Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11–13) by YAC cloning and FISH analysis

Abstract

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.

Tissue-specific and allele-specific replication timing control in the imprinted human Prader-Willi syndrome region.

“…Genes that are expressed differentially on the basis of their chromosomal origin and tissue type, such as SNRPN, offer the ideal system to study these issues. Approximately 3-4 Mb of contiguous DNA including the PWS/ AS region located on human chromosome 15ql 1-13 has been cloned in YACs and 750 kb of contiguous sequences in the SNRPN region have been cloned subsequently in cosmids [Kuwano et al 1992;Mutirangura et al 1993a;Nakao et al 1994). Prior studies on replication in the PWS/AS region were directed at assessing the relationship between genetic imprinting and asynchronous allele-specific replication timing by analysis of a total of eight phage or cosmid clones distributed along the 3-to 4-Mb PWS/AS interval (Kitsberg et al 1993a;Knoll et al 1994).…”

Detection of aberrant DNA methylation in unique Prader — Willi syndrome patients and its diagnostic implications

“…Deletions in PWS and AS typically affect a region of 4-5 Mb, which includes the loci D15S9, D15S11, D15S13, D15S63, SNRPN, D15S10, D15S113, GABRB3, D15S97, GABRA5, D15S78 and D15S12 (7). Recently, we have identified a pair of PWS sibs (family S) who have a deletion of less than 300 kb (8).…”

Fish mapping of a translocation breakpoint at 6q21 (or q22) in a patient with heterotaxia

“…According to a YAC contig map by Centre d'Etude du Polymorphisme Humain (CEPH) (Chumakov et al, 1995), 33 CEPH-YAC clones were chosen from a region covering a breakpoint, 6q21 or q22, of a translocation in the patient. AIu-PCR was performed as described previously (Kuwano et al, 1992). DNA from each YAC was amplified for 30 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 4 rain, using the primers (5'-3'): 2484, AGGAGTGAGCCACCGCACCCAG-…”