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This investigation reorganizes and identifies chromosomal anomalies and delineates the associated clinical findings. The present investigation involved 37 individuals with anomalies of chromosome 22. The clinical profile with the corresponding cytogenetic anomalies was studied.

A gene dosage study was conducted on a rare complete trisomy 22 human fibroblast cell line utilizing three lysosomal enzymes, ∝-iduronidase, ∝-galactosidase B, and arylsulfatase A, whose genes are located on chromosome 22 and two control enzymes, ,β-hexosaminidase A and -- fucosidase, with genes not on chromosome 22. A gene dosage effect was clearly demonstrated for an early passage number of the fibroblasts; however, later passage numbers gave inconclusive results. This study suggests that gene dosage studies must be carefully designed to be conducted only on early, matched passage number cells. ∝-fucosidase gave anomalous results most likely due to pleiotropic effects. The present gene dosage study confirmed the trisomic nature of the cell line studied and suggests that this type of study may be a useful diagnostic tool for small deletions, additions, or unbalanced translocations.

Thesis (PhD (Psychiatry))--Stellenbosch University, 2008. / BACKGROUND: Schizophrenia is a heterogeneous illness resulting from complex gene-environment interplay. The majority of molecular genetic work done has involved Caucasian populations, with studies in these and Asian populations showing 2-32% of sufferers to have chromosomal aberrations. So far the discovery of a specific susceptibility mechanism or gene still eludes us, but the use of endophenotypes is advocated as a useful tool in this search. No cytogenetic studies of this nature have been reported in any African schizophrenia population. AIM: The aim of the study was to combine genotypic and phenotypic data, collected in a homogenous population in a structured manner, with the hope of characterising an endophenotype that could be used for more accurate identification of individuals with possible chromosomal abnormalities. METHODOLOGY: A structured clinical interview was conducted on 112 Xhosa schizophrenia patients. (Diagnostic Interview for Genetic Studies, including Schedules for the Assessment of Negative and Positive Symptoms.) Blood samples (karyotyping and/or FISH analysis) as well as urine samples (drug screening) were obtained and nine head and facial measurements were performed. Descriptive statistics were compiled with reference to demographic, clinical and morphological variables. Comparisons between mean differences for these variables were made.

<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>The array-based form of comparative genomic hybridization (array-CGH) is a new methodology that has shown to be of significant importance. This thesis focuses on the development of array-CGH with the aim to define candidate regions/genes on chromosome 22 in a wide spectrum of cancer-related conditions. In <b>paper I</b>, we developed and applied the first comprehensive genomic microarray, representing human chromosome 22, for analysis of DNA copy number. Using this array-based approach, we identified gene copy number alterations, including heterozygous/homozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of imbalanced translocation, in several 22q-associated disorders. In <b>paper II</b>, we applied the same array to perform DNA copy number profiling of a series of ovarian carcinoma. cDNA arrays were also used in this study to correlate gene expression levels with DNA-copy number. In the course of this analysis, we determined a small 3.5 Mb candidate 22q telomeric region and suggested a number of specific candidate genes. <b>Paper III</b> described the comprehensive and high-resolution analysis of chromosome 22 in a large set of various stage breast cancers. Multiple distinct patterns of genetic aberrations were observed. The smallest identified candidate locus was 220 kb in size and mapped to a gene-rich region in the vicinity of telomere of 22q. Intriguing result of this study was the detection of high frequency (26.6%) of intra-tumoral clonal variation in gene copy number profiles, which should be viewed as a high number, considering that we study in detail only a single human chromosome. In <b>paper IV</b>, we profiled a series of 28 Wilms tumor samples using 22q-array in order to assess specific regions affected with DNA dosage-alterations. The distribution of aberrations defined a complex amplifier genotype and delimited two tumor suppressor/oncogene candidate loci. These results open up for several avenues for continued research of these tumor forms. These findings also demonstrate the power of array-CGH in the precise determination of minute DNA copy number alterations and strengthen the notion that further studies, preferentially in the context of the entire human genome, are needed.</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.