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Positional Cloning of Genes Associated with Human Disease

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Positional Cloning of Genes Associated with Human Disease Positionul Cloning of Genes Associuted wíth Humun Díseuse Scott Anthony Whitmore B.Sc Thesis submitted for the Degree of Doctor of Philosophy Department of Cytogenetics and Molecular Genetics, 'Women's and Children's Hospital, North Adelaide, South Australia. Faculty of Medicine, Department of Paediatrics, University of Adelaide, South Australia. January, 1999. Table of Contents Page Summary I Declaration IV List ofPublications V Abbreviations VI Acknowledgements VIII Chapter 1: Literature Review I Chapter 2z Matenals and Methods 58 Chapter 3: Physical Mapping of Chromosome 16q24.3 92 Chapter 4: Identification of Transcribed Sequences at 16q24.3 120 Chapter 5: Characterisation of the GASI I and CI6orf3 Genes 156 Chapter 6z Characterisation of Transcription Unit 6 (T6) 196 Chapter 7: Cloning of the Gene for Cystinosis 227 Chapter 8: General Discussion and Future Directions 248 References 255 Appendix: Publications Summary Genetic linkage analysis has been successful in mapping the gene responsible for Fanconi anaemia type A (FAA) to chromosome 16q24.3. In addition, loss of heterozygosity (LOH) studies have localised a tumour suppressor gene involved in the development of sporadic breast cancer to the same chromosomal region. The main aim of the thesis was to isolate the gene(s) responsible for these disorders using a positional cloning strategy. At the start of the project there was a lack of cloned DNA and candidate genes located at 16q24.3, therefore a detailed physical map of this region was constructed based on overlapping cosmid, BAC, and PAC clones. The resulting map extends approximately 1.1 Mb from the telomere of chromosome 16q and consists of a minimum overlapping set of 35 cosmids, 2 PACs, and 1 BAC clone. This physical map encompasses the genetic markers that define the region most likely to contain fhe FAA gene and a breast cancer tumour suppressor gene. Refined LOH and linkage analysis by collaborating laboratories was successful in narrowing down the candidate region of both diseases to approximately 750 kb between Dl6S303 and D16S3026. Selected cosmids spanning the region between these markers were used as templates for exon trapping experiments to enable identification of candidate genes. Additional transcript data was obtained from the analysis of ESTs mapped to 76q24 3 as part of the Human Gene Map construction at NCBI, and dbEST database screening of partial cosmid sequence (generated from collaborating laboratories). A total of 71 unique exons were trapped from 26 cosmid templates. Each one was successfully mapped back to its cosmid of origin allowing an integration of the physical and transcript map. Twenty eight exons showed significant homology to ESTs, while 7 corresponded to genes previously mapped to chromosome 16q24.3. A group of five exons could be linked based on their EST homology I and physical map proximity. The corresponding gene (FAA) was later shown by collaborators to be deleted for 2 of these 5 trapped exons in an Italian patient with Fanconi anaemia. Further screening of individuals affected with the disorder have shown a multitude of mutations associated with this gene. However, comparison of normal and breast tumour DNA failed to identify mutations in FAA restricted to tumour DNA. Therefore it was concluded that FAA was unlikely to have a role in breast tumourigenesis. The region between D16S303 and D16S3026, containing at least 20 transcripts, was then screened for the presence of a breast cancer tumour suppressor gene. Two candidate transcripts were examined in detail. This involved the isolation of the complete sequence of the corresponding gene, followed by the identification of its genomic structure. Single stranded conformation polymorphism (SSCP) analysis was used to screen for mutations restricted to breast tumours. One of these transcripts, subsequently referred to as the growth arrest-specific 11 (GASII) gene, was a likely candidate since the mouse homologue was expressed specifically during cell growth arrest. GASII was found to consist of 1l exons, one of which was identified by exon trapping, which span approximately 25 kb of genomic DNA. SSCP analysis of breast tumour DNA failed to identify nucleotide sequence alterations when compared to corresponding normal DNA from the same individuals. In addition, Southern analysis of breast cancer cell line DNA failed to identify homozygous deletions encompassing GASII and RT-PCR eliminated exon skipping as a disease causing mechanism in these cell lines as well as in affected individuals. These results suggest this gene is not involved in breast carcinogenesis. Another gene, Cl6orf3 (chromosome 16 open reading frame 3), was found to lie within intron 2 of GASI1. This gene is expressed at a low level as a 1.2 kb mRN.\ is intronless, and codes for a 125 amino acid protein which exists in two isoforms based on the presence or absence of two copies of an imperfect tetrapeptide repeat. SSCP mutation analysis II also indicated this gene was not mutated in breast tumours Analysis of a second transcript, T6, showed that this gene consisted of l8 exons (5 were identified from exon trapping) which code for a protein of 669 amino acids that does not show homology to any previously characterised proteins. SSCP mutation analysis of T6 again failed to identify SSCP changes specific for breast tumour DNA alone, suggesting this gene is also not involved in breast carcinogenesis. The approach of exon trapping was also applied to a separate collaborative project, the positional cloning of a gene responsible for nephropathic cystinosis. The collaborators successfully constructed a cosmid contig across the -500 kb candidate region on chromosome 17p73 and selected clones were subsequently used for exon trapping to identify candidate genes. A total of eight overlapping cosmids were used to identify 29 exons, 10 of which corresponded to characterised genes previously mapped to this chromosomal region. Of the 15 exons that did not display homology to database sequences, 6 were subsequently shown by collaborators to belong to a gene (CZll,9) which was located within a region of homozygous deletion seen in a cystinosis family. SSCP analysis of individuals affected with cystinosis has subsequently identified 1l different disease-causing mutations within this gene. The technique of exon trapping has therefore enabled the cloning of two disease genes, FAA, and CTNS, from a positional cloning approach. It has also allowed the identification of many other novel transcripts that are candidates for a tumour suppressor gene mapping to I6q24.3 and which have not yet been further characterised. The eventual identification of all genes (including tumour suppressor genes associated with LOH) involved in the oncogenic pathway in breast cancer will improve our understanding of tumour progression in this disease. , m List of Publicøtions Most of the work presented in this thesis can be found in the following publications. A copy of each manuscript is given in the appendix. The FAB Consortium. (1996). Positional cloning of the Fanconi anaemia group A gene. Nctture Genet. 14: 324-328. Ianzano, L., D'Apolito, M., Cen|ra, M., Savino, M., Levran, O., Auerbach, 4.D., Cleton- Jansen, A-M., Doggett, N.4., Pronk, J.C., Tipping, 4.J., Gbson, R.4., Mathew, C.G., Whitmore, S.4., Apostolou, S., Callen, D.F., Zelantê,L., and Savoia, A. (1997) The genomic organisation of the Fanconi anemia group A(FAA) gene. Genomics 4l:309-314. 'Whitmore, S.4., Crawford, L, Apostolou, S., Eyre, H., Baker, E., Lower, K.M., Settasatian, C., Goldup, S., Seshadri, R., Gibson, R.4., Mathew, C.G., Cleton-Jansen, A-M., Savoia, 4., Pronk, J.C., Auerbach, 4.D., Doggett, N.4., Sutherland, G.R., and Callen, D.F. (1998). Construction of a high-resolution physical and transcription map of chromosome 16q24 3 a region of frequent loss of heterozygosity in sporadic breast cancer. Genomics 50: 1-8 Whitmore, S.4., Settasatian, C., Crawford, J., Lower, K.M., McCallum, B., Seshadri, R., Cornelisse, C.J., Moerland, E.W., Cleton-Jansen, A-M., Tipping, 4.J., Mathew, C.G., Savino, M., Savoia, 4., Verlander, P., Auerbach, 4.D., Van Berkel, C., Pronk, J.C., Doggett, N.4., and Callen, D.F. (1998) Characterisation and screening for mutations in breast cancer of the growth arest-specific 11 (GASII) and Cl6orf3 genes at 16q243. Genomics 52.325-337. Town, M., Jean, G., Cherqui, S., Attard, M., Forestier, L., Whitmore, S.4., Callen, D.F., Gribouval, O., Broyer, M., Bates, G.P., van't Hofl W., and Antignac, C. (1998). A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nature Genet. 18.319-324. V Abbreviations BAC: bacterial artificial chromosome. bp base pairs BLAST basic local alignment search tool. C16orf: chromosome 16 open reading frame. oDNA: complementary deoxyribonucleic acid. cM centimorgan cR: centray CGH comparative genomic hybridisation. dbEST: database of expressed sequence tags. dNTP: deoxynucleotide triphosphate DNA: deoxyribonucleic acid DCIS ductal carcinoma in situ EST expressed sequence tag. ET trapped exon. FA: Fanconi anaemia FAA: Fanconi anaemia group A gene. FAB Fanconi anaemia/Breast cancer FISH: fluoresence in situ hybridisation. GAS growth arrest-specific hnRNA. heteronuclear ribonucleic acid. kb kilobase pairs LCIS lobular carcinoma in situ. LOH: loss of heterozygosity Mb megabase pairs. VI l"rg: mrcrogram. prl: microlitre. mg milligram ml: millilitre mRNA: messenger ribonucleic acid NCBI: National Centre for Biotechnolo gy Information. ng nanograms. ORF open reading
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