Gene identification by array CGH

Haploinsufficiency of specific genes is a known cause of disease, both in acquired (cancerous) and congenital disorders. Haploinsufficiency can be brought about by single-base changes or deletions of stretches of DNA. The genome-wide detection of DNA alterations by array CGH can mark genomic sites where genes associated with a particular disease may be located. Previously, Albertson et al. (38) used this approach to identify ZNF217 and CYP24 as putative oncogenes in breast cancer. Similarly, we narrowed down the critical region of deletion for the gene(s) causing congenital aural atresia, located on chromosome 18q22.3-q23 (21). More recently, we have applied this approach successfully to identify the causative gene for

Figure 11.1.

Proposed flow-scheme for application of the array CGH procedure in a clinical setting. As an example DNA from a patient with Prader Willi Syndrome, containing a known microdeletion on 15q11-q13 is labeled and hybridized onto our genome-wide tiling resolution BAC array (~32 000 clones, each printed once onto the microarray slide) in a label swap experiment versus a sex-mismatched reference pool. The normalized log2 test-over-reference ratios (y-axis) are plotted for each clone ordered per chromosome by Mb position (x-axis), from p-ter to q-ter. Chromosome X and Y serve as control for deletion and duplication detection in these sex-mismatch experiments. The results of the individual experiments indicate the presence of potential copy number alterations; however, the combination of both experiments increases the statistical significance of an observed copy number alteration. This is indicated by the solid line at -1 for the microdeletion region (see combined chromosome 15 profile), which means a 100% probability for a downstate (deletion) as computed by a Hidden Markov Model. The gene content of this region can now be studied, if needed array findings can be validated (especially useful for deletions smaller than 1 megabase) and finally parents can be tested to distinguish causative alterations from normal variation.

CHARGE syndrome (39). CHARGE syndrome (OMIM 214800) is a non-random pattern of congenital anomalies including choanal atresia and malformations of the heart, inner ear and retina. In this study a de novo microdeletion of 4.8 megabases was identified on 8q12 by hybridizing genomic DNA from a CHARGE patient versus a normal control on our 1-Mb-resolution BAC array. A literature search identified another CHARGE patient with an apparently balanced chromosome 8 translocation, identified by routine karyotyping (40). Array CGH analysis of this patient revealed another microdeletion partially overlapping with the one encountered in our index patient. Further analysis of 17 additional CHARGE patients on a tiling resolution chromosome 8 array did not reveal additional microdele-tions. As a next step, sequence analysis of nine genes located within the minimal region of deletion overlap identified 10 de novo heterozygous mutations in a novel gene called CHD7, including seven stop-codon mutations, two missense mutations and one mutation at an intron-exon boundary. This latter study showed that array CGH may indeed serve as an effective new approach to localize disease-causing genes. This approach is of particular interest for sporadic malformation syndromes that cannot be tackled by other mapping procedures because of reproductive lethality.

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