Causal role of immune cell traits in stroke: A Mendelian randomization study.

OBJECTIVES: Immune mechanisms play a crucial role in the development of stroke. However, immune-related phenotypes are diverse and their associations with stroke are largely unknown. Here, we aimed to systematically explore the causal role of immune cell traits in stroke and its subtypes by leveraging data from genome-wide association studies (GWASs). MATERIALS AND METHODS: Exposure data were obtained from a recent GWAS on 731 immune cell traits profiled by flow cytometry involving 3757 individuals. By conducting two-sample univariable Mendelian randomization (MR) analyses, each immune cell trait was assessed for causal relationships with stroke outcomes from the MEGASTROKE Consortium (40,585 cases and 406,111 controls). The robustness of the MR results was verified by a series of sensitivity analyses. RESULTS: We identified three significant associations after Bonferroni correction (P < 1.37E-05). Increased CD27 expression on memory B cell (OR = 1.23, 95% CI = 1.14-1.33, P = 2.78E-08), IgD-CD38dim B cell (OR = 1.16, 95% CI = 1.09-1.23, P = 5.98E-06) and unswitched memory B cell (OR = 1.18, 95% CI = 1.10-1.27, P = 1.09E-05) were associated with a higher risk of large-artery atherosclerotic stroke (LAS). Furthermore, expression quantitative trait loci data also indicated elevated blood CD27 mRNA level was a risk factor for LAS (OR = 1.37, 95% CI = 1.02-1.84, P = 0.037). CONCLUSIONS: This study provided genetic evidence of the causal relationship between immune cell traits and stroke, highlighting the role of CD27 on memory B cell as a novel factor for LAS risk.

A Joint Extraction System Based on Conditional Layer Normalization for Health Monitoring.

Natural language processing (NLP) technology has played a pivotal role in health monitoring as an important artificial intelligence method. As a key technology in NLP, relation triplet extraction is closely related to the performance of health monitoring. In this paper, a novel model is proposed for joint extraction of entities and relations, combining conditional layer normalization with the talking-head attention mechanism to strengthen the interaction between entity recognition and relation extraction. In addition, the proposed model utilizes position information to enhance the extraction accuracy of overlapping triplets. Experiments on the Baidu2019 and CHIP2020 datasets demonstrate that the proposed model can effectively extract overlapping triplets, which leads to significant performance improvements compared with baselines.

Effect of carbendazim on honey bee health: Assessment of survival, pollen consumption, and gut microbiome composition.

Gut microbiota and nutrition play major roles in honey bee health. Recent reports have shown that pesticides can disrupt the gut microbiota and cause malnutrition in honey bees. Carbendazim is the most commonly used fungicide in China, but it is not clear whether carbendazim negatively affects the gut microbes and nutrient intake levels in honey bees. To address this research gap, we assessed the effects of carbendazim on the survival, pollen consumption, and sequenced 16 S rRNA gene to determine the bacterial composition in the midgut and hindgut. Our results suggest that carbendazim exposure does not cause acute death in honey bees even at high concentrations (5000 mg/L), which are extremely unlikely to exist under field conditions. Carbendazim does not disturb the microbiome composition in the gut of young worker bees during gut microbial colonization and adult worker bees with established gut communities in the mid and hindgut. However, carbendazim exposure significantly decreases pollen consumption in honey bees. Thus, exposure of bees to carbendazim can perturb their beneficial nutrition homeostasis, potentially reducing honey bee immunity and increasing their susceptibility to infection by pathogens, which influence effectiveness as pollinators, even colony health.

Gene-based association analysis reveals involvement of LAMA5 and cell adhesion pathways in nicotine dependence in African- and European-American samples.

Nicotine dependence (ND) is a chronic brain disorder that causes heavy social and economic burdens. Although many susceptibility genetic loci have been reported, they can explain only approximately 5%-10% of the genetic variance for the disease. To further explore the genetic etiology of ND, we genotyped 242 764 SNPs using an exome chip from both European-American (N = 1572) and African-American (N = 3371) samples. Gene-based association analysis revealed 29 genes associated significantly with ND. Of the genes in the AA sample, six (i.e., PKD1L2, LAMA5, MUC16, MROH5, ATP8B1, and FREM1) were replicated in the EA sample with p values ranging from 0.0031 to 0.0346. Subsequently, gene enrichment analysis revealed that cell adhesion-related pathways were significantly associated with ND in both the AA and EA samples. Considering that LAMA5 is the most significant gene in cell adhesion-related pathways, we did in vitro functional analysis of this gene, which showed that nicotine significantly suppressed its mRNA expression in HEK293T cells (p < 0.001). Further, our cell migration experiment showed that the migration rate was significantly different in wild-type and LAMA5-knockout (LAMA5-KO)-HEK293T cells. Importantly, nicotine-induced cell migration was abolished in LAMA5-KO cells. Taken together, these findings indicate that LAMA5, as well as cell adhesion-related pathways, play an important role in the etiology of smoking addiction, which warrants further investigation.

Demonstration of critical role of GRIN3A in nicotine dependence through both genetic association and molecular functional studies.

Nicotine dependence (ND) is a chronic disease with catastrophic effects on individual and public health. The glutamate receptor subunit gene, ionotropic N-methyl-d-aspartate 3A (GRIN3A), encodes a crucial subunit of N-methyl-d-aspartate receptors (NMDARs), which play an essential role in synaptic plasticity in the brain. Although various variants of GRIN3A have been associated with ND in European-American and African-American samples, no study has been reported for the association between GRIN3A and ND in Chinese Han population. We performed an association study of 16 single nucleotide polymorphisms (SNPs) in GRIN3A with ND in 2616 Chinese individuals. SNP-based association analysis indicated that SNP rs1323423 was significantly associated with the Fagerström Test for Nicotine Dependence (FTND) score after correction for multiple testing (P = 0.0026). Haplotype-based association analysis revealed that Block 3, formed by rs1323423-rs10989591, was significantly associated with the FTND score after correction for multiple testing (global P = 0.0183). Furthermore, luciferase reporter assay demonstrated that the DNA region containing rs1323423 was an enhancer element, the activity of which was significantly impacted by rs1323423 genotype. Considering that rs1323423 is located in a potential enhancer region, we performed GRIN3A editing in HEK293T cells with CRISPR/Cas9 and found that the DNA region around rs1323423 has a regulatory function and the expression of GRIN3A affects the expression of other NMDA subunits. Moreover, we demonstrated that nicotine at a concentration of 100 µM decreased expression of GRIN3A in SH-SY5Y and HEK293T cells at the RNA and protein level, respectively. This study provides novel evidence for the involvement of GRIN3A in ND.

The Crystal Structure of a Maxi/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages.

Cage proteins assemble into nanoscale structures with large central cavities. They play roles, including those as virus capsids and chaperones, and have been applied to drug delivery and nanomaterials. Furthermore, protein cages have been used as model systems to understand and design protein quaternary structure. Ferritins are ubiquitous protein cages that manage iron homeostasis and oxidative damage. Two ferritin subfamilies have strongly similar tertiary structure yet distinct quaternary structure: maxi-ferritins normally assemble into 24-meric, octahedral cages with C-terminal E-helices centered around 4-fold symmetry axes, and mini-ferritins are 12-meric, tetrahedral cages with 3-fold axes defined by C-termini lacking E-domains. To understand the role E-domains play in ferritin quaternary structure, we previously designed a chimera of a maxi-ferritin E-domain fused to the C-terminus of a mini-ferritin. The chimera is a 12-mer cage midway in size between those of the maxi- and mini-ferritin. The research described herein sets out to understand (a) whether the increase in size over a typical mini-ferritin is due to a frozen state where the E-domain is flipped out of the cage and (b) whether the symmetrical preference of the E-domain in the maxi-ferritin (4-fold axis) overrules the C-terminal preference in the mini-ferritin (3-fold axis). With a 1.99 Å resolution crystal structure, we determined that the chimera assembles into a tetrahedral cage that can be nearly superimposed with the parent mini-ferritin, and that the E-domains are flipped external to the cage at the 3-fold symmetry axes.

Association of STAT4 polymorphisms with hepatitis B virus infection and clearance in Chinese Han population.

Genome-wide association study indicates that STAT4 is a plausible candidate for an association study with HBV-related liver diseases. We aimed to examine the roles of STAT4 polymorphisms on HBV-related liver diseases in a Chinese Han population. We selected 13 SNPs in STAT4 based on the HapMap database to investigate their associations in 3,033 participants. SNP rs7574865 was significantly associated with HBV infection [odds ratio (OR) 1.15; 95 % confidence interval (CI) 1.00, 1.31; P = 0.046] and clearance (OR 1.17; 95 % CI 1.02, 1.33; P = 0.028). Further, haplotype-based association analysis indicated that the haplotype CTCTT, formed by SNPs rs8179673, rs7574865, rs4274624, rs11889341, and rs10168266, was significantly associated with HBV infection (OR 0.87; 95 % CI 0.76, 0.99; P = 0.022) and clearance (OR 0.86; 95 % CI 0.75, 0.99; P = 0.018). Bioinformatics analysis of these SNPs predicted that they participate in transcriptional regulation. Taken together, our findings demonstrate that variants in STAT4 play a critical role in HBV infection and clearance in the Chinese Han population.

Meta-Analysis Reveals Significant Association of the 3'-UTR VNTR in SLC6A3 with Alcohol Dependence.

BACKGROUND: Although many studies have analyzed the association of 3'-untranslated region variable-number tandem repeat (VNTR) polymorphism in SLC6A3 with alcohol dependence (AD), the results remain controversial. This study aimed to determine whether this variant indeed has any genetic effect on AD by integrating 17 reported studies with 5,929 participants included. METHODS: The A9-dominant genetic model that considers A9-repeat and non-A9 repeat as 2 genotypes and compared their frequencies in alcoholics with that in controls was adopted. Considering the potential influence of ethnicity, differences in diagnostic criteria of AD, and alcoholic subgroups, stratified meta-analyses were conducted. There existed no evidence for the presence of heterogeneity among the studied samples, indicating the results under the fixed-effects model are acceptable. RESULTS: We found a significant association of VNTR A9 genotypes with AD in all ethnic populations (pooled odds ratio [OR] 1.12; 95% confidence interval [CI] 1.00, 1.25; p = 0.045) and the Caucasian population (pooled OR 1.15; 95% CI 1.01, 1.31; p = 0.036). We also found VNTR A9 genotypes to be significantly associated with alcoholism as defined by the DSM-IV criteria (pooled OR 1.18; 95% CI 1.03, 1.36; p = 0.02). Further, we found a significant association between VNTR A9 genotypes and alcoholism associated with alcohol withdrawal seizure or delirium tremens (pooled OR 1.55; 95% CI 1.24, 1.92; p = 1.0 × 10(-4) ). In all these meta-analyses, no evidence of publication bias was detected. CONCLUSIONS: We concluded that the VNTR polymorphism has an important role in the etiology of AD, and individuals with at least 1 A9 allele are more likely to be dependent on alcohol than persons carrying the non-A9 allele.

Toxicity profiling of water contextual zinc oxide, silver, and titanium dioxide nanoparticles in human oral and gastrointestinal cell systems.

Engineered nanoparticles (ENPs) are increasingly detected in water supply due to environmental release of ENPs as the by-products contained within the effluent of domestic and industrial run-off. The partial recycling of water laden with ENPs, albeit at ultra-low concentrations, may pose an uncharacterized threat to human health. In this study, we investigated the toxicity of three prevalent ENPs: zinc oxide, silver, and titanium dioxide over a wide range of concentrations that encompasses drinking water-relevant concentrations, to cellular systems representing oral and gastrointestinal tissues. Based on published in silico-predicted water-relevant ENPs concentration range from 100 pg/L to 100 µg/L, we detected no cytotoxicity to all the cellular systems. Significant cytotoxicity due to the NPs set in around 100 mg/L with decreasing extent of toxicity from zinc oxide to silver to titanium dioxide NPs. We also found that noncytotoxic zinc oxide NPs level of 10 mg/L could elevate the intracellular oxidative stress. The threshold concentrations of NPs that induced cytotoxic effect are at least two to five orders of magnitude higher than the permissible concentrations of the respective metals and metal oxides in drinking water. Based on these findings, the current estimated levels of NPs in potable water pose little cytotoxic threat to the human oral and gastrointestinal systems within our experimental boundaries.

Integrated analysis of circRNA-associated ceRNA network in ischemic stroke.

Introduction: Stroke, of which ischemic stroke (IS) is the major type, is the second leading cause of disability and death worldwide. Circular RNAs (circRNAs) are reported to play important role in the physiology and pathology of IS. CircRNAs often act as competing endogenous RNA (ceRNA) to regulate gene expression by acting as miRNA sponges. However, whole transcriptome-wide screenings of circRNA-mediated ceRNA networks associated with IS are still lacking. In the present study, we constructed a circRNA-miRNA-mRNA ceRNA network by whole transcriptome-wide analysis. Methods: CircRNAs, miRNAs and mRNAs expression profiles were downloaded from the Gene Expression Omnibus (GEO) datasets. We identified differentially expressed (DE) circRNAs, miRNAs, and mRNAs in IS patients. StarBase and CircBank databases were used to predict the miRNA targets of DEcircRNAs, and mirDIP database was used to predict the mRNA targets of DEmiRNAs. CircRNA-miRNA pairs and miRNA-mRNA pairs were established. Then, we identified hub genes via protein-protein interaction analysis and constructed a core ceRNA sub-network. Results: In total, 276 DEcircRNAs, 43 DEmiRNAs, and 1926 DEmRNAs were explored. The ceRNA network included 69 circRNAs, 24 miRNAs, and 92 mRNAs. The core ceRNA subnetwork included hsa_circ_0011474, hsa_circ_0023110, CDKN1A, FHL2, RPS2, CDK19, KAT6A, CBX1, BRD4, and ZFHX3. Discussion: In conclusion, we established a novel hsa_circ_0011474 - hsa-miR-20a-5p/hsa-miR-17-5p - CDKN1A ceRNA regulatory axis associated with IS. Our findings provide new insights into the pathogenesis of IS and offer promising diagnostic and predictive biomarkers.

Risk factors for stroke outcomes in adults: Stroke in China.

This study aimed to determine the possible risk factors for stroke outcomes based on prospective cohort study in China. A total of 146 stroke patients were recruited and divided into 2 groups, which assessed using the modified Rankin Scale (mRS), good outcomes (mRS <= 2) and poor outcomes group (mRS > 2). Demographic, clinical, and laboratory characteristics of participants were obtained from the medical record. The multivariable logistic regression analysis was employed to assess the risk factors for stroke outcomes. Of 146 participants, 28 (19.18%) were presented with poor outcomes at day 90. As a result of multivariable logistic regression analysis, a significantly increased risk of stroke outcomes was found in patients with Barthel Index (BI) score (stroke (OR 1.50, 95% CI 1.21 ~ 1.85, P < .001) and IS (OR 1.48, 95% CI 1.20 ~ 1.83, P < .001)).

Long-term durability of metastable ß-Fe2O3 photoanodes in highly corrosive seawater.

Durability is one prerequisite for material application. Photoelectrochemical decomposition of seawater is a promising approach to produce clean hydrogen by using solar energy, but it always faces the problem of serious Cl- corrosion. We find that the main deactivation mechanism of the photoanode is oxide surface reconstruction accompanied by the coordination of Cl- during seawater splitting, and the stability of the photoanode can be effectively improved by enhancing the metal-oxygen interaction. Taking the metastable ß-Fe2O3 photoanode as an example, Sn added to the lattice can enhance the M-O bonding energy and hinder the transfer of protons to lattice oxygen, thereby inhibiting excessive surface hydration and Cl- coordination. Therefore, the bare Sn/ß-Fe2O3 photoanode delivers a record durability for photoelectrochemical seawater splitting over 3000 h.

Alanine-shaving mutagenesis to determine key interfacial residues governing the assembly of a nano-cage maxi-ferritin.

The fundamental process of protein self-assembly is governed by protein-protein interactions between subunits, which combine to form structures that are often on the nano-scale. The nano-cage protein, bacterioferritin from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for an alanine-shaving mutagenesis study to discover key amino acid residues at symmetry-related protein-protein interfaces that control protein stability and self-assembly. By inspection of these interfaces and "virtual alanine scanning," nine mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, dynamic light scattering, native PAGE, and temperature-dependent CD. Many of the selected amino acids act as hot spot residues. Four of these (Arg-30, which is located at the two-fold axis, and Arg-61, Tyr-114, and Glu-128, which are located at the three-fold axis), when individually mutated to alanine, completely shut down detectable solution formation of 24-mer, favoring a cooperatively folded dimer, suggesting that they may be oligomerization "switch residues." Furthermore, two residues, Arg-30 and Arg-61, when changed to alanine form mutants that are more thermodynamically stable than the native protein. This investigation into the structure and energetics of this self-assembling nano-cage protein not only can act as a jumping off point for the eventual design of novel protein nano-structures but can also help to understand the role that structure plays on the function of this important class of proteins.

Identification of 34 genes conferring genetic and pharmacological risk for the comorbidity of schizophrenia and smoking behaviors.

The prevalence of smoking is significantly higher in persons with schizophrenia (SCZ) than in the general population. However, the biological mechanisms of the comorbidity of smoking and SCZ are largely unknown. This study aimed to reveal shared biological pathways for the two diseases by analyzing data from two genome-wide association studies with a total sample size of 153,898. With pathway-based analysis, we first discovered 18 significantly enriched pathways shared by SCZ and smoking, which were classified into five groups: postsynaptic density, cadherin binding, dendritic spine, long-term depression, and axon guidance. Then, by using an integrative analysis of genetic, epigenetic, and expression data, we found not only 34 critical genes (e.g., PRKCZ, ARHGEF3, and CDKN1A) but also various risk-associated SNPs in these genes, which convey susceptibility to the comorbidity of the two disorders. Finally, using both in vivo and in vitro data, we demonstrated that the expression profiles of the 34 genes were significantly altered by multiple psychotropic drugs. Together, this multi-omics study not only reveals target genes for new drugs to treat SCZ but also reveals new insights into the shared genetic vulnerabilities of SCZ and smoking behaviors.

A helix swapping study of two protein cages.

Protein cages have been the focus of studies across multiple scientific disciplines. They have been used to deliver drugs, as templates for nanostructured materials, as substrates in the development of bio-orthogonal chemistry, and to restrict diffusion to study spatially confined reactions. Although their monomers fold into four-helix bundle structures, two cage proteins, DPS and BFR, self-assemble to form a 12-mer with tetrahedral symmetry and an octahedrally symmetric 24-mer, respectively. These monomers share strong similarities of both sequence and tertiary structure. However, they differ in the presence of a short additional helix. In BFR, the fifth helix is at the C-terminus and is positioned along the 4-fold symmetry axis, whereas with DPS, an extra helix helps to define the 2-fold axis in the cage and is located between the second and third helices in the monomer bundle. In an attempt to investigate if these short helices govern protein assembly, mutants were designed and produced that delete and swap these minidomains. All mutants form highly helical structures that unfold cooperatively as evidenced by thermal melting followed by circular dichroism. Dynamic light scattering, size exclusion chromatography, and sedimentation equilibrium experiments demonstrated that although many of the BFR mutants do not self-assemble and form lower-order complexes, many DPS mutants could form cages despite their unnatural design. Taken together, our data indicate that the BC helix is less important than the E helix for overall cage self-assembly, suggesting that dimerization may not play a role in nanostructure formation that is as key as previously assumed. Additionally, we found that fusing the minidomain from BFR onto DPS results in a mutant that assembles into a homogeneous population of a novel protein oligomer. This assembled cage while still formed from 12 subunits is larger in overall shape than that of the native protein.

Identification and characterization of SEC24D as a susceptibility gene for hepatitis B virus infection.

Single nucleotide polymorphisms (SNPs) and genes associated with susceptibility to hepatitis B virus (HBV) infection that have been identified by genome-wide association studies explain only a limited portion of the known heritability, indicating more genetic variants remain to be discovered. In this study, we adopted a new research strategy to identify more susceptibility genes and variants for HBV infection. We first performed genetic association analysis of 300 sib-pairs and 3,087 case-control samples, which revealed that 36 SNPs located in 31 genes showed nominal associations with HBV infection in both samples. Of these genes, we selected SEC24D for further molecular analysis according to the following two main lines of evidence. First, a time course analysis of the expression profiles from HBV-infected primary human hepatocytes (PHH) demonstrated that SEC24D expression increased markedly as time passed after HBV infection (P = 4.0 × 10-4). Second, SNP rs76459466 in SEC24D was adversely associated with HBV risk (ORmeta = 0.82; Pmeta = 0.002), which again indicated that SEC24D represents a novel susceptibility gene for HBV infection. Moreover, SEC24D appeared to be protective against HBV infection in vitro. Consistently, we found that SEC24D expression was significantly enhanced in non-infected liver tissues (P = 0.002). We conclude that SEC24D is a novel candidate gene linked to susceptibility to HBV infection.

Implication of Genes for the N-Methyl-D-Aspartate (NMDA) Receptor in Substance Addictions.

Drug dependence is a chronic brain disease with harmful consequences for both individual users and society. Glutamate is a primary excitatory neurotransmitter in the brain, and both in vivo and in vitro experiments have implicated N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor, as an element in various types of addiction. Recent findings from genetics-based approaches such as genome-wide linkage, candidate gene association, genome-wide association (GWA), and next-generation sequencing have demonstrated the significant association of NMDA receptor subunit genes such as GluN3A, GluN2B, and GluN2A with various addiction-related phenotypes. Of these genes, GluN3A has been the most studied, and it has been revealed to play crucial roles in the etiology of addictions. In this communication, we provide an updated view of the genetic effects of NMDA receptor subunit genes and their functions in the etiology of addictions based on the findings from investigation of both common and rare variants as well as SNP-SNP interactions. To better understand the molecular mechanisms underlying addiction-related behaviors and to promote the development of specific medicines for the prevention and treatment of addictions, current efforts aim not only to identify more causal variants in NMDA receptor subunits by using large independent samples but also to reveal the molecular functions of these variants in addictions.

Transcriptome Analysis of Newly Emerged Honeybees Exposure to Sublethal Carbendazim During Larval Stage.

There are increasing concerns regarding the impact of agrochemical pesticides on non-target organisms. Pesticides could cause honeybee abnormal development in response to neurotoxins such as neonicotinoid. However, knowledge of carbendazim, a widespread fungicide in beekeeping practice, influencing on honeybee (Apis mellifera L.) brain development is lacking. Large-scale transcriptome approaches were applied to determine the changes in global gene expression in the brains of newly emerged honeybees after carbendazim exposure during the larval stage. To further understand the effects of carbendazim on the brain development of honeybees, the functions of differentially expressed genes were compared between the treatment and control groups. We found that neuroregulatory genes were down-regulated after carbendazim exposure, which suggest the neurotoxic effects of this fungicide on honeybee nervous system. Carbendazim exposure also altered the expression of genes implicated in metabolism, transport, sensor, and hormone. Notably, larvae in the carbendazim-treated group observed longer time to shift into the dormant pupal state than the control group. Moreover, a low juvenile hormone and high ecdysone titers were found in the treatment group compared to control group. The data is the first report of neurotoxic effects on honeybee caused by carbendazim, and the sublethal carbendazim may disturb honeybee development and is a potential chemical threating the honeybee colonies.

Association and cis-mQTL analysis of variants in CHRNA3-A5, CHRNA7, CHRNB2, and CHRNB4 in relation to nicotine dependence in a Chinese Han population.

Nicotine dependence (ND) is a worldwide health problem. Numerous genetic studies have demonstrated a significant association of variants in nicotinic acetylcholine receptors (nAChRs) with smoking behaviors. However, most of these studies enrolled only subjects of European or African ancestry. In addition, although an increasing body of evidence implies a causal connection of single-nucleotide polymorphisms (SNPs) and epigenetic regulation of gene expression, few studies of this issue have been reported. In this study, we performed both association and interaction analysis for 67 SNPs in CHRNA3-A5, CHRNA7, CHRNB2, and CHRNB4 with ND in a Chinese Han population (N = 5055). We further analyzed cis-mQTL for the three most significant SNPs and 5580 potential methylation loci within these target gene regions. Our results indicated that the SNPs rs1948 and rs7178270 in CHRNB4 and rs3743075 in CHRNA3 were significantly associated with the Fagerström Test for Nicotine Dependence (FTND) score (p = 6.6 × 10-5; p = 2.0 × 10-4, and p = 7.0 × 10-4, respectively). Haplotype-based association analysis revealed that two major haplotypes, T-G and C-A, formed by rs3743075-rs3743074 in CHRNA3, and other two major haplotypes, A-G-C and G-C-C, formed by rs1948-rs7178270-rs17487223 in CHRNB4, were significantly associated with the FTND score (p ≤ 8.0 × 10-4). Further, we found evidence for the presence of significant interaction among variants within CHRNA3/B4/A5, CHRNA4/B2/A5, and CHRNA7 in affecting ND, with corresponding p values of 5.8 × 10-6, 8.0 × 10-5, and 0.012, respectively. Finally, we identified two CpG sites (CpG_2975 and CpG_3007) in CHRNA3 that are significantly associated with three cis-mQTL SNPs (rs1948, rs7178270, rs3743075) in the CHRNA5/A3/B4 cluster (p ≤ 1.9 × 10-6), which formed four significant CpG-SNP pairs in our sample. Together, we revealed at least three novel SNPs in CHRNA3 and CHRNB4 to be significantly associated with the FTND score. Further, we showed that these significant variants contribute to ND via two methylated sites, and we demonstrated significant interaction affecting ND among variants in CHRNA5/A3/B4, CHRNA7, and CHRNA4/B2/A5. In sum, these findings provide robust evidence that SNPs in nAChR genes convey a risk of ND in the Chinese Han population.