Tosoh, Tokyo, Japan) following the manufacturer’s instructions. We did not include hospital death patients who underwent resuscitation. 2.3. Genotyping Peripheral blood was obtained at autopsy, and genomic DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The DRD2/ANKK1 Taq1A polymorphisms were assessed using a Custom TaqMan® SNP Genotyping Assay (Applied Biosystems, Foster City, CA). Polymerase chain reaction (PCR) was performed using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, USA) in 25-μL reaction aliquots containing 11.25 μL of genomic DNA (1–20 ng), 12.5 μL of 2 × TaqMan® genotyping master mix, and 1.25 μL of 40 × TaqMan® SNP genotyping assay mix. The reactions were performed with one cycle at 95 °C for 10 min, 40 cycles at 92 °C for 15 s and 58 °C for 90 s. Allelic discrimination was carried out using CFX Manager software (Bio-Rad, Hercules, USA). The DRD2/ANKK1 –141C Ins/Del polymorphisms were determined as described by Grzywacz et al. [14]. Genotypes of several samples were confirmed by direct sequencing.
4. Discussion This case-control study investigated the association between DRD2/ ANKK1 polymorphisms and fatal MA intoxication. There were significant differences in Taq1A polymorphism genotype frequency between the fatal MA intoxication cases and control cases. In the dominant model, the A1/A1 + A1/A2 genotype was more likely to be found in fatal MA intoxication cases than the A2/A2 genotype (OR = 2.47; 95%CI = 1.12–5.46). The Taq1A polymorphism is located 10 kb downstream of the DRD2 gene and results in a Glu-to-Lys (Glu713Lys) substitution within ANKK11 [5]. The A1 allele of the Taq1A polymorphism is associated with reduced brain DRD2 density [15]. The Taq1A polymorphism was not found within the DRD2 coding region; however, it is likely located in a regulatory region of the DRD2 gene, or linked to the DRD2 functional polymorphism or regulatory region. The Taq1A polymorphism has been reported as associated with decreased aggregation of the ANKK1 protein [16], and it may influence ANKK1 activity and subsequently affect DRD2 expression or the DRD2 signal transduction pathway. The association between Taq1A polymorphism and neuropsychiatric disorders and addiction has been well studied. Meta-analyses found that the Taq1A A1 allele was associated with risk of attention deficit hyperactivity disorder [17] and the Taq1A polymorphism significantly decreased the risk of schizophrenia susceptibility in Asian populations [18]. The Taq1A A1 allele has also been reported to increase the risk of addictive behaviors including alcohol [10,12], cocaine [11], opioid [9], and cannabinoid [19] dependence. The Taq1A A1 carriers (A1/ A1 + A1/A2) were found to be more frequent in a group of MA abusers than in healthy controls [20]; however, other studies did not find an association between Taq1A polymorphism and MA dependence [21,22] or MA abuse [23,24]. Ujike et al. reported that the A1/A1 genotype was
2.4. Statistical analysis Hardy–Weinberg equilibrium analysis was performed using the SNPAlyze software Ver. 8.0.2 (Dynacom, Chiba, Japan), and differences in genotype and allele distribution were assessed using Fisher's exact test. The CSF dopamine concentrations and DRD2/ANKK1 genotypes were compared via nonparametric Wilcoxon rank-sum tests using SAS statistical software (SAS Enterprise Guide, Cary, NC, USA). Differences were considered significant at p-values < 0.05. 3. Results Thirty-seven fatal MA intoxication and 235 control cases were genotyped for both DRD2/ANKK1 polymorphisms. Table 2 shows the results of the allele and genotype frequency analyses for Taq1A and –141C Ins/Del polymorphisms, and the associations of Taq1A and –141C Ins/Del genotypes with fatal MA intoxication. There were no significant deviations from the Hardy–Weinberg equilibrium in fatal 7
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Table 2 Frequencies of Taq1A and –141C Ins/Del genotypes in fatal methamphetamine intoxication and control cases, and their association with fatal methamphetamine intoxication. C: cases where methamphetamine and psychotropic drugs were not detected, MA: fatal methamphetamine intoxication cases. SNP
C
Taq1A
Genotype A1/A1 A1/A2 A2/A2 Dominant A2/A2 A1/A1 + A1/A2 Recessive A2A2 + A1/A2 A1/A1 Allele A1 A2
–141C Ins/Del
Genotype Ins/Ins Ins/Del Del/Del Dominant Ins/Ins Del/Del + Ins/Del Recessive Ins/Ins + Ins/Del Del/Del Allele Ins Del
MA
Taq1A A2/A2 A1/A2 + A1/A1
(%)
n
(%)
30 101 104
(12.77) (42.98) (44.26)
5 23 9
(13.51) (62.16) (24.32)
0.050
104 131
(44.26) (55.74)
9 28
(24.32) (75.68)
− 0.030
1.00 (reference) 2.47 (1.12–5.46)
205 30
(87.23) (12.77)
32 5
(86.49) (13.51)
− 0.798
1.00 (reference) 1.07 (0.39–2.95)
161 309
(34.26) (65.74)
33 41
(44.59) (55.41)
− 0.091
1.00 (reference) 0.65 (0.39–1.06)
158 65 12
(67.23) (27.66) (5.11)
26 10 1
(70.27) (27.03) (2.70)
1.000
158 77
(67.23) (32.77)
26 11
(70.27) (29.73)
− 0.851
1.00 (reference) 0.87 (0.41–1.85)
223 12
(94.89) (5.11)
36 1
(97.30) (2.70)
− 1.000
1.00 (reference) 0.52 (0.07–4.09)
381 89
(81.06) (18.94)
62 12
(83.78) (16.22)
− 0.633
1.00 (reference) 0.83 (0.43–1.60)
of relapse of substance abuse such as of MA [29]. Although the effects of the Taq1A polymorphism on fatal MA intoxication are not clear, the Taq1A polymorphism likely affects dopamine signaling and MA dependence. The frequencies of –141C Ins/Del polymorphisms in fatal MA intoxication cases did not differ significantly. The –141C Del-positive genotypes, Del/Del and Del/Ins, were risk factors for the rapid-onset of methamphetamine psychosis within 3 years after beginning abuse [22]. However, the –141C Ins/Del polymorphism was not associated with fatal MA intoxication in this study. Thus, the –141C Ins/Del polymorphism is likely not related to death by MA intoxication. The CSF dopamine concentration was not associated with Taq1A or –141C Ins/Del polymorphisms in any of the study cases. Postmortem serum, pericardial fluid, CSF, and urine dopamine concentrations have previously been demonstrated to be higher in cases of fatal intoxication, such as by MA and psychotropic drugs, than in cases with other causes of death [13,30]. However, there was no significant association between Taq1A or –141C Ins/Del polymorphisms and CSF dopamine concentrations in MA intoxication cases. These results suggest that the Taq1A and –141C Ins/Del polymorphisms did not affect CSF dopamine concentrations in our fatal MA intoxication cases. This study was limited by its small sample size. Further studies are required to evaluate the effects of the DRD2/ANKK1 genotype on drug intoxication with a larger sample of fatal MA intoxication cases.
–141C Ins/Del Ins/Ins Del/Ins + Del/Del
MA
n
Median (range)
P-value
n
Median (range)
P-value
31
18.8 (8.07–152) 15.8 (0.005–265)
0.262
8
25.1 (5.22–63.2) 36.0 (1.72–198)
0.878
18.5 (1.10–265) 12.3 (0.005–76.5)
0.217
33.0 (1.72–198) 28.6 (3.72–127)
0.943
42
44 29
16
19 5
Odds ratio (95% CI)
n
Table 3 Dopamine concentrations (ng/ml) in autopsy specimens of cerebrospinal fluid from fatal methamphetamine intoxication and control cases. C: cases where methamphetamine and psychotropic drugs were not detected, MA: fatal methamphetamine intoxication cases. C
P-value
a negative risk factor for prolonged MA psychosis after therapy and complication of spontaneous relapse after remission [22]. The A1 allele of Taq1A decreases the DRD2 density, and individuals with a lower DRD2 density have a lower risk for prolonged MA psychosis and spontaneous relapse [22]. Striatal DRD2 binding potential was lower among carriers of the A1 allele [25]. The Taq1A A1 allele was associated with fatal MA intoxication in this study, but it is unclear how reduced DRD2 density affects fatal MA intoxication. The primary mechanism of action of MAs is on the dopamine system in the brain, resulting in high abuse potential and neurotoxic effects [26]. MA, alcohol, and cocaine abusers have significantly lower than normal DRD2 availability, and the consequent effect on neuronal function in the orbitofrontal cortex has been associated with loss of control and compulsive drug intake in addicted subjects [27]. The A1 Taq1A allele has been described as a reward gene [28], and Blum K et al. found that low DRD2 density and polymorphism were associated with an increased risk
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