Inactivation of chromosome X is the transcriptional silencing of one chromosome X in females, providing dosage compensation of X-linked gene expression [1]. The ratio of gene expression from paternal and maternal X chromosomes should be 50:50. As a result, mosaic X-link­ed gene expression in females is observed. But this process is generally random, therefore, some deviation or skewing from an expected mean of X-inactivation pattern is ob­served in the population [2,3]. In females with no history of genetic familial disorders, the percentage of females with extremely skewed X-inactivation [degree of skewing (DS) >90%] varies from 4 to 17% [4]. However, there are a number of observations indicating extremely skewed X-inactivation to be associated with different genetic dis­eases [2,3]. For example, in families with X-linked mental retardation (XLMR), skewed X-inactivation is considered to be a common feature, with incidences of markedly skewed X-inactivation (DS >80%) in about 50% of carri­ers [3]. The contribution of XLMR in congenital disorders is very important, as the occurrence spreads in up to 1 in 600 males [5]. X-Linked mental retardation is an ex­tremely heterogeneous group of genetic disorders; there­fore, investigation of X-inactivation skewing in families with different forms of XLMR is a current problem in medical genetics.

Among XLMR families, Rett syndrome [RTT (MIM #312750)] has particular significance. Rett syndrome is a severe neuro-degenerative disorder, affecting girls almost exclusively. Affected girls are born healthy, they appear to be normal until 6-18 months of age, then loss of ac­quired skills, language disabilities and stereotypic hand movement occur [6]. Frequency of RTT in the population is about 1 in 10,000 to 15,000 girls; it might thus be con­sidered as one of the most common causes of mental retar­dation in females. Rett syndrome-affected girls comprise 2.5% of all mentally retarded children in the Russian population [7]. It has been demonstrated that mutations in the MECP2 gene (Xq28) are the primary cause of RTT; mutations have been identified in 70-90% of sporadic cases and about 50% of familial cases [8]. The study of X-chromosome inactivation patterns in RTT is considered an important and informative milestone of RTT investigation. Firstly, skewed X-inactivation affects clinical appearance of RTT, and can lead to less or more severe manifestation of disease, depending on the direction of X-inactivation skewing [7,9]. Secondly, the presence of a mutation in a child affected by RTT, in combination with skewed X-inactivation in the mother, results in a 50% risk of an RTT-affected child at birth. This fact has to be considered in RTT prevention and prenatal diagnosis [10]. Thus, X-inactivation assay provides important data for genotype-phenotype correlations in RTT carriers and affected girls [7].

A commonly used technique for determining of skewed X-inactivation is a polymerase chain reaction (PCR)-based method for determining methylation patterns of polymorphic (CAG)_n repeats in intron 1 of the human androgen receptor gene (HUMARA) [11]. Recently, a molecular-cytogenetic X-inactivation assay, based on step-wise application of differential replication staining and fluorescence in situ hybridization (FISH) techniques, to identify the inactivation status of the paternal and mater­nal chromosome X in RTT girls, was described [9]. Appli­cation of FISH with an original alphoid chromosome X-specific DNA probe, permits the differentiation of paternal and maternal X chromosomes according to the inactiva­tion status (active or inactive). The aim of the present study was the comparison of these two techniques for the development of molecular-cytogenetic assays, allowing precise analysis of X-inactivation in interphase and meta­phase cells.