The evolution of molecular ruler analysis for characterizing telomere imbalances: from fluorescence in situ...

Submicroscopic telomere imbalances are a significant cause of mental retardation with or without other phenotypic abnormalities. We previously developed a set of unique telomere clones that identify imbalances in 3% to 5% of children with unexplained mental retardation and a normal karyotype. This targeted screening approach, however, does not provide information about the size or gene content of the imbalance. To enable such comprehensive characterization, a "molecular ruler" clone panel, extending up to 5 Mb proximal to the first telomere clone for each chromosome arm, was developed. This panel of clones was successfully used to delineate the size of unbalanced telomere aberrations in a fluorescence in situ hybridization assay. However, the fluorescence in situ hybridization analysis was quite labor-intensive, and for many cases, the imbalance extended beyond our 5-Mb coverage. Therefore, to develop a more efficient and comprehensive method for characterizing telomere imbalances, we developed a custom oligonucleotide microarray consisting of high-density coverage of all telomere regions as well as a whole-genome backbone. Overall, 44 pathogenic imbalances studied by fluorescence in situ hybridization or oligonucleotide array showed a size range of 400 kb to 13.5 Mb. In four of these, the array detected additional interstitial imbalances adjacent to the telomere imbalance, demonstrating the usefulness of added probe coverage. In 10 cases with benign imbalances inherited from a normal parent, the size ranged from 170 kb to 1.6 Mb. These results demonstrate that array comparative genomic hybridization will aid in more efficient and precise characterization of telomere imbalances leading to the development of gene dosage maps at human telomere regions for genotype/phenotype correlations.