Clinical, molecular, physiologic, and therapeutic feature of patients with CHRNA4 and CHRNB2 deficiency: A systematic review.

The α4ß2 nAChRs are crucial ion channels that control neurotransmitter release and play a role in various physiologic and pathologic processes. CHRNA4 encodes the α4-nAChRs, while CHRNB2 encodes the ß2-nAChRs. Recent studies have found different variants of α4ß2-nAChRs in individuals with conditions such as AD, ADHD, ALS, PD, and brain abnormalities. We conducted a scoping review following a six-stage methodology structure and adhering to PRISMA guidelines. We systematically reviewed articles using relevant keywords up to October 2, 2023. In this summary, we cover the clinical symptoms reported, the genes and protein structure of CHRNA4 and CHRNB2, mutations in these genes, inheritance patterns, the functional impact of mutations and polymorphisms in CHRNA4 and CHRNB2, and the epidemiology of these diseases. Recent research indicates that nAChRs may play a significant role in neurodegenerative disorders, possibly impacting neuronal function through yet undiscovered regulatory pathways. Studying how nAChRs interact with disease-related aggregates in neurodegenerative conditions may lead to new treatment options for these disorders.

A novel alcohol+nicotine co-use self-administration procedure reveals sex differences and differential alteration of mesocorticolimbic TLR- and cholinergic-related neuroimmune gene expression in rats.

Although alcohol and nicotine are two of the most commonly co-used drugs with upwards of 90% of adults with an alcohol use disorder (AUD) in the US also smoking, we don't tend to study alcohol and nicotine use this way. The current studies sought to develop and assess a novel alcohol + nicotine co-access self-administration (SA) model in adult male and female Long-Evans rats. Further, both drugs are implicated in neuroimmune function, albeit in largely opposing ways. Chronic alcohol use increases neuroinflammation via toll-like receptors (TLRs) which in turn increases alcohol intake. By contrast, nicotine produces anti-inflammatory effects, in part, through the monomeric alpha7 receptor (ChRNa7). Following long-term co-access (6 months), rats reliably administered both drugs during daily sessions, however males generally responded for more alcohol and females for nicotine. This was reflected in plasma analysis with translationally relevant intake levels of both alcohol and nicotine, making it invaluable in studying the effects of co-use on behavior and CNS function. Moreover, male rats show sensitivity to alterations in alcohol concentration whereas females show sensitivity to alterations in nicotine concentration. Rats trained on this procedure also developed an anxiogenic phenotype. Finally, we assessed alterations in neuroimmune-related gene expression in the medial prefrontal cortex - prelimbic, (mPFC-PL), nucleus accumbens core (AcbC), and ventral tegmental area (VTA). In the AcbC, where α7 expression was increased and ß2 was decreased, markers of pro-inflammatory activity were decreased, despite increases in TLR gene expression suggesting that co-use with nicotine modulates inflammatory state downstream from the receptor level. By contrast, in mPFC-PL where α7 was not increased, both TLRs and downstream proinflammatory markers were increased. Taken together, these findings support that there are brain regional and sex differences with co-use of alcohol + nicotine SA and suggest that targeting nicotinic α7 may represent a novel strategy for treating alcohol + nicotine co-dependence.

Familial Epilepsy Associated With Concurrent CHRNB2 Mutation and RBFOX1 Exon Deletion: A Case Report.

Understanding the genetic basis of epilepsy may lead to an improved understanding of its etiology, more precise medical management, and ultimately improved outcomes. It is imperative for patients with epilepsy to obtain a molecular diagnosis, especially when strong familial epilepsy is discovered. We investigated a multi-generational family with epilepsy. The proband was a 19-year-old female who experienced focal onset seizures, with presenting symptoms of feeling dizzy, disorientation, and loss of consciousness. Her electroencephalography (EEG) studies revealed interictal focal slowing and sharp waves in both the left or right hemispheres independently. EEG monitoring showed that the seizures arose from the left fronto-temporal region and her brain MRI was normal. The proband's sister also suffered from focal onset seizures. Her EEG showed focal epileptiform discharge in the right temporal region, and her brain MRI was unrevealing. Two genetic tests were conducted for the proband: 1) array comparative genomic hybridization (CGH) revealed 16p13.3 deletion but no 22q deletion; and 2) next generation sequencing (NGS) Epilepsy Panel revealed a few variants of uncertain significance (VUS), including in CHRNB2 (c.1423A>G, p.Ile475Val) and RBFOX1 (RNA binding fox-1 homolog 1) (exon1-2 deletion). The proband's sister also carries both the CHRNB2 (cholinergic receptor nicotinic beta 2) variant and RBFOX1 deletion. The proband's father carries the CHRNB2 variant, and her brother and mother carry the deletion of RBFOX1. In this family, the co-expression of the CHRNB2 variant and RBFOX1 deletion may cause the clinical seizures seen in the proband and her sister. It is also possible that the RBFOX1 deletion is associated with an increased risk of seizure disorder with variable expressivity.

BEX2 is poor prognostic factor and required for cancer stemness in gastric cancer.

Gastric cancer is the fifth most common malignancy worldwide. However, targeted therapy for advanced gastric cancer is still limited. Here, we report BEX2 (Brain expressed X-linked 2) as a poor prognostic factor in two gastric cancer cohorts. BEX2 expression was increased in spheroid cells, and its knockdown decreased aldefluor activity and cisplatin resistance. BEX2 was found to upregulate CHRNB2 (Cholinergic Receptor Nicotinic Beta 2 Subunit) expression, a cancer stemness-related gene, in a transcriptional manner, and the knockdown of which also decreases aldefluor activity. Collectively, these data are suggestive of the role of BEX2 in the malignant process of gastric cancer, and as a promising therapeutic target.

CHRNB2 represses pancreatic cancer migration and invasion via inhibiting ß-catenin pathway.

BACKGROUND: Pancreatic cancer is one of the most lethal disease with highly fatal and aggressive properties. Lymph node ratio (LNR), the ratio of the number of metastatic lymph nodes to the total number of examined lymph nodes, is an important index to assess lymphatic metastasis and predict prognosis, but the molecular mechanism underlying high LNR was unclear. METHODS: Gene expression and clinical information data of pancreatic cancer were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Patients in TCGA were averagely divided into low and high LNR groups. Then, Weighted Gene Co-expression Network Analysis (WGCNA) was utilized to build co-expression network to explore LNR-related modules and hub genes. GO and KEGG analysis was performed to find key pathways related to lymph node metastasis. Next, GSE101448 and the overall survival data in TCGA was employed to further select significant genes from hub genes. Considering the key role of CHRNB2 in LNR and survival, gene set enrichment analysis (GSEA) was applied to find pathways related to CHRNB2 expression in pancreatic cancer. The contribution of CHRNB2 to migrative and invasive ability of pancreatic cancer cells was confirmed by Transwell assays. We finally explored the role of CHRNB2 in EMT and ß-catenin pathway via Western Blot. RESULTS: High LNR was significantly related to high T stages and poor prognosis. In WGCNA, 14 hub genes (COL5A1, FN1, THBS2, etc.) were positively related to high LNR, 104 hub genes (FFAR1, SCG5, TMEM63C, etc.) were negatively related to high LNR. After taking the intersection with GSE101448, 13 genes (CDK5R2, SYT7, CACNA2D2, etc.) which might prevent lymph node metastasis were further selected. Among them, CHRNB2 showed the strongest relationship with long survival. Moreover, CHRNB2 also negatively related to the T stages and LNR. Next, knockdown of CHRNB2 expression could acetylcholine (ACh)-independently increase the migration and invasion of pancreatic cancer cells, while CHRNB2 overexpression ACh-independently decrease the migration and invasion of pancreatic cancer cells. For exploring the underlying mechanism, CHRNB2 downregulated ß-catenin pathway might through controlling its upstream regulators such as SOX6, SRY, SOX17, and TCF7L2. CONCLUSIONS: CHRNB2 negatively relates to lymph node metastasis in pancreatic cancer patients. CHRNB2 could inhibit ß-catenin pathway, EMT, migration and invasion of pancreatic cancer cells via ACh-independent mechanism.

The ß2V287L nicotinic subunit linked to sleep-related epilepsy differently affects fast-spiking and regular spiking somatostatin-expressing neurons in murine prefrontal cortex.

Mutant subunits of the neuronal nicotinic ACh receptor (nAChR) can cause Autosomal Dominant Sleep-related Hypermotor Epilepsy (ADSHE), characterized by frontal seizures during non-rapid eye movement (NREM) sleep. We studied the cellular bases of the pathogenesis in brain slices from mice conditionally expressing the ADSHE-linked ß2V287L nAChR subunit. ß2V287L mice displayed minor structural alterations, except for a ~10% decrease of prefrontal cortex thickness. However, they showed a substantial decrease of the excitatory input to layer V fast-spiking (FS) interneurons, despite a concomitant increase in the number of glutamatergic terminals around the cell soma. Hence, prefrontal hyperexcitability may depend on a permanent impairment of surround inhibition. The effect disappeared when ß2V287L was silenced until postnatal day 15th, suggesting that the transgene selectively affects the maturation of glutamatergic synapses on FS neurons. The other main population of interneurons in layer V was constituted by somatostatin-expressing regular spiking cells. When tested with 10 µM nicotine, these displayed larger somatic nicotinic currents in transgenic mice. Thus, during wakefulness, activation of ß2V287L-containing nAChRs by the high cholinergic tone may counteract hyperexcitability by promoting local inhibition by somatostatin-expressing cells and decreasing the effect of glutamatergic deficit in FS neurons. This interpretation was tested in networks disinhibited by 2 µM bicuculline. Slices expressing ß2V287L were more susceptible to develop synchronized activity in the absence of nicotine. Addition of the drug boosted excitability in the controls, but had little effect in ß2V287L. Our findings suggest why NREM sleep favors ADSHE seizures and nicotine can be palliative in patients.

Long Non-coding RNAs Gabarapl2 and Chrnb2 Positively Regulate Inflammatory Signaling in a Mouse Model of Dry Eye.

Purpose: To elucidate the expression profile and the potential role of long non-coding ribonucleic acids (RNAs) (lncRNAs) in a dry eye disease (DED) model. Methods: A DED model was established in C57BL/6J mice with 0.2% benzalkonium chloride (BAC) twice a day for 14 days. The differentially expressed lncRNAs were detected by RNA-seq technology (Gene Expression Omnibus, GEO GSE186450) and the aberrantly expressed lncRNAs were further verified by RT-qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related candidate genes and potential pathological pathways. Cells from a human corneal epithelial cell line (HCECs) were cultured under hyperosmolarity. The regulation of inflammatory factors by silencing potential targeted lncRNAs was verified in vitro in HCECs. Results: In our study, a significant increase in corneal fluorescence staining and a reduction in tear production were observed in DED mice at all follow-ups compared with the controls, and the differences were increasing over time. In total, 2,649 upregulated and 704 downregulated lncRNAs were identified in DED mice. We selected six aberrantly expressed and most abundant lncRNAs and performed RT-qPCR using the samples for RNA-seq. Chrnb2, Gabarapl2, and Usp31 were thereby confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that the neuroactive ligand-receptor interaction signaling pathway was the most enriched, followed by the calcium signaling pathway and cytokine-cytokine receptor interaction. Following treatment of Gabarapl2 siRNA and Chrnb2 siRNA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were significantly downregulated in the HCECs. Conclusion: Our study suggests that Chrnb2 and Gabarapl2 may be involved in the inflammation response by regulating TNF-α, IL-1ß, and IL-6 in DED. These candidate lncRNAs may be both potential biomarkers and therapeutic targets for DED.

Nicotinic Receptors in Sleep-Related Hypermotor Epilepsy: Pathophysiology and Pharmacology.

Sleep-related hypermotor epilepsy (SHE) is characterized by hyperkinetic focal seizures, mainly arising in the neocortex during non-rapid eye movements (NREM) sleep. The familial form is autosomal dominant SHE (ADSHE), which can be caused by mutations in genes encoding subunits of the neuronal nicotinic acetylcholine receptor (nAChR), Na+-gated K+ channels, as well as non-channel signaling proteins, such as components of the gap activity toward rags 1 (GATOR1) macromolecular complex. The causative genes may have different roles in developing and mature brains. Under this respect, nicotinic receptors are paradigmatic, as different pathophysiological roles are exerted by distinct nAChR subunits in adult and developing brains. The widest evidence concerns α4 and ß2 subunits. These participate in heteromeric nAChRs that are major modulators of excitability in mature neocortical circuits as well as regulate postnatal synaptogenesis. However, growing evidence implicates mutant α2 subunits in ADSHE, which poses interpretive difficulties as very little is known about the function of α2-containing (α2*) nAChRs in the human brain. Planning rational therapy must consider that pharmacological treatment could have different effects on synaptic maturation and adult excitability. We discuss recent attempts towards precision medicine in the mature brain and possible approaches to target developmental stages. These issues have general relevance in epilepsy treatment, as the pathogenesis of genetic epilepsies is increasingly recognized to involve developmental alterations.

Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy.

PURPOSE: Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy. METHOD: We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene. RESULTS: We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4ß2 (wild-type), α4ß2-Thr26Met (homozygote), and α4ß2/ß2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation. CONCLUSIONS: This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

Genetic models of focal epilepsies.

Focal epilepsies were for a long time thought to be acquired disorders secondary to cerebral lesions. However, the important role of genetic factors in focal epilepsies is now well established. Several focal epilepsy syndromes are now proven to be monogenic disorders. While earlier genetic studies suggested a strong contribution of ion channel and neurotransmitter receptor genes, later work has revealed alternative pathways, among which the mammalian target of rapamycin (mTOR) signal transduction pathway with DEPDC5. In this article, we provide an update on the mutational spectrum of neuronal nicotinic acetylcholine receptor genes (CHRNA4, CHRNB2, CHRNA2) and KCNT1 causing autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and of LGI1 in autosomal dominant epilepsy with auditory features (ADEAF). We also emphasize, through a review of the current literature, the contribution of in vitro and in vivo models developed to unveil the pathogenic mechanisms underlying these two epileptic syndromes.

Generalized epilepsy in a family with basal ganglia calcifications and mutations in SLC20A2 and CHRNB2.

BACKGROUND: The genetic understanding of primary familial brain calcification (PFBC) has increased considerably in recent years due to the finding of causal genes like SLC20A2, PDGFRB and PDGFB. The phenotype of PFBC is complex and has as of yet been poorly delineated. The most common clinical presentations include movement disorders, cognitive symptoms and psychiatric conditions. We report a family including two sisters with brain calcifications due to a variant in SLC20A2 and generalized tonic-clonic seizures as the principal phenotypic trait. METHODS: The affected siblings underwent whole exome sequencing and candidate variants and cosegregation in the family were validated by Sanger sequencing. RESULTS: Both siblings and their asymptomatic father were heterozygous for a variant in SLC20A2. The siblings also had a variant in CHRNB2, a known epilepsy gene associated with autosomal dominant frontal lobe epilepsy, which they had inherited from the mother. CONCLUSIONS: To our knowledge, the reported siblings represent the third and fourth subjects with confirmed SLC20A2 variants exhibiting epilepsy as a phenotypic trait. Our findings support seizures as part of the phenotypic spectrum of SLC20A2-related PFBC. However, the present phenotype may also result from additional genetic influence, such as the identified missense variant in CHRNB2.

Agonist and antagonist effects of tobacco-related nitrosamines on human α4ß2 nicotinic acetylcholine receptors.

Regulation of the "neuronal" nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4ß2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 µM. At 100 µM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4ß2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 µM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

Mutational analysis of CHRNB2, CHRNA2 and CHRNA4 genes in Chinese population with autosomal dominant nocturnal frontal lobe epilepsy.

OBJECTIVE: The present study aims to investigate the gene mutations of CHRNB2, CHRNA2 and CHRNA4 in Chinese population with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). METHODS: 257 ADNFLE patients (74 sporadic and 32 familial) were collected, including 42 pedigree patients and 215 sporadic cases. Exon mutational screening of CHRNB2, CHRNA2 and CHRNA4 was performed by direct PCR sequencing. RESULTS: No published mutations of CHRNB2, CHRNA4 and CHRNA2 genes were detected in this study. Three kinds of c.SNP (c.66C> T, c.249C> T, c.375A> G) were detected on the 2(nd) and 5(th) exons of CHRNA2; six kinds of c.SNP (c.639T> C, c.678T> C, c.1209G> T, c.1227T> C, c.1659G> A, c.1629C> T) were detected on the 5(th) exon of CHRNA4. Three novel mutations were discovered, respectively locating on the exon 5 of CHRNA4 gene (c.570C> T), 5(th) and 6(th) exons of CHRNB2 gene (c.483C> T and c.1407C> G). The three mutations were absent in 200 healthy controls, indicating that the mutations were very rare. CONCLUSION: CHRNA4, CHRNB2 and CHRNA2 may be not the causative genes of Chinese ADNFLE population. Whether the three novel synonymous mutations were genetic factors of ADNFLE pathogenesis in Chinese Han population needs to be further studied.

CHRNA4 rs1044396 is associated with smoking cessation in varenicline therapy.

BACKGROUND: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4 and CHRNB2 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. METHODS: This cohort study enrolled 483 smoking patients who received behavioral counseling and drug treatment (varenicline, bupropion, and/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196) and CHRNB2 (rs2072660 and rs2072661) polymorphisms were genotyped by high resolution melting analysis. RESULTS: Patients with rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%; p = 0.007, n = 167). The CT or TT genotypes were associated with higher odds ratio for success (OR = 1.67, 95% CI = 1.10-2.53, p = 0.02), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. CONCLUSION: The CHRNA4 rs1044396 is associated with smoking cessation in individuals on varenicline therapy. We suggest that this polymorphism influences the varenicline response, but replications of this finding are needed.

The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non-rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4ß2(*) subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na(+)-dependent K(+) channel), DEPD5 (Disheveled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex.

Association of alpha4beta2 nicotinic receptor and heavy smoking in schizophrenia.

INTRODUCTION: Previously we suggested that the CHRNA7 polymorphism in nicotinic receptor genes, in particular the D15S1360 in CHRNA7, is associated with smoking in schizophrenia. Schizophrenia patients are usually heavy smokers. In this study we hypothesized that high-affinity nicotinic receptors are associated with smoking in such patients. OBJECTIVE: To investigate the role of alpha4 (Ch 20) and beta2 (Ch 1) genes in conferring a risk for smoking and for smoking a large number of cigarettes daily in subjects with schizophrenia. METHODS: Our study sample consisted of 241 white European schizophrenia patients (157 smokers and 84 nonsmokers) from the Toronto area. Current smoking status was assessed by the medical history. We investigated 4 markers located in the CHRNA4 gene and 3 markers located in the CHRNB2 gene. RESULTS: There was no difference in age or ethnicity between the 2 groups and the population was not stratified (lambda=0.4527). We found a significant association between the CHRNA4 rs3746372 allele 1 and a large number of cigarettes smoked daily (p=0.0203). The intragenic interaction between rs3787116 and rs3746372 (p = 0.0050) in CHRNA4 showed a significant interaction for the number of cigarettes smoked. CONCLUSION: Although our findings suggest an association between rs3746372 allele 1 and heavy smoking, further study is warranted to investigate the relation between smoking and high-affinity nicotinic receptor genes in schizophrenia.