Susceptibility genes and neurodevelopmental mechanisms in dyslexia

Developmental dyslexia is a specific reading disability, which is characterised by unexpected difficulty in reading, spelling and writing despite adequate intelligence, education and social environment. It is the most common childhood learning disorder affecting 5-10 % of the population and thus constitutes the largest portion of all learning disorders. It is a persistent developmental failure although it can be improved by compensation. According to the most common theory, the deficit is in phonological processing, which is needed in reading when the words have to be divided into phonemes, or distinct sound elements. This occurs in the lowest level of the hierarchy of the language system and disturbs processes in higher levels, such as understanding the meaning of words. Dyslexia is a complex genetic disorder and previous studies have found nine locations in the genome that associate with it. Altogether four susceptibility genes have been found and this study describes the discovery of the first two of them, DYX1C1 and ROBO1. The first clues were obtained from two Finnish dyslexic families that have chromosomal translocations which disrupt these genes. Genetic analyses supported their role in dyslexia: DYX1C1 associates with dyslexia in the Finnish population and ROBO1 was linked to dyslexia in a large Finnish pedigree. In addition a genome-wide scan in Finnish dyslexic families was performed. This supported the previously detected dyslexia locus on chromosome 2 and revealed a new locus on chromosome 7. Dyslexia is a neurological disorder and the neurobiological function of the susceptibility genes DYX1C1 and ROBO1 are consistent with this. ROBO1 is an axon guidance receptor gene, which is involved in axon guidance across the midline in Drosophila and axonal pathfinding between the two hemispheres via the corpus callosum, as well as neuronal migration in the brain of mice. The translocation and decreased ROBO1 expression in dyslexic individuals indicate that two functional copies of ROBO1 gene are required in reading. DYX1C1 was a new gene without a previously known function. Inhibition of Dyx1c1 expression showed that it is needed in normal brain development in rats. Without Dyx1c1 protein, the neurons in the developing brain will not migrate to their final position in the cortex. These two dyslexia susceptibility genes DYX1C1 and ROBO1 revealed two distinct neurodevelopmental mechanisms of dyslexia, axonal pathfinding and neuronal migration. This study describes the discovery of the genes and our research to clarify their role in developmental dyslexia.

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.

Podocyte molecules in the pancreas and lymphoid tissues and their association to autoimmunity of diabetes

Type 1 diabetes is a disease where the insulin-producing beta cells of the pancreas are destroyed by an autoimmune mechanism. The incidence of type 1 diabetes, as well as the incidence of the diabetic kidney complication, diabetic nephropathy, are increasing worldwide. Nephrin is a crucial molecule for the filtration function of the kidney. It localises in the podocyte foot processes partially forming the interpodocyte final sieve of the filtration barrier, the slit diaphragm. The expression of nephrin is altered in diabetic nephropathy. Recently, nephrin was found from the beta cells of the pancreas as well, which makes this molecule interesting in the context of type 1 diabetes and especially in diabetic nephropathy. In this thesis work, the expression of other podocyte molecules in the beta cells of the pancreas, in addition to nephrin, were deciphered. It was also hypothesised that patients with type 1 diabetes may develop autoantibodies against novel beta cell molecules comparably to the formation of autoantibodies to GAD, IA-2 and insulin. The possible association of such novel autoantibodies with the pathogenesis of diabetic nephropathy was also assessed. Furthermore, expression of nephrin in lymphoid tissues has been suggested, and this issue was more thoroughly deciphered here. The expression of nephrin in the human lymphoid tissues, and a set of podocyte molecules in the human, mouse and rat pancreas at the gene and protein level were studied by polymerase chain reaction (PCR) -based methods and immunochemical methods. To detect autoantibodies to novel beta cell molecules, specific radioimmunoprecipitation assays were developed. These assays were used to screen a follow-up material of 66 patients with type 1 diabetes and a patient material of 150 diabetic patients with signs of diabetic nephropathy. Nephrin expression was detected in the lymphoid follicle germinal centres, specifically in the follicular dendritic cells. In addition to the previously reported expression of nephrin in the pancreas, expression of the podocyte molecules, densin, filtrin, FAT and alpha-actinin-4 were detected in the beta cells. Circulating antibodies to nephrin, densin and filtrin were discovered in a subset of patients with type 1 diabetes. However, no association of these autoantibodies with the pathogenesis of diabetic nephropathy was detected. In conclusion, the expression of five podocyte molecules in the beta cells of the pancreas suggests some molecular similarities between the two cell types. The novel autoantibodies against shared molecules of the kidney podocytes and the pancreatic beta cells appear to be part of the common autoimmune mechanism in patients with type 1 diabetes. No data suggested that the autoantibodies would be active participants of the kidney injury detected in diabetic nephropathy.

Molecular Genetics of Schizophrenia and Related Intermediate Phenotypes in a Founder Population

Schizophrenia, affecting about 1% of population worldwide, is a severe mental disorder characterized by positive and negative symptoms, such as psychosis and anhedonia, as well as cognitive deficits. At present, schizophrenia is considered a complex disorder of neurodevelopmental origin with both genetic and environmental factors contributing to its onset. Although a number of candidate genes for schizophrenia have been highlighted, only very few schizophrenia patients are likely to share identical genetic liability. This study is based on the nation-wide schizophrenia family sample of the National Institute for Health and Welfare, and represents one of the largest and most well-characterized familial series in the world. In the first part of this study, we investigated the roles of the DTNBP1, NRG1, and AKT1 genes in the background of schizophrenia in Finland. Although these genes are associated with schizophrenia liability in several populations, any significant association with clinical diagnostic information of schizophrenia remained absent in our sample of 441 schizophrenia families. In the second part of this study, we first replicated schizophrenia linkage on the long arm of chromosome 7 in 352 schizophrenia families. In the following association analysis, we utilized additional clinical disorder features and intermediate phenotypes – endophenotypes - in addition to diagnostic information from altogether 290 neuropsychologically assessed schizophrenia families. An intragenic short tandem repeat allele of the regional RELN gene, supposed to play a role in the background of several neurodevelopmental disorders, showed significant association with poorer cognitive functioning and more severe schizophrenia symptoms. Additionally, this risk allele was significantly more prevalent among the individuals affected with schizophrenia spectrum disorders. We have previously identified linkage of schizophrenia and its cognitive endophenotypes on the long arms of chromosomes 2, 4, and 5. In the last part of this study, we selected altogether 104 functionally relevant candidate genes from the linked regions. We detected several promising associations, of which especially interesting are the ERBB4 gene, showing association with the severity of schizophrenia symptoms and impairments in traits related to verbal abilities, and the GRIA1 gene, showing association with the severity of schizophrenia symptoms. Our results extend the previous evidence that the genetic risk for schizophrenia is at least partially mediated via the effects of the candidate genes and their combinations on relevant brain systems, resulting in alterations in different disorder domains, such as the cognitive deficits.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Immunodeficiencies, pathogens and sex in upper respiratory diseases

In the first part of this thesis the association of different forms of sinonasal diseases and plasma concentrations of C3, C4, immunoglobulins, immunoglobulin G subclasses, C4A and C4B gene numbers were studied in 287 adult patients and 150 sex-matched adult controls. Patients were well characterized and stratified into groups using strict clinical criteria and females and males were also studied as separate groups. Severe primary antibody antibody deficiencies were rare in patients coming to sinonasal operations. Female patients had more recurrent sinusitis and other mucosal infections and males had more nasal polyposis. Upregulation of complement activity was seen in acute rhinosinusitis patients (high levels of plasma C3, C4, and complement classical pathway activity CH50) and male patients coming to sinonasal operations (high levels of plasma C3 and C4). In females, total and partial C4B deficiencies and lower levels of IgG1 and IgG3 were associated with rhinosinusitis leading to sinonasal operations. C4A deficiencies were found to predispose to severe chronic rhinosinusitis in females and males. In female patients with chronic or recurrent rhinosinusitis with nasal polyposis C4B deficiencies seem to predispose to the disease, but in males with a similar disease C4B deficiencies seem to be protective. This suggests a different pathophysiology between sexes in this form of sinonasal disease. In the second part of this thesis work 213 children coming to elective tonsillectomy were studied and compared with 155 randomly selected school children. An association with recurrent upper respiratory tract infections and hypersensitivity disorders was seen especially in children under 7 years of age. However, this association was not seen in levels of specific IgE to respiratory allergens in the same age group. Both symptomatic respiratory allergy and specific IgE to respiratory allergens became more common in boys than girls over 7 years of age. We were able to show that although both rhinoviruses and bacterial pathogens were found in the tonsils, no association between their presence and clinical forms of tonsillar disease was seen. The ability of GAS to bind complement regulators FH and C4BP did not differ between strains causing tonsillar diseases or septicemia, suggesting that other virulence mechanisms of the bacteria are more important.

α-Synuclein pathology in very elderly Finns : A population-based clinico-neuropathologic and molecular genetic study of Dementia with Lewy bodies

Populations in developed countries are ageing fast. The elderly have the greatest incidence of de-mentia, and thus the increase in the number of demented individuals, increases the immediate costs for the governments concerning healthcare and hospital treatment. Attention is being paid to disorders behind cognitive impairment with behavioural and psychological symptoms, which are enormous contributors to the hospital care required for the elderly. The highest dreams are in prevention; however, before discovering the tools for preventing dementia, the pathogenesis behind dementia disorders needs to be understood. Dementia with Lewy bodies (DLB), a relatively recently discovered dementia disorder compared to Alzheimer’s disease (AD), is estimated to account for up to one third of primary degenerative dementia, thus being the second most common cause of dementia in the elderly. Nevertheless, the impact of neuropathological and genetic findings on the clinical syndrome of DLB is not fully established. In this present series of studies, the frequency of neuropathological findings of DLB and its relation to the clinical findings was evaluated in a cohort of subjects with primary degenerative dementia and in a population-based prospective cohort study of individuals aged 85 years or older. α-synuclein (αS) immunoreactive pathology classifiable according to the DLB consensus criteria was found in one fourth of the primary degenerative dementia subjects. In the population-based study, the corresponding figure was one third of the population, 38% of the demented and one fifth of the non-demented very elderly Finns. However, in spite of the frequent discovery of αS pathology, its association with the clinical symptoms was quite poor. Indeed, the common clinical features of DLB, hypokinesia and visual hallucinations, associated better with the severe neurofibrillary AD-type pathology than with the extensive (diffuse neocortical) αS pathology when both types of pathology were taken into account. The severity of the neurofibrillary AD-type pathology (Braak stage) associated with the extent of αS pathology in the brain. In addition, the genetic study showed an interaction between tau and αS; common variation in the αS gene (SNCA) associated significantly with the severity of the neurofibrillary AD-type pathology and nominally significantly with the extensive αS pathology. Further, the relevance and temporal course of the substantia nigra (SN) degeneration and of the spinal cord αS pathology were studied in relation to αS pathology in the brain. The linear association between the extent of αS pathology in the brain and the neuron loss in SN suggests that in DLB the degeneration of SN proceeds as the αS pathology extends from SN to the neocortex instead of early destruction of SN seen in Parkinson’s disease (PD). Furthermore, the extent of αS pathology in the brain associated with the severity of αS pathology in the thoracic and sacral autonomic nuclei of the spinal cord. The thoracic αS pathology was more common and more severe compared to sacral cord, suggesting that the progress of αS pathology proceeds downwards from the brainstem towards the sacral spinal cord.

VEGF-C and angiopoietins in lymphangiogenesis

Angiogenesis and lymphangiogenesis occur during development as the result of tightly coordinated signalling programs to generate two hierarchically organised vascular systems. All tissues and organs are dependent on a functional blood vasculature for oxygen and nutrients, whereas the lymphatic vasculature functions to collect excess tissue fluid, passing it through lymph nodes for immune surveillance, and returning it to the blood circulation. Effectors that control developmental angiogenesis and lymphangiogenesis are also involved in pathological settings, and therefore potential targets for therapy. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) growth factors, signalling through endothelial VEGFR and Tie receptors, have been established as key regulators of angiogenic and lymphangiogenic processes in development and disease. In this study, we aimed to obtain a clearer understanding of the vascular effects of stimulation by VEGF-C, Ang1 and Ang2, all known to be involved in lymphangiogenesis. In cell culture models, we found that both intrinsic and microenvironmental regulatory mechanisms are involved in the regulation of endothelial cell phenotypes, and distinct responses to VEGF signalling are induced by specific receptor pathways in different endothelial cell types. Surprisingly, we also found that Ang1 induces sprouting lymphangiogenesis in vivo by a VEGFR-3 dependent mechanism, establishing Ang1 as a novel lymphangiogenic factor. Using inducible transgenic mouse models, we found that VEGF-C-induced lymphatic hyperplasia persisted independently of the growth factor, indicating that short pro-lymphangiogenic therapy could lead to lasting improvements in tissue oedema. While VEGF-C had blood vessel effects in embryos, no angiogenic side effects were observed in adult tissues. Furthermore, inducible transgenic expression of Ang2 during embryonic development confirmed Ang2 as an important regulator of lymphatic remodelling and mural cell contacts. The unexpected similarity of the lymphatic maturation defects caused by excess Ang2 to those observed in Ang2 deficient mice demonstrated that correct doses of Ang2 are crucial for the control of lymphatic development. Unlike Ang1, Ang2 did not induce lymphatic sprouting. Although Ang1 has been shown to be able to substitute for Ang2 during developmental lymphangiogenesis, their lymphatic effects are not identical. These findings further our understanding of the basic mechanisms of angiogenesis and lymphangiogenesis, important for the future development of targeted therapies for vascular diseases such as cancer, inflammation, lymphoedema and ischemia. VEGF-C and Ang1 especially emerged as promising candidates for pro-lymphangiogenic therapy.

Fumarate Hydratase and Succinate Dehydrogenase in Neoplasia

Germline mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell cancer (HLRCC). FH is a nuclear encoded enzyme which functions in the Krebs tricarboxylic acid cycle, and homozygous mutation in FH lead to severe developmental defects. Both uterine and cutaneous leiomyomas are components of the HLRCC phenotype. Most of these tumours show loss of the wild-type allele and, also, the mutations reduce FH enzyme activity, which indicate that FH is a tumour suppressor gene. The renal cell cancers associated with HLRCC are of rare papillary type 2 histology. Other genes involved in the Krebs cycle, which are also implicated in neoplasia are 3 of the 4 subunits encoding succinate dehydrogenase (SDH); mutations in SHDB, SDHC, and SDHD predispose to paraganglioma and phaeochromocytoma. Although uterine leiomyomas (or fibroids) are very common, the estimations of affected women ranging from 25% to 77%, not much is known about their genetic background. Cytogenetic studies have revealed that rearrangements involving chromosomes 6, 7, 12 and 14 are most commonly seen in fibroids. Deletions on the long arm of chromosome 7 have been reported to be involved in about 17 to 34 % of leiomyomas and the small commonly deleted region on 7q22 suggests that there might be an underlying tumour suppressor gene in that region. The purpose of this study was to investigate the genetic mechanisms behind the development of tumours associated with HLRCC, both renal cell cancer and uterine fibroids. Firstly, a database search at the Finnish cancer registry was conducted in order to identify new families with early-onset RCC and to test if the family history was compatible with HLRCC. Secondly, sporadic uterine fibroids were tested for deletions on 7q in order to define the minimal deleted 7q-region, followed by mutation analysis of the candidate genes. Thirdly, oligonucleotide chips were utilised to study the global gene expression profiles of uterine fibroids in order to test whether 7q-deletions and FH mutations significantly affected fibroid biology. In the screen for early-onset RCC, 214 families were identified. Subsequently, the pedigrees were constructed and clinical data obtained. One of the index cases (RCC at the age of 28) had a mother who had been diagnosed with a heart tumour, which in further investigation turned out to be a paraganglioma. This lead to an alternative hypothesis that SDH, instead of FH, could be involved. SDHA, SDHB, SDHC and SDHD were sequenced from these individuals; a germline SDHB R27X mutation was detected with loss of the wild-type allele in both tumours. These results suggest that germline mutations in the SDHB gene predispose to early-onset RCC establishing a novel form of hereditary RCC. This has immediate clinical implications in the surveillance of patients suffering from early-onset RCC and phaeochromocytoma/paraganglioma. For the studies on sporadic uterine fibroids, a set of 166 fibroids from 51 individuals were collected. The 7q LOH mapping defined a commonly deleted region of about 3.2 mega bases in 11 of the 166 tumours. The deletion was consistent with previously reported allelotyping studies of leiomyomas and it therefore suggested the presence of a tumour suppressor gene in the deleted region. Furthermore, the high-resolution aCGH-chip analysis refined the deleted region to only 2.79Mb. When combined with previous data, the commonly deleted region was only 2.3Mb. The mutation screening of the known genes within the commonly deleted region did not reveal pathogenic mutations, however. The expression microarray analysis revealed that FH-deficient fibroids, both sporadic and familial, had their distinct gene expression profile as they formed their own group in the unsupervised clustering. On the other hand, the presence or absence of 7q-deletions did not significantly alter the global gene expression pattern of fibroids, suggesting that these two groups do not have different biological backgrounds. Multiple differentially expressed genes were identified between FH wild-type and FH-mutant fibroids, and the most significant increase was seen in the expression of carbohydrate metabolism-related and hypoxia inducible factor (HIF) target genes.

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.

Molecular and clinical characteristics of tricarboxylic acid cycle-associated tumors

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare, dominantly inherited tumor predisposition syndrome characterized by benign cutaneous and uterine (ULM) leiomyomas, and sometimes renal cell cancer (RCC). A few cases of uterine leiomyosarcoma (ULMS) have also been reported. Mutations in a nuclear gene encoding fumarate hydratase (FH), an enzyme of the mitochondrial tricarboxylic acid cycle (TCA cycle), underlie HLRCC. As a recessive condition, germline mutations in FH predispose to a neurological defect, FH deficiency (FHD). Hereditary paragangliomatosis (HPGL) is a dominant disorder associated with paragangliomas and pheochromocytomas. Inherited mutations in three genes encoding subunits of succinate dehydrogenase (SDH), also a TCA cycle enzyme, predispose to HPGL. Both FH and SDH seem to act as tumor suppressors. One of the consequences of the TCA cycle defect is abnormal activation of HIF1 pathway ( pseudohypoxia ) in the HLRCC and HPGL tumors. HIF1 drives transcription of genes encoding e.g. angiogenetic factors which can facilitate tumor growth. Recently hypoxia/HIF1 has been suggested to be one of the causes of genetic instability as well. One of the aims of this study was to broaden the clinical definers of HLRCC. To determine the cancer risk and to identify possible novel tumor types associated with FH mutations eight Finnish HLRCC/FHD families were extensively evaluated. The extension of the pedigrees and the Finnish Cancer Registry based tumor search yielded genealogical and cancer data of altogether 868 individuals. The standardized incidence ratio-based comparison of HLRCC/FHD family members with general Finnish population revealed 6.5-fold risk for RCC. Moreover, risk for ULMS was highly increased. However, according to the recent and more stringent diagnosis criteria of ULMS many of the HLRCC uterine tumors previously considered malignant are at present diagnosed as atypical or proliferative ULMs (with a low risk of recurrence). Thus, the formation of ULMS (as presently defined) in HLRCC appears to be uncommon. Though increased incidence was not observed, interestingly the genetic analyses suggested possible association of breast and bladder cancer with loss of FH. Moreover, cancer cases were exceptionally detected in an FHD family. Another clinical finding was the conventional (clear cell) type RCC of a young Spanish HLRCC patient. Conventional RCC is distinct from the types previously observed in this syndrome but according to these results, FH mutation may underlie some of young conventional cancer cases. Secondly, the molecular pathway from defective TCA cycle to tumor formation was intended to clarify. Since HLRCC and HPGL tumors display abnormally activated HIF1, the hypothesis on the link between HIF1/hypoxia and genetic instability was of interest to study in HLRCC and HPGL tumor material. HIF1α (a subunit of HIF1) stabilization was confirmed in the majority of the specimens. However, no repression of MSH2, a protein of DNA mismatch repair system, or microsatellite instability (MSI), an indicator of genetic instability, was observed. Accordingly, increased instability seems not to play a role in the tumorigenesis of pseudohypoxic TCA cycle-deficient tumors. Additionally, to study the putative alternative functions of FH, a recently identified alternative FH transcript (FHv) was characterized. FHv was found to contain instead of exon 1, an alternative exon 1b. Differential subcellular distribution, lack of FH enzyme activity, low mRNA expression compared to FH, and induction by cellular stress suggest FHv to have a role distinct from FH, for example in apoptosis or survival. However, the physiological significance of FHv requires further elucidation.

Animal model and molecular interactions of Cln5

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited pediatric neurodegenerative disorders, leading to retinal degeneration, death of selective neuronal populations and accumulation of autofluorscent ceroid-lipopigments. The clinical manifestations are generally similar in all forms. The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin) is a form of NCL, especially enriched in the Finnish population. The aim of this thesis was to analyse the brain pathology of vLINCLFin utilising the novel Cln5-/- mouse model. Gene expression profiling of the brains of already symptomatic Cln5-/- mice revealed that inflammation, neurodegeneration and defects in myelinization are the major characteristics of the later stages of the disease. Histological characterization of the brain pathology confirmed that the thalamocortical system is affected in Cln5-/- mice, similarly to the other NCL mouse models. However, whereas the brain pathology in all other analyzed NCL mice initiate in the thalamus and spread only months later to the cortex, we observed that the sequence of events is uniquely reversed in Cln5-/- mice; beginning in the cortex and spreading to the thalamus only months later. We could also show that even though neurodegeneration is inititated in the cortex, reactive gliosis and loss of myelin are evident in specific nuclei of the thalamus already in the 1 month old brain. To obtain a deeper insight into the disturbed metabolic pathways, we performed gene expression profiling of presymptomatic mouse brains. We validated these findings with immunohistological analyses, and could show that cytoskeleton and myelin were affected in Cln5-/- mice. Comparison of gene expression profiling results of Cln5-/- and Cln1-/- mice, further highlighted that these two NCL models share a common defective pathway, leading to disturbances in the neuronal growth cone and cytoskeleton. Encouraged by the evidence of this defected pathway, we analyzed the molecular interactions of NCL-proteins and observed that Cln5 and Cln1/Ppt1 proteins interact with each other. Furthermore, we demonstrated that Cln5 and Cln1/Ppt1 share an interaction partner, the F1-ATP synthase, potentially linking both vLINCLFIN and INCL diseases to disturbed lipid metabolism. In addition, Cln5 was shown to interact with other NCL proteins; Cln2, Cln3, Cln6 and Cln8, implicating a central role for Cln5 in the NCL pathophysiology. This study is the first to describe the brain pathology and gene expression changes in the Cln5-/- mouse. Together the findings presented in this thesis represent novel information of the disease processes and the molecular mechanisms behind vLINCLFin and have highlighted that vLINCLFin forms a very important model to analyze the pathophysiology of NCL diseases.

Sindbis virus and Pogosta disease in Finland

Sindbis virus (SINV) (genus Alphavirus, family Togaviridae) is an enveloped virus with a genome of single-stranded, positive-polarity RNA of 11.7 kilobases. SINV is widespread in Eurasia, Africa, and Australia, but clinical infection only occurs in a few geographically restricted areas, mainly in Northern Europe. In Europe, antibodies to SINV were detected from patients with fever, rash, and arthritis for the first time in the early 1980s in Finland. It became evident that the causative agent of this syndrome, named Pogosta disease, was closely related to SINV. The disease is also found in Sweden (Ockelbo disease) and in Russia (Karelian fever). Since 1974, for unknown reason, the disease has occurred as large outbreaks every seven years in Finland. This study is to a large degree based on the material collected during the 2002 Pogosta disease outbreak in Finland. We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".