Human Molecular Genetics Advance Access originally published online on September 2, 2003
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Human Molecular Genetics, 2003, Vol. 12, No. 21 2817-2825
DOI: 10.1093/hmg/ddg301
© 2003 Oxford University Press
A novel sequence-based approach to localize translocation breakpoints identifies the molecular basis of a t(4;22)
1Division of Human Genetics and Molecular Biology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA and 2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
Received July 2, 2003; Revised August 15, 2003; Accepted August 25, 2003
Low copy repeats (LCRs) located in 22q11.2, especially LCR-B, are susceptible to rearrangements associated with several relatively common constitutional disorders. These include DiGeorge syndrome, Velocardiofacial syndrome, Cat-eye syndrome and recurrent translocations of 22q11 including the constitutional t(11;22) and t(17;22). The presence of palindromic AT-rich repeats (PATRRs) within LCR-B of 22q11.2, as well as within the 11q23 and 17q11 regions, has suggested a palindrome-mediated, stem-loop mechanism for the generation of such recurring constitutional 22q11.2 translocations. The mechanism responsible for non-recurrent 22q11.2 rearrangements is presently unknown due to the extensive effort required for breakpoint cloning. Thus, we have developed a novel fluorescence in-situ hybridization and primed in-situ hybridization (PRINS) approach and rapidly localized the breakpoint of a non-recurrent 22q11.2 translocation, a t(4;22). Multiple primer pairs were designed from the sequence of a 200 kb, chromosome 4, breakpoint-spanning BAC to generate PRINS probes. Amplification of adjacent primer pairs, labeled in two colors, allowed us to narrow the 4q35.1 breakpoint to a 6.7 kb clonable region. Application of our improved PRINS protocol facilitated fine-mapping the translocation breakpoints within 4q35.1 and 22q11.2, and permitted rapid cloning and analysis of translocation junction fragments. To confirm the PRINS localization results, PCR mapping of t(4;22) somatic cell hybrid DNA was employed. Analysis of the breakpoints demonstrates the presence of a 554 bp palindromic sequence at the chromosome 4 breakpoint and a 22q11.2 location within the same PATRR as the recurrent t(11;22) and t(17;22). The sequence of this breakpoint further suggests that a stem-loop secondary structure mechanism is responsible for the formation of other, non-recurrent translocations involving LCR-B of 22q11.2.
* To whom correspondence should be addressed. Tel: +1 2155903856; Fax: +1 2155903764; Email: beverly{at}mail.med.upenn.edu
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