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Gene identification by whole genome array CGH

Gene identification by whole genome array CGH. Richard Barber Richard.Barber@bwhct.nhs.uk 21st February Gene Discovery. Rearrangements. Large genomic rearrangements have been instrumental in identifying disease loci for many syndromes APC RB DMD UBE3A

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Gene identification by whole genome array CGH

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  1. Gene identification by whole genome array CGH Richard Barber Richard.Barber@bwhct.nhs.uk 21st February Gene Discovery

  2. Rearrangements • Large genomic rearrangements have been instrumental in identifying disease loci for many syndromes • APC • RB • DMD • UBE3A • Traditionally found by G banding karyotype with FISH studies to confirm deletions and duplications • This approach relies on microscopically visible rearrangements >5Mb which are rare • The phenotypes of individuals with large contiguous gene deletions are often difficult to relate to a single gene disorder • Some genomic disorders are caused by the action of one gene • CMT1A and HNPP caused by dup/del of the PMP22 gene

  3. Microarrays • Array CGH uses an array of genomic fragments with known physical locations immobilised on a glass slide • Allows higher resolution and automation compared to karyotype • Useful for disease gene identification for sporadic dominant LOF disorders with a limited reproductive potential • Classical linkage could not work for these cases as there is usually only one affected individual in a family • Using aCGH this allows detection of possible de novo deletions/duplications in index which may be pathogenic • Define boundaries of affected region using a second quantitative method then select candidate genes to screen in other affected individuals

  4. CHARGE Vissers et al 2004 Nat Genet 36: 955-957 • Classic example of aCGH identifying a disease gene is for CHARGE syndrome • Sporadic malformation syndrome • This is a pleiotropic disorder showing heart defects, retarded growth and development, genital hypoplasia, ear anomalies and deafness • Research group tested 2 CHARGE patients with a 1Mb resolution genome-wide BAC array • Identified a de novo deletion of 4.8Mb on 8q12 in one patient • Confirmed deletion by FISH • Screened initial 2 patients and a further 16 using tiling resolution array containing 918 overlapping BAC clones • Deletion affected 31 clones, no new deletions found • Tested a previously reported (1991) CHARGE patient with an apparent balanced chromosome 8 translocation • Found a complex deletion partially overlapping 4.8Mb deletion

  5. Gene Discovery • Defined the shortest region of overlap as 2.3Mb • Sequenced all 9 annotated or predicted genes within or close to the SRO in 17 affected individuals • Found 10 heterozygous mutations in the CHD7 gene • 7 stop codons • 2 missense • 1 possible splicing • CHD7 codes for chromodomain helicase DNA-binding protein (CHD) • This type of protein is thought to have pivotal role in embryonic development by affecting chromatin structure and gene expression • Disease mechanism is probably through haploinsufficiency as nonsense mutations were present in other affecteds

  6. Uses of aCGH • For non-genomic deletion syndromes seems to be luck if find a patient carrying a large deletion (genome architecture) • Can increase chances by screening for those individuals with other phenotypes such as mental retardation or a second genetic condition • However, if flanking genes are dosage sensitive and embryonic lethal, drastically reduces the chances of finding a large deletion • Estimated that deletions affecting at least one whole exon may account for ~10% of mutations • If screened every exon in genome for copy number changes (250,000), gives 65% chance of identifying any disease gene among 10 unrelated patients

  7. Uses of aCGH • Can combine an aCGH and candidate approach to identify genes • Sharp et al wanted to identify regions of genome that may be ‘rearrangement hotspots’ • Try and identify new genomic disorders • Found 130 sites (in silico) • Constructed a BAC microarray targeted to these regions (2,007 BACs) • Tested the microarray on 316 NCs • Found 384 sites of copy number polymorphisms (CNP) • Sharp et al Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nature Genetics vol 38 no 9 p1038-2006

  8. Study Method • Analysed 290 children and young adults with idiopathic mental retardation from UK • All cases had normal G-banded karyotypes and most were FRAX-A normal • Most had been tested for cryptic subtelomeric rearrangements by FISH • None had been tested by CGH • Most interested in copy number changes not seen in the NCs • All rearrangements had to be confirmed by a second method such as FISH

  9. Results • Found 7 variations of uncertain significance • Found 16 individuals (5.5%) with microdeletions or duplications that are likely to be pathogenic • 2 unbalanced translocations • Several known genomic disorders • 6 previously unidentified rearrangements that may be new genomic disorders

  10. Results 2 • 4 individuals showed deletion of the same 4 contiguous BACs spanning ~500kb in 17q21.31 • This region is known to have a polymorphic inversion • Confirmed deletions by FISH and was shown to be de novo in one case • The 4 individuals all had marked phenotypic similarities • Probably a previously unidentified recurrent microdeletion syndrome

  11. Results 3 • Used a high density oligonucleotide array which mapped the breakpoints to large clusters of flanking segmental duplication • Found a pair of directly orientated segmental duplications 38kb in length and >98% conserved sequence • The regions did not vary in controls and contained the genes CRHR1 and MAPT • Both of these are highly expressed in brain and have been implicated in several neurodegenerative and behavioural phenotypes • On the basis of phenotype similarities to the 4 cases they found a fifth individual by array CGH

  12. Koolen et al • Screened 360 individuals with idiopathic mental retardation using array CGH 32,477 BACs • They also found a ~600kb deletion in 17q21.31 • They then made an MLPA kit and screened 840 additional cases • Found 2 more cases • Koolen et al A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism. Nat Genet. 2006 Sep;38(9):999-1001

  13. Decipher Database • Decipher - DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources https://decipher.sanger.ac.uk/ • DECIPHER collects clinical information about chromosomal dels/dups/ins/translo/invs and displays this information on the human genome map with the aims of: • Increasing medical and scientific knowledge about chromosomal dels/dups • Improving medical care and genetic advice for individuals/families with submicroscopic chromosomal imbalance • Facilitating research into the study of genes which affect human development and health

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