Association of monoamine oxidase A and serotonin transporter gene variants with attachment to parents in patients with type 1 diabetes

Sladkorna bolezen tipa 1 je multifaktorska avtoimunska bolezen, za katero zbolijo predvsem otroci in mladostniki. V magistrski nalogi smo se osredotočili na povezavo vpliva okolja (navezanost na starša) ter genetske predispozicije (variabilnosti gena za monoamin oksidazo A, in gena, ki kodira serotoninski transporter). Ideja izhaja iz beta-celične ˝stresne˝ hipoteze, po kateri stresni in travmatični dogodki v življenju posameznika vplivajo na razvoj sladkorne bolezni tipa 1. Negotova ali dezorganizirana oblika navezanosti neugodno vpliva na soočanje s stresom pri posamezniku, genotipa s 3.5/3.5 ponovitvami variabilnega števila tandemskih ponovitev v promotorju gena za monoamin oksidazo A in S/S polimorfizem v genu za serotoninski transporter pa sta povezana z nižjo ekspresijo teh dveh genov in skupaj z vplivom navezanosti potencialno vplivata na razvoj psihopatologij. Namen naloge je bil preveriti, ali je variabilnost genov za monoamin oksidazo A in serotoninski transporter skupaj z dezorganizirano ali negotovo navezanostjo na starša povezana z razvojem sladkorne bolezni tipa 1. Biološki vzorci, demografski podatki ter podatki o navezanosti na starša preiskovancev so bili zbrani v okviru raziskovalnega programa Pediatrične klinike Ljubljana. Iz brisov bukalne sluznice smo izolirali DNA zdravih kontrolnih preiskovancev, medtem ko je bila DNA bolnikov s sladkorno boleznijo izolirana iz polne krvi ob njihovih kontrolnih pregledih. Nato smo z metodami verižne reakcije s polimerazo, agarozno gelsko elektroforezo, Sangerjevim sekvenciranjem in dodatnim preverjanjem z metodo kapilarne elektroforeze na čipu preiskovancem določili prisotnost izbranih genetskih sprememb. S statistično analizo smo ugotovili, da med skupinama otrok in mladostnikov s sladkorno boleznijo tipa 1 in zdravih kontrolnih preiskovancev ni značilne razlike v pogostosti določene oblike navezanosti na starša ter v prisotnosti posameznih analiziranih genotipov. Raziskava je kljub negativnim ugotovitvam pomembna za nadaljne študije iskanja vzrokov za nastanek sladkorne bolezni tipa 1, ki se bodo morale osredotočiti na večje število genov, ki so udeleženi pri razvoju psihopatalogij ter odzivu na stres, s poudarkom na epigenetskih spremembah, in na druge okoljske dejavnike, s poudarkom na raziskovanju vpliva stresnih in travmatičnih dogodkov.Diabetes type 1 is multifactorial autoimmune disease that mostly affects children and adolescents. The focus of this master thesis is to understand and explore the connection between environmental (attachment to parents) and genetic predisposition (variability of gene encoding for monoamine oxidase A and gene encoding for serotonin transporter). The idea stems from beta-cell stress hypothesis proposing that stressful and traumatic events affect onset of type 1 diabetes. Insecure or disorganized type of attachment to parents is particularly unfavourable to stress management of an individual. Genotypes with 3.5/3.5 repeats of variable number of tandem repeats in gene encoding for monoamine oxidase A promoter and S/S of polymorphism in gene encoding for serotonin transporter are linked to lower expressions of these genes and combined with an unfavourable type of attachment style potentially influence psychopathology development. The purpose of master thesis was to evaluate the variability of genes, encoding for monoamine oxidase A and serotonin transporter and insecure or disorganized attachment style, and their simultaneous contribution to the development of diabetes type 1. The samples and demographic data of participants in the study, together with data on their attachment styles were collected through the research program of University Children’s hospital in Ljubljana. We isolated DNA from buccal swabs of healthy controls while the DNA samples of participants with type 1 diabetes were previously isolated during their regular check-ups with diabetologist. We used polymerase chain reaction, agarose gel electrophoresis, Sanger sequencing and additional method of capillary electrophoresis on-a-chip to determine the type of genetic variants in individual sample. We carried out statistical analysis and concluded that there are no significant differences between the children with type 1 diabetes and healthy controls regarding a certain style of attachment to parents and analized genotypes. Even though we did not confirm the hypothesis, the results are relevant for future studies on the etiology of the diabetes type 1. Future studies should include larger number of genes involved in psychopatology development and stress response with emphasis on epigenetic modifications and additional environmental causes with emphasis on influence of stressful and traumatic events