title: A novel sequence-based approach to localize translocation breakpoints identifies the molecular basis of a t(4;22).
abstract: 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.
authors.affiliation: Division of Human Genetics and Molecular Biology, The Children's Hospital of Philadelphia, PA 19104, USA.
authors.forename: Manjunath A
authors.initials: MA
authors.lastname: Nimmakayalu
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Gotter
authors.forename: Tamim H
authors.initials: TH
authors.lastname: Shaikh
authors.forename: Beverly S
authors.initials: BS
authors.lastname: Emanuel
mesh_terms: D020856:AT Rich Sequence / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002904:Chromosomes, Human, 21-22 and Y / Q000235:genetics*; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008214:Lymphocytes / Q000648:ultrastructure; D008969:Molecular Sequence Data; D020401:Primed In Situ Labeling; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014178:Translocation, Genetic / Q000235:genetics*