Figure 4

Mapping of PROP1 deletion. (a) Schematic of the PROP1 genomic organization (not drawn to scale). PCR/STS markers (open boxes) that were amenable to PCR amplification are boxed in bold. Upstream of PROP1 the only previously established STS marker that yielded a PCR product and that without any doubt could be assigned to the extended PROP1 locus was WI-16216. Inverse PCR of NspI restriction endonuclease digested and self-circulated DNA fragments revealed preservation of a NspI site 18.2 kb distal of the inverse primer pair R4inv (right-hand gray arrow head). Thus, the upstream breakpoint was narrowed down to a range of 3.8 kb (defined by the lack of the neighboring NspI site, open gray arrow head). (b) Long-range PCR with R4F and three additional PCR reverse probes (green boxes) yielded a product for P17R and P18R, but not for P15R. (c) Direct sequencing of the shortest product (R4F/P17R) disclosed the range of recombination (shown as frame in the electropherogram) within two AluY elements (gray boxes in panel a). (d) PCR products of the same molecular weight were obtained from genomic DNA of case 2, 3 and 4 using R4F and P17R primers suggesting equal deletion boundaries for these patients. PCR of the DNA of a previously published patient from India (PI) as well wild-type DNA (WT) did not result in the 1576 bp fragment.