Chromosomal preparations of 36 DS children and their parents were obtained from blood lymphocyte cell lines. Metaphase chromosomes were prepared using stan­dard techniques of colcemid treatment, hypotonic treat­ment and methanol/acetic acid fixation (3:1) three times for 15 min. before being placed onto slides. Standard karyotyping and detection of chromosomal aberrations with classical GTG-banding were performed. Cytogenetic chromosome analysis was performed on peripheral blood lymphocytes, using the technique described before [6] The morphological pecularities of chromosome 21 were stud­ied (cytologic markers: specificity or presence of satel­lites, short arm (p) form, etc.) in order to determine the parental origin of chromosome 21 (Fig. 1). Centromeric alphoid chromosome 21-specific DNA probe (aRI-6) cor­responding to alphoid array subsets a-21-1 from the col­lection at the Cytogenetic Laboratory, National Center of Mental Health, Moscow, Russia, was used for FISH [9-11].

In situ hybridizaton was performed as described in detail previously [9,11] using a biotinilated DNA probe. Slides with fixed cells were treated for 2 min. with 70% formamide, 2X SSC at 72°C for chromosomal DNA dena­turation, dehydrated in 70, 80, 100% ethanol solutions for 2 min. each and air-dried. DNA probes with a concentra­tion of 20 ng in 10 mL of hybridization solution were denaturated at 100°C for 5 min., placed on ice and then applied to the slides. Fluorescence in situ hybridizations, at high stringency conditions, were performed at 42°C overnight, followed by washing of the slides in 50% formamide, 2X SSC for 15 min. at 42°C. Detection of the biotin-labeled probe was performed by the use of one layer of fluorescein-avidin (Sigma, Moscow, Russia) according to the method of Pinkel et al. [12]. Slides were mounted in antifade solution [0.2% p-phenilenediamine (Sigma) in 80% glycerol, 20 mM Tris-HCl, pH 8.0], and 400 ng/mL propidium iodide and 200 ng/mL DAPI (4',6-diamidino-2-phenylindole 2HCl) as counter-staining. Identification of chromosomes was performed by Q-banding, produced by DAPI.

Figure 1. Detection of chromosome 21 origin by cytogenetic analysis. A) Case of maternal meiosis I or II non-disjunction (family #20). B) Uninformative case (family #1). C) Case of maternal meiosis I non-disjunction (family #10)