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Schwannomatosis (SWN) is a genetic disorder that predisposes affected individuals to develop multiple Schwannomas anywhere in the peripheral nervous system. This can be due to a mutation in the LZTR1 or SMARCB1 genes on chromosome 22. SWN has the defining clinical symptom of chronic pain and a lack of vestibular schwannomas, which sets it apart from other, related disorders such as Neurofibromatosis Type II (NF2). Currently, it is unknown what causes the chronic pain of SWN patients but it is hypothesized that cytokines may have promote the neuropathic pain experienced by patients. This study investigates the presence of the chemokine CCL2 and the cytokine IL6 in human SWN schwannomas and non-SWN schwannomas to determine if there is a difference in the presence of these cytokines between the two tumor types. It was demonstrated that all of the SWN schwannomas expressed both CCL2 and IL6 whereas the non-SWN schwannomas expressed only one or the other protein if either. These results indicate that the presence of these cytokines within the SWN schwannomas is different from non-SWN schwannomas and could be a potential contributing factor in the occurrence of neuropathic pain experienced by SWN which is part of the differential diagnosis for NF2 and SWN.

<p>Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the <i>NF2 </i>gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the <i>NF2</i> gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the <i>NF2</i> locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the <i>NF2 </i>locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the <i>GSTT1/CABIN1</i> locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the <i>CABIN1</i> gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.</p>

<p>Microarray-based comparative genomic hybridization (array-CGH) has emerged as a versatile platform with a wide range of applications in molecular genetics. This thesis focuses on the development of array-CGH with a specific aim to approach disease-related questions through improved strategies in array construction and enhanced resolution of analysis. In <b>paper I</b>, we applied an array covering 11 Mb of 22q, encompassing the <i>NF2</i> locus, for deletion detection in sporadic schwannoma. Hemizygous deletions and tumor heterogeneity were identified. Array-CGH was established as a reliable platform for detection of DNA dosage alterations. <b>Paper II</b> described the construction of the<i> NF2</i> gene-specific microarray for high-resolution scanning of deletions in the <i>NF2</i> locus. We report a novel PCR-based non-redundant strategy for microarray fabrication, which considerably improved the sensitivity and reliability of deletion detection. <b>Paper III</b> reported the first tiling-path array comprehensively covering a human chromosome. The usefulness of the 22q-array was demonstrated by applying it to detect DNA dosage-alterations in 22q-associated disorders. In <b>paper IV</b>, we optimized array-CGH protocols for deletion detection in 22q11 deletion-syndrome. We showed that genomic and cDNA clones are not optimal for analysis of 22q11 locus and that PCR-based non-redundant strategy is reliable for deletion detection in such regions. In <b>paper V</b>, we utilized the 22q-array for understanding the genetic basis of schwannomatosis. Two commonly deleted regions were identified within the <i>IGL</i> and the <i>GSTT1/CABIN1</i> loci. Further investigations using high-resolution arrays, bioinformatic analysis and mutational screening were performed. Missense mutations, specific to the schwannomatosis- and NF2 samples, were identified in the <i>CABIN1 </i>gene. <b>Paper VI</b> described the first array-CGH study for comprehensive and high-resolution profiling of deletions spanning the 17q11 locus. Both typical and atypical deletions were identified in NF1 samples. Bioinformatic analysis revealed novel segmental duplications, which can potentially mediate 17q11 deletions.</p>

<p>Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral vestibular schwannomas (VS). Patients affected by a severe NF2 phenotype also presents with peripheral schwannomas, meningiomas and ependymomas. The closely related disorder schwannomatosis also displays multiple schwannomas, but never VS. Mutation screening of the <i>NF2</i> gene in the above mentioned tumors did not identify mutations in numerous of cases. We analyzed the DNA sequence covering the <i>NF2</i> locus in order to identify evolutionarily conserved non-genic sequences (CNGs) with unknown regulatory function (paper I). The aim was to analyze CNGs for mutations in DNA derived from patients affected by NF2 associated tumors. During mutation analysis of the coding part of <i>NF2</i> and within the CNGs defined in paper I, were mutations detected in 39% of sporadic meningiomas (paper II). Two candidate regions were identified on 22q using array-CGH. Methylation profiling did not identify methylation of the <i>NF2</i> promoter in these tumors. Sporadic schwannomas were profiled for CNV using a 22q genomic array in the search for putative gene(s) that in addition to <i>NF2</i> could be involved in the development of schwannoma and/or schwannomatosis (paper III). The predominant aberration identified was monosomy 22. Terminal and interstitial deletions encompassing the <i>NF2</i> gene were detected in tumor DNA and eight loci affected by CNV in constitutional DNA. Some of these CNVs are unlikely to be phenotypically neutral, considering their size and gene content. Two schwannomatosis candidate regions were identified on 22q using array-CGH (paper IV). These regions were further characterized by a PCR-product based array with higher resolution. Rearrangements of the immunoglobulin lambda (<i>IGL</i>) locus detected were restricted to schwannomatosis patients. In the second candidate region spanning <i>GSTT1</i> and <i>CABIN1</i> genes, was frequent copy number polymorphism at the <i>GSTT1</i> locus identified. We further describe missense mutations in the <i>CABIN1 </i>gene, making this gene a plausible candidate which may contribute to the pathogenesis of these disorders. </p>

Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the NF2 gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the NF2 gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the NF2 locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the NF2 locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the GSTT1/CABIN1 locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the CABIN1 gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.

Microarray-based comparative genomic hybridization (array-CGH) has emerged as a versatile platform with a wide range of applications in molecular genetics. This thesis focuses on the development of array-CGH with a specific aim to approach disease-related questions through improved strategies in array construction and enhanced resolution of analysis. In <b>paper I</b>, we applied an array covering 11 Mb of 22q, encompassing the NF2 locus, for deletion detection in sporadic schwannoma. Hemizygous deletions and tumor heterogeneity were identified. Array-CGH was established as a reliable platform for detection of DNA dosage alterations. <b>Paper II</b> described the construction of the NF2 gene-specific microarray for high-resolution scanning of deletions in the NF2 locus. We report a novel PCR-based non-redundant strategy for microarray fabrication, which considerably improved the sensitivity and reliability of deletion detection. <b>Paper III</b> reported the first tiling-path array comprehensively covering a human chromosome. The usefulness of the 22q-array was demonstrated by applying it to detect DNA dosage-alterations in 22q-associated disorders. In <b>paper IV</b>, we optimized array-CGH protocols for deletion detection in 22q11 deletion-syndrome. We showed that genomic and cDNA clones are not optimal for analysis of 22q11 locus and that PCR-based non-redundant strategy is reliable for deletion detection in such regions. In <b>paper V</b>, we utilized the 22q-array for understanding the genetic basis of schwannomatosis. Two commonly deleted regions were identified within the IGL and the GSTT1/CABIN1 loci. Further investigations using high-resolution arrays, bioinformatic analysis and mutational screening were performed. Missense mutations, specific to the schwannomatosis- and NF2 samples, were identified in the CABIN1 gene. <b>Paper VI</b> described the first array-CGH study for comprehensive and high-resolution profiling of deletions spanning the 17q11 locus. Both typical and atypical deletions were identified in NF1 samples. Bioinformatic analysis revealed novel segmental duplications, which can potentially mediate 17q11 deletions.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral vestibular schwannomas (VS). Patients affected by a severe NF2 phenotype also presents with peripheral schwannomas, meningiomas and ependymomas. The closely related disorder schwannomatosis also displays multiple schwannomas, but never VS. Mutation screening of the NF2 gene in the above mentioned tumors did not identify mutations in numerous of cases. We analyzed the DNA sequence covering the NF2 locus in order to identify evolutionarily conserved non-genic sequences (CNGs) with unknown regulatory function (paper I). The aim was to analyze CNGs for mutations in DNA derived from patients affected by NF2 associated tumors. During mutation analysis of the coding part of NF2 and within the CNGs defined in paper I, were mutations detected in 39% of sporadic meningiomas (paper II). Two candidate regions were identified on 22q using array-CGH. Methylation profiling did not identify methylation of the NF2 promoter in these tumors. Sporadic schwannomas were profiled for CNV using a 22q genomic array in the search for putative gene(s) that in addition to NF2 could be involved in the development of schwannoma and/or schwannomatosis (paper III). The predominant aberration identified was monosomy 22. Terminal and interstitial deletions encompassing the NF2 gene were detected in tumor DNA and eight loci affected by CNV in constitutional DNA. Some of these CNVs are unlikely to be phenotypically neutral, considering their size and gene content. Two schwannomatosis candidate regions were identified on 22q using array-CGH (paper IV). These regions were further characterized by a PCR-product based array with higher resolution. Rearrangements of the immunoglobulin lambda (IGL) locus detected were restricted to schwannomatosis patients. In the second candidate region spanning GSTT1 and CABIN1 genes, was frequent copy number polymorphism at the GSTT1 locus identified. We further describe missense mutations in the CABIN1 gene, making this gene a plausible candidate which may contribute to the pathogenesis of these disorders.