University of Zagreb Faculty of Science Department of Biology Martina Ĉonda Genotyping of families with obsessive-compulsive disorder Graduation thesis Zagreb, 2014 This Graduation thesis, produced at the Neurobiochemistry laboratory of the University Clinics of Child and Adolescent Psychiatry, University of Zürich, under mentoring of Dr. Edna Grünblatt, Assoc. Prof. Department of Child and Adolescent Psychiatry at University of Zürich, Zürich (Switzerland), is subjected for evaluation to Department of Biology, Faculty of Science, University of Zagreb for acquisition of the title master of molecular biology. ACKNOWLEDGEMENTS To PD Dr. Edna Grünblatt from University of Zürich, for warm welcoming to her research group and including me to their research project for the purpose of this Master thesis. To Dr. Jasmin Bartl, Dr. Zoya Marinova and Miryame Hofmann for their selfless guidance during my work for this Master thesis, numerous beneficial advices and patience as well as delightful company. To dr. sc. Domagoj Đikić, for his kindness and cooperativeness as well as useful advices during writing of this Master thesis. To my family and friends for their endless help and support during my studies. BASIC DOCUMENTATION CARD University of Zagreb Faculty of Science Department of Biology Graduation thesis GENOTYPING OF FAMILIES WITH OBSESSIVE-COMPULSIVE DISORDER Martina Ĉonda Roosveltov trg 6, 10000 Zagreb, Croatia Obsessive-compulsive disorder (OCD), a neuropsychiatric disorder, is characterized by the presence of obsessions and/or compulsions. According to previous publications association of genetic variations in glutamatergic genes with OCD are considered that may potentially contribute to the pathophysiology of OCD. The main aim of this thesis was to provide confirmatory evidence for association of five selected single nucleotide polymorphism (SNP); rs301443 (G/C), rs12682807 (A/C), rs11081062 (C/T), rs11663827 (A/G) and rs1556995 (G/A) from a three glutamatergic candidate genes; SLC1A1, DPGAP1, GRIK2 with early-onset OCD using family-based study and case-control study. The current study could not confirm the previous findings of these three candidate genes as risk variants in early- onset OCD. (65 pages, 25 figures, 17 tables, 45 references, original in: English) Thesis deposited in Central Biological Library Keywords: early-onset, glutamate, SLC1A1, DLGAP1, GRIK2, polymorphisms Supervisor: Dr. Edna Grünblatt, Assoc. Prof. (University of Zürich, Zürich, Switzerland) Co-supervisor: Dr. Domagoj Đikić, Assoc. Prof. Reviewers: Thesis accepted: TEMELJNA DOKUMENTACIJSKA KARTICA Sveuĉilište u Zagrebu Prirodoslovno-matematiĉki fakultet Biološki odsjek Diplomski rad GENOTIPIZACIJA OBITELJI OBOLJELIH OD OPSESIVNO- KOMPULZIVNOG POREMEĆAJA Martina Ĉonda Roosveltov trg 6, 10000 Zagreb Opsesivno-kompulzivni poremećaj (engl. Obsessive-compulsive disorder, OCD) je psihiĉka bolest koja je okarakterizirana prisustvom opsesija i/ili kompulzija. Prema dosad objavljenim istraživanjima o uzroku nastanka OCD, smatra se da genetske varijacije, koje utjeĉu na poremećaj homeostaze glutamat neurotransmisije potencijalno sudjeluje u patofiziologiji OCD. Glavni cilj ovog rada je pronaći povezanost pet odabranih polimorfizama jednog nukleotida (engl. single-nucleotide polymorphism, SNP); rs301443 (G/C), rs12682807 (A/C), rs11081062 (C/T), rs11663827 (A/G) i rs1556995 (G/A), i triju gena kandidata glutamat neurotransmisije; SLC1A1, DPGAP1, GRIK2 sa nastankom ranog oblika OCD koristeći uzorke obitelji oboljelih i kontrole. Dobiveni rezultati triju predloženih genskih riziĉnih varijanti ne podržavaju ulogu u nastanku ranog oblika OCD i povezanosti sa kliniĉko patološkim obilježjima. (65 stranica, 25 slika, 17 tablica, 45 literaturnih navoda, jezik izvornika: engleski) Rad je pohranjen u Središnjoj biološkoj knjižnici. Kljuĉne rijeĉi: rano pojavljivanje bolesti, glutamat, SLC1A1, DLGAP1, GRIK2, polimofizam Voditelj: Dr. sc. Edna Grünblatt, izv. prof. (Sveuĉilište u Zürichu, Zürich, Švicarska) Suvoditelj: Dr. sc. Domagoj Đikić, izv. prof. Ocjenitelji: Rad prihvaćen: Table of Contents 1. Introduction ............................................................................................. 1 1.1. Obsessive compulsive disorder ........................................................ 1 1.1.1. Definition and clinical features .................................................... 1 1.1.2. Epidemiology of obsessive-compulsive disorder ........................ 3 1.1.3. Diagnosing of obsessive-compulsive disorder ............................ 3 1.1.3.1. Clinical classification of obsessive-compulsive disorder .... 4 1.1.4. Etiological factors of obsessive-compulsive disorder .................. 5 1.1.4.1. Neuroanatomical brain alterations involved in OCD .......... 6 1.1.4.2. Genetic factors .................................................................. 7 1.1.4.3. Environmental factors ........................................................ 8 1.1.5. Treatment of obsessive-compulsive disorder ............................. 9 1.1.6. Heritability of OCD ...................................................................... 9 1.2. Genome-wide association studies .................................................. 10 1.2.1. Single-nucleotide polymorphism (SNP) .................................... 12 1.2.2. Glutamatergic candidate genes ................................................ 12 1.2.2.1. SLC1A1 gene .................................................................. 13 1.2.2.2. DLGAP1 gene ................................................................. 15 1.2.2.3. GRIK2 gene ..................................................................... 16 1.3. Study design ................................................................................... 17 1.3.1. Family-based study .................................................................. 17 1.3.2. Case-control study .................................................................... 19 1.4. Aim .................................................................................................. 20 2. Materials and Methods .......................................................................... 21 2.1. Materials ......................................................................................... 21 2.2. Methods .......................................................................................... 23 2.2.1. Subject collection ...................................................................... 23 2.2.2. Extraction DNA ......................................................................... 24 2.2.2.1. DNA extraction and purification from saliva ..................... 24 2.2.2.2. DNA extraction and purification from blood ..................... 25 2.2.3. DNA concentration measurement ............................................. 26 2.2.4. TaqMan SNP Genotyping Assay .............................................. 26 2.2.5. SNPman ................................................................................... 29 2.2.6. Statistical analysis .................................................................... 30 3. RESULTS ............................................................................................. 32 3.1. TaqMan SNP Genotyping Assay results ......................................... 32 3.1.1. Analysis of polymorphisms on the SLC1A1 gene ..................... 32 3.1.2. Analysis of polymorphisms on the DLGAP1 gene .................... 33 3.2. Results of case–control study ......................................................... 35 3.2.1. Genotype distribution and Hardy-Weinberg equilibrium (HWE) test of SLC1A1, DLGAP1, GRIK2 genes polymorphisms in the case– control study .......................................................................................... 35 3.2.2. Case-control association analysis ............................................ 37 3.2.3. Male case-control association analysis .................................... 38 3.3. Results of family–based study ........................................................ 40 3.3.1. Transmission disequilibrium test (TDT) analysis ...................... 40 3.3.2. Sib - transmission disequilibrium test (DFAM) analysis ............ 41 3.4. Association of SLC1A1, DLGAP1, GRIK2 genes polymorphisms with clinical-pathological features in OCD patients .......................................... 42 3.4.1. Severity of symptoms analysis (measured by the CY-BOCS) .. 43 3.4.1.1. Analysis of severity of symptoms between the three genotypes using one-way ANOVA test.............................................. 43 3.4.1.2. Analysis of severity of symptoms between risk allele and non-risk allele using independent samples T test .............................. 46 3.4.2. Age of onset analysis ............................................................... 50 3.4.2.1. Analysis of age of onset between the three genotypes using one-way ANOVA test ............................................................... 50 3.4.2.2. Analysis of age of onset between risk allele and non-risk allele using independent samples T test ............................................ 52 4. Discussion ............................................................................................. 55 5. Conclusion ............................................................................................ 62 6. References ............................................................................................ 63 List of Figures Figure 1 Scheme of the OCD cycle. ..............................................................