Identification and Characterisation of Genes Jo Field Monday 9 th November 2009

1. Identification and Characterisation of Genes Jo Field Monday 9th November 2009 • Gene identification through knowledge of the gene function • Illustrate using examples such as ciliopathies, Fanconi anaemia or HNPCC

2. Identification and Characterisation of Genes • Gene identification from knowledge of gene function • For example if the known function of a gene is consistent with the phenotypic effects observed in a genetic disease • human genes can be identified from their homology to an orthologous gene that shows relevant function • e.g. MSH2 in HNPCC • if the gene function is not well-characterised, sequence analysis can give clues to the function of the gene if particular protein motifs are present (e.g. transmembrane regions)

3. Tumour Normal Identification and Characterisation of Genes • Gene identification in HNPCC • HNPCC tumours show microsatellite instability (MSI) phenotype compared to normal tissue • HNPCC also linked to a genetic locus causing MSI • Unable to find mechanism behind MSI from tumour DNA studies

4. Identification and Characterisation of Genes • Gene identification in HNPCC • Previous studies in model organisms showed that bacteria and yeast containing defective mismatch repair (MMR)genes show similar repeat instability • Results suggested that a defect in mismatch repair could be leading to the MSI seen in HNPCC tumours • Mismatch repair in bacteria/yeast • MutHLS mismatch repair system in E. coli • MutS binds mismatched nucleotides in DNA • Similar system seen in S. cerevisiae (yeast) • MutS homologue = MSH2 (also binds DNA containing mismatched nucleotides) • MutL homologues = MLH1 & PMS2

5. Identification and Characterisation of Genes • Gene identification in HNPCC • Fishel et al (1993) and Leach et al (1993) cloned the human homologue of the bacterial MutS/yeast MSH2 (hMSH2) • Leach et al used polymorphic markers to finely map the candidate region identified by linkage in HNPCC families • Used PCR with degenerate primers designed to conserved regions of known MSH2 homologues • used cloned product to probe a human cDNA library • full-length clone showed significant sequence similarity to yeast MSH2 • Expression of hMSH2 in E. coli led to a dominant mutator phenotype, as with MMR genes from other bacteria, suggesting hMSH2 functions in mismatch repair system

6. Identification and Characterisation of Genes • Gene identification in HNPCC • Detected germline mutations in MSH2 in affected individuals from HNPCC families and somatic mutations in MSH2 in sporadic tumours • alterations which significantly altered protein product (frameshift/nonsense mutations) • missense mutations in highly conserved regions of protein • Strongly suggests that MSH2 mutations are responsible for HNPCC and the MSI phenotype of HNPCC and sporadic tumours • Indicates that the MSI phenotype is intrinsically related to pathogenesis. • Similar approach used to identify the MLH1 gene in HNPCC

7. Identification and Characterisation of Genes • Gene identification from knowledge of gene function • Can use phenotypic similarities between diseases – those that show similar phenotypes may be caused by mutations in functionally related genes • functionally related genes may be part of the same pathway (metabolic, signalling, protein modification) • genes/gene products may have similar structure or function • may interact via protein-protein interactions or be part of the same protein complex • may act as part of the same developmental process • Such genes may lead to the same or similar phenotypes when mutated  Syndrome families or genetically heterogeneous disorders

8. Identification and Characterisation of Genes • Gene identification from knowledge of gene function • Predicting disease genes by finding similarities between different clinical phenotypes and identifying possible genes • using bioinformatics to predict candidate genes based on e.g. gene expression pattern, data from mouse mutants • using tools such as yeast two-hybrid assay to identify possible protein-protein interactions • looking at functional genomic data such as co-expression, similar protein domains • to compare and rank candidate genes based on their similarity to known disease genes associated with the disease

9. Identification and Characterisation of Genes • Gene identification in genetically heterogeneous disorders • E.g. Fanconi anaemia (FA) • Autosomal recessive cancer susceptibility syndrome • Progressive bone marrow failure • cancers include acute myeloid leukaemia (AML) and squamous cell carcinoma • FA cultured cells are hypersensitive to DNA cross-linking agents such as mitomycin C, diepoxybutane (DEB) • Cell fusion/complementation experiments allowed FA patients to be classed into several complementation groups (currently = A-M)

10. Identification and Characterisation of Genes • Gene identification in Fanconi Anaemia • Use a known in vitro function of a gene to help gene identification • Can use the sensitivity to crosslinking agents as a functional marker to identify new genes in FA • e.g. by screening a cDNA library for the ability to rescue FA cells from DEB-hypersensitivity – identify FANCC gene (functional complementation) • identified FANCA, FANCG genes in similar way • Found that the proteins identified formed protein-protein interactions with each other

11. Identification and Characterisation of Genes • Gene identification in Fanconi Anaemia • involved in DNA repair in response to DNA damage (e.g. DNA crosslinks) • some FANC proteins (FANCA, FANCG, FANCC, FANCF) form a multisubunit nuclear core complex • others (e.g FANCD2) interact with the FANC core complex and proteins with roles in DNA repair, e.g. BRCA1 • FANCD1 = BRCA2

12. Identification and Characterisation of Genes • Gene identification from knowledge of gene function • Ciliopathies e.g Bardet-Biedl (BBS), Kartagener & Meckel syndromes, primary ciliary dyskinesia (PCD), polycystic kidney disease (PKD) • genetically heterogeneous disorders • caused by genes involved in a shared developmental process/pathway (cilia biogenesis) • gene products may also physically interact (e.g. some BBS gene products form a protein complex) • overlapping clinical phenotypes share some clinical signs typical of cilia-related disease • e.g. cystic kidneys, encephalocele, situs inversus, retinitis pigmentosa

13. Identification and Characterisation of Genes • Gene identification in ciliopathies • Approaches for gene identification in cilia-related disorders • Identify novel ciliary genes in a model organism (e.g C. elegans) and look for human orthologues • Example - using comparative genomics (e.g. looking for transcription factor binding sites common among known ciliary genes) – Chen et al, 2006 • looking at expression patterns (e.g. microarrays) • “ciliopathy candidate exon capture array” followed by large-scale sequencing on Solexa/Illumina platform used to identify PRKACA mutation in nephronophthisis (Otto et al, 2009)

14. Identification and Characterisation of Genes • References • Fishel et al (1993). The Human Mutator Gene Homolog MSH2 and Its Association with Hereditary Nonpolyposis Colon Cancer. Cell 75, 1027-1038 • Leach et al (1993). Mutations of a mutS Homolog in Hereditary Nonpolyposis Colorectal Cancer. Cell 75, 1215-1225 • Oti and Brunner (2007). The modular nature of genetic diseases. Clin Genet 71, 1-11 • Garcia-Higuera et al (2001). Interaction of the Fanconi Anemia Proteins and BRCA1 in a Common Pathway. Molecular Cell 7, 249-262 • Macé et al (2005). 3R coordination by Fanconi Anemia proteins. Biochimie 87, 647-658 • Afzelius (2004). Cilia-related diseases. J. Pathol 204, 470-477 • Eley et al (2005). Cilia and disease. Curr Opin Genet Dev 15, 308-314 • Adams et al (2008). Recent advances in the molecular pathology, cell biology and genetics of ciliopathies • Chen et al (2006). Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics. Genome Biol 7, R126 • Otto et al (2009). Exon capture and large-scale sequencing of 828 ciliopathy candidate genes in patients with nephronophthisis, Senior Loken- and Joubert syndrome. Platform session 216, 59th Annual Meeting, ASHG