Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes

Abstract

BackgroundSmall supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA).To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms.ResultsBy FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19).ConclusionsOur results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype–phenotype correlations.

Small Supernumerary Marker Chromosome May Provide Information on Dosage-insensitive Pericentric Regions in Human

“…No relevant cross-hybridizations or background could be observed. The idea for how to establish this kind of probeset was previously stated in a comment for a paper published by Castronovo and colleagues in 2013 [ 19 , 20 ].…”

“…sSMCs of different sizes present in the same patient [ 14 , 24 ], previously, we established already a probe sets called pericentric-ladder-FISH (PCL-FISH) with a resolution of ~10 Mbps [ 25 ]. Also two similar probe sets were published before in 2007 and 2013 [ 19 , 26 ]. However, a probe set for the pericentric region for narrowing down the dosage insensitive regions of all human chromosome arms was not available yet.…”

Preservation of truncal genomic alterations in clear cell and papillary renal cell carcinomas with sarcomatoid features: An intra‐ and intertumoral, multifocal fluorescence in situ hybridization analysis reveals limited genetic heterogeneity

“…However, prior studies have shown this can occur in an acquired fashion in hematopoietic and solid organ malignancies . Unfortunately, these LOH or copy number‐neutral changes could not be detectable by current FISH platforms, despite using a BAC clone probe (RP11‐572M14, chr3: 10,011,785‐10,180,797) that covers the entire sequence of VHL gene (VHL, chr3:10,141,008‐10,153,670) . Further molecular analysis, particularly by high‐resolution single‐nucleotide polymorphism microarrays, may serve to identify an even higher percentage of alterations in 3p chromosomal regions in future studies …”

“…[70][71][72] Unfortunately, these LOH or copy number-neutral changes could not be detectable by current FISH platforms, despite using a BAC clone probe (RP11-572M14, chr3: 10,011,785-10,180,797) that covers the entire sequence of VHL gene (VHL, chr3:10,141,008-10,153,670). 16,17,25,26,69,73 Further molecular analysis, particularly by high-resolution single-nucleotide polymorphism microarrays, may serve to identify an even higher percentage of alterations in 3p chromosomal regions in future studies. 8,16,17,26,68 RCC with sarcomatoid features is an aggressive, heterogeneous subset of tumors that frequently present at a high clinical stage.…”

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

“…The euchromatic material in sSMC can be detected by a microarray analysis. A set of pericentric core probes for each arm of human chromosomes has been validated for characterizing unambiguously the chromosomal origin of sSMC and the level of mosaicism (Castronovo et al, 2013 ).…”