Causal influence of dietary habits on the risk of major depressive disorder: A diet-wide Mendelian randomization analysis.

BACKGROUND: Extensive observational evidence links diet quality to the risk for major depressive disorder (MDD), while clinical trials show that dietary improvement can improve depressive symptoms. However, due to issues with blinding dietary trials, confirming a causal relationship for diet's influence on MDD requires further research. Thus, we systemically investigated the bi-directional causal relationships between dietary habits and MDD by using two-sample Mendelian randomization (MR). METHODS: We collected publicly available genome-wide association studies' summary statistics for dietary habits from UK Biobank (n = 449,210) and MDD from the Psychiatric Genomics Consortium (n = 142,646). We used a weighted median approach to synthesize MR estimates across genetic instruments. For the robustness of our results, we compared weighted median results with results from the inverse-variance weighted, the weighted mode, and MR-PRESSO. RESULTS: There was moderate evidence that beef intake has a protective effect on MDD. There was weak but detectable evidence that cereal intake has a protective effect on MDD, while non-oily fish intake might increase the risk of MDD. We did not observe any causal effect of MDD on dietary habits. LIMITATIONS: Our study may suffer from the violation of assumptions of MR due to horizontal pleiotropy; therefore, we did several sensitivity analyses to detect and minimize the bias. CONCLUSIONS: In this two-sample MR analysis, we observed that higher beef intake may be protective against MDD. However, MDD did not appear to affect dietary habits. Potential mechanisms need to be further investigated to support our novel findings.

Association of the D-amino acid oxidase gene with methadone dose in heroin dependent patients under methadone maintenance treatment.

Methadone is a synthetic opioid used for the maintenance treatment (MMT) of heroin dependence. It primarily binds to the µ-opioid receptor (MOR; with its gene, namely OPRM1). Methadone is also an N-methyl-D-aspartate (NMDA) receptor antagonist. The role of NMDA receptor in the regulatory mechanisms of methadone dosage in heroin dependent patients is so far not clear. D-amino acid oxidase (DAO) is an important enzyme that indirectly activates the NMDA receptor through its effect on the D-serine level. To test the hypothesis that genetic polymorphisms in the DAO gene are associated with methadone treatment dose and responses, we selected four single nucleotide polymorphisms (SNPs) in DAO from the literature reports of the Taiwanese population. SNPs were genotyped in 344 MMT patients. In this study, we identified a functional SNP rs55944529 in the DAO gene that reveals a modest but significant association with the methadone dosage in the recessive model of analysis (P = 0.003) and plasma concentrations (P = 0.003) in MMT patients. However, it did not show association with plasma methadone concentration in multiple linear regression analysis. It is also associated with the methadone adverse reactions of dry mouth (P = 0.002), difficulty with urination (P = 0.0003) in the dominant model, and the withdrawal symptoms of yawning (P = 0.005) and gooseflesh skin (P = 0.004) in the recessive model. Our results suggest a role of the indirect regulatory mechanisms of the NMDA reporter, possibly via the DAO genetic variants, in the methadone dose and some adverse reactions in MMT patients.

Splice-Site Variants in the Gene Encoding GABA-A Receptor Delta Subunit Are Associated with Amphetamine Use in Patients under Methadone Maintenance Treatment.

Chronic opioid use disorder patients often also use other substances such as amphetamines. The gene-based analysis method was applied in the genomic database obtained from our previous study with 343 methadone maintenance treatment (MMT) patients. We found that the gene encoding gamma-aminobutyric acid type A receptors (GABA-A receptor) delta subunit isoforms (GABRD) was associated with amphetamine use in heroin dependent patients under MMT in Taiwan. A total of 15% of the 343 MMT patients tested positive for amphetamine in the urine toxicology test. Two genetic variants in the GABRD, rs2889475 and rs2376805, were found to be associated with the positive urine amphetamine test. They are located in the exon 1 of the splice variant and altered amino acid compositions (T126I, C/T, for rs2889475, and R252Q, G/A, for rs2376805). The CC genotype carriers of rs2889475 showed a four times higher risk of amphetamine use than those with TT genotype. The GG genotype carriers of rs2376805 showed a three times higher risk of amphetamine use than the AA genotype carriers. To our knowledge, this is the first report that demonstrated an association of the delta splice variant isoform in the GABA-A receptor with an increased risk of amphetamine use in MMT patients. Our results suggest that rs2889475 and rs2376805 may be indicators for the functional role and risk of amphetamine use in MMT patients.

Increased Nectin-4 levels in chronic ketamine abusers and the relationship with lower urinary tract symptoms.

Persistent ketamine use causes susceptibility to addiction and bladder toxicity. We examined the association of lower urinary tract symptoms and levels of Nectin-4, a member of the cell adhesion molecules that is essential for maintaining the urothelium barrier in chronic ketamine abusers. We measured the plasma levels of Nectin-4 in 88 patients with ketamine dependence and 69 controls. Patients with ketamine dependence were assessed for ketamine use variables, psychological symptoms, and lower urinary tract symptoms. We found Nectin-4 levels were increased in ketamine-dependent patients compared to the controls (p < 0.0001). Patients with urinary tract symptoms exhibited lower Nectin-4 levels than those without (p = 0.021). Our results suggest an up-regulation of Nectin-4 following chronic and heavy ketamine use. Patients with ketamine dependence with a compromised upregulation of Nectin-4 are likely to have more severe urinary tract symptoms. The mechanisms underlying the involvement of Nectin-4 in ketamine addiction and bladder toxicity warrant future investigation.

Genetic variants in NECTIN4 encoding an adhesion molecule are associated with continued opioid use.

Methadone is a synthetic opioid used as maintenance treatment for patients addicted to heroin. Skin irritation is one of the adverse events caused by opioid use. 344 methadone maintenance treatment (MMT) patients were recruited with records and measurements on methadone dose, plasma methadone concentrations, and treatment emergent symptom scales (TESS). 15 patients reported with skin irritation. Five SNPs located within the NECTIN4 genetic region were genotyped. The NECTIN4 gene within the adherens junction interaction pathway was associated with methadone dose in pathway-based genome wide association analyses (P = 0.0008). Three highly-linked SNPs, rs11265549, rs3820097, and rs4656978, were significantly associated with methadone dose (P = 0.0003), plasma concentrations of R,S-methadone (P = 0.0004) and TNF-α (P = 0.010) in all 344 MMT patients, and with self-report skin irritation symptom scores (P = 0.010) in the 15 MMT patients who reported with skin irritation. To identify the possible roles of plasma level of Nectin-4 in the responses to MMT and opioid use, additional age- and gender-matched 51 controls and 83 methadone-free abstinent former heroin users were recruited. Plasma level of Nectin-4 was the highest in MMT patients among the three groups. The results suggest involvement of genetic variants on NECTIN4 in methadone dose. Plasma Nectin-4 level is likely an indicator for continued use of opioids.

Adhesion Molecules as Potential Novel Biomarkers for Opioid Dependence.

BACKGROUND: Cell-cell adhesion is essential in maintaining the structure and function of an organ. Several adhesion molecules have recently been identified as associated with heroin dependence in both genetic and peripheral plasma studies. METHODS AND RESULTS: We reviewed literature concerning studies on adhesion molecules in opioid addictions in rodents and human, including human genetic associations in different ethnic groups, and treatment responses to methadone maintenance treatment in heroin-dependent patients. CONCLUSION: Some important and novel findings were summarized and discussed. Adhesion molecules in the peripheral plasma, e.g., cadherin-2 (CDH2), may be biomarkers for both methadone treatment outcome and nectin 4 may be an indicator for continued opioid use. Neural cell adhesion molecule (NCAM) in the central nervous system may regulate opioid withdrawal and analgesic responses. Future studies to uncover the mechanisms underlying the involvement of adhesion molecules in the pathological process of addictions will be an important research direction in the field.

Genetic polymorphisms in the opioid receptor delta 1 (OPRD1) gene are associated with methadone dose in methadone maintenance treatment for heroin dependence.

Delta opioid receptor (DOR) is well known to be involved in heroin dependence. This study tested the hypothesis that single nucleotide polymorphisms (SNPs) in the opioid receptor delta 1 (OPRD1) gene coding region are associated with treatment responses in a methadone maintenance therapy (MMT) cohort in Taiwan. Three hundred forty-four MMT patients were recruited. Diastolic/systolic blood pressure, heart rate, methadone dosage, and plasma concentrations of methadone were recorded. Twenty-five SNPs located within the OPRD1 genetic region were selected and genotyped from the genomic DNA of all 344 participants. After pairwise tagger analyses, tagger SNP rs204047 showed a significant association with methadone dosage (P = 0.0019), and tagger SNPs rs204047 and rs797397 were significantly associated with plasma R, S-methadone concentrations (P < 0.0006) in patients tested negative in the urine morphine test, which indicated patients with a better response to MMT. The major genotype carriers showed a higher methadone dosage and higher plasma concentrations of R, S-methadone than the minor genotype carriers. The results indicated that OPRD1 genetic variants were associated with methadone dosage and methadone plasma concentration in MMT patients with a negative morphine test result.

Serum Level and Activity of Butylcholinesterase: A Biomarker for Post-Stroke Dementia.

Cholinergic neurotransmission regulates the immune response and inhibits cytokine release after stroke. The changes in the level/activity of blood cholinesterase (ChE) in patients with post-stroke dementia (PSD) are less known. This study aimed to examine post-stroke plasma acetylcholinesterase (AChE) and butylcholinesterase (BChE) and determine whether they are biomarkers for PSD. Thirty patients with PSD, 87 post-stroke patients without dementia (PSNoD), and 117 age- and gender-matched healthy controls were recruited. Missense genetic variants AChE rs1799806 and BChE rs1803274 were genotyped. The plasma AChE level did not differ between the PSD and PSNoD groups. However, BChE levels were significantly lower in the PSD than in the PSNoD group (3300.66 ± 515.35 vs 3855.74 ± 677.60 ng/mL, respectively; p = 0.0033). The activities of total ChE, BChE, and AChE were all lower in the PSD group (19,563.33 ± 4366.03, 7650.17 ± 1912.29, 11,913.17 ± 2992.42 mU/mL, respectively) than in the PSNoD group (23,579.08 ± 5251.55, 9077.72 ± 1727.28, and 14,501.36 ± 4197.17 mU/mL, respectively). When further adjusting for age and sex, significance remained in BChE level and activity and in total ChE activity. BChE rs1803274 was associated with reduced BChE activity, while AChE rs1799806 did not influence AChE activity. The level and activity of BChE, but not of AChE, were decreased in PSD patients and may therefore aid in PSD diagnosis.

A Post-hoc Study of D-Amino Acid Oxidase in Blood as an Indicator of Post-stroke Dementia.

Stroke is an important risk factor for dementia. Epidemiological studies have indicated a high incidence of dementia in stroke patients. There is currently no effective biomarker for the diagnosis of post-stroke dementia (PSD). D-amino acid oxidase (DAO) is a flavin-dependent enzyme widely distributed in the central nervous system. DAO oxidizes D-amino acids, a process which generates neurotoxic hydrogen peroxide and leads to neurodegeneration. This study aimed to examine post-stroke plasma DAO levels as a biomarker for PSD. In total, 53 patients with PSD, 20 post-stroke patients without dementia (PSNoD), and 71 age- and gender-matched normal controls were recruited. Cognitive function was evaluated at more than 30 days post-stroke. Plasma DAO was measured using the enzyme-linked immunosorbent assay. White matter hyperintensity (WMH), a neuroimaging biomarker of cerebral small vessel diseases, was determined by magnetic resonance imaging. We found that plasma DAO levels were independently higher in PSD subjects than in PSNoD subjects or the controls and were correlated with the WMH load in stroke patients. Using an area under the curve (AUC)/receiver operating characteristic analysis, plasma DAO levels were significantly reliable for the diagnosis of PSD. The sensitivity and specificity of the optimal cut-off value of 321 ng/ml of plasma DAO for the diagnosis of PSD were 75 and 88.7%, respectively. In conclusion, our data support that plasma DAO levels were increased in PSD patients and correlated with brain WMH, independent of age, gender, hypertension, and renal function. Plasma DAO levels may therefore aid in PSD diagnosis.

GRK5 Is Associated with the Regulation of Methadone Dosage in Heroin Dependence.

Background: There is no countable biomarker for opioid dependence treatment responses thus far. In this study, we recruited Taiwanese methadone maintenance treatment patients to search for genes involving the regulatory mechanisms of methadone dose by genome-wide association analyses. Methods: A total of 344 Taiwanese methadone maintenance treatment patients were included in a genome-wide association study. The involvement of GRK5 in opioid dependence was then further confirmed by gene expression study on lymphoblastoid cell lines derived from 3 independent age- and gender-matched groups: methadone maintenance treatment patients, medication-free former heroin abusers, and normal controls. Results: The results indicated that GRK5, the gene encoding an enzyme related to µ-opioid receptor desensitization, is associated with methadone dose by additive model of gene-based association analysis (P=6.76×10-5). We found that 6 of the 55 single nucleotide polymorphisms from the genome-wide genotype platform and 2 single nucleotide polymorphisms from the 29 additionally selected single nucleotide polymorphisms were significantly associated with methadone maintenance dose in both genotype and allele type (P ≤ .006), especially in patients who tested negative in the urine morphine test. The levels of GRK5 gene expression were similar between methadone maintenance treatment patients and medication-free former heroin abusers. However, the normal controls showed a significantly lower level of GRK5 gene expression than the other groups (P=.019). Conclusions: The results suggested an important role for GRK5 in the regulatory mechanisms of methadone dose and course of heroin dependence.

APBB2 is associated with amphetamine use and plasma beta-amyloids in patients receiving methadone maintenance treatment.

APBB2, amyloid beta (A4) precursor protein-binding family B member 2, has been reported to be associated with opioid dependence. In this study, we reported the first time that the genetic variants in the APBB2 gene were associated with use of amphetamine in opioid dependent patients undergoing methadone maintenance treatment (MMT). 344 heroin-dependent patients undergoing MMT were recruited and assessed for use of amphetamine and opioids by urine toxicology, withdrawal severity, and side effects. DNAs were genome-widely genotyped for all patients. Single nucleotide polymorphisms (SNPs) in APBB2 were selected for association analyses for methadone treatment responses. Gene expression levels of APBB2 were measured by real-time polymerase chain reaction (PCR) in the EBV-transformed lymphoblastoids from patients. MMT patients who used amphetamine showed a significantly higher percentage of positive results in the urine morphine test (P=0.005), and insomnia (P=0.018). In single locus association analyses, SNPs rs3935357 and rs4861075 located at intron 6 were significantly associated with amphetamine use in both genotype and allele type (general linear model (GLM), P=0.0003, and 0.0002 for genotype, and 0.0003, and 0.002 for allele type, respectively). The major allele type carriers had twice risk of amphetamine use compared to the minor allele type carriers. Subjects with the TT genotype of rs4861075 showed significantly higher levels of APBB2 gene expression in both total (P=0.02) and long-form (P=0.037) than those with CC genotype. Detailed mechanisms underlying the association of APBB2 with amphetamine use and level of plasma amyloid beta in MMT patients require further investigation.

Inflammatory chemokine eotaxin-1 is correlated with age in heroin dependent patients under methadone maintenance therapy.

BACKGROUND: Degeneration of central neurons and fibers has been observed in postmortem brains of heroin dependent patients. However, there are no biomarkers to predict the severity of neurodegeneration related to heroin dependence. A correlation has been reported between inflammatory C-C motif chemokine ligand 11 (CCL11, or eotaxin-1) and neurodegeneration in Alzheimer's disease. METHODS: Three-hundred-forty-four heroin dependent, Taiwanese patients under methadone maintenance treatment (MMT) were included with clinical assessment and genomics information. Eighty-seven normal control subjects were also recruited for comparison. RESULTS: Using receiver operating characteristics curve analyses, CCL11 showed the strongest sensitivity and specificity in correlation with age by a cut-off at 45 years (AUC = 0.69, P < 0.0001) in MMT patients, but not normal controls. Patients 45 years of age or older had significantly higher plasma levels of CCL11, fibroblast growth factor 2 (FGF-2), nicotine metabolite cotinine, and a longer duration of addiction. Plasma level of CCL11 was correlated with that of FGF-2 (partial r2 = 0.24, P < 0.0001). Carriers with the mutant allele of rs1129844, a functional single nucleotide polymorphism (Ala23Thr) in the CCL11 gene, showed a higher plasma level of Aß42, ratio of Aß42/Aß40, and insomnia side effect symptom score than the GG genotype carriers among MMT responders with morphine-negative urine results. CONCLUSIONS: The results suggest possible novel mechanisms mediated through CCL11 involving neurotoxicity in heroin dependent patients.

Missense mutation at CLDN8 associated with a high plasma interferon gamma-inducible protein 10 level in methadone-maintained patients with urine test positive for morphine.

We previously reported a high plasma chemokine interferon gamma-inducible protein 10 (IP-10) level and prolonged electrocardiography QT-interval in methadone maintenance treatment (MMT) patients with HIV or HCV infection. The purpose of this study was to evaluate the genetic association of high plasma IP-10 level in the MMT patients. The gene-based and pathway-based association analyses were conducted using a genome-wide association study dataset in 344 MMT patients for identifying genes and pathways associated with plasma IP-10 level. We found that plasma IP-10 level was significantly associated with a pathway in the tight junction (P = 1.01x10-5), where the claudin 8 (CLDN8) gene had the most significant association (P = 6.8x10-5). A functional single nucleotide polymorphism (SNP) rs686364 at exon 1 of CLDN8 showed strong association with plasma IP-10 levels, in the MMT subjects with positive urine test for morphine (dominant model, P = 0.00004). The minor allele type carriers had higher plasma IP-10 levels than the major allele type carriers. Our data support that the tight junction protein claudin 8 exon 1 is a predictor for the plasma levels of IP-10 in MMT patients with urine test positive for morphine.

Diversity-Oriented Synthesis of Coumarin-Linked Benzimidazoles via a One-Pot, Three-Step, Intramolecular Knoevenagel Cyclization.

Diversity-oriented synthesis of coumarin-linked benzimidazoles from N-(2-aminophenyl)-2-cyanoacetamide was achieved via a one-pot, three-step sequential reaction in excellent yields. In situ intramolecular cyclization of the cyanoacetamide afforded benzimidazoles which subsequently underwent a Knoevenagel condensation of the 2-cyanomethylbenzimidazoles with salicylaldehydes promoted by triethylamine to reach the target compounds. An important intermediate, 2-(2-imino-2H-chromen-3-yl)-1H-benzimidazole was characterized by X-ray analysis and further hydrolyzed to 2-(coumarin-3-yl)benzimidazole in acidic condition. Among the synthesized compounds, some were found to be promising inhibitors of porcine kidney d-amino acid oxidase (pkDAO).

Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits.

D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.

The association of genetic polymorphisms in the κ-opioid receptor 1 gene with body weight, alcohol use, and withdrawal symptoms in patients with methadone maintenance.

Methadone is a synthetic opioid that binds to the κ-opioid receptor with a low affinity. This study tested the hypotheses that the genetic polymorphisms in the κ-opioid receptor 1 (OPRK1) gene region are associated with methadone treatment responses in a Taiwan methadone maintenance treatment (MMT) cohort. Seventeen single nucleotide polymorphisms (SNPs) in OPRK1 were selected and genotyped on DNA of 366 MMT patients. Six SNPs from rs7843965 to rs1051660 (intron 2 to exon 2) were significantly associated with body weight (P < 0.007). A haplotype of 4 SNPs rs7832417-rs16918853-rs702764-rs7817710 (exon 4 to intron 3) was associated with bone or joint aches (P ≤ 0.004) and with the amount of alcohol use (standard drinks per day; global P < 0.0001). The haplotype rs10958350-rs7016778-rs12675595 was associated with gooseflesh skin (global P < 0.0001), yawning (global P = 0.0001), and restlessness (global P < 0.0001) withdrawal symptoms. The findings suggest that genetic polymorphisms in OPRK1 were associated with the body weight, alcohol use, and opioid withdrawal symptoms in MMT patients.

Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3')-deoxyribonucleotidase and partially inhibits its activity.

Infection with hepatitis C virus (HCV) is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3) is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3')-deoxyribonucleotidase (cdN, dNT-1) was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

OPRM1 genetic polymorphisms are associated with the plasma nicotine metabolite cotinine concentration in methadone maintenance patients: a cross sectional study.

Majority of the heroin-dependent patients smoke cigarettes. Although it has been reported that the OPRM1 genetic polymorphism is associated with the brain mu-opioid receptor binding potential in cigarette smokers, there is no direct evidence showing the impact of plasma cotinine, a nicotine metabolite, on treatment responses to methadone maintenance treatment (MMT). In this study, we aimed to test the hypothesis that the genetic polymorphisms in the OPRM1 are associated with the methadone treatment responses and the severity of cigarette smoking directly measured by the plasma concentration of cotinine in a Taiwanese MMT cohort. Fifteen OPRM1 single-nucleotide polymorphisms (SNPs) were selected and genotyped on DNA samples of 366 MMT patients. Plasma concentrations of cotinine were measured by cotinine enzyme-linked immunosorbent assay. The plasma cotinine concentration had positive correlation with concentrations of methadone (P = 0.042) and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (P = 0.037). Methadone treatment non-responders, defined by a positive urine morphine test, had a higher plasma concentration of cotinine (P = 0.005), but a lower plasma concentration-to-dose ratio of both R- and S-methadone (P = 0.001 and 0.012, respectively) than the responders. OPRM1 genetic variants, rs1074287, rs6912029, rs1799971, rs12209447, rs510769, rs3798676, rs553202, rs7748401, rs495491, rs10457090, rs589046, rs3778152 and rs563649, were significantly associated with the plasma concentration of cotinine when using recessive model for genotypes (general linear model (GLM), P<0.038; false discovery rate (FDR)<0.035) and additive model for allele types (GLM, P<0.03; FDR<0.049) in association analyses. The G allele carriers of SNP rs1799971 (A118G) on exon 1 of OPRM1 gene had a lower plasma cotinine concentration than the A allele carriers (GLM, P = 0.029). OPRM1 genetic polymorphisms are associated with the plasma concentration of cotinine in a Taiwanese MMT cohort. Carriers with the major allele of SNP rs1799971 had a higher plasma cotinine concentration.

UGT2B7 genetic polymorphisms are associated with the withdrawal symptoms in methadone maintenance patients.

AIM: To test whether the genetic polymorphisms within the gene encoding the UGT2B7 gene may have an impact on methadone treatment. MATERIALS & METHODS: Twelve SNPs in UGT2B7 were selected. 366 methadone maintenance treatment patients in Taiwan were recruited and genotyped. RESULTS: In a genotype recessive model, rs6600879, rs6600880, rs4554144, rs11940316, rs7438135, rs7662029, rs7668258, rs7439366, rs4292394 and rs6600893 showed significant associations with severity of withdrawal symptoms (permutation p < 0.002), pupil size (permutation p < 0.048) and tremor (permutation p < 0.008). Haplotypes of GATCAGCCGC and CTCTGATTCT were significantly associated with pupil size score and tremor score (p < 0.034). CONCLUSION: These results suggest that SNPs of the UGT2B7 gene may play important roles in opiate withdrawal symptoms.

Expression and membrane integration of SARS-CoV M protein.

SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46-68 and 78-100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14-36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis.