Representational difference analysis, RDA, involving human retroviral-related sequences and their possible role in schizophrenia

by Klempan, Timothy Arthur

Abstract (Summary)
RetroWal-related sequences are widespread in the human genome and rnay account for > IO% of total human DNA. The genomic sites for different groups of such sequences appears variable and rnany are inherited as stable, germ line elements. Sites of retroviral Esertion fiequently constitute mutational hot spots which predispose for various DNA rearrangements, while others may transpose, leading to insertional mutagenesis. These processes have been implicated in a number of diseases including schizophrenia, a common disease (1 % prevalence worldwide) with both heritable and environmental components, and devastating coosequences to affticted individuals. It has been lrypo thesized that integration of a novel retroviral element in utero may inactivate or upregulate nearby sequences, leadmg to schizophrenia later in Me. The objective of this study was to examine the nature of endogenous human retroviral sequences in pair-wise cornparisons using a modified version of representutional dzrerence analysis (RDA), a DNA-based methodology capable of disthguishg small clifferences between complex genomes. Retroviral-specinc sequences were amplified from a pair of unrelated individuals and a set of monozygotic twms discordant for schizophrenia. The amplified sequences (amplicons) were studied in paWd combinations such that subtractive and kinetic e~chment would permit the detection of "unique" sequences, ifpresent in the afEected member of pairs. Modification of the RDA protocol established diaing this research identined a number of parameters affecthg the final outcome, hcluding amplicon punty, efficiency of adapto r sequence ligation, proportions of representations used hybridization conditions, stringency of PCR amplification, primer speczcity, and total rounds of RDA carried out. Usbg a redesigned protocol fie rounds of RDA were performed with retrovirai products hm unrelated individuals and dixordant monozygotic twins. A distinct mcrease in amplification eEciency was seen in Merence products from unrelated individuals over five successive rounds while w quantitative change was apparent in products nom twins. The sequence analysis of Werence products revealed eight distinct sequence types iii (contigs) whose diiution is stmtifîed by sample origin and whose proportions change between the first and fifth round of RDA. Of these contigs, the presence of sequences simüar to a multiple sclerosisassociated retrovirus in most combinations and rounds of RDA follows the suggestion that such retro WaCrelated sequences are common in the human genome. A second sequence type (1 17 bp) is identical to a region of the human poly(ADP-nise) polymerase gene, shows proportional increases m the RDA combination of monoygotic t h , and may be associated with schizophrenia. Contig 4 (124 bp), with no demonstrated similarity to database sequences, could indicate a similar association in the combination of unrehted individuais. The inherent cornplexit. of the RDA methodology descnid in this study demands ngorous adherence to a controiled protocol. The possible association of the dexnkd sequences with schizophrenia is an avenue of research which remains to be fùrther investigated. KeywOrds: representational difference analy sis, retrovirus, reverse transcriptase, polymerase chah reaction, Pol gene, schizophrenia, multiple sclerosis
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Source Type:Master's Thesis



Date of Publication:01/01/1998

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