Chromosome 6q deletions in gastric carcinoma : A Positional cloning approach to isolate cDNAs at 6q16.3-q23.3 = Delecções do braço longo do cromossoma 6 em carcinomas gástricos : Isolamento de cDNAs em 6q16.3-q23.3 por clonagem posicional

Dissertação de Doutoramento em Biologia Humana apresentada à Faculdade de Medicina da Universidade do PortoO cromossoma 6 é um dos cromossomas mais frequentemente envolvido em alterações estruturais em tumores gástricos. De todas as anomalias descritas deste cromossoma, a delecção parcial do braço longo representa é a alteração mais vezes detectada, tanto por análise citogenética como por estudos de perda de heterozigotia (LOH).A perda de heterozigotia em 6q ocorre em todos os tipos histológicos de carcinoma gástrico, incluindo tumores precoces.O objectivo "major" deste estudo foi isolar genes localizados em 6q que podem estar envolvidos no desenvolvimento e progressão de tumores gástricos. Em estudos anteriormente realizados pelo nosso grupo, foram definidas duas regiões de delecção mínima uma intersticial em 6q16.3-q23.1 (15 cM) e uma distal em 6q27 (> 30 cM). No entanto, como a extensão destas duas regiões não permitia ainda o início de pesquisa de genes, o nosso primeiro objectivo foi delimitar a região intersticial. Para isso, foram seleccionados novos marcadores localizados na região (marcadores microssatélites), que viriam a ser aplicados na análise de um painel alargado de tumores primários. Do mapeamento detalhado da região foi possível restringir a região intersticial de 15 cM para uma nova região de apenas 2 cM (aproximadamente 3 Mb) (Artigo I).No sentido de ultrapassar os problemas de análise e interpretação decorrentes da contaminação estromal em tumores primários, procedemos ao estudo de tumores xenografados em ratinhos atímicos e linhas celulares derivadas dos xenografos, estabelecidos no nosso Instituto. Este estudo, envolvendo análise de microssatélites e FISH, permitia verificar se as linhas celulares em causa apresentavam as mesmas alterações em 6q detectadas nos tumores primários, o que, a verificar-se, permitiria utilizar este material como para estudos futuros, nomeadamente estudos funcionais.Verificámos que, tal como nos tumores primários, duas linhas celulares apresentavam uma delecção distal (6q27). Estes resultados apoiam a existência d ..

Chromosomal aberrations in hepatocellular carcinoma: a study by comparative genomic hybridization and interphase cytogenetics.

Lee Siu-wah.Thesis submitted in: December 1999.Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.Includes bibliographical references (leaves [106]-116).Abstracts in English and Chinese.Abstract (in English) --- p.iAbstract (in Chinese) --- p.iiiAcknowledgements --- p.vTable of Contents --- p.viList of Figures --- p.ixList of Tables --- p.xAbbreviations --- p.xiChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.2Chapter 1.2 --- Etiology of Hepatocellular Carcinoma --- p.5Chapter 1.2.1 --- Viral Infection --- p.5Chapter 1.2.1.1 --- Hepatitis B Virus --- p.5Chapter 1.2.1.2 --- Hepatitis C Virus --- p.7Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.8Chapter 1.2.3 --- Aflatoxin --- p.9Chapter 1.3 --- Genetic Studies of Hepatocellular Carcinoma --- p.9Chapter 1.3.1 --- Conventional Cytogenetics --- p.9Chapter 1.3.2 --- Molecular Cytogenetics --- p.12Chapter 1.3.3 --- Molecular Genetic Studies --- p.12Chapter 1.3.3.1 --- Proto-oncogenes --- p.12Chapter 1.3.3.2 --- Tumour Suppressor Genes --- p.13Chapter 1.3.3.3 --- Cell Cycle Genes --- p.14Chapter 1.4 --- Background of Study --- p.16Chapter 1.5 --- Objectives of Study --- p.17Chapter Chapter 2 --- Material and Methods --- p.18Chapter 2.1 --- Materials --- p.19Chapter 2.2 --- Analysis of Chromosomal Imbalances by Comparative Genomic Hybridization --- p.23Chapter 2.2.1 --- Comparative Genomic Hybridization --- p.23Chapter 2.2.2 --- MethodsChapter 2.2.2.1 --- Preparation of Normal Metaphases --- p.30Chapter 2.2.2.2 --- Extraction of High Molecular Weight DNA --- p.30Chapter 2.2.2.3 --- Labeling of DNA by Nick Translation --- p.31Chapter 2.2.2.4 --- Labeling Efficiency --- p.31Chapter 2.2.2.5 --- Preparation of Probe --- p.33Chapter 2.2.2.6 --- Hybridization --- p.33Chapter 2.2.2.7 --- Washing and Detection of Signals --- p.35Chapter 2.2.2.8 --- Image Acquisition and Analysis --- p.35Chapter 2.2.2.9 --- Control Experiments --- p.36Chapter 2.3 --- Positional Mapping of Novel Amplicon by Interphase Cytogenetics --- p.39Chapter 2.3.1 --- Fluorescence in situ Hybridization --- p.39Chapter 2.3.2 --- Using Yeast Artificial Chromosomes (YAC) as Probe --- p.41Chapter 2.3.3 --- Methods --- p.48Chapter 2.3.3.1 --- Culture of Yeast Artificial Chromosomes --- p.48Chapter 2.3.3.2 --- Extraction of Total YAC DNA --- p.48Chapter 2.3.3.3 --- Verification of YAC Clones for Chimerism by FISH --- p.49Chapter 2.3.3.4 --- Inter-Alu-PCR --- p.49Chapter Chapter 3 --- Assessment of Genetic Changes in HCC by Comparative Genomic Hybridization (CGH) --- p.57Chapter 3.1 --- Introduction --- p.58Chapter 3.2 --- Materials and Methods --- p.58Chapter 3.2.1 --- Patients and Specimens --- p.58Chapter 3.2.2 --- Comparative Genomic Hybridization --- p.60Chapter 3.2.3 --- Statistical Analysis --- p.60Chapter 3.3 --- Results --- p.61Chapter 3.3.1 --- Overall Copy Number Aberrations in 67 HCC and Surrounding Cirrhotic Tissues --- p.61Chapter 3.3.2 --- TNM Staging --- p.61Chapter 3.3.3 --- Tumour Size --- p.72Chapter 3.3.4 --- Serum AFP Elevation --- p.72Chapter 3.3.5 --- Chromosomal Aberrations in HCC arising from Cirrhotic and Non- cirrhotic Livers --- p.72Chapter 3.4 --- Discussion --- p.73Chapter 3.4.1 --- Recurrent Gains --- p.73Chapter 3.4.2 --- Recurrent Losses --- p.75Chapter 3.4.3 --- Tumour Progression --- p.76Chapter 3.5 --- Conclusion --- p.78Chapter Chapter 4 --- Positional Mapping of a Novel Amplicon on Chromosome 1q21-q25 by Interphase Cytogenetics --- p.79Chapter 4.1 --- Introduction --- p.80Chapter 4.2 --- Materials --- p.82Chapter 4.3 --- Methods --- p.82Chapter 4.3.1 --- Preparation of Paraffin-embedded Tissue Sections --- p.82Chapter 4.3.2 --- Verification of YAC Probes for Chimerism --- p.83Chapter 4.3.3 --- Hybridization Efficiency of Test and Reference Probes --- p.83Chapter 4.3.4 --- Slide Pretreatment and FISH with YAC Probes --- p.88Chapter 4.3.5 --- Scoring of FISH Signals --- p.88Chapter 4.4 --- Results --- p.89Chapter 4.4.1 --- Relative Copy Number Gain --- p.89Chapter 4.4.2 --- Intratumour Heterogeneity --- p.90Chapter 4.5 --- Discussion --- p.90Chapter 4.6 --- Further Studies --- p.104Chapter 4.6.1 --- Fine Mapping of Chromosomal Region 1 q21 --- p.104Chapter 4.6.2 --- Isolation of Novel Genes in the Amplicon --- p.105Chapter 4.6.3 --- Expression Status of the EDC Genes --- p.105References --- p.10

A molecular investigation of the novel gene underlying autosomal dominant retinitis pigmentosa in a South African family

The inherited retinal degenerative disorders are a common cause of severe visual handicap in the W estem world. Retinitis pigmentosa (RP) is a group of retinopathies in which a primary feature is a progressive loss of photoreceptor and retinal pigment epithelium function. Over the last decade, investigations into the patho-physiology of RP have identified numerous disease-causing genes and loci (for a current listing refer to the web site http://www.sph.uth.tmc.edu/Retnet/). A study of a South African family with an autosomal dominant form of RP (adRP) forms the basis of this dissertation. In this family, comprising 44 individuals, the first manifestation of visual disturbance is usually evident between 20 and 30 years of age. Subsequently, another South African adRP family, consisting of 25 members, was also incorporated into this investigation. Genetic linkage analysis facilitated the mapping of the disease phenotype in the two South African adRP families to a 10 cM interval on chromosome 17q22. This novel locus, designated RP17, is the eighth identified for adRP. Haplotype construction in the two kindreds, in conjunction with multipoint analyses subsequently fine mapped RP17 to a 1 cM region between microsatellite markers D17S1604 and D17S948. Although the two families are from ethnically diverse population groups, they share the same disease-associated haplotype spanning 12 cM, which suggests that the disorder may be caused by the same pathogenic mutation in the same gene. The positional cloning approach was utilised in an endeavour to identify the RP17 gene and an attempt was made to construct a physical map of the 1 cM critical region. A contig consisting of seven yeast artificial chromosome (YAC) clones was assembled using sequence-tagged-site (STS) content mapping. In order to close a gap in the YAC contig, a bacterial artificial chromosome (BAC) library was screened and the vectorette PCR technique was used to verify overlapping sequences. This contig should provide a useful tool for the purpose of isolating genes or transcription units within the RP17 critical interval. In this regard, purified YAC DNA was isolated using pulsed-field gel electrophoresis and the cDNA selection technique was employed to generate a transcription map. This approach was applied to YAC 75Ic12 using a foetal brain cDNA library, and two rounds of selection were performed to create a sub-library for enriched cDNAs derived from this clone. Screening for the presence of contaminating sequences in the 480 transformants revealed that (i) approximately 7% of the selected clones contain COT-1 DNA and (ii) none of the clones were contaminated with yeast AB1380 DNA. Ten randomly chosen clones were sequenced and subjected to BLASTN analysis, which revealed the presence of a 23 bp contaminant, known genes as well as novel transcripts. In order to optimise efforts to isolate the adRP gene, four positional candidates residing on 17q were screened for evidence implicating them in the adRP phenotype in the two 17q22-linked families. The genes investigated were: PDEG (gamma subunit of rod phosphodiesterase), TIMP2 (tissue inhibitor of metalloproteinases-2), PKCA (protein kinase C alpha) and retinal fascin. These candidates were chosen on the basis of (i) mapping to 17q, (ii) expression in the retina and/or (iii) potential involvement in the rod phototransduction pathway. Recombination events between the adRP locus and a single strand conformation polymorphism (SSCP) in PDEG, and a restriction fragment length polymorphism (RFLP) in TIMP2 provided evidence for the exclusion of these candidate genes. A novel SSCP detected in the promoter region of retinal fascin was genotyped in the two adRP families and showed a lack of co-segregation with the disease locus. Furthermore, direct DNA sequencing of the coding regions as well as the promoter region of retinal fascin in RP affected family members did not reveal any pathogenic mutations. In addition, data is provided which suggests that PKCA does not reside on any of the YACs and BACs encompassing the RP17 critical interval. This gene is therefore unlikely to be responsible for the adRP phenotype in the two RP17-linked families. Ultimately, the work reported in this thesis may contribute to the body of knowledge on inherited retinal degenerative disorders. Moreover, this investigation should provide the basis for further study of the aetiology of RP in all families linked to the RP17 locus on chromosome 17q22. The immediate application of these molecular findings is the potential for pre-symptomatic testing of at-risk members from the two adRP kindreds

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library

Gene detection with synthetic oligonucleotide sequences

This thesis describes experiments to test the hypothesis that it is possible to detect coding regions in genomic DNA by using short synthetic oligonucleotides. Two types of sequences were targeted. The first consisted of sequences complementary to sites for rare-cutter restriction enzymes, which are often clustered in the CpG rich islands located adjacent to, or within, coding regions. The second were sequences with complementarity to consensus sites for control and regulation within or around coding regions, such as splice sites and transcription factor binding sites. Southern hybridization experiments were first carried out to test the hypothesis that short oligonucleotides, based on G/C rich regions and on consensus splice site sequences, could be used as hybridization probes to detect cosmids or fragments of cosmids containing CpG islands or splice site junctions. The cosmid vector pWE15 which contains two Notl sites and several clones containing the genes for human proteolipid protein, calcitonin/calcitonin gene related peptide-a (CGRP), glutathione-S-transferase and NADH-ubiquinone oxidoreductase were used as model systems for testing this hypothesis. Secondly, the polymerase chain reaction was used to test the hypothesis that short oligonucleotides based on rare cutter sites could be used as specific PCR primers using subclones of the cosmid vector pWE15 and phagemid pSL1180 as model systems, or in a less specific manner to amplify DNA in well characterised clones such as those containing the PLP, calcitonin/CGRP, glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes. In addition the hypothesis that this method could also be used on total human genomic DNA, so that clones enriched for either CpG islands or for coding regions could be obtained, using rare cutter site, splice site and transcription factor site oligonucleotides, was also tested. Several methods including a "TA" cloning strategy were employed in order to generate mini-libraries of the amplification product for subsequent evaluation. Degenerate oligonucleotides with just their 3' ends based on the splice site and translation start site consensus sequences and with cloning sites at their 5' ends were also used on the model clones to test whether coding regions could be identified. Thirdly, experiments were carried out to demonstrate that short oligonucleotides based on rare cutter sites could be used as PCR primers for sequencing directly into CpG islands/coding regions in cloned DNA. Subclones of the four genes mentioned above were used as models to test this. Finally, experiments were carried out to test the hypothesis that the ligation of primer/linkers to rare-cutter restricted cosmids followed by direct PCR-sequencing could be used to obtain direct sequence from putative CpG islands in cloned genes. Several cosmid clones including the glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes were used for this. The experiments showed that some of the hypotheses concerning G/C rich sequence detection could be confirmed. When genomic DNA was used as template, G/C rich oligonucleotides as short as 8-mers could prime PCR amplification and enable "TA" clones to be produced which were enriched 66-fold for CpG rich sequences. In addition, conditions necessary for direct and specific amplification using G/C rich oligonucleotides as short as 7-mers with vector constructs as model target DNA were identified. However, PCR using G/C rich oligonucleotides was not capable of identifying CpG islands within cosmid clones. G/C rich 8-mer oligonucleotides may also be used in certain situations for directed sequencing within cloned genes and could thus be used as random or directed primers in a large volume sequencing project. Primer/linkers for rare-cutter restriction sites may also be used for sequencing into CpG islands within cosmids. Oligonucleotides that have their 3' ends complementary to the splice site consensus sequence can also prime amplification (Degenerate Oligonucleotide Primed-PCR) from some splice sites in some cloned genes, although with low success rate. In conclusion, the development of methods to enrich for CpG islands in genomic DNA was successful, but identifying CpG islands in cosmid clones was not. However a degree of success was achieved in the direct sequencing of CpG islands within clones. The work with splice site sequences was less successful, and it must be concluded that other methods should be employed for the identification of coding regions within cloned DNA

Identification of potential gene coding sequences within large cloned DNA arrays : analysis of zinc finger motif

Imperial Users onl

Genetic mapping of the rat agu gene

In 1993, a mutant strain, AS/AGU arose spontaneously in an enclosed colony of the Albino Swiss (AS) strain of rat. AS/AGU animals exhibit a set of locomotor abnormalities. They display a general instability and whole body tremor, are slow at initiating movement, show reductions in purposeful action, and perform poorly at locomotor tests such as mid-air righting. L-dopa administration or fetal midbrain transplants reverse the majority of the symptoms, resembling the observations made on Parkinson's disease patients. These features make the AS/AGU strain a useful model for movement disorders due in significant part to failure of the dopaminergic transmission system. Crosses of AS/AGU to other laboratory rat strains point to a single recessive mutation with essentially complete penetrance (agu/agu) as the cause of the abnormal phenotype. There is no evidence of sex linkage or maternal inheritance. In the absence of any evidence of the function of the agu gene product, positional cloning of this locus was begun. The first step was the establishment of a genetic map location for the agu locus. A large series of microsatellite markers were analysed and used to identify which of the strains PVG, BN, and F344 differed to a greater extent from AS/AGU. Differences at 43%, 62% and 47% of the loci were recorded, respectively. BN and F344 were therefore selected as the reference strains in backcrosses to AS/AGU, in an attempt to maximise the number of informative markers which could be used to type the progeny

Genome analysis of protozoan parasites. Proceedings of a workshop

This workshop proceeding reviews the current state of some of the major genome programs in the world, reviews recent developments in the genome analysis of the important protozoan parasites such as leishmania, toxoplasma, trypanosoma and theileria; reviews major technological advancements contributing towards eukaryotic genome analysis; identifies major applications of the knowledge gained from the genome analysis projects; identifies ILRAD's (International Laboratory for Research on Animal Diseases) needs for theileria and trypanosoma genome analysis, and explores possibilities of collaboration with an international group of scientists involved in genome analysis projects. Topics of discussion include molecular dissection of genomes, establishing a laboratory for parasite genome analysis, genetic analysis of theileria and plasmodium, genetic analysis of trypanosomes and techniques for physical and functional analysis

Molecular analysis of chromosome 15 in Prader-Willi syndrome

Prader-Willi syndrome is a complex multi-system disorder characterised by mental retardation, obesity with insatiable appetite and hypogonadism. Cytogenetic and molecular studies found that some patients with Prader-Willi syndrome had deletions involving the proximal part of the long arm of their patemally-inherited chromosome 15. Several patients lacking deletions were shown to have inherited both of their chromosomes 15 from their mothers’ (maternal uniparental disomy). A phenotypically distinct disease, Angelman syndrome, was shown to commonly be due to matemally—inherited deletions of the same region of chromosome 15 or occasionally to paternal uniparental disomy. These findings led to the suggestion that genes subject to genetic imprinting, a phenomenon in which alleles of paternal and maternal origin are differentially expressed, were associated with the development of Prader-Willi syndrome and Angelman syndrome. The work described in this thesis was directed towards the characterisation of the molecular abnormalities found in subjects with Prader-Willi syndrome and the eventual identification of genes that might be involved in the pathogenesis of these imprinted conditions. Thirty patients with Prader-Willi syndrome were carefully analysed with a set of DNA markers mapping to chromosome 15 . Densitometry and family studies with polymorphic markers were used to investigate these patients. Eighteen subjects had patemally—inherited deletions involving 15q11-q13, eight subjects had maternal uniparental disomy for chromosome 15, one individual had inherited two intact chromosomes 15 from his mother and was mosaic for the presence of a patemallyinherited chromosome fragment that included 15q11-q13, while the remaining three patients were not found to have any abnormalities involving chromosome 15 . Prader-Willi syndrome patients with the last two sets of findings have not been well characterised in the past. One patient was found to be deleted for a more circumscribed set of DNA loci than those that were in missing in the other individuals with deletions. Consideration of the limited deletion found in this patient together with results from other laboratories suggested that the gene(s) responsible for Prader-Willi syndrome were located between two loci mapping to 15q11-q13, D15813 and DISSIO. Pulsed field gel electrophoresis studies were performed to better characterise the region of chromosome 15 near D158 10. A long range physical map extending over approximately 2 800 kb was constructed and several novel CpG islands were identified. These structures are often associated with transcribed sequences and potentially could mark the location of candidate genes for Prader-Willi syndrome or Angelman syndrome. A series of experiments were performed utilising chromosome jumping libraries and yeast artificial chromosomes that were aimed at cloning DNA sequences associated with these CpG islands. While these attempts met with only limited success, they demonstrated a valid approach to attempting to identify candidate genes for the diseases under study. One probable CpG island was cloned, however, a very high GC-content interfered with attempts to analyse this DNA fragment in detail. A subject with Prader—Willi syndrome and maternal uniparental disomy for chromosome 15 was additionally shown to have a second genetic disorder, Bloom syndrome. This autosomal recessive condition, which is characterised by an increased incidence of a wide range of malignancies, was thought to have occurred because of the inheritance of two disease gene alleles from a carrier mother. Through the identification of a meiotic crossover event involving the distal portion of the matemally-inherited chromosomes 15, it proved possible to establish the location of the Bloom syndrome gene as being 15q25—»qter. This was the first example of this form of analysis being used to regionally localise a disease gene. The work described in this thesis has allowed the better characterisation of the molecular defects found in patients with Prader-Willi syndrome and related disorders. It has also provided important information about the physical structure of the region deleted in patients with Prader-Willi syndrome. It will be necessary to continue such efforts if attempts to identify the genes responsible for these disorders and understand the basis of genetic imprinting are to be successful