Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.

Mutation analysis of TGF-β signaling pathway genes among Finnish patients with primary pulmonary hypertension and hereditary hemorrhagic telangiectasia

Primary pulmonary hypertension (PPH), or according to the recent classification idiopathic pulmonary hypertension (IPAH), is a rare, progressive disease of pulmonary vasculature leading to pulmonary hypertension and right heart failure. Most of the patients are sporadic but in about 6% of cases the disease is familial (FPPH). In 2000 two different groups identified the gene predisposing to PPH. This gene, Bone morphogenetic protein receptor type 2 (BMPR2), encodes a subunit of transforming growth factor β (TGF-β) receptor complex. There is a genetic connection between PPH and hereditary hemorrhagic telangiectasia (HHT), a bleeding disorder characterized by local telangiectasias and sometimes with pulmonary hypertension. In HHT, mutations in ALK1 (activin like kinase type 1) and Endoglin, another members of the TGF-β signaling pathway are found. In this study we identified all of the Finnish PPH patients for the years 1986-1999 using the hospital discharge registries of Finnish university hospitals. During this period we found a total of 59 confirmed PPH patients: 55 sporadic and 4 familial representing 3 different families. In 1999 the prevalence of PPH was 5.8 per million and the annual incidence varied between 0.2-1.3 per million. Among 28 PPH patients studied, heterozygous BMPR2 mutations were found in 12% (3/26) of sporadic patients and in 33% of the PPH families (1/3). All the mutations found were different. Large deletions of BMPR2 were excluded by single-stranded chain polymomorphism analysis. As a candidate gene approach we also studied ALK1, Endoglin, Bone Morphogenetic Receptor Type IA (BMPR1A or ALK3), Mothers Against Decapentaplegic Homolog 4 (SMAD4) and Serotonine Transporter Gene (SLC6A4) using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. Among patients and family members studied, we found two mutations in ALK1 in two unrelated samples. We also identified all the HHT patients treated at the Department of Otorhinolaryngology at Helsinki University Central Hospital between the years of 1990-2005 and 8 of the patients were studied for Endoglin and ALK1 mutations using direct sequencing. A total of seven mutations were found and all the mutations were different. The absence of a founder mutation in the Finnish population in both PPH and HHT was somewhat surprising. This suggests that the mutations of BMPR2, ALK1 and Endoglin are quite young and the older mutations have been lost due to repetitive genetic bottlenecks and/or negative selection. Also, other genes than BMPR2 may be involved in the pathogenesis of PPH. No founder mutations were found in PPH or HHT and thus no simple genetic test is available for diagnostics.

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.

Human Torque teno virus: epidemiology, cell biology and immunology

Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.

The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Time to rest : Signals in shoot apex developmental transitions underlying dormancy

Vuodenajat rytmittävät monivuotisten kasvien elämää pohjoisella pallonpuoliskolla, jolla varmin merkki lähestyvästä talvikaudesta on asteittain lyhenevä päivänpituus. Kun päivänpituus on lyhentynyt tiettyyn raja-arvoon saakka, kasvu hiipuu ja kasvin kehityksessä tapahtuu suuria muutoksia. Väitöskirjatyössäni tutkittiin mekanismeja, jotka liittyvät pituuskasvun päättymiseen, silmujen lepotilan kehittymiseen ja kärkisilmun muodostumiseen hybridihaavan ja koivuntaimilla lyhyen päivänpituuden seurauksena kasvihuoneolosuhteissa. Vain lepotilaiset silmut selviytyvät luonnossa ankaran talvikauden yli, joten etenkin lepotilan kehittymisen tutkiminen on keskeistä pyrittäessä selvittämään monivuotisille kasveille tyypillisen kasvutavan mekanismeja. Jo pitkään on tiedetty, että täysikasvuiset lehdet vastaanottavat tiedon päivänpituudesta ja lähettävät signaaleja varren johtojänteissä ylöspäin kohti kasvin kärkiosaa. Sen sijaan varren kärjen ja kärkikasvupisteen roolia lepotilan kehittymisessä on selvitetty vain vähän. Kuitenkin juuri kärkikasvupisteen selviytyminen vuodesta toiseen on tärkeää, koska sen jakautumiskykyiset solukot tuottavat kasvin maanpäälliset osat. Tässä työssä tehdyissä varttamiskokeissa osoitettiin, että varren kärki ei ainoastaan vastaanota signaaleja lehdistä ja ajoita toimintaansa niiden mukaan, vaan myös kärjellä itsellään on aktiivinen rooli lepotilan kehittymisessä. Erityisesti kiinnitettiin huomiota kärkikasvupisteen eri alueiden, ns. apikaalimeristeemin ja rib-meristeemin erilaisiin tehtäviin ja pääteltiin, että molemmat vaikuttavat lepotilan kehittymiseen. Kokeissa käytettiin normaalien hybridihaapojen lisäksi siirtogeenisiä hybridihaapoja, jotka eivät lopeta kasvuaan lyhyt päivä –olosuhteissa. Siirtogeeniset hybridihaavat ilmensivät voimakkaasti fytokromi A -nimistä valon vastaanottajamolekyyliä rib-meristeemin alueella, mikä saattoi osaltaan vaikuttaa poikkeavaan pituuskasvukäyttäytymiseen. Myös useiden lepotilan kehittymiseen liittyvien geenien ilmenemisessä havaittiin poikkeavuuksia verrattuna ei-siirtogeenisiin kontrolleihin, joiden silmuissa kehittyi lepotila lyhyt päivä –altistuksen seurauksena. Väitöskirjatyössäni havaittiin, että myös kaasumainen kasvihormoni etyleeni toimii viestinvälittäjänä silmujen lepotilan kehittymisessä ja vaikuttaa etenkin lepotilan oikeaan ajoittumiseen. Etyleenillä huomattiin olevan määräävä rooli päätesilmun muodostumisessa: siirtogeeniset koivut, jotka eivät aisti etyleeniä, eivät muodostaneet päätesilmua. Silti siirtogeeniset koivut vaipuivat lepotilaan, joskin myöhemmin kuin ei-siirtogeeniset kontrollit. Tämän perusteella todettiin, että lepotilan ja päätesilmun kehittyminen ovat erillisiä kehitystapahtumia, vaikka ne saattavatkin ajoittua osaksi päällekkäin.

Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.