THE STUDY OF X CHROMOSOME INACTIVATION IN MENTAL RETARDATION: COMPARATIVE ANALYSIS OF MOLECULAR-CYTOGENETIC AND POLYMERASE CHAIN REACTION-BASED TECHNIQUES IN RETT SYNDROME
Iourov IY1, Vorsanova SG2,*, Villard L3, Kolotii AD2, Yurov YB1,*
*Corresponding Author: Professor Yuri B. Yurov and Professor Svetlana G. Vorsanova, Cytogenetic LaboraČtory National Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe shosse 2, Moscow 113 152, Russia; Tel.: +7-095-952-89-90; Fax: +7-095-952-89-40; E-mail: y_yurov@hotmail.com; y_yurov@yahoo. com
page: 33

MATERIALS AND METHODS

Thirty-three patients, diagnosed as having the classical form of RTT [7], fulfilled the diagnostic criteria of RTT [12]. The presence of MECP2 mutations was detected in 70% of the Russian cohort of RTT patients; the data is published elsewhere [13]. Short-term peripheral blood lymphocyte cultures were used for cytogenetic and molecular-cytogenetic study for each of the 33 RTT girls and their parents.

To determine active (early replicating) and inactive (late replicating) X chromosomes, counter-staining of chromosomal preparations with fluorescent dye Hoechst 33258 (Sigma, Moscow, Russia), was done. The fluores­cent staining procedure allows direct visualization of rep­licating patterns of chromosomes after treatment of cell cultures with BrdU and following FISH experiments. Dif­ferential replication of X chromosomes was performed by the method described by Latt [14]. The replicating pat­terns of late replicating X chromosomes were analyzed after cultivation of cells in the presence of 100 mg/mL 5-BrdU (5-bromo-2ĺ-deoxyridine) added 6-7 hours before harvesting, followed by staining of chromosomes by fluo­rescent dye Hoechst 33258 (Sigma).

Molecular-cytogenetic (FISH) analysis was performed using the chromosome X-specific alphoid DNA probe pYAM10-40 [15,16] and the original rapid FISH protocol [16,17]. Cy3-labeled chromosome X-specific DNA probe (at a concentration of 50 ng/mL) in a standard hybridiza­tion mixture (50% formamide, 2X SSC, 10% dextran sulfate) was used. The probe and chromosomal DNA were denaturated simultaneously under a coverslip at 75░C for 5 min. Hybridization signals and differential replication patterns after Hoechst 33258 (Sigma) staining were de­tected simultaneously using a Leitz-Aristoplan fluoresence microscop (Leitz-Wetzlar, Wetzlar, Germany) with objec­tives of 63X or 100X, as described in detail earlier [17]. One hundred cells were analyzed for each patient. DNA was extracted and purified following routine procedures.

The X-inactivation status of X chromosomes was as­sessed by technique described in [11] with some modifica­tions [18]. Primers for PCR were designed in the (CAG)n flanking sequences of the HUMARA gene in intron 1. Primer sequences were: 5'-TCC AGA ATC TGT TCC AGA GCG TGC-3'; AR-P2: 5'-GCT GTG AAG GTT GCT GTT CCT CAT-3'. A total of 400 ng of DNA were digested by HpaII and ethanol precipitated. Polymerase chain reactions were performed with 100 ng of DNA, on both HpaII digested and undigested DNA for all subjects. The PCR conditions were as follows: 0.2 mmol/L dNTPs, 1.25 mmol/L Mg++, and 0.5 U Taq (GIBCO BRL, Gaithersburg, MD, USA) in a final volume of 20 mL. An­nealing temperature was 60░C for 30 cycles and the PCR products were analyzed by a LiCor sequencer (LICOR, Lincoln, NB, USA) using ONE-D Scan software.




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