Genomic sequencing, comparative genomic analysis and mouse transgenics were used to investigate two regions of human chromosome 7 that are associated with chromosomal loss, duplication and disease. Williams-Beuren syndrome (WBS) is a complex neuro-developmental disorder arising from a microdeletion at chromosome band 7q11.23 that results in a hemizygous condition for a number of genes. Within this region we completely characterized 200 kilobases (kb) of genomic DNA sequence containing the genes LIMKI, WBSCRl and RFC2. The orthologous region on mouse chromosome 5 was sequenced and compared to 7q11.23. A previously unidentified gene, ( WBSCRS), was found in the region commonly deleted in WBS patients. Expression patterns and alternative splice variants of WBSCRS and WBSCRl were determined. Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene dense, G-C rich sub-region of 7q22 to the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes (BAC). A 296 kb segment from the physical map, spanning from acetylcholine esterase (Ache) to transferrin receptor 2 (Tfr2), was compared to 267 kb of human sequence. A conserved linkage of twelve genes including arsenite resistance 2 (Ars2) and zonadhesin (Zan) was identified. The paired immunoglobulin-like receptor locus (PILR) at 7q22 shares homology with 7q11.23 and contains the inhibiting PILRA and activating PILRB receptors. These receptors are expressed in myeloid cells and have been established as novel regulators of innate immunity. Expression analysis of the human PILRB gene revealed it has been dramatically affected by the insertion of a segmental duplication that is paralogous to the sequence flanking the WBS critical region. Sequencing and analysis of the orthologous region in the mouse genome revealed a previously unreported paired immunoglobulin like receptor gene (Pilrb2). An evolutionary analysis of the PILR locus in six mammalian genomes revealed that this locus is dynamically evolving by means of gene duplication, insertion, mutation and conversion. Ars2 is a novel gene that is present in a diverse number of eukaryotic organisms and is essential for development in Danio rerio and Arabidopsis thaliana. To address the role of Ars2 in mammals, we characterized its expression in mouse and human tissues and implemented a gene targeting strategy to create a null allele in the mouse genome. Ars2 was found to be transcribed throughout development and was expressed ubiquitously in mouse and human tissues. The Ars2 protein localized to the nucleus. Ars2 null mice have an early embryonic lethal phenotype establishing that Ars2 is necessary for the development of diverse multi-cellular organisms. Zonadhesin (ZAN) is the only mammalian sperm protein that has been demonstrated to bind to intact zona pellucida of the egg in a species-specific manner. We investigated the in vivo role of zonadhesin by creating a mutant mouse line that lacks zonadhesin. Zonadhesin null mice show no obvious phenotype and were determined, through natural mating, to be equally as fertile as their wildtype littermates. A web-based software tool called Laj (Local alignments with java) was developed to display comparative genomic sequence data in an intuitive and informative way. All of our genomic sequence comparisons can be viewed at http:Nweb.uvic.cal-bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/.

In this study the question of nonrandomness in the distribution of human constitutional rearrangements was evaluated. The distribution of breakpoints were analysed in three groups of reciprocal translocations and three groups of inversions, subdivided according to method of ascertainment of cases for study. In addition, one data set of structural aberrations obtained from sperm chromosomes was also analysed. The method of statistical analysis, based on the binomial distribution, was developed specifically to allow testing distributions in chromosome segments as small as chromosome bands. The distribution of breakpoints was analysed in all data sets using this method, in addition to testing for overall nonrandomness using goodness of fit statistics. Nonrandomness in breakpoint distributions was found in reciprocal translocations (rcp) and inversions ascertained through abnormalities and through incidental events. However, random distribution was observed in incidentally ascertained de novo rearrangements as well as in sperm chromosome aberrations. The nonrandomness in the distribution of rcp breakpoints can be largely attributed to a bias in ascertainment of cases based on the phenotypic manifestations of chromosomal imbalance resulting from a rearrangement. A dependence of the probability of producing specific types of balanced or unbalanced progeny on the position of breakpoints is a likely explanation for the nonrandomness produced in breakpoint distributions. However, some bands including, 5q35, 7p22, 9p22, 13ql4, and 17q25, were observed in different ascertainment groups, excluding selection bias as a likely explanation for this observation. These bands may represent true sites of nonrandom rearrangement due to some factor associated with an underlying DNA sequence or structural characteristic of chromatin that predisposes to rearrangement at specific sites. The nonrandomness observed in the distribution of inversion breakpoints is most likely the product of a founder effect. Many identical inversions in apparently unrelated individuals have been found suggesting that a few ancestral mutations have become widespread in the population. A large data set of incidentally ascertained de novo inversions is required to distinguish between sites of frequent breakage and nonrandomness produced by the ascertainment of related cases. All evidence considered together, indisputable predisposition to rearrangement at specific sites was not found in this study. Furthermore, an overall random association of constitutional rearrangement breakpoints in bands with known oncogenes and fragile sites was observed. However, the possibility of oncogenes and fragile sites as factors involved in constitutional rearrangements in a few isolated cases cannot be excluded. Nonrandomness was found when distribution of breakpoints in light and dark G bands was compared. An excess of breakpoints in some light G bands was observed even after a conservative correction for a possible pattern recognition bias which may lead to the overascertainment of breakpoints in light G bands. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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Thèse--Liège. / Includes bibliographical references.

The HBS1L-MYBintergenic region on chromosome 6q23 is a quantitative trait locus (QTL) of haemoglobin F (Hb F) expression. It shows characteristics of a distal regulatory regionwhich may affect expression of neighbouring genes including MYB. However, the exact functional role of this region on Hb F level is still unclear. Recently, a 3-bp deletion site in this intergenic region was found to be linked with high Hb F level and possess motifs for gene regulation, making it a candidate functional site of this QTL. Here we further investigate this region for its functional role in Hb F modulation. Erythroid cultures were used to profile MYBand GYPA expression from human cord blood of different 6q23 haplotypes. Regulatory functions of the 3-bp deletion region were assessed by luciferase assays. Identification of proteins binding to this region was done through electrophoretic mobility shift assay (EMSA) with subsequent mass spectrometry. Results were confirmed by chromatin immunoprecipitation (ChIP). High Hb F haplotype at 6q23 was associated with lower MYB expression and accelerated erythroid maturation when compared to low Hb F haplotype. The 3-bp deletion linked to a high HbF haplotype showed a site-specific suppressive effect on gene expression in luciferase assays. An architectural DNA-bending/looping chromosomal protein, high mobility group protein B1 (HMGB1), was found to bind differentially to this site depending on the presence or absence of the 3-bp deletion. These functional studies confirm a regulatory role of the Hb F QTL at HBS1L-MYB intergenic region on chromosome 6q23. The suppressive effect of high Hb F haplotypes can lead to reduced MYB expression and accelerated erythroid maturation that is known to be associated with a high Hb F level. Identification of HMGB1 binding to this 3-bp deletion site provides a potential mechanism for the action of this QTL on expression of MYB and other important genes in this chromosomal region. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

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The karyotype has been recognized for a member of years as a useful taxonomic criterion in separating groups of plants and animals and to a more limited extent at the species level. More recently, chromosomal aberrations have been associated with human congenital malformations and show promise as a diagnostic aid. The accurate identification of the individual chromosome is, accordingly, becoming more essential to the refined application of cytology to these areas. [...]