Brain tumours account for the most frequent type of solid tumours among children. Despite advances in surgery and chemotherapy, brain tumours are still the main cause of cancer deaths in children. Furthermore, little is known about DNA methylation changes in paediatric astrocytoma. Recent investigations suggest that many tumours are initiated not only by genetic abnormalities, but also caused by epigenetic changes. DNA methylation is a key epigenetic mechanism that controls the regulation of gene expression. Interestingly, unlike DNA mutations, epigenetic abnormalities are reversible. The reversibility of epigenetic abnormalities upon pharmacological unmasking has prompted interest in developing epigenetic therapy with the crucial goal of restoring the expression of aberrantly silenced genes. The focus of this study was to utilise a combination of different microarray strategies to develop an integrative candidate gene approach to identify several novel frequently methylated genes in a cohort of paediatric HGA (High grade glioma) samples. In addition, to investigate the potential of therapeutic efficacy of a DNA methyltransferase inhibitor, 5-Aza-dC in paediatric HGA. There were 147 genes commonly identified to be potentially methylated in IN699 cells using the two different array strategies integration; re-expression array and Illumina Infinium Human Methylation 450k array. Furthermore, using two complementary microarray strategies including methylation 450k array and expression array, this work identified 55 genes that were both methylated and under-expressed in these HGA cultures. Following validation with CoBRA and RT-PCR coupled with the response of hypermethylated promoters to the demethylating agent 5-Aza-dC, six novel genes (CXCL14, PRR5L, ELTD1, ITGA2, KRT8 and NTM) that are frequently silenced in paediatric astrocytoma were identified. This study suggests that re-expression of ii CXCL14 inhibited the colony formation and cell growth and reduces the migration rate significantly in IN699 short term culture and likely have functional significance in the development of paediatric HGA and an excellent candidate gene for further analysis. In parallel, the efficacy of 5-Aza-dC treatment was examined in paediatric HGA aiming to introduce this epigenetic therapy as a potential mechanism in management of this tumours. This study demonstrated that, relatively low dose of 5-Aza-dC sharply reduced the colony formation and inhibited proliferation and not through the apoptotic effect. It is likely that this reduction in proliferation without cell death is due to using relatively low doses that do not acutely kill cells, thus, allow the sustained alterations in both gene expression patterns and appearance of a new phenotype to emerge. Taken together, this work contributes to a more detailed understanding of the effect of epigenetic silencing on paediatric HGA. This investigation also demonstrated the use of epigenetic drug, 5-aza-dC to reverse the gene silencing for the potential treatment of paediatric HGA.

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes. The aim of the study was to describe genetic derangements in endocrine tumors. Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors. Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of  benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine. Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior. Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis. Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.

In chronic lymphocytic leukemia (CLL), the presence of multiple subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has implicated antigen(s) in leukemogenesis. These stereotyped subsets display similar immunoglobulin (IG) gene usage, almost identical complementarity determining region 3’s and may share clinical features. For instance, subsets #1 (IGHV1/5/7/IGKV1-39) and #2 (IGHV3-21/IGLV3-21) have inferior outcome compared to non-subset patients, whereas subset #4 (IGHV4-34/IGKV2-30) display a favourable prognosis. The aim of this thesis was to investigate genomic aberrations, gene expression patterns and methylation profiles in stereotyped subsets and compare epigenetic profiles in CLL and mantle cell lymphoma (MCL). In paper I, we investigated genomic aberrations in subsets #2, #4 and #16 and in non-stereotyped samples (n=101) using high-density 250K SNP arrays. Subset #2 and non-subset #2 IGHV3-21 cases displayed a higher frequency of aberrations than subset #4 cases. The high incidence of del(11q) in both subset #2/non-subset #2 may reflect the adverse survival reported for IGHV3-21 patients. In contrast, the lower frequency of genetic events and lack of poor-prognostic aberrations in subset #4 may partially explain their indolent disease. In paper II, we analysed the global RNA expression in subset #4, #16 and non-subset IGHV4-34 CLL patients (n=25). Subsets #4 and 16 showed distinct gene expression profiles, where genes involved in cell regulatory pathways were significantly lower expressed in subset #4, in line with their low-proliferative disease. In paper III, a genome-wide methylation array was applied to investigate methylation profiles in subsets #1, #2 and #4 (n=39). We identified differential methylation patterns for all subsets and found affected genes to be involved in e.g. apoptosis and therapy resistance. When performing functional annotation, a clear enrichment of genes involved in adaptive immunity was observed. These genes were preferentially methylated in subset #1 when compared to either subset #2 or #4, possibly due to different antigen responses. In paper IV, the genome-wide methylation profiles for 30 CLL and 20 MCL patients were investigated. Distinct methylation profiles were observed, where MCL displayed a more homogeneous profile. Homeobox transcription factor genes showed a higher degree of methylation in MCL, while apoptosis-related genes and proliferation-associated genes were methylated in CLL. In summary, this thesis demonstrates that stereotyped CLL subsets display differences in gene expression profiles, genetic aberrations and methylation patterns, underscoring the functional relevance of subgrouping according to BCR stereotypy. The distinct methylation profiles of CLL and MCL suggests that different epigenetic mechanisms are involved in the pathogenesis of these B-cell malignancies.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) both belong to the group of mature B-cell malignancies. However, MCL is typically clinically aggressive while the clinical course of CLL varies. CLL can be divided into prognostic subgroups based on IGHV mutational status and into multiple subsets based on closely homologous (stereotyped) B-cell receptors. In paper I we investigated 31 MCL cases using high-density 250K single-nucleotide polymorphism arrays and gene expression arrays. Although most copy-number aberrations (CNAs) were previously reported in MCL, a novel deletion was identified at 20q (16%) containing the candidate tumor suppressor gene ZFP64. A high proliferation gene expression signature was associated with poor prognosis, large CNAs, 7p gains and 9q losses. Losses at 1p/8p/13q/17p were associated with increased genomic complexity. In paper II we sequenced exons 4 to 8 of the TP53 gene in 119 MCL cases. 17p copy-number status was known from previous studies or determined by real-time quantitative polymerase chain reaction. TP53 mutations were detected in 14% of cases and were strongly associated with poor survival while 17p deletions were more common (32%) but did not predict survival. In papers III and IV we applied high-resolution genomic 27K methylation arrays to 20 MCL and 39 CLL samples. In paper III MCL displayed a homogenous methylation profile without correlation with the proliferation signature whereas MCL was clearly separated from CLL. Gene ontology analysis revealed enrichment of developmental genes, in particular homeobox transcription factor genes, among targets methylated in MCL. In paper IV we compared three different stereotyped CLL subsets: #1 (IGHV unmutated), #2 (IGHV3-21) and #4 (IGHV mutated). Many genes were differentially methylated between each two subsets and immune response genes (e.g. CD80 and CD86) were enriched among genes methylated in subset #1 but not in subsets #2/#4. In summary, CNAs were frequent and not random in MCL. Specific CNAs correlated with a high proliferation gene expression signature or genomic complexity. TP53 mutations predicted short survival whereas 17p deletions did not. A high proliferation signature was not associated with differential DNA methylation in MCL, which demonstrated a homogeneous methylation pattern. In contrast, genomic methylation patterns differed between MCL and CLL and between stereotyped CLL subsets.

There are more than 7000 described rare genetic disorders; however, the molecular basis underlying approximately half of these disorders is unknown, and the majority are currently untreatable. Stature and growth abnormalities are a common clinical feature of many rare disorders including: Floating-Harbor syndrome (FHS), a short stature syndrome characterized by delayed osseous maturation, language deficits, and unique dysmorphic facial features; Weaver syndrome, an overgrowth syndrome characterized by advanced osseous maturation, developmental delay, and macrocephaly; and Sotos syndrome with cutis laxa, an overgrowth syndrome with marked tissue laxity in addition to the typical Sotos characteristics of developmental delay, macrocephaly, and a unique facial gestalt. The genetic basis underlying these three rare stature conditions were unknown at the outset of this study. We utilized high-throughput exome sequencing approaches to investigate the molecular etiology of these rare disorders and identified truncating mutations in the final exon of SRCAP as the genetic cause underlying FHS, missense mutations in EZH2 in Weaver syndrome, and novel mutations in the Sotos syndrome gene NSD1 in Sotos syndrome with cutis laxa. Next, we investigated the spectrum of SRCAP mutations in FHS and established the clustering of truncating SRCAP mutations in the final exon as being highly suggestive of a non-haploinsufficiency mutational mechanism in FHS. Finally, global methylation array analysis identified a unique methylation ‘epi-signature’ in FHS individuals, providing further insight into FHS disease mechanism and a diagnostic signature. These studies have delineated the molecular etiology of these three rare stature/growth disorders, furthered our understanding of the associated clinical spectrum, and provided biological insight into disease pathogenesis.