Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome.

Abstract

Flow cytometry and recombinant DNA techniques have been used to obtain reagents for a molecular analysis of the Prader-Willi syndrome (PWS). HindIll totaldigest libraries were prepared in X phage Charon 21A from flow-sorted inverted duplicated no. 15 human chromosomes and propagated on recombination-proficient (LE392) and recBC-, sbcB-(DB1257) bacteria. (2)(3)(4)(5)(6).The most common chromosome aberration in PWS patients is a deletion of band 15q11.2 (2); however, the size of these deletions is variable and presumably some are undetectable by using current cytogenetic techniques (see Fig. 1). Other aberrations at 15q11.2 occur in 2-5% of PWS patients (6-8), including translocations and duplications (both tandem and inverted), the latter termed inv dup(15) chromosomes.This multiplicity of chromosome rearrangements involving 15qll->13 in the PWS suggests that the proximal long arm of chromosome 15 contains DNA sequences, such as tandem or inverted repeats, which could predispose this segment to structural instability.To define this region of the human genome on a molecular level, several DNA segments from the proximal long arm of chromosome 15 were isolated from X phage Charon 21A libraries constructed from HindIII-digested DNA obtained from flow-sorted (9) inv dup(15) chromosomes. Because these inverted duplicated chromosomes contain two copies of all sequences within 15pter->15ql3, we were able to more easily isolate DNA segments from the proximal long arm of chromosome 15. Using different cloning and insert screening strategies we determined that many of these sequences are genetically unstable in a recombination-proficient bacterial strain. Electron micrographs of heteroduplexes showed that unstable clones contain inverted repeats, which suggests at least one possible mechanism for their instability. In total, we describe in this report ll different cloned DNA segments that map to 15qll-43; 4 of 8 tested are deleted in one of two PWS patients with a deletion of 15qll.2. These data suggest that there is molecular heterogeneity between 15qll deletions in different PWS patients and that the cytogenetic instability of band 15q11.2 might be explained in terms ofthe types ofDNA sequences it contains. MATERIALS AND METHODSCell Lines. The lymphoblastoid cell lines used were from a karyotypically normal (46,XY) individual (MD-li), two patients with the PWS and different-sized deletions in 15qll.2 (DON-5, and DON-10), and two individuals with inv dup(15) chromosomes of different size [ALD-6 (10) and ALD-24 (11)] (Fig. 1).Bacterial Strains. Strain DB1257 is related to strain DB1170 (12) and was constructed by P1 transduction of CES 200 (13) to kanamycin resistance using a lysate prepared on NK 5857 (Trp::TnS SupF 58). The partial genotype of CES 200 is recBC-, sbcB-. Strain LE392 has been described (14).Construction of the inv dup(15) Library. Charon 21A libraries were created from 50 ng of HindIII-digested DNA from 6 million flow-sorted, inv dup(15) chromosomes, isolated from cell line ALD-24. Chromosomes were...

A molecular deletion study with southern hybridization on typical Prader-Willi syndrome (PWS) patients with various chromosome abnormalities involving 15q11–12 and on an atypical PWS patient with apparently normal karyotype

“…The results of the present molecular study also confirmed the previous findings by Donlon et al (1986) and by , except for the frequency of the molecular deletion. All our patients with an interstitial deletion of 15ql 1.2-q13 had a molecular deletion without any exceptions, while with the same probes, Donlon et al (1986) found a DNA deletion in only one of their two PWS patients with the similar chromosome deletion as seen in our patients.…”

“…The results of the present molecular study also confirmed the previous findings by Donlon et al (1986) and by , except for the frequency of the molecular deletion. All our patients with an interstitial deletion of 15ql 1.2-q13 had a molecular deletion without any exceptions, while with the same probes, Donlon et al (1986) found a DNA deletion in only one of their two PWS patients with the similar chromosome deletion as seen in our patients. A further study using four other chromosome-15 DNA clones (pIR4-3, pIRl0-1, p189-1 and p190-2) as probes by the same group of investigators (Lattet al, 1987) revealed a deletion at a molecular level in three of six PWS patients studied and probably in a PWS-like patient.…”

“…This finding indicates that the patient is not tetrasomic at least for both the pML34 and pTD3-21 sequences, and is consistent with the result of the study on a cell line containing a small inv dup(15) by Donlon et al (1986). One copy of each gene in our patient must have been derived from his one intact chromosome 15, while the other one copy may have come either from a deleted chromosome 15 or from an inverted duplicated chromosome.…”

“…Two probes, pTD3-21 and pML34 (kindly provided by Dr. S.A. Latt, Boston), are HindIII subclones of 2-phage libraries from a flow-sorted inverted duplication chromosome 15 [inv dup(15)(ql3)] (Lalande et al, 1985;Donlon et al, 1986). Both pTD3-21 and pML34 (a 2.2 kb and a 6.4 kb inserts, respectively) were mapped to the region 15q11.2 (Donlon et al, 1986).…”

“…Two probes, pTD3-21 and pML34 (kindly provided by Dr. S.A. Latt, Boston), are HindIII subclones of 2-phage libraries from a flow-sorted inverted duplication chromosome 15 [inv dup(15)(ql3)] (Lalande et al, 1985;Donlon et al, 1986). Both pTD3-21 and pML34 (a 2.2 kb and a 6.4 kb inserts, respectively) were mapped to the region 15q11.2 (Donlon et al, 1986). A 0.67 kb human prealbumin cDNA clone, pPA1 (Mita et al, 1984) assigned to 18qll-q12.3 (Jinno et al, 1986), was used as an internal standard for a comparative analysis of hybridization density.…”