title: Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes.
abstract: 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.
authors.affiliation: Laboratorio di Citogenetica Medica e Genetica Molecolare, IRCCS Istituto Auxologico Italiano, via Ariosto 13, 20145, Milano, Italy. c.castronovo@auxologico.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Castronovo
authors.forename: Emanuele
authors.initials: E
authors.lastname: Valtorta
authors.forename: Milena
authors.initials: M
authors.lastname: Crippa
authors.forename: Sara
authors.initials: S
authors.lastname: Tedoldi
authors.forename: Lorenza
authors.initials: L
authors.lastname: Romitti
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Amione
authors.forename: Silvana
authors.initials: S
authors.lastname: Guerneri
authors.forename: Daniela
authors.initials: D
authors.lastname: Rusconi
authors.forename: Lucia
authors.initials: L
authors.lastname: Ballarati
authors.forename: Donatella
authors.initials: D
authors.lastname: Milani
authors.forename: Enrico
authors.initials: E
authors.lastname: Grosso
authors.forename: Pietro
authors.initials: P
authors.lastname: Cavalli
authors.forename: Daniela
authors.initials: D
authors.lastname: Giardino
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Bonati
authors.forename: Lidia
authors.initials: L
authors.lastname: Larizza
authors.forename: Palma
authors.initials: P
authors.lastname: Finelli