Associations of gene polymorphisms and nutrition with calcium homeostasis and bone mineral density : Studies on skeletal nutrigenetics

Heredity explains a major part of the variation in calcium homeostasis and bone strength, and the susceptibility to osteoporosis is polygenetically regulated. Bone phenotype results from the interplay between lifestyle and genes, and several nutritional factors modulate bone health throughout life. Thus, nutrigenetics examining the genetic variation in nutrient intake and homeostatic control is an important research area in the etiology of osteoporosis. Despite continuing progress in the search for candidate genes for osteoporosis, the results thus far have been inconclusive. The main objective of this thesis was to investigate the associations of lactase, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and parathyroid hormone (PTH) gene polymorphisms and lifestyle factors and their interactions with bone health in Finns at varying stages of the skeletal life span. Markers of calcium homeostasis and bone remodelling were measured from blood and urine samples. Bone strength was measured at peripheral and central bone sites. Lifestyle factors were assessed with questionnaires and interviews. Genetic lactase non-persistence (the C/C-13910 genotype) was associated with lower consumption of milk from childhood, predisposing females in particular to inadequate calcium intake. Consumption of low-lactose milk and milk products was shown to decrease the risk for inadequate calcium intake. In young adulthood, bone loss was more common in males than in females. Males with the lactase C/C-13910 genotype may be more susceptible to bone loss than males with the other lactase genotypes, although calcium intake predicts changes in bone mass more than the lactase genotype. The BsmI and FokI polymorphisms of the VDR gene were associated with bone mass in growing adolescents, but the associations weakened with age. In young adults, the A986S polymorphism of the calcium sensing receptor gene was associated with serum ionized calcium concentrations, and the BstBI polymorphism of the parathyroid gene was related to bone strength. The FokI polymorphism and sodium intake showed an interaction effect on urinary calcium excretion. A novel gene-gene interaction between the VDR FokI and PTH BstBI gene polymorphisms was found in the regulation of PTH secretion and urinary calcium excretion. Further research should be carried out with more number of Finns at varying stages of the skeletal life span and more detailed measurements of bone strength. Research should concern mechanisms by which genetic variants affect calcium homeostasis and bone strength, and the role of diet-gene and gene-gene interactions in the pathogenesis of osteoporosis.

Applications of gene sequence polymorphisms in evolutionary genetic studies of Atlantic salmon (Salmo salar) and other teleost fish species

Evolutionary genetics incorporates traditional population genetics and studies of the origins of genetic variation by mutation and recombination, and the molecular evolution of genomes. Among the primary forces that have potential to affect the genetic variation within and among populations, including those that may lead to adaptation and speciation, are genetic drift, gene flow, mutations and natural selection. The main challenges in knowing the genetic basis of evolutionary changes is to distinguish the adaptive selection forces that cause existent DNA sequence variants and also to identify the nucleotide differences responsible for the observed phenotypic variation. To understand the effects of various forces, interpretation of gene sequence variation has been the principal basis of many evolutionary genetic studies. The main aim of this thesis was to assess different forms of teleost gene sequence polymorphisms in evolutionary genetic studies of Atlantic salmon (Salmo salar) and other species. Firstly, the level of Darwinian adaptive evolution affected coding regions of the growth hormone (GH) gene during the teleost evolution was investigated based on the sequence data existing in public databases. Secondly, a target gene approach was used to identify within population variation in the growth hormone 1 (GH1) gene in salmon. Then, a new strategy for single nucleotide polymorphisms (SNPs) discovery in salmonid fishes was introduced, and, finally, the usefulness of a limited number of SNP markers as molecular tools in several applications of population genetics in Atlantic salmon was assessed. This thesis showed that the gene sequences in databases can be utilized to perform comparative studies of molecular evolution, and some putative evidence of the existence of Darwinian selection during the teleost GH evolution was presented. In addition, existent sequence data was exploited to investigate GH1 gene variation within Atlantic salmon populations throughout its range. Purifying selection is suggested to be the predominant evolutionary force controlling the genetic variation of this gene in salmon, and some support for gene flow between continents was also observed. The novel approach to SNP discovery in species with duplicated genome fragments introduced here proved to be an effective method, and this may have several applications in evolutionary genetics with different species - e.g. when developing gene-targeted markers to investigate quantitative genetic variation. The thesis also demonstrated that only a few SNPs performed highly similar signals in some of the population genetic analyses when compared with the microsatellite markers. This may have useful applications when estimating genetic diversity in genes having a potential role in ecological and conservation issues, or when using hard biological samples in genetic studies as SNPs can be applied with relatively highly degraded DNA.

Genetic polymorphisms and laboratory variables as predictors of blood pressure response to antihypertensive drugs

Hypertension is one of the major risk factors for cardiovascular morbidity. The advantages of antihypertensive therapy have been clearly demonstrated, but only about 30% of hypertensive patients have their blood pressure (BP) controlled by such treatment. One of the reasons for this poor BP control may lie in the difficulty in predicting BP response to antihypertensive treatment. The average BP reduction achieved is similar for each drug in the main classes of antihypertensive agents, but there is a marked individual variation in BP responses to any given drug. The purpose of the present study was to examine BP response to four different antihypertensive monotherapies with regard to demographic characteristics, laboratory test results and common genetic polymorphisms. The subjects of the present study are participants in the pharmacogenetic GENRES Study. A total of 208 subjects completed the whole study protocol including four drug treatment periods of four weeks, separated by four-week placebo periods. The study drugs were amlodipine, bisoprolol, hydrochlorothiazide and losartan. Both office (OBP) and 24-hour ambulatory blood pressure (ABP) measurements were carried out. BP response to study drugs were related to basic clinical characteristics, pretreatment laboratory test results and common polymorphisms in genes coding for components of the renin-angiotensin system, alpha-adducin (ADD1), beta1-adrenergic receptor (ADRB1) and beta2-adrenergic receptor (ADRB2). Age was positively correlated with BP responses to amlodipine and with OBP and systolic ABP responses to hydrochlorothiazide, while body mass index was negatively correlated with ABP responses to amlodipine. Of the laboratory test results, plasma renin activity (PRA) correlated positively with BP responses to losartan, with ABP responses to bisoprolol, and negatively with ABP responses to hydrochlorothiazide. Uniquely to this study, it was found that serum total calcium level was negatively correlated with BP responses to amlodipine, whilst serum total cholesterol level was negatively correlated with ABP responses to amlodipine. There were no significant associations of angiotensin II type I receptor 1166A/C, angiotensin converting enzyme I/D, angiotensinogen Met235Thr, ADD1 Gly460Trp, ADRB1 Ser49Gly and Gly389Arg and ADRB2 Arg16Gly and Gln27Glu polymorphisms with BP responses to the study drugs. In conclusion, this study confirmed the relationship between pretreatment PRA levels and response to three classes of antihypertensive drugs. This study is the first to note a significant inverse relation between serum calcium level and responsiveness to a calcium channel blocker. However, this study could not replicate the observations that common polymorphisms in angiotensin II type I receptor, angiotensin converting enzyme, angiotensinogen, ADD1, ADRB1, or ADRB2 genes can predict BP response to antihypertensive drugs.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Molecular Background of Common Dyslipidemias

The leading cause of death in the Western world continues to be coronary heart disease (CHD). At the root of the disease process is dyslipidemia an aberration in the relevant amounts of circulating blood lipids. Cholesterol builds up in the arterial wall and following rupture of these plaques, myocardial infarction or stroke can occur. Heart disease runs in families and a number of hereditary forms are known. The leading cause of adult dyslipidemia presently however is overweight and obesity. This thesis work presents an investigation of the molecular genetics of common, hereditary dyslipidemia and the tightly related condition of obesity. Familial combined hyperlipidemia (FCHL) is the most common hereditary dyslipidemia in man with an estimated population prevalence of 1-6%. This complex disease is characterized by elevated levels of serum total cholesterol, triglycerides or both and is observed in about 20% of individuals with premature CHD. Our group identified the disease to be associated with genetic variation in the USF1 transcription factor gene. USF1 has a key role in regulating other genes that control lipid and glucose metabolism as well as the inflammatory response all central processes in the progression of atherosclerosis and CHD. The first two works of this thesis aimed at understanding how these USF1 variants result in increased disease risk. Among the many, non-coding single-nucleotide polymorphisms (SNPs) that associated with the disease, one was found to have a functional effect. The risk-enhancing allele of this SNP seems to eradicate the ability of the important hormone insulin to induce the expression of USF1 in peripheral tissues. The resultant changes in the expression of numerous USF1 target genes over time probably enhance and accelerate the atherogenic processes. Dyslipidemias often represent an outcome of obesity and in the final work of this thesis we wanted to address the metabolic pathways related to acquired obesity. It is recognized that active processes in adipose tissue play an important role in the development of dyslipidemia, insulin resistance and other pathological conditions associated with obesity. To minimize the confounding effects of genetic differences present in most human studies, we investigated a rare collection of identical twins that differed significantly in the amount of body fat. In the obese, but otherwise healthy young adults, several notable changes were observed. In addition to chronic inflammation, the adipose tissue of the obese co-twins was characterized by a marked (47%) decrease in amount of mitochondrial DNA (mtDNA) a change associated with mitochondrial dysfunction. The catabolism of branched chain amino acids (BCAAs) was identified as the most down-regulated process in the obese co-twins. A concordant increase in the serum level of these insulin secretagogues was identified. This hyperaminoacidemia may provide the feed-back signal from insulin resistant adipose tissue to the pancreas to ensure an appropriately augmented secretory response. The down regulation of BCAA catabolism correlated closely with liver fat accumulation and insulin. The single most up-regulated gene (5.9 fold) in the obese co-twins was osteopontin (SPP1) a cytokine involved in macrophage recruitment to adipose tissue. SPP1 is here implicated as an important player in the development of insulin resistance. These studies of exceptional study samples provide better understanding of the underlying pathology in common dyslipidemias and other obesity associated diseases important for future improvement of intervention strategies and treatments to combat atherosclerosis and coronary heart disease.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

Insights into the molecular genetics of celiac disease : Applying family- and population-based strategies

Celiac disease, or gluten intolerance, is triggered by dietary glutens in genetically susceptible individuals and it affects approximately 1% of the Caucasian population. The best known genetic risk factors for celiac disease are HLA DQ2 and DQ8 heterodimers, which are necessary for the development of the disease. However, they alone are not sufficient for disease induction, other risk factors are required. This thesis investigated genetic factors for celiac disease, concentrating on susceptibility loci on chromosomes 5q31-q33, 19p13 and 2q12 previously reported in genome-wide linkage and association studies. In addition, a novel genotyping method for the detection of HLA DQ2 and DQ8 coding haplotypes was validated. This study was conducted using Finnish and Hungarian family materials, and Finnish, Hungarian and Italian case-control materials. Genetic linkage and association were analysed in these materials using candidate gene and fine-mapping approaches. The results confirmed linkage to celiac disease on the chromosomal regions 5q31-q33 and 19p13. Fine-mapping on chromosome 5q31-q33 revealed several modest associations in the region, and highlighted the need for further investigations to locate the causal risk variants. The MYO9B gene on chromosome 19p13 showed evidence for linkage and association particularly with dermatitis herpetiformis, the skin manifestation of celiac disease. This implies a potential difference in the genetic background of the intestinal and skin forms of the disease, although studies on larger samplesets are required. The IL18RAP locus on chromosome 2q12, shown to be associated with celiac disease in a previous genome-wide association study and a subsequent follow-up, showed association in the Hungarian population in this study. The expression of IL18RAP was further investigated in small intestinal tissue and in peripheral blood mononuclear cells. The results showed that IL18RAP is expressed in the relevant tissues. Two putative isoforms of IL18RAP were detected by Western blot analysis, and the results suggested that the ratios and total levels of these isoforms may contribute to the aetiology of celiac disease. A novel genotyping method for celiac disease-associated HLA haplotypes was also validated in this thesis. The method utilises single-nucleotide polymorphisms tagging these HLA haplotypes with high sensitivity and specificity. Our results suggest that this method is transferable between populations, and it is suitable for large-scale analysis. In conclusion, this doctorate study provides an insight into the roles of the 5q31-q33, MYO9B, IL18RAP and HLA loci in the susceptibility to celiac disease in the Finnish, Hungarian and Italian populations, highlighting the need for further studies at these genetic loci and examination of the function of the candidate genes.

Genetic studies on recurrent miscarriage

Recurrent miscarriage (RM) is defined as three consecutive pregnancy failures and is estimated to affect ~1% of couples trying to conceive. The cause of RM remains unknown in approximately 50% of cases. In this study, it was hypothesized that some of the underlying factors yet to be discovered are genetic. The aim was to search for mutations in genes AMN, EPCR, TM, and p53 known to cause miscarriage in mouse models and thereby find new genetic causes for unexplained miscarriages in humans. In addition, the mitochondrial genome was studied because mitochondria are involved in processes important in early development. Furthermore, sex chromosome characteristics suggested to underlie miscarriage were also studied. A total of 40 couples and 8 women with unexplained RM were collected for this study and screened for mutations in the candidate genes. Six interesting exonic or potential splice site disrupting variations were detected. However, their phenotypic effects cannot be determined without further investigations. Additionally, an association between the C11992A polymorphism of the p53 gene and RM was detected. The results indicate that women carrying the C/A or A/A genotype have a two-fold higher risk for RM than women with a C/C genotype. This strengthens the results of previous studies reporting that p53 sequence variations may cause miscarriage. The role of variation C11992A in embryonic development is, however, difficult to predict without further studies When screening the mitochondrial genome a heteroplasmic mtDNA variation was found in an unexpected high number of women, as heteroplasmic variations are reported to be rare. One novel variation and 18 previously reported polymorphisms were detected in the mitochondrial genome. Although the detected variations are likely to be neutral polymorphisms, a role in the aetiology of miscarriage cannot be excluded as some mtDNA variations may be pathogenic only when a threshold is reached. Recent publications have reported skewed X chromosome inactivation and Y chromosome microdeletions to be associated with RM. Therefore, these sex chromosome abnormalities in the context of RM were investigated. No associations between skewed X chromosome inactivation or Y chromosome microdeletions and RM in the Finnish patients were detected. Data on ancestral birthplaces of the patients were collected to study any possible geographic clustering, which would indicate a common predisposing factor. The results showed clustering of the birthplaces in eastern Finland in a subset of patients. This suggests a possibility of an enriched susceptibility gene which may contribute to RM.

Molecular genetics of primary open angle glaucoma and exfoliation syndrome

Glaucoma is the second leading cause of blindness worldwide. It is a group of optic neuropathies, characterized by progressive optic nerve degeneration, excavation of the optic disc due to apoptosis of retinal ganglion cells and corresponding visual field defects. Open angle glaucoma (OAG) is a subtype of glaucoma, classified according to the age of onset into juvenile and adult- forms with a cut-off point of 40 years of age. The prevalence of OAG is 1-2% of the population over 40 years and increases with age. During the last decade several candidate loci and three candidate genes, myocilin (MYOC), optineurin (OPTN) and WD40-repeat 36 (WDR36), for OAG have been identified. Exfoliation syndrome (XFS), age, elevated intraocular pressure and genetic predisposition are known risk factors for OAG. XFS is characterized by accumulation of grayish scales of fibrillogranular extracellular material in the anterior segment of the eye. XFS is overall the most common identifiable cause of glaucoma (exfoliation glaucoma, XFG). In the past year, three single nucleotide polymorphisms (SNPs) on the lysyl oxidase like 1 (LOXL1) gene have been associated with XFS and XFG in several populations. This thesis describes the first molecular genetic studies of OAG and XFS/XFG in the Finnish population. The role of the MYOC and OPTN genes and fourteen candidate loci was investigated in eight Finnish glaucoma families. Both candidate genes and loci were excluded in families, further confirming the heterogeneous nature of OAG. To investigate the genetic basis of glaucoma in a large Finnish family with juvenile and adult onset OAG, we analysed the MYOC gene in family members. Glaucoma associated mutation (Thr377Met) was identified in the MYOC gene segregating with the disease in the family. This finding has great significance for the family and encourages investigating the MYOC gene also in other Finnish OAG families. In order to identify the genetic susceptibility loci for XFS, we carried out a genome-wide scan in the extended Finnish XFS family. This scan produced promising candidate locus on chromosomal region 18q12.1-21.33 and several additional putative susceptibility loci for XFS. This locus on chromosome 18 provides a solid starting point for the fine-scale mapping studies, which are needed to identify variants conferring susceptibility to XFS in the region. A case-control and family-based association study and family-based linkage study was performed to evaluate whether SNPs in the LOXL1 gene contain a risk for XFS, XFG or POAG in the Finnish patients. A significant association between the LOXL1 gene SNPs and XFS and XFG was confirmed in the Finnish population. However, no association was detected with POAG. Probably also other genetic and environmental factors are involved in the pathogenesis of XFS and XFG.

Search For Susceptibility Genes In Schizophrenia

Schizophrenia is a severe mental disorder affecting 0.4-1% of the population worldwide. It is characterized by impairments in the perception of reality and by significant social or occupational dysfunction. The disorder is one of the major contributors to the global burden of diseases. Studies of twins, families, and adopted children point to strong genetic components for schizophrenia, but environmental factors also play a role in the pathogenesis of disease. Molecular genetic studies have identified several potential positional candidate genes. The strongest evidence for putative schizophrenia susceptibility loci relates to the genes encoding dysbindin (DTNBP1) and neuregulin (NRG1), but studies lack impressive consistency in the precise genetic regions and alleles implicated. We have studied the role of three potential candidate genes by genotyping 28 single nucleotide polymorphisms in the DNTBP1, NRG1, and AKT1 genes in a large schizophrenia family sample consisting of 441 families with 865 affected individuals from Finland. Our results do not support a major role for these genes in the pathogenesis of schizophrenia in Finland. We have previously identified a region on chromosome 5q21-34 as a susceptibility locus for schizophrenia in a Finnish family sample. Recently, two studies reported association between the γ-aminobutyric acid type A receptor cluster of genes in this region and one study showed suggestive evidence for association with another regional gene encoding clathrin interactor 1 (CLINT1, also called Epsin 4 and ENTH). To further address the significance of these genes under the linkage peak in the Finnish families, we genotyped SNPs of these genes, and observed statistically significant association of variants between GABRG2 and schizophrenia. Furthermore, these variants also seem to affect the functioning of the working memory. Fetal events and obstetric complications are associated with schizophrenia. Rh incompatibility has been implicated as a risk factor for schizophrenia in several epidemiological studies. We conducted a family-based candidate-gene study that assessed the role of maternal-fetal genotype incompatibility at the RhD locus in schizophrenia. There was significant evidence for an RhD maternal-fetal genotype incompatibility, and the risk ratio was estimated at 2.3. This is the first candidate-gene study to explicitly test for and provide evidence of a maternal-fetal genotype incompatibility mechanism in schizophrenia. In conclusion, in this thesis we found evidence that one GABA receptor subunit, GABRG2, is significantly associated with schizophrenia. Furthermore, it also seems to affect to the functioning of the working memory. In addition, an RhD maternal-fetal genotype incompatibility increases the risk of schizophrenia by two-fold.

Molecular Pathways of Disturbed Sleep and Depression: Studies on Adenosine and Gene Expression Patterns

Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.

Amyloid diseases at old age : a pathological, epidemiological, and genetic study

Along with the increased life span of individuals, the burden of old age-associated diseases has inevitably increased. Alzheimer s disease (AD), probably the most well known geriatric disease, belongs to the old age-associated amyloid diseases. The purpose of this study was to investigate the frequency, genetic and health-associated risk factors, mutual association, and amyloid proteins in two old age-associated amyloid disorders senile systemic amyloidosis (SSA) and cerebral amyloid angiopathy (CAA) as part of the prospective population-based Vantaa 85+ autopsy study on a Finnish population aged 85 years or more (Studies I-III), completed with a case report on a patient with advanced AGel amyloidosis (Study IV). The numbers of patients investigated in the studies (I-III) were 256, 74, and 63, respectively. The diagnosis and grading of amyloid were based upon histological examination of tissue samples obtained post mortem and stained with Congo red. The amyloid fibril and associated proteins were characterized by immunohistochemical staining methods. The genotype frequencies of 20 polymorphisms in 9 genes and information on health-associated risk factors in subjects with and without SSA and CAA were compared. In a Finnish population ≥ 95 years of age, SSA and CAA occurred in 36% and 49% of the subjects, respectively. In total, two-thirds of these very elderly individuals had SSA, CAA, or both. However, in only 14% of the population these two conditions co-occurred. In subjects 85 years or older, the prevalence of SSA was 25%. In this population, SSA was associated with age at the time of death (p=0.002), myocardial infarctions (MIs; p=0.004), the G/G (Val/Val) genotype of the exon 24 polymorphism in the alpha2-macroglobulin (α2M) gene (p=0.042) and with the H2 haplotype of the tau gene (p=0.016). In contrast, the presence of CAA was strongly associated with APOE e4 (p=0.0003), with histopathological AD (p=0.0005), and with clinical dementia (p=0.01) in both e4+ (p=0.02) and e4- (p=0.06) individuals. Apart from demonstrating the amyloid fibril proteins, complement proteins 3d (C3d) and 9 (C9) were detected in the amyloid deposits of CAA and AGel amyloidosis, and α2M protein was found in fibrous scar tissue close to SSA. In conclusion, this first population based study on SSA shows that both SSA and CAA are common in very elderly individuals. Old age, MIs, the exon 24 polymorphism of the α2M gene, and H1/H2 polymorphism of the tau gene associate with SSA while clinical dementia and APOE ε4 genotype associate with CAA. The high prevalence of CAA, combined with its association with clinical dementia independent of APOE genotype, neuropathological AD, or SSA, also highlights its clinical significance in the very aged, among which the serious end stage complications of CAA, namely multiple infarctions and hemorrhages, are rare. The report on a patient having advanced AGel amyloidosis added knowledge on the disease and showed that this generally benign condition occasionally may lead to death. Further studies are warranted to confirm the findings in other populations. Also, the role of α2M and tau in the pathogenesis of SSA and the involvement of complement in the process of amyloid beta (Aβ) protein elimination from the brain remain to be clarified. Finally, the high prevalence of SSA in the elderly raises the need for prospective clinical studies to define its clinical significance.

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.