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
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DISCUSSION

The significance of X-inactivation for RTT clinical manifestations and inheritance was commonly reported [9,10,18]. There are a number of results concerning the frequency of cases with skewed X-inactivation in RTT. The frequency of RTT girls with skewed X-inactivation varies from 21% (DS >80%) [10] to 43% (DS >75%) [19], while in normal females, it can vary from 4 to 17% (DS >90%) and from 17 to 38% (DS >75%). These differ­ences could probably be explained by a different interpre­tation of the results rather that technical errors. Some au­thors consider a skewed X-inactivation of 90:10 or more, while other authors chose the levels of 80:20 or 75:25 as the data for skewed X-inactivation [10,19]. For example, our study of a Russian cohort of 28 RTT girls with MECP2 mutations, indicated an extremely skewed X-inactivation (90:10 or more) in only one girl [13]. How­ever, if the level of skewed X-inactivation was lowered to 80:20, six girls (or 21%) should be considered as having skewed X-inactivation [10]. It remains unclear if there is an increase of skewed X-inactivation frequency in RTT Thus, the study of skewed X-inactivation is of great inter­est for further investigations, due to its influence on RTT girl phenotypes.

It was shown that MECP2 mutations are almost exclu­sively of paternal origin [20]. Here, a prevalence of skew­ed X-inactivation in RTT girls with homologous highly heteromorphic X chromosomes and preferential inactiva­tion of the maternal chromosome, were detected by a molecular-cytogenetic approach, and confirmed by the PCR-based technique (seven out of eight girls). Thus, we propose that skewed X-inactivation against maternal (without mutation) chromosome X is a common feature of the classical form of RTT with sporadic incidence of MECP2 mutations.

We have shown that original chromosome-specific DNA probes allow identification of the paternal or mater­nal origin of active (inactive) chromosome X, based on homologous heteromorphism [9]. On comparing the results obtained by FISH and PCR, we found the FISH technique provided practically the same results as PCR. Therefore, molecular-cytogenetic assay using metaphase chromo­somes of individuals with heteromorphic chromosome X could be applied for X-inactivation analysis. This ap­proach could probably be useful for interphase molecular-cytogenetic analysis.

 

 

Figure 1. Fluorescence in situ hybridization with chromosome X-specific alphoid DNA probe pYAM10-40. Chromosome X-specific DNA probe (red colour) was labeled by Cy3-dCTP and visualized by a fluorescent microscope as described earlier. Interphase nuclei and chromosomes were counter-stained by Hoechst 33258 (Sigma) (blue colour). a) (upper) Homologous chromo­some X in an RTT girl without differences in amount of centromeric alphoid DNA (no homologous hetero­morphism). b) (bottom) Chromosome X of an RTT girl with different amount of centromeric alphoid DNA (chro­mosome X heteromorphism). Chromosome X with a small centromeric heterochromatic region replicates late, as indicated by differential staining using the BrdU + Hoechst 33258 (Sigma) technique.

 

Figure 2. Results of the X-inactivation PCR-based assay (numbers 1-8 correspond to the number of patients in Table 1). The lanes on the left are undigested and the lanes on the right are digested by HpaII.

 

 

For future analyses of X-inactivation by molecular-cytogenetic techniques we propose to use two-color FISH: an alphoid DNA probe will be used in order to differenti­ate the parental origin of chromosome X by one color, and the XIST RNA probe will mark the active/inactive chro­mosome X by another color. This potential approach will allow analysis of differentiated cell populations; for exam­ple, in complex tissues containing different types of cells (neurons and glial cell populations in the brain, fetal cells etc.). The use of original DNA probes and FISH as a base for a molecular-cytogenetic X-inactivation assay is appli­cable for further investigation and dissection of XLMR among the heterogeneous group of idiopathic mental retar­dation.




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