Molecular karyotyping in clinical genetics

The microarray format allows for a virtually unlimited flexibility in the choice and amount of genomic fragments to be studied. Many laboratories have started their array CGH studies using low-density custom-made arrays consisting of probes specifically targeted at selected genomic regions. Examples of these are arrays targeting all subtelomeric regions (13, 14), arrays targeting regions known to be involved in microdeletion or microduplication syndromes (10, 15-19), or arrays targeting other chromosomal regions of interest (20-24). These low-density arrays are relatively easy to design and implement and, in contrast to high-density arrays, interpretation can be carried out without extensive statistical expertise. The ultimate power of the microarray-based approaches, however, lies in the genome-wide copy number assessment of patient samples without any a priori knowledge of the genomic regions involved. To this end, high-density arrays have been constructed with the aim of performing such genome-wide analyses, initially with a resolution of 0.75 to 1.4 megabases (11, 12, 25, 26) and, more recently, with a 50-100 kilobase tiling resolution using approximately 32 000 clones (27). In addition to home-made arrays, there are now several companies that offer microarrays for genomic profiling, either for genome-wide analyses or for more targeted approaches (28-30).

Array-based genome-wide copy number screening is expected to have a profound impact on the diagnosis and genetic counseling of patients with congenital mental retardation and malformation syndromes. Its resolution for identifying chromosomal abnormalities reaches far beyond the detection limit of routine chromosome banding techniques (reviews: 1, 31, 32). In addition, the integration of BAC clones into genome browsers like those from Ensemble and UCSC allows for a direct inspection of candidate genes affected by the copy number alterations, which will be of major help in explaining the phenotype. The first attempts to implement these technologies into a routine diagnostic setting are currently ongoing. Initial studies using 1-Mb genome-wide BAC arrays (11, 33) have indicated that this approach can reveal causative microdeletions and/or duplications in 10-20% of patients with unexplained mental retardation and congenital malformations, and this percentage is likely to increase with the introduction of full-coverage 50- to 100-kb resolution arrays. These pilot studies already have provided insight into the quality and reproducibility aspects

Patient DNA

(e.g. DNA from a patient with Prader Willi Syndrome)

Cy3 labeling

Cy5 labeling

Sex mismatch hybridization vs Cy5 labeled reference pool

Sex mismatch hybridization vs Cy3 labeled reference pool

Genome-wide profile

Genome-wide profile

Chromosome 15 profile

Averaged genome-wide profile

Combined chromosome 15 profile

Averaged genome-wide profile

Identify deletions/duplications

Identify target genes on genome browser

Validate array findings (FISH/MLPA)

Test parents for de novo occurrence up of the array CGH procedure, the need for validation of microarray findings by independent technologies such as fluorescent in situ hybridisation (FISH) or multiplex ligation-dependent probe amplification (MLPA; 34), as well as the way to translate these molecular findings into clinical practice.

In this light it is important to note that these pilot studies have indicated that submicroscopic copy number alterations do not always have pheno-typic consequences, as in some of the cases identical alterations were found in either one of the normal parents. This notion has been substantiated by recent studies revealing the presence of large copy number variations (LCVs) in apparently normal individuals (35, 36). In addition, once it has been established that a copy number alteration has occurred de novo in a patient, it may be that this alteration has not been described before in the literature, posing serious problems for genetic counseling. However, in due time increasing numbers of these abnormalities will be documented, either in individual case reports (see for example 37) or in publicly available online databases, and thus further our understanding of the genetic basis of these disorders.

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