A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2 based on high- throughput nonisotopic library screenings

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.     

Molecular analysis of 12 patients with the t(8;21) translocation and M2 acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom cytogenetic abnormality associated with acute myelogenous leukemia of the M2 subtype (FAB classification). The 8q- and 21q+ derivative chromosomes have previously been isolated in somatic cell hybrids and used to map the anonymous sequences D21S65 and D21S17, which were proximal and distal, respectively, to the breakpoint on chromosome 21. DNA from a series of 12 t(8;21) patients and 7 controls was analyzed by pulsed field gel electrophoresis. Physical linkage of probes D21S65 and D21S17 on a 2100 kb NruI fragment was established by partial digestion experiments. In all the patients, the translocation generated a rearranged D21S65 NruI fragment of 650 to 750 kb, suggesting heterogeneity in the breakpoints. This heterogeneity was confirmed by using BssHII, SacII, and EagI enzymes. Our results are consistent with the presence of a 100 Kb breakpoint cluster region on chromosome 21 encompassing the AML1 gene. Interestingly, in half of the patients, demethylation of an NruI site located 7 kb proximal to the last exon of the AML1 gene occurred on the nontranslocated chromosome 21.