Michalski, Antony; (1994) Molecular genetic analysis of a constitutional chromosome translocation in a patient with ganglioneuroblastoma. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study aimed to isolate and characterise the breakpoint junction fragment of a constitutional t(1;13)(q22;q12) from DG, a 5 year old boy with ganglioneuroblastoma, to see whether a gene critical to neural development has been disrupted. Somatic cell hybrids were constructed by fusing the lymphoblastoid cell line from DG with mouse 3T3 cells. One subclone contained a derivative chromosome 1 but no other material from chromosomes 1 and 13. Unique probes were isolated by Alu PCR cloning from a somatic cell hybrid containing 13pter to 13q14 as its only human component. Analysis of 180 clones yielded four probes which flanked the DG breakpoint. The probes were mapped by Southern hybridisation, PCR and FISH using cosmids isolated from a chromosome 13-specific cosmid library. All four probes were located in 13q12; 3'Alu66 mapped above and 3'Alu 78,71 and 62 mapped below, the DG breakpoint. The four 3'Alu probes and an STS for the oncogene, FLT1, were used to screen a YAC library. The fourteen YACs identified with these markers were characterised by PFGE, hybridisation and PCR fingerprinting. PCR analysis using STS for the ends of the YAC identified by FLT1, suggested that this YAC was chimeric, which was confirmed by FISH. Cosmid c12.2, mapping 250 kb distal to 3'Alu66 by FISH, has replaced FLT1 as the closest marker distal to the DG breakpoint. Two YACs were identified with 3'Alu66. An STS from the left hand end of one of the YACs (a 1.3 Mb, non-chimeric YAC) was used to isolated five further YACs. The resulting contig of seven YACs will allow high definition mapping of 13q12 and may include clones that contain the DG breakpoint junction fragment and, hence, a gene important in Nb development.

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