The Role of Genes on Chromosome Locus 4q12 in Human Central Nervous System Tumors

Human central nervous system (CNS) tumors are a heterogeneous group of tumors occurring in brain, brainstem and spinal cord. Malignant gliomas (astrocytic and oligodendroglial tumors), which arise from the neuroepithelial cells are the most common CNS neoplasms in human. Malignant gliomas are highly aggressive and invasive tumors, and have a very poor prognosis. The development and progression of gliomas involve a stepwise accumulation of genetic alterations that generally affect either signal transduction pathways activated by receptor tyrosine kinases (RTKs), or cell cycle arrest pathways. Constitutive activation or deregulated signaling by RTKs is caused by gene amplification, overexpression or mutations. The aberrant RTK signaling results in turn in the activation of several downstream pathways, which ultimately lead to malignant transformation and tumor proliferation. Many genetic abnormalities implicated in nervous system tumors involve the genes located at the chromosomal region 4q12. This locus harbors the receptor tyrosine kinases KIT, PDGFRA and VEGFR2, and other genes (REST, LNX1) with neural function. Gene amplification and protein expression of KIT, PDGFRA, and VEGFR2 was studied using clinical tumor material. REST and LNX1, as well as NUMBL, the interaction partner of LNX1, were studied for their gene mutations and amplifications. In our studies, amplification of LNX1 was associated with KIT and PDGFRA amplification in glioblastomas, and coamplification of KIT, PDGFRA and VEGFR2 was detected in medulloblastomas and CNS primitive neuroectodermal tumors. PDGFRA amplification was also correlated with poor overall survival. Coamplification of KIT, PDGFRA and VEGFR2 was observed in a subset of human astrocytic and oligodendroglial tumors. We suggest that genes at 4q12 could be a part of a larger amplified region, which is deregulated in gliomas, and could be used as a prognostic marker of tumorigenic process. The signaling pathways activated due to gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment. This study also includes the characterization of KIT overexpressing astrocytes, analyzed by various in vitro functional assays. Our results show that overexpression of KIT in mouse astrocytes promotes cell proliferation and anchorage-independent growth, as well as phenotypic changes in the cells. Furthermore, the increased proliferation is partly inhibited by imatinib, a small molecule inhibitor of KIT. These results suggest that KIT may play a role in astrocyte growth regulation, and might have an oncogenic role in brain tumorigenesis. Elucidation of the altered signaling pathways due to specific gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment.

Computational Methods for Detecting Large-Scale Chromosome Rearrangements in SNP Data

Large-scale chromosome rearrangements such as copy number variants (CNVs) and inversions encompass a considerable proportion of the genetic variation between human individuals. In a number of cases, they have been closely linked with various inheritable diseases. Single-nucleotide polymorphisms (SNPs) are another large part of the genetic variance between individuals. They are also typically abundant and their measuring is straightforward and cheap. This thesis presents computational means of using SNPs to detect the presence of inversions and deletions, a particular variety of CNVs. Technically, the inversion-detection algorithm detects the suppressed recombination rate between inverted and non-inverted haplotype populations whereas the deletion-detection algorithm uses the EM-algorithm to estimate the haplotype frequencies of a window with and without a deletion haplotype. As a contribution to population biology, a coalescent simulator for simulating inversion polymorphisms has been developed. Coalescent simulation is a backward-in-time method of modelling population ancestry. Technically, the simulator also models multiple crossovers by using the Counting model as the chiasma interference model. Finally, this thesis includes an experimental section. The aforementioned methods were tested on synthetic data to evaluate their power and specificity. They were also applied to the HapMap Phase II and Phase III data sets, yielding a number of candidates for previously unknown inversions, deletions and also correctly detecting known such rearrangements.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Nicotine Dependence and Smoking Behaviour : A genetic and epidemiological study

The worldwide health burden caused by the tobacco epidemic highlights the importance of study-ing determinants of smoking behaviour and key factors sustaining nicotine dependence. Despite vast-ranging preventive efforts, smoking remains one of the most deleterious health behaviours, and its genetic and environmental factors warrant continuous investigation. The heritability of smoking behaviour and nicotine dependence has been suggested to be relatively high. Earlier smoking behaviour, nicotine dependence, socio-economic position and demographic factors have all been shown to be associated with smoking cessation. This thesis aimed to examine various aspects of smoking behaviour and nicotine dependence from an epidemiological and genetic per-spective. Data for Studies I and IV were obtained from the Older Finnish Twin Cohort, a postal health sur-vey conducted in 1975, 1981 and 1990 on same-sexed pairs and in 1996-1997 on male-female adult pairs. The number of ever-smoking participants was 8941 in Study I and 3069 in Study IV. Data for Studies II and III came from the Family Study of Cigarette Smoking - Vulnerability to Nicotine Addiction. This study is linked to the Older Finnish Twin Cohort with new data collec-tion during 2001-2006 that focused on smoking twin pairs and their family members. The meas-ures included intensive telephone interviews, blood samples and additional postal questionnaires. The numbers of ever-smoking participants was 1370 in Study II and 529 in Study III. Study I examined whether a genetic component underlies smoking behaviour among Finnish adults. Genetic factors were important in the amount smoked and smoking cessation, with about half of the phenotypic differences explained by genetic variance. A novel finding was that genetic influences on amount smoked and smoking cessation were largely independent of genetic influ-ences on age at initiation. This result has implications for defining phenotypes in the search for genes underlying smoking behaviour. Furthermore, even if smoking initiation is postponed to a later age, potential vulnerability to subsequent nicotine dependence cannot be completely inhib-ited. Study II investigated the effect of genetic and environmental factors on nicotine dependence, as measured by the novel multidimensional Nicotine Dependence Syndrome Scale (NDSS). This scale was validated in the Finnish data. The NDSS correlated highly with other established nico-tine dependence scales (FTND and DSM-IV), suggesting that this new scale would be a feasible and valid measure for identifying nicotine-dependent smokers among the ever-smoking popula-tion. About one-third of the phenotypic variation in nicotine dependence in this sample was ex-plained by genetic influences. Study III aimed at identifying chromosomal regions harbouring genes that influence smoking be-haviour and nicotine dependence. Linkage analysis of family data revealed that for smoker and nicotine dependence phenotypes as well as for co-morbidity between nicotine dependence and alcohol use signals on specific chromosome regions (chromosomes 2q33, 5q12, 5q34 7q21, 7q31, 10q25, 11p15, 20p13) exist. Results further support the hypothesis that smoking behaviour phe-notypes have a genetic background. Study IV examined associations of smoking behaviour, socio-economic position and transition of marital status with smoking cessation. Indicators of socio-economic position were important pre-dictors of smoking cessation even when adjusted for previous smoking behaviour. Getting married was associated with an increased probability of cessation in men, a finding confirmed among dis-cordant twin pairs. Thus, having a partner appears to have a positive impact on smoking cessation. In conclusion, nicotine dependence and smoking behaviour demonstrate significant genetic liabil-ity, but also substantial environmental influences among Finnish adults. Smoking initiation should be prevented or at least postponed to a later age. Although genetic factors are important in nicotine dependence and smoking behaviour, societal actions still have a primary role in tobacco control and smoking prevalence. Future studies should examine the complex interactions between genetic and environmental factors in nicotine dependence.