Mechanisms and consequences of small supernumerary marker chromosomes: from Barbara McClintock to modern genetic-counseling issues.

Supernumerary marker chromosomes (SMCs) are common, but their molecular content and mechanism of origin are often not precisely characterized. We analyzed all centromere regions to identify the junction between the unique chromosome arm and the pericentromeric repeats. A molecular-ruler clone panel for each chromosome arm was developed and used for the design of a custom oligonucleotide array. Of 27 nonsatellited SMCs analyzed by array comparative genomic hybridization (aCGH) and/or fluorescence in situ hybridization (FISH), seven (approximately 26%) were shown to be unique sequence negative. Of the 20 unique-sequence-positive SMCs, the average unique DNA content was approximately 6.5 Mb (range 0.3-22.2 Mb) and 33 known genes (range 0-149). Of the 14 informative nonacrocentric SMCs, five (approximately 36%) contained unique DNA from both the p and q arms, whereas nine (approximately 64%) contained unique DNA from only one arm. The latter cases are consistent with ring-chromosome formation by centromere misdivision, as first described by McClintock in maize. In one case, a r(4) containing approximately 4.4 Mb of unique DNA from 4p was also present in the proband's mother. However, FISH revealed a cryptic deletion in one chromosome 4 and reduced alpha satellite in the del(4) and r(4), indicating that the mother was a balanced ring and deletion carrier. Our data, and recent reports in the literature, suggest that this "McClintock mechanism" of small-ring formation might be the predominant mechanism of origin. Comprehensive analysis of SMCs by aCGH and FISH can distinguish unique-negative from unique-positive cases, determine the precise gene content, and provide information on mechanism of origin, inheritance, and recurrence risk.

The chromosomal polymorphism linked to variation in social behavior in the white-throated sparrow (Zonotrichia albicollis) is a complex rearrangement and suppressor of recombination.

Variation in social behavior and plumage in the white-throated sparrow (Zonotrichia albicollis) is linked to an inversion polymorphism on chromosome 2. Here we report the results of our comparative cytogenetic mapping efforts and population genetics studies focused on the genomic characterization of this balanced chromosomal polymorphism. Comparative chromosome painting and cytogenetic mapping of 15 zebra finch BAC clones to the standard (ZAL2) and alternative (ZAL2(m)) arrangements revealed that this chromosome is orthologous to chicken chromosome 3, and that at a minimum, ZAL2 and ZAL2(m) differ by a pair of included pericentric inversions that we estimate span at least 98 Mb. Population-based sequencing and genotyping of multiple loci demonstrated that ZAL2(m) suppresses recombination in the heterokaryotype and is evolving as a rare nonrecombining autosomal segment of the genome. In addition, we estimate that the first inversion within the ZAL2(m) arrangement originated 2.2+/-0.3 million years ago. Finally, while previously recognized as a genetic model for the evolution of social behavior, we found that the ZAL2/ZAL2(m) polymorphism also shares genetic and phenotypic features with the mouse t complex and we further suggest that the ZAL2/ZAL2(m) polymorphism is a heretofore unrecognized model for the early stages of sex chromosome evolution.

Enhanced detection of clinically relevant genomic imbalances using a targeted plus whole genome oligonucleotide microarray.

PURPOSE: Array comparative genomic hybridization is rapidly becoming an integral part of cytogenetic diagnostics. We report the design, validation, and clinical utility of an oligonucleotide array which combines genome-wide coverage with targeted enhancement at known clinically relevant regions. METHODS: Probes were placed every 75 kb across the entire euchromatic genome to establish a chromosomal "backbone" with a resolution of approximately 500 kb, which is increased to approximately 50 kb in targeted regions. RESULTS: For validation, 30 samples showed 100% concordance with previous G-banding and/or fluorescence in situ hybridization results. Prospective array analysis of 211 clinical samples identified 33 (15.6%) cases with clinically significant abnormalities. Of these, 23 (10.9%) were detected by the "targeted" coverage and 10 (4.7%) by the genome-wide coverage (average size of 3.7 Mb). All abnormalities were verified by fluorescence in situ hybridization, using commercially available or homebrew probes using the 32K bacterial artificial chromosome set. Four (1.9%) cases had previously reported imbalances of uncertain clinical significance. Five (2.4%) cases required parental studies for interpretation and all were benign familial variants. CONCLUSIONS: Our results highlight the enhanced diagnostic utility of a genome-wide plus targeted array design, as the use of only a targeted array would have failed to detect 4.7% of the clinically relevant imbalances.

The evolution of molecular ruler analysis for characterizing telomere imbalances: from fluorescence in situ hybridization to array comparative genomic hybridization.

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.

Cobalt enhances DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells.

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.