Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

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).

Molecular Genetic Background of Tumours in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a hereditary tumour predisposition syndrome. Its phenotype includes benign cutaneous and uterine leiomyomas (CLM, ULM) with high penetrance and rarer renal cell cancer (RCC), most commonly of papillary type 2 subtype. Over 130 HLRCC families have been identified world-wide but the RCC phenotype seems to concentrate in families from Finland and North America for unknown reasons. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. FH encodes the enzyme fumarase from mitochondrial citric acid cycle. Fumarase enzyme activity or type or site of the FH mutation are unassociated with disease phenotype. The strongest evidence for tumourigenesis mechanism in HLRCC supports a hypoxia inducible factor driven process called pseudohypoxia resulting from accumulation of the fumarase substrate fumarate. In this study, to assess the importance of gene- or exon-level deletions or amplifications of FH in patients with HLRCC-associated phenotypes, multiplex ligation-dependent probe amplification (MLPA) method was used. One novel FH mutation, deletion of exon 1, was found in a Swedish male patient with an evident HLRCC phenotype with CLM, RCC, and a family history of ULM and RCC. Six other patients with CLM and 12 patients with only RCC or uterine leiomyosarcoma (ULMS) remained FH mutation-negative. These results suggest that copy number aberrations of FH or its exons are an infrequent cause of HLRCC and that only co-occurrence of benign tumour types justifies FH-mutation screening in RCC or ULMS patients. Determination of the genomic profile of 11 HLRCC-associated RCCs from Finnish patients was performed by array comparative genomic hybridization. The most common copy number aberrations were gains of 2, 7, and 17 and losses of 13q12.3-q21.1, 14, 18, and X. When compared to aberrations of sporadic papillary RCCs, HLRCC-associated RCCs harboured a distinct DNA copy number profile and lacked many of the changes characterizing the sporadic RCCs. The findings suggest a divergent molecular pathway for tumourigenesis of papillary RCCs in HLRCC. In order to find a genetic modifier of RCC risk in HLRCC, genome-wide linkage and identical by descent (IBD) analysis studies were performed in Finnish HLRCC families with microsatellite marker mapping and SNP-array platforms. The linkage analysis identified only one locus of interest, the FH gene locus in 1q43, but no mutations were found in the genes of the region. IBD analysis yielded no convincing haplotypes shared by RCC patients. Although these results do not exclude the existence of a genetic modifier for RCC risk in HLRCC, they emphasize the role of FH mutations in the malignant tumourigenesis of HLRCC. To study the benign tumours in HLRCC, genome-wide DNA copy number and gene expression profiles of sporadic and HLRCC ULMs were defined with modern SNP- and gene-expression array platforms. The gene expression array suggests novel genes involved in FH-deficient ULM tumourigenesis and novel genes with putative roles in propagation of sporadic ULM. Both the gene expression and copy number profiles of HLRCC ULMs differed from those of sporadic ULMs indicating distinct molecular basis of the FH-deficient HLRCC tumours.

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.

Novel Insights into the Molecular Genetic Background of Colorectal Tumors

Many of the genes predisposing to highly penetrant colorectal cancer (CRC) syndromes, including hereditary non-polyposis colorectal cancer (MLH1, MSH2, MSH6, PMS2), familial adenomatous polyposis (APC), Peutz-Jeghers syndrome (LKB1), juvenile polyposis (SMAD4, BMPR1A), MYH-associated polyposis (MYH), and Cowden syndrome (PTEN) have already been discovered. Identification of these genes has allowed a more precise classification of the hereditary CRC syndromes and provided a means for predictive genetic testing and surveillance. Some of the genes are also involved in sporadic cancer forms, and therefore the investigation of the rare CRC syndromes has been a breakthrough for general cancer research. Despite the accumulating knowledge on hereditary cancer syndromes, a significant number of familial CRCs remain molecularly unexplained after genetic testing, reflecting the possibility of other predisposing genes or existence of novel syndromes. Moreover, genetic variants conferring low-penetrance risk are still largely unknown. In this study, we examined the role of some new high- and low-penetrance alleles on CRC predisposition. We identified disease causing MYH mutations in a subset (9%) of patients with APC and AXIN2 mutation negative adenomatous polyposis. Due to differences in the pattern of inheritance and clinical manifestation, screening for mutations in MYH is beneficial in view of genetic counselling and surveillance. A novel functionally deficient MYH founder mutation A459D was identified in the Finnish population, and this finding had immediate clinical implications for genetic counselling of at risk families. Many patients with hamartomatous polyposis remain without molecular diagnosis due to atypical phenotypes. We therefore sought to classify 49 patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analyses of PTEN, LKB1, BMPR1A, SMAD4, ENG, BRAF, MYH, and BHD along with revision of polyp histology. Mutations were identified in 11/49 (22%) of the patients. In 6 cases the molecular diagnosis was re-classified guiding surveillance and decisions for prophylactic surgery. Re-evaluation of polyp histology with subsequent more accurate selection of candidate gene analyses is beneficial and can be recommended for patients with unexplained polyposis. Furthermore, germline mutations in ENG underlying juvenile polyposis were described for the first time, characterizing a possible novel genetically defined form of hereditary CRC. Association analyses on two putative low-penetrance alleles, NOD2 3020insC and MDM2 SNP309 were performed in a population-based series of 1042 Finnish CRC patients and in cancer-free controls. In contrast to previous results, NOD2 3020insC did not associate with CRC or age at disease onset in the Finnish population. These data suggest that NOD2 3020insC alone might not be sufficient for CRC predisposition. MDM2 SNP309 was as common in the CRC cohort as in the healthy controls. Interesting trends, however, were observed, which after correction for multiple testing did not reach statistical significance. SNP309 was more common in female CRC patients and a trend towards an earlier age at disease onset was observed in women with SNP309. Subsequent studies have supported this observation and SNP309 could affect gender- or hormone-related tumorigenesis. Finally, a large-scale unbiased effort was designed to characterize the complete mutatome of CRC with microsatellite instability (MSI). Using an approach combining expression microarray and genome database searches, we were able to identify putative MSI target genes. Further characterization of one of the genes suggested that it might play a role also in microsatellite stable CRC and Peutz-Jeghers syndrome pathogenesis.

Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate

Positional cloning has enabled hypothesis-free, genome-wide scans for genetic factors contributing to disorders or traits. Traditionally linkage analysis has been used to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and finally sequencing of regions of interest. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization. Here, we have utilized modern molecular genetic methods combined with the advantages provided by the special population structure in Finland to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as previously identified linkage to 3p14-p24 was replicated in an independent family set of families with Asperger syndrome, but no other ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between autism and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). We also used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure of Finns. We observed significant population substructure which correlates with the known history of multiple consecutive bottle-necks experienced by the Finnish population. We used this information to ascertain a genetically homogenous subset of autism families to identify possible rare, enriched risk variants using genome-wide SNP data. No rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with others in the field. The underlying genetic architecture for this group of disorders appears highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors.

Genetic diversity and microcystin production by Anabaena in the Gulf of Finland, Baltic Sea

Syanobakteerit (sinilevät) ovat olleet Itämeressä koko nykymuotoisen Itämeren ajan, sillä paleolimnologiset todisteet niiden olemassaolosta Itämeren alueella ovat noin 7000 vuoden takaa. Syanobakteerien massaesiintymät eli kukinnat ovat kuitenkin sekä levinneet laajemmille alueille että tulleet voimakkaimmiksi viimeisten vuosikymmenien aikana. Tähän on osasyynä ihmisten aiheuttama kuormitus, joka rehevöittää Itämerta. Suomenlahti, jota tämä tutkimus käsittelee, on kärsinyt tästä rehevöitymiskehityksestä muita Itämeren altaita enemmän. Syanobakteerit muodostavat jokakesäisiä kukintoja Suomenlahdella - niin sen avomerialueilla kuin rannoillakin. Yleisimmät kukintoja muodostavat syanobakteerisuvut ovat Nodularia, Anabaena ja Aphanizomenon. Kukinnat aiheuttavat paitsi esteettistä haittaa myös terveydellisen riskitekijän. Niiden myrkyllisyys liitetään usein Nodularia-suvun tuottamaan nodulariini-maksamyrkkyyn. Itämeren Aphanizomenon-suvun on todettu olevan myrkytön. Vaikka Itämeren kukintoja aiheuttavista Nodularia- ja Aphanizomenon-syanobakteereista tiedetään varsin paljon, on molekyylimenetelmiin pohjautuva syanobakteeritutkimus ohittanut Itämeren Anabaena-suvun monelta osin. Tämän työn tarkoituksena oli syventää käsitystämme Itämeren Anabaena-syanobakteerista, sen mahdollisesta myrkyllisyydestä, geneettisestä monimuotoisuudesta ja fylogeneettisista sukulaisuussuhteista. Tässä työssä eristettiin 49 planktista Anabaena-kantaa, joista viisi tuottivat mikrokystiinejä. Tämä oli ensimmäinen yksiselitteinen todiste, että Itämeren Anabaena tuottaa maksamyrkyllisiä mikrokystiini-yhdisteitä. Jokainen eristetty myrkyllinen Anabaena-kanta tuotti useita mikrokystiini-variantteja. Lisäksi mikrokystiinejä löydettiin kukintanäytteistä, joissa oli myrkkyä syntetisoivia geenejä sisältäneitä Anabaena-syanobakteereita. Myrkkyjä löydettiin molempina tutkimusvuosina 2003 ja 2004. Myrkkyjen esiintyminen ei siten ollut vain yksittäinen ilmiö. Tässä työssä saimme viitteitä siitä, että maksamyrkyllinen Anabaena-syanobakteeri esiintyisi vähäsuolaisissa vesissä. Tämä riippuvuussuhde jää kuitenkin tulevien tutkimuksien selvitettäväksi. Tässä työssä havaittiin mikrokystiinisyntetaasi-geenien inaktivoituminen Itämeren Anabaena-kannassa ja kukintanäytteissä. Kuvasimme Anabaena-kannan mikrokystiinisyntetaasigeenien sisältä insertioita, jotka hyvin todennäköisesti inaktivoivat myrkyntuoton. Insertion sisältäneeltä kannalta löysimme kuitenkin kaikki mikrokystiinisyntetaasigeenit osoittaen, että geenien olemassaolo ei välttämättä varmista kannan mikrokystiinintuottoa. Mielenkiintoista oli se, että inaktivaation aiheuttavia insertioita löytyi kukintanäytteistä molemmilta tutkimusvuosilta. Vastaavia insertioita ei kuitenkaan löydetty makean veden Anabaena-kannoista tai järvinäytteistä. On yleistä, että syanobakteerikukinnoista löytyy usean syanobakteerisuvun edustajia. Myrkyllisiä sukuja tai lajeja ei voida kuitenkaan erottaa mikroskooppisesti myrkyttömistä. Käsillä olevassa tutkimuksessa kehitettiin molekyylimenetelmä, jolla on mahdollista määrittää kukinnan mahdollisesti maksamyrkylliset syanobakteerisuvut. Tätä menetelmää sovellettiin Itämeren kukintojen tutkimiseen. Itämeren pintavesistä ja ranta-alueiden pohjasta eristetyt Anabaena-kannat osoittautuivat geneettisesti monimuotoisiksi. Tämä Anabaena-syanobakteerien geneettinen monimuotoisuus vahvistettiin monistamalla geenejä suoraan kukintanäytteistä ilman kantojen eristystä. Makeiden vesien ja Itämeren Anabaena-kannat ovat geneettisesti hyvin samankaltaisia. Geneettisissä vertailuissa kävi kuitenkin ilmi, että pohjassa elävien Anabaena-kantojen geneettinen monimuotoisuus oli suurempaa kuin pintavesistä eristettyjen kantojen. Itämeren Anabaena-kantojen sekvenssit muodostivat omia ryhmiä sukupuun sisällä, jolloin on mahdollista, että nämä edustavat Itämeren omia Anabaena-ekotyyppejä. Tämä tutkimus oli ensimmäinen, jossa uusin molekyylimenetelmin systemaattisesti selvitettiin Itämeren Anabaena-syanobakteerin geneettistä populaatiorakennetta, fylogeniaa ja myrkyntuottoa. Tulevaisuudessa monitorointitutkimuksissa on otettava huomioon myös Itämeren Anabaena-syanobakteerin mahdollinen maksamyrkyntuotto – erityisesti vähäsuolaisemmilla rannikkovesillä.

The life cycle and genetic structure of the red alga Furcellaria lumbricalis on a salinity gradient

The life cycle and genetic diversity of the red alga Furcellaria lumbricalis (Hudson) Lamouroux were investigated in 15 populations in northern Europe. The occurrence of different life cycle phases and seasonality of reproduction were studied in four brackish populations in the northern Baltic Sea. Furthermore, a new method, based on genome screening with ISSR markers combined with a restriction-ligation method, was developed to discover microsatellite markers for population genetic analyses. The mitochondrial DNA cox2-3 spacer sequence and four microsatellite markers were used to examine the genetic diversity and differentiation of red algal populations in northern Europe. In addition, clonality and small-scale genetic structure of one Irish and four Baltic Sea populations were studied with microsatellite markers. It was discovered that at the low salinities of the northern Baltic Sea, only tetrasporophytes and males were present in the populations of F. lumbricalis and that winter was the main season for tetrasporangial production. Furthermore, the population occurring at the lowest salinity (3.6 practical salinity units, psu) did not produce spores. The size of the tetraspores was smaller in the Baltic Sea populations than that in the Irish population, and there were more deformed spores in the Baltic Sea populations than in the Irish populations. Studies with microsatellite markers indicated that clonality is a common phenomenon in the Baltic Sea populations of F. lumbricalis, although the proportion of clonal individuals varied among populations. Some genetic divergence occurred within locations both in Ireland and in the northern Baltic Sea. Even though no carpogonia were detected in the field samples during the study, the microsatellite data indicated that sexual reproduction occurs at least occasionally in the northern Baltic Sea. The genetic diversity of F. lumbricalis was highest in Brittany, France. Since no variation was discovered in the mtDNA cox2-3 spacer sequence, which is generally regarded as an informative phylogeographic marker in red algae, it can be assumed that the studied populations probably share the same origin.

Genetic and epigenetic mechanisms of tumor predisposition in hereditary non-polyposis colorectal carcinoma and sporadic cancers

Individuals with inherited deficiency in DNA mismatch repair(MMR) (Lynch syndrome) LS are predisposed to different cancers in a non-random fashion. Endometrial cancer (EC) is the most common extracolonic malignancy in LS. LS represents the best characterized form of hereditary nonpolyposis colorectal carcinoma (HNPCC). Other forms of familial non-polyposis colon cancer exist, including familial colorectal cancer type X (FCCX). This syndrome resembles LS, but MMR gene defects are excluded and the predisposition genes are unknown so far. To address why different organs are differently susceptible to cancer development, we examined molecular similarities and differences in selected cancers whose frequency varies in LS individuals. Tumors that are common (colorectal, endometrial, gastric) and less common (brain, urological) in LS were characterized for MMR protein expression, microsatellite instability (MSI), and by altered DNA methylation. We also studied samples of histologically normal endometrium, endometrial hyperplasia,and cancer for molecular alterations to identify potential markers that could predict malignant transformation in LS and sporadic cases. Our results suggest that brain and kidney tumors follow a different pathway for cancer development than the most common LS related cancers.Our results suggest also that MMR defects are detectable in endometrial tissues from a proportion of LS mutation carriers prior to endometrial cancer development. Traditionally (complex) atypical hyperplasia has been considered critical for progression to malignancy. Our results suggest that complex hyperplasia without atypia is equally important as a precursor lesion of malignancy. Tumor profiles from Egypt were compared with colorectal tumors from Finland to evaluate if there are differences specific to the ethnic origin (East vs.West). Results showed for the first time a distinct genetic and epigenetic signature in the Egyptian CRC marked by high methylation of microsatellite stable tumors associated with advanced stage, and low frequency of Wnt signaling activation, suggesting a novel pathway. DNA samples from FCCX families were studied with genome wide linkage analysis using microsatellite markers. Selected genes from the linked areas were tested for possible mutations that could explain predisposition to a large number of colon adenomas and carcinomas seen in these families. Based on the results from the linkage analysis, a number of areas with tentative linkage were identified in family 20. We narrowed down these areas by additional microsatellite markers to found a mutation in the BMPR1A gene. Sequencing of an additional 17 FCCX families resulted in a BMPR1A mutation frequency of 2/18 families (11%). Clarification of the mechanisms of the differential tumor susceptibility in LS increases the understanding of gene and organ specific targets of MMR deficiency. While it is generally accepted that widespread MMR deficiency and consequent microsatellite instability (MSI) drives tumorigenesis in LS, the timing of molecular alterations is controversial. In particular, it is important to know that alterations may occur several years before cancer formation, at stages that are still histologically regarded as normal. Identification of molecular markers that could predict the risk of malignant transformation may be used to improve surveillance and cancer prevention in genetically predisposed individuals. Significant fractions of families with colorectal and/or endometrial cancer presently lack molecular definition altogether. Our findings expand the phenotypic spectrum of BMPR1A mutations and, for the first time, link FCCX families to the germline mutation of a specific gene. In particular, our observations encourage screening of additional families with FCCX for BMPR1A mutation, which is necessary in obtaining a reliable estimate of the share of BMPR1A-associated cases among all FCCX families worldwide. Clinically, the identification of predisposing mutations enables targeted cancer prevention in proven mutation carriers and thereby reduces cancer morbidity and mortality in the respective families.

Dissection of the genetic background of childhood onset progressive myoclonic epilepsies

The progressive myoclonic epilepsies (PMEs) are a clinically and etiologically heterogeneous group of symptomatic epilepsies characterized by myoclonus, tonic-clonic seizures, psychomotor regression and ataxia. Different disorders have been classified as PMEs. Of these, the group of neuronal ceroid lipofuscinoses (NCLs) comprise an entity that has onset in childhood, being the most common cause of neurodegeneration in children. The primary aim of this thesis was to dissect the molecular genetic background of patients with childhood onset PME by studying candidate genes and attempting to identify novel PME-associated genes. Another specific aim was to study the primary protein properties of the most recently identified member of the NCL-causing proteins, MFSD8. To dissect the genetic background of a cohort of Turkish patients with childhood onset PME, a screen of the NCL-associated genes PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8 and CTSD was performed. Altogether 49 novel mutations were identified, which together with 56 mutations found by collaborators raised the total number of known NCL mutations to 364. Fourteen of the novel mutations affect the recently identified MFSD8 gene, which had originally been identified in a subset of mainly Turkish patients as the underlying cause of CLN7 disease. To investigate the distribution of MFSD8 defects, a total of 211 patients of different ethnic origins were evaluated for mutations in the gene. Altogether 45 patients from nine different countries were provided with a CLN7 molecular diagnosis, denoting the wide geographical occurrence of MFSD8 defects. The mutations are private with only one having been established by a founder-effect in the Roma population from the former Czechoslovakia. All mutations identified except one are associated with the typical clinical picture of variant late-infantile NCL. To address the trafficking properties of MFSD8, lysosomal targeting of the protein was confirmed in both neuronal and non-neuronal cells. The major determinant for this lysosomal sorting was identified to be an N-terminal dileucine based signal (9-EQEPLL-14), recognized by heterotetrameric AP-1 adaptor proteins, suggesting that MFSD8 takes the direct trafficking pathway en route to the lysosomes. Expression studies revealed the neurons as the primary cell-type and the hippocampus and cerebellar granular cell layer as the predominant regions in which MFSD8 is expressed. To identify novel genes associated with childhood onset PME, a single nucleotide polymorphism (SNP) genomewide scan was performed in three small families and 18 sporadic patients followed by homozygosity mapping to determine the candidate loci. One of the families and a sporadic patient were positive for mutations in PLA2G6, a gene that had previously been shown to cause infantile neuroaxonal dystrophy. Application of next-generation sequencing of candidate regions in the remaining two families led to identification of a homozygous missense mutation in USP19 for the first and TXNDC6 for the second family. Analysis of the 18 sporadic cases mapped the best candidate interval in a 1.5 Mb region on chromosome 7q21. Screening of the positional candidate KCTD7 revealed six mutations in seven unrelated families. All patients with mutations in KCTD7 were reported to have early onset PME, rapid disease progression leading to dementia and no pathologic hallmarks. The identification of KCTD7 mutations in nine patients and the clinical delineation of their phenotype establish KCTD7 as a gene for early onset PME. The findings presented in this thesis denote MFSD8 and KCTD7 as genes commonly associated with childhood onset symptomatic epilepsy. The disease-associated role of TXNDC6 awaits verification through identification of additional mutations in patients with similar phenotypes. Completion of the genetic spectrum underlying childhood onset PMEs and understanding of the gene products functions will comprise important steps towards understanding the underlying pathogenetic mechanisms, and will possibly shed light on the general processes of neurodegeneration and nervous system regulation, facilitating the diagnosis, classification and possibly treatment of the affected cases.

From Polymorph Screening to Dissolution Testing - Solid Phase Analysis during Pharmaceutical Development and Manufacturing

Solid materials can exist in different physical structures without a change in chemical composition. This phenomenon, known as polymorphism, has several implications on pharmaceutical development and manufacturing. Various solid forms of a drug can possess different physical and chemical properties, which may affect processing characteristics and stability, as well as the performance of a drug in the human body. Therefore, knowledge and control of the solid forms is fundamental to maintain safety and high quality of pharmaceuticals. During manufacture, harsh conditions can give rise to unexpected solid phase transformations and therefore change the behavior of the drug. Traditionally, pharmaceutical production has relied on time-consuming off-line analysis of production batches and finished products. This has led to poor understanding of processes and drug products. Therefore, new powerful methods that enable real time monitoring of pharmaceuticals during manufacturing processes are greatly needed. The aim of this thesis was to apply spectroscopic techniques to solid phase analysis within different stages of drug development and manufacturing, and thus, provide a molecular level insight into the behavior of active pharmaceutical ingredients (APIs) during processing. Applications to polymorph screening and different unit operations were developed and studied. A new approach to dissolution testing, which involves simultaneous measurement of drug concentration in the dissolution medium and in-situ solid phase analysis of the dissolving sample, was introduced and studied. Solid phase analysis was successfully performed during different stages, enabling a molecular level insight into the occurring phenomena. Near-infrared (NIR) spectroscopy was utilized in screening of polymorphs and processing-induced transformations (PITs). Polymorph screening was also studied with NIR and Raman spectroscopy in tandem. Quantitative solid phase analysis during fluidized bed drying was performed with in-line NIR and Raman spectroscopy and partial least squares (PLS) regression, and different dehydration mechanisms were studied using in-situ spectroscopy and partial least squares discriminant analysis (PLS-DA). In-situ solid phase analysis with Raman spectroscopy during dissolution testing enabled analysis of dissolution as a whole, and provided a scientific explanation for changes in the dissolution rate. It was concluded that the methods applied and studied provide better process understanding and knowledge of the drug products, and therefore, a way to achieve better quality.

Work Stress and Early Atherosclerosis: Do Genetic Background and Pre-Employment Risk Factors Explain Conflicting Findings?

According to the models conceptualizing work stress, increased risk of health problems arise when high job demands co-occur with low job control (the demand-control model) or the efforts invested by the employee are disproportionately high compared to the rewards received (effort-reward imbalance model). This study examined the association between work stress and early atherosclerosis with particular attention to the role of pre-employment risk factors and genetic background in this association. The subjects were young healthy adults aged 24-39 who were participating in the 21-year follow-up of the ongoing prospective "Cardiovascular Risk in Young Finns" study in 2001-2002. Work stress was evaluated with questionnaires on demand-control model and on effort-reward model. Atherosclerosis was assessed with ultrasound of carotid artery intima-media thickness (IMT). In addition, risk for enhanced atherosclerotic process was assessed by measuring with heart rate variability and heart rate. Pre-employment risk factors, measured at age 12 to 18, included such as body mass index, blood lipids, family history of coronary heart disease, and parental socioeconomic position. Variants of the neuregulin-1 were determined using genomic DNA. The results showed that higher work stress was associated with higher IMT in men. This association was not attenuated by traditional risk factors of atherosclerosis and coronary heart disease or by pre-employment risk factors measured in adolescence. Neuregulin-1 gene moderated the association between work stress and IMT in men. A significant association between work stress and IMT was found only for the T/T genotype of the neuregulin-1 gene but not for other genotypes. Among women an association was found between higher work stress and lower heart rate variability, suggesting higher risk for developing atherosclerosis. These associations could not be explained by demographic characteristics or coronary risk factors. The present findings provide evidence for an association between work stress and atherosclerosis in relatively young population. This association seems to be modified by genetic influences but it does not appear to be confounded by pre-employment adolescent risk factors.