CANDIDATE GENE STUDY ON NICOTINE DEPENDENCE IN FINNISH SIBPAIRS. Katri Heikkilä - PDF

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CANDIDATE GENE STUDY ON NICOTINE DEPENDENCE IN FINNISH SIBPAIRS Katri Heikkilä Master s Thesis University of Jyväskylä Faculty of Mathematics and Science Department of Biological and Environmental Science

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CANDIDATE GENE STUDY ON NICOTINE DEPENDENCE IN FINNISH SIBPAIRS Katri Heikkilä Master s Thesis University of Jyväskylä Faculty of Mathematics and Science Department of Biological and Environmental Science 2008 PREFACE This study has been performed at the National Public Health Institute, Department of Molecular Medicine, Biomedicum, Helsinki, during April 2004 and June The epidemiological data collection has been performed by University of Helsinki, Department of Public Health. Writing of the thesis has been finalized in I want to thank Professor Leena Peltonen-Palotie for the possibility to participate in this interesting and captivating research. The facilities in Biomedicum created amazing surroundings to work and made everything very comfortable. This project taught me a lot about the Finnish research world and made me understand it much better. Professor Jaakko Kaprio is thanked for enjoyable collaboration and for providing the excellent study sample for this study. The world of epidemiology was totally new for me and prof. Kaprio was able to make it quite easily understandable. My supervisor, Ph.D. Anu Loukola well, how can I thank you enough! It has been a long way until this point and it has been a very memorable time. Your enthusiasm towards this project and science is something I had never seen before. Your drive, usually without coffee, kept also me going and made me try harder, even at the times when everything did not look quite good. All the people at the Department of Public Health, and especially Ulla, thank you for the nice meetings and all the work every one has done for the nicotine project. And Ulla, the secrets of genetics were difficult in the beginning for me too. And then the best workmates ever! Thank you Heidi for helping me to get started and sharing the same aspects on nicotine study project. Marika, William, Juho, Joni, Emma N, Pia, Helena, Annu and all the others, thank you for the coffee breaks and the loud conversations that maybe not everyone could understand. Emma P and Olli, although time has passed and we all are doing different things at the moment, it is amazing how the friendships seem to stay the same. Without you two, this project would have felt much different and the life after it would be little emptier. Last, but not the least, my parents. Thank you for all the support during this project and making it possible to be able to go back to school. And Tommi, thank you for being there in good and bad times. This study has been funded by the Center of Excellence in Disease Genetics of the Academy of Finland and NIH grant DA12854. University of Jyväskylä Faculty of Mathematics and Science Abstract of Master s Thesis Author: Katri Heikkilä Title of thesis: Candidate gene study on nicotine dependence in Finnish sibpairs Finnish title: Nikotiiniriippuvuuden ehdokasgeenitutkimus suomalaisilla sisaruspareilla Date: Pages: Department: Chair: Supervisor(s): Department of Biological and Environmental Science Biotechnology FT Anu Loukola Abstract: Smoking is the most common reason for preventable untimely death. Tobacco products are related to one fourth of the self-inflicted diseases. Smoking is the most important known risk factor of cancers. About 30 % of the diagnosed cancers in Finland are related to smoking. Smoking increases heart attack risk by 2-fold. According to WHO approximately five million people die due to tobacco use every year. It has been estimated that this number will increase to ten million people by Very often the closest relatives of a smoker also become smokers. Therefore, it is believed that also genetic variants are related to smoking. Tobacco consists of over 4000 different chemical compounds, but only one of these compounds, nicotine, is believed to be addictive. Family, twin and adoption studies have revealed a strong genetic component in nicotine addiction. However, surprisingly little is known about the genetics of smoking and nicotine dependence. In this candidate gene study the aim was to identify genes involved in nicotine dependence in Finnish twin families. The study sample consisted of Finnish twins and their siblings born in They were selected based on history of heavy smoking in at least two sibs. Nicotine dependence has been evaluated from a diagnostic telephone interview using the DSM-IV criteria and the Fagerström score. 720 individuals were included in this study. Studied phenotypes were nicotine dependence (DSM-IV score) and regular smoker (smoker or non-smoker). The allelic spectrum of candidate genes was analyzed by using single nucleotide polymorphisms (SNPs). Six candidate genes with functional relevance were selected from the literature: four nicotinic acetylcholine receptor genes and two cytochrome P450 genes. Sequenom s Mass ARRAY technology was utilised to genotype 26 SNPs that were selected based on their genetic location, polymorphic nature and linkage disequilibrium in Finnish families. Family based association analyses were then used to evaluate the role of specific alleles. The results show statistically significant association in three genes with nicotine dependence: CHRNA5 (rs667282, rs ), CHRNA4 (rs , rs ) and CHRNB1 (rs , rs871990) and two genes with regular smoker phenotype CHRNA5 (rs ) and CHRNB1 (rs , rs ). These results with nicotine dependence phenotype are similar to the ones reported earlier. In short, these results show that genetic components play a role in nicotine dependence and give us more perspective in revealing them in more detail. Keywords: candidate gene, nicotine dependence, sibpairs, twins, nicotine, smoking, association analysis, CHRNA4, CHRNA5, CHRNA7, CHRNB, CYP1A2, CYP2A6, single nucleotide polymorphism Jyväskylän yliopisto Matemaattis-luonnontieteellinen tiedekunta Pro gradu tutkielman tiivistelmä Tekijä: Katri Heikkilä Tutkielman nimi: Nikotiiniriippuvuuden ehdokasgeenitutkimus suomalaisilla sisaruspareilla English title: Candidate gene study on nicotine dependence in Finnish sibpairs Päivämäärä: Sivumäärä: Laitos: Oppiaine: Tutkielman ohjaaja(t): Bio- ja ympäristötieteiden laitos Biotekniikka FT Anu Loukola Tiivistelmä: Tupakointi on yleisin estettävissä oleva ennenaikaisen kuoleman syy. Noin neljäsosaan itseaiheutetuista sairauksista syyllisiä ovat tupakkavalmisteet. Suomessa noin 30 % syöpäsairauksista aiheutuu tupakasta. Tupakointi alentaa myös merkittävästi elinkaaren pituutta. WHO:n mukaan tupakoinnista johtuvista syistä joka vuosi kuolee noin viisi miljoonaa ihmistä. Arvion mukaan luku nousee vuoteen 2030 mennessä 10 miljoonaan. Teollisuusmaissa tupakka aiheuttaa joka viidennen kuolemantapauksen. Tupakointi lähes kaksinkertaistaa sydäninfarktin riskin. Tupakoijien lähisukulaisista tulee hyvin usein myös tupakoitsijoita. Tämän vuoksi uskotaan, että henkilön perimä, vanhemmilta peritty geenistö, vaikuttaa osaltaan taipumukseen tupakoida. Vaikka tupakoidessa elimistöön tulee yli 4000 erilaista yhdistettä, on nikotiini se yhdiste joka aiheuttaa riippuvuuden. Nikotiini on hermojen välittäjäaineen, asetykoliinin, kaltainen keskushermostoa stimuloiva aine. Kaksos- ja perhetutkimukset kehittyneitä molekyylibiologisia menetelmiä hyväksikäyttäen, mahdollistavat altistavien geenien selvittämisen. Assosiaatiotutkimusten perusteella voidaan pyrkiä selvittämään nikotiiniriippuvuuden monitekijäistä periytymistä. Assosiaatiotutkimuksissa pyritään osoittamaan että tietty geneettinen osoitin, eli geenissä tapahtunut luonnollinen yhden emäksen muunnos, ja sairastumisalttius liittyvät toisiinsa siten, että osoitin havaitaan useammin sairastuneissa yksilöissä kuin terveissä yksilöissä. Tutkimuksessa oli mukana 720 henkilöä aineistosta, joka koostuu vuosina syntyneistä suomalaisista kaksosista ja heidän sisaruksistaan. Valintakriteerinä on käytetty erittäin runsaan tupakoinnin esiintymistä vähintään kahdessa sisaruksessa samassa perheessä. Ilmiasuina analyyseissä käytettiin sekä nikotiiniriippuvuutta että säännöllistä tupakointia. Nikotiiniriippuvuuden mittarina käytettiin DSM-IV kriteerejä sekä Fagerströmin asteikkoa. Tutkittavia ehdokasgeenejä oli kuusi, CHRNA4, CHRNA5, CHRNA7, CHRNB, CYP1A2 ja CYP2A6. Yhden emäksen monimuotoisuuden osoittimia valittiin geneettisen sijainnin, monimuotoisuuden sekä kytkentäepätasapainon perusteella 26 kappaletta. Osoittimien tutkimiseen tutkimusaineistosta käytettiin Sequenom Mass Array tekniikkaa sekä tietokoneavusteisia analyysejä. Kvalitatiiviset assosiaatioanalyysit osoittavat tilastollisesti merkittävää, p 0.05, assosiaatiota useiden osoittimien ja ilmiasujen väillä, kolmella geenillä nikotiiniriippuvuuden suhteen: CHRNA5 (rs667282, rs ), CHRNA4 (rs , rs ) ja CHRNB1 (rs , rs871990) sekä kahdella geenillä säännöllisen tupakoinnin suhteen: CHRNA5 (rs ) ja CHRNB1 (rs , rs ). Nikotiiniriippuvuusilmiasulla saadut tulokset ovat hyvin samankaltaisia kuin mitä aiemmin on raportoitu. Tutkimus antaa vahvan näytön siitä, että perinnöllisillä tekijöillä on vaikutusta nikotiiniriippuvuuteen. Perinnöllisten tekijöiden tämän kaltaista sekä laajempaa tutkimusta tulisi jatkaa ja tutkittavien ilmiasujen määrää tulisi lisätä. Avainsanat: ehdokasgeeni, nikotiiniriippuvuus, sisarusparit, kaksoset, nikotiini, tupakointi, assosiaatiotutkimus, CHRNA4, CHRNA5, CHRNA7, CHRNB, CYP1A2, yhden emäksen muunnos TABLE OF CONTENTS ABBREVIATIONS 1 INTRODUCTION Smoking and public health Nicotine dependence and addiction Dependence scales Nicotine Twin and adoption studies Linkage studies Association studies Candidate gene approach Smoking and nicotine dependence candidate gene studies Genome-wide association studies (GWA) Nicotinic acetylcholine receptors Metabolising genes Other candidate genes AIM OF THE STUDY MATERIALS AND METHODS Study sample and phenotype information Laboratory methods Blood samples and DNA extraction DNA dilutions Candidate gene selection Assay design PCR primer testing SNP validation Multiplex redesigning and tests Optimisation Throughput testing and genotyping Sequenom MassARRAY genotyping system Template amplification Dephosphorylation hme reaction Sample conditioning and transfer MALDI-TOF mass spectrometry analysis Quality control KariOTyper Hardy-Weinberg equilibrium Linkage disequilibrium Statistical methods LINKAGE format parameter files DOWNFREQ PEDCHECK PSEUDOMARKER...31 3.7.5 LDA RESULTS The study sample The candidate genes and the SNPs Allele frequencies and success rates Quality control Linkage disequilibrium Association studies DISCUSSION...39 APPENDICES...47 ABBREVIATIONS AA African-American BLAST Basic Local Alignment Search Tool cm centi Morgan CNS central nervous system COGA Collaborative Study on the Genetics of Alcoholism dom dominant model DSM Diagnostic and Statistical Manual of Mental Disorders EA European-American EDTA ethylene diamine tetra acetic acid FBAT Family-Based Association Test FTND Fagerström Test for Nicotine Dependence FTQ Fagerström Tolerance Questionnaire hme homogenous MassEXTEND assay HVA homovanillic acid HWE Hardy-Weinberg equilibrium ICD International Classification of Diseases LD linkage disequilibrium LOD score logarithm of odds (a measure of the likelihood of genetic linkage between loci) MAF minor allele frequency MALDI-TOF MS matrix-assisted laser desorption / ionisation time-of-flight mass spectrometry nachrs nicotinic acetylcholine receptors NDSS Nicotine Dependence Syndrome Scale NPHI National Public Health Institute, Finland rec recessive model RTQ Revised Tolerance Questionnaire SAP shrimp alkaline phosphatase SNP single nucleotide polymorphism WHO World Health Organisation Abbreviations of the gene names are provided in the context of each of the genes described in the text 6 1 INTRODUCTION 1.1 Smoking and public health World Health Organisation has estimated that one third of the adult population worlwide smokes. In Finland, 23.5% of the adult population smoke. The annual cigarette consumption in Finland is cigarettes per person. (Mackay and Eriksen, 2002). Smoking is the most common reason for preventable untimely death. Tobacco products are related to one fourth of the self-inflicted diseases. Smoking is the most important known risk factor for cancers. About 30 % of the diagnosed cancers in Finland are related to smoking. Smoking increases heart attack risk by 2-fold. Every other smoker will die of smoking, if they do not quit early enough. Thus, smoking is a significant causing factor in morbidity and work disability. During the last 50 years smoking habit among men has decreased in Finland, but at the same time smoking among women has increased. In the year 2000, 20% and 28% of working aged females and males, respectively, were daily smokers. In the future the increase on women s smoking habit can be seen in the death statistics, where the number of deaths caused by smoking related diseases will increase among women and will even the numbers between sexes. Compared to the rest of Europe the Finns smoke little, but we have as our speciality the problem with very low initiation age. (Patja K and Haukkala A, 2004) According to a study made by WHO, approximately 5 million people die due to tobacco use every year. (Mackay and Eriksen, 2002) It has been estimated that this number will increase to 10 million people by In industrialised countries tobacco causes one fifth of the deaths per year. Smoking related diseases cost to the Finnish society about 2 billion euros per year. A smoker uses the public health care 22% more often than a non-smoker. The Finnish General Medicine association defines nicotine addiction as a chronic disease, which requires treatment. 1.2 Nicotine dependence and addiction Tobacco is the most commonly used heavily addictive substance around the world (Salaspuro M et al., 2003). It generates physiological, social, psychological and emotional 7 addiction. People smoke to maintain nicotine levels in the brain, to avoid the negative effects of nicotine withdrawal and to control their mood. Some smoke to calm them down, to focus better, to solve problematic situations or to cheer themselves up. Tobacco consists of over 4000 different chemical compounds, but only one of these compounds, nicotine, is believed to be addictive. Nicotine addiction is very problematic because of its individual nature. The possible genetic determinants behind the addiction are still largely unknown. According to the international psychiatric classification system nicotine addiction fulfils the criteria for chemical addiction. The affects on behaviour are not as severe as with classical drugs, but the addiction itself can be as severe. Because of the strong addictiveness of nicotine, quitting smoking is very difficult. About 60% of the individuals that smoke in Finland would like to quit, but only 3-8 % of the ones who attempt to quit succeed. (Salaspuro M et al., 2003) Nicotine addiction is a complex multifactorial behaviour with both genetic and environmental determinants (Feng et al., 2004; Niu et al., 2000). 1.3 Dependence scales There are several dependence scales available for the evaluation of nicotine addiction. American Psychiatric Association has established the Diagnostic and Statistical Manual of Mental Disorders (DSM), also containing a diagnostic scale for the recognition of nicotine addiction. Originally this criterion was designed for a wide range of substances and nicotine was added afterwards. In the fourth edition of DSM (DSM-IV) nicotine dependence and nicotine withdrawal are included as disorders. Nicotine abuse is not included since psychosocial problems and intoxication are not clinically significant. The DSM-IV questionnaire consists of seven criteria and three of them must be fulfilled within a twelve-month period. Some example questions are attached in Appendix 1a. (American Psychiatric Association, 1994) WHO created an International Classification of Diseases (ICD) to consistently diagnose diseases clinically worldwide. In the tenth revision of the ICD (ICD-10) also nicotine addiction was included. In ICD-10 there are six criteria, and three or more of them need to 8 be fulfilled simultaneously at some time during the previous year. See appendix 1b for the criteria. (WHO, 1992) In 1978 an eight-question Fagerström Tolerance Questionnaire (FTQ) was introduced, attempting to assess physical dependence on nicotine. FTQ is the most widely used dependence questionnaire for tobacco and nicotine (Fagerström, 1978). FTQ has been revised in 1994 when the eight questions were reduced to six; the revised version is called the Fagerstöm Test for Nicotine Dependence (FTND). The scale was improved by renewal of scoring in questions of time of the first cigarette of the day and number of cigarettes smoked per day. (Heatherton et al., 1991) The maximum score is ten and scores seven or more identify physical dependence on nicotine. The questionnaire is in Appendix 1c. FTQ has been criticized to have somewhat poor psychometric properties. In the Revised Tolerance Questionnaire (RTQ) wording, scale format and modification of some items were enhanced. This scale uses Likert scales, in which the subjects indicate the level of agreement or disagreement using a five-point scale. Maximum score is therefore 30 and this way it gives more accurate response in behaviour. (Tate and Schmitz, 1993) Nicotine Dependence Syndrome Scale (NDSS) evaluates, in a multifactor way, tobacco dependence. It consists of 19 questions and scores are divided in 5 subscales. NDSS measures drive, priority, tolerance, continuity and stereotypy. (Shiffman et al., 2004) 1.4 Nicotine Nicotine as a clean substance is toxic. 60 mg is a lethal dose for an adult. Nicotine increases the pulse rate and blood pressure. As large doses it causes symptoms of toxication and the acute toxicity is so high that even a person who has smoked for a long time typically can smoke no more than 60 cigarettes per day. When the cigarette smoke is inhaled into the lungs, 25% of the nicotine dose is absorbed into the blood stream. It takes only seven seconds for nicotine to reach the brain. The affects in the central nervous system (CNS) are immediate. The nicotine level in the plasma and the brain is very high and this causes a rewarding effect. A remarkable part of 9 the effects of nicotine are due to the increased release of neurotransmitters in the CNS. Nicotine also increases the secretion of glucocorticoids and vasopressin, which has an antidiuretic effect. The positive and negative effects of nicotine are listed in Table I. Table I Positive and negative effects of nicotine in the CNS Positive effects Improvement of cognitive functions Improvement of attentiveness Decrease of anxiety Neuroprotection Analgesia Negative effects Hypothermia Ataxia Cramps Nausea Vomiting Development of addiction The rewarding effect vanishes quite quickly because the nicotinic receptors desensitise and nicotine metabolises. The main metabolite is cotinine, which is metabolised in 24 hours. Compared to the half-life of nicotine, which is 30 minutes, the difference is huge. The amount of smoked cigarettes per day can be measured from cotinine levels in blood or urine. Nicotine binds to nicotinic acetylcholine receptors. These receptors are located in skeletal muscle tissue, medulla, pre-synaptically throughout the central nervous system and postsynaptically in autonomic nervous system. These nervous systems modulate the release of neurotransmitters and ganglionic potentials. (Role and Berg, 1996) 1.5 Twin and adoption studies Two commonly used approaches to examine genetic and environmental components of phenotypic variance are twin and adoption studies. Twin studies examine the concordance rates for traits of interest. They are based on the fact that monozygotic twins share identical genetic material whereas dizygotic twins are genetically comparable to non-twin
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