Figure 1.

Genomic organization of ISL1 and 20-kb deletion. The genomic organization of the six exons of human ISL1 is shown at the top of the Figure. (1) Four MLPA probes were designed to amplify exons 1, 5, and 6 and are designated by arrows as E1, E5, E6_1, and E6_2, respectively. Each exon showed a loss of copy number by MLPA (data not shown). (2) Two array-CGH experiments with Agilent 1 million features microarrays were performed: hybridization with Cy3-labeled test versus. Cy5-labeled reference DNA (Log₂ values plotted by black circles) and Cy5-labeled test versus. Cy3-labeled reference probes (“dye swap” whose Log₂ values are plotted by gray squares). The Log₂(Test/Reference) values were plotted for each microarray oligonucleotide feature at each chromosomal locus. Negative Log₂ values of 14 consecutive probes (black circles) are seen in a zoomed-in array-CGH plot encompassing ISL1 on chromosome 5, indicating a deletion. A nearly mirror image of these Log₂ values is observed from the dye-swap experiment (gray squares). The two outside dashed vertical lines indicate the positions of the innermost oligonucleotide features that showed normal Log₂ values. The two inner dashed vertical lines show the positions of the two outermost of the 14 oligonucleotide features that showed negative Log₂ values. (3) Thirty-four genotyped SNPs are shown relative to the genomic organization of ISL1. (4) We designed several PCRs to identify the chromosomal breakpoints. PCR1: a 21.5-kb product would be expected from the normal chromosome 5 using a primer set, 5p1-F and 3p3-R, indicated by black circles, and whose sequences are listed in Table 2. A 1.3-kb amplicon was generated (see also Figure 2 lane 2) because these primers were brought into proximity of each other by the deletion of the intervening 20-kb chromosomal segment. PCR2 and 3: No PCR product was generated using the same forward primer (5p1-F), in combination with two different reverse primers, 3p2-R and 3p1-R, located inside the right pair of vertical dashed lines. This indicates that these two reverse primers are located within the deleted region.