Macrophage depletion blocks congenital SARM1-dependent neuropathy.

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

Loss of Stathmin-2, a hallmark of TDP-43-associated ALS, causes motor neuropathy.

TDP-43 mediates proper Stathmin-2 (STMN2) mRNA splicing, and STMN2 protein is reduced in the spinal cord of most patients with amyotrophic lateral sclerosis (ALS). To test the hypothesis that STMN2 loss contributes to ALS pathogenesis, we generated constitutive and conditional STMN2 knockout mice. Constitutive STMN2 loss results in early-onset sensory and motor neuropathy featuring impaired motor behavior and dramatic distal neuromuscular junction (NMJ) denervation of fast-fatigable motor units, which are selectively vulnerable in ALS, without axon or motoneuron degeneration. Selective excision of STMN2 in motoneurons leads to similar NMJ pathology. STMN2 knockout heterozygous mice, which better model the partial loss of STMN2 protein found in patients with ALS, display a slowly progressive, motor-selective neuropathy with functional deficits and NMJ denervation. Thus, our findings strongly support the hypothesis that STMN2 reduction owing to TDP-43 pathology contributes to ALS pathogenesis.

Strain-specific altered nicotine metabolism in 3,3'-diindolylmethane (DIM) exposed mice.

Two indole compounds, indole-3-carbinol (I3C) and its acid condensation product, 3,3'-diindolymethane (DIM), have been shown to suppress the expression of flavin-containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO-mediated nicotine N-oxygenation was decreased, whereas CYP-mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N-oxygenation without affecting CYP-mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6 J and CD1 mice fed 2500 parts per million (ppm) DIM for 14 days. In liver microsomes from DIM-fed mice, FMO-mediated nicotine N-oxygenation did not differ from the controls, but CYP-mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM-fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine-N-oxide were measured over 30 minutes. The DIM-fed mice showed greater cotinine concentrations compared with the controls 10 minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP-mediated nicotine metabolism in commonly studied mouse strains.

Beyond cigarettes per day. A genome-wide association study of the biomarker carbon monoxide.

RATIONALE: The CHRNA5-CHRNA3-CHRNB4 locus is associated with self-reported smoking behavior and also harbors the strongest genetic associations with chronic obstructive pulmonary disease (COPD) and lung cancer. Because the associations with lung disease remain after adjustment for self-reported smoking behaviors, it has been asserted that CHRNA5-CHRNA3-CHRNB4 variants increase COPD and lung cancer susceptibility independently of their effects on smoking. OBJECTIVES: To compare the genetic associations of exhaled carbon monoxide (CO), a biomarker of current cigarette exposure, with self-reported smoking behaviors. METHODS: A total of 1,521 European American and 247 African American current smokers recruited into smoking cessation studies were assessed for CO at intake before smoking cessation. DNA samples were genotyped using the Illumina Omni2.5 microarray. Genetic associations with CO and smoking behaviors (cigarettes smoked per day, Fagerstrom test for nicotine dependence) were studied. MEASUREMENTS AND MAIN RESULTS: Variants in the CHRNA5-CHRNA3-CHRNB4 locus, including rs16969968, a nonsynonymous variant in CHRNA5, are genomewide association study-significantly associated with CO (ß = 2.66; 95% confidence interval [CI], 1.74-3.58; P = 1.65 × 10(-8)), and this association remains strong after adjusting for smoking behavior (ß = 2.18; 95% CI, 1.32-3.04; P = 7.47 × 10(-7)). The correlation between CO and cigarettes per day is statistically significantly lower (z = 3.43; P = 6.07 × 10(-4)) in African Americans (r = 0.14; 95% CI, 0.02-0.26; P = 0.003) than in European-Americans (r = 0.36; 95% CI, 0.31-0.40; P = 0.0001). CONCLUSIONS: Exhaled CO, a biomarker that is simple to measure, captures aspects of cigarette smoke exposure in current smokers beyond the number of cigarettes smoked per day. Behavioral measures of smoking are therefore insufficient indices of cigarette smoke exposure, suggesting that genetic associations with COPD or lung cancer that persist after adjusting for self-reported smoking behavior may still reflect genetic effects on smoking exposure.

Pharmacotherapy effects on smoking cessation vary with nicotine metabolism gene (CYP2A6).

BACKGROUND AND AIMS: Evidence suggests that both the nicotinic receptor α5 subunit (CHRNA5) and Cytochrome P450 2A6 (CYP2A6) genotypes influence smoking cessation success and response to pharmacotherapy. We examine the effect of CYP2A6 genotype on smoking cessation success and response to cessation pharmacotherapy, and combine these effects with those of CHRNA5 genotypes. DESIGN: Placebo-controlled randomized smoking cessation trial. SETTING: Ambulatory care facility in Wisconsin, USA. PARTICIPANTS: Smokers (n = 709) of European ancestry were randomized to placebo, bupropion, nicotine replacement therapy or combined bupropion and nicotine replacement therapy. MEASUREMENTS: Survival analysis was used to model time to relapse using nicotine metabolism derived from CYP2A6 genotype-based estimates. Slow metabolism is defined as the lowest quartile of estimated metabolic function. FINDINGS: CYP2A6-defined nicotine metabolic function moderated the effect of smoking cessation pharmacotherapy on smoking relapse over 90 days [hazard ratio (HR) = 2.81, 95% confidence interval (CI) = 1.32-5.99, P = 0.0075], with pharmacotherapy significantly slowing relapse in fast (HR = 0.39, 95% CI = 0.28-0.55, P = 1.97 × 10(-8)), but not slow metabolizers (HR = 1.09, 95% CI = 0.55-2.17, P = 0.80). Further, only the effect of nicotine replacement, and not bupropion, varies with CYP2A6-defined metabolic function. The effect of nicotine replacement on continuous abstinence is moderated by the combined genetic risks from CYP2A6 and CHRNA5 (Wald = 7.44, d.f. = 1, P = 0.0064). CONCLUSIONS: Nicotine replacement therapy is effective among individuals with fast, but not slow, CYP2A6-defined nicotine metabolism. The effect of bupropion on relapse likelihood is unlikely to be affected by nicotine metabolism as estimated from CYP2A6 genotype. The variation in treatment responses among smokers with genes may guide future personalized smoking cessation interventions.

Variants in two adjacent genes, EGLN2 and CYP2A6, influence smoking behavior related to disease risk via different mechanisms.

Genome-wide significant associations with cigarettes per day (CPD) and risk for lung cancer and chronic obstructive pulmonary disease (COPD) were previously reported in a region of 19q13, including CYP2A6 (nicotine metabolism enzyme) and EGLN2 (hypoxia response). The associated single nucleotide polymorphisms (SNPs) were assumed to be proxies for functional variation in CYP2A6. Here, we demonstrate that when CYP2A6 and EGLN2 genotypes are analyzed together, the key EGLN2 variant, rs3733829, is not associated with nicotine metabolism independent of CYP2A6, but is nevertheless independently associated with CPD, and with breath carbon monoxide (CO), a phenotype associated with cigarette consumption and relevant to hypoxia. SNPs in EGLN2 are also associated with nicotine dependence and with smoking efficiency (CO/CPD). These results indicate a previously unappreciated novel mechanism behind genome-wide significant associations with cigarette consumption and disease risk unrelated to nicotine metabolism.

CYP2B6 non-coding variation associated with smoking cessation is also associated with differences in allelic expression, splicing, and nicotine metabolism independent of common amino-acid changes.

The Cytochrome P450 2B6 (CYP2B6) enzyme makes a small contribution to hepatic nicotine metabolism relative to CYP2A6, but CYP2B6 is the primary enzyme responsible for metabolism of the smoking cessation drug bupropion. Using CYP2A6 genotype as a covariate, we find that a non-coding polymorphism in CYP2B6 previously associated with smoking cessation (rs8109525) is also significantly associated with nicotine metabolism. The association is independent of the well-studied non-synonymous variants rs3211371, rs3745274, and rs2279343 (CYP2B6*5 and *6). Expression studies demonstrate that rs8109525 is also associated with differences in CYP2B6 mRNA expression in liver biopsy samples. Splicing assays demonstrate that specific splice forms of CYP2B6 are associated with haplotypes defined by variants including rs3745274 and rs8109525. These results indicate differences in mRNA expression and splicing as potential molecular mechanisms by which non-coding variation in CYP2B6 may affect enzymatic activity leading to differences in metabolism and smoking cessation.

The contribution of common UGT2B10 and CYP2A6 alleles to variation in nicotine glucuronidation among European Americans.

BACKGROUND: To develop a predictive genetic model of nicotine metabolism. UDP-glucuronosyltransferase-2B10 (UGT2B10) is the primary catalyst of nicotine glucuronidation. MATERIALS AND METHODS: The conversion of deuterated (D2)-nicotine to D2-nicotine-glucuronide, D2-cotinine, D2-cotinine-glucuronide, and D2-trans-3'-hydroxycotinine were quantified in 188 European Americans, and the contribution of UGT2B10 genotype to variability in first-pass nicotine glucuronidation assessed, following a procedure previously applied to nicotine C-oxidation. The proportion of total nicotine converted to nicotine-glucuronide [D2-nicotine-glucuronide/(D2-nicotine+D2-nicotine-glucuronide+D2-cotinine+D2-cotinine-glucuronide+D2-trans-3'-hydroxycotinine)] was the primary phenotype. RESULTS: The variant, rs61750900T (D67Y) (minor allele frequency=10%), is confirmed to abolish nicotine glucuronidation activity. Another variant, rs112561475G (N397D) (minor allele frequency=2%), is significantly associated with enhanced glucuronidation. rs112561475G is the ancestral allele of a well-conserved amino acid, indicating that the majority of human UGT2B10 alleles are derived hypomorphic alleles. CONCLUSION: CYP2A6 and UGT2B10 genotype explain 53% of the variance in oral nicotine glucuronidation in this sample. CYP2A6 and UGT2B10 genetic variants are also significantly associated with undeuterated (D0) nicotine glucuronidation in individuals smoking ad libitum. We find no evidence for further common variation markedly influencing hepatic UGT2B10 expression in European Americans.

A compensatory effect upon splicing results in normal function of the CYP2A6*14 allele.

A synonymous variant in the first exon of CYP2A6, rs1137115 (51G>A), defines the common reference allele CYP2A6*1A, and is associated with lower mRNA expression and slower in-vivo nicotine metabolism. Another common allele, CYP2A6*14, differs from CYP2A6*1A by a single variant, rs28399435 (86G>A, S29N). However, CYP2A6*14 shows in-vivo activity comparable with that of full-function alleles, and significantly higher than CYP2A6*1A. rs1137115A is predicted to create an exonic splicing suppressor site overlapping an exonic splicing enhancer (ESE) site in the first exon of CYP2A6, whereas rs28399435A is predicted to strengthen another adjacent ESE, potentially compensating for rs1137115A. Using an allelic expression assay to assess cDNAs produced from rs1137115 heterozygous liver biopsy samples, lower expression of the CYP2A6*1A allele is confirmed while CYP2A6*14 expression is found to be indistinguishable from that of rs1137115G alleles. Quantitative PCR assays to determine the relative abundance of spliced and unspliced or partially spliced CYP2A6 mRNAs in liver biopsy samples show that *1A/*1A homozygotes have a significantly lower ratio, due to both a reduction in spliced forms and an increase in unspliced or partially spliced CYP2A6. These results show the importance of common genetic variants that effect exonic splicing suppressor and ESEs to explain human variation regarding clinically-relevant phenotypes.

Effects upon in-vivo nicotine metabolism reveal functional variation in FMO3 associated with cigarette consumption.

BACKGROUND: Flavin-containing monooxygenases (FMO) catalyze the metabolism of nucleophilic heteroatom-containing drugs and xenobiotics, including nicotine. Rare mutations in FMO3 are responsible for defective N-oxidation of dietary trimethylamine leading to trimethylaminuria, and common genetic variation in FMO3 has been linked to interindividual variability in metabolic function that may be substrate specific. METHODS: A genetic model of CYP2A6 function is used as a covariate to reveal functional polymorphism in FMO3 that indirectly influences the ratio of deuterated nicotine metabolized to cotinine following oral administration. The association is tested between FMO3 haplotype and cigarette consumption in a set of nicotine-dependent smokers. RESULTS: FMO3 haplotype, based on all common coding variants in Europeans, significantly predicts nicotine metabolism and accounts for ∼2% of variance in the apparent percent of nicotine metabolized to cotinine. The metabolic ratio is not associated with FMO2 haplotype or an FMO1 expression quantitative trait locus. Cross-validation demonstrates calculated FMO3 haplotype parameters to be robust and significantly improve the predictive nicotine metabolism model over CYP2A6 genotype alone. Functional classes of FMO3 haplotypes, as determined by their influence on nicotine metabolism to cotinine, are also significantly associated with cigarettes per day in nicotine-dependent European Americans (n=1025, P=0.04), and significantly interact (P=0.016) with CYP2A6 genotype to predict cigarettes per day. CONCLUSION: These findings suggest that common polymorphisms in FMO3 influence nicotine clearance and that these genetic variants in turn influence cigarette consumption.

Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6.

This study demonstrates a novel approach to test associations between highly heterogeneous genetic loci and complex phenotypes. Previous investigations of the relationship between Cytochrome P450 2A6 (CYP2A6) genotype and smoking phenotypes made comparisons by dividing subjects into broad categories based on assumptions that simplify the range of function of different CYP2A6 alleles, their numerous possible diplotype combinations and non-additive allele effects. A predictive model that translates CYP2A6 diplotype into a single continuous variable was previously derived from an in vivo metabolism experiment in 189 European Americans. Here, we apply this model to assess associations between genotype, inferred nicotine metabolism and smoking behaviors in larger samples without direct nicotine metabolism measurements. CYP2A6 genotype is not associated with nicotine dependence, as defined by the Fagerström Test of Nicotine Dependence, demonstrating that cigarettes smoked per day (CPD) and nicotine dependence have distinct genetic correlates. The predicted metric is significantly associated with CPD among African Americans and European American dependent smokers. Individual slow metabolizing genotypes are associated with lower CPD, but the predicted metric is the best predictor of CPD. Furthermore, optimizing the predictive model by including additional CYP2A6 alleles improves the fit of the model in an independent data set and provides a novel method of predicting the functional impact of alleles without direct metabolism measurements. Lastly, comprehensive genotyping and in vivo metabolism data are used to demonstrate that genome-wide significant associations between CPD and single nucleotide polymorphisms are the result of synthetic associations.