Abstract for presentation at 11th International Congress of Human Genetics

Rapid translocation breakpoint mapping by ultra-high resolution array painting facilitates direct amplification and sequencing of junction fragments

  • Miss Deborah Burford, The Wellcome Trust Sanger Institute, Hinxton, Cambs, UK, United Kingdom
  • Dr Susan Gribble, The Wellcome Trust Sanger Institute, Hinxton, Cambs, UK, United Kingdom
  • Dr Elena Prigmore, The Wellcome Trust Sanger Institute, Hinxton, Cambs, UK, United Kingdom
  • Mr Dimitrios Kalaitzopoulos, The Wellcome Trust Sanger Institute, Hinxton, Cambs, UK, United Kingdom
  • Dr Rebecca Selzer, NimbleGen Systems Inc, 1 Science Court, Madison, WI 53711, USA, United States
  • Dr Todd Richmond, NimbleGen Systems Inc, 1 Science Court, Madison, WI 53711, USA, United States
  • Ms Bee Ling Ng, The Wellcome Trust Sanger Institute, Hinxton, Cambs, UK, United Kingdom
  • Dr Nigel Carter, The Wellcome Trust Sanger Institute, Hinxton, Cambs, UK, United Kingdom

    • 1 in 2000 prenatal samples show an apparently balanced de novo reciprocal chromosome translocation, with 6.1% of these being associated with a phenotypic abnormality1. Traditional methods of translocation breakpoint mapping such as fluorescence in situ hybridisation of genomic clones or creating somatic cell hybrids followed by restriction digest and Southern Blotting are laborious, and to date, relatively few breakpoints have been published at sequence level. Recently, the speed of translocation breakpoint mapping has been improved by the procedure of array painting2. In array painting, derivative chromosomes are flow-sorted and competitively hybridised onto a large insert clone array. Analysis of the clones spotted on the array reveals a shift between low/high ratios mapping the chromosome breakpoint at a resolution defined by size and density of probes on the array.
    Here, we report a further development of array painting using custom-made ultra-high resolution oligonucleotide arrays such that the precision of breakpoint mapping is sufficient to allow the direct use of long range PCR (LR PCR) to amplify junction fragments for sequencing.
    We present the mapping and sequencing of reciprocal translocation breakpoints in 4 patients. Sequential array painting using Whole Genome Tile Path arrays to identify breakpoint spanning clones followed by targeted ultra-high resolution oligonucleotide arrays (NimbleGen) refined the 8 translocation breakpoints to regions ranging between 50 basepairs (bp) and 7 Kbp. In all cases, junction fragments were amplified by LR PCR and sequenced. Analysis of the sequence suggested that these rearrangements were mediated by non-homologous end-joining. Two of the 8 breakpoints directly disrupt a gene; the first being a good candidate for the patient’s phenotype, the second being a novel gene with no known function.

    1. Warburton D. Am J Hum Genet 1991;49(5):995-1013.
    2. Fiegler H, Gribble SM, et al. J Med Genet 2003;40(9):664-70.

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