Methylomic profiling and replication implicates deregulation of PCSK9 in alcohol use disorder.

Alcohol use disorder (AUD) is a common and chronic disorder with substantial effects on personal and public health. The underlying pathophysiology is poorly understood but strong evidence suggests significant roles of both genetic and epigenetic components. Given that alcohol affects many organ systems, we performed a cross-tissue and cross-phenotypic analysis of genome-wide methylomic variation in AUD using samples from 3 discovery, 4 replication, and 2 translational cohorts. We identified a differentially methylated region in the promoter of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene that was associated with disease phenotypes. Biological validation showed that PCSK9 promoter methylation is conserved across tissues and positively correlated with expression. Replication in AUD datasets confirmed PCSK9 hypomethylation and a translational mouse model of AUD showed that alcohol exposure leads to PCSK9 downregulation. PCSK9 is primarily expressed in the liver and regulates low-density lipoprotein cholesterol (LDL-C). Our finding of alcohol-induced epigenetic regulation of PCSK9 represents one of the underlying mechanisms between the well-known effects of alcohol on lipid metabolism and cardiovascular risk, with light alcohol use generally being protective while chronic heavy use has detrimental health outcomes.

Association of genetic ancestry with striatal dopamine D2/D3 receptor availability.

Despite ethnic differences in allele frequencies of variants in dopaminergic genes associated with dopamine D2/D3 receptor availability (D2R), no study to date has investigated the relationship between genetic ancestry and striatal D2R. Here, we show that ancestry-informative markers significantly predict dorsal striatal D2R in 117 healthy ethnically diverse residents of the New York metropolitan area using Positron Emission Tomography (PET) with [11C]raclopride (P<0.0001), while correcting for age, sex, BMI, education, smoking status, and estimated socioeconomic status (ZIP codes). Effects of ethnicity on D2R were not driven by variation in dopaminergic candidate genes. Instead, candidate gene associations with striatal D2R were diminished when correcting for ancestry. These findings imply that future studies investigating D2 receptor genes should covary for genetic ancestry or study homogeneous populations. Moreover, ancestry studies on human neurobiology should control for socioeconomic differences between ethnic groups.

Defining the role of corticotropin releasing factor binding protein in alcohol consumption.

The corticotropin releasing factor (CRF) exerts its effects by acting on its receptors and on the binding protein (CRFBP), and has been implicated in alcohol use disorder (AUD). Therefore, identification of the exact contribution of each protein that mediates CRF effects is necessary to design effective therapeutic strategies for AUD. A series of in vitro/in vivo experiments across different species were performed to define the biological discrete role of CRFBP in AUD. First, to establish the CRFBP role in receptor signaling, we developed a novel chimeric cell-based assay and showed that CFRBP full length can stably be expressed on the plasma membrane. We discovered that only CRFBP(10 kD) fragment is able to potentiate CRF-intracellular Ca2+ release. We provide evidence that CRHBP gene loss increased ethanol consumption in mice. Then, we demonstrate that selective reduction of CRHBP expression in the center nucleus of the amygdala (CeA) decreases ethanol consumption in ethanol-dependent rats. CRFBP amygdalar downregulation, however, does not attenuate yohimbine-induced ethanol self-administration. This effect was associated with decreased hemodynamic brain activity in the CRFBP-downregulated CeA and increased hemodynamic activity in the caudate putamen during yohimbine administration. Finally, in alcohol-dependent patients, genetic variants related to the CRFBP(10 kD) fragment were associated with greater risk for alcoholism and anxiety, while other genetic variants were associated with reduced risk for anxiety. Taken together, our data provide evidence that CRFBP may possess both inhibitory and excitatory roles and may represent a novel pharmacological target for the treatment of AUD.

The glucagon-like peptide-1 receptor as a potential treatment target in alcohol use disorder: evidence from human genetic association studies and a mouse model of alcohol dependence.

The hormone glucagon-like peptide-1 (GLP-1) regulates appetite and food intake. GLP-1 receptor (GLP-1R) activation also attenuates the reinforcing properties of alcohol in rodents. The present translational study is based on four human genetic association studies and one preclinical study providing data that support the hypothesis that GLP-1R may have a role in the pathophysiology of alcohol use disorder (AUD). Case-control analysis (N = 908) was performed on a sample of individuals enrolled in the National Institute on Alcohol Abuse and Alcoholism (NIAAA) intramural research program. The Study of Addiction: Genetics and Environment (SAGE) sample (N = 3803) was used for confirmation purposes. Post hoc analyses were carried out on data from a human laboratory study of intravenous alcohol self-administration (IV-ASA; N = 81) in social drinkers and from a functional magnetic resonance imaging study in alcohol-dependent individuals (N = 22) subjected to a Monetary Incentive Delay task. In the preclinical study, a GLP-1R agonist was evaluated in a mouse model of alcohol dependence to demonstrate the role of GLP-1R for alcohol consumption. The previously reported functional allele 168Ser (rs6923761) was nominally associated with AUD (P = 0.004) in the NIAAA sample, which was partially replicated in males of the SAGE sample (P = 0.033). The 168 Ser/Ser genotype was further associated with increased alcohol administration and breath alcohol measures in the IV-ASA experiment and with higher BOLD response in the right globus pallidus when receiving notification of outcome for high monetary reward. Finally, GLP-1R agonism significantly reduced alcohol consumption in a mouse model of alcohol dependence. These convergent findings suggest that the GLP-1R may be an attractive target for personalized pharmacotherapy treatment of AUD.

The contribution of rare and common variants in 30 genes to risk nicotine dependence.

Genetic and functional studies have revealed that both common and rare variants of several nicotinic acetylcholine receptor subunits are associated with nicotine dependence (ND). In this study, we identified variants in 30 candidate genes including nicotinic receptors in 200 sib pairs selected from the Mid-South Tobacco Family population with equal numbers of African Americans (AAs) and European Americans (EAs). We selected 135 of the rare and common variants and genotyped them in the Mid-South Tobacco Case-Control (MSTCC) population, which consists of 3088 AAs and 1430 EAs. None of the genotyped common variants showed significant association with smoking status (smokers vs non-smokers), Fagerström Test for ND scores or indexed cigarettes per day after Bonferroni correction. Rare variants in NRXN1, CHRNA9, CHRNA2, NTRK2, GABBR2, GRIN3A, DNM1, NRXN2, NRXN3 and ARRB2 were significantly associated with smoking status in the MSTCC AA sample, with weighted sum statistic (WSS) P-values ranging from 2.42 × 10(-3) to 1.31 × 10(-4) after 10(6) phenotype rearrangements. We also observed a significant excess of rare nonsynonymous variants exclusive to EA smokers in NRXN1, CHRNA9, TAS2R38, GRIN3A, DBH, ANKK1/DRD2, NRXN3 and CDH13 with WSS P-values between 3.5 × 10(-5) and 1 × 10(-6). Variants rs142807401 (A432T) and rs139982841 (A452V) in CHRNA9 and variants V132L, V389L, rs34755188 (R480H) and rs75981117 (N549S) in GRIN3A are of particular interest because they are found in both the AA and EA samples. A significant aggregate contribution of rare and common coding variants in CHRNA9 to the risk for ND (SKAT-C: P=0.0012) was detected by applying the combined sum test in MSTCC EAs. Together, our results indicate that rare variants alone or combined with common variants in a subset of 30 biological candidate genes contribute substantially to the risk of ND.

A preliminary study suggests that nicotine and prefrontal dopamine affect cortico-striatal areas in smokers with performance feedback.

Nicotine and tonic dopamine (DA) levels [as inferred by catechol-O-methyl tranferase (COMT) Val158Met genotype] interact to affect prefrontal processing. Prefrontal cortical areas are involved in response to performance feedback, which is impaired in smokers. We investigated whether there is a nicotine × COMT genotype interaction in brain circuitry during performance feedback of a reward task. We scanned 23 healthy smokers (10 Val/Val homozygotes, 13 Met allele carriers) during two fMRI sessions while subjects were wearing a nicotine or placebo patch. A significant nicotine × COMT genotype interaction for BOLD signal during performance feedback in cortico-striatal areas was seen. Activation in these areas during the nicotine patch condition was greater in Val/Val homozygotes and reduced in Met allele carriers. During negative performance feedback, the change in activation in error detection areas such as anterior cingulate cortex (ACC)/superior frontal gyrus on nicotine compared to placebo was greater in Val/Val homozygotes compared to Met allele carriers. With transdermal nicotine administration, Val/Val homozygotes showed greater activation with performance feedback in the dorsal striatum, area associated with habitual responding. In response to negative feedback, Val/Val homozygotes had greater activation in error detection areas, including the ACC, suggesting increased sensitivity to loss with nicotine exposure. Although these results are preliminary due to small sample size, they suggest a possible neurobiological mechanism underlying the clinical observation that Val/Val homozygotes, presumably with elevated COMT activity compared to Met allele carriers and therefore reduced prefrontal DA levels, have poorer outcomes with nicotine replacement therapy.

A CHRNA5 allele related to nicotine addiction and schizophrenia.

Schizophrenia and nicotine addiction are both highly heritable phenotypes. Because individuals with schizophrenia have a higher rate of smoking than those in the general population, one could hypothesize that genes associated with smoking might be overrepresented in schizophrenia and thus help explain their increased smoking incidence. Although a number of genes have been proposed to explain the increased smoking risk in schizophrenia, none of them have been consistently linked to smoking and schizophrenia, and thus difficult to explain the increased smoking in schizophrenia. A functional smoking-related nicotinic acetylcholine receptor α5 subunit gene (CHRNA5) nonsynonymous single nucleotide polymorphism (SNP) rs16969968 (Asp398Asn) has recently been discovered and replicated. As such, we tested whether this variant contributes to smoking in schizophrenia in a sample of 313 schizophrenia patients and 525 controls. The Asp398Asn risk allele is significantly associated with smoking severity independently in schizophrenia patient smokers (P = 0.001) and control smokers (P = 0.029). Furthermore, the same risk allele is significantly associated with schizophrenia in both Caucasian (P = 0.022) and African-American (P = 0.006) nonsmoker schizophrenia patients compared with control nonsmokers. Intriguingly, this SNP was not significantly associated with smoking status (smokers vs. nonsmokers) in either schizophrenia patients or controls. Therefore, our study identifies a genetic variant that is simultaneously linked to smoking and schizophrenia in the same cohort, but whether this SNP contributes to the increased smoking prevalence in schizophrenia patients requires additional studies.

A novel form of autosomal recessive pure hereditary spastic paraplegia maps to chromosome 13q14.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disorder characterized by a progressive weakening and spasticity of the lower limbs. HSP is classified according to the presence or absence of accompanying neurologic problems and by the mode of inheritance. Currently, 17 loci have been linked to the various forms of HSP. OBJECTIVE: To determine the chromosomal location of a gene causing pure autosomal recessive spastic paraplegia. METHODS: Genotyping using fluorescently labeled microsatellite markers was performed on three affected individuals and three unaffected individuals from a family displaying pure autosomal recessive HSP (ARHSP) and sensorineural deafness. All family members were then included in the analysis to narrow the genetic interval. Candidate genes were screened for the presence of mutations by heteroduplex analysis. RESULTS: The paraplegic trait linked to a 1.8-Mb region of chromosome 13q14 flanked by the FLJ11712 gene and the microsatellite marker D13S270. The deafness did not link to this region and did not cosegregate with the paraplegic trait. CONCLUSION: The HSP that this family had represents a novel genetic form of pure ARHSP as no other form of HSP (autosomal dominant or recessive) has been linked to chromosome 13.

Mutation at the anophthalmic white locus in Syrian hamsters: haploinsufficiency in the Mitf gene mimics human Waardenburg syndrome type 2.

Mutations in MITF (microphthalmia transcription factor) cause Waardenburg syndrome type 2 (WS2A) in humans, an autosomal dominant disorder consisting of deafness and hypopigmentation. Phenotypes vary significantly within WS2 pedigrees, and there is generally no correlation between the predicted biochemical properties of mutant MITF proteins and disease severity. We have identified a nonsense mutation in the Mitf gene of the anophthalmic white Wh) Syrian hamster that destabilizes its mRNA and prevents the encoded basic helix-loop-helix leucine zipper (bHLHzip) protein from dimerizing or binding DNA target sites. Although the resulting polypeptide does not act as a dominant-negative species in vitro , the Wh mutation is inherited as a semi-dominant trait. It thus more closely resembles WS2 than comparable Mitf alleles in laboratory mice and rats, which are expressed as purely recessive traits.

Mutations in microphthalmia, the mouse homolog of the human deafness gene MITF, affect neuroepithelial and neural crest-derived melanocytes differently.

The mouse microphthalmia (Mitf) gene encodes a basic-helix-loop-helix-zipper transcription factor whose mutations are associated with abnormalities in neuroepithelial and neural crest-derived melanocytes. In wild type embryos, Mitf expression in neuropithelium and neural crest precedes that of the melanoblast marker Dct, is then co-expressed with Dct, and gradually fades away except in cells in hair follicles. In embryos with severe Mitf mutations, neural crest-derived Mitf-expressing cells are rare, lack Dct expression, and soon become undetectable. In contrast, the neuroepithelial-derived Mitf-expressing cells of the retinal pigment layer are retained, express Dct, but not the melanogenic enzyme genes tyrosinase and Tyrp1, and remain unpigmented. The results show that melanocyte development critically depends on functional Mitf and that Mitf mutations affect the neural crest and the neuroepithelium in different ways.

Antiviral determinants of rat Mx GTPases map to the carboxy-terminal half.

Rat Mx2 and rat Mx3 are two alpha/beta interferon-inducible cytoplasmic GTPases that differ in three residues in the amino-terminal third, which also contains the tripartite GTP-binding domain, and that differ in five residues in the carboxy-terminal quarter, which also contains a dimerization domain. While Mx2 is active against vesicular stomatitis virus (VSV), Mx3 lacks antiviral activity. We mapped the functional difference between Mx2 and Mx3 protein to two critical residues in the carboxy-terminal parts of the molecules. An exchange of either residue 588 or 630 of Mx2 with the corresponding residues of Mx3 abolished anti-VSV activity, and the introduction of the two Mx2 residues on an Mx3 background partially restored anti-VSV activity. These results are consistent with the facts that Mx2 and Mx3 have similar intrinsic GTPase activities and that the GTPase domain of Mx3 can fully substitute for the GTPase domain of Mx2. Nevertheless, the amino-terminal third containing the GTP-binding domain is necessary for antiviral activity, since an amino-terminally truncated Mx2 protein is devoid of anti-VSV activity. Furthermore, Fab fragments of a monoclonal antibody known to neutralize antiviral activity block GTPase activity by binding an epitope in the carboxy-terminal half of Mx2 or Mx3 protein. The results are consistent with a two-domain model in which both the conserved amino-terminal half and the less-well-conserved carboxy-terminal half of Mx proteins carry functionally important domains.

Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.

The more than 20 different Mitf mutations in the mouse are all associated with deficiencies in neural crest-derived melanocytes that range from minor functional disturbances with some alleles to complete absence of mature melanocytes with others. In the trunk region of wild-type embryos, Mitf-expressing cells that coexpressed the melanoblast marker Dct and the tyrosine kinase receptor Kit were found in the dorsolateral neural crest migration pathway. In contrast, in embryos homozygous for an Mitf allele encoding a non-functional Mitf protein, Mitf-expressing cells were extremely rare, no Dct expression was ever found, and the number of Kit-expressing cells was much reduced. Wild-type neural crest cell cultures rapidly gave rise to cells that expressed Mitf and coexpressed Kit and Dct. With time in culture, Kit expression was increased, and pigmented, dendritic cells developed. Addition of the Kit ligand Mgf or endothelin 3 or a combination of these factors all rapidly increased the number of Dct-positive cells. Cultures from Mitf mutant embryos initially displayed Mitf-positive cells similar in numbers and Kit-expression as did wild-type cultures. However, Kit expression did not increase with time in culture and the mutant cells never responded to Mgf or endothelin 3, did not express Dct, and never showed pigment. In fact, even Mitf expression was rapidly lost. The results suggest that Mitf first plays a role in promoting the transition of precursor cells to melanoblasts and subsequently, by influencing Kit expression, melanoblast survival.

Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences.

Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.

The microphthalmia (mi) gene appears essential for pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities.

Cloning of MITF, the human homolog of the mouse microphthalmia gene and assignment to chromosome 3p14.1-p12.3.

The mouse microphthalmia (mi) gene encodes a basic-helix-loop-helix-zipper protein whose mutations may lead to loss of pigmentation in the eye, inner ear and skin, and to reduced eye size and early onset deafness. Mice with mutations at mi serve as models for human pigment disturbances in skin and eye that may be combined with sensorineural deafness. We have now obtained cDNA and genomic clones of the human homolog of mouse mi, identified a restriction fragment length polymorphism in the gene, and mapped the gene by somatic cell hybrid and fluorescence in situ hybridization techniques to a region of human chromosome 3 that shows a disrupted syntenic conservation with the region on mouse chromosome 6 to which mi maps. These studies will help to verify if any of the hereditary pigment disturbances in humans are due to mutations in this gene.

Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.

Mice with mutations at the microphthalmia (mi) locus have some or all of the following defects: loss of pigmentation, reduced eye size, failure of secondary bone resorption, reduced numbers of mast cells, and early onset of deafness. Using a transgenic insertional mutation at this locus, we have identified a gene whose expression is disrupted in transgenic animals. This gene encodes a novel member of the basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein family of transcription factors, is altered in mice carrying two independent mi alleles (mi and miws), and is expressed in the developing eye, ear, and skin, all anatomical sites affected by mi. The multiple spontaneous and induced mutations available at mi provide a unique biological resource for studying the role of a bHLH-ZIP protein in mammalian development.