Gene identification by whole genome array CGH Richard Barber 21st February Gene Discovery

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

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

CHARGE Vissers et al 2004 Nat Genet 36: 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

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

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

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 p

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

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

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

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

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 Sep;38(9):

Decipher Database Decipher - DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources 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