Lior Greenbaum

Why do young women smoke? I. Direct and interactive effects of environment, psychological characteristics and nicotinic cholinergic receptor genes.

Despite the health hazards, cigarette smoking is disproportionately frequent among young women. A significant contribution of genetic factors to smoking phenotypes is well established. Efforts to identify susceptibility genes do not generally take into account possible interaction with environment, life experience and psychological characteristics. We recruited 501 female Israeli students aged 20–30 years, obtained comprehensive background data and details of cigarette smoking and administered a battery of psychological instruments. Smoking initiators (n=242) were divided into subgroups with high (n=127) and low (n=115) levels of nicotine dependence based on their scores on the Fagerstrom Tolerance Questionnaire and genotyped with noninitiators (n=142) for single nucleotide polymorphisms (SNPs) in 11 nicotinic cholinergic receptor genes. We found nominally significant (P<0.05) allelic and genotypic association with smoking initiation of SNP rs2072660 and multilocus haplotypes (P<0.007–0.05) in CHRNB2 and nominal (P<0.05) allelic or genotypic association of SNPs in CHRNA7 (rs1909884), CHRNA9 (rs4861065) and CHRNB3 (rs9298629) with nicotine dependence. Employing logistic regression and controlling for known risk factors, the best-fitting model for smoking initiation encompassed a 5 SNP haplotype in CHRNB2, neuroticism and novelty seeking (P=5.9 × 10−14, Nagelkerke r2=0.30). For severity of nicotine dependence, two SNPs in CHRNA7 (rs1909884 and rs883473), one SNP in CHRNA5 (rs680244) and the interaction of a SNP in CHRNA7 (rs2337980) with neuroticism, were included in the model (P=2.24 × 10−7, Nagelkerke r2=0.40). These findings indicate that background factors, psychological characteristics and genetic variation in nicotinic cholinergic receptors contribute independently or interactively to smoking initiation and to severity of nicotine dependence in young women.

Why do young women smoke? V. Role of direct and interactive effects of nicotinic cholinergic receptor gene variation on neurocognitive function

Previous work suggests that young women who smoke cigarettes regularly, or did so in the past, manifest a neurocognitive profile that is characterized by small but significant impairments of response inhibition and attention. The present study sought to determine whether variation in nicotinic cholinergic receptor (nAchR) genes impacts upon cognitive function in these domains by overall or differential effects on the performance of current, former and non‐smokers. The study sample consisted of 100 female college students, current or past smokers, and 144 who had never smoked. All performed a computerized neurocognitive test battery and were genotyped for 39 single nucleotide polymorphisms in 11 nAchR genes. The results, derived from linear or logistic regression, show significant direct and interactive relationships between single nucleotide polymorphisms and haplotypes in several nAchR genes and performance on the Matching Familiar Figures Test (MFFT) Stroop test, Continuous Performance Task (CPT) and Tower of London (TOL) test. Response inhibition (MFFT, Stroop, CPT Loading Phase, TOL) was associated with variants in CHRNA2, CHRNA4, CHRNA5, CHRNA7, CHRNA9, CHRNA10, CHRNB2 and CHRNB3. Selective attention (Stroop) was associated with CHRNA4, CHRNA5, CHRNA9 and CHRNB2. Sustained attention (CPT Boring Phase) was associated with CHRNA4, CHRNA5, CHRNA7, CHRNA10 and CHRNB3. Up to 37% of the variance among the smokers and up to 47% of the variance among the non‐smokers on the test measures was explained. Differences between smokers and non‐smokers in neurocognitive function, putatively implicated in susceptibility to nicotine dependence, may be modulated by variants in nAchR genes, with potential implications for prevention and treatment.

Association of the RGS2 gene with extrapyramidal symptoms induced by treatment with antipsychotic medication.

Objectives To investigate the role of genes encoding regulators of G protein signaling in early therapeutic response to antipsychotic drugs and in susceptibility to drug-induced extrapyramidal symptoms. As regulators of G protein signaling and regulators of G protein signaling-like proteins play a pivotal role in dopamine receptor signaling, genetically based, functional variation could contribute to interindividual variability in therapeutic and adverse effects. Methods Consecutively hospitalized, psychotic patients with Diagnostic and Statistical Manual of Mental Disorder-IV schizophrenia (n=121) were included in the study if they received treatment with typical antipsychotic medication (n=72) or typical antipsychotic drugs and risperidone (n=49) for at least 2 weeks. Clinical state and adverse effects were rated at baseline and after 2 weeks. Twenty-four single nucleotide polymorphisms were genotyped in five regulators of G protein signaling genes. Results None of the single nucleotide polymorphisms were related to clinical response to antipsychotic treatment at 2 weeks. Five out of six single nucleotide polymorphisms within or flanking the RGS2 gene were nominally associated with development or worsening of parkinsonian symptoms (PARK+) as measured by the Simpson Angus Scale, one of them after correction for multiple testing (rs4606, P=0.002). A GCCTG haplotype encompassing tagging single nucleotide polymorphisms within and flanking RGS2 was significantly overrepresented among PARK+ compared with PARK− patients (0.23 vs. 0.08, P=0.003). A second, ‘protective’, GTGCA haplotype was significantly overrepresented in PARK− patients (0.13 vs. 0.30, P=0.009). Both haplotype associations survive correction for multiple testing. Conclusions Subject to replication, these findings suggest that genetic variation in the RGS2 gene is associated with susceptibility to extrapyramidal symptoms induced by antipsychotic drugs.

Association of the Type 2 Diabetes Mellitus Susceptibility Gene, TCF7L2, with Schizophrenia in an Arab-Israeli Family Sample

Many reports in different populations have demonstrated linkage of the 10q24–q26 region to schizophrenia, thus encouraging further analysis of this locus for detection of specific schizophrenia genes. Our group previously reported linkage of the 10q24–q26 region to schizophrenia in a unique, homogeneous sample of Arab-Israeli families with multiple schizophrenia-affected individuals, under a dominant model of inheritance. To further explore this candidate region and identify specific susceptibility variants within it, we performed re-analysis of the 10q24-26 genotype data, taken from our previous genome-wide association study (GWAS) (Alkelai et al, 2011). We analyzed 2089 SNPs in an extended sample of 57 Arab Israeli families (189 genotyped individuals), under the dominant model of inheritance, which best fits this locus according to previously performed MOD score analysis. We found significant association with schizophrenia of the TCF7L2 gene intronic SNP, rs12573128, (p = 7.01×10−6) and of the nearby intergenic SNP, rs1033772, (p = 6.59×10−6) which is positioned between TCF7L2 and HABP2. TCF7L2 is one of the best confirmed susceptibility genes for type 2 diabetes (T2D) among different ethnic groups, has a role in pancreatic beta cell function and may contribute to the comorbidity of schizophrenia and T2D. These preliminary results independently support previous findings regarding a possible role of TCF7L2 in susceptibility to schizophrenia, and strengthen the importance of integrating linkage analysis models of inheritance while performing association analyses in regions of interest. Further validation studies in additional populations are required.

Further evidence for association of the RGS2 gene with antipsychotic-induced parkinsonism: protective role of a functional polymorphism in the 3′-untranslated region

RGS2 (regulator of G-protein signaling 2) modulates dopamine receptor signal transduction. Functional variants in the gene may influence susceptibility to extrapyramidal symptoms (EPS) induced by antipsychotic drugs. To further investigate our previous report of association of the RGS2 gene with susceptibility to antipsychotic-induced EPS, we performed a replication study. EPS were rated in 184 US patients with schizophrenia (115 African Americans, 69 Caucasian) treated for at least a month with typical antipsychotic drugs (n=45), risperidone (n=46), olanzapine (n=50) or clozapine (n=43). Six single nucleotide polymorphisms (SNPs) within or flanking RGS2 were genotyped (rs1933695, rs2179652, rs2746073, rs4606, rs1819741 and rs1152746). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. Our results indicate association of SNP rs4606 with antipsychotic-induced parkinsonism (AIP), as measured by the Simpson Angus scale, in the overall sample and in the African-American subsample, the G (minor) allele having a protective effect. ORs for AIP among rs4606 G-allele carriers were 0.23 (95% CI 0.10–0.54, P=0.001) in the overall sample, and 0.20 (0.07–0.57, P=0.003) in the African-American subsample. In the previously studied Israeli sample the OR was 0.31 (0.11–0.84, P=0.02). We completely sequenced the RGS2 gene in nine patients with AIP and nine patients without, from the Israeli sample. No common coding polymorphisms or additional regulatory variants were revealed, suggesting that association of the rs4606 C/G polymorphism with AIP is biologically meaningful and not a consequence of linkage disequilibrium with another functional variant. Taken together, the findings of the current study support the association of RGS2 with AIP and focus on a possible protective effect of the minor G allele of SNP rs4606. This SNP is located in the 3′-regulatory region of the gene, and is known to influence RGS2 mRNA levels and protein expression.

Oxytocin and vasopressin genes are significantly associated with schizophrenia in a large Arab-Israeli pedigree.

We have previously studied the genetics of schizophrenia in a large inbred Arab-Israeli pedigree and found evidence for linkage on chromosome 20p13. This locus harbours four strong candidate genes for schizophrenia: atractin (ATRN), pantonate-kinase2 (PANK2), oxytocin (OXT) and arginine-vasopressin (AVP). In this study we further explored the association of these genes with schizophrenia in the pedigree and searched for the disease-causing variants. A mutation screening of affected individuals from the pedigree was performed by using intensive sequencing in these four genes of interest. Then, we studied the prevalence of the identified variants in all family members (n=56) as well as in Arab-Israeli nuclear families (n=276) and a Jewish case-control sample (n=545). We also studied the possible functional role of these variants by examining their association with gene expression in the brain (n=104). We identified seven genetic variants in the OXT-AVP cluster in affected individuals from the pedigree. Three of these variants were significantly associated with schizophrenia in this pedigree. A 7-SNP haplotype was also significantly associated with disease. We found significant association of some of these variants in the two samples from the general population. Expression data analysis showed a possible functional role of two of these variants in regulation of gene expression. Involvement of OXT and AVP in the aetiology of schizophrenia has been suggested in the past. This study demonstrates, for the first time, a significant genetic association of these neuropeptides with schizophrenia and strongly supports this hypothesis.

DOCK4 and CEACAM21 as novel schizophrenia candidate genes in the Jewish population

It is well accepted that schizophrenia has a strong genetic component. Several genome-wide association studies (GWASs) of schizophrenia have been published in recent years; most of them population based with a case-control design. Nevertheless, identifying the specific genetic variants which contribute to susceptibility to the disorder remains a challenging task. A family-based GWAS strategy may be helpful in the identification of schizophrenia susceptibility genes since it is protected against population stratification, enables better accounting for genotyping errors and is more sensitive for identification of rare variants which have a very low frequency in the general population. In this project we implemented a family-based GWAS of schizophrenia in a sample of 107 Jewish-Israeli families. We found one genome-wide significant association in the intron of the DOCK4 gene (rs2074127, p value=1.134×10⁻⁷) and six additional nominally significant association signals with p<1×10⁻⁵. One of the top single nucleotide polymorphisms (p<1×10⁻⁵) which is located in the predicted intron of the CEACAM21 gene was significantly replicated in independent family-based sample of Arab-Israeli origin (rs4803480: p value=0.002; combined p value=9.61×10⁻⁸), surviving correction for multiple testing. Both DOCK4 and CEACAM21 are biologically reasonable candidate genes for schizophrenia although generalizability of the association of DOCK4 with schizophrenia should be investigated in further studies. In addition, gene-wide significant associations were found within three schizophrenia candidate genes: PGBD1, RELN and PRODH, replicating previously reported associations. By application of a family-based strategy to GWAS, our study revealed new schizophrenia susceptibility loci in the Jewish-Israeli population.

Evidence for association of the GLI2 gene with tardive dyskinesia in patients with chronic schizophrenia

Tardive dyskinesia (TD) occurs in approximately 20% of patients exposed to long‐term antipsychotic treatment and may be influenced by genetic predisposition, in addition to clinical risk factors. In this study, we implemented a two‐step approach to identify susceptibility genes for TD. First, we performed a secondary analysis of the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) genome‐wide association study (GWAS) dataset to identify candidate genes for TD severity. A total of 327 schizophrenia patients treated with antipsychotics who participated in the CATIE trial were included in a TD severity GWAS (approximately 495,000 SNPs). Cases were defined as demonstrating involuntary movements of a mild degree in two or more body regions or of a moderate to severe degree in at least one body region on at least two separate evaluations, whereas controls were completely free of abnormal involuntary movement on all evaluations. Using logistic regression and controlling for population stratification and relevant clinical risk factors, none of the associated SNPs reached GWAS significance; however, several promising SNPs were identified for follow‐up investigation. In the second step, we performed an association study of the top 25 SNPs in an independent sample of 170 Jewish, Israeli, schizophrenia patients (retrospective, cross‐sectional design). Association of the SNP rs3943552 T allele in the GLI2 gene with TD was observed in a subsample of Ashkenazi Jewish patients (N = 96, P = 0.018; P = 6.2 × 10−5 in the CATIE sample). The GLI2 gene encodes a transcription factor that participates in the development of the dopaminergic system during embryogenesis. Taken together, our findings support a possible contribution of GLI2 to TD susceptibility.

Relationship between Rgs2 gene expression level and anxiety and depression-like behaviour in a mutant mouse model: serotonergic involvement

RGS2 is a member of a family of proteins that negatively modulate G-protein coupled receptor transmission. Variations in the RGS2 gene were found to be associated in humans with anxious and depressive phenotypes. We sought to study the relationship of Rgs2 expression level to depression and anxiety-like behavioural features, sociability and brain 5-HT1A and 5-HT1B receptor expression. We studied male mice carrying a mutation that causes lower Rgs2 gene expression, employing mice heterozygous (Het) or homozygous (Hom) for this mutation, or wild-type (WT). Mice were subjected to behavioural tests reflecting depressive-like behaviour [forced swim test (FST), novelty suppressed feeding test (NSFT)], elevated plus maze (EPM) for evaluation of anxiety levels and the three-chamber sociability test. The possible involvement of raphe nucleus 5-HT1A receptors in these behavioural features was examined by 8-OH-DPAT-induced hypothermia. Expression levels of 5-HT1A and 5-HT1B receptors in the cortex, raphe nucleus and hypothalamus were compared among mice of the different Rgs2 genotype groups. NSFT results demonstrated that Hom mice showed more depressive-like features than Rgs2 Het and WT mice. A trend for such a relationship was also suggested by the FST results. EPM and sociability test results showed Hom and Het mice to be more anxious and less sociable than WT mice. In addition Hom and Het mice were characterized by lower basal body temperature and demonstrated less 8-OH-DPAT-induced hypothermia than WT mice. Finally, Hom and Het mice had significantly lower 5-HT1A and 5-HT1B receptor expression levels in the raphe than WT mice. Our findings demonstrate a relationship between Rgs2 gene expression level and a propensity for anxious and depressive-like behaviour and reduced social interaction that may involve changes in serotonergic receptor expression.

Identification of new schizophrenia susceptibility loci in an ethnically homogeneous, family-based, Arab-Israeli sample

While the use of population‐based samples is a common strategy in genome‐wide association studies (GWASs), family‐based samples have considerable advantages, such as robustness against population stratification and false‐positive associations, better quality control, and the possibility to check for both linkage and association. In a genome‐wide linkage study of schizophrenia in Arab‐Israeli families with multiple affected individuals, we previously reported significant evidence for a susceptibility locus at chromosome 6q23.2‐q24.1 and suggestive evidence at chromosomes 10q22.3‐26.3, 2q36.1‐37.3 and 7p21.1‐22.3. To identify schizophrenia susceptibility genes, we applied a family‐based GWAS strategy in an enlarged, ethnically homogeneous, Arab‐Israeli family sample. We performed genome‐wide single nucleotide polymorphism (SNP) genotyping and single SNP transmission disequilibrium test association analysis and found genome‐wide significant association (best value of P= 1.22×10‐11) for 8 SNPs within or near highly reasonable functional candidate genes for schizophrenia. Of particular interest are a group of SNPs within and flanking the transcriptional factor LRRFIP1 gene. To determine replicability of the significant associations beyond the Arab‐Israeli population, we studied the association of the significant SNPs in a German case‐control validation sample and found replication of associations near the UGT1 subfamily and EFHD1 genes. Applying an exploratory homozygosity mapping approach as a complementary strategy to identify schizophrenia susceptibility genes in our Arab Israeli sample, we identified 8 putative disease loci. Overall, this GWAS, which emphasizes the important contribution of family based studies, identifies promising candidate genes for schizophrenia.—Alkelai, A., Lupoli, S., Greenbaum, L., Giegling, I., Kohn, Y., Sarner‐Kanyas, K., Ben‐Asher, E., Lancet, D., Rujescu, D., Macciardi, F., Lerer, B. Identification of new schizophrenia susceptibility loci in an ethnically homogeneous, family‐based, Arab‐Israeli sample. FASEB J. 25, 4011–4023 (2011). www.fasebj.org