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

BACKGROUND: Small 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...

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Autores principales: Castronovo, Chiara, Valtorta, Emanuele, Crippa, Milena, Tedoldi, Sara, Romitti, Lorenza, Amione, Maria Cristina, Guerneri, Silvana, Rusconi, Daniela, Ballarati, Lucia, Milani, Donatella, Grosso, Enrico, Cavalli, Pietro, Giardino, Daniela, Bonati, Maria Teresa, Larizza, Lidia, Finelli, Palma
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176193/
https://www.ncbi.nlm.nih.gov/pubmed/24171812
http://dx.doi.org/10.1186/1755-8166-6-45
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author Castronovo, Chiara
Valtorta, Emanuele
Crippa, Milena
Tedoldi, Sara
Romitti, Lorenza
Amione, Maria Cristina
Guerneri, Silvana
Rusconi, Daniela
Ballarati, Lucia
Milani, Donatella
Grosso, Enrico
Cavalli, Pietro
Giardino, Daniela
Bonati, Maria Teresa
Larizza, Lidia
Finelli, Palma
author_facet Castronovo, Chiara
Valtorta, Emanuele
Crippa, Milena
Tedoldi, Sara
Romitti, Lorenza
Amione, Maria Cristina
Guerneri, Silvana
Rusconi, Daniela
Ballarati, Lucia
Milani, Donatella
Grosso, Enrico
Cavalli, Pietro
Giardino, Daniela
Bonati, Maria Teresa
Larizza, Lidia
Finelli, Palma
author_sort Castronovo, Chiara
collection PubMed
description BACKGROUND: Small 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. RESULTS: By 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). CONCLUSIONS: Our 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.
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spelling pubmed-41761932014-09-27 Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes Castronovo, Chiara Valtorta, Emanuele Crippa, Milena Tedoldi, Sara Romitti, Lorenza Amione, Maria Cristina Guerneri, Silvana Rusconi, Daniela Ballarati, Lucia Milani, Donatella Grosso, Enrico Cavalli, Pietro Giardino, Daniela Bonati, Maria Teresa Larizza, Lidia Finelli, Palma Mol Cytogenet Research BACKGROUND: Small 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. RESULTS: By 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). CONCLUSIONS: Our 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. BioMed Central 2013-10-30 /pmc/articles/PMC4176193/ /pubmed/24171812 http://dx.doi.org/10.1186/1755-8166-6-45 Text en Copyright © 2013 Castronovo et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Castronovo, Chiara
Valtorta, Emanuele
Crippa, Milena
Tedoldi, Sara
Romitti, Lorenza
Amione, Maria Cristina
Guerneri, Silvana
Rusconi, Daniela
Ballarati, Lucia
Milani, Donatella
Grosso, Enrico
Cavalli, Pietro
Giardino, Daniela
Bonati, Maria Teresa
Larizza, Lidia
Finelli, Palma
Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
title Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
title_full Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
title_fullStr Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
title_full_unstemmed Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
title_short Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
title_sort design and validation of a pericentromeric bac clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176193/
https://www.ncbi.nlm.nih.gov/pubmed/24171812
http://dx.doi.org/10.1186/1755-8166-6-45
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